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Synthesis of three oxytocin analogs related to [1-deaminopenicillamine]oxytocin prossessing antioxytocic activity

Raymond J. Vavrek, Martha F. Ferger, G. Ashley Allen, Daniel H. Rich, Alfred T. Blomquist, and Vincent Du Vigneaud
J. Med. Chem., 1972, 15 (2), 123-126• DOI: 10.1021/jm00272a001 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.acs.org on April 25, 2009

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036

Journal of Medicinul Chemistry
0 Coprrighr I072 bb rhe American ChemicalSocrer)

VOLUME 15, NUMBER 2

FEBRUARY 1972

Synthesis of Three Oxytocin Analogs Related to [ 1-Deaminopenicillamine]oxytocin Possessing Antioxytocic Activity?
Raymond J. Vavrek, Martha F. Ferger, G. Ashley Allen,.$ Daniel H. Rich? Alfred T. Blomquist," and Vincent du Vigneaud*
Department of Chemistry, Cornel1 University, Ithaca, New York 14850. Received July 21, 1971

[ 1-Mercaptodimethylacetic acidloxytocin, [l+'-mercapto-a,a-dimethylpropionic acidloxytocin, and [ 1P-mercapto-0,P-diethylpropionic acidloxytocin, analogs of deaminopenicillamine-oxytocin, have been synthesized from a common protected octapeptide resin intermediate and purified by sequential gel filtration on Sephadex G-15 in 50% AcOH and 0.2 N AcOH. The 3 compounds were devoid of oxytocic and avian vasodepressor activities, but all showed a significant degree of inhibition of the effects of oxytocin on the isolated rat uterus and on avian blood pressure. Each compound showed approximately the same inhibitory potency in both biological systems when compared to that of deaminopenicillamineoxytocin. [I-Mercaptodimethylacetic acidloxytocin and [ 1-0-mercapto-&,a-dimethylpropionicacidloxytocin had about 20% and 3 3 to 47%,respectively, of the inhibitory potency of deaminopenicillamineoxytocin. [I-0-Mercapto$,P-diethylpropionic acidloxytocin had approximately twice the inhibitory potency of deaminopenicillamine-oxytocin ( [ l -P-mercapto-p,P-dimethylpropionic acidloxytocin in both systems. In a series of studies bearing on the specificity of the halfcystine residue in position 1 of oxytocin (Figure 1) in relation to the pharmacol behavior of the hormone, this residue was formally replaced with an L-penicillamine (L-P,pdimethylcysteine) residue.' The resulting [l-~-penicillamine]oxytocin was devoid of oxytocic activity but instead turned out to be a potent inhibitor of the oxytocic activity of oxyt ~ c i n . ' -[l-D-Penicillamine]o~ytocin'-~ ~ was also found to possess antioxytocic activity, but to a lesser extent. Both diastereoisomers also showed an inhibitory effect on the avian vasodepressor (AVD) effect of oxytocin. Following the unexpected finding that the presence of two Me groups on the B-carbon of the half-cystine residue converts oxytocin to an antioxytocic agent, studies were initiated on related analogs to see what modifications in the 1 position would enhance or diminish the antioxytocic activity. Since the formal replacement of the free amino group of oxytocin with H gives an analog, deamino-oxytocin ([l-0-mercaptopropionic acidloxytocin), that is even more potent in its oxytocic activity than ~ x y t o c i n ,the ~,~ deamino analog of penicillamine-oxytocin was prepared.' Deaminopenicillamine-oxytocin ([1-p-mercapto-p,P-dimethylpropionic acidloxytocin) had as potent an antioxytocic effect as L-penicillamine-oxytocin and likewise showed an anti-AVD effect.'-' It is of interest that [l-deaminopenicillamined8]oxytocin, in which the deaminopenicillamine residue has been replaced with a fully deuterated deaminopenicillamine residue, has the same degree of antioxytocic activity as the protio analog.6
?This work was supported in part b y U. S. Public H e a l t h S e M G Grant HE 11680, by NIH Training Grant 5-T01-GM00834 and by a grant from the Mobil Foundation. All optically active amino acid residues are of the L variety unless otherwise indicated. SNIH predoctoral fellow. SNIH training grant predoctoral fellow.

To ascertain whether both p-Me groups are necessary to produce antioxytocic activity, [1-LQ-mercapto-@methylpropionic acidloxytocin and [1-D-0-mercapto-0-methylpropionic acidloxytocin were studied.' Neither of these diastereoisomers having only one p-Me substiuent showed antioxytocic or anti-AVD activity, but both possessed 5-10% of the oxytocic and AVD potencies of deamino-oxytocin. With the demonstration that two p-Me substituents are required for antioxytocic activity, we became interested in whether two such substituents on the a-C would have a like effect. We have therefore synthesized [l-P-mercapto-a,a-dimethylpropionic acidloxytocin, as described in the Experimental Section. The compd, which had no detectable oxytocic# or AVD** activity, was found to possess about half of the antioxytocic potency and about one-third of the antiAVD potency of deaminopenicillamine-oxytocin. Although this compd is not as potent as the P-substitued analog, the results demonstrate that the antioxytocic activity is not confined to substitution on the 0-C. It is to be noted that the analogs so far discussed all possess the 20-membered cyclic disulfide ring present in oxytocin and deamino-oxytocin. It has been found that the 19membered ring analog of deamino-oxytocin, namely [l -mercaptoacetic acidloxytocin, possesses 25 units/mg of oxytocic and 4 units/mg of AVD activity.l 3 Deamino-oxytocin possesses 803 and 975 units/mg of oxytocic and AVD activity, r e s ~To . ~ find out whether substitution of Me groups
#The oxytocic response t o oxytocin o f isolated uteri from Sherman albino rats (200-250 g) in natural estrus was measured by the method o f Holton,'as modified by M u n ~ i c kwith , ~ the use o f Mg-free van Dyke-Hastings s o h as the bathing fluid. Isotonic contractions were recorded with a Harvard heart/smooth muscle transducer and Grass polygraph Model 5. **Avian vasodepressor responses were measured o n conscious roosters (-2500 g) by the method o f Coon," as described in "The Pharmacopeia o f the United States of America,"" as modified by Munsick, et al.

123

124

Journal of Medicinal Chemistry. 1972, Vol. 15,No. 2
CaHdOH NH, 0 CHr-CH-C--NH

I tlu tigneaud, ot ai

I

II

CHa 0

1

CrHs CH--CH,

-CH--C --NH-CH
2

I

I1

I I

s I
S

i
0
6

L o
0 NH
4

I

6 CHI-CH-NH-C-CH-NH-C-1

I/

11

r.=n - CHi-N,

I

C Ht
1

I

CH--(CHz)n-CONH*

I

I

CONHI
I1

\ '

CH-C-.-N

8 I1 9 H-CH-C-NH--CH~-CONHa
I
I

0

/

C Hs-C Hr

C HZ
CH(CIIa)a

I

Figure 1. Structure of oxytocin, with numbers indicating the position of the individual amino acid residues.

for the hydrogens in the 1 position of the 19-membered ring analog would convert the latter to an antioxytocic agent, we have synthesized [I -mercaptodimethylacetic acid]oxytocin. T h s analog, which had no detectable oxytocic or AVD activity, turned out to be only 20% as potent as deaminopenicillamine-oxytocin as an inhibitor of the oxytocic and AVD activities of oxytocin. Thus, the results with this compd demonstrate that antioxytocic activity is not confined to 20-membered ring analogs, Returning, then, to a consideration of substituents in the 0position, we decided to see whether increasing the size of the substituents would influence the degree of antioxytocic activity. We therefore synthesized the diethyl analog, [I$mercapto-0,P-diethylpropionic acidloxytocin. T h s compd, which was devoid of oxytocic and AVD activities, possessed an enhanced antioxytocic activity which was twice that of deaminopenicillamine-oxytocin. The anti-AVD activity was enhanced to about 70% more than that of deaminopenicillamine-oxytocin. It is obvious that further increase in the size of the substituents on the 0-C might prove interesting, and furthermore that substituents on both aand &carbons would warrant investigation. Detailed data on the inhibitory properties of the analogs under consideration are given in Table I, along with the corresponding data for L- and D-penicillamineoxytocin and deaminopenicillamine-oxytocin, which have not previously been evaluated by the method used in this paper. The inhibitory activities of the analogs were detd and expressed as PA, values as defined by Schild14(see footnote a to Table I) in the antioxytocic and anti-AVD systems. Isolated rat uterus and conscious chicken prepns were used,

under the normal conditions of the oxytocic" and AVL)** assays, resp. A dose of synthetic oxytocin (x units) which would give a moderate, reproducible response (R) was first selected. Then 2x units were administered inimediately foilowing an injection of antagonist. The latter procedure was repeated until two levels of antagonist were found, one o i which would reduce the effect of 2x units of oxytocin t ( J slightly more than and the other to slightly less than K. Concns of antagonist were calcd on the basis of ;t 1O-inl tissue bath in the antioxytocic studies and o n the basis of a n assumed blood vol of 150 in1 in the AVD studies. After plotting, according to Schild, the 2 concns of antagonist 011 a logarithmic scale against response, one interpolates between them to the molar concn (M) which corresponds to the response R. The negative logarithm of this concti to the base 10 is termed pA2. Average M ( f f )from a given series of assays and the corresponding pA2 values are listed i n Table I for each analog. . Also presented in Table I are 2 columns ( @ ~ ~ p / i C l ~ ~ ~ l ~ , ~ ! which relate the antioxytocic and anti-AVD potencies of the different analogs to those of deaniinopenicillaminc-oxy tocin. These columns provide a profile of the group of analogs as inhibitors and make it evident that in most cases the ratios show a close parallelism between the two inhibitoi y systems studied. It is interesting that the only analogs in Table 1 which do not fit in with the parallelism shown are the two (Land D-penicillamine-oxytocin)whicli bear a free a-NH2 group. As earlier reported," the 22-membered ring analog of' de. amino-oxytocin, namely [I -7-niercaptovaleric acid] oxytocin, possessed no oxytocic or AVD activity, but did possess antioxytocic activity. This compd has now been found t a have anti-AVD properties also, and a v a l u e s (expressed as in Table I) have been measured for both inhibitions: 3.22 ( 6 ) ,o = 1. I 4 (antioxytocic); 3.97 (7), o = 0.87 (anti-AVD). The corresponding pA2 values are 6.49 and 7.40, resp. Thiis for this analog also we find that its antioxytocic and antiAVD potencies relative to the corresponding potencies ot deaniinopenicillamine-oxytocin are very nearly equal (aDAP/aarralog = 0.36 and 0.33, resp). The marked parallelism between the antioxytocic and anti-AVD profiles of the compds studied may be significant. One may speculate that there is some basic similarity in the receptors involved j n the two tissues. Possibly further comparisons of the relative inhibitory potencies of groups of compds in 2 or more biol systems may yield important information that can be related to the types of receptors involved.
__l-.._-lI _ -

Table I. Inhibitory Properties of Oxytocin Analogs Related t o [I-Deaminopenici1lamine)oxytociri antioxytocic Analog M,a M x MijDAPIaafanalogb

_ . I _ _

1.16 (36) 1 .oo 7.88 1.31 (27) 1.oo a = 0.61 a = 0.51 0.83 7.50 3.18 ( 8 ) 0.4 1 6.86 1.39 (7) [ I-1,-Penicillamine]oxytocin a = 0.40 = 1.05 0.24 6.78 16.8 (7) 0.08 6.32 4.81 (6) [ 1-u-Penicillamine]oxytocin a = 4.0 a = 1.87 0.1 7 7.21 6.18(6) 0.21 [ 1-a-Mercapto-cupdimethylaceticacidloxytocin 6.16 6.91 (10) (J = 1.71 a = 3.56 [ 1-p-Mercapto-a,&-dimethylpropionic acidloxytocin 6.60 2.49 ( 9 ) 0.47 7.41 3.93 (6) 0.33 a = 1.09 a = 0.98 [ 1-p-Mercapto-p,pdiethylpropionic acidloxytocin 7.24 0.58 ( 9 ) 2.0 8.11 0.78 (8) 1.68 u = 0.17 a = 0.12 ___. apA, values (see Schild") represent here the negative log t o the base 10 of the av molar concn &)of an antagonist which will reduce the response of the uterine horn or the chicken t o 2x units of a pharmacologically active compd (agonist) t o the response to x units of the agonist. In these studies synt oxytocin was the agonist used. The number of individual detns is given in parentheses: and 17 is the s t d deviation. h R a t i o of molar concn of [ I-deaminopenicillamine]oxytocin t o molar concn of other analog.
[ 1-Deaminopenicillamine]oxytocin (DAP)

-

PA,

M

anti-AVD ~.. lo' MDAP/fianalog
. . -

--_

--

6.94

p-diethylpropionate (60. DMF).a-dimeth~lpropionyl-Tyr(Bzl>lle-GlnAsnCys(Bz1)-Pro-Leu-Gly-NH. a flow rate of 9 ml/hr. ( V ) .~. described earlier.H. b p 75-76" (4 mm). as detd by nmr. m p 217-218". and oxidn with ferricyanide4was carried out keeping the pH at 6. The residue in the flask was extd with Et.O. Recrystn (1. The tubes corresponding t o the peak were pooled. and the residue was dissolved in 50% AcOH (2 ml)." The analog was eluted at j-thilelting points were detd in open capillary tubes and are uncorrected. H..Cl. DMF).S04 (200 ml) was added. Three equivs each of I and DCI were used in the coupling step. The material moved as one spot on silica gel tlc in Sa and sb. and extd with Et. (111).5 mg.AsnCys(BzI)-Pro-Leu-Gly-~. and evapd t o dryness. [a]*'D -44. The solvent was distd from the reaction mixt until the vapors registered 100" and the distillate was clear. The DCI couplings were allowed t o procede for 4 hr and the p-nitrophenyl ester reactions were allowed t o procede overnight. Anal. (25 g) in a mixt of MeOH (300 ml) and H.O.. The aq soln was extd with toluene and then acidified with HCl.O.8 g) from pentane (25 ml) gave analytically pure IV (1. S.. This was added dropwise t o a large vol of H. This ester (46 g) was dehydrated by refluxing with P.H.s b . neutralization. Crystn from hexane gave 13. The MeOH and NH. 0. and the resulting milky suspension was stored overnight at 5".S)C. [ a I Z Z D-31. H.Cl. found..[I -Deaminopenicillamine]oxytociiis Journal of Medicinal Chemistry. The lyophilisate was dissolved in 50% AcOH (2 ml) and applied t o a 2. (30 g) in C.7" (c 1.8" (c 1 .. The clear. The analog gave the correct ratios of amino acids and NH. and coupling using 3 equivs each of the acid IV and DCI as described earlier.3" (C 1. (3:s).O in the centrifuge tube. ethyl bromoacetate (83. (175 ml) was added dropwise t o granular Zn (33 g) with heating.. wt 370 mg. DMF). preceded by a small shoulder of uv.. and the aq layer was washed with Et. (C. for 15 min.H. chloropivalic acid (pchloro-a. (C. Anhyd NH. AcOH.8" (c 1. Boc-glutamine and Boc-asparagine were added as their p-nitrophenyl esters in 3 times the equiv amts and coupled in DMF. Distn of the residue gave 17. p-(S-Bzl-mercapto)-p.10 X 1 0 3 t m column of Sephadex G-15 which had been equilibrated with 50% AcOH..O.S.. on amino acid analysis. (40 ml) in a 100-mlMerrifield reaction flask. dried in vacuo over KOH. which was crystd from hexane. packed ppt was frozen and lyophilized..N. N: Calcd.3 g. Amino acid analysis gave the correct ratios for the amino acids and NH. (C66H8J. were removed in vacuo.Cl..6 g) was refluxed with benzylmercaptan (21 g) in piperidine (40 ml) for 24 hr.O layer was dried (Na. [aIz3D +17. (C6. all as -43. (200 ml) for 24 hr. The Et.) and evapd t o dryness in vacuo. cooled. A mixt of 3-pentanone (43 g). deblocking..wdimethylpropionic acid) (13. EtOH.7. the resin was washed with CH. and lyophilized. 2 125 Experimental Section?? S-Bzl-mercaptodimethylaceticAcid (I)..zo grade which was distd through a 1. and ammonolytic cleavage of the peptide from the resin were carried out as described for the synthesis of 111 t o give 357 mg of the protected nonapeptide amide V.2.66 g. m p 225".). Three equivs each of Boc-amino acids and DCI were used in the coupling steps. colorless filtrate was lyophilized.p-diethylpropionic Acid (VI). neutralization. DMF).O with stirring. p-(S-Bzl-mercapto)-orpdimethylpropionic Acid (IV). and a second coupling with 3 equivs of IV and DCI was allowed t o procede overnight. which represents 88% of the theoretical wt gain based on the amt of Boc-glycine esterified t o the resin.1 mole) was added t o a s o h of NaOEt (0. This soln was applied t o a 1. Anal.. mp 95-%. No.1 mole) was added. 1972.15 g) in anhyd EtOH (100 ml) and stirred under N. dild with 1 vol of H. the mixt was refluxed for 4 hr. ordimethylpropionic Acidloxytocin. The soln was acidified (pH 2).3 g of a yellow oil." the substituted resin was subjected to an addnl deblocking and neutralization sequence. and 10% H. H. S. Anal. then dried overnight in uacuo over KOH.. The NH.75 g) was added dropwise in anhyd EtOH (40 ml) and the soln was refluxed for 1 hr under N. Benzyl mercaptan (12. wt 49. and the AcOH s o h was lyophilized t o give 402 mg of compd 111.H.N. and the residue was dissolved in H. and the residue was dissolved in 0. N. and finally with several portions of anhyd EtOH. bp 158-167" (0.H.20 X 110-cm column of Sephadex G 1 5 which had been equilibrated with 0. p-diethylpropionyl-Tyr(Bz1)-Ile-Gln.76 mmole of glycine per g of resin was swelled overnight in CH. H. H. concd t o a small vol.18 Boc-glycyl resin" (4 g) contg 0.6 g (62%) of the acid IV. Anal. The contents of the tubes corresponding t o the main fractions were pooled.. The pro- . the org s o h was dried (MgSO.SO. and the solvent was evapd.2 g) was added t o a warm soln of Na (1. The protected octapeptide-resin I1 ( 1 g) was swelled overnight in CH.. [CY]~*D Amino acid analysis gave the correct ratios for amino acids and NH.O (150 ml).. The material moved as one spot o n silica gel tlc plates in solvents Sa and s b . H.The analog was eluted at a flow rate of 4 0 ml/hr and emerged as a sym peak centered at 400 ml.S) C. m p 51-51. Anal. DMF).O (50 ml).2 g). [I -p-Mercapto-a.O. The protected nonapeptide amide 111 (100 mg) was dissolved in anhyd NH.. and after the first 3-hr coupling period the peptide-resin was subjected t o an addnl treatment with 3 equivs of the acid and of DCI.3 mm).O. and the analog was isolated and purified as described earlier.). and lyophilized t o give 4 0 mg.101zS2) C .-absorbing material.. Solvent systems:lSa. The protected nonapeptide amide V (100 mg) was reduced with Na in liq NH.2 X 30-cm column packed with glass helices.O. N..CO. H. A portion of this mixt (15.O (20 ml) and EtOH (50 ml) was added t o the h o t s o h . The suspension was filtered. and the residue was refluxed with K.).S. H. 11.O (160 ml). After 15 min.and ferrocyanide ions were removed with AG3-X4 resin (trifluoroacetate cycle). The resin was sucked dry. N. CH.O. acidified with HCl. It was homogenous on silica gel tlc in solvents Sa and sb. The ppt was collected by centrifugation and washed several times with H. were within *0. After the initial reaction had subsided.. 0. the resulting product was oxidized with K.5" (cor).O (100 ml).OH-CHCI. the resin was rinsed with several portions of DMF. Anal.."Where elemental analyses are indicated only by symbols of the elements. and PhCH. The residue was distd at reduced pressure t o give ethyl p-hydroxy-p. After the Boc-O-benzyltyrosine was coupled t o the peptide.03% trifluoroacetic acid (300 ml). The soln was adjusted t o pH 6. and coupling as described in the The AcOH was reagent synthesis of [8-phenylalanine]oxytocin. Ir and nmr data were consistent with the acid IV. The wt gain of the resin was 2. C..H. followed by ammonolysis and purification.S) C. analytical results obtained for the elements.H.O.N." allowing the blue color t o persist for 15 sec before destroying excess Na with NH4C1.SJ C. the lyophilisate was dissolved in AcOH and filtered. er al. N. (10 ml) and subjected t o one 21-step solid-phase cycle as described previously.. and the product was extd with Et.. the soln was refluxed for 1 8 hr under N.8. The solvents were removed in uacuo.O was removed. washed on the filter with H 0 and air-dried. The protected octapeptide-resin compd I1 (1 g) was swelled overnight in CH.9" (c 1. Ir and nmr data were consistent with the structure VI.O.O.6 g..C1.O) C.5 g).p-diethylpropionic Acid]oxytocin. Anal.yunsaturated ester. The moist. Before cleavage with methanolic NH. and the residue was stirred with DMF for 3 hr. 11. (10 ml) and subjected t o a sequence of deblocking. Ir and nmr data were consistent with I. At the end of the 4hr coupling period. Benzylmercaptan (6. and hydrolysis was carried out at reflux for 6 hr. This compd was synthesized by the solid-phase method of Merrifield. and the basic aq layer was washed with Et. The Boc-glycyl resin was then subjected to seven 21-step cycles of deblocking. [ 1-MercaptodimethylaceticAcid]oxytocin. concd t o 5 ml. The residue was dissolved in H.H. and the Et.4 g (needles).. the resin was washed with CH. distd from Na.. (C. Boc-Tyr(Bzl)-Ue-Gln-Asn-Cys(Bzl>Pro-Leu-Gly Resin (11). [ 1-p-Mercapto-p.p-unsaturated ester and 75% of the p. The s o h was cooled. Crystn from hexane give 4. The Et. (C.8. m p 75-76". n-BuOHAcOH-H.O was removed from the dried soln. The flask was stoppered and the suspension was stirred at 0" overnight.)C. (C. Amino acid analysis of an acid-hydrolyzed sample gave the correct ratios of amino acids and NH. p(S-Bzl-mercap to)-a.5 g). The ppt was collected.2 mole) in abs EtOH. (C45H69N1101zSZ) C. the H.2 N AcOH.O (75 ml) for 24 hr.4% of the theoretical values.Cl. An acid-hydrolyzed sample of this analog gave the expected ratios of amino acids and NH. was bubbled into the suspension for 3 hr at 0". with coupling allowed t o procede overnight. The soln was adjusted t o pH 7. Amino acid analyses were performed o n a Beckman/Spinco amino acid analyzer Model 116 b y the method of Spackman. H. (25 ml) and reduced with Na at the boiling point of NH. Ethyl a-bromoisobutyrate" (9. The protected nonapeptide amideVII moved as one spot on silica gel tlc plates in solvents Sa and sb.. Vol.O being removed by centrifugation and decantation.O (100 ml) was added t o the cooled s o h and the org layer was sepd and dried (MgSO. was removed in vacuo.Fe(CN).(VII) was synthesized by condensation of I1 (1 g) with VI. p(S-Bzl-mercap to)$. Anal. S-Bzl-mercaptodimethylacetyl-Tyr(Bzl)-Ile-Gln-Asn-Cys(Bzl)Ro-Leu-Gly-NH. Subsequent steps for washing.. 15. The org layer was dried (MgSO. H. E x a m of the fractions by uv absorption (275 mp) showed a sym peak centered at 46 ml. (200 ml). The ferri. and the combined DMF s o h was concd t o a small vol. Distn of the residue after evapn of the solvent gave a mixt of 25% of the a. A soln of NaOH (2 g) in a mixt of H. and then swelled for 3 hr in cold (0") anhyd MeOH.O (4: 1: 1). [ a j z 2 D -32. m p 235".

Baxter. 81. J. for very helpful discussions relating to the biological studies. Endocrinology. 30.)-ON~’~ to give BOC-Glu(NH&NrPht-Dbu diphenylmethyl ester (4). SOC. Schild.Fe(CN). V. M. (3) W. J. Brit. and S. and the analog was -37. Spackman. Chem. approximately 0. New York.3NL. Oxytocin analogs which bear a Me. D. 90. IS. Brazil. 647 (1966). Med. M. Biol. Bethesda. G. B. l4 to yield BOC- Glu(NHz)Nr-Pht-Dbu-S-Bzl-Cys-Pro-Leu-Gly-NHz (6). It is concluded that [ 5-a. Chem. to Mrs. SOC. Marquet. Allen. M Baxter. Pharmcol. 6 6 . R. 328 (1948).. Amino acid analysis gave the correct ratios of amino acids and NH. P. 0. M. is reported. Hase. which was subsequently deesterified to give 5 . 92. J. H? N. Anal. Received July 15.y-Diaminobu tyric acid]oxytocinf $ S. S. 1190 (1958). Louis L. and that this effect is a result of an increase in the conformational flexibility of this analog as compared with oxytocin. 4 5 1 (1960).. Further Studies of the Role of the Asparagine Residue in Oxytocin. Janet Huisjen of this department for her highly capable performance in carrying out the bioassays.. Anal.y-diaminobutyric acid] oxytocin and on the determination of its biological properties in order to assess . Amer. Chem. du Vigneaud. H. Jarvis and V. D..No.. 5015 (1966). B. J. Stein. and V. 189 (1947). respectively. J. Endocrinology. H.~ y . DMF). Chem. T. R. Jarvis. P.4 equiv of DCI and 2. 1563 (1962). A. 3. J.4. Marshall and R.. J.’” 0-carboxa n i i d e e t h ~ lhydroxymethyl. Ferger. All naturally occurring neurohypophyseal hormones of known amino acid sequence possess an Asp(NHz) residue in position 5. The amino acids (except glycine) are of the L configuration. Meador. p 469. 396 (1968). (9).03 unit/mg of avian vasodepressor activity. 242. /Abbreviations used have been suggested in J. Y. H.. Y. New York 10029. Peptide 9 was successfully dephthalylated with either hydrazine or hydrazine acetate” to yield 10. C. Schulz. On assay for biological activity this analog was found to possess 0.03 unit/mg of oxytocic activity... Bodanszky and J. Blomquist. G. J. Ind. R. 2 Hase. W. 79 (1939). Fr. Synthesis and Biological Properties of [ S-cu. N-ZSBzl-CysO-Bzl-Tyr-Ile-Glu(NH2)-Nr-Pht-DbuS-Bzl-Cys-ProLeu-Gly-NH. 3592 (1969). du Vigneaud.y-diaminobutyric acidloxytocin possesses a low affinity for the neurohypophyseal hormone receptors. J. 1970. which was fully deprotected by treatment with ’’ . Bioorg.678The elucidation of the conformation of oxytocin’ confirmed the central role of the Asp(NHz) residue.. Chem.Erit.H. 62.y-diaminobutyric acidloxytocin. Chromatogr. Holton.. Merrifield.. Sheehan. Chan. an analog of oxytocin which contains an a. 1966. Merrifield. 678 (1958). W. Wuu. Ferrier. (C4. 1423 (1 964). H. Pharmacol. References (1) H.zSz) Acknowledgments. R . the above solvents. Murti. Sao Paulo. Endocrinology. 1972. Biol. and to Dr. W. The free tetrapeptide. Int. [aIz3D (c 1. Chem. A. Department of Pharmacology. and V. 2394 (1965).126 Journal of Medicinal Chemistry. (4) D... 1.y-diaminobutyric acid residue in place of the asparagine residue in position 5 . W. “The Pharmacopeia of the United States of America. Chem. Munsick. Chem. Sawyer. 1571 The synthesis of [ 5-cu. I. du Vigneaud. J. 8. of mammalian pressor and antidiuretic activities. The hexapeptide (6) was elongated stepwise to yield the fully protected nonapeptide. 1768 (1967). Walter* Mount Sinai School of Medicine of the City University o f N e w York. (London). J. du Vigneaud. J. ‘The authors are greatly indebted to Dr. Munsick.. W.. for use of his laboratory in this aspect of the work.a m i n ~ p r o p y land . Chem. Biol. Cornel1 University Medical College. Vol...’ . and R. which in turn was esterified with PhzCHN2. Amer.. Arch. 240.” 18th rev. U. Hope. Schulz and V. 1267 (1967).. M.. ?This work was supported in part b y United States Public Health Service grants AM-13567 and A M . ( 5 ) B. in the following manner: the acid 1 was converted to its p toluenesulfonate salt (2). Schwartz.. R .6” isolated and purified as described earlier. H. R. F. SOC. J. and A. du Vigneaud. and less than 0. 1030 (1970). S. 2149 (1963). Y. and R. The analog was incapable of inhibiting the oxytocin-induced responses in the 4 biological systems tested. van Dyke. Biochemistry. Sifferd and V. and A. M. 2. and V. abstract 449. and J. Chan. Amer. H.0 equiv of N-hydroxysuccinimide according to the procedure of Weygand. 1323 (1968). The dipeptide 5 was secured from Nr-Pht-Dbu*H. H. the capability of qy-diaminobutyric acid (Dbu) to replace successfully the Asp(NHz). Chim.. W. Med. the resulting product was oxidized with K. 123 (1971). ibid. 4264 (1965).1 0 0 8 0 . J. 38. du Vigneaud. Coon. C. (6) V.. ibid. Soc. 9. 237. B. T.16-’8 the resultant ester 3 was allowed to react with BOC-G~U(NH. wt 31 mg. M. 111 International Pharmacology Congress. H..0. V. Md. L.O (l).. Chem. In the present communication we report on the synthesis of [ 5-a. Takashima. and H. B. Manning. ~ i-Pr5 side chain in position 5 instead of the carboxamidemethyl moiety all exhibit an exceedingly low potency with respect to the activities characteristic of oxytocin-a finding which led us to focus on the importance of position 5 for the conformational stability of the hormonal molecule. Chan. Jacques.001 unit/mg. V. Fraefel and V. and Walter H. B. Soc. Merrifield. Nangeroni. 3. du Vigneaud. and W.88. Biochemistry. Moore. du Vigneaud. Weidmann-Hattier. et al. 8 A (1 970). et al. 7. d u Vigneaud. It is critical for the maintenance of both /3-turns comprised of the sequences -Tyr-Ile-Glu(NHz)-Asp(NH2)and -Cys-Pro-Leu-Gly(Figure 1). and V. A. M. D. du Vigneaud. tected nonapeptide amide VI1 was reduced with Na in liq NH. obtained after phthalylation of the dihydrochloride salt of the free acid by the general procedure of Nefkens. (2) W. S-Bzlwas elongated with BOCCys-Pro-Leu-Gly-NH. Sawyer. 860 (1960). Glu(NH2)-Nr-Pht-Dbu ( 5 ) in the presence of 1. Manning. Fear. Chem. 108. D. Schwartz. Pharmacodyn. 1385 (1964). Bull. 753 (1935). 13(5). Schulz and V. The analog moved as 1 spot on silica gel tlc plates in C.01 and 0. J.-. New York State Veterinary College at Cornell University. 6 6 .85. du Vigneaud. Chem. Amer.

. 15 (2).alpha.1021/jm00272a002 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. Hase.acs. I. 1972.. and R.. L. Chem. DC 20036 . Schwartz.Role of the asparagine residue in oxytocin. Med.W.gamma.org/10. Walter J.-diaminobutyric acid]oxytocin S. Synthesis and biological properties of [5-. 1155 Sixteenth Street N.1021/jm00272a002 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society.org on April 25.doi. Washington.. 126-128• DOI: 10. 2009 More About This Article The permalink http://dx.

Spackman.16-’8 the resultant ester 3 was allowed to react with BOC-G~U(NH. Jarvis.03 unit/mg of avian vasodepressor activity. Sawyer. V. Amer. 328 (1948). H. 237. 240. M.y-Diaminobu S.Erit. and V. U. (2) W. R .. A. and R. Biochemistry. Meador. and less than 0. Coon.. and A.. J.. to Mrs.’” 0-carboxa n i i d e e t h ~ lhydroxymethyl. and Walter H. M.. p 469. DMF). 13(5). an analog of oxytocin which contains an a. Chem... 647 (1966). J.-.85. Chem. S. B. Pharmacodyn.No. which was fully deprotected by treatment with ’’ . and J. 4264 (1965). The dipeptide 5 was secured from Nr-Pht-Dbu*H. 62. Baxter.y-diaminobutyric acid residue in place of the asparagine residue in position 5 . for use of his laboratory in this aspect of the work. Ferger. Biochemistry. H. du Vigneaud. Sao Paulo. J. (3) W. R. The analog was incapable of inhibiting the oxytocin-induced responses in the 4 biological systems tested.0. 30. and V. Chan.zSz) Acknowledgments.. IS. ibid. J. H? N. Pharmacol.y-diaminobutyric acid] oxytocin and on the determination of its biological properties in order to assess . the capability of qy-diaminobutyric acid (Dbu) to replace successfully the Asp(NHz). Endocrinology. R. Weidmann-Hattier. Murti. A. Glu(NH2)-Nr-Pht-Dbu ( 5 ) in the presence of 1. M. which was subsequently deesterified to give 5 . 123 (1971). 7. et al. L. Ind. S-Bzlwas elongated with BOCCys-Pro-Leu-Gly-NH. Med.. Munsick. New York.O (l). 8 A (1 970). 1563 (1962). 90.0 equiv of N-hydroxysuccinimide according to the procedure of Weygand. du Vigneaud. Takashima. 678 (1958). Med. 0. 753 (1935). I. Chem. 6 6 . du Vigneaud. C. Pharmcol. et al. Soc.6” (c 1. The hexapeptide (6) was elongated stepwise to yield the fully protected nonapeptide. Sheehan. Marquet. Janet Huisjen of this department for her highly capable performance in carrying out the bioassays. W. V. the above solvents. In the present communication we report on the synthesis of [ 5-a. of mammalian pressor and antidiuretic activities. G. J. W. Biol. Amer. wt 31 mg.. Chem.. van Dyke.. Chromatogr. Bodanszky and J. It is concluded that [ 5-a. Merrifield. Biol. 242.)-ON~’~ to give BOC-Glu(NH&NrPht-Dbu diphenylmethyl ester (4). 81. Bull. and V. Peptide 9 was successfully dephthalylated with either hydrazine or hydrazine acetate” to yield 10. du Vigneaud. 1970. and A.. H. W. Chem. and the analog was isolated and purified as described earlier. (4) D. Moore.. Chem. M. M. and W. obtained after phthalylation of the dihydrochloride salt of the free acid by the general procedure of Nefkens. du Vigneaud. l4 to yield BOCGlu(NHz)Nr-Pht-Dbu-S-Bzl-Cys-Pro-Leu-Gly-NHz (6).. 1267 (1967). Chan. is reported. Y. 108. 79 (1939). and R. 9. Bethesda. J.’ . 4 5 1 (1960). J. 189 (1947).. 2149 (1963). D. Fear. Anal. Stein. V. 1. Schild.. du Vigneaud. References (1) H. Jarvis and V. A. Merrifield. Further Studies of the Role of the Asparagine Residue in Oxytocin. D. It is critical for the maintenance of both /3-turns comprised of the sequences -Tyr-Ile-Glu(NHz)-Asp(NH2)and -Cys-Pro-Leu-Gly(Figure 1). T. 88. Wuu. All naturally occurring neurohypophyseal hormones of known amino acid sequence possess an Asp(NHz) residue in position 5. H. and to Dr. ( 5 ) B.. W. 1768 (1967). 1323 (1968). for very helpful discussions relating to the biological studies.y-diaminobutyric acidloxytocin possesses a low affinity for the neurohypophyseal hormone receptors. d u Vigneaud. G. Schulz. W. Biol. New York State Veterinary College at Cornell University. Fr. 1423 (1964). Blomquist. SOC. J. M. M Baxter. Int. and that this effect is a result of an increase in the conformational flexibility of this analog as compared with oxytocin.. Manning. Synthesis and tyric acid]oxytocinf $ Biological Properties of [ S-cu. Schulz and V. approximately 0. D. Schwartz.. Marshall and R. D. Soc. Brazil. Received July 15. B. Cornel1 University Medical College. Endocrinology. Oxytocin analogs which bear a Me. 3592 (1969). 2394 (1965). Chem. Endocrinology.. Hase.678The elucidation of the conformation of oxytocin’ confirmed the central role of the Asp(NHz) residue. Manning. ‘The authors are greatly indebted to Dr. B. 8. 2. Y. the resulting product was oxidized with K. J.. The amino acids (except glycine) are of the L configuration.a m i n ~ p r o p y land . Schwartz. 3. and H. Merrifield. J. Department of Pharmacology. Amer. 5015 (1966). J.001 unit/mg. and V. The analog moved as 1 spot on silica gel tlc plates in C. 396 (1968). Holton. Chem. Chem. Anal.03 unit/mg of oxytocic activity. H. Schulz and V. P. H.y-diaminobutyric acidloxytocin. J. Chan. R . T. Ferrier. 92. Munsick. (9). Arch. On assay for biological activity this analog was found to possess 0. du Vigneaud. Hope. (C4. SOC. 1030 (1970). Vol. 1190 (1958). J. Brit. The free tetrapeptide.126 Journal of Medicinal Chemistry. Walter* Mount Sinai School of Medicine of the City University o f N e w York. which in turn was esterified with PhzCHN2. /Abbreviations used have been suggested in J. B...Fe(CN). abstract 449. Bioorg. H. Md. Chem. Y. 38.” 18th rev. F. 111 International Pharmacology Congress. Chem.. and S. ibid.4. C. Sawyer.4 equiv of DCI and 2. SOC. Nangeroni.~ y . J. 2 Hase. 860 (1960). Chem. R. 1972.3NL. Jacques. H. (6) V. du Vigneaud. B. 3. J..01 and 0. du Vigneaud.H. R. respectively. W. 1966. New York 10029. S. Amer. “The Pharmacopeia of the United States of America. 1385 (1964). Amino acid analysis gave the correct ratios of amino acids and NH. Allen. Fraefel and V. N-ZSBzl-CysO-Bzl-Tyr-Ile-Glu(NH2)-Nr-Pht-DbuS-Bzl-Cys-ProLeu-Gly-NH. ~ i-Pr5 side chain in position 5 instead of the carboxamidemethyl moiety all exhibit an exceedingly low potency with respect to the activities characteristic of oxytocin-a finding which led us to focus on the importance of position 5 for the conformational stability of the hormonal molecule. 66. tected nonapeptide amide VI1 was reduced with Na in liq NH. du Vigneaud. P. 1571 The synthesis of [ 5-cu. Chim. (London). ?This work was supported in part b y United States Public Health Service grants AM-13567 and A M .1 0 0 8 0 . Sifferd and V. Louis L. in the following manner: the acid 1 was converted to its p toluenesulfonate salt (2). [aIz3D -37. M.

9 mmole) in the presence of N-methylmorpholine (0.O.. Anal. Dbu . 1. 23324 ml). N. 572 mg (93. m-Pht-Dbu p-Toluenesulfonate ( 2 ) . 0.5 ml) as described above.9 mmoles) in a mixt of MeOH (70ml) and THF (20 ml) was subjected to catalytic hydrogenation using 0.O (6. Anal. i n vucuo unless otherwise noted.55 g. yield. (C. Anal.005'.75 mmole) in the presence of N-methylmorpholine (0.O (250ml) contg EDTA (1 g).. H. AcOH). and dried over NaOH. which was suspended in acetone and collected by filtration after a ] ' O D + 9 .mp 236240" dec.O.3 g (50.O gave 0. mp 2052 0 8 ' dec. using Beckman custom research resin PA-28. 0. Experimental Proceduref W-Pht-Dbu*H. The Cu salt was sumended in a s o h of EDTA (18.H (2. (C80H96N1z015SZ) C. 19 mmoles) in DMF (10 ml) was 0 ' . 0.N. 4. yield. This enhances the conformational ambiguity of the acyclic tripeptide. yield 4. 1. The crude product was purified by boiling with 80% EtOH (50 ml) and collected by filtration after cooling.7" (c 1.50 ml) contg AcOH (0.68 mmoles) in DMF (5 ml) was stirred into a soln of S-Bzl-Cys-Pro-Leu-Gly-NH. N.CCO. was purified by boiling with 80% EtOH. [a]"D -70. 20 mmoles) was dissolved in warm H.O.N.O (15 ml) together with p-TsOH (3. Tenn.3' (c 1. purified by partition chromatography26using 2 solvent systems. 2. Compd 6 (562mg. 0. It exhibited approximately 0.05 mole) in H. (C.5 ml) as described in the synthesis of 7. yield. H.O ( 1 ) . is unable to stabilize the oxytocin conformation. The crude product was repeatedly pptd from 90% EtOH.O. It is concluded that Dbu.mp 176-177'dec. mp 205-206" dec.S) C.H. analytical results obtd for the elements were within f0.4mmole) was removed with F3CC0. (9). Carb~ethoxyphthalimide'~ (13.2HC1(9.56 ml) for 24 hr.CN (100ml) and then from DMF-EtOAc gave fine needles. Conformation of oxytocin in solution as proposed by Urry and Walter. Anal. Anal. Vol. 2.O.O) C. Optical rotations were detd with a Carl Zeiss photoelectric precision polarimeter set at 0. [ 2. The predominant factor may be that Dbu-in contrast to Asp(NH2)-lacks the carbonyl group in the side chain and thus is unable to form an H bond with the amide NH of the residue in position 8. 1. Gly-NH. unlike the Asp(NH2) residue. yield. 6.. The analog did not inhibit the oxytocin-induced responses in any of these 4 biological test systems. which resulted upon trituration of the oil with Et. The peptide trifluoroacetate (535 mg) dissolved in DMF (5 ml) was allowed to react with N-BOC-U-Bzl-Tyr-ONpJ0(370 mg.)-m-Pht-Dbu Diphenylmethyl Ester (4). A soln of 3 (2. After 23 hr EtOAc (SO ml) contg a small amount of AcOH (0.. The resultirig trifluoroacetate (525 mg) was dissolved in DMF (5 ml) and was allowed to react with N-Z-S-Bzl-Cys-ONp" (268 mg. The crude product was recrystd from EtOAc.1.33.O.2%). and the resulting o i l was crystd from a mixt of EtOAc (10 ml) and Et.8' (c 1.) C. Elementary analyses were carried out b y Galbraith Laboratories. 0.2. yield. 5 mmoles) in the presence of Et3N (0.4 mmoles) 2 2 ' . BOC-Glu(NH.81 g (92. H. yield.025 mole) was added.03 unit/mg of avian vasodepressor activity. 615 mg (54. yield. yield. Compd 1 (5.6 mmole) was treated with F3CC0.O.O.H.mp 265-268" dec. Anal.5%). The crude product was recrystd from boiling H. 1.N.1 ml) was added to ppt the crude product. H.H8.O (1:2). washed with Et.O. 0. DMSO). H. BOC-lle-Glu(NH. BOC-Glu(NH. The soln was concd to form a cryst residue. MeOH).yDiaminobutyric acidloxytocin n I Journal of Medicinal Chemistry. 1972.)-ONp (1.. (C.Cl. and hence is not an effective substitute for the Asp(NH2) residue in neurohypophyseal hormones. The BOC group of 8 (521 mg.)-~-Pht-DbuS-Bzl-Cys-Pro-Leu-Gly-NH. Recrystn from MeOHEt.6 K. [a]"D -42. 2 ' (c cooling. The intermediary dithiol was oxidized to the disulfide by action of aq [ 5-a.01ml).31g.83g (54.O (100ml) and 4 N NaOH (37. DMF). et al.5" (c 1..6%).25g.H (2..5 g of 10% Pd/C as catalyst. and Et.2mmoles)..83g. 1.. [C~]'~D -12.0.9%).08ml) for 20 hr.mp 159-160' dec. mp 148-150' (sintg 146"). which was purified by repptn from 96% EtOH. (C. EtOH.SH. mp 170-171' dec.yield. [a]"D +14. 5. 563 mg (86. N.H. (C.2mmoles).O.1 ml). 4 mmoles) in DMF (10 ml) was allowed to react with BOC-Glu(NH. was filtered.1' (c 1. Anal. A soln of diphenyldiazomethane (3.. The crude product (615 mg). [ffIz0D -73. 21 mmoles). Anal. 11. Y.O. which factor in turn also appears to affect the conformation of the 20-membered ring adversely. CH.0 g (92.22g.MeOH).N.76 g (58. stirred into a soln of 2 (6.8%)..O. 5 (572 mg. [a]"D -54. H. 395 mg (62. AcOH).H.H.N. A soln of DCI (346 mg. The reaction mixt was stirred at 2 2 ' for 1 in DMF (30ml) at hr and at room temp for 18 hr and then filtered and the filtrate was concd to an oily residue which solidified upon trituration with a mixt of EtOAc-Et. 0. yield.. Evaporations were performed under reduced pressure. 660 mg. After 60 min the Cu 'salt of W-Pht-Dbu was filtered. Recrystn from CH.5 ml). Peptide hydrolysates were chromatographed on a Beckman/Spinco Model 12OC amino acid analyzer.. ( 7 ) .O. This indicates that the greatly diminished capability of this compound to evoke the responses characteristic of oxytocin resides in its low affinity for the oxytocin receptors. AcOH).32g. . and an aq s o h (20 ml) of CuSO. 0.81. (C. H. Where analyses are indicated only by symbols of the elemehts. 1. BOC-Glu(NH. All melting points were detd with a Thomag-Hoover capillary melting point apparatus and are not corrected.O.O (100ml).1ml).H (1 ml) for 30 min. N. N. A Soh of 4 (1. (8).[S-cu. Anal.O. H: calcd. 2 127 Figure 1. yield. 15 mmoles) in DMF (5 ml) at 5 After 10 min the reaction mixt was concd.. N. DMF). Upon bioassay [ 5-qy-diamin~butyric acidloxytocin was found to possess 0.5 mmold) was removed with F.86 g.. and the mixt was stirred vigorously for 60 min and then refrigerated for 2 hr.)-W-Pht-DbuS-B~l-Cys-Pro-Le~-G~-~z (6). N-ZS-BzlCys-O-Bzl-Tyr-Ile-Glu~H.0 (200 $All reactions were carried out at room temp and all products were dried at room temp over P. yield.8' (c 1.55g. IS.9%).H.4% of the theoretical values. 570 mg.03 unit/mg of rat uterotonic activity. (573 mg. The material was filtered and washed repeatedly with EtOAc. (5 g. 7. The reaction mixt was concd to an oily residue which was crystd from Et. This salt was dissolved in DMF (3 ml) and allowed to react with BOC-llebNpZ9(317 mg. N.N.)-~-Pht-DbuS-BzlCys-RoLeu-Gly-NH.05 mole) were added to the mixt with strong stirring.. and Et. 13. mp 148-150" dec.1ml) was added to the reaction mixt to ppt the crude product.5%).06 mole) and NaHCO. [a]"D +7.S) C. [a]'OD +14.9%). washed successively with H. 1 N HC1).70g.EtOH.17g.S) C. and less than 0.found. (C.)-W-Pht-Dbu (5). After 3 hr the catalyst was removed by filtration and the filtrate was concd to a residue which was crystd from Et. yield. mp 203-206" dec. yield.05 mole) was dissolved in a mixt of H. pptd with EtOAc (35 ml) contg AcOH (0.9g.83 g (92. washed successively with H.g HF as described by Sakakibara.N.01 unit/mg of rat pressor activity.) C. The volatile components were removed and the ppt.3' (c 1.. and N-hydroxysuccinimide (276 mg.001 unit/mg of rat antidiuretic activity. 0.9%).6mmole) in the presence of N-methylmorpholine (0.y-Diaminobutyric acid] oxytocin was K3Fe(CN). Knoxville. 0.''$'* The white ppt was collected by fittration. After 24 hr a mixt of EtOAc and Et.2" (c 2.1%) of product. H. less than 0.99g. The BOC group of7 (5 25 mg..O (l:l.No.1 g.34g. 7. W-Pht-Dbu Diphenylmethyl Ester p-Toluenesulfonate (3).S) C. 0.

” B. du Vigneaud. 1972. S.A. Nat. Walter. 21. . (33) D. L. Czech. Boers-Boonekamp. yield. I. and I.2164 (1967).O contg 3. Amer.935 (1959). 24. 21. (C. 239. Chem. du Vigneaud.H. SOC. Peptides and Proteins. 2nd. du Vigneaud. Spackman. (C. Costopanagiotis. L. and B. 42. G.21) was detected by Folin-Lowry color detn. Chem. 348. and J. Reel. S. Biol. Y. Endocrinology. ~ modified . and D. Med. King. G.451 (1960).. A. Following the removal of resin the filtrate was concd to ea. Trav. Wiley.. and ferro.O and then applied to a short column of CM-Sephadex (H+ form) in a mixt of DMSOMeOH-H.O. Ressler and V.O. 201. R. Sakakibara. and R. E. H. (31) M. and R. Miller. Trav. Int. 2 Hase. M. J. Wiinsch.-A. Tyr (0. G. 0. H.. Collect. R. Sawyer.Oz) C..265 (1951). Leu (1. J. Chem. 38. Overweg. and Walter N-%S-Bzl-Cyd)-Bzl-Tyr-Ile-Glu(NH. . D.01). Howard. Effluent and washings of the same solvent were combined (ea.472 (1964). Chim. Dravarek.. Hope. and E. Chim. W. Munsick. Randall. Urry. Chem.5% pyridine (7:2:9).1 mmole) was dissolved in anhyd HF (ea.96). S. Bull. Naturforsch. et ~ 1 Assays activity were performed on anesthetized. Yamashiro. 1601 (1962). 87.00: Cys (1. M. 1190 (1958). and R. R.5%pyridine (7:2:9). Zahn. 241. I S . W. Nature (London).89). to result in a residue. C. M. Soc.328 (1948).. N. Smith and K. Moore. Watanabe. Schwartz. Bontekoe.Fe(CN). J. (23) S. Symp. Sheehan and V. R. 79. L. Anal.OH and treated with excess 0. 78. Biol. in “Chemistry and Biochemistry of Amino Acids. J. The soln was adjusted to pH 8. Chimia. 250 ml). A small amt of insol materials was removed by filtration and AcOH (0. Mack Publishing Co. Y. D. 1 N AcOH).O. After 15 min the pH was adjusted to 6. They are also grateful for the technical assistance rendered by Mrs.. N.O (20 ml) was filtered. SOC.. (34) P. I. Y.. Bitter.S . Acta.00). Frank.Gly (1..Jap. 161. Bull. Amino acid analysis33after hydrolysis in 6 N HCl at 110” for 23 hr gave the following molar ratios with glycine taken as 1. Biol. The authors are indebted to Mr. hydrated Sprague-Dawley male rats according to the method of Jeffers. No.76 (1964). Sieber. Urry and R. J. 0. Chim. Schwartz. D. C.3z The fractions corresponding to the principal peak (Rf 0.O contg 3..3-ml fractions were collected.. Biol. van den Brink-Zimmermannovi. 295 (1962). Pays-Bas. 50. New York.00). and H. J. 63... the product was eluted with aq AcOH increasing from 50 to 80%. J. P. Naturforsch. Koch. (32) 0. although in the former case a small amount of by-product was detected. Trav.1% AcOH (100 ml) and the soln was extd with Et. A. Chim. Boissonnas. Okada. H. Chem. and H. The mixt was concd to a small vol and the product was isolated by lyophilization. Schwyzer. who was involved in the early phase of this work.The column was eluted with the upper phase and 90 9. Y. A.H.Jap. S.. Amer. Chem. Chem. (28) S.O (4:1:5) (0.5% AcOH and 1. Shimonishi. 1626 (1963). (35) R. et al. Glu (1.A sample for analysis was dried at 100” for 8 hr. Sugihara. R. Chem. p 749. B.... Sakakibara..3 mg (18%).. N. L. F a r . Acknowledgments. 0.763 ( 1966). H. Fruton.36) were pooled and dild with excess H. Danko. [5-a. A. Avian vasodepressor assays were performed on 3 conscious chickens according to the . yield. C. A. 20. Exp. Chem.0” (c 0. Sei. Ledger and F. (25) D. Anal. (29) S. dild with H. 45. J. Holton. Chim.. Boissonnas. after azeotropic removal of the org phase. Amer.H (10). van Dyke. ~ for~ antidiuretic procedure employed by Munsick. W. SOC.O (3:3:1). SOC. washed with H. 68. V.. Marcel Dekker. Vol.5469 (1965).) C. 111. ibid. 1955. Zaoral and J. H.49 for Tyr). (26) D. Proc. Chem. yield. After elution with the upper phase a single peak (Rf 0. H. Silverman. Jeffers. A.The soln was applied to the same column of Sephadex G-25 which had been equilibrated with both phases of the solvent system. [cY]~’D -45. Helv.426 (1966). The product pptd by addn of H. (27) R. 0. W. J. References St. Sci. 1970. Bioassay Methods. J. (39) “The Pharmacopoeia of the United States of America. was used to measure the antidiuretic activity.O. concd and. The dried material was dissolved in 0. J. 342 (1968). Pharmacol. H. (22) R.2 ml) was added to the filtrate. 10 ml and applied to a column (3 X63 cm) of Sephadex G-25 (100-200 mesh).2 with 1 N NH. This material was dissolved in the upper phase (3 ml) of the solvent system BuOH-PrOH-H.5 with 1 N AcOH.13 mmole) in DMSO (3 ml). Acta. 5500 (1966). Schwartz. 66. HF was evapd with N. US.8. Sawyer. Acad. B. F. B.Oi. 776 (1948). Aboderin. Chem.5% AcOH and 1.34 with upper phase of BuOHAcOH-H. [CY]~’D -34. Pharmacol.O (4:1 :l ) . D.” 17th rev. Assays were carried out on 6 rats.01 N K..2 mmole) and the reaction was carried out at room temp for 6 hr. A. J . Stein. B. Walter. Bodanszky and V. Schwartz. Fractions 71-83 were combined. Endocrinology. which had been equilibrated with the lower phase of BuOH-EtOH-H. 46.694 (1958). Helv. Pays-Bas. US. H. Lowry.02). I. Brit. Darnell. and P. C. Weygand. Munsick.55 by tlc on silica gel G (Merck) in a solvent system of BuOH-AcOH-H. H. W. and V. 237. I . Nefkens. p 5 1 . Yamashiro. Dbu (1. A soln of 1 M hydrazine hydrate in DMSO (0. and J. and R. B. in “Organic Syntheses. 87. 257 (1968). N. I. 3107 (1954). A.5% AcOH and 1..)-Dbu~-Bzl-Cy~~oLeu-Gly-NH. M. 147 mg (77%).. Mintz and Mrs. by M ~ n s i c k with ~ ~ the use of Mg-free van Dyke-Hastings soln as the bathing fluid.676 (1965). H. L. 88. Reel. The aq layer was passed through a column (3 X 8 cm) of Dowex 1-X2 (acetate form).. H. Amer. 1971. yield of [ 5-ol. 18. Walter.. (30) H.N. Chem.. Gillessen. M.02). 351. J . Protein and Polypeptide Horm. Both methods gave identical product showing an R f 0. 2. L. 16. R.” Collect.42 for Tyr). Aust. N. 15 ml) together with anisole (0. 1355 (1971). Compd 10 (146 mg. J. Kishida. C. 3. C.Sz ’ CzH. 68. 0. f37) W. In an alternative experiment a soln of 2 M hydrazine acetate in DMF (2 ml) was used for the removal of the phthalyl group of 9 (306 mg.5%pyridine (7:2:9) (0. 162. Rudinger. and W. and S.5 ml). J.NH. Delpierre.91.O ( 4 : l : l ) (0. Walter. 2504 (1959).. urethane-anesthetized male rats as described in the United States Pharma~opeia. (24) S. du Vigneaud. J.and excess ferricyanide ions were removed by treatment of the soln with AG3X4 (Bio-Rad) in the CF form.. and V. Denning. Vol. Schwartz. M. which was dried in a desiccator over NaOH for 3 hr.y-Diaminobutyric acidloxytocin (1 1).. (36) . I .O contg 3.O. Amer. 184 (1942): (38) W. Y. New York. 155 mg (53%). Chan. 2. V. Walter. Austin. Chem. J. B. SOC. Chem. Biol. Proc. 1965. 30. Chem. lyophilized. V.46 for Tyr). 50 mg. Soc..08 with BuOH-AcOH-H. 40. M. After washing the column well with H. Hoppe-Seyler’s Z. J.Exp. Kidd and F.688 (1960).Ile (0.1183 (1955).H. A. The reaction mixt was stirred vigorously at 75” for 3 hr.The eluates were dild with H. A. Easton. Proc. H.O (100 ml).Physiol. R f values on tlc on silica gel G (Merck) with different solvent systems gave the following values: 0. A. Amer. Jaquenoud and R.128 Journalof Medicinal Chemistry. Amer.933 (1965).32 with upper phase of BuOH-EtOH4. L. Rat pressor assays were carried out on 5 atropinized.~’ The biological activities were measured against the USP Posterior Pituitary Reference Standard.O.181 (1971). The reaction mixt was stirred at 20” for 60 min. J. Ed.J.” as modified by Sawyer. Nat. Chem.04). Livezey... Aliquots from every second fraction were taken for detn of Folin-Lowry color values. 1836 (1969). G. 1310 (1966). Oxytocic assays were performed on 6 isolated uterine horns from 3 rats in natural estrus according to the method of H ~ l t o n .76.CCO. A. 19. Tesser. 1856 (1949). D. Weinstein. Beyerman. S . T.6” (c 0. and H.O and lyophilized. 193. Ther. Murti. Anal.. Commun.I. Beyerman. 1355 (1967).. Proc. 71. SOC. Dubois. and dried. H. I. Rudinger. Kuwata and H. 66..6.N. Hornle. J. Med. (2. F.. 653 (1967). 81. S. Walter and I. L. J. Chem.02). J.mp 211-216” dec. The product was pptd with H. Gutte. N. 1563 (1962). Rosebrough. not more than 6 hormone injections were given to each animal. Walter.C. L. J.Pro (1. J. and A. p. Nature (London). du Vigneaud. Pays-Bas.560 (1959). Pa.956 (1971). Havran.. DMF). Hoffmann.y-diaminobutyric acid] oxytocin as monoacetate. Acad. Soc. Nivard?R e d . Steward.4 ml) was added to a suspension of 9 (200 mg.. C. Guttmann and R. in contrast to average duration of the response. Org. 860 (1960).38 maximal depression of urine flow. R. Schwartz.

2009 More About This Article The permalink http://dx. Adamantane and analogous spiro-3'-pyrrolidines K. Paerels. Washington. Peters J.org on April 25. 129-132• DOI: 10.W. B. Med.. 1. L. J. Chem. Lundahl. and A..1021/jm00272a003 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. DC 20036 . 1155 Sixteenth Street N. J. A. G.org/10. 15 (2).doi. M.acs. Schut. 1972.1021/jm00272a003 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. Schlatmann.Synthesis and antiviral activities of adamantane spiro compounds.

perhydro-4. with subsequent saponification and decarboxylation. R=CJIii R=CH2C6H5 R = CHzCHzOH Scheme I1 3 L Sa Discussion The antiviral activity of theN-methyl derivative 6b has been the subject of a previous communi~ation.~ Compared with 1-adamantanamine this compound is about 3 times more active in vivo against Influenza A2 Japan and A2 Hong Kong. In vitro it shows a broader antiviral spectrum: 6b was demonstrated to be active against Coxsackie A2 1 and Rhino 2 (HGP) in qualitative (Table IV) and quantitative experiments: whereas 1-adamantanamine was inactive.3.H. Received May 1. Compd Sa could also be synthesized by treatment of the dinitrile 3 with HBr and hydrolysis of the reaction product5 (Scheme 11). 2-Adamantanone (1) was condensed with cyanoacetic ester using Cope conditions4 to give 2 (Scheme I). R = H d. 2 129 Synthesis and Antiviral Activities of Adamantane Spiro Compounds.10-4 mole/kg per day in comparative experiments with I-adamantanamine? Virus challenge (by aerosolation) took place between the two administrations of the compounds on the first day of the experiment. c. feasible. This paper deals with the synthesis and antiviral activity of a number of adamantanespiro-3’-pyrrolidines.7-methanoindane. parainfluenza Sendai. In particular smaller alkyl-substituted derivatives displayed interesting antiviral action against influenza A. R = CH. R = CH. I Journal of Medicinal Chemistry. Paerels.3 Synthesis. 6i was formed by alkylation of 6a with acetone and subsequent reduction.1 0-4mole/kg per day or 1. Coxsackie A2 1. The adamantanespiro-3 -pyrrolidines 6a-6f (Table 11) were synthesized by reduction of the corresponding diones with LAH.Adamantane Spiro Compounds. HOAc HCN 1 2 3 4 Sa. 2-cyano-2-cyanomethyladamantane (3). J. Compd 6h was formed by acetylation of 6a and subsequent reduction. The synthesis and antiviral properties of related adamantane spiro heterocyclic compounds will be the subject of a further paper. The dose was 5. Philips-Duphar Research Laboratories. The synthesis of the spiropyrrolidine derivatives of bornane. f. d.5-dione (Sa). Addition of HCN to 2 in strongly alkaline medium proceeded smoothly and gave. and cyclohexane has been achieved by the same methods (Table 111). 15. The Netherlands. R = CHZCHZOH b. A. No. In vitro data (Table IV) were obtained by the plaque inhibition method. perhydro-4.3. e. R = CHI C.’ The compounds were added just before virus inoculation.H NaOAc. The adamantanespiro-3’-pyrrolidines which have smaller alkyl sub- . Vol. The discovery of a suitable method for the synthesis of 2-adamantanone2 furthered the possibilities of substitution at a bridge atom of the adamantane nucleus and made the synthesis of secondarily substituted adamantanes. The N-methyl adamantanespiro-3 -pyrrolidine is superior to l-adamantanamine in level and spectrum of activity. In view of the interesting antiviral properties of l-adamantanamine’ it was deemed worthwhile to study the influence of structural modifications on antiviral activity. Scheme I COOC. Adamantane and Analogous Spiro3’-pyrrolidines K. R = CHzC& f.CH=CH* 6a. L.7-methanoindane. Weesp. M.* and A. including spiro compounds. R = H b. The importance of the adamantane nucleus for antiviral activity was investigated by comparison with spiropyrrolidines derived from bornane.COOC. Schlatmann. Reaction of 4 with primary amines gave the corresponding substituted pyrrolidines diones Sb-Sf (Table I). NCCH. 1. and rhinovirus. Lundahl. 1972. and cyclohexane. G . This activity was compared’with that of spiropyrrolidines derived from other alicyclic compounds. Schut.CH=CH. The in vivo antiviral activities against Influenza A2 Japan are presented in Tables I1 and 111.l]nonane. In vivo experiments were carried out using mice (Swiss SPF) weighing 19-21 g each. The substances were administered orally twice a day for 5 days. Peters N. R=CH. B. V. R = C 6 H i i e . Compd 6g was obtained by treatment of 6f with S0C12. Hydrolysis of 3 afforded the adamantanespiro-2’-succinic anhydride (4) which was converted by treatment with NH3 into adamantanespiro-3’-pyrrolidine-2. Catalytic reduction of 3 followed by pyrolysis of the diamine dihydrochloride6 7 led to an alternate synthesis of 6a (Scheme 111). bicyclo [3. J.1]nonane. 19 71 Adamantanespiro-3’-pyrrolidineand several N-substituted derivatives were synthesized. bicycle [3. Biological Results.

bN: calcd. H.H.. HCI C. Increasing size of the substituents at the N-atom tends tQ diminish in vivo activity against Influenza A2 Japan.N. "C 3 Sa b c e f H CH. "C Yield... %ee reference 8..5 254 dec 77 92 87 60 83 47 85 80 94 ++ ++ ++ + + - + + + 'Prepd by procedure B described in the Experimental Section unless otherwise stated. Of the analogous spiro-3'-pyrrolidines activity is highest for 9 structurally most closely related to adamantane.63.. CH. Table 111.No. 15. H. CH2C6H5 d e f g CH.5 144-145 91 85 81 70 90 63 6a 'Prepd by procedure A described in the Experimental Section. C. 1972. HCl 83-8Sd stituents generally are comparable to 6b in level and spectrum of activity (Tables I1 and IV).NO. qrocedure given separately in the Experimental Section. H. 0 7 187-189 214-216 84.H. C1 C.. H. N. b++ = activity comparable to that of l-adamantamine or better. HCl C. N.C. N. N. CH.N..5"): C.NO. "c: calcd. Adamantanespiro-3'-pyrrolidine-2'.. Both compounds were less active in vivo against Influenza A. R Formula Analyses Mp...and -oct-2-enamines. 0 C.. C.N. ..13. HCl C. % An tiviral act. 0 H. eAnalysis for picrate (mp 171-171. N C.N.NO * HC1 C. C1 H. 0. H. Japan than 6b. Table 11. H. 77.CH. making an estimation of antiviral activity in vitro impossible in the procedure used. but comparable to 1-adamantanamine..N. H.. Adamantanesuiro-3 "molidines' ~ ~~ ~ ~~ No. % Paerels. H..H.= inactive.5-144 138-138.. 2 Scheme 111 Yield. H. C. CH." In general cytotoxicity is found with 6d-6g.OH C. The synthesis of Sa has been described separately. HC1 C.H.N hydromaleate C. **Note Added in Roof. 6.130 Journal of Medicinal Chemistry. fC: calcd.5 11 "See footnote b. Analogous Spiro-3'-pyrrolidines No. et al..18..H.litc mp 151". 61. C1 C..H. found. HCI Cl6HZ5N. N. + = significant activity but less than that of 1-adamantanmine.2]octan. 77. Table I. HCI 186-188 70.. HCl C..found. N. the diamantanespiro-3'-pyrrolidineand its N-methyl derivative were prepared. m NH ' HCIC C.H. H.N03 C...H..H. "C Yield. N..OH CH.74.N.H3. Vol. . N.H.. C.H. 0 C.N..5'.NO. 0. Structure Formula Analyses MP. Cb C.H.5-87. dpicrate mp 148-149". This stresses the importance of the adamantane nucleus for antiviral activity in this series. C. Table 11. H.H...5 142. R Formula Analyses MP.CH.NO.H.2. A similar trend has been observed in a series of bicyclo[2.N.CH.** In general no activity was found against B strains of Influenza neither in vitro nor in vivo.CH=CH.CH=CH. 266-267.5 10 HCl C.CIC C.NO. H. After completion of this manuscript.H..H. 0.N.06.H.ClN~ HCl C.. N.H. N. H. N. N C. Nevertheless 9 is less active than 6b.63. 62. C1 C. ~f 253-254 144-145d 266-268 273-276 256-260 dec 262-264 302-306 236-237. HCI 219-221 81. in vivob 6a b H CH3 C2H5C h i C i-C3H7C CH.The cyclohexane derivative 11 does not in fact possess any activity at all.. N. 0 C. in vivoa 8 C. dMp of hydrochloride.. N e C. 5. HCl 246-247 90 + ++ + 9 C. % An tiviral act. found..5 '-dionesa No.

) H. Bernhardt. ethyl bicyclo[ 3. a soln of KCN (64 g. Yields and properties are given in Table I. After cooling. N-Isopropyladamantanespiro-3'-pyrrolidine Hydrochloride (6i). + = 40-70% inhibition. 2-Carboxy-2-carboxymethylbomane. 6a crystd after addn of Et. (1 g) at 50". T = cytotoxic. 64%. H. 6020 tube voltmeter.) to give Sa (see Table I). the solid was filtered off.45 g of 6i. Adamantanespiro-3'-pyrrolidine Hydrochloride (6a).O (140 ml) was added. and mass spectroscopic data are fully in accord with the structures proposed. H. N: calcd. The temp rose to approx 110". Procedure of Scheme II. The solid carboxamidecarboxyadamantane was heated under N. After cooling the reaction product was converted into the free base by treatment with 2 N NaOH and was extd with CH.llnon-9ylidenecyanoacetate was converted into the corresponding dinitrile: yield. No. 76.5 1. After cooling.. ceased.H.8 g.34. HeLa cells. Bontekoe.H. H. Bicyclo [ 3.05-0. Acknowledgments. and to Miss J. 77.O in a usual way. S.." The bases were converted into the hydrochlorides and crystd from EtOH. Me. mp 226-229'. solvent CH. 26.)C.:4 yield. A . found.. Anal. Ethyl Perhydr0-4. was then evapd and C. 3 (76 g.6 g (76%). (C.Hl6NZ) C.1 1. After cooling. General Procedure A.l]nonane-9-spiro-3'-pyrrolidine Hydrochloride (9).3. see Table 111.l]non-9-ylidenecyanoacetate. column 5 X 150 cm) with C6H6yielding 10. 0. Adamantanespiro-3'-pyrrolidines (6). Following the procedure used for the prepn of 4.O. see Table 11. Pascher.Perhydro-4. see Table 11. (C13~. (3. A further crop was obtained by concn of the mother liquor. Anal.H. f = 20-40% inhibition. . Prepd according to method A: yield. er al. Anal. 2-Carboxy-Zcarboxymethylbornane (11 g. N-Methylbornane-2-spiro-3'-pyrrolidine-2'. The solvent was evapd.1 Inonane. 0. 0. 0. W.65 g of the anhydride as a waxy substance. Anal. 0.1]nonan-9-0ne'~ (13. 2Cyano-2-cyanomethyladamantane (3). (C1. The crystals were stirred in a 0.4% of the theoretical values.) for 2 hr at 80". Anal. (C.). 5. 14. 72.. found.5-136'.-EtOH-25% NH. 2 (100 g.. analytical results of those elements were within *0. Ethyl Bicyclo [ 3. The mixt was stirred and kept at 65" for 16 hr. H.NO.) C. and F. .H.H. After 6-8 hr the uptake of H.63. Adamantanespiro-2'-succinicAnhydride (4).5 g. being passed in continuously (4 l.5'dione. Vol. 0. N-Methylbomane-2-spiro-3'-pyrrolidine hydrochloride (8) was prepd according to procedure B (see Table 111).24 mole) was melted and heated at 230" for 3.02 g of 6g.05-0. 75. Germany. mp 81-82'. Procedure of Scheme 111.68.98 mole) in H. the residue was crystd from EtOH (0.N. H.5-87". column 5 X 150 cm) with C. 0: calcd. fl-AA = 1-adamantanamine. N.OH ( 3 : 3 : 1 v/v). 3 (10 g. M. After cooling. NH. CIn calf kidney cells. By concn of the filtrate some further crops were obtained. 0.3 g (46%) of cryst product: mp 50-51".Cl. J . Bonn.. Concn of compounds 10-4M. 14. and crystd from C. C. N. C: calcd. washed. Germany. 2-Cyano-2-cyanomethylbornane.$ 3 was converted into Sa according to ref 5 : yield.0.3.096 mole) was condensed with ethyl cyanoacetate by the method used for the prepn of 2..7-methanoindane was obtd by the method used for the prepn of 3: yield. 26.1 g. 8 (7 g. Bicyclo[3. with stirring. cording to tlc.NO. (0. at 320" for 5-10 min. 10 was obtd from 5-cyano-5-cyanomethyl perhydro-4.5-4 hr. I Table IV. to Mr. 5 was reduced with LAH in refluxing THF according to known methods. 67%.: yield. Where analyses are indicated only by symbols of the elements. Mikroanalytisches Lab. mp 135. the ppt was filtered with suction. Yields and properties are given in Table 11. N-p-Chloroethyladamantanespiro-3'-pyrrolidine Hydrochloride (6g).2 N KOH soln (500 ml). N.15 mole) was dissolved in hot C.57. 4 (33 g. Adamantanespiro-3'-pyrrolidine-2'.88.H.5".8 g (38%) of a colorless oil. H.H.. Following the procedure of scheme 111 for the prepn of 6a. 5-Cyano-5-cyanomethyl perhydro-4.05 mole) and PtO. The free base of 6a (2.7-methanoindan-5-one# (30 g. washed with H. 51%..CO (4 ml. The catalyst wos filtered off and a colorless soln resulted in which 6a and the diamine 7 were present ac?Melting points were measured in closed capillary tubes in an electrically heated aluminum block. After the evoln of gas had ceased the product was distd in vacuo to yield 7. By the method of Scheme 111 for the prepn of 6a.) C. The mixt was neutralized with 50% NaOH.. the mixt was cooled.8 The solvent was evapd and the resulting residue was heated under N.2-cyano-2-cyanomethylbornane (10.O.) C. chick embryo fibroblasts. eSlightly toxic. H. 0: calcd.O.) H. van der Heeden for measurements and interpretation of the spectra. van Deursen and Miss M.) C. After standing for 0. yielding 18. Japanb SendaiC A.2 mm.2 g (63%). 0. and dried: yield. A. (C..3. in concd HCl (2. found.3. (750 ml). 93%. C: calcd. Plekkenpol for cooperation in the synthesis of several compounds. filtered./hr). N-Ethyladamantanespiro-3'-pyrrolidine Hydrochloride (6h). H. 14. 7. D.H. Anal.Cl.).15 mole) in C. After removal of the catalyst the soln was acidified with 1.H.60..4 N dry HC1 in EtOH (12 ml) and evapd.S'-dbnes (5). crystd from EtOH. 90%. mp 126-127". found.41 mole) was dissolved in EtOH (800 ml).. Mikroanalytisches Lab. llnonane (see Table 111). (50 ml) and with vigorous stirring RNH.Adamantane Spiro Compounds. 1972. Anal. 4 (5 2. A further crop was obtained by addn of H.. Bomane-2-spiro-2'-succinic Anhydride. The resulting oil was purified by dry column c h ~ o m a t o g (Merck '~ silica gel 0.2 mm. (3 X 20 ml) was added and evapd to 7. 0. We are indebted to Mr.03. According to the procedure used for the prepn of 3. The free base of 6a was acylated with Ac.38 mole) was dissolved at 90" in concd H.2 mole) was condensed with ethyl cyanoacetate as in the prepn of 2. for 15-30 min at 220-225'.5 g (96%).H. F. The acetamide was reduced with LAH in refluxing THF in 16 hr: yield. 87%. N.H.O) gave 2. the residue was crystd from EtOH. nmr. Elbach uber Engelskirchen. mp 86. Temperature was indicated b y a chromel-alumel couple on a Philips G. 13. Ethyl born-2-ylidenecyanoacetate" was converted into 2-cyano-2-cyanomethylbornane according to the procedure described for 3: yield. 0.045 mole) was heated at 180" for 20 min under N.026 mole) was dissolved in SOC1.2 g.Sfdione (Sa). 0.) C. W. The resulting red oil was purified by dry column chromatogI4 (Merck silica gel 0. Experimental Section? Ethyl adamant-2-ylidenecyanoacetate(2) was prepd from 2adamantanone according to Cope.. M. (30 ml) was added at 60-65". Sekbt for supplying the antiviral i n vitro results.4 g) were added. (C..052 mole) was converted into 5. 9 10 11 1-AA +e + - f +e f f + ++ - T f T f - a++ = >70% inhibition.07.NO. $This synthesis was performed by Mr. the residue dissolved in MeOH (30 ml) and the soln acidified with concd HCl(5 ml).4 g (61%) of 2-carboxy-2-carboxymethylbornane: mp 138-140". N.= <20% inhibition. 0. 0. In Vitro Antiviral Properties of the SDir0-3'-uyrrolidines Hydrochloridesa Influenza Parainfluenza Coxsackie No. 9-Cyano-9-cyanomethylbicyclo [ 3. tlc-plates silica gel F 2 5 4 . and the mixt was hydrogenated overnight at 4.H. The moist substance was heated. 9 was obtained from 9-cyano-9-cyanomethyl bicyclo[ 3. Anal. mp 99-99. found. E. 15.3. Adamantanespiro-3'-pyrrolidine-2'.55. and the ppt was filtered with suction and washed with H. mp 152-153". 0. 0.01.5'dione (Sa). 72. fjMerck.3.7-methanoindane (see Table 111).015 mole) was dissolved in EtOH (10 ml). Crystn (EtOH-Et. (C.05 mole) in EtOH (250 ml) and concd HCl(l0 ml) was hydrogenated with PtO. The soln was shaken for 5 min and poured on ice (approx 10 1. Anal. W.5 hr at 60-65". Microanalyses were performed b y A. (14 ml) and refluxed for 30 min.SO. (C. Perhydro-4-7-methanoindane-S-spiro-3'-pyrrolidine Hydrochloride (10). (C. #Obtained from Aldrich Chemical Co.ld 6a 6b 6h 6i 6c 8 Journal of Medicinal Chemistry.2 kg/cmz. Adamantanespiro-3'-pyrrolidine-2'. and the ppt was filtered and washed (C. 96%.7-methanoind-S-ylidenecyanoacetate. Ir.2 131 Rhino 2(HGPId + ++ + +e ++e f T + - + + f ++ ++ ++e T T ++e T f - i.N.$I160.O: yield.N. The SOC1. General Procedure B.

(19641. and M. Bull.2-bis(cyanomethyl)adamantane. Cragoe. A. Arch.. G . Virusforsch. Asinger. Davies. Geluk and J.2. R. R. S.. T. Science.* A. leading to heterocyclic spiro compounds. also with cyanoacetic ester as reagent. Starting with adamantanespiro-2'-succinic anhydride' we were able to prepare adamantanespiro4'-perhydroazepine XVIII and adamantanespiro-3'-piperidine XV via another route (Scheme 111). The synthesis and antiviral properties of adamantanespiro3'-pyrrolidine and derivatives have been described in a previous paper. Soc. Chem. L.and 7-membered ring analogs. Geschickter. Goodman. and E. W. (14) B. Org. VI11 = a. Th. M. J. W. Peters. Treatment of I with malononitrile did not give a Michael reaction but instead led to the formation of adamantylidenemalononitrile in high yield (Scheme I). Hardenbergh. J. 2 R. hlcGahen..' For that we had to prepare 2. Chem. T. J. A14. C.' Because of the strong antiviral activities found in the pyrrolidine series.442 (1887). 132 (1972). Synthesis. 1969. F.. Loev.. . H. Finally reduction with LAH gave the spiro compound IX.- van Hes. Peters N. 15. and F. F. 32. 'T. Hofmann. Med. J. A. which could be converted into the diacid by hydrolysis. E. K. Goedemans. Smit. The antiviral activity of these compounds is discussed. W. International Congr. J. B. L. A . which could be converted into the lactone V with TsOH. The cyclic imide VI11 was obtained by treatment of the anhydride with MeNH2 in C&IH. Roland. C. Sprague. Ind. Sheth. (12) E. A. L. Tobey. Amer. K. 19. Vol. 19 71 The synthesis of adaniantanespiro-3'-piperidine (XV) and adamantanespiro-4'-perhydroazepine (XVIII) is reported. and C.381 (1950). 13. 190 (1966). Peters. B. M. Kralt. Proc. M. Med. Med.132 Journal oj Medicinul Chemistry. S. A. E. G C : O H CH COOCH. Treatment of V with KCN in DMSO resulted in the formation of VI. Chemother. Re- ~ o VI1 z F J - J p = 0 CH. (10) J. R. Chem. et al.862 (1 964). C. 15. Watts. C. C. C. Grunert.R. 12. Chem. W. A . These compounds can be prepared from the key intermediate 2-bromomethyl2-(&bromoethyl)adamantane (XII). E. Schlatmann. M. A possible explanation may be steric hindrance. Johnson.. Chem. Hoffmann. Smit. 1003 (1968). V. 3452 (1941). Rice. Chem. Fell. Peters.3325 (1967). de Bock. A. (8) H. Seide. 197. LAH IV e . J. Tetrahedron Lett. 15.\ . R. J. A. E. J.. Treatment of adamantanespiro-2'-succinic anhydride' with MeOH gave the half-ester 111. Sette.. Chzm. Paerels. we continued our investigations in this field by preparing 6. Treatment of the diol X with 48% HBr or with PBr3 did not produce the de- . van Hes. M. With the latter several other reactions have been carried out. Following Scheme I1 it was possible to prepare the 6-membered ring analog. Najer. Whitney. Giudicelli. W. G. References W. Synthesis and Antiviral Activities of Adamantane Spiro Compounds. IX duction of the anhydride with LAH in THF gave the diol X as well as 12% of the hydroxy acid IV. Ladenburg.11. and J. M. FzoH V VI I11 ried out the reaction under various conditions. Paulshock. Wood. and W. Schlatmann.. 24. A. Org. 63. and A. Neumayer. We carScheme I CN Scheme 11 No ___. Received May 4.. Hermann. Philips-Duphar Research Laboratories. Cheni. Reduction with LAH in Et20 yielded the hydroxycarboxylic acid IV. Hermann. (1 1) L. and E. 5361 (1968). Snyder. and A. Nasutavicus. and C. Gregory. and F. J. van Hes.. Tetrahedron. Refluxing the diacid with Ac20 gave the anhydride VI1 in high yield. J. R. 144. (13) B. Soc. J... W. but failed to obtain the desired condensation product. Fr.. and J. Jr. Scott. C. R. Ber. J . Weesp. M. M. A. 2 2572 _ . 71 (1970). (London). and J. Kralt. R. Robb. M . (9) A . Cope. C.. Ges. Wyckhoff. The Netherlands. Haff. C. We first tried to obtain the 6-membered analog by an analogous route as for the adamantanespiro-3'-pyrrolidine. Kauer. Benson. No. 254 (1970). 2026 (1967).126 (1969). 20.

Washington. Chem. and A. 2 R.. 1155 Sixteenth Street N.org on April 25. 2009 More About This Article The permalink http://dx.1021/jm00272a004 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. Smit. Med. T.. A. Van Hes. Peters J. 132-136• DOI: 10.acs.doi. 15 (2).W.org/10. 1972. Kralt.1021/jm00272a004 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society.Synthesis and antiviral activities of adamantane spiro compounds. DC 20036 .

K. (14) B. 32. leading to heterocyclic spiro compounds. Org. 19. S. Ind. and E. G . M . W. References W. R. and C. L. Chem. Benson.. A. and J. These compounds can be prepared from the key intermediate 2-bromomethyl2-(&bromoethyl)adamantane (XII). R. Hoffmann. We first tried to obtain the 6-membered analog by an analogous route as for the adamantanespiro-3'-pyrrolidine.. Reduction with LAH in Et20 yielded the hydroxycarboxylic acid IV. A.132 Journal oj Medicinul Chemistry.. and E.. Peters. and M. The Netherlands.. No. but failed to obtain the desired condensation product.. (9) A . M. Med.. A. G. Goedemans. R. Sheth. Weesp. Paulshock. A . Kauer. The cyclic imide VI11 was obtained by treatment of the anhydride with MeNH2 in C&IH. which could be converted into the diacid by hydrolysis. L. Sette. 1969. 1003 (1968).. J. Scott. M. Peters.862 (1 964). Refluxing the diacid with Ac20 gave the anhydride VI1 in high yield. 20. Re- VI ~ o VI1 z F J - J p = 0 CH.R. C. L. Amer. J. Smit. C. (12) E. Loev. J. Smit. R. 2026 (1967). Ges. Whitney. W.442 (1887). J . J. Treatment of the diol X with 48% HBr or with PBr3 did not produce the de- . W. van Hes. Goodman. which could be converted into the lactone V with TsOH. Soc. Philips-Duphar Research Laboratories. H. A possible explanation may be steric hindrance. E. W. Tetrahedron. Nasutavicus. FzoH V I11 ried out the reaction under various conditions. and C.126 (1969). Fell. 132 (1972). also with cyanoacetic ester as reagent. Treatment of V with KCN in DMSO resulted in the formation of VI. G C : O H CH COOCH. Roland. A. C. Snyder. Synthesis. Peters N.' For that we had to prepare 2.. and J. van Hes. Chem. 2 R. Treatment of adamantanespiro-2'-succinic anhydride' with MeOH gave the half-ester 111. Proc. 12. Geluk and J. Chem. Th. Cragoe. . S. (1 1) L. C. Bull.2. We carScheme I CN Scheme 11 No ___. 19 71 The synthesis of adaniantanespiro-3'-piperidine (XV) and adamantanespiro-4'-perhydroazepine (XVIII) is reported. Cope. Watts. Seide. Hermann. and J. J. and A. F. 254 (1970). A. (London). Schlatmann.2-bis(cyanomethyl)adamantane. Med. Hofmann. C. M. 197.* A. Grunert. Hermann. Hardenbergh. Chzm. K. 15. Wood. Cheni. 2 2572 _ . hlcGahen. T. Kralt. and W. Treatment of I with malononitrile did not give a Michael reaction but instead led to the formation of adamantylidenemalononitrile in high yield (Scheme I).\ . Tetrahedron Lett. E.and 7-membered ring analogs. 3452 (1941). M. R. (13) B. M. Haff. Neumayer. 144. M. The synthesis and antiviral properties of adamantanespiro3'-pyrrolidine and derivatives have been described in a previous paper. C.. Chem.3325 (1967). VI11 = a. J. C. J. LAH IV e . J. B. W. Fr. A. Schlatmann. Paerels. Science. The antiviral activity of these compounds is discussed... V.. Vol.- van Hes. et al. R. T. 71 (1970). Chem. Ber. and A. IX duction of the anhydride with LAH in THF gave the diol X as well as 12% of the hydroxy acid IV. J. Geschickter. de Bock. C. 5361 (1968). Asinger. Ladenburg. A. W.' Because of the strong antiviral activities found in the pyrrolidine series. F. A14. Arch. Kralt. and F. 63. Rice. Peters.. B.(19641. E. Org. Jr. Tobey. M. M. Giudicelli. we continued our investigations in this field by preparing 6. R.11. 190 (1966). Davies. (10) J. 15. Virusforsch. J. International Congr. (8) H. 'T. Finally reduction with LAH gave the spiro compound IX.. Robb. Following Scheme I1 it was possible to prepare the 6-membered ring analog. C. Starting with adamantanespiro-2'-succinic anhydride' we were able to prepare adamantanespiro4'-perhydroazepine XVIII and adamantanespiro-3'-piperidine XV via another route (Scheme 111). A. Wyckhoff. J. Najer. and F. Received May 4. Chemother. Gregory. Sprague.381 (1950). Synthesis and Antiviral Activities of Adamantane Spiro Compounds. 24. 13. A . Soc. With the latter several other reactions have been carried out. 15. E. Chem. Johnson. Med.

The 7-membered ring analog XVIII could be obtained from XVI by two different methods. XXVIII). to give the imide XX. As mentioned above.-H CH. HCl CH.2 The reaction of the dibromide with NaCN (technical grade) in methyl Cellosolve yielded a mixture of two products. H ~ O H ~ ~ ~ NH. The dibromide could be cyclized with NazS in DMF to XXV. the results are much better (Scheme IV).Antiviral Adamantane Spiro Compounds. Treatment of the dibromide with primary amines resulted in the formation of spiropyrrolidine compounds (XXVII. was about 90%. No. which cyclized to XV upon treatment with KOH in n-BuOH. . followed by NaH. Reduction of XVI with Pt02-Hz in EtOH-HCI resulted in the formation of XVII. (CH. Reactions with the Dibromide XII. Vol.. Treatment of a solution of the dibromide XI1 in DMSO with NaCN.NH. which with Me1 could be converted to the sulfonium iodide XXVI in good yield. HCl XXIV XXIIl . Reduction with LAH afforded the desired compound XVIII. The 2 compounds could be separated by chromatography. Refluxing of the dibromide with dried NaCN then led to the spiro compound XXI by a Thorpe-Ziegler ring closure of the intermediate dicyanide XVI (see also “Reactions with the Dibromide XII”).KOH ___c 2. HCl XVII A xx V 0 V XIX sired dibromide XI1 but the ether XI.).-H. . The bromocyano compound XI11 could be reduced with PtOzHZin EtOH-HCI to XIV. Heating of the latter at about 290” caused ring closure.CH.Br XIV 1. If the reaction is performed in DMSO4 with NaH. gave the cyclic product XXI in a yield of 75%.NH. Pt 0. HCl HCl - NH . In the other method3 the dicyanide was treated with HBr-EtzO. Reaction of XXII with HzNOH resulted in the formation of XXIII. followed by hydrolysis. 2 133 Scheme I11 X XI XI1 PtO. XXI on hydrolysis5 was converted in high yield to the ketone XXII.). 1972. the bromocyano compound XI11 in about 30% yield and the dicyano compound XVI in 50% yield. isolated in the absence of the NaH step. 1.NH ‘ HCI “““i MC I XI11 (CH. HCI ? ’7 fl 1 XV XVIII . because of the slight reactivity of one of the Br atoms (neopentyl structure) of the dibromide.Br DMSO NaCN D __c [0~ XVI XI1 I 2. 15. HCI N ~ HzNoH - C . By reduction of XXIII with Na in EtOH the amine XXN was obtained. The latter comScheme IV CH. The yield of dinitrile.CH.Br CH. XVIII was obtained in low yield (16%). 2 Journal of Medicinal Chemistry. The latter could be converted into the dibromide XI1 with Ph3P and Brz in benzonitrile. the dibromide could be cyclized to XXI by treatment with NaCN in methyl Cellosolve in a low yield.NH.

The ppt was filtered and crystd from Me. but it still is less active than corresponding pyrrolidine compound.) and evapd. and in the laboratory of Dornis und Kolbe.CH.41 g of malononitrile in 10 ml of pyridine was stirred at room temp for 23 hr.O. West Germany. After cooling. 2 van Hes.NH. In in vitro experiments these compounds generally appeared to be cytotoxic except the two piperidine derivatives IX and X V . the in vivo activity. / / XXVI Experimental Section (In collaboration with Mr. et (11 Scheme V idine derivative XV is about the same as that of I-adamantanamine. A mixt of 2 g of I and 2. nmr spectrum (CDCl. XXX and XXXI (Scheme VI). mp 174-177'. The reaction of the dibromide XI1 with H2"H2 * H 2 0 gave 2 crystalline products. and poured into 500 ml of H. Substitution at the N atom by Me (IX) reduces the activity. The acid s o h was extd with Et. XXXIV XXXII XXXIII pound was converted to the salicylideneimide XXIX (Scheme V). 3200. Nmr spectra were measured on a Varian HA-100 instrument (Me. This also followed from the reaction of XXX with salicylaldehyde to a Schiff base. A m ixt of 15.5. 2'-Oxoadamantanespiro-3'-tetrahydrofuran 0.O.CH.O: yield 16 g (90%) after / / H. cooled. Heym) Melting points were determined in open capillary tubes in a Buchi apparatus and are uncorrected.O soln was extd with 2 N KOH. and excluded the other possible isomer XXXIV.).SO. after which the solvent was evapd in vacuo and the residue dissolved in 2 N KOH. found 223.mp 108-110". The alkaline s o h was washed with Et. The structure of XXXI was proved by catalytic hydrogenolysis which led to the formation of adamantanespiro-3'-pyrrolidine.34 and 4.NH. or reduced.O s o h and crystn of the residue from hexane. 4 9 of the theoretical values. 2 triplets centered a t 82.5 g of KCN and 5. ir spectrum (KBr). mp 101-104". respectively. W.H. The reaction mixt was poured into 250 ml of 2 iV HCI. and 90 ml of Et.' Change of the basic function t o a sulfonium or hydrazino group (XXVI: XXX) destroyed.O formed was removed by distn. ['. respectively (CH. Estimation of in vitro antiviral activities was therefore not possible by the used method. The Et. 2-Carboxy-2-(p-hydroxyethyl)adamantane (IV). A mixt of 5.CO. - XXVIII XXIX Scheme VI xv i XXXI xxx 1 H. mass spectrum. mp 188-191".CH.H. calcd 224. found 247. The ppt was ffltered and crystd from EtOH: yield 1. West Germany. Mulheim a. The recording and interpretation of the spectra were carried out under the supervision of Mr. 2-Carboxy-2-methoxycarbonylmethyladamantane (111). Analyses were carried out in the laboratory of A. M' 206.8 g (94%). The compd was identical with an authentic sample. soln was dried (K.19 g (13%). analytical results obtained for those elements were within t 0 . No.81 g of 111. Ruhr. equiv wt. cooled. Crystn from Me. and then the alkaline s o h wa. equiv wt. A mixt of 1.O). Ir and mass spectra were recorded. Bernhard. 900 mg of LAH. 2-Carboxy-2-(/3-cyanothyl)adamantane (VI). Antiviral Properties. was refluxed and the H.62 g of adamantanespiro-2'-succinic anhydride. and extd with Et. the C. 1972. The perhydroazepine compound XVIII is about twice as active as l-adamantanamine. Elbach uber Engelskirchen.d.O. decompd with 2 N H. H.O and then acidified with 2 N HCl.17.. The in vivo antiviral activities against influenza A2 Japan are present in Table I. 1760 cm-' (7-lactone). and 15 ml of pyridine was boiled for 3 hr.54 g of V in 110 m l of dry DMSO was refluxed 4 hr.CO-hexane: yield 1. on a PE 337 and a MS 9 AE 1 spectrometer.' 250 ml of MeOH. A suspension of IV (9 g) and 50 mg of TsOH in 150 ml of C. 15.-PtO. and 3135 cm-'. van Deursen. Where analyses are indicated only by symbols of the elements. NCH. respectively.28 g (80%). Vol. 2-hydroxymethyl-2-(phydroxyethyl)adamantane (X) was isolated as a by-product: yield 0. After acidification with .NCH.134 Journal ofMedicina1 Chemistry. The in vivo activity of the piper- crystn from Et. Adamantylidenemalononitrile (11).5 g (94%): mp 180-182". calcd 252.CO-hexane afforded pure product: yield 7.O was refluxed 10 min. The ir spectrum of XXX in CC14 displayed bands at 3380. The cyclopentane derivative XXIV has an activity of the same order as that of the N-methyladamantanespir0-3'-pyrrolidine. acidified. characteristic6 of an >NNH2 group. After evapn of the Et.$).

54 (CH.Br~): 2-Bromomethyl-2-(p-cyanoethyl)adamantane (XIII) and 2-Cyanomethyl-2-@-cyanoethyl)adamantane (XVI).94 and 3. On completion of the addn. 3'-Cyano-4'-aminoadamantanespiro-3'-cyclopentene (XXI).79 (CH. was stirred and heated for 3 hr at 110-115'..H.CI.78 (CH.00 and 2.nmr spectrum (CDCI. 59..93 g of NaCN (dried in vacuo at 110") in 50 ml of methyl Cellosolve was refluxed for 96 hr.Antiviral Adamantane Spiro Compounds. singlet at 63. H.CH.1 g of NaCN (tech grade) in 50 ml of methyl Cellosolve was stirred and refluxed for 5 hr. H. NMethyladamantanespim3'-piperidine Hydrochloride OX). 15. HC1 C15H26CIN NH . mp 270-277" after crystn from EtOH.07 g (28.51. a. After cooling. (Cl.CI. H.O soln. a.9%). VI (6 g).CI. + = significant activity but less than that of 1-adamantanamine. For test conditions see previous paper. respectively.. HC1 C14H24aN c..O was refluxed for 5 hr.= inactive.52 g of 2-carboxy-2-(p-carboxyethyl)adamantane Ac.4 ml of 96% H.55 g (98%).H.). 6.5 hr.H.5 g (86%) C.. Anal. Crystn from C.7 g (92. and the residue (69.05 g of XI1 and 2. after crystn from hexane. with petr ether (bp 40-60') as eluant: yield 63. and 2700 ml of 1 N NaOH. the soln was acidified with 2 N HCI. found 128. H. the reaction mixt was heated to 125".H~.7.O and 200 ml of THF was added slowly. (C. . 1972..).O and extd with four 100-ml portions of CH.O was added and the mixt was extd with four 100-1111 portions of CH. Br. 2 N HCl the ppt was filtered and crystd from C.82 (CH. After sepn. N Cp H. 69.CP. Then the solvent was evapd in vacuo.25 g) in 100 ml of dry Et.5 g) qnd LAH (0.O) and 2 triplets centered at.8 g of XI1 and 5.5. and then 42. A soln of 0. The product was a brown oil: yield 50.3 g of Ph. A mixt of VI11 (0. The H. 2-Carboxy-2-(p-carboxyethyl)adamantane..).SO.57: found. salt was filtered. H. 3390 (NH). Vol. Fraction II.mp 236-239'.O). with CH2CI. mp 150-152"..O layer was acidified with concd HC1 and the ppt filtered: yield 8. The combined exts were washed with a 10% NaHCO. was refluxed and MeNH. Then the Ac.. ir spectrum (KBr).SO. and filtered.. was evapd... 50 ml of H. 2-Hydr6~ymethyl-2-@-hydroxyethyl)adamantane (X). respectively. The MeNH.CI. and 11. After cooling. (C. and the residue was chromatogd over a silica gel column. calcd 126. the CH.2 g of XI was added in 0. had mp 75.CIN): C.CI.84 and 3.5 g of VI1 in 60 ml of dry C.N.5 g) was heated in a sublimation apparatus under N.CO-hexane.). followed by 500 ml of 2 N H.O was refluxed for 2 hr. (C15H. HC1 XXVI c14H231s XXX 1 ' 3H23CIN2 "++ = activity comparable to that of 1-adamantanamine or better. k : calcd.7 g of X. 2-BrOmomethy1-2-(p-bromoethyl)adamantane (XII). A mixt of 7. N NH .8%) of XIII. .O. mp 301-306" after recrystn from EtOH-C. at 250-300".H.61 g (49%) of XVI. 59.O. The CH.01 (NCH. Thereupon a mixt of 50 ml of H. 24NMethyl-p-carboxamidoethyl)-2-carboxyadamantane (0. .). Anal. 1770 (C=O) and 1805 cm-' (C=O). dissolved in H. The ppt was filtered and sublimed at 250-300' (15 mm): yield 687 mg (41.. 1690 (COOH).mp 126-128" after crystn from Me. 2 Table I.ClN~ 0.calcd 233. in vivo" IX xv XVIII XXIV c c 0 0 + ++ NCH.N.5-77 after crystn from hexane. 2-(N-Methyl-~carboxamidoethyl)-2-carboxyadamantane. as eluant.70 (CH.).: yield 0. ir spectrum (KBr).H. soln was evapd and the residue sublimed at 200-300" (15 mm). 6 1. Crystn from EtOH-H.3 ml of Br. A mixt of 16. N An tiviral act.CI.6'-Dioxoadamantanespiro-3'-tetrahydropyran (VII).CI. the THF was evapd. had mp 116-118" after crystn from C. m).. 4. N. Adamantanespiro-2'-succinic anhydride (73 g) was added t o 1300 ml of dry THF.O layer was extd once with 250 ml of CH. A mixt and 30 ml of of 1. The THF soln was washed with a satd NaCl soln and dried (MgSO. 1615 cm-' (NC=O).. then the reaction complex was decompd with H. 2'. 60.7 g (85. 2'. mp 80-81'. the H.P in 245 ml of PhCN was treated slowly at 0' with 12. nmr spectrum (CDCI.): C. was refluxed 18 hr. equiv wt.6%). - A Formula Cl. After evapn of the solvent under reduced pressure the residue was suspended in 100 ml of H. The ppt was filtered and crystd from EtOH-C. Adamantane Spiro Compounds Journal of Medicinal Chemistry. . followed by slow addn of 21. N.4. then cooled.O and extd with Et. 61.32 g (93%). yielding 59.: yield 6 g (96%).CI. The mixt was boiled for 16 hr.25 H. singlet at 63. a.O was evapd in vacuo: yield 1.O) and 2 triplets centered at. Br.95 g (12%) of IV. After being dried (MgSO.C=O). Sublimate and residue were suspended in CH. A stirred soln of 62. After evapn of the CH. 70. Adamantanespiro-3'-tetrahydrofuran A mixt of 114 ml of 48% HBr. nmr spectrum (CCI.H.H.~N Analyses C.6'-Dioxo-N-methyladammtanespiro-3'-piperidine (VIII). COOH).O afforded pure VIII: yield 360 mg (78%). Fraction I. Cl.9 g of solid was obtained. No. and the ppt was filtered: yield 6 g (93%). found 232. The solids were filtered with suction and washed with THF.. singlet at 63.H.NBr): H. EtOH-HCl was added to the dried Et.O).-hexane. Anal. HCl ++ ++ C.) and 2 triplets centered at 62. found.H. the reaction mixt was chromatogd over a silica gel column. soln and dried (MgSO.. after which the temp was maintained at 125" for a further 3 hr. The undissolved product was sublimed again. 2500 (broad.4 g) was added to a mixt of 3800 ml of CH.H. C: calcd. was introduced simultaneously up to satn. and acidified with 2 N HCl. Anal. N NH. 2 135 No.O C. dissolved in 250 ml of 2 N NaOH. mp 119-121'.6%) of pure X: mp 109-11l". H.CH.equiv wt.3 g of LAH. After cooling.

After drying (MgSO. filtered.): C. J.Cl.O was evapd in vucuo.3 g (94%) of a yellow oil.. Anal. The combined Et.136 Journal of Medicrval Uiernistry.O.NNH.01 (CH. 8 ml of xylene.25 g of H.CS). until the solid material had dissolved.CI. 0 . (C. 1 ml of concd HCl.. Org. After the mixture had cooled.H.28 g (83%). and A. To the cooled soln. H.Hz31S): C. at 80" for 75 min.O and 0. (C.O was added.O: yield 0.. Ao. 26. EtOH-HC1 was added.5-94.7 g of dry NaCN.NH. C: calcd. Anal. A mixt of 85 mg of XXXI. A mixt of 0.O and an increasing amount of EtOH being used as eluant.54 g of XXV and 30 ml of Me1 was stirred for 24 hr at room temp. (C.41 g (93%). Spectrochim. calcd 228.O.79. and evapd to dryness in vucuo. mp 50. A soln ot 8. (C. After cooling.23 g of XIV in 300 ml of n-BuOH was added slowly t o a stirred and refluxing s o h of 1. and dried: yield 3. J . The structure was proved by redn of XXXI.O.. 100 ml of Et. After filtration and evapn of the solvent. 0. filtered. H. 129 (1972) (part 1). Med. H.. With EtOH-HCI in THF the HCl salt was obtained: mp 193. and 50 ml of EtOH was stirred for 15 min. with Et.O..O was evapd.O and extd with six 40-ml portions of CIi.O was treated Nith 3. yielding 560 mg (10. After cooling. S. 25A.7 g of FYO. H.t. The combined Et.O.O): C. yielding 2.[ N '-Salicylidene-(P-aminoethyl)] adamantanespiro-3'pyrrolidine (XXIX).4 g of LAH in 12 ml of THF was r e fluxed for 4 hr under N. AdaniantanespUo-4'-perhydroazepineHydrochloride (XVIII). After standing for 17 hr./-Aminopropyl)-2-bromomethyladamantane Hydrochloride W V ) . Jforded 1. The H. (C. J.3 g (95%) of a yellow-orange oil. 70. and the mixt extd with four 3 0 " portions of 0..2 g of PtO. nip 290-792".. (4) J . ir spectrum (KRr). yielding 50 mg of XXVII: mp 285-290".)..) and the addn of EtOH-HCI. 86.' N-(p-Aminoethyl)-adamantanespiro-3'-pyrrolidine (XXVIII).CH.. most of the EtOH was evapd.H...O): C.CI.mp 238-240'.3280 (1961).7g of KOH. and filtered. HBr was introduced into a mixt of 5 g of XVI and 200 ml of Et.mp 259-263". H. and added to a s o h of 1.). References ( 1 ) K. C. Fraction I1 was obtained as 500 mg (40%) of compd XXX (oil). After addn of Et.O was refluxed for 15 min.H.5" after crystii from EtOH-H. J ..O. XXXI . N. After the reaction mixt had cooled. Chem. 100 ml of EtOH. Then the H.H.48 g (83%).CI. J.O and ice (about 300 ml) were added and the reaction mixt was extd with three 100-ml portions of CH. ( 6 ) D. 2-(-y-Aminopropyl)-2-(P-aminoethyl)adamantane Dihydrochloride (XWI). was added and the mixt was refluxed under N. for 4 hr. C I .5 g of XI1 and 50 ml of H. The residue was chromatogd over a silica gel column.ClN. and 10 ml of 4. Schlatmann.5-195... and 75 ml of dry DMSO was stirred at room temp for 0.CI. 160 mg of salicylaldehyde. washed with Et.O.CIN): H.4 ml of 15%NaOH were added slowly. A mixt of 320 mg of C. Adamantanespiro-3'-aminocyclopentaneHydrochloride (XXIV). The residue was dissolved in 50 ml of H. Freenor.1 g (90%). .. Baldwin. Treatment of the oil with EtOH-HCl in THF resulted in the formation of the di-HC1 salt: mp 255-258". 3070 (NII).O layer was made alkaline and extd with three 10-ml portions of Et. G. and the ppt was filtered with suction: yield 3. 160 ml of EtOH.. nmi spectrum (CDCI. 1. mass spectrum. M + 247.CIN): C.68 g of KOH in 200 ml of n-BuOH.NNH.+JN. After evapn of the solvent in vacuo the residue was boiled with 65 ml of C. After drying of the org layer (&SO. mass spectrum. the residue was suspended in 50 ml of H. 15.H.. Cl. N. 10 ml of H. H.O s o h was made alkaline and extd with four 50-in1 portions of CH. A s o h of 3. 97 (1969).NOH.A mixt of 2.N'-Bis(adamantanespiro-3'-pyrrolidine)(XXXI) and NAminoadamantanespiro-3'-pyrrolidine (XXX).58 g (94%). 2273 (1964). Conipd XVII (190 mg) was heated in a sublimation apparatus at 290' until sublimation ceased. Luundahl. Bloomfield. N. L. J. A mixt of 2.O and then extd with four 100-ml portions of Et.88 g of NaH (50%dispersion in oil) was added.2 N HCl. The ppt was filtered.5 hrt followed by stirring at 50" for 105 min. was evapd. Adaniantanespiro-3'-tetrahydrothiophene(XXV). HCl. Chem. J. ( 3 ) W. an oil was obtained which was chromatogd over a silica gel column.O and extd with three 100-ml portions of Et. The ppr was filtered. This product was identical with that obtd by heating of XVII. C1. Then THF was added..S ' 9H. N.4%)of XX: mp 234-236" .O and 20 ml of concd HCl and then extd with three 50-ml portions of CH. 2367 (1967).1 g (75%).1 g of XXII in 15 ml of EtOH. H.O s o h was made alkaline and extd with four 30-ml portions of E. 5037 (1964). Anal. with Et. and evapd: yield 2. and 60 mg of PtO.. and then the reaction mixt was evapd to dryness: yield 1. 1. A mixt of 2.42.. and a few mg of NaI was refluxed for 48 hr.S). 69. and 1 ml of EtOH was refluxed for I hr. mp 252-255". which was treated with Et. triplet centered at 2.S). Anal. (5) S..05 g of XII: 2. 3175.This compd was identical with the compd described in part 1' and prepd via a different route. Nasutavicus ai:d F. et al. Anal.PO.O.O: C.9 g of XI1 and 45 ml of DMF. the s o h was evapd.25 ml of 4. H. (C. singlet at 62. (2) A. Br. N-Salicylideneamino)adamantanespiro-3-pyrrolidine (XXXII) was prepd in the same way as XXIX: yellow needles. N. mp 214-215". m s s spzctruni. J. was treated in a Parr apparatus with H..O solns were washed with a satd NaCI s o h and dried (MgSO.$I.O was stirred and heated for 24 hr at 120". The oil obtained was treated with Et. The solvent was evapd and the residuc treated with 150 ml of H. (C. After refluxing for 1 hr. and extd with three 20-in1 portions of Et.H. After being refluxed for 12 hr the reaction mixt was cooled.O. dried (MgSO. A s o h of 3.CIN~ 0.8 g of XVI.O. the reaction mixt was poured into ice-H. Anderson and F. then cooled.O.4 g of XIII.). and triplet centered at 2.BrCIN): C. The ppt was filtered and washed with THF. A s o h of 325 mg of XXI in 10 ml of AcOH and 0. The reaction mixt was filtered and most of the EtOH evapd. The ppt was filtered and crystd from EtOH-Et. The cryst solid was filtered and washed with Et. Adamantanespiro-3'-piperidine Hydrochloride ( X V ) . Tetrahedron Lert. N. M. B. had mp 117-1 19" after crystn from hexane.1 N EtOH-HCI was reduced in a Parr apparatus in 17 hr. 300 mg of XII. The CH.0. N-Benzyladamantanespiro-3'-pyrrolidine ( W I I ) .6 ml of H. in 300 ml of EtOH was refluxed for 68 hr.5-51.5 g of XXI (31%):mp 239-241".NH.O and washed with Et. N. N.5".9 g of oxime XXIII in 6 0 nil of dry EtOH was treated with 8 g of small pieces of Na in about 2 hr under N. and the ppt was filtered: yield 82 mg (80%). H. Chem. Johnson. Org Chrm.O and EtOH-HCI: yield 0. Vol. A better method for prepg XXI is the following. After addn of EtOH-HCl the THF was evapd i n vacuo.9 g (91%).81 (CCH. A mixt of 230 mg of XXVIII.. found. A mixt of 6. the reaction mixt wa5 poured into 300 ml of H. Amer.. Anal.. Adamantanespirocyclo3'-pentanone Oxime (XXIII). M + 380.5 hr.N.mp93. The residue was suspended in 100 ml of H. 0.): C.O acidified with a few drops of 48% HBr. Adamantanespiro-3'-cyclopentanone (XXII). H.O at O". 0. ti. Paerels.83 (CH. the Et. Anal. After drying of the combined exts. A mixt of 1. and 4.0 g of XI1 and 45 ml of H. and 2 hr at 110". made alkaline. washed with H. and the residue was stirred for 1 hr at 100" in 1 7 ml of H.25H20): C. 1680 cm-' (C=O). 2 vat2 Hes..O was added.O. and then the Et.H. 1972.O and the ppt was filtered with suction: yield 250 mg (85%).7'-Dioxoadamantanespiro-4'-perhydroazephe(XX).1626. AdamantanespUo4'-perhydroazepineHydrochloride (XVIII). The H. Ki. Acta. mp 255-262". a yellow oil was obtained. A.H.mp 181-183" (from EtOH).O in 9 ml of H. Anal.5" after crystn from THF-MeOH. Soc.O in vacuo.1624. 200 mg (17%) of compd XXXI. (C. the solvent was evapd in vacuo and the residue was crystd from hexane: yield 1. (Ci5H30CIzN. the ppt was filtered and dried: yield 2. EtOH-HCI was added. 32. H? I.H. After evapn of most of the H. 2'.N. the reaction mixt was cooled and poured into 500 ml of cold H.N. Ana!.O..Cl. Schut. A refluxing soln of 0. I S . 4 0 mi of EtOH. After drying and evapn of the CH. H. After drying and evapn of the Et.5".5". 0. Anal.5%).). A mixt of 560 mg of XX and 0. mp 127-129. N.NCH.CH. After filtration..O: yield 2. C1. H. Thereupon 1.63 g (68%) after crystn from EtOH-THF.O.O exts were washed with H. soln to 25 ml it was cooled. After cooling. After concn of the C.O as eluant. Fraction I. This mixt was maintained at room temp for 15 min and then refluxed for 1 hr. then 4 ml of 85% H.. Jan.O and EtOH-HCI.mp 281283". the cryst ppt was filtered: yield 235 nig (71%). Q.H. k715 (C'=O). HCI.5 N EtOH-HC1 was reduced in a Parr apparatus in 17 hr.4 g of Na.. The ppt was filtered..6 ml of H. A. Anal. Hadzi and J. mp 108-108. Et. S-Methyladamantanespiro-3'-tetrahydrothiophoniumIodide (XXVI). The residue was shaken with THF and filtered: yield 26 mg (16. Peters.H. after being stirred for another 1. This compd was identical with an authentic sample.): C. The suspension was heated to 95" in 15 min and.O. nip 213-215'. for 21 hr. The sublimed product was dissolved iri 25 ml of H. 2-(. exact mass found 228.

org/10.org on April 25. Chem.W. DC 20036 . Identification and synthesis of pyrrolnitrin metabolites Patrick J. 137-139• DOI: 10. Med. Washington.. and Terry L.Biological inactivation of pyrrolnitrin.doi. 15 (2).1021/jm00272a005 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N. 1972.acs.. Williams J.1021/jm00272a005 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. 2009 More About This Article The permalink http://dx. Murphy.

2 2-3 2.3 containing an amount of radioactivity corresponding to 100 pg of pyrrolnitrin was found to be devoid of pyrrolnitrinlike antifungal activity (level of detection >0. Pyrrolnitrin was readily metabolized in vitro using rat liver microsomes supplemented with NADPH. urine.[2-nitro-3-chlorophenyl]pyrrole (l). This product is 4-chloro-3-( 3-chloro-2-nitropheny1)maleimide (5).4 Total 52. Microbiological assay of a sample of urine (0. hr Per cent radioactivity excreted 1 1. 3-chloro-4. Results and Discussion Excretion. 1972.7 2 8.8 ml) Table 11. b4. The absence of pyrrolnitrin in the bile samples was further indicated by miciobiological assay.4 pg of pyrrolnitrin. 4-chloro-3-(3-chloro-2-nitropheny1)maleimide ( 5 ) . no pyrrolnitrin could be detected by glc (limit 0. Williams The Lilly Research Laboratories. Six rats (150 g) were given a dose of ['4C]pyrr~lnitrin (4 mg. Indianapolis. Antibiotic activity was rapidly destroyed in vivo. Each of the compds has been found to be devoid of pyrrolnitrin-like antifungal activity. When radioactive pyrrolnitrin was used. Bile. A fourth product has been identified by "trapping" the metabolite as a HS(CH2)20H adduct and identifying this adduct by comparative tlc. 2 137 Biological Inactivation of Pyrrolnitrin.4 ml) containing an amount of radioactivity equivalent to 100 pg of pyrrolnitrin showed no detectable pyrrolnitrin-like antifungal activity. excretion. The results obtained from iv administration of ['4C]pyrr~lnitrin (Table I) indicate that after 24 hr approximately 30% of the radioactivity is excreted in the urine. minimal activity is noted after oral administratione2Gastric acidity has been mentioned as a possible cause of this decreased activity.2 X lo6 dpm/mg) sc.3 3-4 5. When [14C] pyrrolnitrin was administered to rats having cannulated bile ducts. However. The oxidation products include a potent alkylating agent. Three of the products have been isolated and identified by comparison to chemically synthesized standards. no radioactive zone corresponded to unchanged pyrrolnitrin. After the cannulae were in place and the bile flow resumed. Bile was collected during the indicated periods.Metabolism of Pyrrolnitrin Journal of Medicinal Chemistry. 4-chloro-3-(3-chloro-2-nitrophenyl)-3pyrrolin-2-one (3). Extn of the bile with PhH removed between 5 and 10%of the total radioactivity from the aq phase. The ext was concd and chromatogd by tlc. The tlc plate was analyzed by radioautography. but active metabolic degradation has not been thoroughly investigated.2 mg of pyrrolnitrin administered (6 X lo6 dpm/mg). 15. as a topical preparation.4 15. and the extract was analyzed by glc. Although 29% of the administered radioactivity was excreted in the urine in 24 hr and 50% in the bile in the same time period there was no pyrrolnitrin-like antifungal activity found in either plasma. One third of the plasma was used for bioassay. Identification and Synthesis of Pyrrolnitrin Metabolites Patrick J. In vitro studies have shown that the pyrrole ring of pyrrolnitrin is readily oxidized by enzymes having the properties of mixed-function oxidases.4 2.1 4 7.O to 4. In the course of our studies on the pharmacology of pyrrolnitrin we have examined its metabolism in the rat using both in vivo and in vitro techniques. The plasma was isolated by centrifugation.2 5.3 pg). or bile. a number of metabolites were detected in the plasma. 1971 The absorption. is a broad-spectrum antifungal agent first reported by Arima and coworkers. Biliary Excretion of Radioactive Metabolitesa % radioactivity recovered Time interval.0 X 10' dpmlmg) in 0. and an aliquot was used for liquid scintillation counting. and the parent compd cannot be detected in either the urine or the bile after ip administration.8 24 29. c3. 1. Urine. while the remainder was extracted. We have found that pyrrolnitrin disappears rapidly from plasma.6 6. Although a minimum of 6 radioactive materials were present. hr lb 2c 0-1 0. The results obtained from the plasma as well as from urine and bile indicated that pyrrol- . Tlc of PhH extracts of urine from rats that had been given ['4C]pyrrolnitrin by iv injection showed no unchanged pyrrolnitrin. and metabolic conversion of [ ''C]pyrrolnitrin (1) have been examined in the rat using both in vivo and in vitro techniques. Urinary Excretion of Radioactive Metabolitesu Time after injection.4 48. No. 5-22 35. the rats were given a single dose of [ ''C]pyrrolnitrin by injection into the stomach. Metabolic Conversion of [ ''C]Pyrrolnitrin in vivo. approximately 50% of the radioactivity was excreted in the bile in the first 24 hr (Table 11). The radioactive materials excreted in the bile were extd and cxamined by tlc. Blood samples were taken at intervals from 30 min up to 24 hr. Indiana 46206. nor was there any detectable pyrrolnitrin-like antifungal activity. Pyrrolnitrin. These compounds are 3-(3-chloro-2-nitrophenyl)succinimide (2).1 12.7 uThe bile ducts of two 200-g rats were cannulated.7 6. Murphy* and Terry L. and 4-chloro-3-( 3-chloro-2-nitrophenyl)-5-hydroxy-3-pyrrolin-2-one (4). The chemical synthesis of each of these compds is described.2 ml of polyethylene c 1 1 glycol.0 uEach of four 200-g male rats was given an iv injection of 2 mg of ['4C]pynolnitrin (6. none of which possessed pyrrolnitrin-like antifungal activity.8 1-2 2.0 mg of pyrrolnitrin administered (6 X lo6 dpm/mg). Analysis of radioactivity indicated that the extracts contained the equivalent of from 1. Vol.7 ~~~ ~ ~~ 4-5 6.' Although this antibiotic is effective Table I. A bile sample (0. Received August 18.01 pg). Plasma.

138

Journal of Medicinal Chemistry, 1972, Vol, 15,No. 2

Murphy and Williams

fRAClION nitrin is rapidly converted to a series of metabolites that are N U M B E R $. t ' i i &A devoid of antifungal activity. In Vitro Conversion of [ ''C]Pyrrolnitrin. In order to learn more about the metabolic conversion products of pyrrolnitrin in vitro incubations were conducted using various liver subcellular components. The mitochondrial and 100,OOOg supernatant fractions had no effect on pyrrolnitrin. The microsomal fraction had a minimal ability to alter the drug when incubated without cofactors, but in the presence of NADPH or (less effectively) NADH there was a significant conversion (up to 15%) of added pyrrolnitrin in 30 min. Tlc indicated that a minimum of 6 products were TUBE N U M B € R formed during these incubations. Because a number of these t ELUANT t t t metabolites seemed to correspond chromatographically to El~O.MeOH 0enrene 0en~ene-EI,O E1,O 4:ll 4:, the in vivo metabolites. further studies were undertaken Figure 1. Column chromatography of pyrrolnitrin and pyrrolnitrin using large-scale incubation of pyrrolnitrin with isolated rat metabolites. The concentrated extract was dissolved in benzene and liver microsomes. placed atop a 240 X 22 mm column of silicic acid. The eluant was The metabolites of pyrrolnitrin were isolated by column changed as indicated; each fraction was 10 ml. The radioactive fractions were combined as indicated by fraction numbers 1-7. chromatography of the extracts. The distribution of the metabolites is shown in Figure 1 . The individual peak fractions were combined, concd, and analyzed. S l o n d o r d - Compound I l l Rodiooctirity Fraction 1 was found to contain only unchanged pyrrolnitrin. Fraction 2 contained a component, R f(5% MeOHSolvent S y s t e m Solvent System S o l v e n t System i-Pr20) 0.38. This material was isolated by prep tlc. The 3 1 2 mass spectrum of the purified material was identical with that of 3-(3-chloro-2-nitrophenyl)succinimide(2). Fractions 3 and 4 were not analyzed further. Fraction 5 was purified by prep tlc in 10%MeOH-i-Pr20. Two radioactive bands were observed (Sa,R f-0.32; 5b, R f -0.44). These bands were eluted separately, and the purified compds were subjected to mass spectrometric analyses. The mass spectrum of Sa was identical with that of 4-chloro-3-(3-chloro-2-nitrophenyl)-3-pyrrolin-2-one (3). The mass spectra of 5b was identical with that of 4-chloro-3-(3-chloro-2-nitrophenyl)-5hydroxy-3-pyrrolin-2-one(4). Fractions 6 and 7 were chromatographed in a number of solvent systems and were found to contain a mixture of components. None of these metabolites has been identified. The route of metabolism indicated by the identified comA B C A B C A B C pounds suggested that structures such as 5 and 6 might be
1:;

*

c 1

H

c1

H
6

5

Figure 2. Thin-layer chromatographic analyses of extracts from in with rat liver microsomes: A, vitro incubation of [ 14C]pyrr~lnit~in pyrrolnitrin + NADPH; B, pyrrolnitrin + NADPH + HS(CH,),OH; C, pyrrolnitrin + HS(CH,),OH. Solvent system 1, 15 cm in PhH followed by 10 cm in i-Pr,O-MeOH (9:l); solvent system 2, 15 cm in PhH followed by 10 cm in i-Pr,O-HOAc (95:5); solve$ system 3, 15 cm in PhH followed by 10 cm in EtOAc-PhH (1:l). The R fof pyrrolnitrin in aII 3 systems is 0.6.5.

readily formed by common oxidation reaction. Since both of these compds are substituted maleimides, their generation in a biological system might be expected to lead to rapid binding to proteins and/or reaction with biological SH compds. A further study of 5 was undertaken in order to assess this possibility. When 5 was allowed to react with HS(CH&OH, a quantitative formation of 7 occurred. With the hope of trapping intermediates such as 5 a series of incubations were performed with pyrrolnitrin in the presence of 6-mercaptoethanol with and without NADPH. The ex-

c 1
7

tracts were then cochromatographed with 7 in 3 solvent systems. The results are shown in Figure 2. In the presence of P-mercaptoethanol and NADPH a number of radioactive zones are formed which are not present in the extracts from incubations containing either of these compds alone. One of the compds formed cochromatographs with 7 in each of the solvent systems tested. Thus it seems that at least one, if not more, maleimide type compounds are generated during the microsomal oxidation of pyrrolnitrin. A summary of these metabolic conversions of pyrrolnitrin by rat liver microsomes is given in Figure 3. All of the metabolites identified were oxidized pyrroles. The pyrrole ring seems to be particularly swceptible to oxidatiqn, which may account for the rapid degradation of pyrrolnitrin in vivo. Compds 2 , 3 , 4 , 5, and 6 were found to have no sig nificant antifungal activity. The generation of a substituted maleimide by microsomal oxidation is most interesting in

Metabolism of Pyrrolnitrin

Journal of Medicinal Chemistry, 1972, Vol. 15, No. 2

139

R =

rl-NO2 CI

Figure 3. Metabolic conversion of pyrrolnitrin in vitro by rat liver microsomes.

light of the known reactivity of maleimides with proteins and SH groups. To our knowledge the conversion of a pyrrole to a maleimide has not been previously reported. Whether significant amounts of the types of maleimides reported herein are formed in vivo can only be assessed by further study. Experimental Section
[ 14C]Pyrrolnitrin labeled in the 2 position of the pyrrole ring was prepd biosynthetically from [ 14C]tryptophan by the method of Hamill, et al.' The isolated material was assayed for purity by tlc and exhibited a single radioactive zone corresponding to authentic pyrrolnitrin. All enzyme cofactors were obtained from Boehringer-Manheim Corp. Silicic acid (100-200 mesh) was purchased from Bio-Rad Laboratories. Assays. Plasma samples were assayed for radioactivity by digestion with a mixt of 0.2 ml of HC10, (70%) and 0.4 ml of 30% %O, at 70" for 45 min in Teflon-capped vials. The samples were cooled rapidly in ice and dissolved in 20 ml of a soln contg 11 ml of PhMe, 9 ml of methyl Cellosolve, and 6 mg of PPO. All other samples were dissolved directly in 10 ml of dioxane-based scintillation fluid prepd by combining 104 g of naphthalene, 75 ml of Permafluor (Packard Instruments), 425 ml of PhMe, 500 ml of dioxane, and 300 ml of MeOH. Radioactivity was detd using a Packard liquid scintillation counter with external standard. Microbiological activity was assayed using Neurospora crassa (Lilly M45-846) as the test organism and pyrrolnitrin as the standard. Glc was carried out according to the method of Hamill, et al., Urinary Excretion. Four male 200-g Sprague-Dawley ,rats were given a single injection of 2 mg of [ I4C]pyrrolnitrin (6 X 10-6 dpm/ mg) in 0.2 ml of polyethyleneglycol iv. The urine was collected for the indicated periods, and total radioactivity was detd by counting an aliquot of the sample. Biliary Excretion. Male Sprague-Dawley rats (200 g) were anesthetized with Et,O and their bile ducts were cannulated. A single dose of [ 14C]pyrrolnitrinin 0.2 ml of PEG,,, was administered by injection into the stomach. Bile was collected over the indicated period, and then the total radioactivity was detd by analyzing an aliquot of the sample. In Vitro Incubation. Rat liver microsomes were prepd by differential centrifugation of 10% liver homogenates prepd in 0.25 M s u c r o ~ eThe . ~ 100,000gpellet was resuspended in 0.05 M phosphate buffer, pH 7.0. NADPH was added at 5 X 10-4M. [ I4C]Pyrrolnitrin was dissolved in 0.05 ml of 0.1% Tween 80 in acetone prior to addn to the incubation medium (final concn, 0.1 mM). Following the addn of the enzyme prepn (1.5 ml) the final vol was adjusted to 2 ml with 0.05 Mphosphate buffer, pH 7.0. After the indicated time the reactions were stopped with 2 ml of Me,CO and then were extd with three 1-ml portions of PhH. Under these condns the recovery of unchanged pyrrolnitrin averaged 90%.The recovery of the metabolites varied due to production of nonextractable material in the course of metabolic degradation. Large-scale incubations were performed using male rats that had been treated for 5 days with phenobarbital (40 mg/kg, ip). Microsomes were prepd in the usual manner and were resuspended in 0.05

Mphosphate buffer, pH 7.0 (200 m1/10 g of liver). Microsomal suspension (30 ml) was placed in a 125-ml Erlenmeyer flask. An NADPHgenerating system consisting of isocitrate dehydrogenase, NADP+, and isocitrate was added to give a final concn of NADPH equal to 2.5 X lO-,M. [14C]Pyrrolnitrin (1 mg in 0.1 ml of 0.1% Tween 80Me,CO) was added to each flask. The reaction mixt was incubated 45 min at 37" with vigorous shaking in air. The reaction was stopped with an equal vol of Me,CO. The protein ppt was collected by centrifugation, and the aq phase was extd with PhH. The exts were concd and used for column chromatog. Column Chromatography. In one series of incubations a total of 100 mg (0.6 X lo6 dpm/mg) of [ ''C]pyrrolnitrin was utilized. The PhH exts contd 1 X lo' dpm. The concd PhH ext was placed atop a silicic acid column (42 g, 240 X 22 mm) packed in PhH. The column was eluted with increasing concns of Et,O and MeOH. The peak fractions were pooled and concd. Chemical Syntheses. Oxidation of F'yrrolnitrin with 00,HOAc. Pyrrolnitrin (2.5 g) was dissolved in 100 ml of glac AcOH. A soln of CrO, (2 g) in 80% AcOH (200 ml) was added over a 1.5hr period with stirring at room temp. After 15 min of add1 stirring 10 ml of abs EtOH was added, and the mixt was stirred for 15 min. The dark green soln was concd in vacuo at 50". The residue was triturated with PhH and H,O, and the aq phase was extd 3 times (PhH). The extracts were washed with H,O and concd. Upon concn a ppt formed which was isolated and recrystd from EtOH to yield pure 4-chloro-3-( 3-chloro-2-nitrophenyl)-5-hydroxy-3-pyrrolin-2one (4). The structure was confirmed by mass spectrometry, ir, nmr, uv, and elemental analyses. The PhH supernatants were then placed atop a column of silicic acid (240 X 22 mm). The column was eluted with PhH. After elution of unchanged pyrrolnitrin a fraction was eluted which contd 2chloro-3-(3-chloro-2-nitrophenyl)maleimide ( 5 ) . This material pptd upon concn and was washed (PhH) and analyzed by nmr, mass spectrometry, uv, ir, and elemental analyses. Oxidation of Pyrrolnitrin with Chloroperbenzoic Acid. Pyrrolnitrin (10 g. 39 mmoles) was dissolved in 60 ml of C b C l , . Solid Na,HPO, (22.4 g) was added. A CH,C1, s o h of m-chloroperbenzoic acid (7.8 g in 60 ml of CH,Cl,) was added slowly (75 min) to the stirred slurry at room temp. After this time the CH,Cl, slurry was treated with a 2% aq Na,S,O, until a neg peroxide test was obtd with starch-iodide paper. The soln was then extd with 3 vol of H,O and dried (MgSO,). The org layer was taken to dryness. The residue was extd with Et,O leaving a blue polymeric ppt. The Et,O ext was taken to dryness and then the residue was triturated with PhH and placed atop a column of silicic acid packed in PhH. The column was eluted with PhH followed by increasing concns of EtOAc. The initial benzene eluate contd pyrrolnitrin followed by a yellow compd, which was chromatog distinct from pyrrolnitrin. Mass spectral, nmr, ir, and uv evidence indicated that it was 343chloro-2-nitropheny1)maleimide (6). The fraction eluted with 5% EtOAc-PhH was concd, and the crude ppt was recrystd from EtOAc(2). The strucPhH to yield 3-(3-chloro-2-nitrophenyl)succinimide ture was confirmed by nmr, ir, uv, and mass spectrometric data. The fraction eluted with 10%EtOAc-PhH was concd and a ppt appeared. This was washed (PhH) to give essentially pure 4-chloro-3(3-chloro-2-nitrophenyl)-3-pyrrolin-2-one (3). The structure was assigned on the basis of mass spectral, ir, uv, nmr, and elemental analyses. The assignment of the carbonyl position was supported by chemical reactivity of the 4-C1 substituent. Although a number of other products were observed, none was sufficiently purified for identification.

Acknowledgment. We would like to thank Dr. R. Hamill, Mr. C. B. Carrell, Mr. J. A. Mabe, and Dr. D. R. Brannon for preparing pure [ 14C]pyrrolnitrin for use in the above studies. We would also like to thank Mr. J. Westhead for the microbiological analyses and Mr. H. Sullivan for assistance in the glc analyses. References
(1) K. Arima, H. Imanaka, M. Kousaka, A. Fukuda, and G. Tamura, J. Antibiot., Ser. A , 18, 201 (1965). (2) M. Nishida, T. Matsubara, and N. Watanabe, ibid., Ser. A , 18, 211 (1965). (3) R. Hamill. R. Elander. J . Mabe, and M. Gorman, Antimicrob. Ag. Chemother., 1967,388 (1968). (4) R. L. Hamill, H. R. Sullivan, and M. Gorman, Appl. Microbiol., 18,310 (1969). (5) G. H. Hogeboom,MethodsEnzymoL, 1, 16 (1955).

.,

Equilibrium and kinetic effects of sixteen compounds on the forms of horse heart ferricytochrome c
George H. Czerlinski, and Rita A. Galuszka
J. Med. Chem., 1972, 15 (2), 140-144• DOI: 10.1021/jm00272a006 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.acs.org on April 25, 2009

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IS.6. The spectral change was observed ' ' ?Supported by a Grant from the Chicago and Illinois Heart Associations (N69-67 plus C70-66). imidazole). benzimidazole. thiazole. 9. for definitions).4. Differences between the oxidized and reduced forms of cytochrome c have been known for some time. and further purified according to Margoliash and Lustgarten. amytal. ef Subsequent "pH-switching'' experiments (see type IV experiments below) led to two apparent rate constants. Czerlinski* and Rita A.4 is generally somewhat smaller than the original absolute change in absorption (for pH 7. With these numbers for the 2 interconversion constants and the apparent protonic dissociation constant of the overall reaction of pK1= 9. pyrazole. the oxidized protein having a more open and flexible conformation than the reduced form. The time constant in presence of hydantoin-5-carboxylic acid probably exceeds the time resolution of the experimental arrangement (0. barbituric acid." adding initially an equimolar ami of sodium ferrihexacyanide. Scheme A. benzimidazole. which suggested the reaction cycle shown in Figure 1. exceptions are imidazole (fastest reappearance). thiophene-3-carboxylic acid. 3 compounds showed an intermediate increase in absorption (ATP. thiazole). 1972. obtained rate constants from temperature jump experiments. The values in presence of all other compds lie in between. in presence of benzimidazole) substantially smaller. et al. 1971 Equilibria and kinetics of the forms of horse heart ferricytochrome c wzIe investigated in the pH range 7-1 0 in the presence of a variety of substances with the intent to find effectors which bind preferentially to one of the several forms. Scheme A.140 Journal of Medicinal Chemistry. thiazolidine-4-carboxylic acid. 2. which is characteristic of the specific compound. Preferential binding would then be indicated either by an increase or a decrease in the absorption. If the pH of horse heart ferricytochrome c in buffer is quickly changed back from 10 t o about 7. The absorption band at 695 mu was used as indicator and is attributed to the form of ferricytochrome c which participates in electron transfer.0 Q pH < 10. In presence of the various test substances. pyrrolidine. pyrrolidine). All experiments involved the simultaneous observation of transmission changes at 633 (a high shoulder of the 695 band) characteristic of protein conformation6 and of pH changes. For this specific electron transfer Brandt.2 sec).4 to 10. 4 showed an absorption increase of more than 20% (ADP. pyrimidine. The original intent of the present investigation was t o verify Scheme A of Figure 1 by using "effectors"..1. 2." Watt and Sturtevant16 determined the enthalpy of oxidation of ferricytochrome c over the pH range 6.6 The equilibrium constant between components 2 and 3 favors component 3 and the interconversion constant was estimated4 at 3. but larger in the presence of imidazole.4dinitrophenol. Many of the listed substances are known to have medicinal effects. ~ proton release upon oxidation of ferrocytochrome c by ferrihexacyanide was subsequently investigated in detail by Czerlinski.No. and thiophene3-carboxylic acid). ATP. If the pH of horse heart ferricytochrome c in buffer is quickly changed from 7. Received July 1.' Among recent reports in this connection are studies on the optical rotary dispersion*-" and on circular d i ~ h r o i s m ' ~ . 2 C z erlinski and Galuszku Equilibrium and Kinetic Effects of Sixteen Compounds on the Forms of Horse Heart Ferricytochrome e t George H.8 (see Figure 1. . thiazolidine-4-carboxylic acid. a value which can be derived from the earlier data of Schejter and George. pyrimidine. The 695 band reappears generally slower in the presence of a test substance (slowest for thiophene-3-carboxylic acid) than in its absence. et a l . hydantoin-5-acetic acid. and antimycin A (the last 2 in 40%EtOH). Urry. . The time constant for the disappearance of the 695 band is increased by about a factor of 10 in presence of AMP. Since these were equilibrium experiments. and pK. the same quick pH change reduced the 695 band to an extent and with a time constant. This interconversion is proton coupled and only the form(s) stable at pH 7 are quickly reducible by ferrohexacyanide. barbituric acid. blown over the s o h to diminish CO.ls and Myer and Harbury.1). hydantoin-5-acetic acid. Chicago." = 7.' demonstrated that the pH dependence of the redox potential of horse heart cytochrome c is due to a rather slow interconversion of different structural forms of the protein.. using a thermostated Pyrex vessel with N. NaN3. The equilibrium constant between components 1 and 4 of this scheme of Figure 1 is 20. These changes were observed at pH 7. The changes were smallest with imidazole and largest with thiazolidineCcarboxylic acid.4 -+ lo). Some of the substances developed demonstrated considerable ligand-induced conformational changes (either enhancing or counteracting the previously shown pH-induced conformational change). Vol.pickup (7.4 within a few seconds after injection of cytochrome c into the reaction mixture. which would hopefully exclusively bind t o either form 1 or form 3. Brandt.4-dinitrophenol) and 2 compds an intermediate decrease (hydantoin-5-acetic acid. in some cases ( e .0-10. one obtains pKH' =. 75% of the 695 band disappears with a time constant of about 1 sec. The absolute value of the change back to pH 7. which are in reasonable agreement with results of Sutin and Christman2 and of H a ~ s t e e nThe . Experimental Section Horse heart cytochrome c Type 111 was obtd from Sigma Chemical Co. Illinois 6061i. the 695 band reappears with a time constant of close to 8 sec.9 and were also able to derive thermodynamic parameters for the interconversion between component l and 3 of Scheme A of Figure l . while 2 showed an absorption decrease of more than 20%(aniline. 5 compds were within 5% of the value of ferricytochrome c in buffer (AMP. g. pyrazole. they had no information as to intermediate steps betwen components l and 3. Of a total of 16 tested compds. NaN3. No such exclusive binding was revealed upon studying the following possible complexing agents: AMP. The above mentioned isomerization of ferricytochrome c at alkaline pH t o form a species with altered electron-transfer properties was also discussed by Greenwood and Palmer. ADP. Galuszka Department of Biochemistry. of ' ~the protein. and t hiazolidine-4-carboxylic acid. It was therefore also of interest to look at the effects of these substances upon the most thoroughly characterized memeber of the electron transfer chain of the inner mitochondrial membrane: ferricytochrome c. imidazole. PhNH2. Northwestern University Medical School.

experiments. more and/or stronger NaOH was needed to reach pH 10 (compare discussion of Table 11). but in these surveying experiments this was assumed in first approximation. with a small He-Ne laser. in some cases. if necessary. The rate constants. A S 3 represents the observed equilibrium signal change associated with this last injection.2 mM) in the above buffer and 0. Figure 2 introduces also the various symbols used in the evaluations. was adjusted to pH 7. convertible to mV). followed after equilibration (as for type I experiments above). Spectral and pH changes were recorded simultaneously. as the protonic step proceeds much faster than the isomerization step. Test substance (4. the equivalents of H. To illustrate the sequence of these Figure 2.40 (or 10.. as all signals).1 M) thorughout all types of experiments. “Add cyt c” represents the quick injection of 500 pl of (nominally) 2 mM cytochrome c stock into 4. AS.SO. 1972 Vol.2 mM). was then injected quickly. NaOH (1 N . Schematic demonstration of an experimental sequence.”’ However.) to its s o h in such an amt that twice the molarity of cytochrome c could have been reduced.SO. 40 pl of 1 N H. Nevertheless. Upon equilibration.Effects on Forms of Ferricytochrome c Journal of Medicinal Chemistry. Oxidized cytochrome c (500 pl. associated with this injection (measured in mV.10M test substance (or 0. The signal change for the linearizable case (relatively small transmission changes) is given by As = V ( C * + c2) (3) It is assumed in this equation that a signal derives only from components 1 and 2 and both components have the same coefficient I) (in mV/ruM).00). The control contd Na. Spectral changes were calibrated by a “blank” of type IV (below). The 4 types of experiments are briefly described in the next paragraphs. “Add NaOH” represents the quick injection of enough 1 N NaOH. to reach (or exceed) pH 10 (type IV experiments). Fifty pl of 10 mM H.SO. A. Na. IS. a stepwise pH change from 10 to 7 cytochrome c alone (that is without test substance present) should directly lead to the rate constant k.00) was quickly injected. utilizing a beam splitter and 2 Ge photodiodes.. 0. the relevant differential equations may easily be linearized.SO. Oxidized cytochrome c (5. The oxidation of reduced cytochrome c was performed with sodium ferrihexacynanide. adjusted to pH 7. The magnitude of the total signal change upon a stepwise pH change is a function of the initial and the final pH. Figure 2 is presented.SO. As the abscissa is directly recorded in seconds. were injected after equilibration to calibrate the pH changes. in the absence of inhibitor. and Radiometer pH-meter Model 26.Fe(CN).030M Na. University Laboratories Model 200. To observe slow changes of pH after the rapid change in pH. were adjusted t o pH 7. 2 141 SO .40 (or 10. Type IV. associated with this injection. to bring the pH back to 7..00) within the thermostated vessel (25”). low in Fe.40 (or 10.SOJ.40 or 10. et ~ l . Type 111.4 (type IV experiments). 40 pl) was injected quickly. the apparent rate constant A is given in sec-’ One obtains in zero approximation . Most frequently. in place of the test substance.SO. (22 mM. Reduced cytochrome c (5 ml. “Add H. Odd Figure 1. This is not necessarily so. but differently labeled here for later discussions. Type 11. 2 mM) in buffer (ionic strength adjusted to 0.00 ml.No.” represents the quick injection of enough 1 N H. x = X exp(-At) t Q with 3 equal to the equilibrium change. Most of our kinetic evaluations. The s o h was then dialyzed 3 times against the buffer mixt and spectrophotometrically tested for purity (98%or more in the reduced form). These are calibrated again by injections of 50 p l of 10 mN H. adjusted to pH 7.10M test substance and the above buffer with Na. and signal changes were recorded. was substituted for the test substance (compare type I experiments).SO. followed by type IV experiments. one could only expect to obtain a limiting value for the rate constant k. The system was weakly buffered by 10 mM sodium phosphate and 4 mM glycine in all s o h . was then quickly injected and changes recorded. The signals were fed into different channels of a Beckman Type RB Dynograph (with Type 9853 plug-ins).00) at 25”.SO. proper biasing of the channel measuring pH changes is necessary. this lower limit is of value in further discussions of the applicability of this scheme.SO. The pH change was observed with a combination glass electrode. in 0. to bring the pH to about 10. for pH switching from component 1 to 3.1 with Na.SO. Oxidized cytochrome c (5.SO. was quickly injected. indicating 2 recorder traces and defining the various signal changes. I’ and I” are one and the same inhibitor I. The change of the trace on the graph is given by d x l d t = -xX (1) with x = particular ordinate value on the graph (in mm. Fifty pl of 22 mM test substance in buffer.40 (or 10. to return the pH to 7. If Scheme B of Figure 1 applies. Type I.00) at 25”. The constant term a (with a < 3 ) takes care of any (initial) rapid change. B. however.. added generally equaled the equivalents of NaOH added before. for the control) were adjusted to pH 7.40 (or to 10. A reduced scheme.030 mM Na. see Table 11) in the above buffer and enough Na.00 ml of 0. represents thus the equilibrium signal change. . 50 ml) in the above buffer (pH 7.SO.00) within the thermostated vessel (25”). odd odd I I . 0. Reaction scheme of Czerlinski. Na. extrapolated back to t = 0. This scheme is based on both equilibrium and kinetic experiments.. implying only oxidized cytochrome c and inhibitor I reacting with any 1 of the 3 presented forms of cytochrome c. Cytochrome c was reduced by the addition of powdered sodium dithionite (purified.’* The above equations imply that the time-dependent change can be properly represented by a single exponential decay curve. Experiments of type IV could easily follow the other 3 types of experiments.5 ml of a solution of the test substance (type I1 experiments).depicting .SO. 0. to maintain constant ionic strength (0.1. to adjust the ionic strength to 0. A semilog arithmic plot results in a straight line with a slope of A.4.4 (or 10. represents the observed equilibrium signal change.1 1M) (for deviating concns. derived from a stepwise pH change for the cytochrome c system are dependent upon the final pH value. Type 14073 of Instrumentation Laboratories. So represents the signal before addition of cytochrome c and obtained such that the light beam through the test vessel was shut off by a piece of black paper. we carried out first type 11.00).2 mM) in the above buffer and 0. AS. Injection of H. of Fisher Scientific Co. was adjusted to pH 7. ~ 4 different forms of oxidized cytochrome c.50 ml. were performed by using a computer program of Berman. As the observed changes in pH and in optical transmission are quite small. except for those where the test substance has a PKH in the range under investigation. Evaluation of Kinetic Data. In the control.

Two to six tests of the type shown in Figure 2 Table 1. The equations for the reduced cycle are easily obtained by settingK11 = 0 in eq 5 and 6. mV A S . A . aSl'/AS. Case 1. 2 Czerlinski and Galuszka The reaction scheme may be written in the general form (not considering effectors). One derives. ponent 10 has the same q as components 1 and 2). ASl/aSo = 0.469 11 5 f 0.07 10.049 2 Azide (Na) 1550 81 7. as well as the ASi. the ASi in thistable are normalized for identical initial signal (reference So= 1500 mV) such that the experimentally obtained (ASi)obsd is multiplied with the factor (1500/So). The three pH values refer to the pH measured after the new equilibrium value is reached and are thus subscripted like the preceding AS.mV AS". mV PH.where a* represents a computergenerated constant to optimize the fit of the theoretical curve t o the data. Thiophen-3-carboxylic acid. mV ha*. * .635 0. 4. The apparent rate constant of this slow change was found to be a function of the analytical concn of the thiazolidine derivative and of pH. The pH before injection of cytochrome c is generally within 0. KII' < K I I .1W So. which was the principal reason for our use of 0.120 f 0.7 from Table I1 for the hydantoinderivative.44 and AS3/ASo = 0.007 7. the standard error is also listed together with these values.04 sec-' for the control from Table I together with Al'/A1 = 4.5 times this amount for aniline. Only those results were used for further evaluation.31 i 0.440 42 37 r 0. AS*. Table I summarizes the results from the control (line 1) and 3 representative inhibitors.For this reduced scheme one may easily derive for the relative signal change 1 . the stronger is the effect by the test substance. The limiting overall response time of the experimental arrangement is close to 0.' and &. no pH change could be detected.2 0.*.070 f 0. 1 None 53 7.' should be compared with the corresponding values for the controls. with AI* 1. Any hl'/Al between 1 and 5 may be corrected for this overall response time (omitted here). .371 9 8.93 0.440 46 44 f 0.2 1. Summary of ReDresentative Result9 were generally conducted on each test substance.02 11. However. Table I1 summarizes the results obtained on all materials tested for preferential binding in an abbreviated manner.5 0.' and k2'.469 28 30 10 1 1 * 0. 15.sec-' PH. apparently due to the buffering capacity of these compounds since upon injection of the calibrating acid. Table I1 does not reveal that the systems with the thiazolidine-4-carboxylic acid undergo a slow change. the experimental error could not be reduced below 0. where the curves indicating the pH change most closely approximated a stepwise change.04 t 0.A =-c=1 t F2 One obtains for the apparent rate constant h = 1/7 Case 11.425 1535 24 22 f 0. sec-' uH. which is in good agreement with the largest AI* found experimentally. the color of the reduced cytochrome c appeared. Deviations within +lo%cannot be considered sig nificant.2 sec. mV A. NaOH (1 2 N . the system of eq 4 may be treated as if the upper cycle is not present at all (assuming that com. Results The experimental results are listed below in two tables. K p ' = K I I .02 9. 15 pl) was needed for imidazole and twice this amount for hydantoin-5-acetic acid. around 30 pl of 1 N acid or base was used. These larger amounts of acids and bases are needed for those compounds. All the compounds in Table I were tested for equivalent proton release upon injection of cytochrome c. We suspected that the S of thiazolidine might have replaced the S Effector No. mV AS.142 Journal of Medicinal Chemistry. When the reaction mixture of the thiazolidine derivative with ferricytochrome c was kept long enough.003 7. Four parameters in the table are superscripted by an asterisk to indicate that the listed values have been obtained by an iterative computer program. This condition leads automatically to the fact that kz'/k.42 f 0. . (10) - KH' 1 6 1 KH" One may then consider two cases. The subscript refers to the same (equilibrium) signal change as for Mi. is defined in Figure 2. Generally.6 0. (0.02 43 7.*exp(-Xj*t) + a*.960 3 Benzimidazole 1550 62 7. * .63 f 0.067 t 0.150 4 Imidazole 1525 7. l .015 OThe initial signal S. dinitrophenol.5 times t h s minimum amount was needed for pyrrolidine and 2. almost all inhibitors showed a change of pH which corresponded to not more than the given error. = K z .. Upon the injection of cytochrome c. k.2 equiv.02 M of this test substance in the experiments listed in Table 11. There were 3 exceptions. no proton change was detectable with imidazole and pyrimidine. These results are needed for Table 11.007 7. however.9 sec-'. No.7 0. This condition allows one to neglect K I I ' .' = k . but twice this amount was needed for the 3 adenosine phosphates and for benzimidazole.6 0. 1. Unfortunately.'/&. 1972. which have protonic dissociation constants at or near the experimental range.460 28 29 f 1 1. For further details see the Appendix. Controls were measured on every day on which materials were tested for preferential binding.03 10. and PhNH2 showed a proton release of 1 equiv within the experimental error. A S .38.*.If also k. The larger the deviations from these control values are.05 unit of pH. The data in Tables I and I1 do not reveal that the vol of injected base (or acid) was not always the same.3 f 0.Xi* represents a pHdependent (apparent) rate constant and appears as parameter in ASi(t) = AS. Vol.

28 0. essentially the same apparent rate constants were obtained except for imidazole and dinitrophenol. This result eliminates case 11. A secondary reaction of imidazole i s reflected in the rather small values for ASl'/AS~and LLS.38 0. On the other hand./AS.'/AS.4-Dinitrophenol 0. except for the highly charged adenosine phosphates. Discussion The various materials tested can be classified into those which have practically no effect and those which do effect the red band (695-633 mp) of cytochrome c.4-dinitrophenol(0.42 0.6 1 0. Vol. listed above under eq 6.37 0.27 0.44 0.228 0. which was 10.38.26 0.40 0. However.28 0.110 0. AMP shows the slowest apparent rate constant in Table 11.110 Barbituric acid 0.43 0. the 2 compounds which also needed the largest amount of base for obtaining the proper change in pH. 0. although their specific action regarding electron transfer is not quantitatively established in this paper.1 0.38 0.44 and AS. The former ones may also be labeled activators.970 0.38 10.21 Imidazole 1. The thiophene derivative is also associated with the slowest apparent rate constant for a stepwise pH change from 10 t o 7.64).50 10. this slow reaction was not further investigated. Fastest for this latter change are imidazole and hydantion-5-acetic acid.9 0.79 2.531. Earlier experiments of Czerlinski.205 0. in that sequence (but all rather close together).921 0.IAS. (compare Table I for definition) was 7.4 to 10.5 2 Thiophene-3-carbox acid "The concns of effector were 0. the given ratio of voltage changes should result in the equilibrium constant between components 2 and 3. leading to decreasing increments of voltage changes.1.34 10. however. 2 143 Effector aS. which was present at pH 7. To test this idea.50 10.o 0. With respect to the results summarized in Table 11.35 11. The spectrum of pur compound with thiazolidine (high concentrations) was identical with that of reduced cytochrome c. No. The initial pH.'/ASd is somewhat large for AMP and ASd/aSo is somewhat large for ADP. the possibility of measuring such a substantial additional change suggests that a protonic dissociation is not associated with component 2 of scheme A of Figure 1.39 9. The subscripts are otherwise those of Table 1.' had shown that exactly 1 H' is released at pH 10. if the pH is changed stepwise from 7. In crude approximation. was 7.= 0.49 Thiazole 1.except for 2.49 Hydantoin-5-acetic acid 0.1 M. The pH dependence of the observables are therefore presumably given by eq 5 and 6. The equilibrium signal changes in presence of effector are denoted by a prime for distinction from those in absence of effector. labeled PKHII. at which point we measured a signal change of 30 mV (compared to the signal.158 0.57 0.4. This change is due to the second dissociation constant.67 0.'/A$.44 ADP 1. In fact.167 to 0.97 0. as their presentation would not provide much new information.37 10.26 0. There is also the trivial solution that the red absorption band is diminished by the reductive action of the test substance upon cytochrome c. However.4. this experiment was only comparatively successful with the thiazolidine derivative. as indicated below.45 ATP 0. the final pH.54 9.07 units in all cases.21 10.5 8 0.33 0. Scheme B of Figure 1 in fact represents a simplified mech- .64 Thiazolidine-4-carbox acid 1.120 0. inhibition may be considered as removing this S to a smaller or larger extent from this ligand position. We changed the pH stepwise to 11.4. as outlined in scheme B of Figure 1.58 0. suggesting that the process of oxidizing reduced cytochrome c in the presence of these compounds does not proceed along a single path. namely that the binding for the duration of the experiments is generally weak. the kinetic parameters for these 3 adenosine phosphates differ considerably. as depicted thus far.150 Benzimidazole 1.960 0. Results from experiments of types I and 111 are not explicitly tabulated. However.45 0.015 0.8 1 4. observing at 633 mp in our thermostated vessel). Some type 111 experiments were conducted at pH 7 and also at pH 10. and thiophene-3-carboxylic acid (7.10 0.17 2. Effectors would influence these pH dependencies. Certainly these are crude approximations as the protonic dissociation constants have to be taken into account quantitatively.28 10. the analytically detd concns of cytochrome C varied only from 0.29 0. well within the error of the detnsof AS.16 0. but only with component 3. Summary of Evaluation Result9 Joumal of Medicinal Chemistry. for simplicity.54).8 2.020 0.1 0. Those which do effect the red band of cytochrome c may be divided into those which enhance the absorption band and those which result in a decrease of the absorption band.2 0.16 11. giving Xi&.5 6 0.39 0.67 0.7 1. Most ideal would have been a type I experiment as the perturbation of the system by the minute injection could certainly be neglected. pH1 is the intermediate pH.60 2.91 0.4 1 10.1 units except for amiline (7. hydantoin-5-acetic acid (8. ef al. The equilibrium results of the 3 adenosine phosphates are rather similar to those of the control. a star is omitted for the h parameters.46 0.62 Pyrazole 0.26 10.949 0.34 9.48 0.76 0.'/ASd.56). the observable effects for type I experiments were quite small and merely gave marginal confirmations of the results already listed.35 0.57 Azide(Na) 1.01 M ) and thiazolidine4carboxylic acid (0. producing the reduced form.63).0 0.46 AMP 1-21 0.96 1. The ratio ASo'/ASo would reflect any differences in cytochrome c concentrations.4 within 0.40 within 0.Effects on Forms of Ferricytochrome c Table 11.4.3 1.Activation may thus be considered as strengthening the binding of this group to the heme Fe. 1972. benzimidazole (7..13 0. Next to this slowest value follow the thiophene derivatives. pyrimidine (7.69 1. we conducted a series of experiments on the Cary recording spectrophotometer.13. located in the sixth ligand position of Fe+3.001 0. the latter ones inhibitors.02M).0. 15. One derives easily from line 1 of Table I that for the control AS. Otherwise. although aS. of methionine 80 in the sixth ligand position.99 0.39 Aniline 1. totaling 10 mV.2 0.140 0.47 10. ADP and the thiazolidine derivatives.234 0.33 0.' PHl 0.47 10. In most cases. = 0. % ' / A 1 &'/A3 ASl'/ASi AS. one may distinguish compounds which seem not to effect cytochrome c from those which either enhance the red gbsorption band or diminish it. The ionic strength was 0. It is by now well established that the 695 band is caused by the S atom of methionine 80.46 Pyrrolidine 1. as defined in Table I.5 0 Pyrimidine 0.170mM.

Acad. A. E. The results for this study significantly support the additivity concept assumed by the Free-Wilson approach. Biochemistry. G. Biochemistry. The substituent constants for groups at the para position of the 2-Ph ring were found to correlate significantly with both Hammett’s meta u constants and Hansch’s 7~ values for those substituents.lg Eq 6 presents thus an expression of the former derivations. P. and G. P. the substituent constants for the 16 different aminoalkyl side chains failed to correlate with 71. Y. Sei U. Amer. and G. M. Vol. P. Acta Chem.. 1045 (1964). Free-Wilson Analysis of 2-Phenylquinoline-4-carbinols Paul N. 8 7 . Scand. Hess. J.. Urry. Fed. AF and AK in Table I1 are set equal to 0 . 120 Stout Rd. U S . Substituent constants for groups at position 8 of the quinoline ring correlate with 71 values for these substituents. No. A. Appendix Equation 6 with KII = 0 was previously derived. D. 83. Sturtevant. Sei. 3. Lustgarten. Chem. 2 Craig N. Stacy and B. Soc. G. Doty. We would also like thank Dr. 674 (1967). Pennsylvania 19101. Margoliash and A. 240..An Introduction toTheory and Application of Stepwise Perturbation. Expressions for apparent rate constants for the two-step scheme of Figure 1. R. P. Acad. G. The significance of these results is discussed. Nat. Detailed measurements aiming at exact values for individual constants. Ambler.144 Journal of Medicinal Chemistry.17.. Brandt. Ed. 7. Craig* Smith Kline & French Laboratories. obtained from the Walter Reed computer record system.. Czerlinski. Biol. .. 765 (1968). 113 (1966). Biochemistry. New York. Philadelphia. Advan. J. Biochemistry. N. Proc. 19. Exp. 243. Palmer. 21. Y. anism for the combination of a ligand-induced (represented by I. C. R. R R. Burford. 26. Substituent constants for groups at position 6 and on the meta position of the 2-Ph ring failed to correlate with u or 71 values. R.Proc. rather than 1. AB. D. Biol. Structure-Activity Correlations of Antimalarial Compounds. David Wojcinsky for technical assistance with the electronic circuits and Dr. K. Chem. 1227 (1965). Urry and P. Havsteen. and R. Amer. 1. D. Ulmer. and Iff) with a pH-induced conformational change. Chem. Vol2. 1773 (196 1). Y. H. 2756 (1 965). and the overall “average” log 1/C value is 3. Greenwood and G. P. U. P. 640 (1965).. Substituents on the 7 position of the quinoline ring correlate well with para u and 71 values.39. ibid. including H.. 1965. Substituent constants at R. Dar.. E.” R. Protein Chem. G. Christman.. Parks. Nat. 4 . Acknowledgment. N. Myer. Academic Press. More details on the derivation of eq 5 and 6 are also obtainable from a monograph on chemical relaxation” (see especially Tables 8. M. or 71 and 71’. 54. 8. Received April 23. Chem. D. Czerlinski. 9 0 2 (1965). The correlation coefficient for the analysis is 0. 3660 (1965). Inc. Hugh J. Sei. G. 347 (1967). 231. G. The authors would llke to thank Mr. Soc. Chem. 15. Proc. Dar. J. Results Following the reported method’ the matrix listed in Table I1 was used as input to be solved by a matrix inversion program developed by Free and coworkers. Amer. W. 1’. Pa. 56.t The ?This is the theoretical value for a hypothetical molecule in which none of the 6 substitutable positions contains any substituent group.4180 (1966). Acta. Nat. and was converted to the customary log l/Cvalue. Biophys. New York. 241. Myer and H.S. Schejter and P. Musky and P.905. for loaning us a Beckman Type RB Dynograph with plug-in units.4567 (1969). could be related to H as 0 by arbitrarily setting the ma& so that columns Q. in “Computers in Biomedical Research. Theor. gave data obtained by Rane and coworkers by the reported methods6 The dose in milligrams *Address correspondence to Craig Chemical Consulting Services. Fed. 2115 (1968). Margoliash and J... as well as Chapter 7). References (1) K. Bio. Biochim. 1966. 19002. Czerlinski and K. Watt and J. 1972. Waxman.. Berman.... at least 3 graded doses were given to 5 animals per dose. . Hess. H-C-OH which cured 50% of the animals was obtained by extrapolation of the number of cures found for each of the doses tested. W. B.359. Viera Bracokova Czerlinski for assistance in the evaluation of the data. Y. Inc. J. 3397 (1964). H.” Marcel Dekker.. The Free-Wilson method of structure-activity correlation’ has been applied with varying degrees of success in recent This paper reports a successful application of the technique to compare the antimalarial test results in mice for 69 substituted 2-phenylquinoline carbinols of general structure I. Biol. G. George. C. J. Czerlinski. J.. I The biological test reports. 222 (1966). Myer. US.57 (1971).. are now in progress. Acad. Harbury. the standard deviation is 0. 234. 19 71 Sixty-nine 2-phenylquinoline-4-carbinols which had been tested in the mouse for antimalarial activity were studied by the Free-Wilson method for structure-activity correlation. 17.. Biol. where C = moles/kg test animal. For every compd. D.. Soc. The substituent constants (sc) w h c h resulted from the regression analysis are listed in Table I. “Chemical Relaxation. Sutin and D.2 and 8. Proc..3. 54. Schejter. Y. eq 3. Czerlinski. heavy lines of part B (and KH’ defined as in part A) are easily obtained from eq 6 by setting k3 = k4 = 0 (as well as KU 0). 1391 (1965). Chapter VII. George.

Washington.W. 1. 1155 Sixteenth Street N.Structure-activity correlations of antimalarial compounds.org on April 25.. Chem. 144-149• DOI: 10.. Free-Wilson analysis of 2-phenylquinoline-4-carbinols Paul N. 2009 More About This Article The permalink http://dx. 1972. 15 (2).1021/jm00272a007 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society.org/10. DC 20036 . Craig J.1021/jm00272a007 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.doi. Med.acs.

Philadelphia. Ulmer. Theor. for loaning us a Beckman Type RB Dynograph with plug-in units. 231. M. 9 0 2 (1965). Scand. Substituents on the 7 position of the quinoline ring correlate well with para u and 71 values. 7. Ambler. W. 765 (1968). as well as Chapter 7). Lustgarten.t The ?This is the theoretical value for a hypothetical molecule in which none of the 6 substitutable positions contains any substituent group. 3397 (1964). Proc. Myer.905. The substituent constants (sc) w h c h resulted from the regression analysis are listed in Table I. 2 Craig N. 19 71 Sixty-nine 2-phenylquinoline-4-carbinols which had been tested in the mouse for antimalarial activity were studied by the Free-Wilson method for structure-activity correlation. 1391 (1965). 1773 (196 1). Chem. 1966. Hess. heavy lines of part B (and KH’ defined as in part A) are easily obtained from eq 6 by setting k3 = k4 = 0 (as well as KU 0). 243. 1. 26. 1227 (1965). Vol. David Wojcinsky for technical assistance with the electronic circuits and Dr. W. Y.57 (1971). Burford.” Marcel Dekker. 241.. J. eq 3. Doty. Bio.. R R. and R. The correlation coefficient for the analysis is 0. The results for this study significantly support the additivity concept assumed by the Free-Wilson approach. ibid. 120 Stout Rd. Amer. Academic Press. Brandt.. New York.. George. Structure-Activity Correlations of Antimalarial Compounds. J. Acad. and was converted to the customary log l/Cvalue.17. H. 4 . Inc. Biol. 113 (1966). M. Y.. .359. C. Viera Bracokova Czerlinski for assistance in the evaluation of the data. Appendix Equation 6 with KII = 0 was previously derived. 240. 222 (1966). Parks. Biochemistry.. 674 (1967). Amer. 83. I The biological test reports. Sei. P. Acta Chem. J.39. 640 (1965). 2756 (1 965). P. E. Czerlinski. P. Sutin and D. Czerlinski and K. Amer. Proc. J. Chem. Chem.. 21. Chem.. gave data obtained by Rane and coworkers by the reported methods6 The dose in milligrams *Address correspondence to Craig Chemical Consulting Services. The significance of these results is discussed. G. New York. The Free-Wilson method of structure-activity correlation’ has been applied with varying degrees of success in recent This paper reports a successful application of the technique to compare the antimalarial test results in mice for 69 substituted 2-phenylquinoline carbinols of general structure I. Soc. 234. 8 7 . could be related to H as 0 by arbitrarily setting the ma& so that columns Q.. Substituent constants for groups at position 8 of the quinoline ring correlate with 71 values for these substituents. H-C-OH which cured 50% of the animals was obtained by extrapolation of the number of cures found for each of the doses tested. H. Biochemistry. 19.. Proc.. Ed. R. 54. 3. Results Following the reported method’ the matrix listed in Table I1 was used as input to be solved by a matrix inversion program developed by Free and coworkers. Acta. The authors would llke to thank Mr. Y. Received April 23. Chapter VII. Detailed measurements aiming at exact values for individual constants. 347 (1967). Musky and P. and G. Biophys. Vol2.lg Eq 6 presents thus an expression of the former derivations.Proc. Myer and H. 1045 (1964).. Christman. P. We would also like thank Dr. Protein Chem. Sei. R. U. B. 3660 (1965). Czerlinski. R. 19002.144 Journal of Medicinal Chemistry. Hess. Y. Czerlinski. Nat. N. Palmer. P. 56. G.. K. Harbury. are now in progress. Soc. 1’. Free-Wilson Analysis of 2-Phenylquinoline-4-carbinols Paul N. 1965. Nat. Havsteen. Soc.. Hugh J. D. “Chemical Relaxation. Sei U. Expressions for apparent rate constants for the two-step scheme of Figure 1.S. at least 3 graded doses were given to 5 animals per dose. The substituent constants for groups at the para position of the 2-Ph ring were found to correlate significantly with both Hammett’s meta u constants and Hansch’s 7~ values for those substituents. Fed. G. the standard deviation is 0.” R. or 71 and 71’. Acad. Schejter. A F and AK in Table I1 are set equal to 0 . Acad. Biochemistry. rather than 1. J. Advan. Acknowledgment. C. D. U S . D.2 and 8. Substituent constants for groups at position 6 and on the meta position of the 2-Ph ring failed to correlate with u or 71 values. Stacy and B. US. Craig* Smith Kline & French Laboratories. E.. in “Computers in Biomedical Research.4567 (1969). J. Pennsylvania 19101. .. Biol. 2115 (1968). AB. Greenwood and G. Substituent constants at R. Y. P. Biol. including H. Margoliash and A. Watt and J.. G. G. No. Chem.3. Biol. Fed. 17. 15. G. Biochemistry... 54. 1972. and Iff) with a pH-induced conformational change. Nat. For every compd.4180 (1966). Biochim. More details on the derivation of eq 5 and 6 are also obtainable from a monograph on chemical relaxation” (see especially Tables 8. 8. Waxman. N. George. Schejter and P.. Pa. D. and G.. Inc. Urry and P. obtained from the Walter Reed computer record system. Berman. Margoliash and J. Urry. A. D. A. Czerlinski. the substituent constants for the 16 different aminoalkyl side chains failed to correlate with 71. Dar. References (1) K. Myer. and the overall “average” log 1/C value is 3.. anism for the combination of a ligand-induced (represented by I. where C = moles/kg test animal. Exp. Sturtevant. Dar.An Introduction toTheory and Application of Stepwise Perturbation. G.

N (i-C5H.15 0.17 0.04 -0.397 0.168 45 0 0 0 0 4 -0. 15. FMaM = 2.54 0. These points are illustrated in Tables I and 11.04 0.906** -0.311 35 0 0 0 0 an = number of examples.487** 0.16 0.361 0.774** 7 1. F34. Groups E. R3 CH. The same antimalarial data have been analyzed by the Hansch multiple parameter method.43 1* * 32 CH. but because their sc values were much closer to that for group C.0785 0.N(CH. I.11 0.10 0.NH(cyclopropyl) CH.143 0.37 F 0. Group A is especially interesting.209 0.-piperid yl CH.OCH.70 0. for arom substituents. but its sc was so close to that for group C that it.270 0. 1972. The sc values are listed in Table I.N(C.17 1.31 4.17 1.27 0.17 0.036 -0.C$ CH. cu constants (ref 17).573 -0. this can only be gauged by the overall F value and standard deviation for the entire regression.N(C6H1 0.193 -0.uc p .0554 0 0 0 0 -0.No.l)2 CH.123 0. by CND0/2 method.uc ERd 0. too.Antimalarials.27 0.N(CH.145 1.562 0. and P (identified in Table 11) gave sc values which are sig nificantly different from that of group C (the reference group).H." the results w i l l be compared with those obtained by the Free-Wilson method in paper 2 of this series. bn constants for R.34 0. CH.-morpholino OCH3 .19f H -0.07 -0.37 6.697* -0.4.23 0.43 0.18 5.12 0.43 0.973** 0.115 -0.55.54 0.07 -0.38 3.06 f 14 0.H7) CH. The most important information to be gained from a FreeWilson analysis is the relative rank of substituent group constants at each position.70 0. ) .37 0.23 0.31 -0.06 0.16 0.077* -0.27 0.N(C.58 0. -0.3 1 2.). even though they were represented by only one example each.Wilson Analysis Journal of Medicinal Chemistry. gFor the overall significance at the 1%level.N(C8Hl7 ) n CH.04 0.10 0.150 0 0 0 0 -0.52 -0. obtained from aliph series (ref 22).02 1.359.) (CC.35 0. and the reference sc for each position is in italics.10 10 -0.52 -0. CH. If the two sc values are very close.323 Rl Substituent 6-Methyl-2-piperidyl 2-Piperidyl CHZN(C4HJ 2 CH.34 0.CH.34 for the 1% level is 2.10 H .45 1* -0. was not significantly different from it.0437 0. This is summarized in Table I for Table I. eone asterisk indicates significance at the 5% level. Vol.174 -0.06f 19 0.19f 0. -0.03 0.CH. Hansch.240 0.76 NCH.43 0. private communication. except where noted.435* 6 1.23 0.16 0. d E values ~ taken from ref 20. This reference group is chosen as either the most frequently studied group.221 1.23 0.37 0. The absence of a T test asterisk does not signify a lack of statistical significance for the particular sc. Groups F and L were represented by two examples.07 -0.N U . 5) CH.115 0.10 8 -0.31 2.06 -0. is very different from the reference sc.115 -0. na 1 9 25 1 14 2 1 2 4 3 2 1 1 1 1 1 2 7 58 2 1 1 34 5 15 10 3 nb m .06f CF3 R4 I 0. unless the sc for a group.489 -0.43 0. Also.11 -0.115 0. Thus the basic assumption of the Free-Wilson method (additivity of group effects) is confirmed for these biological data.0. it is unlikely that they will be significantly different.11 WH3 CH.54 0.55.17 which are made between each sc value (other than the reference sc) and the sc value for the reference group at that position. c l -0. or as the hydrogen analog. Rhee and C.0641 -0.88 0.262 -0. Substituent Constantsg Substituent constant (scIe 0.574 -0.14 0.54 0.39.16 -0.12 0.28 0.00 7.03 H -0.H.284 -0. s = 0.23 4 0.4 32 * * -0.10 0.905.16 28 0.NH(l-adamantyl) CH.06 f g 3 0. represented by only one or two examples. no significant difference should be expected.3 1 1. mp = 3.71 -0. F = 4.203 -0. Two asterisks indicate R calculated by J.445 1. in that it occurred in nine examples.NH-CH. 2 145 F ~ t a t i s t i c 'for ~ the correlation is 4.11 0.70 0.70 0.16 0. r = 0.321** 0. ~ E values regression. Free. It must be emphasized that this does not reflect upon the reliability of the sc value for group A. hence the correlation is highly significant.26 0. they were not significantly different from C.18 2.37 0. Discussion Differences between the additive constants for groups at each position can be checked for statistical significance by T tests.927** 2 1.37 . it only means that the distinction between that sc and the reference sc is not significant. n values obtd from ref 21.4 for 1%.239 0 25 0 0 0 0.

146 Journal of Medicinal Chemistry.3 3 3 3 3 3 - 3 3 3 .3 3 3 .4 3 - 3 3 3 3 - 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 i i 3 3 3 i 3 - 3 3 3 3 3 3 3 3 3 3 3 3 3 . 1972. Vol. No. - 3 4 3 - i - 3 3 3 3 3 3 i $& 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3-333333333 r i d - 3 3 3 3 i i - 3 1 0 .3 . 15. 1 3 3 3 3 3 3 3 3 3 3 4 3 3 3 3333 3 3 3 3 3 3 3 3 3 - 3 3 3 3 3 3 3 3 3 3 .3 3 \ Id 3 i 3 3 M i 3 3 . 2 Craig 3 3 3 3 3 3 .

15.3 v ) N 3 33 3 2 m 3 3 3. Vol. Free.Antimalarials. 3 3 333 N v) 3 3 3 m 3 4 3 3 m . 2 147 * d 3 3 3 z 33v) 33 33- 33 3333 33333- 3 m 3 33 3 d N 3 3 33 36 3 3 3 3 3 3 v) 3 3 33 .3 d -3 3 3 3 0 3 w.?. 3 3 3 3 3333 v) 3 3 3 3 3 3 3 33 333 33333333333433300 v) 3 3 3 N 3 3 3 d 3 3 3 N 3 3 3 1-4 3 3 33 3 b.r 33333 3 33 3 3 Y . No. 1972.Wilson Analysis JournaIofMedicinal Chemistry. 3 33 3 3 3 3 3 00: N 3 33 3333 3 .

et al.123 0.978(+0. Clayton. M.397) (8) sc = -0.190) (7) sc = -0. andT.61* Fl. (14) E.83 7). ibid. J.61* Fl. rather than the exception. Mead.39* 7. Rapport. Vol. ibid.= 6. 11. 11.68. 7. 1813 (1946). since a similar conclusion is drawn from a comparison of the ranges of substituent constants at positions R7 and R4 (1. A.318)n + 0.676(+0. A.232(+0. C. two asterisks. Fujita.157 0. (4) W.” and Hansch’s n values.94* F. Saggiomo. P. No. (7) D. B.s =6.” hence. R. Osdene...s = 6. 2 I _ _ Ouig Table Ill.” Otsu’s Er constants.81 X 0. 908 from the Army Research Program on Malaria. b Comments No significant correlations obtained between ?i or 17’ and n. These results point out possible linear relationships between polar effects of substituent constants in both benzenoid rings.232) Position 6 Position 7 (6) sc = -0. ibid. (9) E. and antimalarial activity. Ann Arbor. Free. M. .338(* 1. The problem of differentiating between these possibilities when one is working with a limited set of substituents is discussed in a companion paper. from eq 5.808 0. Jr.827 0. for CF3 = 0. 1.322 0. J. J. gives a direct comparison of the relative effect upon antimalarial activity of changes in polar effects at these two substituent positions. (15) F. Chem. P. Rane. R. The present approach allows predictions to be made outside of the matrix of compounds employed in the Free-Wilson analysis.and is Contribution No.= 0.811(*0. Chem. 178. Table 111.. Patel.8). . Jacobus. polar effects of substituents at R7 affect the antimalarial activity about 7 times more than do similar polar changes at R4 on the 2-Ph ring (antilog of 0. and J.787)u-meta + 0.Purcell and J.1** F..589(*2.938)o-meta+ 0.148 Journal of Medicirial Chetmstry. 199 (1968). P.010(+0. 824 (1963). No correlation was found when the values for n and nz were studied.592)n + 0. References (1) S. Jr.1* Fl.= 6.220(+0. Biophys.3= 34.635)u-para + 0.837) Position 8 (9) sc = -0. 395 (1964).. 11. Lutz.820)o-meta + 0.537 (1970).198(+0. 1223 (1968).981 0.. S.966 0. Army Research and Development Command Contract No. 277 (1968). 49. Chem.. M. reported in Table I.93. ibid. and R.75 27. 1. and Dr. and J. all positions studied. What is needed to resolve this problem is a substituent group such as CH3S02. 12.72. Jr. Atkinson and A. Russell. G. Soc.136)~-para + 0.61* F..366 (1968). E. antilogg5. J. R.895 0. 13. E. This work was supported by a US.201 (1965). the prediction is to be considered as a maximum value.. Kaiya. Med.. The author wishes to acknowledge helpful discussions with Dr. Ed. Acknowledgments. Atkinson and A.sa Fa. difference = 0. Free.942 0. The larger coefficient for up at R7.566) + 0. not necessarily fully achievable. P.2= 18.654)o-para t . should be approximately equivalent in activity. Rothe and D.174) (3) sc = -0.= 10. see Table 1. W.. W.7** 23.34 8.959(*0.. B.. Puttick. 10.. (13) J. 11. Table 111. .38.. ( 6 ) T.. csc = substituent constants obtd by the aReference 17.. Jr. and R.. 19411945. Thus.151 77.8 1. Parisitol. 175 (1969).9* Fl. It is noteworthy that no simple dependence of antimalarial activity was found for n values of the substituent groups at R1.. 0.457(+0. The application of eq 7. the preparation of the CF3S02 analog at R7 would be expected to result in an increased antimalarial activity of about 5-fold over the CF3 analog (a pard for CF3S02. Gillespie.06* Fl.. regression. The results listed in Table I11 now allow one to predict maximum values of log l / C which might be expected for compds bearing as yet unstudied substituents. Lutz.626(+0. J. at R4. and R.651)n + 2. F. antilog 14.50* 8. Jr. and L.353) 0. DADA 17-69-C-9106.41 and 0. (10) A. 68. 11.Wiselogle. Kato. E. Campbell. J.780)~ 0. Of course it is possible that too great a change in u could lead to a parabolic relationship.. S. i972. as compared to 0.112 0. ibid.297) (5) sc = -0. at the 1% level. However. (12) R. Biochim. E.041(*0. ibid.98 for u .431 (1967).395(+0. Rowlett. J. Boykin.. Corwin H.. would lead to a prediction that the CF3S02 analog of a CF3 member of structure I.170(+0. J.21~22 The results are presented in Table 111. ibid. E.361(*0. M. Koepfli. ibid. if significant correlations with standard parameters can be obtained for the de novo sc values.. to help resolve which paranieter is really the important one. (16) M. Of course it is not necessary to study the regression results in Table I11 to gain this information.8* 8. Acta.236) (2) sc = -0. Purcell.863 0. 11. in an attempt to gain more information from the analysis than just rank. The original presentation of the Free-Wilson method emphasized the relative ranking of substituent groups at each position. Wilson. (11) W. R.53* 4. (2) W. 273 (1968). Senear. (8) W. Ban and T. Mich. J. Amer. K. Chem.23where the judicious selection of one or two additional substituents is discussed.127(+0.5 = 6. CorrelationsObtained by Regression E~uation ~~ P - .55.232) (4) sc = -0. Med.213 0.191(+0.3).” Edwards Bros.162 0.. Hansch. involving 71 instead of u. Also possible are relationships between the 71 values for some of the position substituents and antimalarial activity.94* No significant correlations obtained + 1.38 = 0. P.which is high in u and very low in n. correlations were sought by regression analysis for linear relationships between the sc values and the following parameters: Hammett u constants (meta and para). Davis. J. More activity would be expected from increased electron withdrawal at R7 than at any of the other aromatic ring positions. Med. this was tried since the Hansch method has shown that such relationships are the rule.775 0. “Survey of Antimalarial Drugs..168 0.685. (3) 3.998(+0. Y. 353 (1969). Thus.133 9.220(+0. Puttick. Purcell.425 (1968).794 0. Beasley and W. (5) T. difference = 0.5* asterisk indicates significance at the 5% level. 105.. S. 15. SL‘ No significant correlations obtained Position 1 Position 3 Position 4 (1) scc = -0.

. Hansch. 191 (1953). Non-drugtreated mice survived about 6 days following infection with P. Before ~ this study none of the reported azaindoles would be considered as candidate antimalarial target compounds. (22) J..9 0. 7-azaindole having a p K a = 4. *' CNumber of deaths/5 mice. Mild Mn0.3 0. (18) C. mg/kg 640 640 640 640 640 640 10 IMST. Synthesis of 1 -p-Chlorobenzyl-7-azaindole-3-cx-piperidylmethanol as a Potential Antimalarial Agentf Anthony J. Chem. Also. e. (19) H. 86. An intermediate in the 4-step synthesis. A pharmacological profile of the unsubstituted azaindoles comparing the activities of these compounds with indole." and 1-p-chlorobenzyl-7-azagramine. The basis for exploring the 7-azaindoles rests on the rationale that the structure of 1-pchlorobenzyl-7-azaindole-3a-piperidylmethanol is comparable to the quinoline-5-apiperidylmethanols. Res. The yields in this reaction were consistently good and the product was pure enough for synthetic purposes.Antimalarial Azaindole-3. California 91024. Only starting material was recovered when the carboxylic acid 9 was treated with 2-PyLi in an attempt to prepare the ketone.8 0. 1966.. Otsu. Hansch. Soc. Chem. 14. (23) P. 1971 A single diastereoisomer of 1-p-chlorobenzyl-7-azaindole-3-a-piperidylmethanol was found to have antimalarial activity about 0.6 Toxic deathsC 0 0 0 0 0 0 0 0 1 2 3 4 5 6 8 8a 9 10 11 12 40 160 320 640 640 640 160 320 640 640 640 0 0 2 0 0 0 3 5 0 0 "Test data supplied by Walter Reed Army Institute of Research. 232 (1969).5 0. daysb 0.5 0.~$' A better method to prepare this aldehyde was discovered whereby 7-azaindole is 3-formylated with hexamethylenetetramine in refluxing aq AcOH. The second isomer was extremely hygroscopic and apparently inactive as an antimalarial agent. whose assistance is gratefully acknowledged. None of the substituted 7-azaindole intermediates synthesized in this study showed appreciable activity.. Fujita. J.82 is isolipophilic with respect to quinoline which has a p K a = 4. berghei. ibid.5 0.680 (1971).03 $ Chemistry. Army Medical Research and De." 3nitro-7-azaindole. diazaindoles.5 0. 53. (17) G. i.3 0.' Antimalarial activity in plain indoles with antimalarial type and other miscellaneous side chains has not been ~ b s e r v e d . J. Very little biological activity has been published for any of the azaindoles other than 3-azaindole (benzimidazole) derivatives. N. Ind. 15. Snedecor.150 (1965). and purine was reported. In addition. J. Amer.I4 n-BuLi is known to metalate indoles on the 2 position" and the reactive 2-PyLi l6 may have formed a 2-lithio-7-azaindole in our reaction sysTable I. ~ x i d a t i o n 'of ~ the aldehyde 3 gave 1pchlorobenzyl-3-carboxy-7-azaindole. 787 (1967). 839 from the Army Research Program on Malaria.95 and log P = 2. Received July I . Chem.' The condensation of 3 with 2-pyridyllithium gave 1-p chlorobenzyl-7-azaindole-3-a-piperidylmethanol as a labile oil which tends to decompose back to its aldehyde and ?Contribution No. No. SOC.7 0. Osdene. H.was prepared in good yield by a Duff reaction. pyridyl components.5 that of quinine when tested in mice against Plasmodium berghei. For details of this test see T.' The key aldehyde 3 was also prepared by first 1-alkylating' and then formylating under Vilsmeier reaction conditions. Iwasa. Med. 8. Following procedures for the plain indoles6 1-alkylation provided 1-p-chlorobenzyl-7-azaindole3-carboxaldehyde plus some quaternary product. Verbiscar Instihite of Drug Design. 2. (21) T." Iowa State University Press. The goal of this study was to explore the potential of 7-azaindole as a new antimalarial nucleus.. and C. velopment Command Contract No. This experiment confirms earlier observations about Vilsmeier formylation of 7-azaindoles. 2697 (1946). and the latter compounds show antimalarial activity. Amer." Low pressure hydrogenation of the side chain pyridyl in its pyridinium salt form provided 1-p chlorobenzyl-7-azaindole-3-ar-piperidylmethano1 as mixed diastereoisomers..1 3. Chemical therapy was initiated 3 days postinfection. the low activity of the mixed diastereoisomers indicated that one of the isomers was inactive. $The log P of 7-azaindole and this comparison with quinoline were provided by Professor Corwin Hansch.7 3. Craig. supported by the U. and C. W. S. Fractional crystallization separated one of the diastereoisomers as stable microcrystals. S. h x e a s e in mean survival time between Plasmodium berghei infected mice administered the drug sc and controls. "Statistical Methods. . that this reaction is facilitated by 1-substitution.' The primary interest in azaindoles has been as potential antimetabolites to naturally occurring indole derivatives. Chem. This is the first example of any antimalarial activity in the 7-azaindole class. DADA1 7-69-C-9082. Fujita.7 0. Iowa. et al.3 0.. Iowa City. Unsubstituted 7-azaindole is not formylated under normal Vilsmeier reaction conditions and 7-azaindole3-carboxaldehyde has been made from 7-a~agramine.. Iwasa. 1972.68.9 1. Yamamoto and T. Rev. Antimalarial Activity and Toxicity of 7-Azaindoles in Mice" Compd Dose. Hansch. 5175 (1964). Sierra Madre. H. Chem.piperidylmethanols J. Jaffe. and this isomer was tested for antimalarial activity. which represents a new and facile method for introducing the 3-formyl group on an N-unsubstituted 7-azaindole.0 0.3 0. These included 3-bromo-7-azaindole. T. Vol. Journal of Medicinal Chemistry.59 and logP = 1. Several other 3-substituted 7-azaindoles were prepared as possible intermediates with "handles" for the introduction of side chains. 7-azaindole-3-carboxaldehyde. Accounts Chem. 2 149 (20) T.

Washington.Synthesis of 1-p-chlorobenzyl-7-azaindole-3-. 149-152• DOI: 10. Med.alpha. 1972.org/10.1021/jm00272a008 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. Chem..doi. 1155 Sixteenth Street N.org on April 25.W. Verbiscar J.acs. 15 (2).1021/jm00272a008 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. DC 20036 . 2009 More About This Article The permalink http://dx..-piperidylmethanol as a potential antimalarial agent Anthony J.

e. Craig. Med.5 0.I4 n-BuLi is known to metalate indoles on the 2 position" and the reactive 2-PyLi l6 may have formed a 2-lithio-7-azaindole in our reaction sysTable I. *' CNumber of deaths/5 mice.5 0. Vol. Following procedures for the plain indoles6 1-alkylation provided 1-p-chlorobenzyl-7-azaindole3-carboxaldehyde plus some quaternary product. h x e a s e in mean survival time between Plasmodium berghei infected mice administered the drug sc and controls. Fujita. Ind. 5175 (1964).9 0. Osdene. This is the first example of any antimalarial activity in the 7-azaindole class.5 0... mg/kg 640 640 640 640 640 640 10 IMST. $The log P of 7-azaindole and this comparison with quinoline were provided by Professor Corwin Hansch. 7-azaindole having a p K a = 4. 787 (1967). N. Non-drugtreated mice survived about 6 days following infection with P. 86.7 3. i. 15. 2697 (1946).150 (1965). which represents a new and facile method for introducing the 3-formyl group on an N-unsubstituted 7-azaindole.. DADA1 7-69-C-9082.82 is isolipophilic with respect to quinoline which has a p K a = 4.68. Iowa. Amer. 53. and this isomer was tested for antimalarial activity. Journal of Medicinal Chemistry. 191 (1953). H. "Statistical Methods. J. (19) H. (23) P. SOC.3 0.' The primary interest in azaindoles has been as potential antimetabolites to naturally occurring indole derivatives. Received July I . (21) T.3 0. H. Jaffe. In addition. 839 from the Army Research Program on Malaria. Chemical therapy was initiated 3 days postinfection. Very little biological activity has been published for any of the azaindoles other than 3-azaindole (benzimidazole) derivatives. Chem.95 and log P = 2. Sierra Madre. For details of this test see T. W.' Antimalarial activity in plain indoles with antimalarial type and other miscellaneous side chains has not been ~ b s e r v e d .piperidylmethanols J. Fujita. These included 3-bromo-7-azaindole. S. Antimalarial Activity and Toxicity of 7-Azaindoles in Mice" Compd Dose.5 0. ibid.. Mild Mn0. The yields in this reaction were consistently good and the product was pure enough for synthetic purposes. Unsubstituted 7-azaindole is not formylated under normal Vilsmeier reaction conditions and 7-azaindole3-carboxaldehyde has been made from 7-a~agramine.7 0. Snedecor. Synthesis of 1 -p-Chlorobenzyl-7-azaindole-3-cx-piperidylmethanol as a Potential Antimalarial Agentf Anthony J. . ~ x i d a t i o n 'of ~ the aldehyde 3 gave 1pchlorobenzyl-3-carboxy-7-azaindole. berghei. An intermediate in the 4-step synthesis. The goal of this study was to explore the potential of 7-azaindole as a new antimalarial nucleus. Hansch.5 that of quinine when tested in mice against Plasmodium berghei. and C. Hansch. et al. Also." and 1-p-chlorobenzyl-7-azagramine." Iowa State University Press.' The key aldehyde 3 was also prepared by first 1-alkylating' and then formylating under Vilsmeier reaction conditions. Only starting material was recovered when the carboxylic acid 9 was treated with 2-PyLi in an attempt to prepare the ketone. 232 (1969). California 91024.Antimalarial Azaindole-3. Iwasa. Verbiscar Instihite of Drug Design. (17) G. Iowa City. 1972. T.3 0. No. Chem. (22) J.' The condensation of 3 with 2-pyridyllithium gave 1-p chlorobenzyl-7-azaindole-3-a-piperidylmethanol as a labile oil which tends to decompose back to its aldehyde and ?Contribution No. Before ~ this study none of the reported azaindoles would be considered as candidate antimalarial target compounds. and purine was reported. Army Medical Research and De. pyridyl components. (18) C. Otsu.. 2 149 (20) T. 2. J. S. diazaindoles. Soc. This experiment confirms earlier observations about Vilsmeier formylation of 7-azaindoles. and C. Yamamoto and T.7 0.8 0.3 0." Low pressure hydrogenation of the side chain pyridyl in its pyridinium salt form provided 1-p chlorobenzyl-7-azaindole-3-ar-piperidylmethano1 as mixed diastereoisomers.0 0.9 1.. Hansch.59 and logP = 1. Accounts Chem. 1966. Several other 3-substituted 7-azaindoles were prepared as possible intermediates with "handles" for the introduction of side chains. J.~$' A better method to prepare this aldehyde was discovered whereby 7-azaindole is 3-formylated with hexamethylenetetramine in refluxing aq AcOH.03 $ Chemistry. The basis for exploring the 7-azaindoles rests on the rationale that the structure of 1-pchlorobenzyl-7-azaindole-3a-piperidylmethanol is comparable to the quinoline-5-apiperidylmethanols. The second isomer was extremely hygroscopic and apparently inactive as an antimalarial agent.was prepared in good yield by a Duff reaction. Amer.. 1971 A single diastereoisomer of 1-p-chlorobenzyl-7-azaindole-3-a-piperidylmethanol was found to have antimalarial activity about 0. that this reaction is facilitated by 1-substitution. Rev.8. Fractional crystallization separated one of the diastereoisomers as stable microcrystals." 3nitro-7-azaindole. 14. whose assistance is gratefully acknowledged..1 3. daysb 0. Res. A pharmacological profile of the unsubstituted azaindoles comparing the activities of these compounds with indole. Chem.680 (1971).6 Toxic deathsC 0 0 0 0 0 0 0 0 1 2 3 4 5 6 8 8a 9 10 11 12 40 160 320 640 640 640 160 320 640 640 640 0 0 2 0 0 0 3 5 0 0 "Test data supplied by Walter Reed Army Institute of Research. Chem. velopment Command Contract No. 7-azaindole-3-carboxaldehyde. Chem. Iwasa. None of the substituted 7-azaindole intermediates synthesized in this study showed appreciable activity. supported by the U. the low activity of the mixed diastereoisomers indicated that one of the isomers was inactive. and the latter compounds show antimalarial activity.

§ Also.O day at 640 mg/kg. Anal. elements are within +0.1%.150 Journal of Medicinal Chemistry. Where analyses are indicated only b y symbols of the elements. 12..5 days at 640 mg/kg indicating that the second diastereoisomer is inactive.6 days.H. Compds 1-4. and 1 1 were tested vs. 9. which were sol in H. In the mosquito screen vs. All of the 7-azaindoles. This is about 0. Vol. The mixed diastereoisomers of this structure were tested as the dibenzoyl-d-tartrate salt 8a. N 7-Azaindole-3-carboxaldehyde. Compd 8 has definite CNS effects in mice showing stimulant and possible antidepressant activity. which had an IMST of 0. E. Compds 1 and 9 were inactive.40 mole. None of the other 7-azaindoles had an IMST > 1.The resulting clear yellow s o h was dild with 500 ml of H.) C . the first example of a 7-azaindole derivative with antimalarial activity. compared to 490 mg/kg for 7-azaindole and 3 16 mg/kg for indole.gallinaceum a t 120 mg/kg administered sc in chicks and found to be inactive.1 days. H.5 mg/kg.# Experimental Section** 7-Azaindole was prepd by a known methodlg in an overall yield of 46%.O. For example.O. suppression of oocysts was registered by 4 (75%). Compd 2 (86% deaths) was classified as toxic to the mosquitoes. of AcOH and 168 ml of H.O).20 mole) of 7-azaindole and 42 g (0.6 g (0. P. There were no toxic deaths noted for these 6 compounds. 2 Verbiscar Scheme I H 1 1 la. dibenzoyl-d-tartrate tem.5 the activity of quinine sulfate which at 160 mg/kg has an IMST of 3. (C. simple carbcxylic acids have failed to form ketones with MeLi. P. **Melting points are corrected. Biological Results. This lack of toxicity is notable because several of the other azaindoles are quite toxic. At 320 mg/kg the single crystalline diastereoisomer of 1-p-chlorobenzyl-7-azaindole-3a-piperidylmethanol(8) increased mean survival time by 3. and 12 (50%). therefore. X = Br R 9 CHO -N' equiv R R 7 R 8 8a. This is. Except for 8 and 10 they were also not noticeably toxic at this dose level. Lutz. \ H I X R R 4 COOH I H 11. and for 6-azaindole it is 12 mg/kg.H. 1.30 mole) of hexamethylenetetramine was refluxed with stirring for 6 hr in 250 ml of 33% AcOH (84 g. mp 216-218" (re#These effects were noted by Professor Leo Abood whose assistance is gratefully acknowledged. A mono-L-tartrate salt formed and was recrystd from EtOH as white needles. 11 (50%).9 g (50%) of long white needles. 1972. This would decrease the probability for forming a dianion at the COzH function which appears to be an intermediate in the condensation sequence leading to the ketone.O. L-tartrate I R 5 R 6 / \ $H. mp 175-177". analytical results obtained for those of the theoretical value.NMe. A s o h of 23. gallinaceum at a dose level of 0. 1-12.N. were tested for antimalarial activity against Plasmodium berghei in mice. the LDS0in mice for 5-azaindole is 16. No. C. 15.* §Private communication f r o m Professor R." Attempts to prepare 7-azaindole-3-carbinoleamines by treating 3 with dimethylsulfonium methylide to form an oxirane intermediate" were not successful.40/0 . and the product was allowed to cryst in the refrigerator overnight. Recrystn of the crude product from H.O gave 14. X = NO.

H.14 g (25%) of colorless microcrystals.072 mole) of p-chlorobenzyl chloride.010 mole) of l-p-chlorobenzyl-7-azaindole.CO was stirred under reflux for 10 hr.. but a suitable dibenzoyl-d-tartrate salt of the mixed diastereoisomers was formed. mp 104-1 06".33 g (0.88. After this time a tlc monitor showed that the starting aldehyde was consumed.O providing 3.7%) of a solid. Na. 95:5 PhH-MeOH developer. and the solvent was evpd leaving a solid.43. The oil was sol in Et.35 g. It seems likely that steric hindrance by the bulky p-chlorobenzyl group interferes with salt formation and acid solubility of this compd.32. The EtOAc phase was dried (Na. H. as long white needles. It was crystd from PhMe to give 1.and dihydrochlorides were too hygroscopic to serve as derivs. On cooling the suspended oil crystd. After removing the EtOAc from the oil. It is insol in dil mineral acid but forms an HBr salt. The clear soln was warmed at 40-55" for 75 min and then cooled.O was added and the mixt was brought to boiling for 3 min.9 ml(O.ClO~C. and 70 g of ice water were added as a solid pptd.9 kg/cmz until 3 equiv of H. It was sol in dil HC1 where it gradually decompd. and H.O.ClO) C.: 2.6. Partial identification was obtd by ir analysis of the carbinol: in CHCl.H. to yield 15 g (81%) of fine white needles. (C. Following the addn of 1 ml of concd HCl the undissolved oil was extd into Et.10 mole) of KMnO.66 p (arom).16 g (0.SO.O soln contg the desired product was dried (Na.013 mole) of 1-p-chlorobenzyl-7-azaindole3-carboxaldehyde in 20 ml of THF was added over 4 min as the soln became a clear orange.8 g (0.5%..CO was evapd and the residue was taken up in H.88 g (0.. and crystd from CCl.5" dec). 5. Crystn from EtCOMe gave 0. three 50-ml portions of 3% HOAc. under H.) C.N. Crude yields were consistently in the range of 51-63%. and PhMe but it was insol in i-Pr. An analytical sample was crystd to mp 107-108°. This product is colorless in aq acid in which it is highly soluble. N.082 mole) of K..CO-MeOH as the developer.N.11. Its punty was about 60%.6 g (0. (C. dried.).O. Anal. The Et. (C.045 mole) of POCl.CO and also 3: 1 Me. mp 142-144".H. The solvent was partially removed on a rotary evaporator as a n oil appeared.1 g of crude yellow oil. 5.O again.O. H. C1 l-p-Chlorobenzyl-7-aaindole-3-a-pyridylmethanoL A 2-PyLi reagent was generated in the usual manner in 60 ml of Et. mp 130-145'. The oil crystd from 60-110" petr ether resulting in a 29.5 mm). corresponding to the two diastereoisomers. It was collected.7 g (0. H. a soln of 24. Method B.94 g (88%) yield of pure white needles.ClO) C.Clz) C. 15.H. to free 1. The product is insol in dil HCl and can be crystd from this solvent. H. tlc showed the resulting product contd the double spot corresponding to the two diastereoisomers plus several impurities.. 11.N. 1-p-Chlorobenzyl-7-azaindole-3-a-pyridylmethanol (6.Antimalarial Azaindole-3-piperidylmethanols ported5 214.341pyridine-3-carboxaldehyde as described below. mp 200-204'. and the a c e tate. C1 The mono. H.O. N 1-p-Chlorobenzyl-7-aagramine Hydrochloride. then allowed to come to room temp during another hour of stirring..Cl to break an emulsion.15 mole) of 7-azaindole in 100 ml of DMF over 15 min with stirring.0685 mole) of 7-azaindole-3carboxaldehyde.lN. mp 202-205" dec. which oils out in H. It was homogeneous on tlc using Eastman chromagram 6060 sheets with Me. and then dried (Na. which was extd into Et.43 g (0. The mixt was filtered and the ppt was washed well with 1: 1 Me.07 and 0. An acetate did not cryst.SO.CO.341pyridine.16 mole) of 57% N& in oil was washed with petr ether and the last traces of solvent were evapd in vacuo. (C. The dihydrochloride was hygroscopic. l-p-Chlorobenzyl-7-aaindole-3-a-piperidylmethanol Dibenzoyld-tartrate. tlc.28 g of white powder. Crystn from CC1. Anal. bp 157-160" (0.O.7 g) was hydrogenated as in the previous expt. This pyridylcarbinol was prepd using a 2. were absorbed.90 g (30%) of fine white needles.).COH.341pyridine. in 220 ml of H. It formed a phenylhydrazone as yellow plates.H.041 mole) of l-p-chlorobenzyl7-azaindole in 10 ml of DMF was added over 5 min as the temp remained below 20".020 mole) of 2-b~omopyridine.56.O. Tlc on Eastman chromagram sheet 6060 using 3: 1 Me$@ MeOH developer showed 2 spots at R f 0. N 7-p-Chlorobenzyl-7H-pyrrolo [2. A sample was crystd tomp 154-156" for elemental analysis.N.3 g (0. Anal. Anal.O. dry Et. No. which was extd into EtOAc and dried (NaSO.). followed by a s o h of 17.O. (C.5". Anal.).CIO) C. During this addn the temp of the reaction mixt was kept at 35-40'' using an ice bath. The aq acidic portion was made basic with K.5%) of small yellow needles.25 M BuLi in hexane and 3.47 g (0. After hydrogenation and work-up.. N 7-p-Chlorobenzyl-7H-pyrrolo[ 2. After removing the solvent the resulting product was taken up in 150 ml of H.O and EtOAc. The soln was stirred outside the bath for 10 min and then a soln of 10 g (0.67. N.Cl) c.341 pyridine-3-carboxaldehyde.in CHCl.2 g (50% recovery) of l-p-chlorobenzyl-7azaindole.: 5.H1. 64.O and extd 3 times with EtOAc using NH. * 6. Addn of 1 equiv of HCl in EtOH to this oil and removal of the solvent provided a cryst salt. Eventually a dibenzoyld-tartrate salt was formed in Et.6.. H : 5.NH.. (C.95 g (0.94 (OH). The hydrochlorides of the oil were cryst but extremely hygroscopic. 0. 4: 1 EtCOMe-MeOH developer.71 g (54%) of tan needles.CO. N l-p-Chlorobenzyl-3-carboxy-7-azaindole. Anal. The EtOAc phase was extd with 200 ml of 1N HCl in 3 portions to remove 7-pchlorobenzyl-7H-pyrrolo [ 2. The reaction as reported here was run 7 times with variations in reagent and solvent ratios and with quantities of 7-azaindole as small as 0..47 p.O and worked up to give 1. 6.56 mole) of NaOH in 60 ml of H. Vol.CO there was added over 15 min with good stirring a soln of 15. Stirring was then contd for another 45 min outside the bath.O soln with dil HOAc cleanly removed 7-p-chlorobenzyl-7H-pyrrolo[ 2. Method A.020 mole) of 2. N l-p-Chlorobenzyl-7-azaindole-3-carboxaldehyde.O.11 g (16.O..5-215'). The HCl extract of the above reaction mixt was made basic to free 3. Found: C.15 mole) of p-chlorobenzyl chloride in 75 ml of DMF was added over 10 min and the mixt was stirred a t room temp for 3 hr. The solvent was evpd leaving a solid which oiled out upon addn of 50 ml of H. A tlc monitor indicated the presence of 2 products.6 g (0. 5... This was crystd twice from EtOH to give 1. 1-p-Chlorobenzyl-7-aaindole. mp 120-122". The oil can be extd ifito Et. 0.SO. and 11. The pyridylcarbinol was taken on directly to the hydrogenation step. (C. acetate Rf 0. The HOAc ext from the prepn of 1-p-chlorobenzyl-7-azaindole was basified to yield 1. Merck silica gel G. and 45 ml of n-BuOH was refluxed for 1 hr.O was added to the residue and the dark oil was extd into Et. Evapn of the solvent gave 5 g of the carbinol as a glass that could not be induced to cryst. Crystn attempts were generally unsuccessful but the product seemed to be a reasonably pure mixt of the two diastereoisomers.2 g (0.O. washed with H. 2 151 EtOH giving 7.013 mole) of crude l-p-chlorobenzyl-7-azaindole-3-a-pyridylmethanol in 125 ml of EtOH contg 1 molar equiv of HCl was shaken with 300 mg of PtO. CCl. (C.. mp 178-181. 6.050 mole) of l-p-chlorobenzyl-7-azaindole-3-carboxaldehyde in 300 ml of Me.22. H. giving a 9. carbinol Rf 0. A critical condn in the prepn of this pyridylcarbinol is that just enough 2-PyLi reagent must be generated to react with the aldehyde function.5: 1 mole ratio of 2-PyLi to aldehyde and contd several major impurities due to excess reagent. mp 105-107'. Neither diastereoisomer could be induced to cryst from this mixt.20 and 0.Cl) C.82 g of fine yellow needles. A soln of 21. stopped. H. To 14 ml of DMF cooled to 0" there was added over 3 min with stirring 6.62 g (4.. mp 151-156'.90. Most of the DMF was distd off in vacuo. apparently back to the aldehyde and pyridine (tlc monitor). It is insol in acid probably due to both steric hindrance of the p-chlorobenzyl stibstituent and the electron-withdrawing effect of the 3-CHO which lowers basicity of the nucleus. Anal. .H.O using 8. The Me.H.N.CO.-63. mp 230-233' dec (reported5 mp 231232.. showed 2 spots of about equal intensity at Rf 0.24 g of a second oil which could not be induced to cryst. A tlc check at this point showed a very strong spot corresponding to about 90% of product plus about 5% of starting aldehyde as the major impurity. A tlc of this soln on Merck silica gel G.45 p (7-azaindoles).N. A soln of 2. and the residue distd in vacuo giving 31.22 and 6.32. This was crystd from CCl. To a s o h of 13. It is sol in dil base. However. The mixt was dild with EtOAc. A mixt of 10 g (0.O. at 2..N.74 (acetate).-petr ether gave 1.71. in 500 ml of Me. It was stirred for 1 hr at -60'. l-p-Chlorobenzyl-7-azaindole-3-a-piperidylmethanoL A soln of 5 g (0.016 mole) of Me. This product was crystd from Journal of Medicinal Chemistry.*Cl-. The basic aq layer was acidified with HOAc to ppt a solid. H.O was added and the slower moving diastereoisomer eventually crystd from the soln as a powder. 1972. Excess 2-PyLi appears to react elsewhere in the 7-azaindole nucleus. washed well with H.82 g of a yellow solid.HiJ'J. The clear light orange aq soln of acid solubles was basified to release an oil.10. 6. H. The flask was then cooled in an ice-n-PrOH bath and 150 ml of DMF was added. It tends to decompose on standing for 1 month.53. No attempt was made to improve the yield. 6.52 g (0. Following known procedures. 6. An extn of the Et. Anal.32. mp 35-37'. soln.7 g (81%) yield of l-pchlorobenzyl-7-azaindole.011 mole) of paraformaldehyde. a shorter reflux time (3 hr) and gradual addn (30 min) of hexamethylenetetramine lowered the yield to 12.ClOz) C. When the evoln of H. the solvent evapd.'~ After stirring the reagent at -60" for 30 min a soln of 3.6 g (0.08.

ibid. A. Robison and B. Uritskaya. Otto.2626 (1964). Ann Arbor. Chem. Chem. W.648 (1966). J. A. Clindamycin. Lincomycin (I) is an antibiotic produced b y Streptomyces ‘9’ It has an antibacterial spectrum similar t o ~>~ t h a t of erythromycin and was claimed t o be ~ u p e r i o r because o f its effectiveness against b o t h erythromycin-susceptible and -resistant strains of Gram-positive coccal organisms. P. These can b e rationalized by t h e one (e. lincomycin Ri = C H j R2 11. Patel.. 1964. G. V.2603 (1964). Soc. The possibility that lincomycin blocks 2 separate sites in a metabolic sequence whereas the 7(S)-halogenated compds block only t h e latter in t h e sequential biochemical process. Szmuszkovicz. Chem.. 163 61959).. W. J. (b) J..o r 111-affected cultures have t h e same functional dependencies on drug concentrations as lincomycinaffected cultures in phase I b u t are different from those of I in phase I1 generation. (c) M. Amer. Archer. Abstr. 87. 10. Rubtsov. USSR. (a) E. it was subsequently t h a t there was a “dissociated type” of cross-resistance in Staphylococcus aureus between erythromycin and lincomycin. T. 77. Y. (b) L. J. Butler and B. J. Chem. A. and I11 against E. (b) t h e increase in size of t h e alkyl group a t the C-4’ i n t h e pyrrolidine nucleus which in- Zincolnensis. 34. deoxylincomycin] Ri CHs Ra C. 743 (1959).. T.J. Chem.. (5) M. Chem. 2833 (1962). 1651 (1954).8016 (1967). Russel. M. Nickel. 81. Yakhontov.152 Journal of Medicinal Chemistry. K. Corey and M. 81. Acta Chem.. N. lincomycin) allosterically modifying t h e receptor site for the other lincosaminide antibiotic. The effects of changes in pH o n t h e activity of I. 15. Trav. E. B. 375 (1953). 906-916. P. 6554 (1955). Farm. Izobret. Duncan. and S. Colwell. P. geterocycl. L. A. Abstr. ibid. No. G . M. R. 1247 (1956). Jackson and A. R. Yakhontov. H.g. Zh. 73 (1967). J. (8) (a) M. 457 (1955). R. Heinzelman.. L. Chem.Indian J. Amer. combinations of I1 o r 111 with I show antagonistic effects which depend o n the level of activity.75. Garrett* The Beehive. Amer. Wiselogle. J.. Smith. Khim. Chim. 11. 9. W. J. P.510. Robison and B. 1946. L. clindamycin [ 7(S)-chloro-7- OH X i =H Xi = CsH. Heman-Ackah and Edward R. Medd. (6) (a) W. Russ. Julia and P. Albert. Butler. A. Ghein. Adler and A. University of Florida. (c) halogen substitution of the 7-(S) configuration of lincomycin molecule which potentiated antibacterial effects. Org. C. Williamson. Lorenz... Chem. 87. and F. Lincomycin was therefore modified’ chemically to serve as a basis for t h e understanding of structure-activity relations. Tegner. (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) Kinetics and Mechanisms of Drug Action on Microorganisms. B. Part 1. 27 (1968). Henry. 1221 (1968).. ibid. The generation rate constants of 11. (2) T. Chaykovsky. (3) (a) F.. Med. and A. V. and P. A. (7) (a) M. However. and J. J. 4 . 5516 (1964). 25. Scand. 1353 (1965). 153 (1966). analogs of enhanced potency and broadened antibacterial spectrum were prepared.2032 (1965).Pleininger. 1972. Received June 1 1 . Org. Robison. have only one phase of steady-state generation.. M. 70. Wibaut. Soc. (b) Merck and Co. Thus. J.. clindamycin (11) and U 24729A (111). (9) (a) L. College of Pharmacy. VoE.M.. Robison. Obshch. Acknowledgment. Advan. P. (b) W.. B.. M. (4) W. Robison. Rubtsov and L. and (d) t h e need of CH IR I.(b) A. coli suggest t h a t t h e unprotonated fraction of t h e drug concn contributes t o the activity. Shirley and P. 11. Ph. R. Chem. We are grateful t o Drs. SOC. 7 8 2 (1952). E. 65.79. Robison. N. Willette... Rubtsov. J. Zhur. D. 37. D. H.. Chem. Robison and B. Chem. H. Burger. SOC. W. 5686 (1957).5 times as active as I calcd on t h e basis of molar equivalency.. E. Red. 5. Authors Certificate USSR No. Ya. C. B. V. and U 24729A against Escherichia coli Samuel M. J. P. E. Mich. Steck and R.” Vol. Saxena. Heterocycl. (b) L.J. Chem. Roussel. M. Robison. Amer. Ber. X L -. 30. J Org. M. and D. F..6 times and I11 is 28. creased lipophilicity and t h e activity.. 77. 4 8 0 (1963). I971 Escherichia coli cultures in b r o t h affected b y sub-completely-inhibitory concns of lincomycin ( I ) exhibit 2 phases of steady-state generation while those affected b y t h e 7( S)-halogenated compounds. Edward Publishers. Robison. 6 . Koelsch.2573 (1957). 6 . Van der Voort.H I1 U24729A [ 1’-demethyl-rt‘-de- propyl-rt’(R) and ( S ) n-pentylclindamycirl R. References (1) (a) R. Med..~IO. (c) H. R. De Jonge. Chem.. Chem. However. 13711 (1966). 11. 2 Heman-Ackah and Garrett 34. =CI . I1 is 6. = C5H.. Comparative Studies on Action of Lincomycin. and M. Pays-Bas. The combined actions of I1 and I11 are not antagonistic at a n y level of activity and can be quantitatively predicted from the separate equivalent dose-response curves of either drug. 70. 1. Anthony. F. Rev. W. “A Survey of Antimalarial Drugs. Khim. (b) M. N. C. N. M. ibid. Boykin. Osdene. J.. N. Amer. Bader and W. 1054 (1951). 4 5 9 (1967). Gainesville. D. Chem. Lutz. 13.551 (1968). Chem. J. Tullar. The main structural effects claimed for in vitro activity’ were (a) t h e variation o f the alkyl substituent a t the N‘ a t o m o f t h e pyrrolidine nucleus w h c h changed t h e antibacterial spectrum of activity. 127 (1954). R. Chem. Chem. 431 (1967). Robison. and Leo Rane.H. S. Florida 32601. R. Chem. Med. 2531 (1965). V. 1553 (1960). L.. E. 162535 (1964): Byull. C... Oroshnik. xj = kl x . Scott. 79. Chem. No. J. pp. Amer. Lyttle. Soc. 28. can rationalize these phenomena. L. L. Strube of Walter Reed Army Institute of Research for suggestions and encouragement in this work. F. Whalley. Gen.J. Norsk. Soc. Soc. Yakhontov. H. Soc. = H x*= c1 R. P.. Yakhontov and M. Netherlands Application 6. Selsk. 66.

acs. 1972. Heman-Ackah. Chem. clindamycin.org/10. 1155 Sixteenth Street N. 152-163• DOI: 10. Garrett J.W.Kinetics and mechanisms of drug action on microorganisms. Med.. and Edward R.. DC 20036 . 2009 More About This Article The permalink http://dx.doi. Washington. 15 (2). and U 24729A against Escherichia coli Samuel M.org on April 25. 13.1021/jm00272a009 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. Comparative studies on action of lincomycin.1021/jm00272a009 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.

Mich.J. Chem. The generation rate constants of 11. Med. Comparative Studies on Action of Lincomycin. K. 9. Org. Amer. Lincomycin (I) is an antibiotic produced b y Streptomyces ‘9’ It has an antibacterial spectrum similar t o ~>~ t h a t of erythromycin and was claimed t o be ~ u p e r i o r because o f its effectiveness against b o t h erythromycin-susceptible and -resistant strains of Gram-positive coccal organisms. (9) (a) L. A. Chem. Nickel... Chim. B.J. 2 Heman-Ackah and Garrett 34. (3) (a) F. W. Amer. 2531 (1965). V. J Org. S. 1946. Robison and B. Anthony. 1964. Chem. 70. College of Pharmacy. Robison and B. Ya. R. J. Garrett* The Beehive. H. and U 24729A against Escherichia coli Samuel M. Lorenz. 1353 (1965). W. Chem. X L -.. Robison.M. Soc. (4) W. C. (b) W. J. 70. 6 . L.. Thus. No. Burger. Boykin..75. Roussel.5 times as active as I calcd on t h e basis of molar equivalency. Lincomycin was therefore modified’ chemically to serve as a basis for t h e understanding of structure-activity relations. Ghein. ibid. combinations of I1 o r 111 with I show antagonistic effects which depend o n the level of activity. creased lipophilicity and t h e activity. 1553 (1960). N. 13711 (1966). (8) (a) M. (b) L. R. 163 61959). Medd. pp. Chem. B..459 (1967). Yakhontov. Chem. J. = H x*= c1 R. Zh. and (d) t h e need of CH IR I. Florida 32601.. 11. SOC. 1972. Patel. A... Acta Chem. P. However.. (b) J. Advan. Corey and M. Tullar. A. Chem.2626 (1964). D. Y. Robison.. P. T. E. Scand.. (2) T. Wiselogle. 1. Chaykovsky. 11. Rubtsov.152 Journal of Medicinal Chemistry. ibid. Heterocycl. Russ. Smith. geterocycl. J. E. Amer. Med.. The main structural effects claimed for in vitro activity’ were (a) t h e variation o f the alkyl substituent a t the N‘ a t o m o f t h e pyrrolidine nucleus w h c h changed t h e antibacterial spectrum of activity. L. Jackson and A.Indian J. P. M. V. E. (5) M. R. lincomycin) allosterically modifying t h e receptor site for the other lincosaminide antibiotic. 25. Osdene. We are grateful t o Drs. Butler and B. 87. R. M. it was subsequently t h a t there was a “dissociated type” of cross-resistance in Staphylococcus aureus between erythromycin and lincomycin. N. C.J. 27 (1968). (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) Kinetics and Mechanisms of Drug Action on Microorganisms. Lutz. (c) M.. De Jonge. Koelsch. 87. G. Scott. Steck and R. Soc. University of Florida..79.(b) A. G . 81. W. 6554 (1955)... Yakhontov and M.. B. Chem. Oroshnik. C. Rev. 743 (1959). Heman-Ackah and Edward R. xj = kl x . Rubtsov and L. Amer. F. Amer. and M. D. =CI . P. Chem. I971 Escherichia coli cultures in b r o t h affected b y sub-completely-inhibitory concns of lincomycin ( I ) exhibit 2 phases of steady-state generation while those affected b y t h e 7( S)-halogenated compounds. M. Selsk. J. Ph. 1054 (1951). 10. Khim. Received June 1 1 . 5. H. References (1) (a) R. Albert. 127 (1954). and I11 against E. clindamycin [ 7(S)-chloro-7- OH X i =H Xi = CsH. Zhur. “A Survey of Antimalarial Drugs. and F. M. The possibility that lincomycin blocks 2 separate sites in a metabolic sequence whereas the 7(S)-halogenated compds block only t h e latter in t h e sequential biochemical process. H.. The effects of changes in pH o n t h e activity of I.. B. ibid.551 (1968). L. Clindamycin. The combined actions of I1 and I11 are not antagonistic at a n y level of activity and can be quantitatively predicted from the separate equivalent dose-response curves of either drug. Julia and P. and Leo Rane. analogs of enhanced potency and broadened antibacterial spectrum were prepared. Chem.. Pays-Bas. F. N. A. Org. and D. USSR. Acknowledgment. M. Chem. (7) (a) M. Heinzelman. Obshch.. = C5H. These can b e rationalized by t h e one (e. Wibaut. 15. 375 (1953). J. Chem. Amer. Butler. Whalley. A. 73 (1967). Soc. Williamson. Robison. Henry. Bader and W.J. Robison and B.o r 111-affected cultures have t h e same functional dependencies on drug concentrations as lincomycinaffected cultures in phase I b u t are different from those of I in phase I1 generation. J. 457 (1955). Archer. Med. and S. Ber. 11. Chem. No. 65. Trav. VoE. L.H. (6) (a) W. Duncan. Van der Voort. Soc. Izobret.. 79. (b) M. M. Szmuszkovicz. Chem. 4.2032 (1965). J.. 34. Part 1. 431 (1967). L. I1 is 6. 153 (1966). and P. lincomycin Ri = C H j R2 11. Saxena. ibid. Soc. Robison. Soc. Robison.Pleininger.. 2833 (1962). J. However. Yakhontov. R. 77. Willette. 66. Rubtsov. SOC. Edward Publishers. Shirley and P. R..g.. 1651 (1954).. 6.. J. M. D. 81. Russel. 5686 (1957). A. (c) H. deoxylincomycin] Ri CHs Ra C. Tegner. 28. clindamycin (11) and U 24729A (111).480 (1963). Robison.~IO. Netherlands Application 6. 7 8 2 (1952).” Vol. P.2603 (1964). Norsk. E. J. V. J. H. Ann Arbor. Strube of Walter Reed Army Institute of Research for suggestions and encouragement in this work. (c) halogen substitution of the 7-(S) configuration of lincomycin molecule which potentiated antibacterial effects. Lyttle. V. J. Chem. (b) Merck and Co. Gen.2573 (1957). P. 162535 (1964): Byull. 906-916. N. Uritskaya. W. Colwell. P. C. R. Chem. and J. Abstr. L.H I1 U24729A [ 1’-demethyl-rt‘-de- propyl-rt’(R) and ( S ) n-pentylclindamycirl R. Chem. (a) E.648 (1966). 1221 (1968). Yakhontov. have only one phase of steady-state generation. W. Authors Certificate USSR No. Gainesville. 1247 (1956). 37. (b) L.510. N. Chem. E. Chem.. Adler and A. Khim. 77. T. Otto.. 5516 (1964).8016 (1967). 30. (b) t h e increase in size of t h e alkyl group a t the C-4’ i n t h e pyrrolidine nucleus which in- Zincolnensis.6 times and I11 is 28. coli suggest t h a t t h e unprotonated fraction of t h e drug concn contributes t o the activity. Farm. Chem.. Red. 13. and A. Robison. Abstr. F. can rationalize these phenomena.

00 ml were withdrawn a t 20-min intervals from the cultures. possibly peptidyl "transferase" which appears to be an integral part of the 50s ribosomal subunit and is also the binding site for the amino acylIn contrast." Samples of 1. The settings were: aperture current of 5.and dru affinity constant.5 ml was then dild 100-fold into fresh medium.5' for 20 min with intermittent shaking.28 105k. Effect of Antibiotic Concentration on Generation Rates Fresh solns of the respective antibiotics were aseptically prepd for each experiment.55 0 62. They were dild to obtain counts within a range of 10.97 i.06 25 0 300 80 6.9 1 22. constant temp water bath equipped with a shaker.00 39. is in ml/pg sec. various amounts of Millipore-filtered 1." a factor which promotes the movement of a peptidyl tRNA elongated by a single amino acid residue from its acceptor site to a donor site.HC1. and 0 to 4 pg/ml of III.63 10'kbc 1.69 100 5..5' in an incubator.0 29. amplification of 8.' The sequence of events occurring during protein synthesis" and the steps which might be inhibited by antibiotics have been discussed by Cundliffe and McQuillen.77 3. They were sufficiently dild so that aliquots of 0. No.07 40 46.33 17. respectively.62 3.14 68.66 5.5".5-ml culture vols yielded the desired drug concns (Table I).1' in a 50-gal.HC1 111.37 0 61. The instrument was equipped with a 30-p orifice.Lincomycin Antibiotics Journal of Medicinal Chemism.2.. colilml in drug-free and in sub-completely-inhibitory. Model B (Coulter Electronics Co. colr In Various Concentrations of Lincomycin and Its Analogs at pH 7. z where Cis from 0 to 100 pg/ml of I.23 200 7.5 44. Bacto Antibiotic Medium 3 (Difco Laboratories.62 7. Total Count Method. and III.7 N HC1 and 2 N NaOH. 0 to 20 pg/ml of II..05 at 37.0 40.HC1. other workersI5 have claimed that I binds to "translocase. Kalamazoo.39 24. Florida).k.21 60 33.kap ) YS. in fact. The apparent conflict may be due to an attempt to correlate the results of two distinctly different experimental tests:I6 (a) the puromycin reaction which is specific for detecting an inhibition of peptide bond formation and (b) the guanosine triphosphate (GTP) dependent G-factor catalyzed reaction for determining release of tRNA.63 0.5 ml) of the "seeded" broth were then aseptically transferred from the Calab pourer into replicate loosely capped erlenmeyer flasks. ClindaLincomycin. and Other Derived Constants for Generation of E.O. . C in accordance with the equation: kapp = ko .'~~ Lincomycin is an inhibitor of bacterial protein synthesis. Vol.55 10. This method has been previously described. lower threshold of 13.5' 0.05 k0. The coincidence of total (Coulter count) and viable (colony count) numbers of E.HCl.05 with the exception of those that were used to study the antibacterial activity as a function of pH. concn.5" in the log phase at an organism population of about 1. Mielck and Garrett" observed from their study of I action by microbial kinetics that I.10 10 45.58 3. The solns were added to the cultures generating at 37.11 60 8. Hialeah. Samples were withdrawn every 20-30 min and counted by * Table I.) I. The media were filtered twice through Millipore 0.0 20.32 7.HCI (895 pg of base equiv/mg).67 pg/ml of II. Bacterial Cultures. Aliquots (49. of The Upjohn Co. Apparent First-Order Generation Rate Constants.0 49.01 50 10. Thus the translocation of tRNA from one ribosomal site to the other is presumed to be inhibited by I. The pH of the media was 7. coli ATCC 12407 (referred to as strain B/r in previous publications1s-*' were used in all experiments. 'Of ~ course. pg/ml Phase I PRase I1 pg/ml 10'kapp pg/ml 105kapp 0 62.96 14. and dild in the same way as the sample.50 9. K.HCl(838 pg of base equiv/mg).61 10. An inhibition of the latter reaction does not necessarily imply inhibition of the former reaction per se.18 40 14.. This communication presents the results of testing this hypothesis by microbial kinetics..HC1 or for C > 25 @g/mlIII. An aliquot (5 ml) of culture medium was inoculated from a fresh slant.20 51.45 p HA-filtered aq soln of 0. Jr.. bReciprocal of the intercept of a plot of C / ( k . gain of 10.0 14.0 X lo6E.03 0. The diluent used was a Millipore 0. Bkeciprocal of the slope of such a plot within the limits of footnote b in sec-I.5-ml Calab pourer.HC1 during phase I.26 2.HC1 duringphase I and for 0 < C < 350 pg/ml of I during phase 11." There was no significant evidence of kill superimposed on normal inhibition of generation in the presence of subcompletely-inhibitory concns of the drug. %e quotient of slope and intercept of such plot within the limits of footnote b in ml/pg. The total counts were corrected for the background count of the particular batch of media used.(Q for C > 100 pg/ml of equation: C / ( k . Mich. or for C > 16. C in accordance with the = l/ka +%b/k. G. but would cause peptidyl tRNA to remain in the acceptor site from which the peptide moiety could not be removed by puromycin. 1 m1/100 ml of broth) was added to a bulk amount of broth contd in a Pyrex flask fitted with a 49. 15. kilpp in sec-'. Culture Media. The generation of the culture was followed up to 2 X lo' E. YS. . The similarity in chemical structure of I. A sample of 0.96 68. in accordance with derivations for eq 3.. Mich.5 ml added t o 49. The slants had been prepared from a single colony and were stored in a refrigerator at 4". The flasks were maintained at 37.43 10' ka/kbd 68. Whitfield. or the product of drug partition constant.000-30. and I11 implies that the latter 2 analogs should have mechanisms of action similar to I but with differences in intrinsic bjolog ical activities." I has been reported to inhibit peptide bond formation by competitive binding on a ribosomal site.80 34.HC1 mycin.06 13. To obtain media with a pH in the range of 5. and upper threshold at maximum. it is possible that the action of I and the 7 4 s ) halo analogs might show kinetic parameters of different functional dependencies from that of either drug alone. Replicate slants of E. Detroit.45 p HA filters and autoclaved at 120' for 15 min. so that the combined action of I with such analogs should be quantifiable on a kinetically equivalent basis as has been shown for chloramphenicols" and sulfona m i d e ~ . and where kc is in ml/pg sec.64 5 53. If this were the case. lincomycin-treated cultures had been previously demonstrated.85%NaCl and 1% CH.0 9. The references to concns of drugs throughout this paper refer t o these samples of antibiotics.00 105kab 0.50 30 2. 2 153 maintaining the LY configuration of the thioglycoside to maximize antibacterial activity.0 8.k . Clindamycin (11) and U 24729A (111) are 7(S)-C1 analogs which are claimed to be more than 4 times as active as the parent antibiotic (I)* against a variety of Gram-positive and Gram-negative organisms.70 20 32. were added to the culture media aseptically before the sterilization.) was rehydrated according to the specifications of the manufacturer to peptone broth USP. 1972.82 70 8. colilml. and the culture was allowed to grow for 12 hr at 37. The "seeded" broth was kept in an incubator at 37. The background counts in general did not exceed 1000 counts per 50 pl.HCI 105k.75 15. Antibiotic.HC1 (1000 pg of base equiv/mg) were supplied by courtesy of Dr. Assayed samples of I.30 15.0 54.57 0.72 80 28. .66 10.15 350 2.. possessed 2 modes of action which they attributed to (a) an impairment in the functioning of the tRNA by binding of the drug to the 50s ribosomal site and (b) a possible interference in the synthesis and utilization of a stored metabolite.a 1.04 53. 1 1 .06 30 20. K.50 13.24 150 15. where k. B. An aliquot of this culture (Le. it is possible that the enhanced antibacterial activity' obtained by halosubstitution at the 7 4 s ) configuration of I in the order C1< Br < I may be associated with stereoselective binding at a different site.87 24. II. colilml.000 counts per 50 pl on the Coulter Counter. Experimental Section Organism.96 'Talcd as the slope of a plot of k.34 100 6.1-8.

7. Coulter counts were obtd from samples withdrawn every 20-30 min. were maintd at 37.5 ml) were added to replicate cultures to achieve the desired concns of antibiotics.5' and pH 7.HC1 on a wt/wt basis. Drug solns (0.5" until the organism population had reached 10' E.5-m1 vols of each pH constant broth. No.5'. k.HCl is presumed to be 25 times as potent as I*HCl(phase I) on a wt/wt basis. I S . set contd no drug.HCl and lincomycin ' HCl fractions. coli in steady-state generation were treated . 6 replicate 49. coli at pH the apparent generation rate constants.HCl is presumed to be 4. Vol.HC1 is presumed to e ! 6 times as potent as 1-HCl (phase I) on a wt/wt basis.05 and 37.154 L Journal of Medicinal Chemistry. Similar experiments were performed for 0-100 pg/ml of II. (a) II.HCl fractions. Effect of varied (a) clindamycin .05 were obtd (Figure la). coli culture in the log phase of generation.20.HCl and 0-13 pg/ml of 1II. Generation rate curves of E.HCl and 11' HCl fractions in equipotent mixts at 3 different potency levels on (sec-I) of E. (b) III. 2 Heman-Ackah and Gawett a la ' 40 - 1 lo/ 100 50 80 IOOX Lincomycin 20 0 % Clindomycin 50 Composition of Equipotent Mixtures I 80 60 Drug d 1 0 0 50 200 300 400 Minutes lo9 b I 1 0 0 80 50 50 80 20 i 0 O X Lincomycin 0 % U24729A I Composition of Equipotent Mixtures /fDruq added 106 0 1 0 0 200 Minutes 300 400 lo Figure 1. The 6th replicate in each Figure 2.5-m1 samples of cultures contg 106/mlE.HCl. (b) III.16 times as potent as II. (c) III. Action of Equipotent Mixtures Composed of Different Fractions of Lincomycin and Its Analogs. Effect of pH on Drug-Affected Generation Rates. and (c) 1II.)in absence of drug.05 in the absence and presence of graded (a) lincomycin. inoculated with E. One culture without drug was studied in each experiment as control to obtain the generation rate constant (k. OO 100 i 20 80 50 50 80 20 1 0 0 VO Clindamycin 0 % U24729A Composition of Equipotent Mixtures the Coulter method. coli at 37. Sufficient amts of 1 N HCl and 2 N NaOH were added to broth to obtain pH values 6. 1972. The curves are labeled according to the drug concn in pg/ml. colilml. Replicate 49.HCl and I.10-8. The generation curves for 0-300 Mg/ml of I-HCl at pH 7.HC1 and (b) clindamycineHC1 concns.

L.67 pg/ml) is added to the phase I lincomycin-affected culture of curve C. HCI (4 pg/ml) is added to the clindamycinaffected culture of curve C. The resultant generation curves for the action of equipotent concns of 16.60. 3.0.HCI(l6.34 pg/ml of 11-HClis added to the culture of curve A and curve H is generated when equipotent 200 pg/ml of I .HCl(100 pglml). 175 min after the addn of I in Figure 5 5 other replicates were treated with similar concns of the clindamycin (curves I. Effects of Clindamycin and I11 on Lincomycin-Affected Cultures in phase I and Phase I1 Generation. Equipotency of action is shown by coincident or parallel generation curves of the drugaffected cultures having the same generation rate constant. The resultant generation is given as curve D.5-m1 samples of cultures contg 106/ml of E.5 ml) of graded concns of I1 were added t o each of 5 replicates so that the total antibiotic concn maintd in the cultures would be a priori 1. E.HCl (Figure 3b). J 200 Minutes 1 0 0 300 0 1 0 0 Minutes 200 300 Figure 3.\ 0 W lo7 /0p DO*. Again. and M). coli in steady-state generation (curve A in Figure 4) were treated with aliquots (0. coli in steady-state generation (curve A in Figure 3a) were treated with aliquots (0.HCl(4 pg/ml) and II.0. K. Fifty min after the clindamycin-affected culture of curve B had settled to a new steady-state generation. The separate effects for equipotent concns of I1 and I (phase I) on the generation of cultures are shown. Curve G is generated when 8 pg/ml of IIIeHCl is added to the culture of curve A or the coincident curve H is generated when equipotent 33. 10. Replicate 49.67 pg/ml of II. No. When the lincomycin-affected cultures of curve Chad settled to steady-state phase I generation. added to the culture of curve A. Coulter counts were obtd from samples of the cultures withdrawn every 20-30 min. aliquots (0. Replicate cultures of curve A were also treated with aliquots of a mixt of equal parts of the equipotent concns of I1 and I (curve F) which was prepd to be a priori as equipotent as the corresponding I1 (curve G) or I (curve H) alone. 16.67 pg/ml) added to the culture of curve A. G . Curve B is for generation of culture in the presence of 4 pg/ml of II.Lincomycin Antibiotics Journal of Medicinal Chemistry.HC1 and lincomycineHC1 and (b) of equipotent III.HC1 or for equi- potent concns of 16. I and 111 (Figure 2b). 1972. and 200 pg/ml of I. 2.0.20. The r e sultant generation curves are given as D. Coulter counts were obtd on samples of the cultures withdrawn every 20-30 min. Curve B is for generation of culture in the presence of 16.O with NaOH at an ionic strength of 0. 2. Curve D is generated when equipotent 1. Curve F is generated for a mixt of equipotent concns of III. J. The experiment was repeated in like manner for equipotent concns of 4 pg/ml of III.5" on a Radiometer automatic titrator Model 111 Lc-SBR 2c-SBUI. The effects of I11 on lincomycin-affected cultures in phase I and phase I1 generation were detd in a similar manner and sequence (Table 11).HCl is added to the culture of curve A. when the cultures of curve Chad entered into steadystate generation phase 11. Effects of order of addn of equipotent (a) clindamycin. i.HC1 and 100 pg/ml of I. respectively. HCI (100 pg/ml) is added to the clindamycin-affected culture of curve B and curve E is generated when equipotent 11.67 pg/ml of II*HCl. Effect of the &der of Addition of Lincomycin and Its Analogs in Combination on Microbial Generation.67 pg/ml) is added to the 111-affectedculture of curve B or the coincident curve E is generated when equipotent 111.67 pg/ml of 11-HCIand curve C is generated when in the presence of equipotent 100 pg/ml of 1. A similar treatment of the lincomycin-affected culture of curve C with equipotent amount of I1 resulted in ultimate generation given as curve E.and 4 pg/ml of III. respectively. Cultures were treated with equipotent mixts of I and I1 (Figure 2a). 80 min after addn of I in Figure 4. Curve D is generated when equipotent II. and 3. and 0% concn of 1 antibiotic at 1 dose level and the residual percentage of equipotent concn of the other antibiotic to maintain the anticipated equipotency at that dose level.34 pg/ml of 11-HC1is added to the culture of curve A. respectively. The equipotent concns were considered as 100 pg/ml of I'HC1. Determination of the pKa' Value of 11 and 111. with aliquots (0. 0 .5 ml) of equipotent solns of I and its analogs. .67 pg/ml) and I.67 pg/ml of II. Curve F is generated for a mixt of equipotent concns of II. as curves B and C in Figure 4.HC1 and 11-HClon generation rates of E. I1 and I11 (Figure 2c). Le. Vol. 100. I S .5 ml) of solns of I and its analogs and in combinations thereof. HCl(l6.HCl. Coulter counts were obtained from samples of cultures withdrawn every 20-30 min and apparent generation rate constants (ka p) detd.90. coli (a) Curve A is for generation of culture in the absence of drug. (b) Curve A is for generation of culture in the absence of drug.' values of I1 and I11 were detd by titration in H.2. Replicate 49.67 pg/ml of IIeHCl. The pK.5 ml) of equipotent mixts of antibiotics. These studies were repeated at levels of activity corresponding to the equipotencies of 50.HCl and 4 pg/ml of III.HCl(l6.50.5-ml samples of cultures contg 106/ml of E. respectively.HCl(16.80. F. 2 155 E .e. Curve G is generated when 33. and H. an equipotent amount of I was further added.5 times as equipotent as the concn of the I initially present in the culture (Table 11).HCl or the coincident curve C is for generation of culture when in the presence of equipotent 16..HC1..40.HC1 and 100 pg/ml of I*HCl(phase I) are given as curves B and C. Curve N is for the effect of a mixt of equal parts of the equipotent concns of 11 and I which was prepared to be a priori as equipotent as the corresponding I1 (curve 0) or I (curve P) alone. The mixts consisted of 100.122 Mand temp of 37.

5 ml of fresh broth.67pg/ml are.5 ml of a soln of II.5 ml of fresh broth. kapp is the generation rate constant for culture af- .HCI and 8 pg/ml of III. concn (Table I) is given in Figure 7.34 pg/ml of II. respectively.HC1 (16. in broths contg enough I. Vol. N of E.HC1: 16.5 ml) of a drug-free culture in the log phase of generation and contg 10' E.HC1 to achieve a final concn of 33. so that the organism population was dild 10-fold (curve A' in Figure 6) and 100-fold (curve A" in Figure 6).5-m1 vol of broth contg 10' E.HCl(lOOpg/mI) added t o the culture of curve A. respectively.k c C Results Shape of Generation Curves for Drug-Affected Organisms. time.Curves D. in accordance with the equation In N = kappt t In No (1 1 Effect of Antibiotic Concns on Generation Rates. Coulter counts were obtd on samples withdrawn every 15 min. At the same time. when concns of II.HC1 respectively.5 ml) were added to 45 and 49. The ka values are linearly dependent on drug concns up to l o r p d m l of I. aliquots (5 and 0.67 pg/ml) and I.67pg/ml of II*HCIand curve Cis for generation when in the presence of equipotent 100 pg/ml of I*HCl.0. coli/ml (curve A in Figure 6 ) were added t o 45 and 49. 20. coZi/ml vs.HC1 so that drug concn was restored to 16. The curves for this latter compound were similar to those for clindamycin. so that both the organism and drug concns were dild 10-fold (curve C in Figure 6) and 100-fold (curve D in Figure 6).156 Journal of Medicinal Chemistry. from coulter counts. No. respectively. and 4. 2 Heman-Ackah and Garrett 1 1 0 0 200 300 400 500 Minutes Figure 4. respectively. and 111. and 41. are given for graded concns of I (Fig ure la).HCI is added to the culture of curve A. Curve A is for generation of culture in the absence of drug.G . Lincomycin exhibits the characteristic 2 phases of steady-state generation previously reported" while I1 and I11 have only 1 phase of steady-state generation. E. When the clindamycin-affected culture of curve B was in a steadystate generation and had reached an organism population of lo' E. 16 pg/ml of I1 . 33. obtained (2) where ko is the generation rate constant for the drug-free culture.34. Curve 0 is generated when 33. 25. Reversibility of Action of I and Its Analogs.0 p d m l of III. Aliquots (5 and 0. Curve N is for a mixt of equipotent concns of II. I1 (Figure lb).34 pg/ml (curve B in Figure 6). 15. The plots of log of numbers of E. Curve B is for generation of culture in the presence of 16. The experiment was repeated in like manner and sequence for cultures affected with equipotent concns of 200 pg/ml of I. in accordance with the expression kapp = k o .HC1. A plot of kapp us. The apparent first-order generation rate constants (kap in sec-') are obtd from the slopes of the linear portions o f t h e plot of log of numbers. F. coli per milliliter against time. respectively. and H are for generations of phase I lincomycin-affected cultures of curve C. added after 80 min of initial addn of the I. coli/ml.67. 1972. t. A 49.HC1 (phase I growth).17 wg/ml. Demonstration of antagonistic action of I1 on phase I generation of lincomycin-affected cultures. aliquots of the culture of curve B were dild 10-fold (curve E in Figure 6) and 100-fold (curve F in Figure 6).HC1 is added to the culture of curve A and curve P is generated when equipotent 2OOpglml of I. colilml in log phase generation (curve A in Figure 6) was treated with 0.0.HC1.

nonlinear decrease of the ka with drug concn is observed for I in phase I1 generation tiroughout the concn range studied.HCl= 4.HC1 (phase I).34. CAt 175 min after the initial addn of the I.HC1 is added to the culture of curve A. C. C / ( b . Curve B is for generation of culture in the presence of 16.67 pg/ml of II.67 4. K. d l O O Mg/ ml I. Concentrations of Clindamycin' HCI or III.0 E J 20.HC1 and curve C for generation of culture in the presence of equipotent 100 pg/ml of IsHCl.25. Figure 8 gives a plot of C/(ko vs. IIeHCl.0.00 4. estimated in accordance with eq 2 is approximately of Thus the order 1:6:25 as I. and III. Curves I. bAt 80 min after the initial addition of I. On the other hand. 1972.HC1).kapp = c(kb/ka) + 1 /ka (3) where ka and k b are constants of proportionality related to drug availability in the biophase and drug affinity for receptor or binding sites.HCI (100pg/ml) added to the culture of curve A.0 H M 41. Curve A is for generation of culture in the absence of drug.5 G L 33. Demonstration of antagonistic action of clindamycin on phase I1 generation of lincomycin-affected cultures.34 8.HC1 is added to the culture of curve A. concn of the drugs (Figure 7) is obtained by multiplying the actual concns of 11-HCl and 111-HC1. (in ml/pg sec) is the inhibitory rate constant for the drug.34 pg/ml of II. 11. . 20.2 F K 25.0 pg/ml of III.8 2. and k. and 111.5 OAs indicated in Figures 4 and 5. L.HC1 Added to Phase I and Phase I1 Generations of Cultures Affected by 100 pg/ml of Lincomycin' HCI Concn (pglml) of analogs added to phase Ib and phase I I C lincomycinFinal relative equipotent Reference affected cultures concnd of total antibiotics CUNeSa Clindamycin I11 added a b c c 1. Vol.67 pg/ml are. added after 175 min of initial addn of the lincomycin.HC1 in the plots of Figure 8 in accordance with eq 3 makes the plots coincident.67. 15. Curve 0 is generated when 33. Above these concn ranges. Curve N is for a mixt of equipotent concns of II.in the data of Table I by factors of 6 and 25. respectively.0 2.67 pg/ml) and I.kWp) Action of I. No. respectively. Applicability of Saturable Receptor Site Model to the .Lincomycin Antibiotics Journal of Medicinal ChemistTy. The relative potency of the drugs on a weight basis.and 41. Adherence to the model is observed from the I" 0 1 0 0 200 300 Minutes 400 500 Figure 5 . Similar consideration of the potency estimates for I. coincidence of the plots of kaqp vs. and M are for generations of phase I1 lincomycin-affected cultures of curve C when concns of II.0 D I 16.HCI= 16.0 2.0 3. 2 157 fected with drug concn. C in accordance with a p r e v i o ~ s l y " ~derived '~ saturable receptor site model Table 11.HCI (16. and curve P is generated when equipotent 200 pg/ml of I.HC1 (phase I)-II~HCl-III~HC1.67 pg/ml of II.0.HC1: 16. the curves slowly approach asymptotes.67 10.0 3.33.00 6. J.

Application of saturation kinetics to the actionof 1. coli at 37. .34 pg/ml of II. HCl. and 111. HCl (phase I) > 100 pg/ml. C x f l p g / m l ) I I00 ""Minutes 300 400 Figure 6 .HC1 (curve C) on the growth of E. the closed triangles are for concns of II.67 pg/ml.0 linear plots obtained for concns of drugs greater than 100 pg/ml of I.HC1. C. for the effect of I. t to. 11. Curves E and F are after diln of the culture of curve B. coli cultures at 37. .HC1 at higher concns.0 for II'HCl.05 on equipotent concns.7 5. The values of the constants ka and k b calcd from the slopes and intercepts of such plots are given in Table I. 1 : l O and 1:lOOO with broth contg sufficient amts of II.*K. a n d f = 25.* is the intrinsic inhibitory rate constant of the unprotonated drug and K. 15. Curve B represents the application 'mens of I ' HCl (phase 11) at 0 < C < 350 Mg/ml. kap for E. Dependence of the log of the apparent inhibitory rate constant.HC1 to the culture of curve A to a final concn of 33. Curves A' and A" are after diln of the culture of curve A. = k. Vol. HC1.0 pg/ml of II. and wheref= 1.5'. 16.HC1. C. and f = 25. 2 t Heman-Ackah and Garrett I . 1:lO and 1:lOO with broth t o final drug concns 3. The values for I were obtained by Mielck and Garrett" at similar pH values using the same organism and experimental technique.HC1. II.HCl.The dashed line. and the . C. in the lower concn ranges. HCl (phase I). respectively.HC1 [phase I ] (curve A). coli at 37. Thus. Figure 8. respectively. The drug-free generation rate constants were invariant with pH within the range studied.HCl. {of I*HCl.10-8. Curve A is without drug.HC1.k. The curves are plotted from the data of Table I according to eq 3.f= 6. and where f = 1.'/(K.0 PH 7. on the pH of the broth medium. K l .34 pg/ml of II.HCl. on the apparent generation rate constants.HC1.HCl (phase 11) at 0 < C < 350 pg/ml.5' and pH 7. on drug concn at low drug concns.0 8. HCl.HCl. and 111. The drugaffected generation rate constants at a specified concn of each drug.' + [H+]).HC1 (curve B). larger amounts of a drug are required for the same . + 1 i 0 00 200 Concentration. the open circles are for 11. Curve A represents this application. 300 /" / I 4 - Figure 7. Semilog plots of reversibility studies of E. I (phase 11) adheres to eq 3 at all concns studied. Curve C represents the depenllnce of the kapp on concns. and 4 p d m l of III. respectively. so that the final drug concn is restored to 33.34 pg/ml.HC1 (phase I). ka. kapp (sec-I) of E.0 I 6. II.0 for I.II. and III. Effects of pH on Drug-Affected Generation Rates. The apparent first-order generation rate constants (kapp) were obtained at different pH values (6.HC1. decrease significantly with increased pH values. HCl.HC1> 4 Mg/ml. The drawn lines are consistent with the expression: k. closed circles are for III. C x f ( h q I ml I Concentration. 1972.0 for III*HCl. where the open triangles are for 1.158 Journal of Medicinal Chemistry. where the open circles are for concns of 1. where k. coli generation with time on addition of II. in all cases. 1 : l O and 1:lOO with broth.0 for 111. Curve B is after addn of II. Curve A represents this dependence. Figure 9. The k. demonstrates the linear dependency of k .HC1 and diln of the cultures with broth. is obtd from the expression: kapp = k. Curves C and D are after diln of the culture of curve B. E 5' 0 1 W - - 0 too zoo I 300 400 Concentration.5". of I . respectively.HC1 > 16. and the open triangles are for concns of 111.20) of the culture media in the absence and presence of graded concns of I.' is the dissociation constant for the protonated drug concn. curve B.34 and 0.f= 6 for I I . Deviations occur.0 for I.C/(1 + k&). Demonstration of the coincidence of the dependencies of the apparent generation rate constants. No. and III.

67 pg/ml of II.122 are 7. an equipotent amount of 11.5" and ionic strength of 0. &See ref 17. The effect is less striking at the lowest concn studied. The generation rate constants.HC1 (curve G). kapp. and I1 and I11 in Figure 2c.67% of its a priori potency.) + l/kw g a l c d from the quotient of the slope Zfyntercept of the plot according to the equation in footnote a. Whitfield. curve G has about the same slope as curve 0 or the initial portion of curve P.34 pdml of II. the order of addition of these 2 antibiotics produces no significant change on the ultimate generation inhibition.. the addition of I after 50 min to a clindamycin-affected culture of curve B. is obtd from the equation fitting the apparent generation rate constant.05 for I11 (Table 111). The value of 7.80-6. The slope of curve F is higher than that of curve G or the initial portion of curve H. J.768 7. The addition of graded concns of I1 to I-affected cultures in phase I steady-state generation (curve C) results in graded responses as shown by decreasing slopes of growth curves with increasing concns of I1 (curves D.80 and 6. 200 pg/ml of I. The constants. In both cases the order of addition of the antibiotics produces significant effects on the ultimate generation inhibition and shows antagonism of the action between I and I1 (or 111). G . The null slopes of the plots of the kapp for all the a priori equipotent mixtures of I1 and I11 (Figure 2c) demonstrate the indifference22 of effects. I and I11 in Figure 2b. CAsdetd by titration. and H have the same slopes. and H. pH however.76 has been reported for I. This unequivocally demonstrates antagonism22 of effects between I and its analogs in their combined action against E..HC1 with an equipotent concn Of 4 pdml of III.10.67 p (curve F) in Figure 3b and that of the a priori equipotent concn of either drug alone.00-7. This demonstrates antagonism of effects between I (phase I) and 11. and H). The equipotency of action for I1 (curve B) and I (curve C) is indicated in Figure 4 by coincident or parallel growth curves of cultures affected with the drugs. There are however. in all cases.67 pglml of II. A mixture of equipotent concns of I1 and I (curve N) is less active than the a priori equipotent concn of either the I1 (curve 0) or I (curve P) alone since curve N has a higher slope than curve 0 or the initial part of curve P. respectively.94 7. Curve N possesses some elements of the biphasic characteristics observed for I action alone.00 7.50 for I (phase I). Mixtures of I11 with I produce patterns that are similar to the effects of I1 on lincomycin-affected cultures. Also.HC1. (kapp) of cultures affected by equipotent mixtures of I and I1 are shown in Figure 2a. For example.HC1 (curve H) or 8 pg/ml of III.kaP) vs. respectively. B. i. 1972. cation from Dr. Thus.14 6. The determined pKa' values at 37. The mixtures were prepared so as to be a priori equipotent in their combined action on E. Le. 15. Vol. i. values at pH 5. On the other hand. from data of Mielck and Garrett. the data of Table I1 show that the total drug concn in the culture of curve G is about 1. However. L.5 times as equipotent as the II in the culture of curve 0 or the I in the culture of curve P. Curves D and E have the same slopes as curves F. of the drug-affected cultures at the hi er concns.00 1. No. There are however no significant differences in the effective inhibition of generation produced by the action of a d m l of 11-HCl and 4 pg/ml of III. C in accordance with eq 3 at different pH values are plotted as log ka against pH in Figure 9. The slopes of the plots of log ka vs. 6. G. This indicates that the combination of I and I1 in the culture of curve H has about 1/1." eReported by Herr and Bergy. as the pH approaches the respective pKa' value of the drug. There are no significant differences among the generation inhibition produced by the action of 100 pg/ml of IaHCl in phase I (curve C) and the equipotent concn 16.43 7.10-6. added after 50 min to a lincomycin-affected culture of curve C. ." Table 111. and M in Figure 5). The values of ka increase 10-fold for unit increase in the pH over the range 5.e.57d 7.2 fPersonal communi. The Upjohn Co. produced by an equipotent amount of the I1 added after 50 min to 111-affected culture (curve D) or by an equipotent amount of I11 added after 50 min to 11-affected culture (curve E).00 for I1 and 8.0f 'As detd from the slopes and intercepts of plots of l/ka vs.HC1 mixture of 16. ka. Antagonistic Action of I1 or 111 on Phases I and I1 Generation of Lincomycin-Affected Cultures.70 for 11. coli generation in accordance with Figure 7 at 3 different levels of action. dBased on k. C of drug.e.HC1 in phase I (curve H) or 33. Mch.. Values of pK. coli. G. significant differences in the effective inhibition of generation produced by the action of a mixture of 100 pglml of I. Kalamazoo. and 7. In both cases the antagonism of effects produced by combinations of I1 or I11 with the I on the generation of cultures appear to be greater in phase I1 than in phase I of the lincomycin action.HC1 and 16. I and I1 mixtures or I and I11 mixtures.HC1 (curve G). However. the ultimate steady-state generation rates of lincomycin-affected cultures in phase I1 are not sig nificantly changed by addition of graded concns of I1 (curves I. on the other hand. where k . there are no significant differences in the effective inhibition of generation. tend to lessen. 2 159 fractional inhibition of microbial growth as the pH of the medium is decreased. produces an ultimate steady-state generation rate (curve E) which is the same as that of the culture affected initially with I. PKa' of Lincomycin and Its Analogs.. derived from plots of c/(ko. where curves F. E.' and Intrinsic Inhibitory Rate Constants (ka*) for Lincomycin and Its Analogs Cited Kineticallyb ExperimentallyCliterature 105ka*a determined determined values of Drug (mlhgsec) PKa PKa ' PKa ' Lincomycin 1.. respectively.HC1 (curve F) and that of the a priori equipotent concn of either drug alone. C / ( k .k ) = C(kb/k. The ultimate generation rates represented by the final slopes of curves D through H are less than would be expected a priori from the additivity of equipotent concns of either drug alone and shows antagonism. Effect of the Order of Addition of I and Its Analogs on Microbial Generation. 33.79 8.34 pg/ml of II. . G.5 or 66.6e Clindamycin 7.Lincomycin Antibiotics Journal of Medicinal Chemistry. Jr. Also. [H'] in accordance with the equation: l/ka = l/ka*Ka'[H+]+ l/ka*. K. The mixtures consisted of 100 to 0%of 1 antibiotic and the residual percentage of equipotent amount of the other antibiotic.6f 111 125. produces an ultimate steady-state generation rate (curve D) which is the same as that of the culture affected initially with the I1 (curve B) but shows nothing of the I (phase 11) effect. This pattern of response is likewise observed for combinations of 100 p d m l of I. Effect of Equipotent Mixtures Composed of Different Fractions of I and Its Analogs on Generation Rates. exhibit lessened activity as indicated by higher kapp values than the equipotent antibiotic used alone (Figures 2a and 2b). F.60 for 111. This is most readily manifested at the higher potency levels.HC1 (curve B) in Figure 3a since the initial portion of curve C has the same slope as curve B.05 8.

4. 15. This suggests that the analogs have a mechanism of action similar to I (phase I). which does not occur with cultures affected by the other two analogs (Figure Ib). and [H'] is the H+ concn. equipotent mixtures of I (phase I) and I1 (Figure 2a) or I (phase I) and I11 (Figure 2b) demonstrate unequivocal antagonism. The corresponding molar ratio of potencies would be 1 :6. These apparent differences in potency can be attributed to differences in degrees of partitioning across the bacterial cell membrane and/or to differences in the affinities of the drug for the receptor site.*K. the I (phase 11) action is eliminated. On this presumption. Arithmetical transformation of eq 4 yields 1 k.160 Journal of Medicinal Chemistry.. the correspondingly diluted drugfree cultures showed a very short lag period. In the latter case. 11. The steady-state generation of lincomycin-affected culture changes after several generations of growth to a new steady state. The steady-state generation of the drug affected cultures reverted to new steady-state generation with predictable rate constants when diluted 10-fold and 100-fold.200 pg/ml of I. respectively in F i g ure 6 ) . However.05. Anomalous Antagonisms among Lincosamide Antibiotics. Equipotent mixtures of I1 and I11 are equivalent in their action against E.0. the order of addition of the drugs in a combination has no significant effect on the ultimate steadystate generation of drug-affected cultures (Figure 3b).HC1.HC1 to attain a new steady-state phase of generation.HC1 (curve B in Figure 6). 1972. [H']. which therefore demonstrates the lack of any significant bacteriostatic antagonism or synergism (by the definitions of Garrett22)in the sub-completely-inhibitory range similar to t f a t observed21 for combinations of I (phase I) and erythtomycin. and I11 have similar functional dependencies on drug concns (Figure 7). = ka*f = k. This suggests a similar mode and locus of action for the 2 drugs. However. The extent of generation inhibition by lincomycin (phase I). It took 15-20 min for cultures affected with equipotent concns of 33. 11.5. k. as one possibility. 20 and 2 pg/ml of I.34 pg/ml of II. Kat is the dissociation constant of the protonated base. These latter curves were practically coincident with those of I1 so they are not presented here. on concn for I (phase 11) suggests a different mechanism of I action (Figure 7) which dominates the latter stages of lincomycin-bacterial interaction.g. phase I1 (Figure la). No.' and a slope of 0 when Kat > [H'] (Figure 9).HC1 (curves E and F in Figure 6). before addition of the mixture to a culture is greater than that obtained by adding an equipotent amount of one drug after an interval of time to a culture pretreated with an equipotent amount of the other drug.HC1.* is the intrinsic inhibitory rate constant of the unprotonated drug. pH approaches a slope of unity when [H+] > K. 5-10 min to revert to their new steady states. pH Effect on Activity of Lincosaminide Antibiotics. and I11 against E. respectively. the new steady state was attained after an interval which was dependent on the dilution factor. CT I11 . when I is added to a culture pretreated with I1 or 111.6:28. The relative intrinsic activity of the uncharged drugs on a weight basis is estimated to be approximately of The ratios the order of 1 :5. but 60-80 min for the 1 : 100 diluted 11-affected cultures to attain the new steady states. Discussion The data reported in this paper show that the generation rate constants (kapp) of E. 2 Heman-Ackah and Garrett Reversibility of Drug Action.0:87. It is therefore concluded that eq 4 holds and that it is the unprotonated fraction of the drug concn that is responsible for the antibacterial activity of I and its analogs. 200 pg/ml of I. since the mixtures are less effective on the generation of E. it took 30-40 min for the 1:10 dilution. the similarity in functional dependenc. The functional dependency of the k . A reasonably good agreement is obtained between the experimentally determined PKa' values by potentiometric titrations for the proline rings of the drug species and the kinetically determined values from eq 5 . in terms of molar concns are for the uncharged drugs 1:5. Thus no significant difference is observed between the reversible action of I. e. coli cultures affected by I (phase I).4 as I~HCl-II~HC1-III~HCl. In contrast. Cultures inhibited by low concns of the drugs.67 and 0.8 and 0. coli. coli cultures than the a priori equipotent concentration of either drug alone. In addition.HC1 were further inhibited by addition of more drug to final concns of 16.c> of the kappfor cultures affected with I (phase I). and/or to differences in the intrinsic efficacies of the drug bound to the site.167 pg/ml of II. W+I1 + _1k. Similar lag periods were observed for corresponding dilutions of I-affected and 111-affectedcultures.67 pg/ml of II. that: I (phase I) may have a different binding site from that of I! (or 111) and hence their mechanisms of actionz2 are not t b same Therefore. the extent of generation inhibition produced by the combined action of I (phase I) with I1 (Figure 3a) or I (phase I) with I11 depends on the order of addition of the drugs to a culture. This antagonism of effects obtained by various combinations of I with I1 (or 111) suggests. 1.g. For instance.HC1. Yol.. 11.. f is the fraction of the drug concn unprotonated. respectively (curves C and D. For instance. or I11 increases as some function of pH values of the broth medium. and 8 pg/ml of I11 . 11.*2 Furthermore. the ratio of the biological potencies of these on a weight basis drugs are 1:6:25 as I~HCl-II~HC1-III~HC1 at pH 7. vs. (ml/pg sec) as defined in eq 3 for various pH values (Figure 9) adhered to the e x p r e ~ s i o n ' ~ -7 I k . The equilibration of I and its analogs between nutrient medium and biophase is readily achieved. Le. the ultimate generation inhibition produced by admixing an equipotent amount of I (phasc 1) with an equipotent amount of I1 or of I (phase I) with an equipotent amount of 111.2:94. the prolonged lag period observed for the drugaffected cultures t o attain a new steady-state generation upon dilution suggests that either the rate of dissociation of drug-receptor complex in the biophase is less than the rate of formation of the complex and/or the rate of diffusion of drug from biophase to the broth medium into the biophase through the cell m e m branes. The plot of log k. If the time required for rejuvenation of the cells or consequence of shock on dilution is considered. a n d 8 pg/ml of III.HC1.* Ka K a t + [H'] where. The constants k . ka* Values of ka* and PKa' of the drugs derived from the slopes and intercepts of linear regressions obtained by plotting l/ka vs. are given in Table 111. e.08 pg/ml of III.HC1. at the same relatively short equilibration time of 15-20 min. coli (Figure 2c) and their effects can be quantified on the basis of additivity from the separate doseresponse curves of either drug alone (Figure 7).

e. when no such catalysis exists and A and B compete for such sites.26-28 This can be expressed schematicallyZ9 by The equilibrium constant for the interaction of drug B with the free receptor site. can be inserted into eq 1 0 and 1 1. their inhibitory action differs only by a potency factor.. Vol. k ~ ’ 0. not reacted with either drug A or B is fR = 1 . neither of these 2 mechanisms can explain the observed antagonism between I (phase I) and I1 or 111.(A‘R)]/(A‘R) = kA/k-A = 1/KA ’ (9) Equation 8 can be rearranged to explicitly define the reciprocal of the fraction of receptor sites occupied by the drug B as and eq 9 can be similarly rearranged to define the reciprocal of the fraction of receptor sites occupied by the drug A as (1 1) Equivalent values may be obtained by solving eq 10 and 11 for R.R/(B’R) = B’[RT . (8) (A’)*R/(A’R) = (A’)[RT .(A‘R)] /(B’R) = (kB -t kB ’(A’))/k-B = 1 -t (~B’/~B)(A’)/KB’ and similarly for drug A. The dissociation of one receptor-drug complex. occupied by drugs A and B. such allosteric modifications should result in antagonism for such a combination. the fact that the separate action of each of these several antibiotics is relatively rapidly reversible on dilution denies any significant inaDility of either drug to reach or leave the biophase. l / e B = [ 1 t K A K (A) ~ t (~B’/~B)KA(t A) Kg’ [ 1 + (kB/ka)(A’)] metabolites Ikm (kB’/kB)KA) (KA(A))’ KA -t KBKB‘(B)I /KBKB’ (B) (1 5 ) and I/eA = for the competition of 2 drugs. No. on this presumption.Lincomycin Antibiotics Journal of Medicinal Chemistry. f ~ . Le. though different. nevertheless. Certainly.e. dM/dr. these phenomena would be more readily understood if the one drug inhibited the transport of the other to the receptor site where both acted. respectively. if the separate sites were engaged in “sequential” or “convergent” metabolic proce ~ s e which s ~ ~ lead to a common end product. However. Such would be the case. in effect.(B’R) . the fraction of receptor sites.6B = 1/(1 t KAKA’(A) t KBKB’(B)) (18) Thus the ratio of the fraction of free receptor sites. A Possible Kinetic Model to Rationalize Antagonistic Action. Thus.6 ) . 15. 1972. 2 161 on drug concns could be attributed to the fact that their binding sites. the combined action of I1 (or 111) with I (phase I) would either cause a “sequential blocking”24 of the metabolic pathway to produce “synergism” or cause a “concurrent blocking”2s to produce “additivity” (or equivalence) of effects. can be implicitly defined in B’. should be higher from the combination than would be predicted on the basis of drug equivalency.6A . and since (13) 1/6A = RT/(A’R) = 1/KA’(A’) t 1 t @%)/(A%) (A’) = KA (A) (B’) = KB (B) (14) where (A) and (B) are the respective concns of the drugs in the media. &B ’ / k)KA ~ (A)( 1 -t KAKA’(A))1 fR = 1 . that results in generation is proportional to the fraction of these unreacted receptor sites as per dM/dr = k m ( l . i. (B’R) is additionally catalyzed by the available concentration. RT. change its apparent partition coefficient K 1 or apparent activity.(B’R) . (7) where 6A and 6 B are the fractions of total receptor sites.6B = [I (kB’/kB)KA(A)]/[l +KAKk(A) +KBKB’(B)+ (17) which equation. This fact makes it difficult to accept the conjecture that the presence of a molecule of one drug may sterically block or selectively inhibit the entrance of a molecule of the other to the biophase and. i. R. A and B for the same receptor site. One possibility for rationalizing these antagonisms is that the presence of one drug quasipermanently modifies the allosteric configuration of the receptor site so that it diminishes the effective affinity constant of the other drug for that receptor site without significantly affecting the receptor site-substrate interaction necessary for microbial growth and generation. (A’R) and (B’R).. However.g. eq 7. R. fR. Thus.6A . The (A’) and (B’) values are the drug concns in the biophase in equilibria with the respective extracellular (A) and (B) concns and the respective concns of bound receptors. It should follow that the number of occupied receptor sites in the presence of n equipotent units of a mixture of A and B will be less than the number in the presence of n equipotent units of A or B alone.e. of the other drug in the biophase.6A . When these two rearranged expressions are equated. respectively. if the dissociation of the B’R complex is = is transformed to not catalyzed by the drug A. is . [I +KAKA’@) (kB’/kB)KA(A) t (16) ( k ~PB ’ KA’ (KA(A))~ + KBKB’(B)I / [ 1 -t (kB’/kB )KA (A) 1 KAKA’ (A) Thus.. to the fraction of free reP ceptor sites. where the steady state metabolic rate. or its reciprocal.. (A’). the metabolic rate which is porportional to a fraction of free receptor sites. Hence. for the case when drug A catalyzes the dissociation of receptor site com lex of drug B. are functionally linked. fR . the ratio (A’R)/(B’R) can be obtained and is (A’R)/(B’R) = [ 1 t (~B’/~B)(A’)]KA) (A’)/KB’(B’) (12) This value.

is the apparent generation rate constant in I (phase I) action definedz9by kapp. the “unfavorable” configuration for I may be induced by the other drugs. is acted upon by the receptor site. Consider the metabolic sequence and the apparent generation rate constant. and thus unavailable for reaction in the metabolic sequence of eq 21 leading to the Mz vital for microbial growth and generation. 1972. ~ ~ developments of eq 6-19 are based only on drug A catalyzing the dissociation of the (B’R) complex.k&iKRzL/(l = ko/(l -t KiKRZL) K&R2L) (26) where L is the I concn and ko = qkm = ka/kb. whereas the subsequent dihydrofolate reduc tase inhibition of trimethoprin has a relatively immediate effect on microbial generation.(i - . a “catalytically active” site and an “allosterically modifying site. of generating organisms. L / ( ~-t K ~ K R .koBi(1 .kapp) is greater for phase I1 than for the phase I action of I at each level of concn over the range 60-350 pg/ml of I. since less receptor sites are occupied by drug than would be anticipated on the basis of simple equiv. ’The ~ depletion where the substrate. One feasible proposition could be that 2 possible binding sites exist for one lincosaminide molecule. R1. = = kik2KRIKR2SRiRz =kikzKEIKE. N. becomes kapp. then. or both may induce such configurations for the other. S(EI)T (E. I (phase 11) action may be rationalized as a different mechanism from that of I (phase I) and I1 (or 111) action. Rz. . in a “sequential b l ~ c k i n g ” ’of ~ protein synthesis t o enhance generation i n h i b i t i ~ n .ei>(l . The fact that I does have a second mode of action is of interest with regard to this hypothesis in that it may be speculated that the phase I1 of lincomycin action may be related to its possible ability to allosterically modify the receptor site for the other lincosaminide antibiotics.8A .S(Ri)T(R2)T(1 = k. will always be greater for such allosterically modifying components of combinations (Figures 2a and 2b) than can be predicted a priori on the premise of drug equivalence of the components (Figure 2c and eq 19). such a model as eq 6 to represent the modification of an allosteric configuration of a receptor site for one drug by the presence of another results in an antagonism of antibacterial action.kaL( 1 -t kbL) = ko .=ko(l .HC1. but which becomes effective only after a finite time of drug-bacteria interaction (Figure la). The data of Table I show that the extent of generation inhibition ( k o. S . These fractions of receptor sites treated with I may be defined” as 8 1 = K ~ K R . since it has been observed7 that replacement of the 7(R)-OH group of I by a halogen substituent in the 7(R) configuration had little or no effect on in vitro antibacterial activity but a halogen substitution in the 7(S) configuration increased the activity 4. Since the generation rate is proportional to the metabolic rate leading to generation and the number. k l k Z K ~Rz(SR1) . No. If I competes with M 1for the receptor site Rz in phase I action and competes with S for R1 in phase I1 action. A Possible Kinetic Model to Rationalize the Biphasic Action of Lincomycin. Mz. kapp.’rZ6-28 The high activity of I1 (or 111) relative to I (phase I) s u g gests the possibility that I (phase I) may exert an allosteric effect which modifies the affinity of itself and the others for the catalytically active site.) = ko . the new generation rate constant. The 8 and tlz are the respective fractions of these sites that are treated with I. and eq 26 are considered.KRZL) (28) and thus the apparent generation rate constant kapp for phase I1 lincomycin action will always be less than the apparent generation rate constant. to transpose it to the metabolite. Similar expressions can be developed for the simultaneous catalysis of the dissociation of the (A’R) complex by the drug B. When phase I1 action comes into play. (1 - 6.)(r .0 2 ) e. then dMz/dt = kZ(R2M1) = k z K ~RzMl= .e2) (22) where (23) km = k I ~ ~ KKR2 R . and the total amounts of receptor sites ( R l h and ( R ~ ) T are constant. 15.to 32-fold in the order C1< Br < I. W / d t = q W / d t = qk. respectively. The metabolite or its successors is possibly utilized by the enzyme (or receptor site) that is also the binding site for I (phase I) action.162 Joumal of Medicinal Chemistry. vital for microbial growth and generation. Of course. The binding of I (phase I) at the “allosteric site” possibly may produce an extensive conformational change that causes an “unfavorable” configuration of the “active” site of the receptor and thereby decreases the net reactivity of the “catalytically active” site for all of these drugs. These facts may indicate that the 7(R) and 7(S) epimers of I bind differently on the same receptor site (or enzyme).) T since it may be postulated that the amount of substrate.el)(l - 8&V= kapp.8 generation lag for its effect to be observed after drug addition. = qk.62) = ko/(1 -t KiKRIL)(l = kapp1/(1 KiKR2L) K. This model may be considered as similar to the synergistic action of sulfonamide-trimethoprin combinations on the tetrahydrofolate utilization sequencez9 where the prior sulfonamide action on folic acid synthesis has a 5. for phase I lincomycin action. Vol.. L ) (24) where kapp. 2 Heman-Ackah and Garrett Thus. alency of action of each drug of the c o m b i n a t i o r ~The .. An explanation that has been proposedi7 for the lincomycin (phase 11) action was that I may block an additional receptor site engaged in the synthesis of an essential metabolite.N (27) and when k o = qk. as in eq 18.01 - 4 + B1&)=kappI . then from eq 7 dN/dt = q(dM/dr)N = qkm( 1 . S . to produce the metabolite M1 necessary for the receptor which is site.(l . k.8B)N = kappN (20) of this necessary metabolite or its precursors during successive microbial generations imposes a new rate-limiting factor and a new and lessened steady-state generation (phase 11) is established. Steric configuration may play an important role in this type of drug-receptor reaction. It implies an action which is interdependent with the initial I (phase I) action.

563 (1962). Microbiol.603 (1964). 10. Antibiot. Ag. IS. (24) V. kappII. J. (14) J. Mol. B. 54.. Antimicrob. J. Gen. 1969. B.. Elion. (22) E. (5) M. (29) E.25. Biol. Biophys. 1972. The parameter values are ki = 2.Lewis. S. Cundliffe and K. Nut. M. R.Chang. coli cultures a t 37.. Oleinick. Magerlain. R. Barber and P.is the generation rate constant for lincomycin-affected cultures m phase I1 steady-stage generation and where kc may be the product of the drug partition constant K. J. J. N. Sih. Biol. Birkenmeyer. and J. Vol.203 (1966). Miller. ibid. 32$493(1968). W. ’ ~ Whatever ’~ the mechanisms of action. Weisblum. Biol. In this case. Bacteriol.03 X b = 1. 560 (1962). E.Garrett. Ag. Garrett and 0. ibid. (4) C..>L (1) J. R. the common action of I (phase I) and I1 (or 111) could be related to the inhibition of the “translocation” of peptidyl-tRNA in polypeptide synthesis and that of I (phase 11) to the kappIlkapp. The authors are greatly indebted to Mr. Birkenmeyer. Miller. H. E. E. and C. 570 (1962). 727 (1966). Med. H. J. 1448 (1970). for the lincomycin at the site of its ml/bg and phase I1 action. (19) E. 0.. Garrett and G. it can be conjectured that the common action of I (phase I) and I1 (or 111) is related to the inhibition of the “peptide bond formation” reported by other worker^'^"^ using cell-free extracts. and F. The ratio of the apparent rate constants for phase I and phase I1 lincomycin action can be derived from eq 28 (29) and is shown to be reasonably valid by the plots of these ratios against I concn in Figure 10 which clearly demonstrates a slope of K I K R .. Wright. 30. Wilhem. (6) J. Weisblum and J. The intercept.L + b. Linearity of the plot of the ratio kappI/kapp I vs. Med.. (20) E.” Modem Scientific Publications. Magerlain. = 1 + (KIKR. for excellent technical assistance. P. Garrett. (12) F. Biol.271 (1971). J. BioL. 1576 (1967). 9. (16) K. 2. is slightly greater than unity which may be attributed to the fact that k d k b of eq 27 and 28 may not be exactly equal to. L. 208.499 (1969). 239. and E’. BioL.431 (1966). ibid. (15) R.the generation rate constant in the absence of The precise physiological significance of these mechanisms cannot be ascertained from the available kinetic data. R. and A. M.Lincomycin Antibiotics Journal of Medicinal Chemistry. L. Chicago. R. (28)J. Mielck and E. M. D.. Chemotherapy. R. This investigation was supported in part by Public Health Service Research Grant ROI AI 10058-01.Mason. however. I does antagonize the action of its 7 4 s ) C1 analogs against E. Vazquez. and F.337 (1969). Watenvorth. Ag. 145. Monro and D. Garrett. although they are proportional to. 155 (1967). (21) E.. and K. Vazquez and R. Sr. Bergy. Sci. W. H. Perry. ibid. 8. B. W. Mol. Smith. Mol. Hitchings. Wright... 15. Commun... R. Antimicrob. Proc. U..p 36. Sei. E. (23) B. R. 28. Med..50in accordance with the equation.. (13) D. Kagan. k. H.. A. L. Lacey. J. and G.477 (1954). N. (25) G. 2 163 inhibition of “peptide bond f ~ r m a t i o n . 59. Acta. Biophys. Igarashi.. Umbarger. Med. G. Alternatively. 723 (1967). (17) J. H. Proc. ibid. (18) E. Ill. and B. (2) R. Antimicrob.. George L. . J. Clapp. 37. 56.. W. 6. 236 (1967).. Res. Monod. Chemother. deBoer. Monro. E. Chemother. N. R. M. Dietz.Potter. (27) H. R .8 (1958). Davies. J. where kapp1 is the generation rate constant for lincomycm-affected cultures in the phase I steadystate generation. Garrett. Soc. Pharm. Chem.. 142. concn for lincomycin-affected E. J. Acud.41 (1951). Chem. Soc. J. However. ibid. National Institutes of Health. Jacob. Biochem.355 (1967). Mason. (7) B. Singer. Herrell. (8) B. Chem. Chemother. Heman-Ackah.. Changeux. (10) B.674 (1964). Science. R. K. Biochem. kappI/kappII = k.No.247 (1958). (9) W. 55. J. (11) E. 554 (1962). References ____ 1 0 0 200 300 Concentration ( / a ml-’) Figure 10. McQuillen. 76. I (phase 11) action may be associated with the inhibition of a synthesis of some essential amino acid or derived peptide precursors not normally available in the broth medium. Grady. R.Frieden. and J. coli. S . 137 (1967).Ishitsuka.. R. Kagan.427 (1965).Garrett. Arzneimittelforsch. E. (26) C. D.. Chem. Perry.. K. D. Corcoran. Kaji. C. Rev.. Inc. Chemother. Acknowledgments.. Treick. Duncan. Fortschr. Symp. and the drug affinity constant K. R. Exp.306 (1963). D. 14. Brit. 8. “Lincomycin.161 (1967). (3) L. Hanka. and R. S. J.3522(1964). J. and G. Herr and M. Burch.

1021/jm00272a010 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. Bloch J.org on April 25. 15 (2).doi.1021/jm00272a010 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. Bobek.Synthesis and biological activity of pyrazines and pyrazine ribonucleosides as pyrimidine analogs M. Chem. Washington. 164-168• DOI: 10.acs. 1972..org/10.. Med. DC 20036 . 1155 Sixteenth Street N. and A. 2009 More About This Article The permalink http://dx.W.

New York State Department o f Health. New York 14203. cytosine. we undertook the synthesis of this compound by a new and facile route.13 Because of the low yield of the reported procedure (overall yield based on pyrazine-2-carboxamide was 4%) and our need for the compound as a precursor of the nucleoside.. but competitively by orotate. Finally.lo 1. Modifications in the heterocycle of the natural nucleosides have resulted in a number of analogs. Roswell Park Memorial Institute.~ 6azathymidine .g. A preliminary account of these data has been presented. Among these. substituted in such a way as to provide analogs of the natural pyrimidines. the growth inhibition exerted by the orotate analog was prevented noncompetitively by these metabolites. The inhibitory activity of the regrowth of these organisms at 6 X lO-'M and 4 X maining analogs ranged from 1o+ M to 4 X 1o-' M. Attempts to prep the Noxide deriv of VI11 or IX by oxidn with m-ClC&CO&I or . emimycin. The inhibitory effects of emimycin and its ribonucleoside were reversed competitively by uracil. an alternative route for the preparation of 1. starting from the readily available 1. Bobek* and A.4¶' and 6-azauridine ! T 8 An examination of possible further alterations within the pyrimidine ring led us to consider the pyrazine ring. The ribonucleosides of this analog and of 1. each inhibiting 50% of the growth of Streptococcus faecium and Escherichia coli at 8 X 10-6M and 1 X lO-'M.2-dihydro-2-oxo-6-carboxypyrazine M.' S-a~acytidine?.2-dihydro-2-oxo-6-carboxypyrazine 4-oxide (V). and which structurally resembles uracil.2dichloroethane afforded only the N-glycoside. but not to the orotate analog. I 9 71 Because of the marked antitumor activity which derives from structural modification of the heterocycle of the natural pyrimidine nucleosides (e. Vol. The position of the oxide function of IVa-c and V was established by comparison of the uv absorption spectra of IVa-c and V with those of 1. 5-. Over this range.2-dihydro-2oxopyrazine 4-oxide was considered. 2-thio-. their possible use in antibacterial chemotherapy appears worthy of exploration. Wagner and Frenzel16 and later Reisser and Pfleiderer" have shown that the direct glucosidation of the Ag or Hg salts of 1.2-dihydro-2-oxopyrazine in the presence of TiC1418 in boiling 1. Sazacytidine. and examined its effect upon various cell systems.2-dihydro2-oxopyrazine were prepared by ribosidation of the corresponding trimethylsilyl derivatives in the presence of TiCl4. particularly with respect to drug resistance. and 6-Me analogs. Because of the selectivity of the pyrazine derivatives for bacterial cells. interfered with the respectively. The 2-benzoyloxy derivs (1Ia-d) were oxidized with m-chloroperbenzoic acid. tumor cells. Bloch Department of Experimental Therapeutics. Investigation revealed the existence of an antibiotic. Included among these are compounds such as 3-deazauridine and 3-deazacytidine. 3-deazauridine. and their nucleosides over the concentration range of 1Od3 to 10-6 M .5-tri-O-acetyl-/3-D-ribofuranosyl)-2-oxopyrazine (VIII) was deacetylated by MeONa in MeOH at 22' to give 1. We also prepared other substituted pyrazines. 4-oxide.2dihydro-2oxopyrazine 4-oxide (emimycin) starting from pyrazine-2carboxamide via 2-aminopyrazine 4-oxide has been reported by Terao" and independently by Palamidessi and Bernardi. faecium resistant to emimycin or its ribonucleoside were cross-resistant to each other. The nucleoside IX has been assigned the /3 configuration on the basis of the trans rule. The syrupy 1. among them the 6-carboxy-. we have prepared the nucleoside derivative of emimycin. and have compared its biological activity with that of the antibiotic. various pyrazine 4-oxide.2-dihydr0-2-oxopyrazine 4-oxides (IVa-d). analogs of pyrimidines were prepared. As a result.2-dihydr0-2-oxopyrazine a compound which has been isolated as the antibiotic emimycin. reversal of the emimycin inhibition of Escherichia coli by pyrimidines confirmed the structural analogy. 2 Bobek and Bloch Synthesis and Biological Activity of Pyrazines and Pyrazine Ribonucleosides as Pyrimidine Analogs M. Similarly.2-dihydr0-2-oxopyrazine furnishes the 0-glucoside and only traces of N-glucoside. 6-azauridine). Ribosidation of the Me& deriv of 1.3. involving the oxidation of benzoyloxypyrazine. Also synthesized were a number of derivatives of emimycin.'' 1. a strain resistant to the latter compound retained its sensitivity to the uracil and uridine analogs. The orotate analog." Since no data have been published on the effect of emimycin on mammalian. which was purified by column chromatog on silica gel. which have demonstrated marked activity against a variety of experimental tumors. and the 1-. none of the compounds inhibited the in vitro showed no inhibitory activity in these systems.2-dihydro-2-0xopyrazine 4-oxide. Their attempts to rearrange the 0-glucoside to the N-glucoside were unsuccessful. The corresponding compounds lacking the N-oxide M. At growth of leukemia L 12 10 or Ehrlich ascites cells in excess of 10-20%. Strains of S.2-dihydro-2oxo-3-isobutyl-6-sec-butylpyrazine 1-oxide. respectively. was synthesized by an improved method. Treatment of Ia-d with PhCOCl in pyridine gave cryst IIa and syrupy IIb-d. to give 1. 1972. the uracil analog 1. Emimycin and its ribonucleoside were equally active.2-dihydr0-2-oxopyrazine 4-oxide. 15. Buffalo. Removal of the protecting Bz group from I I I a d with MeONa in MeOH gave 1.2-dihydro-2-oxopyrazine (Scheme I). in particular.'~'~ which is the 1. The synthesis of 1.6-dimethylpyrazine 4-oxide. No.2-dihydro-l-(2. The results of these studies are provided in this paper. Received July 21.14 and 1.164 Journal of Medicinal Chemistry. Indeed. among which the orotic acid analog showed particularly marked biological activity. No 0-glycoside was detected by tlc in the reaction mixt.12 Results and Discussion (a) Chemical Results.2-Dihydro2-thiopyrazine 4-oxide (VI) was prep from 2-chloropyrazine 4-oxide by treatment with NaHS at room temp.2-dihydro2-0~0-3. to furnish the corresponding cryst 2-benzoyloxy 4-oxides (Ma-d). a different route was chosen by us for prepn of the riboside. Hydrolysis of the Me ester function of IVd was accomplished by treatment with KOH soln at room temp. 1.2-dihydro-1-(/3-D-ribofuranosyl)-2-oxopyrazine (IX). since nucleoside derivatives frequently offer metabolic advantages over the corresponding bases.

SiO Acowc Hou Hov/ Acokd TiC1.H. R = CH. R = H. Deacetylation of X with MeONa in MeOH at 22" gave 1. a-d. _* OAc OAc o k c OAc VllI OH OH IX AcOCH. R.2dihydro-2-oxopyrazines proceeds only with difficulty. which cannot strictly be considered analogs of the natural pyrimidines. The third class of compds is made up of nucleoside derivs of some of the analogs in the first and second categories.2-dihydro-2-oxopyrazine 4-oxide. Treatment of 1.3. d. The spectral data pertaining to the newly prepd compds are summarized in Table I . R = H.2-dihydro-1 -(@-D-ribofuranosy1)-2-0~0- pyrazine 4-oxide (XI). carry an 0 atom at the 4 position. 15. demonstrating that the oxidn of N-1-substituted 1. (b) Biological Results. R' = H c. The second group includes 2-substituted derivs which. 1972. This finding prompted us to investigate the glycosidation of the 1. R' = CH.r"] I S N H VI 0 0 I CH3 CH.3.. marked inhibition of cell growth resulted (Table 11). R' = COOCH. The trcarment of both VI11 and IX with peracids under various conditions" resulted in the gradual loss of uv absorption and yielded complex mixts.2-dihydro-1-methyl-2-oxopyrazine4-oxide (VII). The same relationship applies to .2. The compds lacking the 4-oxide did not exert any inhibitory effects on the microbiol test systems used.).5-tetra-O-acetyl-P-D-ribofuranose in presence of TiC14 in boiling 1. The structure of XI was established on the basis of its uv and ir absorption spectra and its elemental analysis.CO 0 ? 0 .5-tri-O-acetyl-~-D-ribofuranosyl)-2-oxopyrazine 4-oxide (X). OAc 0 1 ) okc 0 1 0 I 0 1 d --+ OAc OAc okc OH OH X XI F3CC0& were unsuccessful. One comprises compds with substituents at the 2 position only. 2 165 0 fxRH la-d R' R"O XX" R' Ila-d 0 0 0 1 R"O N 1IIa-d R' 0 H IVa-d L N L N H V 4 COOH a. VI1 0 1 IVa -----C (CH.2-dichloroethane afforded a low yield of 1. Vol.2-dichloroethane produced 1. The pyrazine derivs synthesized fall into 3 categories. in analogy with uracil.2-dihydro-l-methyl-2-oxopyrazine with m-ClC&CO& in boiling 1. Reaction of the Me3Si deriv of IVa with 1. No. R" = C.Pyrazine Ribonucleosides Scheme I Journal of Medicinal Chemistry. which was purified by column chromatog on silica gel.2-dihydro-1(2.R' = H b. whereas in the presence of the N-oxide.

2-Dihydr0-2-oxo-l-methyl >io4 >10-3 1. The extent of growth inhibition produced in the microbial system varied according to the nature of the substituents on the ring. faecium. Derivative of pyrazine 4-oxide M M _1.2dihydro-l-(p-D-ribofuranosyl)-2-oxopyrazine. the 5-Me deriv of emimycin could not replace thymine for growth of the organism in the basic medium free of folate and contg adenine as the purine source.2-Dihydro-2-oxo-5-methyl 1.2-Dihydro-2-oxo-6-carboxy 6 x lo-' 4x 1.2-Dihydro-2-oxo 8X 1X 1.2 e x -I Q a V g i the nucleoside derivs. similarly. but likely exerts its effect along the de novo path leading to UMP. whereas a 3-fold decrease in activity occurred when the hydroxyl group was replaced by C1. emimycin and its ribonucleoside deriv were equally active. but was as active as the antibiotic against E.faecium. but not in E. faecium and E. This analog retained full activity in the emimycin and emimycin ribonucleoside resistant strains of S. coli. coli.166 Journal of Medicinal Chemistry.2-Dihydro-2-oxo-6-methyl 8 X low4 9x 10-~ I E m -' 1 % aThe corresponding compds without the N-oxide function. duce any growth inhibition at 0oooornoo U & . but was less active than the antibiotic against E. The introduction of Me into the 1 position of emimycin abolished the inhibitory activity of the antibiotic. that the strains resistant to 10-3M of the nucleoside are cross-resistant to the base. coli. suggesting that they exert their activity via a common metabolic intermediate. Over this concn range. 15. faecium. coli. faecium. suggesting a different metabolic path for its activation or a different site of action. including 1.2-Dihydr0-2-thio 7x 5x 2 x 10-5 3 x 10-5 2Chloro 2 x 10-5 1 x 10-5 2-Ethoxy 1. Vol. At IOm4M. In both test systems. inhibited the growth of L-1210 cells in vitro by more than 10-20%. This suggestion received support from the observation that strains of S. Of marked interest is the finding that the pyrazine analog of orotate was more effective than emimycin against S. Growth Inhibitory Effect of Pyrazine Analogs of Pyrimidines0 Concn for 50% growth inhibition of S. coli resistant to 10-3M emimycin are cross-resistant to the emimycin nucleoside and. 1972.2-Dihydro-2-oxo-l-(p-D-ribofuranosyl) 8 X l o . 2 Bobek and BIoch Table 11. indicating that the pyrazine analog of orotate does not interfere with the conversion of exogenous uracil or uridine to UMP and t o further metabolic intermediates. the N-oxide being required for activity. none of the compds which showed inhibitory activity in the bacterial systems. E. No. faecium showed (Table 111) that the inhibitory effects of emimycin and its ribonucleoside were prevented competitively by uracil and cytosine and their nucleosides at concns ranging from to 10-6M. did not proM. faecium. the inhibition exerted by the orotate analog was reversed in a noncompetitive (product) manner. whereas Me at its 5 or 6 position allowed for some inhibitory activity in S. An inhibition analysis carried out with S.to 10-fold. Further support for this deduction comes from the ." 1 x lo-' 1. As detd in the S. Replacement of the 2-oxo group of emimycin by = S decreased the inhibitory activity of the antibiotic in the microbiol systems from 5.2-Dihydro-2-0~0-6-carbome thoxy 4 x io-s >io1. The 2ethoxy deriv was approx 3 times less effective than emimycin against S. faecium system. and the bacterial species used for the assay.

2 161 Table 111.005 mole) and 4ClC. BzCl (3.1130. At 10-3M.N-tO).20 g.01 mole) was dissolved with stirring in a soln of NaHS. by Pyrimidines.to 2-fold. 0. 1. The solvent was removed.O. and dried (Na. v/v.2-Dihydro-2-oxo-6carboxypyrazine 4-oxide Noncompetitive Noncompetitive Noncompetitive Noncompetitive Noncompetitive Noncompetitive 2.2-Dihydro-2-oxopyrazine 4-Oxide (IVa).01 mole) following the procedure described for the prepn of IV.022 mole) was added dropwise with stirring to a mixt of Idla (3. and dild with CHCl. (80 ml). The reaction mixt was heated at reflux temp for another 2 hr. because of this pronounced selectivity.O (15 ml). The mixt was then evapd to dryness. 0. G N ) . 1972.2-Dihydro-l-methyl-2-oxopyrazine 4-Oxide (VII). The soln was cooled to 3040" and addnl 4C1C6H. 5-Methyl-l.4 2. Reversal. whereas. cooled to 20".)." It remains to be detd why.to 3-fold reversal at 10-3M Thymine 2.2820 (broad) (NH. the s o h was applied t o a column (1 X 20 cm) of Dowex 50 (Ht) resin. 1. the orotate analog being reversed more effectively than emimycin or its ribonucleoside. An amber oil sepd which was extd with PhMe (80 ml). the residue was. ClC. and dried (Na. Experimental Section? 2-Benzoyloxypyrazine 4-Oxide (IIIa). The column was washed with H. 1660-1620 ( G O .l-dichloroethane (C. 0.2-Dihydro-2-thiopyrazine 4-Oxide (VI). IVd (0.H (1. Uv spectra were obtained on a Cary Model 14 recording spectrophotometer.v/v). (80 ml) and then with H. It might be added here.O (60 ml).1250 (N-+O).&-EtOAc (6: 1.obtainable in a possibly limited amount.O and the acid fraction was collected and evapd. might mean that the uracil analogs act at the base or ribonucleoside level per se. The eluate was evapd to dryness and the residue was recrystd twice from EtOH furnishing 400 mg of 1. v/v). 825 cm-' (N+O.02 mole) was added to dry pyridine (20 ml). 0.H ( 5 g) was added to the soln and the work-up proceeded as described for the prepn of IIIa. The resin was filtered and washed with MeOH (80 ml). 1. IIIa (2.1 g.2dihydro-2-oxopyrazine2' (2. After 2 hr at 22".02 mole) was dissolved in dry 1.CO. with substrate concns ranging from lo-. 1730 ( G O ) . coli by emimycin was also found to be competitively reversed by uracil.Pyrazine Ribonucleosides Journal ofMedicinal Chemism.022 mole) was added dropwise with stirring. 2-Benzoyloxy-5-methylpyazine 4-Oxide (IIIb). 2-Chloropyrazine 4oxide23(1. It was concd to an oil. 1630 (broad) (eo. (80 ml) and H.2-Dihydro-2-oxo-6-carbomethoxypyrazine 4-oxide (IVd) was prepd from IIId (2.O: imax 3500 (OH).Cl. and the pH of the mixt was adjusted to 4-5 with HCl.001 mole) was dissolved in a KOH (0.5 Cy tosine 0.0 .H. 0.O. at the highest concn of lo-' M. To this soln. faecium by Pyrazines Inhibition indexa obtained with 1.Cl.O (80 ml). The reaction mixt was stirred at 22" for 15 hr and evapd.1105 cm-' (ES. was poured into 50 ml of icecold H.H.01 mole) in the same manner as described 3160.2825 (NH.3080. washed successively with a satd soln of NaHCO. This relatively small extent of reversal suggests a sparing effect of thymine or thymidine on de novo pyrimidine synthesis.H.to 15fold in a noncompetitive manner. 4+Melting points were taken on a Fisher-Johns apparatus and are uncorrected.this metabolite prevented the growth inhibition exerted by emimycin and its nucleoside by only 1.-.H.H. and the remaining syrupy product was dissolved in Et. A soln of 1.4 g) soln in H. 1. 1.2-Dihydro-2-oxopyrahe ranosyl)-2-oxopyrazie Substrate 4-oxide 4-oxide 20 Uridine 20 11 2'-Deoxyuridine 10 0. 2-Benzoyloxypyrazineao (4.2% concn in MeOH. 4C1C6H. Concn of the eluate containing Id (as shown by tlc) furnished cryst substance. Solvent concn was conducted under reduced pressure in a rotary evaporator. thymine or thymidine reversed the inhibitory effect of the analogs from 4.to 3-fold reversal at lO-'M 10-fold reversal at 10-3M 6 observation that orotate reversed the inhibitory effect of the orotate analog in a competitive manner. the potential use of emimycin and its derivs in antimicrobial chemotherapy appears worthy of exploration.2940.COEt. the residue suspended in EtOH (50 ml) and reevapd to a solid. after standing at 20" for 15 hr. C=C.550 g.). CH). The ir absorption spectra were detd in pressed KBr disks with a Perkin-Elmer Model 5 2 1 spectrophotometer. Cryst IIIa which sepd from the soln after standing for several hours at 20" was collected by filtration and recrystd from MeOH. MeOH-washed Dowex 50 (H') resin (10 ml) was added to the soln with stirring. 2960 (CH). to lo.H (4 g) was added. the highest concn used. The MeOH s o h was evapd. Cy). from exogenous orotate.Cl. This ext was washed with H.C0. To this mixt BzCl(3. 1740 (C=O carfor IVc: imaX boxyl). However. which was recrystd from Me. Addnl4-ClC.H.5. PhMe was removed by evapn and the remaining oily residue was dissolved in xylene (50 ml). The fact that orotate does not reverse the emimycin or emimycin riboside action extensively. 2840 (NH. (60 ml) was heated at reflux temp for 2 hr and was then cooled to 30-40". 0.C0. 50 ml). 0.H4C0. or that competition for their activation via a pyrophosphorylase or kinase is exerted more effectively by the exogenously supplied uracil or uridine than by UMP. and the mixt was heated at reflux temp for 2 hr. 15. the inhibition of the growth of E. 1260 .0 g) in C.H. 4ClC.to 3-fold reversal at lO+M 10-fold reversal at 10-3M Thymidine 10-fold reversal at 10"M 2-fold reversal at 10'SM 2-fold reversal at 10-3M Orotic acid "[I]/[ SI for 50% growth inhibition.2-dihydro+methy1-2-0xopyrazine~~ (0.30 g. The filtrate was concd t o 50 ml and allowed to stand at 5" for 4 hr. The column was subsequently washed with Me.CO.O (4 X 100 ml) and dried (Na. 0. that.1230. No.02 mole) and dry pyridine (20 ml). except that a larger vol of MeOH (200 ml) was used because of the poor solubility of both IIIc and IVc. prepd by satg a soln of 0.S at 5".SO. 2-Benzoyloxy-6-carbomethoxypyrazine 4-Oxide (IIId).00 g. CH). faecium.340 g. in analogy with our observations in S.01 mole) was dissolved in MeOH (100 ml) and a 2 N MeONa-MeOH soln was added dropwise with stirring until pH 9-10 was achieved. This soln was concd to a thick oil which was dissolved in C. as compared to their effect on the bacteria.SO. G C . The product was collected by filtration and recrystd from 96% EtOH: pmaX 3125.CO. of the Inhibition of Growth of S.7 0. The reaction mixt. The soln was washed with a satd s o h of NaHCO.5 g) was added. the residue was suspended in EtOH-H. C=N) 1295.OOg) was added and the reaction mixt was heated at 55-60' for 15 hr.46 g Na in EtOH (50 ml) with H.30 g. the analogs are only marginally active against the mammalian cells.2-dihydro-2-oxo6-carbomethoxypyrazine 4-oxide (IVd): Vmax cm-' 3 110. which was chromatogd on a silica gel column in C.2-~ihydr0-2-0~0-6-methylpyrazine~' by the procedures described under IIIb.74 g.16 g. 2-Benzoyloxy-6-methylpyrazine 4-oxide (IIIc) was prepd from 1. 3080. (80 ml). 1275.2-Dihydro-2-oxo-5-methylpyrazine 4-oxide (IVb) was prepd from IIIb in the same manner as described for IVa.O (1:1. 0. 0. 1. 3090. COC).extd with boiling 98% EtOH (3 X 150 ml) and filtered while hot. 1.1260. The residue was recrystd from H.SO..2945.8 Cytidine 0. and the mixt was worked up as above.9 2 2'-deoxy cytidine 1 0. which was recrystd from 96% EtOH.2-Dihydro-2-oxo-6-methylpyrazine 4-oxide (1Vc) was prepd from IIIc (2.1025.1 g.H (2. 1745 (C=O). 0. To this soln mchloroperoxybenzoic acid (85% product. Vol. 1250. 5 .H (1.O. 1.).CO-MeOH (6: 1. Optical rotation are equilibrium values and were detd on a Jasco ORD/UV Model 5 instrument at 0.2-Dihydro-l -@-D-ribofu1.1215.9 Uracil 0. 40 ml).2-Dihydro-2-oxo4-carboxypyrazine 4-Oxide ( V ) . M.3080.1 g. The soln was then cooled to 20". 1245 cm-' (broad) (N-tO).

and A. The syrupy 1. Bloch. E. Ser. Biochem. The techniques used for these detns have been published p r e v i o ~ s l y .2". Bloch. 13.O. Med.. this soln was added 1.J. D.242 (1960).CO (7:1. After cooling to 22".5-Tetra-O-acetylQ-D-ribofuranose (9. Chem. 30 ml). Terao. 123 (1964). and A. and evapd. K. 20.A. this antibiotic showed marked antitumor activity: but the severe local toxocity in man4 which it produced limited its clinical usefulness.. Vesely. J. R. and crystn occurred after solved in Me. Biol. washed with H. 20. Amer.Ochiai.PhMe (80ml) was added.CO-MeOH (4: 1. Chem. Cihak. ibid. 4 -Thiotoyocamycin retained full inhibitory toyocamycin. Welch. 1. J.2043 (1949)..NH (3 ml). The mixt was cooled to 22" and poured slowly with vigorous stirring into a satd soln of NaHCO. Yonehara. Tynan. which was subsequently washed with CHCl.01mole) and TiC1..3300 was recrystd from 96% EtOH: [a]"D -204.3-d]pyrimidine. H. Buffalo. B. The solvent was evapd. It was filtered through a Celite pad. Received July 30. (13) G.5 X 8 cm) of silica gel. and the residue was recrystd from EtOH-Et. Whistler.the other involved. The soh was cooled to 22". Prusoff. L. E.5hr at 90-95"with stirring and exclusion of atm moisture.343.3. activity against a strain of S t r e p t o c o c c u s faecium resistant to I O .CO was removed by evapn and the remaining solid Vmax 3400. 1971 The 4'4hio analog of the antibiotic toyocamycin was prepared b y condensation of 2. O'Donnell. 1.CO as the eluent.2-Dihydrol-(p-D-ribofuranosyl)-2-oxopyrazine 4-Oxide ( X I ) . the soln was neutralized with HCl and evapd.1580 (1952). 0. with vigorous stirring.Cl. Umezawa. 28.830cm-' (N-+O). K.209 (1956).5-tri-O-acety1-4thio-D-ribofuranosyl chloride with the chloromercuri derivative of 4-acetaminod-bromo-S-cyanopyrrolo[ 2. J. Pfleiderer.045 mole). 30. (17) F.2. and the reaction mixt was heated at reflux temp with stirring and exclusion of atm moisture for 4 hr.. 1660 (GO). (250ml). Bloch.5-tri~-acetyl-p-D-ribofuranos~l)-2-oxopyrazine 4-oxide (X)(480mg) obtd by evapn of the solvent was dissolved in MeOH (50 ml). Sawlewics. Karasawa. Chem.3-d]pyrimidine.785 (1958).. Frenzel. Ed.SiCl (3 drops) was heated for 0. I n one.Karmas and P.). and the soln was concd to an oil. Yale J . Biological Assays.. Z . D. and the reaction mixt was heated for 5 hr at reflux temp with stirring and exclusion of atm moisture.3280(OH). Terao. 22. A mixt of IVa (1. Bobek and A.55g. Klein.2-Dihydro-l~p-D-ribofuranosyl)-2-oxopyr~~e (IX). Roswell Park Memorial Institute. Welch. New York State Department of Health.O. (16) G.3. the ring 0 of the carbohydrate moiety was replaced b y S. Org. (4) W. 5. Experientia. v/v). A.5-tetra-O-acetylQ-D-ribofuranose 0. Chem. L. (20) K. (18) U. Vesely. Int. Currie. A Me. 1967. 2 Robek. Palamidessi and L.2925(CH). (9) M .om.3. (6) J. the substitution of the 4 and 6 position of the heterocycle with C1 a n d amino groups.* R. Tanaka. and H.. 14. Acta. washed with H. Piskala. Sorm and I .. followed b y removal of the protecting groups with MeOH-NH3 and removal of Br with HJPd catalyst.Cl. Ser. Ass.12g. Purdue University. (1500ml). 0. "Aromatic Amine Oxides. Biophys. 125. (11) J. Reisser and W. N. Proc.' In exptl systems. which was dissolved in dry C. To (3.73 (1969).Sorm and H. (14) B. Bobek. H. and Me.182 (1963). Netherlands. t w o structural modifications of the toyocamycin molecule were made. Spoerri. (500ml).. 386 (1969). DeZeeuw and E. Ital. Acta Polon. to which is attached a CN group. 1645 cm'' (GO). 10. The resin was filtered and washed with MeOH (20 ml). and Department of Biochemistry. L. E. The fraction contg IV (as shown by tlc) was evapd.. Pharmacol. A . ~ ~ g) was added.NH (4 of 1.840. 3400. and M. Antibiot. Keilova.CO (1:1. 99. When treatment with MeOH-NH3 was carried out a t 1 2 0 . Wagner and H. (100 ml). 13. v/v) mixt was applied to the column and the eluate was evapd.5 hr at 22" the soln was neutralized with Dowex 50 (H+) resin. Med.6diamino-5-cyano-7-(4-thio-~-~-ribofuranosyl)pyrrolo [ 2.SiC1(3 ml) was heated at reflux temp with stirring for 1 hr. SOC.2". J. The syrupy residue was dissolved in MeOH (5 ml) and applied to a dry column (2. 411 (1970).Si). Recrystn from EtOH gave pure IX: [a]"D -21. The results obtained in in vitro . Welch.CO-EtOH (1:2. and dried (Na. The syrupy residue was disv/v.3-d]pyrimidine. (2) F. and A. (5) R. No. Angew. and the reaction mixt was heated at reflux temp for another 2 hr. 4.548 (1957). effected removal of the protecting groups and nucleophilic substitution of the Br group to furnish 4-chloro-6-amino-5-cyano-7-(4-thi~-/3-Dribofuranosyl)pyrrolo[ 2.O. J. Lafayette.H. ibid.. (22) H. (3 ml). Gordon.3g. R. (12) M. Dutcher. 461 (1970). The org layer was sepd. Jaffe. 0. T h e SYNPremaining after removal of the solvent was purified by column chromatog on silica gel with CHC1. The mixt was filtered through a Celite pad. Prusoff.W. A mixt (4.. standing of the soln overnight at 22". SOC.168 Journal of Medicinal Chemistry.401(1960). (21) G. into a satd s o h of NaHCO.2. (7) F. It was cooled to 22" and poured slowly. Chem. 2 . Ber. Cheeseman. Hetman.-Me.Neoplasma.J. H. Amer. The org layer was sepd.2-dihydro-2-oxopyrazine ml). 93. Robins. (23) B. resp. 1682 (1963).215 (1958). Chem. Experientia. followed b y treatment with MeOH-NH3 a t 5 . Bobek. Handschumacher. 2945-2920 (CH).. Robins. Gazz.dild with dry PhMe (40ml). (OH).Si). 0. Klein. v/v) as the eluent. (3) F . 29. The 4'-thio derivatives proved to be effective inhibitors of the growth o f leukemif L . (Me. which was then washed with CHC1. (Me. (10) M." Elsevier. N.SO.2-dihydro-l-(2.71. and chromatogd on a silica gel co!umn with PhH-Me. Science.. (25) M. L. The remaining solid was evapd once more from dry PhMe (40 ml). Bernardi. E. and J. 2960. 1963. Bloch Department of Experimental Therapeutics. and the residue was dissolved in MeOH (100 ml). R. 1. D. Schaaf and P. Condensation with the chloromercuri deriIative of 4-chloro-6-bromo-5-cyanopyrrolo[2. 2623 (1964). (15) J. and Bloch syrup remaining after removal of the MeOH was dissolved in 96% EtOH and purified by column chromatog on silica gel with Me. E. Biol.168 (1957). 1230.. Pharmacologist. M. (19) E.I 2 1 0 cells in vitro. 1972. and A. and dried (Na$O. Ser. J. 15. Amsterdam. A . Chim.01 mole).concd to 5-8 ml.542(1966). (8) W. and Me.H. 11. 16. H. J. Chem..Niedballa and H.03 mole) and TiC1. Chem. G. 13. (3 ml) were added to this soln.O'Donnell. (24) G.. Schindler and A.341pyrimidine was formed. Me. Whistler.~ M The antibiotic toyocamycin' is an analog of adenosine in which N-7 of the imidazole ring is replaced by C. Org. 74. A .. 221 (1959). In an attempt at decreasing this toxicity.24 (1965). Vorbriiggen.20g. The resulting soln was cooled to 22". their concn for 50% reduction o f growth ranging f r o m 4 X lo-' to 5 X 10-6M. Cancer Res. Synthesis and Biological Activity of 4'-Thio Analogs of the Antibiotic Toyocamycin M. Foks and J.260 (1971). New York. 23. Lajtha. Indiana.5 hr at 22".437 (1966). Biochem. 232. The References (1) M. 202 (1964). and A. J. Spoerri. E. R . in addition to this replacement. Vol.. A catalytic amt of MeONa was added to this soln and after 0. A catalytic amt of MeONa was added to this soln and after 0.H. and J.). Sorm. Chem. 9. Whistler.3. and dissolved in dry C. ibid. Pharm.

.. Bloch J.doi.W. 1155 Sixteenth Street N. Chem.acs. DC 20036 . Whistler. Bobek. R.org/10. 168-171• DOI: 10. L. 2009 More About This Article The permalink http://dx.Synthesis and biological activity of 4'-thio analogs of the antibiotic toyocamycin M.1021/jm00272a011 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. and A. Washington.1021/jm00272a011 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. Med. 15 (2).org on April 25. 1972.

3-d]pyrimidine.om. Science.01 mole). A mixt of IVa (1. (5) R.03 mole) and TiC1. Org. Chem. into a satd s o h of NaHCO. v/v) as the eluent. N. followed b y removal of the protecting groups with MeOH-NH3 and removal of Br with HJPd catalyst.' In exptl systems. 1682 (1963). 20. Vol. 5. Synthesis and Biological Activity of 4'-Thio Analogs of the Antibiotic Toyocamycin M. 232. Bobek and A.CO as the eluent. The resin was filtered and washed with MeOH (20 ml).Si). Indiana. Antibiot. J. 1963..CO (7:1.CO (1:1. Robins. Acta.. Biophys. and the residue was recrystd from EtOH-Et. which was then washed with CHC1.. (6) J.3. L. It was cooled to 22" and poured slowly. and the reaction mixt was heated at reflux temp with stirring and exclusion of atm moisture for 4 hr. 9. A Me.2925(CH). Chem. (16) G. Pharmacologist. Acta Polon. (Me. Ser.O. and dried (Na.concd to 5-8 ml.182 (1963). Schaaf and P. J. Pharm. H.Sorm and H. 13. 1660 (GO). Bloch.1580 (1952). 2945-2920 (CH). T h e SYNPremaining after removal of the solvent was purified by column chromatog on silica gel with CHC1... Int. (20) K. their concn for 50% reduction o f growth ranging f r o m 4 X lo-' to 5 X 10-6M. A.5hr at 90-95"with stirring and exclusion of atm moisture. ibid. Gazz. 16. J.5-tetra-O-acetyl~-D-ribofuranose 0.840. (23) B..3. Bloch. 1967. The techniques used for these detns have been published p r e v i o ~ s l y .. (24) G. K. 20. (14) B.55g. Biochem.. Frenzel.5 X 8 cm) of silica gel. 1972. v/v) mixt was applied to the column and the eluate was evapd. Me. The syrupy residue was disv/v. D. R. Keilova.12g. ibid.H.01mole) and TiC1. and M. Yale J .3280(OH). 461 (1970). the soln was neutralized with HCl and evapd. A catalytic amt of MeONa was added to this soln and after 0.5-Tetra-O-acetylQ-D-ribofuranose (9. and Me. (17) F.Neoplasma. t w o structural modifications of the toyocamycin molecule were made. The resulting soln was cooled to 22". resp.O.PhMe (80ml) was added.SO. J. 28. 4 -Thiotoyocamycin retained full inhibitory toyocamycin.). Prusoff. Amer. Buffalo. this antibiotic showed marked antitumor activity: but the severe local toxocity in man4 which it produced limited its clinical usefulness. 2623 (1964). activity against a strain of S t r e p t o c o c c u s faecium resistant to I O . Welch. Welch.24 (1965). ~ ~ g) was added. (500ml). A .Cl.2-dihydro-2-oxopyrazine ml).Cl.O'Donnell. E. (4) W. Whistler.I 2 1 0 cells in vitro. R . Foks and J.2. Bobek. Med. Tynan. E. DeZeeuw and E.. Vesely. Bernardi.830cm-' (N-+O).3-d]pyrimidine.20g.W. A . (100 ml). Palamidessi and L. Chem. and H.). 3400. Bobek. effected removal of the protecting groups and nucleophilic substitution of the Br group to furnish 4-chloro-6-amino-5-cyano-7-(4-thi~-/3-Dribofuranosyl)pyrrolo[ 2. 1. Org.2-Dihydro-l~p-D-ribofuranosyl)-2-oxopyr~~e (IX).045 mole). Whistler.-Me. Gordon.548 (1957). Umezawa.2". and the reaction mixt was heated at reflux temp for another 2 hr. Cihak. (13) G. 411 (1970).260 (1971). The results obtained in in vitro . Sorm and I . 202 (1964). Chem. with vigorous stirring. (1500ml). 22. (18) U. The mixt was filtered through a Celite pad. (19) E. (15) J. 2960.437 (1966).3g.71. 1971 The 4'4hio analog of the antibiotic toyocamycin was prepared b y condensation of 2. Klein. Chem.5-tri~-acetyl-p-D-ribofuranos~l)-2-oxopyrazine 4-oxide (X)(480mg) obtd by evapn of the solvent was dissolved in MeOH (50 ml). R. Received July 30. and evapd. L. Biological Assays.* R.J. Ed. which was dissolved in dry C.3300 was recrystd from 96% EtOH: [a]"D -204. and Me. Cancer Res. The soh was cooled to 22". and dissolved in dry C. H. Amsterdam. 2 Robek. Ital.. Hetman. SOC. 74.dild with dry PhMe (40ml). and the soln was concd to an oil. E.341pyrimidine was formed. The References (1) M. Wagner and H. Klein. (Me. 13. J. 1645 cm'' (GO). To (3. which was subsequently washed with CHCl. the ring 0 of the carbohydrate moiety was replaced b y S. 30 ml). Recrystn from EtOH gave pure IX: [a]"D -21. and A.. SOC. No. H. B. G.73 (1969). 125.NH (3 ml). Robins. Schindler and A. When treatment with MeOH-NH3 was carried out a t 1 2 0 . 0..209 (1956). 4. Lajtha. Ass. L.242 (1960). and the reaction mixt was heated for 5 hr at reflux temp with stirring and exclusion of atm moisture. followed b y treatment with MeOH-NH3 a t 5 . Currie.215 (1958). Ber.343. ibid. Purdue University. Terao.Niedballa and H. Chem. The mixt was cooled to 22" and poured slowly with vigorous stirring into a satd soln of NaHCO. Piskala. L. The syrupy residue was dissolved in MeOH (5 ml) and applied to a dry column (2.Karmas and P. 1.2-dihydro-l-(2. Vesely. E. 1. washed with H. The fraction contg IV (as shown by tlc) was evapd. 221 (1959). J. Tanaka.2". J.. and Bloch syrup remaining after removal of the MeOH was dissolved in 96% EtOH and purified by column chromatog on silica gel with Me.2.CO-MeOH (4: 1. Whistler.A. Experientia.2-Dihydrol-(p-D-ribofuranosyl)-2-oxopyrazine 4-Oxide ( X I ) . 99. Sorm. Netherlands.H. (250ml). 13. 10.Ochiai. and crystn occurred after solved in Me. 93. R. and dried (Na$O. 0.H. and A. Lafayette. Biol. 14. (2) F.. Biochem. the substitution of the 4 and 6 position of the heterocycle with C1 a n d amino groups. Ser. E. (21) G. Pfleiderer. Pharmacol.NH (4 of 1. Angew. A . and the residue was dissolved in MeOH (100 ml).785 (1958). this soln was added 1. 1230..5 hr at 22". (9) M .2043 (1949).. K.3-d]pyrimidine. in addition to this replacement. standing of the soln overnight at 22". M. (10) M.6diamino-5-cyano-7-(4-thio-~-~-ribofuranosyl)pyrrolo [ 2.5-tri-O-acety1-4thio-D-ribofuranosyl chloride with the chloromercuri derivative of 4-acetaminod-bromo-S-cyanopyrrolo[ 2. J. In an attempt at decreasing this toxicity. Dutcher. Chim. The remaining solid was evapd once more from dry PhMe (40 ml).. 15. v/v). Proc. 29.. and Department of Biochemistry. (3 ml). (3 ml) were added to this soln.Si). (12) M. Z . 0. New York State Department of Health. Biol. Condensation with the chloromercuri deriIative of 4-chloro-6-bromo-5-cyanopyrrolo[2. 23.O. Bloch Department of Experimental Therapeutics. to which is attached a CN group. A catalytic amt of MeONa was added to this soln and after 0.. Chem.SiC1(3 ml) was heated at reflux temp with stirring for 1 hr. Spoerri.CO was removed by evapn and the remaining solid Vmax 3400.the other involved. and A.5 hr at 22" the soln was neutralized with Dowex 50 (H+) resin. and A.. A mixt (4. Terao. "Aromatic Amine Oxides. Sawlewics. Roswell Park Memorial Institute. (8) W. Reisser and W. Karasawa. (OH). The org layer was sepd. Welch. Experientia.3. The solvent was evapd. 2 .168 Journal of Medicinal Chemistry. The syrupy 1." Elsevier. (3) F .3. The 4'-thio derivatives proved to be effective inhibitors of the growth o f leukemif L . Spoerri. Ser. Med. (25) M. 11. 30. and A. The org layer was sepd. washed with H. H. Chem. New York. 0. E. Vorbriiggen.542(1966). Jaffe. Amer. D.168 (1957).~ M The antibiotic toyocamycin' is an analog of adenosine in which N-7 of the imidazole ring is replaced by C. Yonehara. N.SiCl (3 drops) was heated for 0. D. and chromatogd on a silica gel co!umn with PhH-Me. (22) H. (7) F. Cheeseman. Handschumacher.J. 386 (1969). and J.CO-EtOH (1:2. After cooling to 22".401(1960). O'Donnell. It was filtered through a Celite pad. Prusoff. (11) J. and J. Chem. Bloch. 123 (1964). I n one.

Application ~ of the procedure employing trimethylsilyl derivs to pyrrolo [2.30-isopropylidene4-thio-P-D-ribofuranosyl)pyrrolo [2.341 pyrimidine (VI). As a result of substitution of S for 0 in the ribofuranosyl moiety.5tri4-acetyl-4-thio-D-ribofuranosyl chloride in dry PhMe to give 4-chloro-6-bromo-5-cyano-7-(2. and ir spectra. 3 d ]pyrimidine (Ib).“ The inhibitory activity of the analogs in the mammalian cell system was found to differ from their effect on the bacteria primarily by the fact that toyocamycin was approx 10 times more potent an inhibitor of L-1210 growth than was the 4’-thio analog or its 6-amino deriv. Dehalogenation of Ib with H2/Pd gave 4-amino-5-cyano-7-(4-thio-fl-D-ribofuranosyl)pyrrolo [2. The structure of this product was established as 4-acetamino-6bromo-5-cyano-7-(2.341 pyrimidine (11.6diamino-5-cyano-7-(4-thio-fl-D -ribofuranosyl)pyrrolo[2. 3 4 ]pyrimidine (V).4%. since treatment of 6-bromo-4-chloro-5-cyano-7-(2.faecium system.R’=NHe Treatment of 2.5-tri-O-acety1-4-thio-D -ribofuranosyl chloride with the chloromercuri deriv of 4-acetamino-6bromo-5-cyanopyrrolo[2. unlike its inactivity in the bacterial system. The effect of the 4’-thionucleosides on the growth of Sfreptococcus faecium and leukemia L-12 10 cells is summarized in Table I. the fusion procedure has been employed for the prepn of a number of pyrrolopyrimidine nucleosides in yields varying from 3 to 20% (based on the sugar ~ o m p o n e n t ) .’ Since the chloromercuri procedure had been used by us successfully for the prepn of 4’-thio purine nucleoside^. been used for the prepn of pyrrolopyrimidine nucleosides.341pyrimidine (111).R~=NH~ R=CI . the uv absorption maxima for I1 in EtOH.5-tri-O-acetyl0-D -ribofuranosyl)pyrrolo [ 2 . No.7 The susceptibility of the 6-Br of V to nucleophilic substitution is presumably due to the ability of the S atom to accommodate both positive and negative charges. The 6-Br deriv was inactive at 10m3M. Treatment of Ib with MeOH-NH. Various methods have. to furnish the 4-chloro-6amino-5-cyano-7-(4-thio-P-D -ribofuranosyl)pyrrolo [2. To evaluate the influence of certain substituents in the pyrrolopyrimidine ring structure on the yield. exhibit small bathochromic shifts relative to those obsd for toyocamycin. at 115” produced 4. Of importance for the potential chemotherapeutic use of these analogs is the finding that the active 4’-thio derivatives retained their full activity against a strain of S. 1.5. pmr.’ Similar bathochromic shifts were exhibited by the 9.6-diamino-5-cyano-7(4-thio-0-D-ribofuranosyl)pyrrolo[ 2 .34]pyrimidine (IV). The site of attachment of 4-thio-D-ribose was assigned to N-7. On the basis of the trans rule.3. and the site of ribosidation. R ’ = Br IL R=NH~. The yield of V was 25. and TsOH produced 4-amino-5-cyano-7-(2. 1972. in the past. The site of glycosidation of V was assigned at N-7 on the basis of the uv spectra.34]pyrimidine nucleosides (Scheme I).^ we attempted to apply this method to the synthesis of 4’thiopyrrolo [2. could not be completely characterized. Removal of the protecting groups from V was accomplished with MeOHNH3 at 5’. Biological.3.RI=H m X I R=NH~.5-tri0-acetyl-4thio-0-D-ribofuranosyl)pyrrolo [ 2 . which was isolated as a cryst substance by column chromatog. the anomeric configuration was presumed to be P. because of the low yield obtained (less than l%). 4’-thiotoyocamycin was approximately 10 times more effective an inhibitor than was toyocamycin. Scheme I I HgCl R = NHAc or R = CI OAc Io R = N H A c - P R=CI Hoed OH OH I b R:NH2. which was identical in every respect with the compd prepd from Ib. In the bacterial test system.4’-ThioAnalogs of Toyocamycin Journal ofMedicinal Chemistry.34] pyrimidine furnished a 20% yield of the acetylated syrupy nucleoside (Ia).34] pyrimidine and 4-acetamino6-bromo-5-cyanopyrrolo[ 2 . These properties and the procedures used for the synthesis of the compds are described in this paper. was condensed with 2. faecium resistant to lO-. 2. while the 6-amino deriv was 3 times more potent. This variance in activity constitutes a parallel to the marked growth-inhibitory activity of 8-aminoadenosine and the inactivity of 8-bromoadenosine in the S.341pyrimidine. Furthermore.3.3.3. Chemical. for example. prepd in 85% yield. 3 4 ]pyrimidine (111). 3 4 ]pyrimidine with MeOHNH3 at 110’ affected removal of the blocking groups with concomitant displacement of the 4-C1 to give 4-amino-6bromo-5-cyano-7-(P-D-ribofuranosyl)pyrrolo[ 2 . Ribosidation of the chloromercuri salt of 4-amino-5-cyanopyrrolo [2. 4-chloro-6-bromo-5-cyanopyrrolopyrimidine was used in the condensation reaction. Treatment of V with MeOH-NH. pH 1 and pH 12. Vol. 2 169 systems showed that these deriv had biol properties different from those of the parent compd.’ The heavy metal salt procedure was also used in an attempt t o synthesize tubercidin. 3 4 ]pyrimidine is the same. produced a nucleoside material which. Treatment of I1 with Me2C0. but the desired nucleoside was not obtained. 4’-thiod-bromotoyocamycin was markedly inhibitory of the tumor cell growth.M toyocamycin or to the related analog . 3 4 ]pyrimidine.34]pyrimidine. A. This finding was rather unexpected. 9. at 1 15-120’ produced 4.6 Recently. indicating the presence of a cis diol. thus proving that the site of ribosidation of the chloromercury deriv of both 4-chloro-6bromo-5-cyanopyrrolo[2. on the basis of uv absorption.4’-thio(3-D -ribofuranosyladenine ring.3d]pyrimidines has furnished mixts of isomeric nucleosides in high yield. Elemental analysis and spectral examn of the product isolated indicated that nucleophilic displacement of the 6-Br by NH2 had occurred.5-tri~-acetyl-4-thio-P-D -ribofuranosy1)pyrrolo [ 2 . The chloromercuri salt of 4-chloro-6-bromo-5-cyanopyrrolo[ 2. 3 d ]pyrimidine on the basis of its uv. B.4’-thiotoyocamycin). Deacetylation of Ia with methanolic NH3 at 5’ gave crystalline 4-amino-6bromo-5-cyano-7-(4-thio-P-D-ribofuranosyl)pyrrolo [ 2 .2-dimethoxypropane.

.5H.3' (solvent b). EtOH (50 ml) was added to 237 (17.H.5H. decompn products..N. v/v) and (b) EtOH. Anal. Pmr spectra were obtd on a assays have been published previo~sly. dried in a desiccator under reduced pressure. Optical rotations are equilibrium values 239 (20. Anal.O.6 g. 224 mp (21. The cooled soh was evapd to dryness.O) C. The soln was evapd to dryness and the residue anose) in 350 ml of dry PhMe was added.5H20) C.8 g (85.1' D (solvent a). I1 (133 mg) was dissolved in and 6 g of Celite was added. Model 337 spectrophotometer. a 10% NaOH soln was added dropwise with stirring mp (11. 0.O (8 ml) was added to the (Nujol) 2235 cm-l (C=N). N.2-dimethoxypropane (0.06.800).5" (solvent This soln was washed with 30% KI (2 X 80 ml) and once with H.N.3.002 mole) was dissolved in [a]''D -88.3%).510). was no longer evident. the mixt was stirred for an additional 30 min.4%). 1735 cm-' until a clear soln was achieved.O. N. C1.. 1.7 g of this mixt. and (38.42 g (0. .680). 228 mp concn of the filtrate to near dryness. and MeOH was added to the residue and then removed until the odor of NH.5-tetra-O-acety1-4-thio-p-D-ribofurwith 30 ml of Me.O) C. v/v) as the developing solvent sepd the nucleoside from the (C=N). Model 14 spectrophotometer.CO (10 ml).35 g (25.. (100 ml) and dried (Na. 201" dec. mp 208-210" (H. Recrystn from H. V (2. 208 (22.4201.3-d]pyrimidine (VI). S. The solvent was evapd and the residue was recrystd from H.510). 275 (sh) (18.O afforded the product in 43. and the mixt was hydrogenated at 25" at 136-137. Melting points were taken on a Fisher-Johns 316 ( E 11.5 ml). 284 (14. 210 mp (18. Method 1.15 (CCH.31 g): ?Where analyses are indicated by the symbols of the elements.02 mole) of HgCl. 288 (13. [ c ~ ] . (C. 0. 15. (pH 12) 292 (sh) (9980).) 2235 ( G N ) .3-d]pyrimidine7 (2. uv (EtOH) 293 ( E 9920).7901.004 mole) was covered with 40 ml of methanolic NH. 213 (21.5 136". EtOH (8 ml) was added. . Anal. 1755 cm-' (OAc).O: yield. mp 112-113'. The pH of the mixt was adjusted to 7. [2. H.490).02 mole) of 2.N. 36 mg. (pH 12) 289 (14.. 240 (19.4" (solvent a).O) C .P-D-ribofuranosyl yield 713 mg.S . Ia (1.9801.83 (C. Inhibition of Cell Growth by 4'-Thiotoyocamycin and Derivatives Concentration (M) for 50% growth inhibition of S.5% yield (675 mg): mp 199mixt was stirred with exclusion of moisture at 85-90" for 30 hr. yield 6. (C.3-d] was dissolved in 10 ml of MeOH. uv max (EtOH) analytical results for those elements were within *0.01 mole) and 288 ( e 12. mp 195-197" dec.. faecium resistant to Comvound S.670). (C.11 g.5-tri-O-acetyl-4-thio-a. 350 mg (87.650).180).O (150 mg). ~-103. The uv spectra and the chrobromo-5-cyanopyrrolo[ 2.810). 1765 (OAc).5H. The reaction mixt was allowed to stand at residue was dried by azeotropic distn with xylene. ir (CCl.3. 235 (9690).320).3. 0.O. Anal. Recrystn of this solid from EtOHride (3. 0. 0.~-d]pyrimidine (V). of the solvent was purified by column chromatog on silica gel with 4-Chloro-6-bromo-5-cyano-7-(2. To a mixt of 4-chloro-6ml of MeOH and evapd to yield 2.C1N503S ' H.8% of Ia: uvmax (EtOH) bromo-5-cyanopyrrolo[2. S.O mixt afforded an analytical sample: mp 202-205'.01 mole) in 40 ml of 20% EtOH were added. [aIz5D-119.670). After removal of the catalyst by filtration and 228 (34. and the mixt was evapd to dryness. and the mixt was g (0.080).28 d ( J .5-tri-0-acetyl-4-thio-cw.S.BrClN.46 g (34.. (pH 1) 285 yellow syrup. ir the residue and evapd to dryness.p-D-ribofuranosyl stirred for 1 hr. (pH 1) 292 (sh) (14. 4-Acetamino-6was recrystd from H. (C. Recrystn from an 4Chloro-6-amino-5-cyano-7-(4-thio-~~D-~bofuranosy~)py~o~oEtOH-H.3. Vol. N. 234 (14. Leukemia L-1210 4x 4 x 10-~ 6 X lo7 5 x 1F6 > 10-3 7 x 10-6 4 x 10-5 >io-.080). 6.5-tri-O-acetyl-4-thio-~Dat 115-1 20". S. pmr (CDCl. and Bloch Table I. Uv spectra were taken on a Cary defined medium free of purines but containing 1 mg/ml of folate.) 2235 (C=N). H.30 22" for 12 hr.H. 2.000). mp 134mixt of MeOH (100 ml) and Pd black (100 mg). H. and the reaction H. giving a pale ( E 10.880). uv max (EtOH) 292 ( E 12.~ that the 4'-thio derivative of 6-mercapto-9-(/3-D-ribofuranosyl)purine retained its activity against a strain of S.060). Column chromatog on silica gel with hexane-EtOAc (12.H.8101. (pH 1) 284 (12.H.064 g. 218 (16. uv max (EtOH) 294 ( E 19.. the mixt was allowed to stand at 5' for 4 hr. (satd at O"). faecium Toyocamycin Tubercidin Toyocamycin 4 '-Thiotoyocam ycin 4'-Thio-6-aminotoyocamycin 4'-Thio-6-bromotoyocamycin 9 x 10-5 7x 4 x 10-5 >io4 . Anal.5". dried by azeotropic distn with xylene. apparatus and are corrected. Solvent concn was carried out at reThe resistant strains were selected by serial transfer in increasing duced pressure in a rotary evaporator.CO (8:2.02 mole) was matographic mobilities were identical with those of the product preadded to 300 ml of H. uv max (EtOH) 293 (sh) ( E 13. crystals sepd pyrimidine (4'-Thiotoyocamycin) (11). (pH 1) 298 (15.O (1 :4.01 mole) in 100 ml of dry PhMe. 2. [ a I z 5 D -38.O mixt afforded the product in 87. 7 x 104 x 10-5 > 10-3 > 10-3 tubercidin (7-deazaadenosine). The evapd to dryness and the residue was recrystd from a H. 0.O b).090). 1972.O (150 ml).5-t~-O-acetyl-4-thio-~-D-riboPhH-Me. Dry column chromatog utilizing silica gel and CHC1.0.faecium resistant to the corresponding 6-mercapto-9-(/3-D-ribofuranosyl)purine..2.1 concn in (a) DMF(C. Whistler.). 0. and were detd on a Jasco Model ORD/uv-S at 0.O-EtOH solids were removed by filtration and washed with 200 ml of EtOAc. by filtration gave 11: yield.S.780). The soln was then evapd to dryness. ir (Nujol) 2220 cm-' ( e N ) . (satd at 0") and the reaction mixt was heated in a sealed tube for 14 hr at 115-120".580). The TsOH ' H.3-d]pyrimidine7 (5.. Removal of the crystals (C.002 mole) was covered with and Celite was then collected by filtration. S. 282 (18. v/v) as the eluent.. in 100 ml of EtOH.6-D~mino-5-cyano-7-(4-thio-~~D-ribofur~osyl)py~olo [2. atm pressure for 4 hr. and was concd to dryness at 40-45". 285 (14. H. This finding parallels the observation reported by us p r e v i o ~ s l yshowing .0901. The syrup remaining after removal of the solvent 4-Amino-5-cyano-7-(4-thio-p-D-ribofuranosyl)pyrrolo [ 2. A soln of 6. H.080).3-d]pyrimidine (Ib).15 g. No. (pH 1) 277 (11. 2. The syrup was dissolved in 50 furanosyl)pyrrolo[2.ir (Nujol) 2235 cm-' (6:4.126 g.). mp 221-222". The mixt of the 4-Amino-6-bromo-5-cyano-7-(4-thio-pD-ribofuranosy1)pyrrloHgCl deriv of 4-chloro-6-bromo-5-cyanopyrrolo [ 2. mixt: yield.4% of the theoretical values. and the reaction mixt was extd with 5 portions (20 ml each) of CHC1. added dropwise with stirring until a clear soln was achieved (approx 4-Amino-S-cyan0-7-(2. was was triturated with EtOH.H. Ib (505 mg) was added to a which were collected by filtration: yield.. J . The remaining solid material To 7. 120 mg (80%).5a mixt of Me.2" (solvent a). H. [aIz5D-15. Whether these observed biol differences between toyocamycin and the 4'-thio derivs result in an improvement of their toxic properties remains to be detd.3.S~ 0.7%).070). and to this mixt 10%NaOH soln was pared by method 1.CO. Ib (60 mg) was covered with 5 ml of MeOH-NH..2 g (19.O: yield. residue.341pyrimidine (111).~ S.95. 8. 281 mp (14.690). washed with H. 298 H.530). satd at 0") was added.H. S.SO. (satd at 0") and allowed to stand at 5" for 15 276 (sh) (14. (0.H).58 g.O. H. allowed to stand at 5' for 24 hr. N. The techniques used for the microbial Elmer.72 g. (pH 1) 314 (10. uv max (EtOH) 297 (sh) It was worked up as described for the prepn of Ia. 50 ml of MeOH-NH.S) C. IV (1.5501. N. The cooled soln was evapd to dryness and the residue ribofuranosy1)pyrrolo [2. S . H. anosyl)pyrrolo[2. Method 2.1% yield (0.BrN50. Then. was added. Anal.2%).2001. 237 (19. then 450 mg of KHCO. N.-MeOH (4: 1. 213 mp (23. The solids were removed by filtration and washed chloride" prepd from 1.7901. and crystals were collected by filtration: chloadded a soln of 2. = 7. After 5 hr at So.6 g of Celite and a soln of (NHAc).960).280). Recrystn from MeOH afforded an analytical sample: mp ml. ir (CCl.3-0isopropylidine-4-thio-p-D-ribofur8 ml). 205 (17. The syrup remaining after removal 234 mp (12. and after 4 hr the cryst compd was collected by filtration. A soln of 5.O) C.170 Journal ofMedicina1 Chemistry. Experimental Section? (satd at 0") and the reaction mixt heated in a sealed tube for 14 hr 4-Acetamino-6-brmo-5-cyano-7-(2. 0. 0. 2.340).341pyrimidine (Ia).860). (2.3-d]pyrimidine (IV).3-d] pyrimidine [2. Ir spectra were detd on a PerkinBiological Assays.____-.510). MeOH-NH. 4. and 8 with 10% NaOH soln.) 1. hr.O.5 HgC1.140). v/v) as the developing solvent resolved the nucleoside and decompn products. faecium was grown in a Varian A-60 instrument (Me$). A mmp showed no depression.O and 50 ml of MeOH-NH. 283 (14.O). The solvent was was stirred under exclusion of moisture at 90-95" for 5 0 hr. and Hz). 0. 2 Bobek.

Amer. A carbocyclic analog. Bloch. Chem. Robins. R. Chem. Amer.to ninefold. Fukuoka. Synthesis and Antimicrobial Activity of a Carbocyclic Puromycin Analog. and F. B. C.343 (1961).-alanylamino ) ]cyclopentyl)purine? Susan Daluge and Robert Vince* Department o f Medicinal Chemistry. This study was aided by grants CA-12585 and 12422 from the U. R. Med.arbocyclic h r o m y c i n Analog Journal ofMedicina1 Chemistry. and L. j ~ ~ ?This work w a s generously supported by Grant AI 08142 from the US. Puromycin (l). 87. have precluded the use of puromycin in the treatment of human or animal infectious diseases or neoplasm^. Townsend. The cultures are incubated at 37" for 40 hr. and it has been demonstrated that the antibiotic causes premature release of the polypeptide chains from the ribosome. Sameyoshi. Org. 6-Dimethylamino-9. 56. A. J. However. J. 9. W.Wilson. ( 3 ) M. Amer. with an average viability of 99%. The excellent technical assistance of Miss Ginger Dutschman and Mr. K. The removal of the 5'-OH group from puromycin and its analogs would be desirable from a toxicity standpoint. 1..I (1966). E. and N. No. K. 13. and A. L. puromycin has been used extensively as a tool in the investigation of protein biosynthesis. (5) E.. Ohkuma. Heterocycl. Heterocycl. R.. Shinaoka. Urbas and R. Tokuzen. Robins. University of Minnesota. Hendess. C. 60-62 (1956). Taylor and R. Maue is gratefully acknowledged.5658 (1964). Tolman. Nichol.34 (1964). L. Chem. Le. L. Katagiri. (11) A. J. it may be possible to modify 1 within the region outlined by the dotted line and still retain the activity of the antibiotic. (1968). 52. the classical nucleoside compounds introduce two undesirable structural features into the puromycin analogs which have not been demonstrated as essential for biological activity. Antibiotics. Whistler. 86. Goodman.. K. the furanosylo and the 5'-OH group. Soc. Received July 23. after which the viable cells are counted by trypan blue exclusion. Also. all of these structures have been of the classical nucleoside type in which an N-substituted amino sugar is attached to a purine or pyrimidine ring through a glycosidic linkage.2102(1969).''* This difficulty could be circumvented by replacing the furanosyl ring with a cyclopentyl system which sterically simulates the sugar moiety and provides a hydrolytically stable C-N bond." It has been s u g 1 A variety of analogs and isomers of puromycin have been prepared to define the structural requirements for inhibition in an attempt to further understand its mode of a c t i o n . J. Chem. M. Bloch. Bobek. Ass. and R. (8) R. Ser. K.. 6-dimethylamino9-(R-[ 2R-hydroxy-3R-(p-methoxyphenyl-~-alanylamino)lcyclopentyl )purine (2). R. Robins. Soc. The in vitro antitumor assays were carried out by our microassay technique which involves the introduction of 0. Tolman. A .. Chem. L. was synthesized and evaluated for antimicrobial activity. W.Public Health Service and T 4 3 6 from the American Cancer Society. Acknowledgment. 31. Minneapolis.Public Health Service..^ The nephrotic syndrome results from small amounts of aminonucleoside produced by the hydrolytic removal of the amino acid moiety from administered p u r ~ m y c i n .. (6) C. Gueffroy. 411 (1970). J. including renal lesions. Rep.813 (1966). Cancer Chemother.. Whistler. 1972..' For this reason. 7. (10)E.' has been found to inhibit protein synthesis in a wide variety of organisms. K. Minnesota 55455.799 (1970). L. A . many nucleosides which may be effective chemotherapeutic agents become ineffective in vivo because they are rapidly destroyed by cleavage into a purine or pyrimidine and a carbohydrate moiety. (12) B. followed by 0.J. Soc. Vol. ibid. J. and L. R. D. Kozaburo. (7) R. College of Pharmacy. Robins. Nishimura. (2) K. and L.{ R. J.219 (1965).[ 2R -hydroxy-3R-( p-methox yphenyl-r. G. Cancer Res. R. Toxic manifestations. an antibiotic with antitumor activity. and also circumvented the nephrotic syndrome associated with puromycin by releasing a nontoxic aminonucleoside upon hydrolysis. During this time the cell number in the controls increases approximately eight. Amer. The carbocyclic analog exhibited antimicrobial activity comparable to puromycin. Chem. 91. Its structure has a striking resemblance to that of the aminoacyl-adenyl terminus of aminoacyl-tRNA. S.36 The difficulties encountered in preparing 3aminoribosyl nucleosides have severely limited the availability of these compounds. References (1) H.301 Illus. Proc. Thus. I971 An assessment of the requirement for the furanosyl 0 and the CH'OH moiety in the puromycin molecule was undertaken by the synthesis of a novel puromycin analog. 15. Mayama. E.Noell and R. Whistler. Ser.Recent ~ studies demonstrate that the aminonucleoside is first monodemethylated" and subsequently converted to the 5'-nucleotide. L. (4) W. J. Tolman. L. Chem. The diastereoisomer (19) of 2 was also isolated and found to be devoid of antimicrobial activity. 2 171 concns of inhibitor. Since ribonucleosides are easily cleaved hydrolytically or enzymatically. . S. Reist.5 ml portions of medium contg 3 X lo5 L1210 cells. Townsend. 1995 (1965). B.5 ml aliquots of the medium (RPMI 1630 + 10% calf serum) contg the various concns of the analog into 16 X 125 mm screw cap culture tubes. J. G a m . (9) M. 14.Mihich. 7..

Med.. 2009 More About This Article The permalink http://dx. 1155 Sixteenth Street N.Synthesis and antimicrobial activity of a carbocyclic puromycin analog. 171-177• DOI: 10. 15 (2). DC 20036 .org on April 25. Washington..acs.1021/jm00272a012 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society.org/10. 6-Dimethylamino-9-[R-[2R-hydroxy-3R-(pmethoxyphenyl-L-alanylamino)]cyclopentyl]purine Susan Daluge. Chem.1021/jm00272a012 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.doi.W. and Robert Vince J. 1972.

Sameyoshi. 6-Dimethylamino-9. Thus. have precluded the use of puromycin in the treatment of human or animal infectious diseases or neoplasm^. The cultures are incubated at 37" for 40 hr. (1968). Ser.. R. R. K. Its structure has a striking resemblance to that of the aminoacyl-adenyl terminus of aminoacyl-tRNA..36 The difficulties encountered in preparing 3aminoribosyl nucleosides have severely limited the availability of these compounds. Whistler. J.5658 (1964). 1972. the furanosylo and the 5'-OH group. (11) A. Robins. Soc. Received July 23. and L. A . Vol.Public Health Service and T 4 3 6 from the American Cancer Society. 1995 (1965). L. D.. and N. Urbas and R.. 13. W.Noell and R. Fukuoka. E. W. Minneapolis. (9) M. 14. S.''* This difficulty could be circumvented by replacing the furanosyl ring with a cyclopentyl system which sterically simulates the sugar moiety and provides a hydrolytically stable C-N bond.5 ml aliquots of the medium (RPMI 1630 + 10% calf serum) contg the various concns of the analog into 16 X 125 mm screw cap culture tubes. K.' has been found to inhibit protein synthesis in a wide variety of organisms. A . J. 87. Amer. J. 86. Since ribonucleosides are easily cleaved hydrolytically or enzymatically..343 (1961). 9.Mihich.813 (1966). was synthesized and evaluated for antimicrobial activity. L. C. Gueffroy..5 ml portions of medium contg 3 X lo5 L1210 cells. (10)E. followed by 0. and L. Robins. B. Amer. 15.{ R. and L.^ The nephrotic syndrome results from small amounts of aminonucleoside produced by the hydrolytic removal of the amino acid moiety from administered p u r ~ m y c i n . The carbocyclic analog exhibited antimicrobial activity comparable to puromycin. (8) R. with an average viability of 99%. Mayama. Org. L. Whistler. ibid. (12) B.. 411 (1970). No. Le. During this time the cell number in the controls increases approximately eight. Puromycin (l).[ 2R -hydroxy-3R -( p-methox yphenyl-r. Ohkuma. G a m . and F. Cancer Res. Bobek. Chem. I971 An assessment of the requirement for the furanosyl 0 and the CH'OH moiety in the puromycin molecule was undertaken by the synthesis of a novel puromycin analog.. Shinaoka. Maue is gratefully acknowledged. an antibiotic with antitumor activity. (5) E. J. J. Ser. Heterocycl. it may be possible to modify 1 within the region outlined by the dotted line and still retain the activity of the antibiotic.Public Health Service. B. A.J. and it has been demonstrated that the antibiotic causes premature release of the polypeptide chains from the ribosome. 7. Robins. J. Heterocycl. The diastereoisomer (19) of 2 was also isolated and found to be devoid of antimicrobial activity. The removal of the 5'-OH group from puromycin and its analogs would be desirable from a toxicity standpoint. Tokuzen. Proc. R. J.2102(1969).219 (1965). 56. Also.Tolman. Robins. A carbocyclic analog. puromycin has been used extensively as a tool in the investigation of protein biosynthesis. Acknowledgment. Chem.301 Illus. Soc. Antibiotics. L. E.799 (1970). L. and A.. Tolman. M.. Chem. References (1) H. (2) K. Whistler. many nucleosides which may be effective chemotherapeutic agents become ineffective in vivo because they are rapidly destroyed by cleavage into a purine or pyrimidine and a carbohydrate moiety. The in vitro antitumor assays were carried out by our microassay technique which involves the introduction of 0. J. all of these structures have been of the classical nucleoside type in which an N-substituted amino sugar is attached to a purine or pyrimidine ring through a glycosidic linkage. ( 3 ) M. Townsend.Recent ~ studies demonstrate that the aminonucleoside is first monodemethylated" and subsequently converted to the 5'-nucleotide. K. Chem.I (1966).Tolman. 31. R. R. Chem. Cancer Chemother. Synthesis and Antimicrobial Activity of a Carbocyclic Puromycin Analog. (4) W. and R. 52.-alan ylamino ) ]cyclopentyl)purine? Susan Daluge and Robert Vince* Department o f Medicinal Chemistry. Amer. Hendess.. G. College of Pharmacy. and also circumvented the nephrotic syndrome associated with puromycin by releasing a nontoxic aminonucleoside upon hydrolysis." It has been s u g 1 A variety of analogs and isomers of puromycin have been prepared to define the structural requirements for inhibition in an attempt to further understand its mode of a c t i o n . Amer. L. 7. Townsend. Bloch. However. . Taylor and R. C.34 (1964). Reist. Chem.Wilson. (7) R. (6) C. 60-62 (1956). 2 171 concns of inhibitor. the classical nucleoside compounds introduce two undesirable structural features into the puromycin analogs which have not been demonstrated as essential for biological activity. including renal lesions. R. The excellent technical assistance of Miss Ginger Dutschman and Mr. University of Minnesota. Chem.arbocyclic h r o m y c i n Analog Journal ofMedicina1 Chemistry. Katagiri. Bloch. 1. j ~ ~ ?This work w a s generously supported by Grant AI 08142 from the US.to ninefold. Soc. J. Ass. Med. Goodman. S. Minnesota 55455. Nichol. K.' For this reason. K. Kozaburo. Rep. after which the viable cells are counted by trypan blue exclusion. 91. Toxic manifestations. 6-dimethylamino9-(R-[ 2R-hydroxy-3R-(p-methoxyphenyl-~-alanylamino)lcyclopentyl )purine (2). L. Nishimura. This study was aided by grants CA-12585 and 12422 from the U.

in dioxane by the method of Vander Werf. R = Ac 14. or transketalization in acetone with pTsOH gave only recovered 10. I 9 72. Vol. cryst solid.l5 and the resulting azide reduced catalytically to crystalline trans-3-amino-2-hydroxycyclopentanone ethylene ketal (7) in 76% yield (from 5 ) .§ The proximity of a protonated $The corresponding bromohydrin of cyclohex-2-enone ethyleneketal prepd b y this method was a stable. In view of these observations. 8 Unpublished results. R = H 13a. The resulting bromoh drin 4 decomposed on storage and was not characterized.. R = n 19a. No.OH'HCl 0 HON 8 OH AcON OAc 9 10 11 12 12 1 . The epoxide was opened with NaN. The ketal function of 10 proved unusually resistant to hydrolysis. 15.H. mp 96-98'.6-dichloropyrimidine. The carbocyclic analog (2) of puromycin was synthesized by the route shown in Scheme I. which was characterized. .8. The requirement for the 2'-OH in puro- mycin has not been demonstrated. Attack by N3. R = cbz gested that this 5'-nucleotide is responsible for the nephrotic syndrome.9 Hz). (Structures 318 depict only one enantiomer of the racemic form actually obtained. as a stable colorless liquid. unpublished results. 2 Daluge and Vince Scheme I Et. epoxide 5 was formed in 73% yield (from 3 ) .rWhen bromohydrin 4 was immediately treated with NaOH in refluxing benzene. I 1 . The purine moiety was formed via a standard method. THF Ac.17 Condensation of 7 with 5-amino-4.O 0 dioxane EtOH 3 4 5 6 7 NMe. the antimicrobial activity of the carbocyclic analog. This difficulty has been encountered in the hydrolysis of other ketals and acetals of similar purine derivatives.O-H. R = Cbz OMe z R=H Z .R= H 14a. it is required for the acceptor activity of aminoacyl adenosines12 which are thought to bind at the ribosomal site in the same manner as puromycin. et al.) Cyclopent-2-enone ethylene ketal (3)13 was treated with NBS in Et. we decided to synthesize the carbocyclic analog 2 which retains the structural reauirements that have thus far been demonstrated to be esskntial for activity. 2.O-H. Attempted hydrolysis by refluxing HC1 at pH 3 . However. R = Ac CbzCl Ba(0H) l 3 Tq++ DM F HN cbz 17 I o n DMF W HN)(o 0 18 :-> 15 co RHNfH I RHNfH d 6 HN OH co I OMe 19. and H-3 appears upfield as a multiplet at T 7. 2 represents a new class of puromycin analogs which should produce a nontoxic aminonucleoside upon hydrolysis of the amino acid moiety. B. NH. I NMe. . Thus.. refluxing aq NH4Cl.36. Chemistry. followed by ringclosure of the resulting crude pyrimidine 8 with triethyl orthoformate in the presence of EtS03H gave the 6-chloropurine 9 in 84% yield (from 7).O-p yridine 13. In addition.at C-3 of epoxide 5 would be expected.O by a modification of the procedure of Guss and RosenthalI4 in which NaHC03 was used to buffer the reaction mixture. would provide information relating to the 'and 5' oxygens of purostructural requirements of the 4 mycin for biological activity. The 6-dimethylaminopurine 10 was formed in 75% yield when 9 was treated with refluxing Me2NJ3.53 (J = 7.I6 The structure of 7 was confirmed by the nmr spectrum in which H-2 appears as a doublet at T 6.172 Journal of Medicinal Chemistry.

unpublished results.121) m/e 300 (M . The mass spectra of 2 and 19 are almost identical. Even in the presence of HONH2*HCl. et a123The resulting carbobenzoxy blocked diastereomers 2a and 19a (97% by method A. but that below pH 1. In particular. As it a p peared that conditions necessary for hydrolysis of 10 effected decomposition of the product(s). Since attempted separation by chromatography or recrystallization resulted in considerable losses. HONH2 appears to prevent decomposition.24the fragmentation is dominated by the aminoacyl moiety. were separated by chromatography and characterized. It is hoped that further studies now in progress on the diborane reduction of analogs of 10 will provide information on the mechanism of this highly stereospecific reduction. The cis stereochemistry of the H2N and OH groups was further confirmed by facile formation of the cyclic carbamate 18 on treatment of the carbobenzoxy derivative 17 with NaOMe in DMF. partial recovery of 10 (69%) and considerable darkening of the reaction solution resulted.20 This mixture was acetylated in Ac20 and the resulting acetamides.HCl along with sufficient NaOH to give a pH of 6.18) and prominent peaks at m/e 318 (M . and there is a minor (M . the cyclohexyl analog of 12. An aq soln of 10 and a 5-fold excess of HONH2 -HC1 adjusted to pH 1 with HCl was warmed (75") for 3 hr.18) #In the nmr (DMSO-d. Identical conditions at a pH of 3 gave only recovered 10.0 Hz for 11 and 9. probably by direct attack on the resonance-stabilized carbonium ion formed on ring . Similarly. 1972.I?. However." It thus seems likely that the protonated purine moiety accounts for the difficulty of hydrolysis of compounds such as 10. The two coupling methods resulted in samples of 2 and 19 with identical optical purities. a modification of the mixed anhydride method suggested by Anderson.121 __ _______-- **Further details concerning this reaction will be published. the appearance of H-2 as a doublet (J2. dark tar and a lowered yield of oxime (43%) were obtained if a pH slightly lower than 1 was used. could be useful in the synthesis of amino sugar nucleosides.5 is reached). As with puromycin. . 2 113 amine has been found to hinder acetal hydrolysis. Vol.) of trans-2-acetoxy-3-(6-dimethylamino-9puriny1)cyclohexanone 0-acetyloxime. differing slightly only in the relative intensities of some ions. and then adding HONH2. The diacetyl derivatives 13a (39%) and 14a (3%) were isolated when the mixture of amino alcohols from reduction of 12 was treated with Ac20-pyridine. characterized as its AcOH salt 16. the dicyclohexylcarbodiimide-N-hydroxysuccinimide method21'22and B. The AcNH and OH groups were shown to be cis in 13 and trans in 14 by acyl migration studies (see Experimental Section).opening of the protonated ketal. 77% by method B) could not be separated.121 . treatment with Amberlite IR-120 resin at 60" for 2 hr led only to decomposition. 3.77. separation of diastereomers 2 and 19 by chromatography was possible.3= 9. comparable with the overall yield via 13 and 16.Carbocyclic Puromycin Analog Journal ofMedicina1 Chemistry. in the molecular ion M Iaccounts for the m/e 289 (M . if general. The 0-acetyl oxime 12 was reduced to a mixture of amino alcohols with diborane in THF by a modification of the procedure used by Feuer and Braunstein for the conversion of cyclohexanone 0-acetyloxime to cyclohexylamine. Cleavage of the benzylic bond followed by loss of H20 accounts for the base peak at m/e 300 .150) peak. and 13a was converted to 13 on treatment with NH3 in MeOH. The molecular ion (m/e 439) is relatively small. giving a yield of 2a and 19a. The diborane reduction of 10 appears to be the first reported example of the reduction of an oxime to an amine in the presence of a purine ring.18) peak due to loss of H20 involving the OH group. Fission of the bond a to the C=O B HNH OH I .121) and m/e 121.1. The nmr spectra of 11 and 12 confirm the structures shown. oxime 11 was synthesized directly from 10 under what appear to be unique conditions for oxime formation.18) ion is somewhat less abundant for puromycin. When 10 was refluxed in HC1 at pH 1. the mixture was acetylated without separation. Following hydrogenolysis of the Cbz group. There is a metastable peak for the transition 3 18' + 300+t 18. These results suggest that hydrolysis of the ketal of 10 requires a pH lower than 3.** The carbocyclic aminonucleoside analog 16 was coupled by to N-benzyloxycarbonyl-pmethoxyphenyl-~-alanine~ 2 methods: A. Tlc of 11 indicated it t o be a mixture of syn and anti oximes. On neutralization of the colorless reaction solution.150) m/e 439 (MI) B B B Hi I OH HN OH HN I ?=O . No.5 Hz for 12) considerably downfield from the H-3 multiplet# indicates that the oximation conditions did not result in isomerization via an enediol. pure 11 precipitated immediately in almost quantitative yield (solid forms as pH 3. giving a 70% yield of a chromatographically homogeneous diacetyl derivative (12). Hydrolysis of 13 with Ba(OH)2 gave a carbocyclic analog of the puromycin aminonucleoside.) m/e 318 (M . u c=o CH I I c=o CH I I m/e 439 (M. This would be expected as the puromycin (M . Predominant attack of ~~~ ~~~ hydride from the purine side of the cyclopentyl ring.+ CH-NH. In a rather narrow pH range. when oxime formation was attempted by warming a solution of 10 at pH 1.121 . The (M . after chromatography. The mixture of amino alcohols from the reduction of 12 was also coupled to N-benzyloxycarbonyl-pmethoxyphenylL-alanine by method A. . H-2also appears as a doublet ( 7 .'* The neighboring OH would not be expected to affect the rate of hydrolysis of the ketal significantly. 10 or its hydrolysis products is unstable and decomposes rapidly before hydroxylamine may attack. I B I HN OH c=o+ I/ e= + m/e 289 (M . 15. black tar formed on neutralization.B= 10. 13 (45%) and 14 (4%).5 Hz) downfield from the H-3 multiplet at 7 5.121 . Structure 2 is assigned to the diastereomer having [aI2'D of -83" and structure 19 to the diastereomer having [(uI2'D of -8" (see Results and Discussion).

Experimental Section?? 6-Oxabicyclo[3. equipped with a direct inlet probe.* Best overall yields of epoxide were obtd by using the crude oil immediately. are as follows (mM):Staphylococcus aureus (NRRL B-3 13). with a Varian A-60D spectrometer. Analytical results are within f0.. A growth curve for S. Merck. Escherichia coli (NRRL B-2 10) 0.: .4% of the calcd values.060 and 0.485. The aq layer was extd with addnl Et. Bacillus subtilis (NRFX B-549. 190 (B t 28). 2% MeOH-CHCI. this oil was partially solidified from petr ether (bp 30-60°).O (2 X 100 ml). Vol. This carbocyclic analog and others which are under preparation are being evaluated for in vitro inhibition of protein biosynthesis in an attempt to explore the requirements for binding to ribosomes. 20% MeOH-CHCI.1.O (240 ml) were stirred vigorously for 6. Preliminary studies with 2 and 19 on an E.0]hexan-2-one Ethylene Ketal ( 5 ) . The mixt was filtered.0 mmoles).244. In addition. In another run. 0. nmr. at which time all of the solid had disappeared and the pH was approx 7. (37. TICwas run on silica gel (Eastman chromagram sheets with fluorescent indicator) in these solvent systems: A.120.114 Journal of Medicinal Chemistry. which incorporates the ribose ether that replacement of 0 by CH2 results in a shift of 2 mass units to (B t 28)...5 hr. D. Thus. Celite is a diatomaceous earth product of Johns-Manville. Fission of the bond between the aminoacyl group and the cyclopentyl ring accounts for the m/e 228 ion. There is a metastable peak for the transition 300’ + 228’ t 72. 1972. - . e. Prep tlc was done on 20 X Z O cm glass plates coated with 2 mm of silica gel F 254 (E. These ions. and 134 (B t H . Such a lag period is consistent with the mechanism of action of puromycin2 since the antibiotic would be expected to be consumed as it is incorporated into the growing peptide chains.: & -t . 1 5 % MeOH-CHCI.-& - 0 24dnU Results and Discussion Antimicrobial testing of diastereomers 2 and 19 revealed that 1 isomer was completely inactive while the other exhibited growth inhibition on the same order of magnitude as puromycin.485 and 0. 2 Daluge and Vince c ~ mle 228 mle 245 mle 271 0 4 / r - 0 06lnM 0- 18) ion has several favorable routes for further fragmentation which these carbocyclic analogs lack. with a Perkin-Elmer 237B spectrophotometer.030 and 0.O layer then sepd. The aminonucleoside 16 was tested for nephrotoxicity in rats at a dose of 33 mg/kg under the same conditions that are required for puromycin aminonucleoside to cause severe nephrotic syndrome at 15 mg/kg. aureus in the presence of different concns of puromycin or the carbocyclic compound is illustrated in Figure 1. (4. 1200-1040 cm-’ (CO). or corresponding ones.O (240 ml). B. 0. Optical rotations were measured at ambient temps with a Perkin-Elmer 141 automatic polarimeter. Evapns were carried out in vacuo with a bath temp of less than 45’ unless otherwise noted. 50-eV mass spectra.’. the removal of the CHzOH moiety is not detrimental to activity and at the same time provides 2 with a resistance to kinase activity upon re- lease of the aminonucleoside. As with puromycin.g _. low-resolution. with a Hitachi Perkin-Elmer RMU-6D mass spectrometer (ion source temperature 250°.. loss of the elements of CHzO involving the 5’-OH.36 g).. The minimum inhibitory concns by a 2-fold serial dilution test in broth for puromycin and the carbocyclic analog 2. giving gummy white crystals: mp 42-47”.. The absolute stereochemistry of the active isomer has tentatively been assigned that of structure 2 on the basis of its biological activity and in accordance with the stereochemistry of puromycin. 0. Klebsiella pneumoniae (NRRL B-l17). 10%MeOHCHCI. ir (Nujol) 3450 (OH). 0. NaHCO. giving a forerun whiqh ir showed to be a mixt of 3 and unketalized material (1. are prominent in the spectra of adenosines” and are also abundant in the spectrum of puromycin.20 g. Evapn left crude bromohydrin 4 as a yellow oil (70 g). as indicated by an appropriate metastable peak. This resistance to phosphorylation circumvents the nephrotic syndrome associated with puromycin aminonucleoside.CH3N). the ribofuranosyl ring can be replaced with the more hydrolytically stable cyclopentane ring without loss of activity.300 mole). AR. 15. Other fragmentations directed by the purine moiety account for ions of m/e 206 (B t 44).g. Et. respectively. The combined Et. No.. Crude 4 was dissolved in PhH (600 ml) and refluxed with powd NaOH (36 g) for 1.O layers were washed with satd NaCl and dried (CaSO. uv.26 No nephrotoxicity was observed even after 17 days of treatment with 16. Darmstadt) and column chromatog on silica gel (Baker.0 hr. This solid could not be recrystd and darkened on standing. coli ribosomal system are consistent with the antimicrobial activities. C. The details of these experiments will be the subject of a future paper.40 g. It is consistent with the structure proposed for the (B t 30) ion in adenosines.. 50. . The novel puromycin analog 2 provides a molecule with the structural features required for puromycin-like antimicrobial activity. The aq layer was satd with NaCl.85 g. 0.). NBS (53. and the Et. Fission of the m/e 300 ion on either side of the amide carbonyl accompanied by proton transfers accounts for the m/e 245 and 271 peaks. _- -~ . Cyclopent-2-enone ethylene ketal (3)’. A lag period is observed with both compounds when the concns are lower than those required for complete inhibition. The method of prepn of 4 is a modification of that of Guss and Rosenthal. 162 (B). Evapn of the combined PhH soln and wash left a pale yellow liq which was distd.300 mole)..060. the m/e 300 peak further fragments to give a prominent m/e 164 (B t 2H) peak characteristic of the dimethylaminopurine moiety. and the black solid washed with addnl PhH (200 ml). E. ir.accelerating potential ISOOV). 5 % MeOH-CHCl. ??Melting points were detd on a Mel-Temp apparatus and are corrected. with a Cary 14 recording spectrophotometer. 60-200 mesh).244 and 0. Solid samples were dried in vacuo (<1 mm) at 56’ before analysis. and H. 163 (B t H).

. 5.3 and the singlet at 7 -0. Anal.).. ethylene 1555 cm-' (purine). 20. Evapn left a mole of 5 gave a 54% yield of 7 (after crystn from PhH).50 (d. Vol.57 (d. 0 zH.). trans-2-Acetoxy-3-(6dimethylamino-9-purinyl)cyclopentanone 0-Acetyloxime (12).) gave fractions Ethylene Ketal (10).77 (s. in THF layer.53 (d.. Contd elution with 3% MeOH-CHCl..75 hr. 5. tlc indicates $$Spectra of this solid (uv. mp 164. a meter).). which crystd Ketal(9). 0. H. 1972.0 mmoles) in H. nmr (DMSb-d. Elution with 2% MeOH-CHCIt (2 1.).) 7 6. 32. Anal. After evapn. which crystd from EtOAc (1.N.00 g (2. The (44. Rf 0. 2. 13. and the mixt was cooled. and elemental analysis (C. followed by fractions contg Me. 6. The white sample. et al.0 hr. amide).H (1. J = 4.1640 (amide C=O). (2.4. 1. The resulting mixt was refluxed with purine (14). 99%).0 hr. Anal.) gave 13 as a pale yellow glass mmoles) in n-BuOH (160 ml) was refluxed under N..H. 75%). (1.0 Hz.85 g. Rf 0. At this time solid had formed.6 (m) overlected and washed with hexane (100 ml).5-7.84 g. Anal.33) and numerous minor contaminants (CDCl.). purine H-2 and H-8 and NHC=O). = 9.9 Hz. The doublet at 7 4.). giving 11 (8.O. and the aq layer was extd with addnl dioxane (50 ml). giving the analytical sample H-2 and H-8). and nmr as expected.9 . The mixt was cooled (5") for Ac. appeared immediately on addn of D. Separation of (+-)-9-[@(3a-Acet5 (2.O. H. the EtOH was evapd.72 (both s) overlapped by 1.78 mmoles) of 12 was stirred in NaOH.5-164'. N(CH. N(CH. mp 151.68 disappeared on addn of D.NH (100 ml) was refluxed 3. (1..5-2. nmr (DMSOd. ir and nmr were as expected.47 g.5 mm) left 6 as a pale yellow gummy for 12.) (C. H-3). The THF was evapd (25'.01 (s. mp 171-171. at ambient temp for 15 hr.30. 6.1-5.~. redn.18 was dissolved in Ac. 15.) trans-3-(6-Chlor~9-purinyl)-2-hydroxycyclopentanone Ethylene gave a pale yellow glass (290 mg). 1. 265 Elution with 3% MeOHCHC1.0 hr combined dioxane layers were washed with satd NaCl(50 ml) and under N. Such a Hz. (C.5-2.0-7.75 (both s.50 in solvent B. redn of 1. formula of C. 7. No. 7.46 g). nmr Rf 0. 4.C=O). deterioration.5. and Et..0 Hz. 6. This gummy solid (C.0 (measured on noted for 13.). H.O. 1. Crystn from EtOAc (500 ml) gave tan needles (22. ir (Nujol) 3300 (OH). ir ion of 332.) 7 8. 4. H. Anal. 2 CH. NH. ir (CHC1. 7 8. N(CH.O.1-4. NH. After filtration.O (140 ml) was added amide NH required several hours. (2.76 disappeared. NH). The solid was collected. Evapn (25'. The soln was stirred at 70-75' The diacetyl derivatives 13a and 14a were obtd when the mixt for 3. mp 98-99' (10 mm).8 sharpened to a doublet at 7 4. prep tlc and appeared to carbonate on standing.37 g. Crystn from F%H gave 7 as pale yellow crystals (2. tlc still shows 2 spots.. mp 152-154'. Crystn of this solid from EtOAc gave 14 as white crystals (159 (CaSO. C=NOH).) uv.O could account for The analytical sample of 11 was prepd by crystn from EtOActhis minor product. ir identical with that of the analytical 3100 (OH. followed by 5 (31. 2 175 which was cryst from EtOAc. and the multiplet at 7 5.66 (both s.). 32.1 of 9 as white needles.28 in solvent and extd with EtOAc (3 X 100 ml).H.8-1.Carbocyclic Puromycin Analog Joumal ofhfedicinal Chemistry. gummy solid (3.). purine H-2' and H-8').). Anal. To a stirred mixt of 10 (10.8 (m. and H-3').0 hr at ambient temp. 3. The basic eluent (500 ml) was mp 97-98'.95 g. 1562 cm-' (amide). mp 208-209' dec. 3.0 g) was added to the filtrate.2-4. 3. H-1'.35 in solvent B. mp 15 1-152'.25 hr.. followed b y redn of the N-acyl moiety and subsequent acetylation of treatment with Ac.2 mm) left a brown oil (42 g) contg 8 which was 45% from 12). (m.73 and 1.) 7 8.1550 (purine. l . mo 156-158" :Rf 0. in contrast to the rapid exchange 2 N HCl (approx 28 ml) dropwise until the pH was 1. 0.3200.4 mmoles) in Ac 0 (200 ml) was stirred at 60-65' for 4.) 7 8.N.16 g.63 (d.) 7 8.03 g. and air-dried. 1.5' . Anal. 88..52 (s. and it was stirred ir (KBr) 3300-3050 (OH. J % .O. 1.) The method of prepn of 6 is that of Vander Werf. Recrystn of such a sample from EtOAc gave the analytical (CHCI.08 (s) and DMSO-d. 5.3 (m.) C. e.. a mass spectrum molecular a mixt of syn and anti oximes. hexane. 7.1 vigorous stirring for 48 hr. 1610. OH). 4. White crystals of 10 were collected mg.2CH2 and CH.). Evapn (SO".38 g. (C..0 (m. nmr (DMSO-d.3-6.6. 3. 2CH. Evaun left several hours.8 having greater Rf values.7-6. 6. 0. purine trans-3-Aminp-2-hydroxycyclopentanone Ethylene Ketal (7). 2CH.57 disOxime (11). N. A soln of 9 (3.39 in solvent C. The soln was concd to 20 ml only 14 as an amorphous white solid (247 mg).. and ir as expected. mp 202-204" dec.0 mmoles) in aq 25% contg both 13 and 14 (124 mg. 1600. After cooling the reaction soln (ice solid (3.).09 g). 5-amine4. The EtOAc ext was dried B. approx 130 mequiv of hydride) over a period of 1.20 mmoles).6-difrom EtOAc-hexane to a white solid (64 mg).5-7. giving 12 as white crystals (8. and white solid began to ppt. Redistn of fingerprint region from that of the analytical sample.(10. ir differs slightly in the (7 mm).42 g). ir.74 and 1.ir (KBr) 3250. 2.NO. washed with H.91 g) which ir showed to contain no and passed slowly through a column of 100 ml of Amberlite IRAazide.O. Evapn left a tan solia .1 (m.4-5. NH). 3%). overSamples of 5 (distd once) were stored for months at 5' without laps DMSO-d. 400 resin (OH-) packed in MeOH. (C.CO).7-5.0 hr.OH. N. mp 181-183". The soln was stirred for an addnl 3.O (50 ml). 1. 6.tt ir. A amido-2a-hydroxy)cyclopentyl]. nmr (DMSO-d.08 (s) and DMSO-d.Cl) C. Rf 0.5-88" 70%). ir as expected. ( C ~ 4 ~ o N 6 C.). and OH). (7. N.dimethylaminopur~e (13) qnd soln of NaN.63 g. N) suggeSt a molecular differs slightly in the fingerprint region from the analytical sample. which tlc in solvent tion of such a sample at 90-100" (0. 73%). (C.N. lapping 8. H-2'. bp such a sample 3 times from FtOAc gave 12 as white prisms. The multiplet at 7 4. (s.Hi. N(CH. 1660. The tan solid was col(purine).5 with 6 N from the BH. ir identical with that of the analytical sample.O and stirred at 60' for 1. Rf 0.$$ chloropyrimidine (14.O (8.3.OJ C.98. Hexane 1555. 1535 (purine. ir identical with that of the analytical shaken with triethyl orthoformate (200 ml) for a few min. An 0 --t N acyl migranmr identical.). H. H-2' and H-81. H.99 g.) C. 1.63 and the multiplet at 7 2. 1562 cm-'. Anal.N (37 ml. 0.04 g. H-2).41 g. N. Rf 0. 1600 (200 ml) was added. (7.5 mm).7 (m. ir sample. 6. . 3. brown glass (3.45 in solvent A. smaller spot at Rf 0. tion during the BH. H. 1600. cooled (0-5') soln of 12 (4. . ir identical with that of the analytical sample. amide).. and H-3'). The pH of the hot soln was then adjusted to 6. 2CH. Complete exchange of the NH. ) N.$.9-7. ir (KBr) 3250 broad (OH.H. 6. ir identical with that of the analytical sample.65 g.82 (s.28 g. The dioxane layer was sepd from the aq mmoles) in dry THF (100 ml) was added a 1M soln of BH. NOCOCH. leaving a yellow. N. Rf 0. OH).) 1660 (amide C=O). = 9. When run on a larger scale. J . giving white needles.47 and 0. 1. (100 mg) and the residue was stirred with 2 N HCl(110 ml) at ambient temp under H.5 hr. The singlet at 7 7.N. 4.00 g.5". H-2).20 mm) gave the analytical E indicated to be largely a mixt of amino alcohols (major spot at sample of 7 as white needles: mp 99-100'.46 in solvent B. 76%). COCOCH. bp 28-86' (7 mm).).0 hr at 0-So.45 in solvent A. bp 86.HC1(11. disappeared within 5 min of addn of D. 6.0 (5 ml)-Dyridine (10 ml) at ambient temo for 18 hr. 88.5-166. overlaps DMSO-d.Rf 0. Sublimaevapd. uv as expected.): 1660. H-3).8 (m. nmr (DMSO-d.N.) C. 6. 2. l.J.76 (s.5-152.6 (m) sample of 10 as white crystals.9 (m.q and the structure a. 6.0-4. and CH. The doublet at 7 4. H-1'.).6. 7.54 in solvent B: overlapping 8. giving crude 9 (24. Such samples of 11were sufficiently pure for use. EtS0.5 1. trans-~-~6-Dimethylam~o-9-purinyi)-2-hy~oxy~clopentanoneJ = 4. (50 psi) for 20 hr.49 (s. 1638 (amide C=O). for 44 hr. 2093 cm-' (N.H. 89%) did not dissolve and was removed by sample of 13 as white crystals. trans-3-(6-Dimethylamino-9-purinyl)-2-hydroxycyclopentanone C. purine sample was recrystd twice from EtOAc. A soln of 7 (14. 3. The amino alcohols could not be separated by ketal). (C. filtration. 3.3 ml) was added. A soln of 11 (8. -0. 1. 6. Recrystn of such s sample from EtOAc gave the analytical solid EtgH'Cl. . the yield of 7 was lower. This material bath). 25.O. NHC=O).H.).20 mmoles).4698.uv as expected. A soln of Diborane Reduction of 12.. To a stirred.82 g.1 (m. 164 mequiv).- 1 .5 Hz. mp 156-158'.8 mmoles). 1. Recrystn of such a sample gave the analytical sample of 5 as a colorless liq. nmr).O ml. the residue was dissolved in MeOH (50 ml) leaving pale yellow solid (2.4 (m.8 (m. ir as expected.O. for 3. was dissolved in abs EtOH (75 ml) and shaken with PtO.74 g) which was chromatogd on a column of silica gel (200 g) packed in CHCI. 2. = 7.O (10 ml) was added to the (*)-9-[a-(3a-Acetamido-2@hydroxy) cyclopentyI] 4-dimethy laminorefluxing soln over 1. with somewhat different relative intensities. 1. and H. N(CH. H-2'. 12.O. H. 84% from 7 ) .0 mmoles) in dioxane (40 ml) was brought to reflux.g.82 and 1. H-3).49 uv.%4N. and then dried (CaSO.3 (m. H-2). 6.) and concd to 50 ml. 1600.(3 1.9 (m. 1.68 (s.1-4. C. 4%). H.).53 (s. nZoD1.26 g).8 mp 165-167'.OH).95 g.54 (i. on addn of D. ir as expected. 1. and all solid had dissolved. 162.

Anal. llO(110. 420 (0.. The diacetyl derivative 13a was converted to 13 in 85% yield on treatment with a satd s o h of NH. 418 ( O S ) .77 (s.O. giving white powder. giving a mixt of 2a and 19a. 4.N.7).). The soln was cooled to 0". giving a white powder (452 mg.9 disappeared on addn of D. N.436 mmole.[2R-hydroxy-3R-(p-methoxyphenyl-~alanylamino)] cyclopentylburine (2) and 6-Dimethylamino-9.7 (m. assigned structure 2. could not be a brown glass (800 mg) which was chromatogd on a column of silica gel (50 g) packed in CHC1. Anal. approx 0. Evapn of the combined filtrate and wash (35". (1 atm) for 10 min. (200 mg.O (2 X5 ml).41 and 0.O. 5. Method A.N. 403 (0. The multiplets at 7 8.472 mmole).2. In another run 17 and 18 were separated on a silica gel column eluted with 3% MeOH-CHCl. H.30 ml(0.43 ml. ir as expected.) C..2). 134 (23. 173. (C. 40 ml) for 3.3300-3100 (OH. 8. Evapn left a white solid.05 ml.2.{R-[ 3R-(Benzyloxycarbonyl-p-methoxyphenyl-L-alanylamino)2R -hydroxy]cyclopentyl}-6dimethylaminopurine (2a) and [ 3S-(Benzyloxycarbonyl-p-methoxyphenyl-L-alanylamho)9-{S2S-hydroxy]cyclopentyl}-6dimethylaminopurine (19a).0 hr. Anal. With 13. 3.8). 6. uv and ir as expected. 190 (12.l f .1 N HC1) 270 mp (log E 4.307).) C.HZ2N. N.8). purine H-2 and H-8).9 (m.) 1660 (2 amide.7-8. and the combined filtrate was evapd. The glass having R f0.444 N ethanolic HC1 (7.606 mmole) in dry THF (10 ml) was cooled to -10". A soln of the residue in EtOAc (5 ml) was cooled a t 0" and then filtered. 77%).28) in approx equal amts.O. [a]436 -16. 89.5 (173. and then N-benzyloxycarbonyl-p-methoxyphenyl-L-alanine. N. 3 CH. (850 ml) gave a yellow glass which crystd from EtOAc giving 13a as tan crystals (377 mg.7 (89.) C.52 in solvent B.0 hr.4 mg.5). The Cbz derivative 17 was eluted as a glass which crystd from abs EtOH. 2. 3.H-2'. purine H-2 and H-8). The basic MeOH eluent (250 ml) was evapd. 1.176 Journal ofMedicina1 Chemistry. (C. in 30% yield (from 12).8" ( c 0.50) and 18 (Rf 0. (2. filtered. N€&. which was resolidified from EtOH-Et. 6.). mp 180-182" dec. evapd to dryness. tlc and ir identical with those of the analytical sample. Evapn left a white solid (740 mg). a white solid. H-l'.10" for 10 min.4 mg. 318 (23. After stirring at 0" for 30 min. 0. mp 161-161. (other bands were unreacted starting materials) gave a mixt of 2a and 19a as a solid foam ir. Evapn left the free amine as a white solid (118 mg.4 mmole) of an approx 1.2 (m. mp 226-227" .) single carbamate band at 1760. (C. 18 was isolated in 74% yield (from 16). 1.6).).472 mmole) and N-hydroxysuccinimide (54. N. 0. and evapd. 300 (loo).N (61.93 mmole) in DMF (5 ml) was cooled to 5 " . 1670 and 1655 (2 amide C=O). A s o h of 16 (100 mg.5' . H.H) C.) C.H..5 ml).ir (KBr) 3417. " A s o h of Et. 4.O.) 1650. 121 (59. 0. 2. ir and nmr as expected. Evapn of the combined filtrates left a colorless glass which was triturated with H. 2.1 N NaOH) 277 (4. crystd from abs EtOH. giving 16 as a white powder (685 mg.52 (s. 0. 1655 (amide C=O). 9 [a]436 -103. The mixt was filtered. 0. 89%).O.5-4.CO.O) 277 (4.32 mmoles) of 12 was treated directly with the mixed anhydride of N-benzyloxycarbonyl-p-methoxypheny1-Lalanine in this manner.18 (s) overlapped by 8. and NCHC=O).. 9. 2. then H.7-4.H. mp 218-220'. and 0. and H-3'). 405 (0. N. ir. NHC=O). 3. -&lo. ir (CHCI. Recrystn from MeOH (5 ml) gave a 1: 1 mixt of 2 and 19 as white crystals. When the mixt of 17 and 18 was treated with NaOMe in DMF as described below.O. H. 120 (5.). which was dissolved immediately in DMF (5 ml). The BaCO.404 (1. 3.6)..O.9). 0. mp 1. assigned structure 19. 0.H4). leaving a mixt of 2 and 19 as a white solid (176 mg.51 in solvent C. 271 (8. mp: part at 170-172" and the rest at 200-205" .). mass spectrum (probe temp ca. The filtrate was dild to 15 ml with EtOAc.).8).3).) C.5-4. 0. H-2'. Condns were chosen which have been shown to give 63% migration with cis-2-benzamidocyclopentanol and no migration with trans-2-benzamidocyclopentanol.N.5).13 ml. (C. ir identical with that of the analytical sample prepd as described below.0 hr and then stored at 4" for an addnl 24 hr. Anal. 3'-Carbamate (18). Evapn of the dried EtOAc soln left a mixt of 2a and 19a as a white solid foam (250 mg. NH. uptake had ceased. which tlc in solvent B showed to be a mixt of 17 (Rf 0. NH.. mp 163-164. 122 (10. NH: and OH). B. 0.31.25 mmoles) was dissolved in DMF (10 ml). 7.30 mequiv) and stirred at 25" in a stoppered flask for 18 hr. by which time H. Crystn from EtOAc-hexane gave white solid (32 mg. ir (CHCI.0 calcd for 318t + 300+). ir (KBr) shows no acetate C=O band. OH).26. The 2 bands were each stirred for 18 hr with 20% MeOH-CHCI.43. the soln .Rf.5". 1. H. H. 7..5 ml).73 (s.9). Extn of the major band with 20% MeOH-CHCI. Method B .5 1. mp 95-100" with frothing. 148 (9.0 (m. 163 (16.8-4. 1600. 82 (9.CO. A s o h of this glass in MeOH was passed slowly through a column of 10 ml of Amberlite IRA400 resin (OH-) packed in MeOH. and DCC (97. and the soln was cooled t o -10" and added to the mixed anhydride.2 (m. The purine and methoxy nmr resonances indicate a mixt.314). (0. (C.450 mmole) in MeOH was passed slowly rhrough a column of 10 ml of Amberlite IRA-400 resin (OH-) packed in MeOH.. The soln was stirred for 1 min. In the chromatog of 13 and 14. The N-Ac deriv 13 (730 mg. The diastereomers 2 and 19 were sepd by prep tlc (50-70 mg per plate) in solvent D. H. 301 (21. [ a ] 5 8-45. 2.8).53-154".436 mmole) was dissolved in glacial AcOH (15 ml) and shaken with 10% Pd/C (125 mg) under H.41 in solvent B. With 14. It was filtered. Further recrystn of such a mixt did not change the ratio of 2 to 19. 2 Daluge and Vince was allowed to stir at ambient temp for 20 hr. 3310 (OH.H.O. on evapn of EtOH.O. (+-)-9[p-( 3a-Amino-2ol-hydroxy)cyclopentyl]-6dimethylamhopurine Acetate (16).3 mg.2-7. NH). 2.).7 calcd for 300'. TheN-Ac derivative 13 (50 mg.164'). 6.7-4.606 mmole) was dissolved in DMF.95 (q.5-196.6). after chromatog.2 calcd for 163++ 134'). 422 (0.5210" dec. H. H .3 mg.2 disappeared on addn of D. The procedure is that used by Baker and Joseph" to prepare the 2'. mp 100-110" with frothing.). Rf 0. Elution with 2% MeOH-CHCl.419 ( O S ) . R f 0. The CHCI. ir (KBr) 1737 cm-' (acetate C=O indicates presence of 15). N(CH. tained from method A. H.3'-carbamate of the puromycin aminonucleoside. 0. The multiplet at 7 4.25 (s. 0.).O (20 ml) was added.312)..6).310 mmole) and Et. and 2.. mp 223224" . (0. NO.7-8.41. 0. in MeOH.5 1 (s. and nmr identical with those of the mixt ob(266 mg. 165 (8. 1.52 in solvent B. Crystn from abs EtOH gave white crystals.472 mmole) was added. Vol.H..1 N HCl) 270 mp (log E 4. 92%). 6. The glass having R f0. The diastereomers could not be sepd by crystn or chromatog.N (0. 0.7). along with N-benzyloxycarbonyl-p-methoxyphenyl-i-alanine4 (155 mg. CHCI.5 mmole) was added. ir and nmr as expected.6".) C. (C.NH).N.O.606 mmole) was added and the mixt stirred at .2" (c 0. mp 184-186" dec. nmr identical with those of the samples described above. mp 195-196'. nmr (DMSO-d. then one-half satd NaHCO. giving white crystals.{S[2S-hydroxy-3S-~-methoxyphenyl-L~alanylam~o)]cyclopentyl 1purine (19). 6-Dimethylamino-9-{R.). giving white needles. 70%).ir and nmr as expected.323). and chromatogd by prep tlc on a plate developed in solvent C.). NH. 109 (5. as well as 13a and 14a.. 39%).3-1. metastable transitions: 283.) 7 8. 15. NH. The soln was dild with EtOH (50 ml) and treated with excess Dry Ice.. 2. 6. N(CH. CH.). Evapn left 18 as a glass which solidified from abs EtOH (10 ml).00 g (8.. An analytical sample of the mixt was prepd by solidification from MeOH.. (H.0 (m. tlc gave 2 spots with R f 0.O (5 ml). (700 ml) gave 14a as a colorless glass (154 mg). 250") m/e above 80 (relative intensity) 439 (0. 0. 228 (11. Ethyl chlorocarbonate (72. ~C. Rf. 4.2"zz .H. 6. Carbobenzoxy chloride (0. Recrystn of such a sample from EtOAc gave the analytical sample of 13a as white needles. confirming the assignment of structure of 13a and 14a Acyl Migration Studies.164 mmole) was dissolved in 0. 97%).6).2' A. and the s o h was stirred at ambient temp for 1. the dicyclohexylurea was washed with EtOAc (20 ml).301).. N. A mixt of 2a and 19a prepd by method A (250 mg. 3%). The resulting mixt was stirred at -10" for 1.-and 2CH. and H-37. uv max (0. OC. m p 207. Anal. and the oil which formed was extd into CHC1.4). H.) 7 8.) C.667 mmole) was added. A sample of 17 purified by chromatog (890 mg.CH.4). Treatment of 14 with ethanolic HC1 as in part A gave.O .) and evapd. N. A sample of the free amine obtd by resin treatment of 16 as in method A (159 mg. CHCI. dark materials remained on the silica gel columns after all of the acetylated compds had been eluted.nmr (DMSO-d.450 mmole). 4.H. N. mp 194. The soln was stirred at 100" for 2.0 (283. 1972. Anal. OCH. At this time tlc showed complete conversion to 18.5-196". 1600 cm-' (aromatic.3).N. uv max (0.1 N NaOH) 277 (4. 1757 and 1733 (carbamate C=O).0 hr. (H.O) 277 (4.322). Rf0. nmr identical with that of pure 19.4 N s o h of NaOMe in MeOH was added. The crude mixt of amino alcohols resulting from the diborane redn of 3.O (2. 4. ir (CHCI.Anal.N.N. was removed by filtration through Celite. 4s o h of 16 (145 mg. Contd elution of the column gave 18 as a white solid. 164 (82.5'. NH.4). Anal. 150 (14. (+)-9-[p-( 3cl-Amino-2cl-hydroxy)cyclopentyl]-6dimethylaminopurine 2'.1).2 (m. giving almost quant recovery of the pure diastereomers as colorless glasses.0-5. 0.. extd with H. 1600 cm-' (arom. Elution with 1%MeOH-CHC1. C=O): 1605 (arom. mp 194-196". ir (KBr) 3275 broad (NH).8-7. ir (KBr) 3420. leaving a colorless glass (96 mg).3 calcd for 3 0 0 + + 228+).H. nmr as expected.5 mm) left a colorless glass. 3309.40 mmoles) was refluxed in a satd aq soln of Ba(OH). mp 194. The mixt was filtered through Celite and the Celite was washed with addnl AcOH (10 ml).2 (m. 421 (1. exts were dried (CaSO. (approx 0.5 N.6). and the solid collected was washed with addnl DMF (5 ml). (C.. (3 X 20 ml)..

J. L. and F. [ a ] . l ) . 422 (1. P.. ir (CHCI. D. PP 6-7: (18) S . W.. Sci. and Carl S. edema.2549 (1955). (51 . (27) G. Med. and H. 1590 cm-’ (arom. hypercholesterolemia.. Desiderio. C. T h e 3’-N-acetylated derivative of t h e metabolite. Tong. 89. and the Departments of Medicinal Chemistry and of Medicine.. and references therein. 0. J.2). R. Haladova. (26) H. 300 (loo). R. J. 93. J.77. 1597. S . Baker and J.2y3exhibits trypanocidal as well as a n t i t u m o r proper tie^. University of Minnesota.8).. J. David and A. as shown by comparison of t h e urinary metabolites of l a with chemically synthesized 3’-N-methylated derivatives of l a. Vander Werf. (19) T. 0. 148 (12. Wagner. 125. 89. F. or i p routes. E. 271 (8. Nathans in “Antibiotics I. (15) C.4” (c 0. Shaw. Williams. Lee. 164 (76. 1. does n o t appear to be reflected in differential . Ed.1589 (1952).. 5541 7. The Organic Chemistry of the Active Site. M. Tetrahedron Lett. Kmetec and A. pp 79. Biedron.O-D-ribofuranosyl)-6-dimethylamino-9H-purine ( l a ) . J. “Design of Active-Site-Directed Irreversible Enzyme Inhibitors. pp 259-277. 134 (23.. CIBA Found. Biol. Roll. ibid. Baker.8).336 (1967). Braunstein. E. Chem. Chem. Atlantic City. Pharmacol.14.1 N NaOH) 277 (4. Biochem.” W. (16) R..7 7 . Baker.. U.) 1650. Chem. 2 177 (9) D. W. T.439 (1956). W.405 (1. D. (8) B. New York.1). (13) E. Vol. 94 3’-met hylamino-3’-deoxy-~-D-ribofuranosyl)-6-dimethylamino-9H-purine ( 1 c). Zimmerman and G. BioL.H.317). Drying at 56’ (0. guinea pigs.7). 76. R. W. Amer. N. (4) B. 3808 (1970). 109 (4. No.. S. viz. Rychlik. Shaw. R. and L. Org.. Toxicol. New York. (6) J. 15. Identification and Synthesis of the Major Nucleoside Metabolite in Rabbit Urine after Administration of Puromycin Aminonucleoside’?Herbert T. 1968. 10. A.. Tirpack. SOC. Res. Chem. or rabbits...). 59.7151 (1967). (0. References (1) B. J.O.0). E. t h o u g h reversible. S . V. Acknowledgments. S . when administered to rabbits is m o n o d e m e t h y l a t e d at t h e 6-N position to give 943‘am~no-3’-deoxy-~-D-ribofuranosyl)-6-methylamino-9H-purine (9). (12) I. Rosenthal. McEwen.308).. Goodman.7). Ed. 318 (34. J. Soc. The authors also are indebted to Dr. Y. 11. and Grant 65G078. Gottlieb and P. NH. 13 (1969). ir (KBr) 3375 broad (OH.6). 1493 (1970). J. F... t h e latter constituting t h e major nucleoside metabolite of l a in the urine. Nagasawa. L.O). Schwender in “Synthetic Procedures in Nucleic Acid Chemistry. 5 1 . K. m / e above 80 (relative intensity) 439 (l. Amer. Proc.. 190 (17. 30. 1972.) C.-82. rabbits d o n o t metabolize l a by m e t h y l a t i o n o n t h e 3’-amino g r o u p of the a m i n o ribose moiety. C. Schaeffer and C. N. Hutchings. and A. (23) G . hyperlipidemia. Derr. J. SOC. J.” D. H. Zemlicka. Chem.. Y. (2) D. Vol. Zimmerman.8). N.05 mm) for 24 hr gave a white solid foam. metastable transitions same as those of 19. The excellent technical assistance of Maxine Palm is acknowledged. D. 319 (6. JournalofMedicinal Chemistry. Brown. S . 1968.6”.. (14) C. Heisler. H. 34. and L.2510 (1970). Tsuboyama. NH.. Chem. N. Rev.O)277 (4. Org. (7) P. 1817 (1969). l (1955). Org. Amer. Guss and R. (22) W. Amer. (28) B. (24) S. Chladek.N.J.1 N HCI) 270 mp (log E 4.S.77. Soc.5). Jr. J. Carbohyd. 122 (6.. Swingle. Chem. G. and J. W..61. Chem. Soc.2). Garbisch. I971 9~3’-Amino-3’-deoxy. W. after sepn by chromatog. W. Isaac. and 9~3’-dimethylamino-3’-deoxy-~-D-ribofuranosyl)-6-dimethylamino-9H-purine ( 1b).21. (25) S . NH). 92. 150 (13.. M.” Wiley. Parker and N.. Alexander. 19.0). Springer-Verlag. A. degradation products. Interscience Publishers. Appl. Hawtrey. 1076 (1963). Anderson.5). 228 (11. Chem. Tipson. Anal. 163 (17. I.317). Chem. 121 (39. and K. Neidle. 420 (1. and J.* Frances N. Minneapolis.777 (1957). The aminonucleoside l a produced by the Edman degradation of the antibiotic puromycin and i n d e p e n d e n t l y synthesized. Amer. N. (C. mass spectral fragments) t o 8 synthesized chemically by methylation of 9~3’-acetamido-3’-deoxy~-D-ribofuranosyl)-6-amino-9H-purine ( 6 ) o n t h e 1 posit i o n with MeI.5). Nagasawa. had [a] within experimental error of those of samples prepd by method A. Med. Symp.. (10) R. Herb Nagasawa for providing the data on nephrotoxicity studies. Moffatt. 775 (1970). 260’). (20) H. Feuer and D.41. Biol.81.7). Carroll. 5 8 5 (1964). t h e aminonucleoside of puromycin. Goodman. 403 ( l .. Eggers. 13. l a elicits a nep h r o t i c syndrome characterized by hypoproteinemia.. Kiss.250 (1971). M. sc.3). ministered to rats by oral. Exp.. L.. Alexander Medical Research Laboratories.New York. R. 43. E. J. Nathans and A. Fisher. H. CHC1. Piszkiewicz. J. April 18. 301 (22. W. 1967.Proc. Purines. American Heart Association. 36. SOC. United States Public Health Service. T. and J. 120 (5. P. J. H. 3568 (1967). Nathans. a n d ascites-a syndrome that is clinically indistinguishable f r o m the kidney disease frequently observed in children. Chem. 220.Metabolism of Puromycin Aminonucleoside crystd. 8 . Nagasawa. (21) J. Joseph. Chem. Blackford.89. 3271 (1966). Biol. (3) D. 1967. Chem. Shirota. Bruice and D.404 (1. Contrary to earlier speculations.248 (1967).5). J. Nut. Pharmacol. J. (17) H.. Nature (London). Verheyden. mass spectrum (probe temp ca. and W. J. Hydrogenolysis of a mixt of 2a and 19a prepd by method B gave a 91% yield of 2 and 19 which. b y Baker. 35 (1969). F. 419 (3. H. J.7).e. Y. and Z.^" The appearance of massive. 197. . followed b y rearrangement in dil NH40H. 82 (9. G. Fodor and J. Chem. B. (H. SOC. 165 (7.. Mol. Lee. 1956. Anderson.uv max (0. Chem. Veyrieres. Cerna.* The striking species susceptibility to toxicity b y l a m a n ifested b y its lack of n e p h r o t o x i c i t y in mice. Y. [a]436 -188. Alexander. C. 418 (2. M.’ l a has since been utilized extensively for the experimental i n d u c t i o n of this disease in rats. proteinuria.. (11) E. Amer. SOC. 289 (6. 9. Weinfeld. E. e t aZ. and G. Zorbach and R.. Amer. Minnesota. 35. Received June 4. 2109 (1964). J. and was presented in part at the 52nd Annual Meeting of the Federation of American Societies for Experimental Biology. Chem.. Acad. J. SOC. McCloskey. Minneapolis Veterans Hospital. 421 ( l S ) . Mechanism of Action. 15 (1955). 1231 (1954). Org.).5012 (1967). 94 3‘-acetamido-3’-deoxy-~-D-ribofuranosyl)-6-methylamino-9~-punne ( 8 ) was identical in all respects (tlc patterns. 737 (1959). proteinuria frustrated clinical trials of l a as a t u m o r c h e m o t h e r a o e u t i c agent in mane6When ad?This work was supported in part by Grant HE-04983.. S . P.8).8). Joseph. Callahan. 1650 (amide C=O). i.

Chem..org/10. 177-181• DOI: 10.1021/jm00272a013 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. and Carl S. Shirota.doi. DC 20036 .1021/jm00272a013 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. 2009 More About This Article The permalink http://dx.Identification and synthesis of the major nucleoside metabolite in rabbit urine after administration of puromycin aminonucleoside Herbert T. 15 (2). Med. 1972. Washington.W. Nagasawa. Frances N.org on April 25..acs. Alexander J. 1155 Sixteenth Street N.

(21) J. 122 (6. New York. Chem.5012 (1967). l ) . SOC. Schwender in “Synthetic Procedures in Nucleic Acid Chemistry. E.250 (1971). Zimmerman. Amer. Carbohyd.. Neidle. Soc. Baker.). Soc. and was presented in part at the 52nd Annual Meeting of the Federation of American Societies for Experimental Biology. and H. [ a ] . i. [a]436 -188. J. pp 79.. Alexander. 82 (9. (7) P. Mol.14. 11. (12) I. K. Zorbach and R. or i p routes..05 mm) for 24 hr gave a white solid foam. New York. Med.0). 43. 120 (5. ibid. Amer. M... pp 259-277. 134 (23. Lee. t h e aminonucleoside of puromycin. J. S . ministered to rats by oral. Vol. Wagner. B. metastable transitions same as those of 19. Acad. had [a] within experimental error of those of samples prepd by method A. PP 6-7: (18) S . Contrary to earlier speculations. The excellent technical assistance of Maxine Palm is acknowledged..77. McCloskey. 30. J. 0. SOC. Med. Nathans in “Antibiotics I. 121 (39. C. D. 35 (1969). Heisler. Brown. Chem. Baker. J. Acknowledgments. United States Public Health Service.7). “Design of Active-Site-Directed Irreversible Enzyme Inhibitors. Joseph. W. 289 (6. when administered to rabbits is m o n o d e m e t h y l a t e d at t h e 6-N position to give 943‘am~no-3’-deoxy-~-D-ribofuranosyl)-6-methylamino-9H-purine (9). T h e 3’-N-acetylated derivative of t h e metabolite.. Piszkiewicz. Goodman. 3271 (1966). edema. and K. viz. (C.. NH. Lee. A. J. 59.. (8) B. Biol. Guss and R. 94 3’-met hylamino-3’-deoxy-~-D-ribofuranosyl)-6-dimethylamino-9H-purine ( 1 c). G.5). (0. JournalofMedicinal Chemistry. Shaw.7). Atlantic City.1589 (1952). 125. E.439 (1956). P. Tirpack.6). SOC. Chem. Hawtrey. 5 8 5 (1964).8). Swingle. Parker and N. Pharmacol. 5 1 . Chem. The authors also are indebted to Dr. Nature (London). Chem. 1590 cm-’ (arom. 19. 420 (1... E. 2 177 (9) D. sc. The Organic Chemistry of the Active Site. J. (2) D. J. N.N...317).New York.5). McEwen.” W. References (1) B. S . 421 ( l S ) . Toxicol. 9. 8 . 260’).2).7). Amer.8). L. 93.O)277 (4. Jr. Goodman. 1968. (14) C.. Chem. 318 (34.405 (1. Minnesota. R. (10) R.7151 (1967). Braunstein. R. (25) S . Appl.7). H.. and 9~3’-dimethylamino-3’-deoxy-~-D-ribofuranosyl)-6-dimethylamino-9H-purine ( 1b).O-D-ribofuranosyl)-6-dimethylamino-9H-purine ( l a ) . Rychlik. 3808 (1970). Chem. (H.” Wiley.O.H. SOC. 36.) 1650. Blackford. Amer. m / e above 80 (relative intensity) 439 (l. 197.8).. SOC. Feuer and D. 1967. Chem. F. (24) S. Springer-Verlag.O). Ed. Haladova. BioL. 1076 (1963). P. H. S . J. The aminonucleoside l a produced by the Edman degradation of the antibiotic puromycin and i n d e p e n d e n t l y synthesized. 13.61. E. Purines. Y. rabbits d o n o t metabolize l a by m e t h y l a t i o n o n t h e 3’-amino g r o u p of the a m i n o ribose moiety. 89. Mechanism of Action. L. . H. J. Tipson. (6) J. (15) C. 1968. Exp. Chem. T. and J.. Derr. T. Amer. (4) B. 1493 (1970). Roll. Hydrogenolysis of a mixt of 2a and 19a prepd by method B gave a 91% yield of 2 and 19 which. 271 (8. CIBA Found. Y. W. l a elicits a nep h r o t i c syndrome characterized by hypoproteinemia.. (26) H. J.41.J. M. R. Fodor and J.. (27) G. W. Chem. CHC1. N.308). S .uv max (0. 76. (23) G . Chem. S. Org.* The striking species susceptibility to toxicity b y l a m a n ifested b y its lack of n e p h r o t o x i c i t y in mice.404 (1. V. J. Eggers.. Proc. University of Minnesota. Anderson. D. N. (22) W. F.) C. J. 1231 (1954).. and A. Anderson. Joseph. Org. Fisher. Identification and Synthesis of the Major Nucleoside Metabolite in Rabbit Urine after Administration of Puromycin Aminonucleoside’?Herbert T. 164 (76. Vander Werf. Tetrahedron Lett. J. followed b y rearrangement in dil NH40H. 190 (17. H.777 (1957). 220. J. mass spectral fragments) t o 8 synthesized chemically by methylation of 9~3’-acetamido-3’-deoxy~-D-ribofuranosyl)-6-amino-9H-purine ( 6 ) o n t h e 1 posit i o n with MeI. and L. Schaeffer and C. (28) B. Pharmacol. E. 403 ( l .4” (c 0.-82. 228 (11. 2109 (1964). 89. S .. P.. Chem. Kiss. 419 (3. Nathans and A. J. M. Minneapolis. 34.. 94 3‘-acetamido-3’-deoxy-~-D-ribofuranosyl)-6-methylamino-9~-punne ( 8 ) was identical in all respects (tlc patterns. Nagasawa.. (20) H. 148 (12. G. Y. Desiderio. l (1955). (16) R. I971 9~3’-Amino-3’-deoxy. N. 737 (1959).5). Nathans. degradation products. ir (CHCI. guinea pigs. 418 (2. 92. Herb Nagasawa for providing the data on nephrotoxicity studies.8). SOC. W. L.0). (17) H. 301 (22.Proc. U.81. W. Nagasawa. Anal. 150 (13. and Grant 65G078. Drying at 56’ (0. 109 (4. Zimmerman and G. Rosenthal. Alexander. Moffatt. hypercholesterolemia. b y Baker. J. (51 ... Shaw. t h e latter constituting t h e major nucleoside metabolite of l a in the urine. Y. 13 (1969).. Callahan. Org. Minneapolis Veterans Hospital. 775 (1970). Res. Gottlieb and P. Carroll. 15 (1955). e t aZ.6”. a n d ascites-a syndrome that is clinically indistinguishable f r o m the kidney disease frequently observed in children. Baker and J.2). Chladek. and J. R.e. Amer. Amer. NH. Garbisch. 165 (7. 15. A. M.336 (1967).” D. W. J. Biedron. 1956. H. does n o t appear to be reflected in differential . Chem.3). Nagasawa.317). Sci..* Frances N. W. April 18. 300 (loo). 1967. 1. and J. and references therein. Interscience Publishers. C... as shown by comparison of t h e urinary metabolites of l a with chemically synthesized 3’-N-methylated derivatives of l a. J. 1817 (1969). No.77. Shirota.1).8). mass spectrum (probe temp ca. ir (KBr) 3375 broad (OH..Metabolism of Puromycin Aminonucleoside crystd. Biol. (11) E. and Carl S. and L.2549 (1955).). N. David and A.. and Z.7 7 .2y3exhibits trypanocidal as well as a n t i t u m o r proper tie^. and W. Chem.’ l a has since been utilized extensively for the experimental i n d u c t i o n of this disease in rats. NH). and F. Tong. Soc. 163 (17.89. Williams.248 (1967). (13) E. Nut. 1650 (amide C=O). Vol. J.21. 3568 (1967). Received June 4. Isaac. Biol. 1972. 5541 7. F. Cerna. J. C. Bruice and D. proteinuria. proteinuria frustrated clinical trials of l a as a t u m o r c h e m o t h e r a o e u t i c agent in mane6When ad?This work was supported in part by Grant HE-04983. (3) D. hyperlipidemia. (19) T. 319 (6. 422 (1.. W.1 N NaOH) 277 (4. Hutchings. Weinfeld. Zemlicka. J. Rev.^" The appearance of massive. and the Departments of Medicinal Chemistry and of Medicine. Veyrieres. I.1 N HCI) 270 mp (log E 4. American Heart Association. 35. Ed. after sepn by chromatog.2510 (1970). Kmetec and A.5). R. 0. Verheyden. Org.S. Tsuboyama. t h o u g h reversible.. 10. Alexander Medical Research Laboratories. Symp. or rabbits.. D. Chem. Biochem. and G. Chem. 1597.

and this N-methyl transferase. or guinea pig. 215. l b and IC. et al. Catalytic hydrogenolysis of the benzyl group gave IC. 15. Shirota. 8.178 Journal ofhfedicinal Chemistry. Thus. et aZ.. Although one of the two metabolites appeared to have low mobility such as would be expected for 3'-amino-3'deoxyinosine.19 The latter has been synthesized previously by Baker. The benzilidine derivative 2 of the aminonucleoside l a was reduced t o the benzyl analog 3 with NaBH4 in MeOH. Methylation of 6 with Me1 in DMA gave the 1-methiodide (7) which rearranged8 in dil NH40H to 8.21 . which are refractory to toxicity by la. Vol. is absent in the lung fractions of the rat. and 3 methylated with CH20-HC02H to the benzyl methyl derivative 4. and Alexander Scheme I1 HOCH /N\ RI lul 0 OH R2 NH2 On NH I on 6 5 cow. the antitumor principle isolated from the broth of Helminrhosporium sp. the major nucleoside metabolite of la in the rat. 7 J I '\c-J *z 9 b-u' NH OH COW) 8 metabolic handling of the drug by the different species. guinea pig. " 3 F! N CH II OH NH 1 2 CH3 TH2 CH3 I OH 3 '6% / '6'5 of l a with formaldehyde under catalytic hydrogenation condition^'^ led to mixtures of the 3'-N-dimethyl (lb) and 3'-N-monomethyl (IC) derivatives as revealed by tlc. Metabolism and Degradation Studies. the mouseg-" has previously been prepared by solvolysis of the halogen in the corresponding blocked 6-chloropurineaminonucleoside with MeNHz folIowed by debl~cking'~-" and by enzymatic demethylation of la. mi~e). No. et aL. An unambiguous synthesis of the 3'-N-monomethylated compd IC is depicted in Scheme I. the metabolic fate of l a follows similar courses. unless the catalyst was prereduced prior to addition of the Schiff base. / Nagasawa. and speculated that perhaps rabbits detoxified la by methylation of the 3'-amino group on the aminoribose moiety. After administration of l a t o rabbits. Eschweiler-Clarke methylation of l a also gave l b in good yield and free from IC. viz.this product was present only in small amounts and our inter$Lee. spect ively. No. qualitatively and quantitatively. §This rearrangement very likely proceeds by opening of the pyrimidine ring followed by recyclization in a manner similar to the rearrangement of 1-methyladenosine and 1-methyl-2'-deoxyadenoresine t o 6-N-methyladenosine and 6-N-methyl-2'-deoxyadenosine. although direct chemical evidence was not presented. on cow. Chemistry. The preparation of l b by reductive methylation Scheme I CH \" / CH3 cq3 . mouse. in both susceptible (rat) and nonsusceptible species (guinea pigs. 1972.$ 9-(3 '-Amino-3~-deoxy-~-D-ribofuranosyl)-6-methylamino9H-purine (9).'* In the present study we have attempted t o clarify this point by administration of l a to rabbits and examining their urines for the 3'-N-methylated products.. Synthetic 3'-N-substituted dimethyl (lb) and monomethyl (IC) derivatives of l a were required for comparison with the metabolites of l a isolated from rabbit urine.~3'' Wilson. the nucleoside fraction isolated from urine by ion-exchange chromatography contained 3 aminonucleoside components separable and detectable by tlc (Figure 1A). 2o Treatment of 5 in aq soln with Ac10 selectively acetylated the 3'-amino group to give 6 ." have recently reported the synthesis of l b and I C by somewhat different procedures. This possibility is not without merit as rabbits are known to possess an enzyme system uniquely capable of N-methylating a variety of structurally unrelated amines quite nonspecifically. One of these was unchanged la. 2 CH CH3 \ . w h c h is found predominantly in lung tissue. but none of the metabolites corresponded in R fto the 3'-N-methylated derivatives l b and IC prepared above. by an alternate method (Scheme 11) starting from 3'-amino-3'-deoxyadenosine (S)." We have prepared 9 acetylated on the 3'amino N. have suggested that rabbits. metabolize this compound differently from rats.

88% HC0.. It follows that the metabolite of l a isolated from rabbit urine is. clearly establish the purine base moiety of the molecule as 6-methylaminop~rine.. (f) 3'-amino-3'-deoxyinosine. 9. est was focused on the other metabolite which predominated. = red after spraying with aniline phthalate reagent and heating at 110" for 0..Metabolism of Puromycin Aminonucleoside Journal ofMedicinal Chemistry. The tlc patterns of the hydrolysate compared against 6-methylaminopurine and synthetic 3-amino-3-deoxy-D-ribose in several solvent systems (Figures 1A and 1B) confirmed their identity. terns were essentially reversed on Silicar tlc 7-GF with 88%HCO.. The series of ions (Table I) which include the purine base fragment b. (d) synth 8 (hyd). the base plus various portions of the sugar skeleton.and gave a positive test with ninhydrin under alk conditions... (b) l a (hyd). 1972.. and alkaline AgN03 reagents. (d) D-ribose..5 hr.H-abs EtOH-H. in fact. C. which was isolated by prep tlc. (c) acetylatedmetabolite(hyd). Silica gel HF.5374 lamp. 236 nm). ( e ) 6-methylamino purine. The aminonucleoside metabolite exhibited a molecular ion at m/e 280. This major nucleoside metabolite. o = Fluorescence-quenching under 2. Acetylation gave a product no longer chromogenic with ninhydrin reagent. 9. ( e ) l a (hyd). while the acetylated product showed the expected molecular ion at m/e 322.. ( e ) 5 . Thus. exhibited a uv absorption maximum at 266 nm (minimum.. (0 acetylated la (hyd).1 Figure 1. 2 179 (a) 1 - . (c) crude nucleoside fraction from rabbit urine after administraThe pattion of la.. (a) Synth 3-amino-3deoxy-D-ribose. la) (b) ( c ) id) (e) if) .O (3:16:3). thereby fully identifying the latter as 8.. aniline phthalate. Vol. Hydrolysis of either the metabolite or its acetylated derivative gave a sugar moiety which tested positive with ninhydrin.O (3:32:6). Further evidence was adduced from analysis of the mass spectra of the metabolite and its acetylated derivative (Table I).30) peaks (loss of CH20) at m/e 250 and mle292. B.. CMA-I.~~ Synthetic 8 had essentially identical mass spectral fragmentation patterns (Table I) as well as identical Rfvalues on tlc in several solvent systems as this acetylated metabolite.59) peak (loss of CH3CONH2)22y23at m/e 263 in the acetylated derivative verifies the chemical evidence that the acetylation occurred on the 3'-amino N. CMA-11. I/ e (b) ic) id) (e) i f ) (gi ih) 0) (1) . . The (M . (c) major nucleoside metabolite (hyd). and a purine base whose uv absorption spectra in acid and alkali resembled that for 6-methylaminopurine. Silicar tlc 7GF. leading t o the conclusion that this metabolite was 9(3'-amino-3' -deoxy-fl-D-ribofuranosyl)-6-methylamino-9ipurine.. The presence of unsubstituted 5'-OH groups in both compounds was indicated by the (M . i. Silicar tlc 7 G F .Habs EtOH-H. (a) Ib. 6 ) adenine.e. (b) IC. the early metabolic transformations of l a in the rabbit appear to be similar to that observed in the other . A. (h) synth 3amino-3-deoxy-D-ribose. (g) 6-dimethylaminopurine. (f) 6-dimethylaminopurine. (b) major nucleoside metabolite (hydrolyzed = hyd).29) and m/e 120 @tH-29). Tlc patterns of I a metabolites and their degradation products. No.. (d) la.(i) D-ribose.. (a) 6-Methylaminopurine.. and the ions m/e 121 (b t 2H . 15.

which was no longer chromogenic with ninhydrin indicating that the 3'-amino group was now acetylated. ir. m/e 350 ( 1. The daily urine collections were adjusted t o pH 8. 6-methylaminopurine. . Spectrophotometers used were: uv. ( 5 : 2 : 1.6 X 14. (50:20:3) (CMA-I)..O gave a product. The eluate was concd to dryness. Woodside.02 N NaOH with an authentic sample. were not chromogenic with ninhydrin but gave the characteristic red color for pentoses with aniline phthalate reagent. (C.3 ml of freshly distd PhCHO in 30 ml of abs EtOH was heated on the steam bath under reflux for 2 hr. **Melting points were taken on a Fisher-Johns mp apparatus and are corrected. and no qualitative differences in the metabolic handling of l a were observed in young or mature rabbits. Beckman 1R-10.I4 mp 184.9) t o remove the free purine bases. After centrifugation of the urine at 2500 rpm at +5" for 20 min. An analytical sample recryst from CH. Proteinuria gradually subsided thereafter and disappeared by day 41.O.O was added followed by an addnl50 ml of 95% EtOH-HOAc.40 mmoles) of l a and 1.or Galbraith Laboratories. species examined the 6-N-monodemethylat ed product of la. A.CN gave 122 mg (76% yield) of l b . By Reductive Methylation.15 was sepd from l a and from the small amount of unknown product near the origin by preparative tlc twice on silica gel with CMA-I1 and eluted with 0. Purification and Acetylation of the Major Nucleoside Metabolite.5". Evapn of the solvent and recrystn of the residue from CH. H. and some unchanged nucleoside was still detectable by tlc after the prescribed period (Silicar tlc 7GF. the following compds were also subjected to the conds of this hydrolysis. the metabolite. incubation of la with S-adenosylmethionine and the soluble fraction from rabbit lung containing the N-methyl transferase described by Axelrod'* failed to give l b or IC as determined by tlc. predominating. but only the sugar resulting from la. Vol. The reaction mixt was dild with 2 vols of abs EtOH and concd to incipient dryness in vacuo.O) 266 nm.O. lower phase) (CMA-11).H. IC. Treatment of this metabolite in H. or if the catalyst was Pd black.NH and heated under reflux for 1 hr. tests.or acetylated l a did not. Beckman DK2A.05 M ammonium formate buffer (pH 5. colorless crystals.. mp 236-237" after recrystn from MeOH-Et.60. Ascites and edema were absent in these animals even at day 25. mp 183-1234".-95% EtOH. mp 184-185" (lit. Rats sacrificed on days 29 and 30 had 1-2 g of ascitic fluid in the peritoneal cavity. The 3-dimethylaminoresult3-deoxy-D-ribose and the 3-methylamino-3-deoxy-D-ribose ing from the hydrolysis of I b and IC. acetylated metabolite. This product on tlc or paper chromatog gave a blue-violet color with ninhydrin reagent at a level of 5 pg. 1b. mp 162.. uv max (H. Knoxville. 70 eV.9). and synthetic 8 had identical R f values in (Figtwo solvent systems as synthetic 3-amino-3-deoxy-D-ribosez7 ures 1B and 1C). acetylated metabolite. Recrystn of the crude product from EtOAc gave 519 mg (80. N. adjusted to pH 1 with concd HC1. the catalyst then was removed by filtration (Celite). A second crop. TICsolvents: CHCl. and the solvent was evapd in vacuo.O with Ac. total yield. PtO.2 M ammonium formate buffer (pH 7. 15. min 236 nm..OH. This metabolite was therefore unsubstituted on the 3'amino group of the amino sugar moiety. This latter fraction was adjusted to pH 1 and desalted by (a) charging onto a fresh column of AG 5@X4 (NH. uv max (MeOH) 274 nm ( E 19. Toxicity Studies. This was repeated until the odor of HOAc and CH.5 ml of 37% aq CH.5.5 NNH. la. 184. 2 is therefore comparable in nephrotoxicity to puromycin itself. sc) to male albino rats weighing 45-50 g caused the appearance of mild proteinuria commencing at day 14. Control urine samples collected for 6 days prior t o administration of l a were separately pooled and treated similarly in the following description.5 cm column of Bio-Rad Ag 50-X4 cation-exchange resin (100-200 mesh.01 N HC1 and 0. viz. NH:). mp 238-241". The column was washed with 500 ml of 0.6%) of lb.0 ml of Et. the clear amber supernatant was dild five. the filtrate was concd to dryness. (cyj*'D -60. 106 mg. (C.50 mmole) in 2. The sugars liberated from all of the above nucleosides gave positive alk AgNO. uptake ceased (5 hr). the residue was dissolved in abs EtOH. 5.. viz. as did 1.N. mixts of the mono (IC)and dimethyl (1 b) derivatives were formed requiring extensive chromatog manipulation for sepn.180 JournalofMedicinal Chemistry. l b was not completely hydrolyzed under these conds. ms.) C. and charged on a 3.800). Hitachi-Perkin Elmer RMU-6 (ionization energy. 2 Nagasawa.9310 Experimental Section** Isolation of Nucleoside Metabolites of l a from Rabbit Urine.O was heated under reflux for 2 hr. N.00 g (3. of 0.1 were pooled and lyophilized. and 8 prepd synthetically (vide infra).^^^# The benzylidine derivative 2 (dose: 5 1 pmoles/kg per day for 24 days.O. The purines liberated from this hydrolysis were compared with 6-dimethylaminopurine. Anal. however. The metabolite with Rf -0.. acetylated la. Both New Zealand white and Shingler strain rabbits gave the same results. 0. Anal. The metabolite was hydrolyzed in 0. and the excess HCO.. l a (147 mg. and (c) eluting the nucleoside metabolites with 1.5-186"). and the results (Figure 1B) showed the product from the metabolite to be 6-methylaminopurine. and control #Doses are based on the weights of the animals on day 1 . and Alexander urine samples did not show the presence of any of these components. This crude isolate contd 3 uv-absorbing compds as detd by tlc.1 N NH.5-164.CN gave the same mp. N. pooled. 625 mg (97%). The glassy residue which was contaminated with the 5'-formyl derivative of I b [ir (KBr) M ' ) ] was dissolved in MeOH contg 1730 cm-' (ester). 5% (NHJ. optical rotations were detd in a Perkin-Elmer Model 141 polarimeter. 9-(3'-Benzalim~o-3'-deoxy-p-D-ribofu~no~l)~dimethyl~~ no-9H-purine (2).02 mmoles) of l a in 5 ml of 37% aq CH. A mixt of 1. respectively.O were removed as azeotropes by successive addn of PhMe and EtOAc followed by evapn. IC.). Furthermore.1' (c 1. The mixt was carefully equilibrated t o the atm (caution: f i e hazard) and 594 mg (2. Hydrolytic Degradation Studies.N.0 t 0. uv max (MeOH) 270 nm ( C 23. The solvent was evapd to incipient dryness in vacuo and the glassy residue was crystd from EtOAc-petr ether t o give 1.26No evidence of nephrotoxicity to rats was observed with l b (dose: 5 1 pmoles/kg per day for 23 days). Where analyses are indicated only by symbols of the elements. and adenine. For comparative purposes.H and CH. The desired nucleoside fraction was obtained by elution with 3 1. Shirota. analytical results obtained for these elements were within *0. None of these spots corresponded in R f t o synthetic l b and IC. If the catalyst was not activated by prereduction. confirmed by comparison of its uv spectrum in 0.MeOH-1 N NH.20 g (92% yield) of 2. but lb.H. (500 mg) was prereduced in 50 ml of 95% EtOH-HOAc (1:l) at 1.) C. (b) washing the column with distd H.H and 0. The mixt was hydrogenated at room temp until H. l a as a 1% aq soln was administered sc to male New Zealand white rabbits housed in metabolism cages (dose: 30 mg/kg per day in divided doses to 2 different sites) for 6 days. 90:7 or 90:3).-MeOH-2. mp 184-185" (lit. B.t o tenfold with distd H. The nephrotoxicity of some of the intermediates and products synthesized in the course of this work was evaluated in rats by measuring their daily urinary protein excretion after administration of the compounds according to previously established protocol^.5-186').O. and no evidence for the presence of 3'-N-methylated metabolites of l a could be found in the urine of rabbits dosed with la. and stored refrigerated under toluene. 9. Y.700). and the residue was recrystd from MeOH to give colorless crystals.05 kg/cm2 for 30 min. Py). No.5 ml of 2 N HC1 for 3 hr at 100".O was no longer detectable. of 0. which is 7-10 days later than the usual onset of massive proteinuria following treatment with equimolar doses of la.0 ml of 88% HCO.01 N HCl and then approx 6 0 ml of the discolored resin at the topmost portion of the column was withdrawn and transferred t o a smaller column contg about 20 ml of fresh resin. N. By Eschweiler-CLatke Methylation.CO. one of which was unchanged l a (Figure 1A). ion source temps as indicated). The new column was washed with 3 1. Anal.'. rats given IC(dose: 51 pmoles/ kg per day for 27 days) developed slight proteinuria commencing on day 23 which persisted until the drug was withdrawn on day 28. Tlc indicated that l a had completely reacted by this time. Eluates having uv absorbance of >0. . 1972. acetylated la. Microanalyses by Schwarzkopf Microanalytical Laboratory.0 N NH. The mass spectra of these products are recorded in Table I. and 3'-amino-3'-deoxyinosine. was obtained by concn of the mother liquor. CHC1. Tenn. 94 3'-Dimethylamino-3'-deoxy-~D-ribofuranosyl)-6-dimethylamino-9H-purine (Ib).O.4% of the theoretical values.

uv max (MeOH) 274 nm ( E 19. 6 .. Py).. Chemother. Fed. 356 (1964).O. F. *H20) c. Antonowicz. S .. 6 0 0 mg of cryst NaBH. W.. Robins. was found to be complete within 1 hr. .N. S. J. Lechevalier.8175 (1959). T. hoc. Blackford. P. M. American Cyanamide Co. and reduced in vol when 3 crystd spontaneously. 52. S. 35. 77. and the solid residue was dissolved in 25 ml of H.. Exp. Baker. D. 77. N. 1595 cm-' (C=N). The residue was Journal of Medicinal Chemistry. Jones and R. E. (9) H... K.O). Joseph. Amer.) C. 258 (1969). Soc. Joseph. and G. Current Res. Recrystd from MeOH-Et. The solvent was evapd. and D. Hawtrey. R. Wilson. Patent 2. 228 (1960). (C.. and tlc was relied upon. Anal. the solvent was evapd to dryness in vacuo. Swingle. Acta.97. Amer. S. J.6 ml of 88% HCOOH.600). Chem Soc. D. C.. mp 219-223' 229dec. J. 265 (1968). Gerber for an inoculum of Helminthosporium sp No. I. 336 (1967). Kissman. This product was dissolved in 1 N NH." [or]"D -44". ir (KBr) 3275 (OH).1958). (2) B. Baker. A soln of 154 mg (0.462 mmole) of 4 in 6 ml of 95% EtOH-HOAc (1: 1) was heated on the steam bath for a few min with a spatulaful of Pd black. mp 212-214" (lit. Bennett. and the reaction mixt was stirred at room temp until 6 disappeared (19 hr) as revealed by tlc (CMA-11). E. 693 (1955). Exp. Suhadolnik. Elmer Reist for experimental quantities of chemically synthesized 5. McMahon for the mass spectra.8174 (1959). J. Schaub.O.852. (26) B. Py). Alexander. 0. Hewitt. 1. I. Swingle. and J. Chem. and J. Chem. Exp. Kessner. Desiderio.. Alexander. 9 4 3'-Acetamido-3'-deoxy-p-D -ribofuranosyl)-6-methylamino9H-purine (8). obtained from the mother liquor melted at 158-161". (25) H. Shaw. and H. Williams. 345 m (86. Schaub. R. J. Tetrahedron Lett. Amer. (C13H. J. uv max (MeOH) 274 nm (E 19.800). Exp. 138. Coleman for technical assistance in the toxicity screening and J. L.. Gerber and H. and the residue was triturated with ether to give 127 mg of crude product.. Derr. Org. 1.. but the mp (dec pt) appears to be a poor criterion for assessing purity. 193 ( 1963). Chem. ibid. W. L. Chem.) C. 2938s (1958).O. R.[3'-(N-Methyl~-benzyl)amino-3'-deoxy-p-D-ribofuranosyl]6-dimethylamino-9H-purine (4). K. Bwchem PharmcoL. The white hygroscopic product which pptd on addn of Et. Cancer Res. Vol. J.3808 (1970). W. and J. Py) (lit. Nagasawa.1" (c 0. H. Chem Soc. Ther. Proc. 17. Goldman and J. and H.4N. M.. 247" dec). The reaction mixt was poured into 250 ml of petr ether. The tubes having uv max of 265 nm and min below 235 nm were combined and lyophilized. C.Metabolism of Puromycin Aminonucleoside (C. Burchenal. Nagasawa. N.5).) C. Pediatrics. Tong. 1972.5-218"). and H. K. (C.. H. H. 11. and J. C. Wallace. Proc. Lederle Laboratories Division. and L.. mp 165-167" (lit. Marsico. 75 mg. T. Goldthwait. and the residual glass was subjected to the action of a mechanical vac pump for 18 hr. The fluffy white solids were recrystd from EtOAc-petr ether. W. 25. H. Williams.. (23) S. Biochim Biophys.0 ml of 95% EtOH was heated under reflux until tlc showed no trace of remaining 3 (10 hr). This procedure was necessary to remove traces of impurities which poisoned the catalyst.25 mole of H.J. Med.37 mmole) sample of 5"in 1. Anal.852. Abstr. dried (Na.. 53. 27. C. Med. R. 85. F. A. U. Swingle and M. 3125 (NH). Chem. 1731 (1962). Abstr. H. 3271 (1966). Biol.O.506 (Sept 16. (C1$24N60J C.3 ml of 37% aq CH.2" (c 1. uv max (MeOH) 247 nm ( E 19.O. G. Biol. (14). (18) N. S. The residue was dissolved in abs EtOH and the EtOH evapd to remove traces of H. (27) B..HI6N. (b) the identity of their ir spectra in CHCl. The mixt was evapd to dryness in vacuo and the residue recrystd from abs EtOH to give 57 mg (50%yield) of 6. 53. Res. Anal. ) N. Soc. J. Goodman.. N. Cancer Chemother. (5) S. Baker. 89.. ~ .0 mmole) in a mixt of 0. Biol.. F. (C. Schaub. To 74 mg (0.465 (1968).6%). N. (7) S.O. (16) L. Anal. Amer. and K.. (10) R. 123. and 3. J. Recrystn from abs EtOH gave 117 mg (82%) of IC.424 (1955). (11) S. J.O). 857 (1958). and A.400). R. Derr. J. and the yellow oil that settled out was taken up in Me. 9 4 3'-Methylamino-3' deoxy-p-D-ribofuranosyl)-6dimethylamino9H-purine (IC). SOC.2510 (1970). U. We are indebted to Dr. A.5 hr and filtered. D. Anal. the combined EtOAc exts were washed (H. P. Pharmcol. 1565 cm-' (amide 11). and L. R. S..23 g (3. and the catalyst removed by filtration. A second crop.O. mp 208-212" dec. 9 4 3'-Acetamido-3'-deox~p-D-ribofurano~l)-6-amino-9~-purine (6). After removal of the catalyst.12 (1955). homogeneous by tlc. and passed through a column containing 10 g of 50-100 mesh Dowex-3 ( O H ) resin at a rate of 12-15 drops/min collecting 10-ml fractions. Pharmcol. Nagasawa. E. Axelrod. (13) British Patent 782. T. That this was a dimorphic cryst form of the higher melting product was indicated by (a) the identity of their R f values on tlc (CMA-11). Frenk. ibid. SOC. 16.. 4. R. We also thank K..&I. F.CO. Chem.22 mmoles) of 2 in 50 ml of MeOH was added slowly at room temp. mp 137-139". and R. Amer. F. W. Alexander. 1 6 (1956). ir (KBr) 1690 (amide I).~ . Nagasawa. and the EtOH was evapd in vacuo. Crop 2 (33 mg) was obtd by diln of the mother liquor with Et. Biedron.&. washed (petr ether). Oleson.Exp. 1957). Baker.. W. (15) B. Soc. M. L. 1222 (1954). Antibiot. 21 5 as well as a sample of 5 isolated from this organism and t o Dr. After multiple extns with EtOAc.60. H. S. Metcalf. (24) S. This process was repeated until the odor of HCOOH was no longer detectable. H. 9 4 3'-Benzylamino-3'-deoxy-~D-ribofurano~l~6-dimethylamino-9H-purine (3). Soc. C.. F. and J." 216. Dickie. E. and (c) the elemental analysis. N. Acknowledgments. [a]''D -49... Appl. [ a ] 1 5 D -32. R. 2211 (1967). Goldman and J. G. (20) B.440 (Sept 4. Abs EtOH was added to dissolve the residue. Schaub. (19) N. Joseph. Chem. R.. J. A. Prog. The highest melting sample obtd was 258-259" dec 263-265'. A 100-mg (0. T. Gamble. N.O?) C. Baker. and J. Anal. Drug. (21) J. 7. Amer. (22) R.505 (Sept 16. C.2 mg (73%) of crude 8. Dubach. 27. J. W. The filtrate was concd to incipient dryness. Med. H. H. mp 250-252".O gave crystals melting at 122-125" or slightly lower. To a stirred soln of 1. 7 (1955). (12) J. Lee. B. Both samples behaved similarly in the tlc system described in the Experimental Section. monitored by tlc (CMA-I).O. Soc. 77.0 ml of H. Williams. R. Org. J. l(195. Fed.O was collected. S. Abstr. P. and the mixt was hydrogenated at room temp in an all-glass micro hydrogenator until tlc (CMA-11) indicated complete removal of the benzyl group (3 hr). Proc. 421 (1966). T.06 g (86%yield). Williams.. Borowsky. In some runs recrystn of the crude product from EtOAc-petr ether or MeOH-Et. Eggers. References (1) H. Halliday. S . Fresh Pd black catalyst (78 mg) was added to the filtrate. Nagasawa. (C~&ON~O c.SOJ.24 mmole) of 6 dissolved in 5 ml of dimethylacetamide was added 500 pl of Mel. K. No.2 181 then extd several times with hot EtOAc and the combined EtOAc extracts concd to give 58._ (3) B.O was acetylated in the cold by addn of 200 pl of freshly distd Ac. provided generous samples of puromycin aminonucleoside . Craig. and R. (6) J. R. Patent 2. M. 15. 1958). L.. M. Loechler. Chassy. A. Toxicol. The reaction. P. (17) L. The reaction mixt was stirred at room temp for 15 min. 179. H.. 92. Chem. J.$... 3 (384 mg. (4) R. Med.I3 162-164'. F. 28 (1962). H. Alexander. W. Derr.93. S. then heated under reflux on a steam bath for 2. Norton. Recant. Chem. Heymann.N. 15.O. ~ H. Nancy N. (8) U. and C. 4 . Tsuboyama. R. H. Waller. Biol. 413 (1963). Ne 9. and dried to give 11 mg of crude 7. and J. mp 235-237" 230" and 233" for compd with 0. Alexander. McCloskey.5911 (1955). 1660 (C=N). 7 7 .N60. Marsico.

1021/jm00272a014 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.. DC 20036 ..1021/jm00272a014 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. Washington. 1972. 15 (2). Jeanette Thomas J. 1155 Sixteenth Street N.Nucleosides of 2-azapurines and certain ring analogs John A. 2009 More About This Article The permalink http://dx. Montgomery.doi. Med.org/10. Chem.W. 182-187• DOI: 10.acs. and H.org on April 25.

R = p-Parabinofuranosyl e series. However. Kettering Foundation.Alabama 35205. Vol. et al. Our initial studies were carried out on a model c o m 14 15 16 a series. It occurred to us that an adaptation of the procedure developed by us for the synthesis of 5-amino. Birmingham..5-d]-v-triazine. No." and later Leonard. R = @-Pnbofuranosyl c series. et al. no imidazole intermediates were isolated. 19 71 A convenient synthesis of 2-azapurine nucleosides (7-glycosylimidazo[ 43-d 1-v-triazines) in 5 steps from the corre- sponding purine nucleosides is described. R = 2-deoxy-pD-ribofuranosyl d series. Received August 20.5-&-triazine) nucleoside has not previously been described. 2-Azaadenosine. Azapurines have shown anticancer activity and other interesting biological properties. and 2-azaadenosine has shown consistent activity against L1210 leukemia on both chronic and single-dose schedules. 9-cyclopentyladenine (la).' The ribonucleosides of certain 8-azapurines (v-triazolo [4.182 Journal of Medicinal Chemistry. Thus Taylor. Chemotherapy. 13b). which was benzylated in the usual manner12 to give 1-benzyl-9-cyclopentyladenine hydrobromide (2a)and converted to its I-oxide (5a) by the procedure of Stevens. $A preliminary communication describing part of this work has appeared. such an approach to the preparation of imidazolecarboxamidines from adenine nucleosides was complicated by the ease of reclosure of the proposed intermediates of the Dimroth rearrangement of 1-substituted adenines. Jeanette Thomas Kettering-MeyerLaboratory. National Cancer Institute. l3 Treatment of 2a with reflux- . NIH-71-2021. R = p-Pxylofuranosyl ?This work was supported by funds from the C. and 4-amino-1-P-D-ribofuranosylpyrazolo-4-carboxamidine are all cytotoxic at low concns.5-d]pyrimidines) have been and have shown biological activity as the intact nucleoside^." observed that 1-substituted adenines rearrange quantitatively in boiling water to N-substituted adenines. 1972.'^^ 3 but the synthesis of a 2-azapurine (imidazo [4. Southern Research Institute. 2 Montgomery and Thomas Nucleosides of 2-Azapurines and Certain Ring Analogs? John A.1-0-D -ribofuranosylimidazole4carboxamide (15b)9 might provide an intermediate suitable for the preparation of nucleosides of 2-azaadenine.$ Stevens. described the preparation of 2azaadenosine l-oxide but were unable to reduce this material to 2-azaadenosine (4-amino-7-0-D-ribofuranosylimidazo [4. R = cyclopentyl b series. Contract No. National Institutes of Health. Montgomery* and H. F. 15. 2 -deoxy-2-azaadenosine.8 pound.

Treatment of 1la with methanolic NH3 (satd at 0”) at 80” for 2 days gave the desired 5-amino-N-benzyloxy-1-cyclopentylimidazole-4carboxamidine (12a) in good yield. ’ ~ 12b then gave 5-amino-1-P-D -ribofuranosylimidazole-4-carboxamidine (9b).2 Table 1. No. 13c). and the neutral EtOH-H20 solution (2-deoxy-0-D -erythro-pentofuranosylimidazo [4 . Base hydrolysis of the 1-oxide Sa gave 1cyclopentyl-5-formamidoimidazole4carboxamidoxime (6a).16we found that aq base treatment of Sa opened the pycisity of the N-benzyloxyamidine (1 lb) compared to the rimidine ring but did not cause deformylation of the resultant formamido compound 6a. et al. however.SI-v-triazine (17) exists in equilibrium with 3. 9-/3-D-arabinofuranosyl-2-azaadenine (4occurred to give N-benzyloxy-1-cyclopentyl-5-formamidoamino-7-0-D -arabinofuranosylimidazo[4.6-triaminopyrimidine (16. whereas hydrogenolysis with Pd/C gave primarily 9-cyclopentyladenine 1-oxide (Sa). and 4-aminoformylate l l a with methanolic HCl gave only l-benzyloxy9-cyclopentyladenine (loa). a result also in general 13d). N-benzylamidine is essential for successful deformylation.2-Azapurine Nucleosides Joumal ofMedicinal Chemistry.0 [3.541-Vwas allowed to stand 2 days at room temp. . were unable to reduce 2-azaadenosine 1Ra Ni-catalyzed hydrogenolysis of the benzyloxy group of oxide to the desired 2-azaadenosine (13b). which was isolated as the HCl salt and converted to 9-cyclopentyl-2-mercaptoadenine (4) by treatment with CS2 in DMF and to 9-cyclopentyl-2-azaadenine (4-amino-7-cyclopentylimidazo [4. 1972. refluxing a neutralized EtOH-H20 solution of 10b gave N-benzyloxyadenosine (14b). Since aq base is known to convert adenosine to 4. It seemed probable that. 1-benzyloxy-9-cyclopentyladenine hydrobromide (loa) could be converted directly to 12a by the methanolic N H 3 treatment. altions” giving N-benzyl-9-cyclopentyladenine though a better yield was obtained if the reaction was carried out in aq EtOH containing only 1 equiv of NaOH. 15. It seemed possible that deformylation of 1l a might be accomplished by a transamidation reaction that could compete effectively with the intramolecular ringclosure reaction. Stevens.22 <2. R = H) exclusively. Also. 13e). its absence indicated that if there is an equilibrium between 13a and the corresponding diazo compound. but the use of methanolic NH3 is also necessary (see above).5-d]-v-triazine.5 4.5. prepared by the method described above. ED.. while standing 3 days at room temp gave the imidazole l l b . despite the precedent of the Dimroth rearrangement. an uncarboxamidines requisite for conversion to 2-azaadenine nustable compound that could be purified by careful recryscleosides (13) can now be obtained from the corresponding tallization from EtOH. no intermediate was detected.. imidazole-4-carboxamidine (1la). 9-/3-D-xylofuranosyl-2-azaadenine (4-amino-7-0-Dagreement with the findings of Fujii.0 49 9-p-D-Arabinofuranosyl-2-azaadenine 9-0-D-Xylofuranosyl-2-azaadenine 4-Amino-7-p-D-ribofuranosylpyrazolo- >loo >loo 0.§ Thus. 7a could also be produced directly from 6a by acid treatment. ring opening triazine. If. Treatment of the aminoamidine (9b) with NaN02 in the free base from 10a was converted toN-benzyloxy-9cyaq AcOH gave 2-azaadenosine (13b). umolelLa 1. which gave age. behaved exactly as the cyclopentyl compound. in contrast to the Dimroth re§This result was anticipated.H ing aq ethanolic NaOH (excess) caused the Dimroth rearrangement to occur in agreement with previous observa(3a). since treatment of 1-benzyl-9-P-D arrangement obtained in boiling H 2 0 (loa or l l a + 14a). This procedure was clopentyladenine (14a). however. these results indicate that the v-triazine ring is more labile to base than the pyrimidine ring. Cytotoxicity of 2-Azaadenine Nucleosides ComDound 2-Azahypoxanthine 2-Azaadenine 9-Cy clopent y l-2-azaadenine 2-Azaadenosine 2’-Deoxy-2-azaadenosine 183 pentyladenine (la). dil aqNaOH caused cleavage of both rings of 13a to give approximately equal amounts (1Sa)9 of 5 -amino-1-cyclopentylimidazole-4-carboxamide and 4. resulting from hydrogenolysis of the N-0 bond with Ra Ni and of the C-0 bond with Pd/C with concomitant ring closure in both cases.1-cyclopentylimidazole4carboxamidoxime (7a). it is not amening procedure or the benzyloxy group permitted ring-opening followed by deformylation rather than reclosure (Dimable to nucleosides since the acidic cleavage of the pyrimiroth rearrangement). we prepared 1-benzyladenosine and dine ring would also result in cleavage of the glycosyl linksubjected it to the methanolic NH3 treatment. the crude bromide was also used to prepare 2’-deoxy-2-azaadenosine (4-amino-7carefully neutralized.5-e]-s-triazolo [ 1 . in contrast to the results of Stevens. et N-benzyladenosine exclusively. and this turned out to be the case. ribofuranos lpurine-6(1H)-thione in a similar manner gave N-benzylHydrogenolysis of 1l a with Ra Ni gave primarily 9-cycloadenosine. which could be deformylated with methanolic NH3 to 12b. Thus. In order to establish whether the particular conditions of this ring-openAlthough the route described above provided the desired model compounds for our nucleoside work. in agreement with the results of F ~ j i i . 13a) by treatment with aq NaN02.14 Catalytic reduction of the carboxamidoxime 7a with Ra Ni gave 5-amino-1eyclopentylimidazole-4carboxamidine (9a). adenine nucleosides (1) by a relatively simple 4-step procaused debenzylation back to the 1-oxide. the 5-amino-1-glycosylimidazole-41-benzyloxy-9-cyclopentyladenine bromide (loa).17 An attempt to dexylofuranosylimidazo[4. Hydrogenolysis of the benzyloxy group of 12a with Ra Ni then gave 5-amino-l-c~clopentylimidazole-4-carboxamidine (sa).5-d]-v-triazine. Vol. Boiling in EtOH or H20.. It was not possible to prevent cyclization of l l a or its reduction products (6a and Sa).5-diamino-6-cyclopentylamino-v-triazine (16a).. Hot. Furthermore.”we examined the ir spectrum of 13a in CF3C02Hfor the presence of a diazo band.[ 5(4)-diazoimidazol-4(5)-yl]-striazole (18) and that only 18 was detected in CF3C02H solution of 17.4+-triazine 20 “The concn required to inhibit the growth of treated cells to 50% of that of untreated controls as measured by colony counts. In either case. It was 17 18 also possible to convert 10b directly to 12b. the decreased baa1. which was deformylated in acid to himino. In boiling H20 cedure. Since previous work from this laboratory showed that imidazo[4.’ . The conditions employed in this route to 9a are completely compatible with nucleosides. 1-Benzyloxyadenosine hydrobromide (lob).5-d]-v-triazine. as mentioned above.I6 Benzylation of the 1-oxide Sa gave. the equilibrium favors 13a almost exclusively.

8-diazaadenosine (7-amino-3-P-D-ribofuranosyl-v-triazolo[4. 21b) from 8-azaadenosine (19b)20 via the corresponding pyrazole (20a) and triazole (20b).1N S O 5 'QH1 5N504 C9H I 5N 5'3 C9H 'gH1 1 SN 5'4 5N504 UAnalyzed for C.Og C. CRecrystn of picrate. Calkins. indicating that neither enzymatic sugar cleavage nor deamination followed by sugar cleavage appear to be important to the activity of this nucleoside. X = CH b series. Biophys. 2-Azaadenosine (13b) given at levels of 75-400 mg/kg on day 1 only increased the life-span of BDF. the 2-aza analog of the ribonucleoside of 4-aminopyrazolo[ 3. It was about equally active at levels of 8-23 mg/kg per dose when given chronically qd 1-9. 9-Pentofuranosyl-2-azapurines Crude yield. X = N 7-~-D-ribofuranosyl-7H-pyrazolo[ 3 . 5-Amino-1-pentofuranosylimidazole-4-carboxamidines (9b-e) p-D-Ribofuranosyle 48 H..'20 times as toxic as 2-azaadenine. Chem.Biochim.Ok p-D-Xylofuranosyl 28 H20k CloHl. eAnalytica1 sample of picrate obtd.20 C16H21N505 1OH 13N50$ C16H21N504 C16H21N~05 16H?.* 0.Og p-D-Arabinofuranosyl' 57 MeOHl p-D-Xylofuranosyli 33 H. Free base obtd by treatment of the picrate with Dowex 1-X8 (carbonate) ion-exchange resin. J. Med. 4-Amino-7-fl-D-ribofuranosylpyrazolo[3. Sample was obtd by evapn of a s o h of the compd in this solvent.Og p-D-Xylofuranosyle 54 H. 4-amino-lp-D-ribofuranosylpyrazolo-4-carboxamidine (20a).N505*0. and L.20 C9H 1SN5'4 p yrazole-4-carboxamidineb 4-Amino-7-P-D-ribofuranosyl- 207-208 C9H12N604 __ pyrazolo [3.4-d]v-triazine (21a). Experimental Section Melting points were detd with a Mel-Temp apparatus and are not corrected.. having an ED5.N. C9H12N604 240-241 203-204 236-238 234-235 C9H 1Z N 6 0 . mice injected ip with lo5leukemia L1210 cells by 30-35%. but its EDS0valuehas not been accurately determined. The cytotoxicities of the 2-azapurines to human epidermoid carcinoma cells No.Hl. "C Formula" 74 69 60 HZO MeOH-H. 9-Pentofuranosyl-2-azaadenines (13b-e) p-D-Ribofuranosyl 64 HZO 2-Deoxy-p-D-nbofuranosyl 31 H.Oi D. 2-Azaadenosine (13b) is 5 times as toxic as 8-azaadenosine. 1972.OC 233-234 190-1 9 1 175.1-0 dine (19a)19 and 2. . R % ture' are given in Table I. N. fAlso prepd by deformylation in MeOH-NH. 4-Amino-7-p-D-ribofuranosyl-7H-pyrazolo[3. 10. None of the other 2-azapurines or the intermediates leading to them that have been tested has shown any significant activity against leukemia L 1 2 10. at 78" of N-benzyloxy-5-formamido-l-~-D-ribofuranosylimidazole-4-carboxamidine.184 Journal ofMedicinal Chemistry.25H20 C. eIsolate as the picrate.0. Biologic Evaluations.N504.4il]-v-triazine (21a) Compound 4-Amino-l-~-D-ribofuranosylpyrazolo[ 3.Ok p-D-Arabinofuranosyl 43 H. iDid not cryst. and about 7 times as toxic as 2-azahypoxanthine.5-d]-v-triazine. "C 233-235 218-220 220-2 2 2 173-175 146-150h 175-177h 166-16gh 192-194'' 150h 1 $1 Formula" 3N 5'3 - A. 130 (1967). kIsolated by chromatog on a thick silica gel plate before recrystn. 2 Montgomery and Thomas HO 19 OH HO 20 OH HO 21 OH a series. bSee Ref 13. bAnalyzed as the picrate.25H.. fAnalytica1sample was not obtd. Of the other nucleosides of 2-azaadenine.D-Xylofuranosyld B. H.176d C10H13N505 C16H.4-d]-v-triazine (21a) -D -ribofuranosylpyrazolo[3.9 pmoles/l.. gRecrystn of picrate. CH. C9H12N603 'gH lZN6O4 Table 111. Klenow and S. N. 384 (1961). hMp of picrate. Frederiksen. Reist. F. Goodman.Og 2-Deoxy-p-D-ribo furanosy li 31 MeOHi p-D-Arabinofuranosyle 100 H.O H.Og 2-Deoxy-p-D-ribofuranosyle 25 H. is quite cytotoxic. H. Vol.005 as toxic. J.4H.4-d]pyrimidine 5oxide 5-Amino-N-benzyloxy-l-p-Dribofuranosylpyrazole-4carboxamidine 5-Amino-1-p-D-ribofuranosyl- Crude yield. IS.0.No. but the synthetic intermediate to 21a.D-Ribofuranosyled 97 H. % Recrystn solvent MP. dE. 2 in culTable 11. 9-Pentofuranosyladenine 1-oxides (5b-e) 30 HZO p-D-Ribofuranosylb 2-Deoxy-p-D-ribofuranosylC 64 H2O p-D-Arabinofuranosyld 67 95%EtOH 74 HZO p. value of 1. Acta.4-d]-v-triazine 32 H P "Anaiyzed for C. D. dMp of picrate.4d]pyrimidine (4-APP) is only 0.4-d ]pyrimifrom 4-amino. 5-Amino-N-benzyloxy-l-pentofuranosylimidaole-4-carboxamidines ( 12b-e) p. only 2'-deoxy-2-azaadenosine (13c) appears to be cytotoxic. Uv spectra were detd in aq s o h with a Cary Model 14 Recrystn solvent MP. 52.

A soln of l-cyclopentyl-5-formamido-N-benzyloxyimidazole-4-carboxamidine (11. H.3181-182d C8H14N604 triazole-4-carboxamidineb 40 H P 2.0 mmole) and benzyl bromide (342 mg.1 N HCl.. dMp of picrate. 1660. 2950.HC1(970 mg.H. HCl . I88 mg (17%). and dried at 78": mp 256-257". The combined CHCl. 1640.50). A. after which it was stirred for 20 hr. The analytical samples were dried over P. A.0 mmoles) was stirred for 10 min before the addn of CS. Anal. refluxed for 24 hr. 278 (9. 5-Amino-l-cyclopentyl-4-imidazolecarboxamidoxime (7a).260 (12. 2 7 1 (20.0 mmoles) in MeOH (350 ml) contg Ra Ni catalyst (2 g) was hydrogenated for 24 hr at room temp and atm pressure. 267 (9. pH 7. 3170 (NH).N. 284 (12. Vol. without purification. ext was dried (MgSO.1 N NaOH.52). 1. 1450 (CHI. 305 (3. the ext was evapd to dryness. 0.60). (2 X 20 ml).. 2. 266 (9. Chromatog analyses were carried out on tlc plates of silica gel H (Brinkmann). A soln of 2a (150 mg. obtd from a previous run by recrystn from EtOH.3).) and evapd to dryness in vacuo.2865 (aliph CH).2865 (aliph CH). 521.1 N HCl.1 N NaOH. 0. 262 (13. 100 mg (45%). The combined fiitrate and washes were evapd to dryness in vacuo. 60 mg (53%). extn of the residue from the aq soln gave another 20 mg: total yield. 9-Cyclopentyladenine (la).26 mmole) in 98% formic acid (2 ml) was refluxed for 4 hr and then evapd to dryness. C1.09 g. When a soln of the n H.).2-Azapurine Nucleosides Table 1V. The pH of a soh of the residue in H.N. hmax nm ( E X lo-') 0. (C.0.9). 1. Amax nm (E X lo-') 0. 1530.0 mmole) in EtOH (150 ml) contg 5% Pd/C (100 mg) was hydrogenated at room temp and atm pressure for 20 hr during which the theoretical uptake of H.No. ext of the residue was dried (MgSO. and 621 spectrophotometers. A. A soln of 5-amino-1-cyclopentylimidazole4carboxamidine (9).Hl. 391 mg (39%) of starting compd was obtained. Anal.3295 (NH). A soln of Sa (16. mp 292' dec. A. 0. 1580. (5 ml) was kept for 10 days with exclusion of light and then evapd to dryness in vacuo below 40". hmax nm (E X lo-'): 0. 261 (13.N50). (5 ml).8-Diazaadenosine 7.H. (C. 30 mg (57%). and the soln was evapd to dryness.). A soln of the residue in MeOH was acidified with concd HCl and evapd to dryness. 234 (19. 22 mg.4 mmole) in 95% EtOH (20 ml) and 1 N NaOH (2 ml) was refluxed for 3 hr. * 0.5). 1510 (purine ring stretch). highly concentrated). and the fiitrate was evapd to dryness in vucuo. Further CHCl.2870 (aliph CH).6 mmoles) in 3 N HCl(322 ml) was refluxed for 10 min and evapd to dryness in vacuo. and evapd to dryness in vacuo.58 mmoles) in 1N NaOH (10 ml! was refluxed for 30 min. Compound spectrophotometer. 0.730. Most of the chromatog purifications were carried out on Mallinckrodf SilicAR-7 with the solvents indicated. (C&. N.N.05: mp 265-266'.2). The residue crystd from EtOH as an HCl salt: yield.O. H. 2. 144 mg (65%) The uv. 73.60). 246 mg (87%). hmax nm (E X lo-.8-Diazaadenosine (21b) Journal of Medicinal Chemistry. omax (cm-') 3300-2600 (br) (NH+. Anal.0 mmole) in EtOH (75 ml) andH. 233 (43.. ring stretch). 298 (sh) (3. Ymax (cm-l) 3310.0). The CHC1. Omax (cm-') 3295. After trituration with ether. A soln of Sa (1. 4. and pmr spectra of this material indicate that it is impure Sa. 68 mg (31%).1495 (imidazole ring stretch).1490 (purine ring stretch).63). H. 1575. The catalyst was then removed.04 UAnalyzed for C.0 mmoles) in DMA (20 ml) was heated for 20 hr at 110" and evapd to dryness in vacuo. 283 (11. 3140 (arom CH).07 mm) for 16-20 hr a t the temps given. 3300-2200 (br) (OH). The analytical sample was obtd by recrystn from EtOH.Oe 193-195 C8H11N. The residue crystd from C. The residue crystd from EtOH (125 ml) as a white solid: yield. 10. 730.1).2). ir and uv spectra) with an authentic sample of 9-cyclopentyladenine. was refluxed for 10 min in 3 N HCl(4 ml).3050. N. Anal.1).2870 (aliph CH). CH).N.O (200 ml) was neutralized with 1 N NaOH. ir. N. neutralized with 1N HC1.CO. mp 102-103".1 N NaOH.327 mg. (C&. This material was identical in all respects (mp. 0. H. B.. was ohserved. The catalyst was removed. The soln was neutralized with dil NaOH and extd with CHCl. pH 7. (CpH. 273 (10.45). 0. Evapn of the aq mixt gave a residue that crystd from 20% aq MeOH as a yellow solid: yield.1565. The process was repeated. was dried at 78'.. and then poured into H. 1.O.3190. 10. A second crop was obtd from the soln of the analytical sample and dried at 100': hmax nm (E X lo-') 0. N. 1625.94). B..) C.4HC1 (230 mg. 1685 ( G N ) .O (60 ml) Crude yield.1 N HCI. 0.HBr) C. C. The spots were detected by uv light after spraying the plates with Ultraphor (WT. Bmax (cm-') 3435.3020 (arom CH).O (150 ml).1575 (C=NH$ and imidazole ring stretch).O (100 ml) was neutralized with 1 ml of 1N NaOH. 2960. and dried at 78": mp 192-194".67HZO) C. The identity of the fractional hydrates was further confirmed by conversion to 9-cyclopentyladenine (see above). (276 mg.2).45). 2950.. 10.1 N HCI. From the filtrate.).2870 (aliph CH).1).). 9-Cyclopentyl-2-mercaptoadenine (4a). 216 f28. 1485 (purine and C.90 mmoles) in H. 232 (27.3075..H. B . 2 185 Formulas MP.1 N NaOH (20.11. % Recry ste solvent .HC1. 295 (2. 1. The analytical sample was obtd in a previous run by recrystn from EtOH and dried at 100" (0.00 g. N. 2.O).1 N NaOH. chemical shifts quoted in the case of multiplets are measured from the approximate center. (TMS) with a Varian A-60A spectrometer. bAnalyzed at the picrate. This material was identical in ir and uv spectra with an authentic sample of 5-amino-l-cyclopentyl-4-imidazolecarboxamidoxime (7). 2950. 268 (19. the residue crystd from EtOH: yield. N. The analytical sample. A soln of 9-cyclopentyladenine (203 mg. and hydrogenation was resumed for another 24 hr. A. N. and extd with CHCl. neutralized with concd HCl.0 mmoles) in glacial AcOH (50 ml) and 30% aq H.94 mmole) in DMF (25 ml) contg a suspension of anhyd K.* I-Benzyl-9-cyclopentyyladenine Hydrobromide (2a). The residue crystd from EtOH as a white solid: yield.0. 3125. 1495. IS. 268 (sh) (12.4). hmax nm (E X lo-. 2. and evapd to dryness in vacuo.N.05 g. The product.. 1565.5H20 5-Amino-l$-D-ribofuranosyl-l. CRecrystn of picrate.. A soh of 7a . 263 (7.0 mmole) in abs EtOH contg sponge N i catalyst (50 mg) was hydrogenated at atm pressure for 24 hr.O (15 ml) was raised to 10 with concd NH40H before it was extd with CHCI.) and evapd to dryness.) 0.3-triazole-4-carboxamidine 54 MeOH 138-140 CI6H. 1630. The residue crystd from EtOH as a white solid: yield.37 g (67.O. H. The soln was filtered. 9-Cyclopentyladenine 1-Oxide (Sa). Anal. 268 (8.8).1).18 g (90%). The residue crystd from MeOH as a white solid: yield. 1225 (N-.2.1 N HCl.690 (C.1645. BmaX (cm-') 3265 (NH). as a white solid: yield.N. Anal. 2960. 260 (9.70 g (78%). the prodresidue i uct pptd as a white solid: yield.6).695 (C.07 mm) over P.N." B. The CHCl.) EtOH. After drying (MgSO. This material was identical in all respects with that obtd from reaction A.44H20) C.1.N5S) C.1 N HCI. 241 mg (66%). (100 ml).1580 (purine ring stretch).5 H. 37%. A s o h of 9-cyclopentyladenine (2. B. The residue crystd from EtOH as a white solid: yield. 'C 9Q-D-Ribofuranosyl-8-azaadenine 1-oxide 78 MeOH 208-210 'gH 1a N 6 0 5 5-Amino-N-benzyloxy-1-p-D-ribofuranosyl-l. pmr spectra in DMSO-d.11). The catalyst was removed by filtration and washed with MeOH. The analytical sample from DMF was dried at 100": mp 321-323'.8).03 g. A soln of 1-cyclopentyl-N-benzyloxy-5-formamidoimidazole-4-carboxamidine (327 mg.99): Bmax (cm-'): 3365 3260.H. 1630 ( G N ) . 1450 (CH). fresh catalyst (50 mg) was added. The material was identical with an authentic sample of 9-cyclopentyladenine ..O) C. pH 7. H. A soln of 9. (0. 263 (13. 2. pH 7. 1490.3080 (NH). Amax nm (E X lo-') 0. The analytical sample was obtd in a previous run by recrystn from EtOH. (Cl. eIsolated by chromatog on a thick silica gel plate before recrystn.H.1 N NaOH. (4 X 25 ml). 1. pH 7 . 0. N-Benzyl-9-cyclopentyladenine (3a). The residue crystd from aq EtOH (80%): yield. A soln of 7a (2. H. 284 (11.) and evapd to dryness in vacuo.H.H. 1972.4HC1(64 mg.2. 0. A soln of 2a (374 mg. 1560.3160 (NH.5%). ext was dried (MgSO. Ir spectra were detd in pressed KBr discs with Perkin-Elmer Models 221-G.3140. pH 7. H. (CI. 257 (9. The residue crystd from EtOH: yield. 5-Amino-l-cyclopentyli1nidazole-4-carboxamidine (9a).0). 1. Spectral data indicated that this material is J -cyclopentyl-5formamidoimidazole-4-carboxamidoxime (6a). Anal. 283 (11.

Jr. (11) N.. then cooled.1 N NaOH. Thomas... Anal.H. and members of the Molecular Spectroscopy Section of Southern Research Institute for the spectral and most of the microanalytical data reported. 204 mg (66%).4). It was dried at 78": mp 177-182". YmaX (cm-') 3490. The authors are indebted to Dr. (7) L.4 HCl): yield.2865 (aliph CH). Biophys. 703 mg (73%).H..1 N HCl.7 (m. was obtd by recrystn from EtOH and dried at 78": mp 275" dec. The product was isolated by chromatog on a silica gel plate as a white glass that crystd from H. Ind. 1690. Both ir and uv data of these two crops are in agreement with those given in A above. 2940.8-diazaadenosine (21b) were all prepd in essentially the same manner as 9-cyclopentyl-2-azaadenine ( l a + 5a + 10a + 12a + 9a + 13a). 3147 (1960).3000 (arom CHI. Loeffler. Upon cooling. A.S.I-d]-v-triazine(2 la). obtd by recrystn from EtOH. J. 5-Amino-1-cyclopentylimidazole-4-carboxamide (15) and 4.95 mmole) in H. H. to Mrs. H. Jr. A. (C. L. Commun.O (40 ml) and EtOH (15 ml) was neutralized with 1N NaOH.0 mmoles) in H.H. 458 (1969). 266 (sh) (10.O (60 ml) and EtOH (10 ml) was refluxed for 27 hr.O (40 ml) was added slowly a soh of NaNO. 0. 1650. The combined CHCl..O (15 ml).. and to Dr. N. Atlanta. J. 2945. (C. The middle band also gave a white solid: yield. 1590. and F.9). 2-Aza-9-cyclopentyladenine (13a).186 Journalof Medicinal Chemistry. The 9-pentofuranosyl-2-azapuMes (13b-e). Brown. 1972. 3190. 19th Southeast Regional Meeting of the American Chemical Society. 1. 830. 257 (7. No. A s o h of 9-cyclopentyladenine 1-oxide (218 mg. (6) J. 3340.0 mmole) in DMA (20 ml) contg PhCH. Montgomery and H. and J. Szweykowska.. kmax nm ( E X lo-'): 0 .72). Anal. Chem. after 2 triturations with Et. 1075.2755 (1958). and then refluxed for 8. H.H2.N. The analytical sample was obtd by recrystn from EtOH and dried at 78": hmax nm ( E X i o n 0 . U. the second crop was a 1. (690 mg. Evapn of the filtrate to 10 Acknowledgment.84). 2910. H. 2950. N. 1460 (imidazole ring stretch). stirred s o h of 5amino-1-cyclopentylimidazole4carboxamidine . 1695 (C=O). 297 (6. Martha Thorpe for her help in the interpretation of the pmr spectra. References (1) J..69 (1970). Georgia. 1 N HCI. 289 (9. Montgomery. Chem.H. and L. E. Lee. This material was identical in all respects with that from the previous run. 3190 (NH). 118 mg (16%). 1. 4amino-7-0-D-ribofuranosylpyrazolo [ 3 . A s o h of 10a (827 mg.O (150 ml) to which 2 ml of 1N NaOH was added was refluxed 9 hr and then evapd to dryness.y The fast moving band gave a solid (6 mg. Leonard. Thomas and J. Margaret H. 2920.0~ 0. 8.4 (m. Biochem.2): omax (cm-') 3100 (br). 1. identified as 4. Amax nm ( E X lo-.N. pH 7. (London). 125 mg (36%). I-Benzyloxy-9-cyclopentyyladenine Hydrobromide (loa). 1. 20. H. W. The residue crystd from EtOH.1719 (1966). 2. Smith. A. The ppt that formed in the remaining aq s o h was collected as a white yolid: yield..1 N NaOH. 4. B. Two recrystn from EtOH gave the product as a white solid: yield. 1962 (1971). 264 (10. 695 (C. Chem. 1.0 mmole) in H. and evapd in vacuo without heating to remove the alc. A s o h of 10a (390 mg.O) C.0 mmoles) in H. 690 (C. 1700. Davoll. The soln was filtered and evapd to dryness. 1565 (purine ring stretch). (C.6). L.&N. A soln of l l a (6. Repeated CHC1. G. Q. 1510 (azaadenine ring stretch).4). W. K. hmax nm ( E X lo-)) 0.$.).. Pharmacol. J. Amer. S. Jr.).3). 1590.5C.H.). A s o h of 1-benzyloxyadenosine hydrobromide (908 mg. Effect of Base o n 2-Aza-9-cyclopentyladenine. h o g . 80.02) C. The residue. N-Benzyloxyadenosine (14b). 269 (17. (9) H.O (60 ml) was neutralized with 1 N NaOH (0. 0. 735. dild with EtOH (30 ml) to give a clear s o h . 255 (sh) (6..88). 4. (14) M.37). 279 (10. 163 mg (23%).54 g. (3) W. SOC. J.) and evapd to dryness in vacuo.1040 (CO). 1495 (C. Stevens.3140. A.O: yield. Lee. A. 374 (5. Frederikson. pH 7. Omax (cm-') 3430. 2 7 0 (17.90). Montgomery and H.NJ . extn of the resulting cloudy s o h gave a clear aq layer.). 15. A. 145 mg (41%). ibid. 2.1 N NaOH (30 ml) was refluxed with stirring for 20 hr. Bennett.. Vail. hmax nm ( E X 0.3080.1 N HC1.685 (C. Biochem. Chem. 204 (13.1 N NaOH.15 g. 1490 (C.3345. SOC. N. W.I.0 mmoles) was kept for 4 days at 25" and evapd to dryness in vacuo without heating. J. The analytical sample of this material. M.95 ml). CancerRes. 1.1593 (1958). J. Dmax (cm-') 3400-3000 (br) (NH).7 mmoles) in H. Martinez. Magrath.1 N HCl. The residue. Hamzi.2HC1: yield. M. (12) J. Nucleosides. (satd at 0") (800 ml) was heated in a stainless steel reaction vessel at 80" for 2 days and then evapd to dryness. Anal. 3025 (NH). a white cryst solid. N. 0.1505 (C=N. 1645.20).. 3160. 15.5. Montgomery and H. 1575 (ring stretch). Heterocycl.85). 1590.H. 262 (12. 258 (11.H50H) C. Taylor and P. Montgomery. 0. 22. 2950. 4. 34002400 (br) (NH:). Montgomery. The details for each compd are given in Tables 11-IV. H. 255 (8. Tong. 112. A.&.3400.BrN. An EtOH s o h of the residue was acidified with concd HCl and evapd to dryness. 1630 (C=N). Abstracts. 318 (4. W. N-Benzyloxy-9-cyclopentyladenine (14a). 10. 252 (18. 254 (7. P. Nat. 1680.0. A s o h of the residue in EtOH was acidified with concd HCl and evapd to dryness.). 735. pH 7. 1525.82.92). (10) E. Montgomery. 3380.0 mmole) in H. Dmax (cm-') 3450. 2860 (aliph CH). Thomas.. (CyHl. 2007 (1963).. Thomas.30). 0.N. 318 (4. 6 in ppm.2860 (aliphatic CH).1500 (C. Chem. 107 mg (37%). W. NH and NH. 284 (13.3). The slower moving band gave a white solid. L. 5-Amino-N-(benzyloxy).H. N. J.Br (684 mg. 1525 (purine ring stretch). Vail for the cytotoxicity data reported.3020. H. The ppt that formed was collected after standing 2 hr in the cold: yield. A. mp 169-171'. and G. 236 (25. Med.5-Diamino-6-cyclopentylamino-v-triazine (16).76 (1965). 36.0). H.. N. 1680 (C=NH:). 1500. C=C). 528 mg (79%).8).5diaminod-cyclopentylamino-v-triazine (16).2865 (aliph CHI. Skipper..1 N NaOH.1). a cryst solid pptd: yield. and S. 7.) C.. 0. Acad. Anal. S. To a cold.. Anal. pH 7 ._ " C. 257 (7.690 (C. pH 7. (5) J. 268 mg (82%).l-cyclopentyliidazole-l-carboxmidine (12a). vmax (cm-I) 3380 (OH). N. J. C. Brown.O.). N. 730. omax (cm-') 3400 (OH). 1205 (COC).). W.O. Tong. A. Chem.2860 (CH). Loeppky. Chem. Chem. Goodman.5 hr. L. 262 (12. 264 (10. 740. 1610. 235 (25. The analytical sample was obtd in a previous run by recrystn from H. and H. was dissolved in EtOH (50 ml) and hydrogenated at room temp and atm pressure for 20 days in the presence of Ra Ni catalyst. Proc. l-Cyclopentyl-5-formamido-N-benzyloxy~idazole-4-car~xamidine (1 la). The analytical sample was obtd by recrystn from EtOH and dried at 78": mp 132-133". Anal. R. This product was identical in all respects with that from the previous run. B.1).. Sci. A s o h of 1l a (327 mg.H.N.2920.1 N NaOH. 1600. 1. Anal.3320 (NH).7).1 N HCl. L. Each was extd with hot MeOH. 4.3300 (NH).504 (1962). A. CH). R. kept for 2 days at 25". Purification of this material was effected by chromatog on a thick plate using CHC1. 1680 (amide I). (13) M. 2 Montgomery and Thomas ml caused the pptn of another cryst solid that was identified by its uv and ir spectra as 1cyclopentylJ-formamido-N-(benzyloxy)imidazole-l-carboxamidine: yield.84 mmoles) in 0. (C. ext was dried (MgSO. It was identified by spectral and analytical data as 5-amino-1-cyclopentylimidazole4carboxamide ( 15). D. Laster for the leukemia L1210 screening data. 3200-2200 (br) (NH. A.-MeOH (9: 1) as developing solvent.80. mp 129130". yield.). was a white solid: yield. 709 (1966). Schabel. Amer. A soln of the residue in EtOH was acidified with concd HCl and evapd to dryness.7). (satd at 0") (200 ml) was heated in a stainless steel reaction vessel at 80" for 2 days and evapd to dryness in vacuo. Skoog. The white solid that pptd on cooling was collected by filtration: yield. Stevens and G.1 N nm ( E X NaOH.27).1530. J. An 18% recovery of starting material was obtained from the filtrate.O~ HC1) C. F. (C. 1645.) 0. pH 7.3100 (arom CH). The residue crystd in 2 crops from EtOH as a white solid (1. Chumley. K. 1%)that was identified as 9-cyclopentyl-2-azaadenine (13).1 N NaOH. 269 (16. 111 mg (36%).9).H.1 N HCl.9).02 g. A. 303 mg (78%). hmax 0. (C. Arch.) C. Grimm.4HC1(1. 2870 (aliph CH). (4) G. The analytical sample was obtd by careful recrystn from EtOH. (8) J. 0. 1610. 3 bands were obtd. B.8). crystd from EtOH as a light yellow solid: yield. H.O and dried at 78": mp 212-215". CH. A s o h of 2-aza-9-cyclopentyladenine (375 mg.. SOC. . Carraway. H.). Achmatowicz. 143 mg (21%).2759 (1958).06).0 mmoles) in methanolic NH. A. J.17). C.0 mmoles) in methanolic NH. 287 (13. 274 (14.H. Vol.12). and 2. mp 179-181" dec. Coburn. The analytical sample. 2945. 0. 293 (4.. to Mrs. (60%).). Org.7 (m. W. H. and the white cryst solid that pptd was collected by filtration (27 1 mg). 5. was made basic (pH 9) with 1N NaOH. 1 N HC1. A. 1530 (amide 11). Abstr 74 (1967).37).). 1615. hmax nm ( E X 0.0). 1450 (CH). Goodman. 56. 256 (12. 1650 (CH=NH$). 286 (7. 297 (6.790 (C.9). pH 7. 115 (1964). (2) J. A s o h of 10a (413 mg. 286 (7.

Nb-Substituted Adenosines
(15) C. Temple, Jr., C. L. Kussner, and J. A. Montgomery, J. Org. Chem., 32,2241 (1967). (16) M. A. Stevens, H. W. Smith, and G . B. Brown, J. Amer. Chem. SOC.,82, 3189 (1960). (17) T. Fujii, C. C. Wu, T. Itaya, and S . Yamada, Chem. Znd. (London), 1598, 1967 (1968). (18) J. A. Montgomery and H. J. Thomas, J. Org. Chem., 28,2304

Journal ofMedicina1 Chemistry, 1972, Vol. 1.5,No. 2

187

(19)

(1963). J. A. Montgomery, S. J. Clayton, and W. E. Fitzgibbon, Jr., J. Heterocycl. Chem., 1,215 (1964). (20) J. A. Montgomery, H. J. Thomas, and S . J. Clayton, ibid., 7 , 215 (1970). (21) J. A. Montgomery and C. Temple, Jr., J. Amer. Chem. Soc., 80,409 (1958).

N6-SubstitutedAdenosines: Synthesis, Biological Activity, and Some Structure-Activi ty Relationships
M. H. Fleysher
Department o f Experimental Therapeutics, Roswell Park Memorial Institute, Buffalo,New York 14203. Received July I 7, I 9 71

Nucleosides of N6-substituted adenines, which possess cytokinin activitz, inhibit the growth of tumor cells, while the corresponding adenines are relatively inactive. Addnl N -substituted adenosines have been prepd and tested to secure information regarding structure-activity relationships, if any. The new compds include N6-butyl-, N6-n-2-propoxyethy1, N6-n-2-butoxyethyl-, N6-cyclohexyl-, N6-cyclopropylmethyl-, N6-tetrahydrofurfuryl-, N6-geranyl-, N6-farnesyl-, and @-a-pyridoxyladenosine. They were prepd from 6-chloropurine riboside by nucleophilic substitution with the appropriate amine. The known cytokinin compds, 2-methylthio-N6-isopentenyladenine, cis-6<~-chloro-2-butenylamino)purine, and trans-6-(~-chloro-2-butenylamine)purine and their ribosides, and trans-zeatin riboside were examd for other biological activity. The alkylated adenosines show optimal cytokinin activity when the N6substituent contains a double bond. In Escherichia coli the compds were active at 10-6-10-4M with the trans isomers showing greater activity than the cis compds. As inhibitors of mouse adenocarcinoma cells (TA-3) in culture, some of the compds were active at 10-5-10-6M but in sarcoma $180 cells in culture they were all less active. Redn of the double bond in the side chain lowered activity of these compds in the tumor cell cultures. The trans isomers are more active against tumor cells in vitro than the cis analogs, paralleling their activity as cytokinins. The presence of an OH group in the side chain diminished antitumor activity. A moderate increase in survival time of mice bearing leukemia L-1210 was produced by the compds bearing an ether linkage in the side chain. We have previously reported',' the synthesis of a series of N6 -substituted adenine ribosides which are potent cytokinins. Many of these compds inhibited the growth of neoplastic cells in vitro at concns of about 1OW6M. Most of these adenosine analogs had different effects on various leukemic cells and no effects on lymphocytes in vitro. It was also noted that at lower concns (e.g, 10-8-10-7M) some stimulation of human leukemic (line 6410) cell growth took place in contrast to the inhibitory effects that occurred at higher concns. The corresponding free adenines were relatively inactive against tumor cells. The more active tumor inhibitory nucleosides were found to be N6benzyl-, N6-furfuryl-, N64hoxyethyl-, N6-phenyl-, and N6thenyladenosines. Like N6-(3-methyl-2-butenyl)adeno~ine'~ (IPA), these analogs are also active as cytokinins. The present communication? is an extension of our previous work. In order to secure further information on the structure-activity relationships in this series of adenosine derivatives, addnl analogs were prepd and examd for their biol properties. These include N6-n-Bu- (I), N6-n-2-propoxyethyl- (11), N6-n-2-butoxyethyl- (111), N6-cyclohexyl- (IV), fl-cyclopropylmethyl- (V), N6-tetrahydrofurfuryl- (VI), N6geranyl- (VII), N6-farnesyl- (VIII), and N6-cu4-pyridoxyladenosines (IX). The compds were tested for cytokinin activity in the tobacco callus bioassay, and in microbial and tumor systems. This series of compds was augmented by truns-N64-hydroxymethyl-2-butenyladenosine (zeatin riboside), a potent cytokinin that was prepd in this work by the method of Shaw, e t aL4 The compds were chosen for synthesis and examn of properties for the following reasons.
t A portion of this material was presented by the author at the 160th National Meeting of the American Chemical S ~ c i e t y . ~

(I) The n-Bu fragment represents the shortest C chain among a series of N'mbstituted adenines resulting in really good cytokinin a ~ t i v i t y(11,111) .~ The propoxyethyl and butoxyethyl compds are homologs of N6-2-ethoxyethyladenosine, a compd with antitumor activity in vivo.' (IV, VI) The cyclohexyl and tetrahydrofurfuryl compds are satd derivatives of unsatd analogs which showed biol and antitumor activity. (V) The cyclopropylmethyl derivative was prepd to examine the biol effect of the smallest satd ring structure. (VII, VIII) The geranyl and famesyl side chains were chosen to det the effects of multiple isopentenyl fragments on a single side chain. (IX) The cu4-pyridoxy1 side chain was included to evaluate the effect of a strong hydrophlic substituent which contains a biol active moiety. Zeatin riboside (trans-N6-(4-hydroxymethyl-2-buteny1)adenosine was included because it is about the most active known cytokinin of the N6-adenosine ~ e r i e s . ~ The , ~ compds were prepd by condensing 6-chloropurine riboside (6-chlorog-P-D-ribofuranosyl-9H-purine) with the corresponding amines by nucleophilic substitution in boiling EtOH, using CaC03 or E t a as ancillary acid acceptors.' The compds were isolated and purified by crystn. The purity of the products was confirmed by chromatog, elemental analyses, and by uv spectra. The physical data are given in Tables I and 11. The new compds were examd for cytokinin activity in a tobacco pith assay system by the method of Murashige and Skoog.8 As shown in Table 111, the compds vary in activity. The N6-butyl and propoxyethyladenosines have good activity, although somewhat less than that of zeatin riboside. The other compds displayed marginal or no cytokinin activity noted since high concns had to be used for initial response.

N6-Substituted adenosines. Synthesis, biological activity, and some structure-activity relations
M. H. Fleysher
J. Med. Chem., 1972, 15 (2), 187-191• DOI: 10.1021/jm00272a015 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.acs.org on April 25, 2009

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Nb-Substituted Adenosines
(15) C. Temple, Jr., C. L. Kussner, and J. A. Montgomery, J. Org. Chem., 32,2241 (1967). (16) M. A. Stevens, H. W. Smith, and G . B. Brown, J. Amer. Chem. SOC.,82, 3189 (1960). (17) T. Fujii, C. C. Wu, T. Itaya, and S . Yamada, Chem. Znd. (London), 1598, 1967 (1968). (18) J. A. Montgomery and H. J. Thomas, J. Org. Chem., 28,2304

Journal ofMedicina1 Chemistry, 1972, Vol. 1.5,No. 2

187

(19)

(1963). J. A. Montgomery, S. J. Clayton, and W. E. Fitzgibbon, Jr., J. Heterocycl. Chem., 1,215 (1964). (20) J. A. Montgomery, H. J. Thomas, and S . J. Clayton, ibid., 7 , 215 (1970). (21) J. A. Montgomery and C. Temple, Jr., J. Amer. Chem. Soc., 80,409 (1958).

N6-SubstitutedAdenosines: Synthesis, Biological Activity, and Some Structure-Activi ty Relationships
M. H. Fleysher
Department o f Experimental Therapeutics, Roswell Park Memorial Institute, Buffalo,New York 14203. Received July I 7, I 9 71

Nucleosides of N6-substituted adenines, which possess cytokinin activitz, inhibit the growth of tumor cells, while the corresponding adenines are relatively inactive. Addnl N -substituted adenosines have been prepd and tested to secure information regarding structure-activity relationships, if any. The new compds include N6-butyl-, N6-n-2-propoxyethy1,N6-n-2-butoxyethyl-, N6-cyclohexyl-, N6-cyclopropylmethyl-, N6-tetrahydrofurfuryl-, N6-geranyl-, N6-farnesyl-, and @-a-pyridoxyladenosine. They were prepd from 6-chloropurine riboside by nucleophilic substitution with the appropriate amine. The known cytokinin compds, 2-methylthio-N6-isopentenyladenine, cis-6<~-chloro-2-butenylamino)purine, and trans-6-(~-chloro-2-butenylamine)purine and their ribosides, and trans-zeatin riboside were examd for other biological activity. The alkylated adenosines show optimal cytokinin activity when the N6substituent contains a double bond. In Escherichia coli the compds were active at 10-6-10-4M with the trans isomers showing greater activity than the cis compds. As inhibitors of mouse adenocarcinoma cells (TA-3) in culture, some of the compds were active at 10-5-10-6M but in sarcoma $180 cells in culture they were all less active. Redn of the double bond in the side chain lowered activity of these compds in the tumor cell cultures. The trans isomers are more active against tumor cells in vitro than the cis analogs, paralleling their activity as cytokinins. The presence of an OH group in the side chain diminished antitumor activity. A moderate increase in survival time of mice bearing leukemia L-1210 was produced by the compds bearing an ether linkage in the side chain. We have previously reported',' the synthesis of a series of N6 -substituted adenine ribosides which are potent cytokinins. Many of these compds inhibited the growth of neoplastic cells in vitro at concns of about 1OW6M. Most of these adenosine analogs had different effects on various leukemic cells and no effects on lymphocytes in vitro. It was also noted that at lower concns (e.g, 10-8-10-7M) some stimulation of human leukemic (line 6410) cell growth took place in contrast to the inhibitory effects that occurred at higher concns. The corresponding free adenines were relatively inactive against tumor cells. The more active tumor inhibitory nucleosides were found to be N6benzyl-, N6-furfuryl-, N64hoxyethyl-, N6-phenyl-, and N6thenyladenosines. Like N6-(3-methyl-2-butenyl)adeno~ine'~ (IPA), these analogs are also active as cytokinins. The present communication? is an extension of our previous work. In order to secure further information on the structure-activity relationships in this series of adenosine derivatives, addnl analogs were prepd and examd for their biol properties. These include N6-n-Bu- (I), N6-n-2-propoxyethyl- (11), N6-n-2-butoxyethyl- (111), N6-cyclohexyl- (IV), fl-cyclopropylmethyl- (V), N6-tetrahydrofurfuryl- (VI), N6geranyl- (VII), N6-farnesyl- (VIII), and N6-cu4-pyridoxyladenosines (IX). The compds were tested for cytokinin activity in the tobacco callus bioassay, and in microbial and tumor systems. This series of compds was augmented by truns-N64-hydroxymethyl-2-butenyladenosine (zeatin riboside), a potent cytokinin that was prepd in this work by the method of Shaw, e t aL4 The compds were chosen for synthesis and examn of properties for the following reasons.
t A portion of this material was presented by the author at the 160th National Meeting of the American Chemical S ~ c i e t y . ~

(I) The n-Bu fragment represents the shortest C chain among a series of N'mbstituted adenines resulting in really good cytokinin a ~ t i v i t y(11,111) .~ The propoxyethyl and butoxyethyl compds are homologs of N6-2-ethoxyethyladenosine, a compd with antitumor activity in vivo.' (IV, VI) The cyclohexyl and tetrahydrofurfuryl compds are satd derivatives of unsatd analogs which showed biol and antitumor activity. (V) The cyclopropylmethyl derivative was prepd to examine the biol effect of the smallest satd ring structure. (VII, VIII) The geranyl and famesyl side chains were chosen to det the effects of multiple isopentenyl fragments on a single side chain. (IX) The cu4-pyridoxy1 side chain was included to evaluate the effect of a strong hydrophlic substituent which contains a biol active moiety. Zeatin riboside (trans-N6-(4-hydroxymethyl-2-buteny1)adenosine was included because it is about the most active known cytokinin of the N6-adenosine ~ e r i e s . ~ The , ~ compds were prepd by condensing 6-chloropurine riboside (6-chlorog-P-D-ribofuranosyl-9H-purine) with the corresponding amines by nucleophilic substitution in boiling EtOH, using CaC03 or E t a as ancillary acid acceptors.' The compds were isolated and purified by crystn. The purity of the products was confirmed by chromatog, elemental analyses, and by uv spectra. The physical data are given in Tables I and 11. The new compds were examd for cytokinin activity in a tobacco pith assay system by the method of Murashige and Skoog.8 As shown in Table 111, the compds vary in activity. The N6-butyl and propoxyethyladenosines have good activity, although somewhat less than that of zeatin riboside. The other compds displayed marginal or no cytokinin activity noted since high concns had to be used for initial response.

whereas the cis isomers.O (680:170: 144). 18.and famesyladenosines).1 17. coli N6€ompound p-n-But yladenosine ~-n-2-Propoxyethyladenosine Molar concn for 50% growth inhibition N6-Tetrahydrofurfuryladenosine N6-Geranyladenosine N6-Farnesyladenosine N 6 e 4Pyridoxyladenosine N6-(4-Hydroxy-3 methyl-trans-buteny1)adenosine (zeatin riboside)* usee reference 8. mp E x 10-3 N6-n-2-Butoxyethyladenosine N W y clohexyladenosine "Cy clopropylmethyladenosine N6-Tetrahydrofurfuryladenosine N6-Geranyladenosine N6-Farnesyladenosine N6-(r4-Pyridoxyladenosine 26 3 26 3 263 265 263 26 5 266 266 274 19. Vol. The a4-pyridoxyl-. nBuO~)H~ACOH-H.0.66 0. The N6-3-chloro-2-butenyladenosines and adenines are less active than the N'4sopentenyl analogs but their trans isomers are distinctly more active than the pH 7. possessing more steric bulk. wheat germ. 9.6 17.59 2.72 0. followed by the n-butyl-.00 1. trans isomers impart the highest order of cytokinin activity. 2-amino.46 adenosine (trans-zeatin riboside)b Solvent s y s t e f i B C D 0.O (100032.0.0 16.90 0.72 0. This nucleoside and other 2-substituted N6-isopentenyl and zeatin ribosides have been synthesized and examd for cytokinin activity! While 2-methylthio. M.28 1. No.24 7. 15.0 pH 12. coli.7 18.2 Fleysher Table I.00 1. perhaps for the same reason.0 E X lo-' h max. D. a fact which has been obsd previously.41 0. et al.67 0. this effect may be due to the presence of an OH group in the side chain causing loss of antitumor activity.72 0.9 19. An @-adenosine analog isolated from sol RNA of E. lo They concluded that planar side chains.20 4. 18.5:300) (cloudy upper phase).67 0. Zeatin riboside is not active in this system.6 18.84 1.OH (7:2: 1). i-PrbH-1% aq (NH SO.2 18.57 0. J.3 18.85 0. CSee reference 11.87 0. n-2-butoxyethyl-.51 0. and yeast. In another study.210 267.90 1. 18.83 0.4 17.00 2. (2: l). 211 267.80 0. 267 267 267 268 26 7 26 7 26 8 269 270. having cytokinin activity has recently been described by Leonard and his coworkers' and identified as N6-isopentenyl-2-methylthioadenosine.07 1.76 0.95 5.9.7 19. *See reference 10.7 h max.2.4 18. Growth Inhibition in E..13 1. 210 267. . Table 11. 19.89 0.4 17. coli are summarized in Table IV.8 17. cyclohexyl-. dSee refence 12. i. and 2-chloro substituents in the original compd had very little influence on cytokinin effectiveness. and cyclopropylmethyladenosineswere inactive.7 20.52 0.86 0.17 3.89 0.79 0.00 at concentrations Compound N h B u t y ladenosine N6-n-2 Propoxyethyladenosine N6-n-2 Butoxyethyladenosine N W y clohexyladenosine N6Cyclopropylmethyladenosine 10 ve/l 25 ~ d 200 l udl 2.6.0 17.83 0.0.89 0.3 20. 18.00 1.8 18. $ The activities of all the compds discussed as inhibitors of the growth of E. b e e reference 4. i-PrOH-H 0-NH.81 0.90 0. mp e x adenines and adenosines were investigated by Hecht. isopentenyl-.73 7.6 3 uThe solvent systems used for descending chromatog (Whatman No. the 2-hydroxy substituents greatly lowered cytokinin activity6 showing that an OH group in the structure has marked effects. Pyridoxyladenosine is inactive.72 0.81 0. geranyl-.3 20.80 2.72 0. Cytokinin Activity of the New N6-Substituted Adenosines on Tobacco Bioassay4 Relative growth compared to control as 1. E.72 Table I V .81 0.1 18. Because of these cytokinin variations some of these compds were tested in this work in addn to the new ones prepd here.2 Against sarcoma S-180 cells (Table V) most of the new compds other than N6-isopentenyladenosine were inactive paralleling their cytokinin effects. B.0 Compound N6-n-Butyladenosine 2-propox yethyladenosine N6-nA max.41 1.4 18. 210 268.41 3.325 18. C.86 0. Leonard for generous gifts of these materials. and tetrahydrofurfuryladenosines. bSee reference 4.3 267.72 0.60 0. The most active inhibitors were N6-2-propoxyethyladenosine and trans-zeatin riboside.87 0.73 0.79 0. Hecht and Professor N. The N6-a4-pyridoxyladenosine is inactive.77 0. The results show the effects of N6 side chain satn and of very low water solubility (geranyl.1 19.68 0. cis compds paralleling their cytokinin activity. The low water solubility shown by N6-isopentenyl-2thiomethyl compds possibly accounts for their minimal activity in this system. Paper Chromatography and R f Values of N'-Substituted Adenosines Compound 6-Chloropurineriboside N6-n-Butyladenosine N 6-n-2-Propoxyethyladenosine N 6-n-2-Butoxyethyladenosine N 6-Cyclohexyladenosine N 6Cyclopropylmethyladenosine N 6-Tetrahydrofurfuryladenosine N 6-Geranyladenosine N 6-Farnesyladenosine N 6a4-Pyridoxyladenosine A 0.68 E 0.63 0.80 0.1 4.86 0.05 1.74 0. mp 10-3 19.and butoxy~~~~ $We are indebted to Dr.00 1. 1 paper) (measured by vol): A.83 0.29 N6-(4-Hydroxy-3-methyl-trans-butenyl)0.52 2.13 1.210 267.5 Table 111. the effects of side-chain planarity in a series of " h b s t i t u t e d 5x 5x N6-n-2-Butoxyethyladenosie 5x N'-Cyclohex yladenosine 4 x 10-5 fl-Cyclopropylmethyladenosine None N'-Tetrahydrofurfuryladenosine 8X N'Geranyladenosine 8X (in suspension) (in suspension) N6-Farnesyladenosine 3x 1 x 10-3 N"-a4-Pyridoxyladenosine N'-trans-Zeatin riboside 5x N6-Isopentenyl-2-thiomethyladeno~ine~ 5 X (in suspension) N6-3-Chloro4s-2-butenyladenosine* 1 x lo-% 7X N6-3Chloro-trans-2-butenyladenosine* (in suspension) Nb-Isopentenyl-2-thi~methyladenine~ 5 X N6-3Chloro-cis-2-butenyladenineb None P-3-Chloro-frans-2-butenyladenineb 8X "-1 sopentenyladenine4d I x 10-3 N6-IsopentenyladenosineCtd 9 x 10-5 %ee reference 9. were less active.62 4.e.40 5.5.87 4. i-PrOHkoncd Ha-H.188 Journalof Medicinal Chemistry. Uv Absorption Spectra of N6-Substituted Adenosines pH 1. farnesyl-.59 0. 1972. The adenines are less active than the corresponding ribosides. however.72 0.72 1. S.3 16.48 0. 16.48 1.79 0.4 19.53 0. EtOAc-n-PrOH-H 0 (4: 1:2) (upper phase).12 1.70 0. The propoxyethyl.210 268 26 9 268.309 18.0.

but the cis and trans chlorobutenyl adenosines are active. .3 9.8 8. When the dose was reduced to 2 mg/kg per day the compd was inactive.5 X None at lo-' (suspension) None at 7.2 9.In Vivo Activity against Mouse Leukemia L-1210 Compound (N6-adenosine analog) Control n-Bu n-2-Propoxethyl n-2-butox yethy >io-' 2.~ 2.6 6.3 x 1 0 .8 The cyclohexyl analog proved to be extremely toxic.2 8. The compds were given ip daily for 6 days at the dosage indicated. Also in this case the trans analogs are more active than their cis isomers paralleling their cytokinin effects. The effects of these compds on mice injected with lo6 cells of leukemia L 1210 are shown in Table VI.9 8.0 8. paralleling their cytokinin activity. paralleling their cytokinin activity. N6-butyl (135% increased lifespan).0 6. of mice used 39 5 8 3 8 11 5 8 8 6 5 5 Dose. a more sensitive system.4 3.6 3.3 6. and N 6 tetrohydrofurfuryl(l25% increased life-span) analogs have the best activities. It may be mentioned that the ether side chain analogs (N6-alkoxyethyladenosines) show far more antitumor activity in vivo than in vitro. as previously detd.5 9.3 6. No. The N6-2 butoxyethyl derivative is less effective. days 7. The geranyl. Growth Inhibitory Activity against Sarcoma 180 Cells and Carcinoma TA-3 Cells in Vitro Molar concentration for 50% growth inhibition Compound Sarcoma 180 Cells TA-3 Cells N6-n-Butyladenosine N6-n-2-propox yethyladenosine N6-n-2-Butoxyethyladenosine N6Cyclohexyladenosine N6€yclopropylmethyladenosine N6-Tetrahydrofurfuryladenosine N6-(kranyladenosine N6-Farnesyladenosine N6-dPyridoxyladenosine N6-trans-~4-Hydroxymethyl-2-butenyladenosine (zeatin riboside) > 10-4 None at l o w 4 None at None at 9 x 10-5 None at lo-* None (suspension) None (suspension) None at 1.5 X lo-' >io-' None at lo-' (suspension) None at (suspension) 7.5 x 10-4 >10-4 >10-4 N6-Isopentenyl-2-methylthioadenosine' N6-3 Chlorosis-2-butenyladenosineb N6-3 chlorotrans-2-butenyladenosineb N6-Isopentenyl-2-thi~methy ladenine~ N6-3 Chlorosis-2-butenyladenineb N6-3 Chloro-trans-2-butenyladenineb N6-Isopentenyladenosinecd (IPA) N6-IsopentenyladenineGd a-dSee footnotes a-d.5 x 1x 1 0 .~ Slight at IO-' 3.25 X lo-' 2. and were found to be more active than against sarcoma 180 (Table V).4 9.0 6. Of the compds tested.5 5. Cyclohexyladenosine and cyclopropylmethyladenosine are slightly active in this system. as in sarcoma 180.and farnesyladenosines have very low solubility in aq systems which may be the reason why they are inactive. mg/kg per day 75 100 200 100 200 300 100 200 5 2 75 100 100 Mean l i f e span.9 7. Table VI.2 8. 1972. N6cyclopropylmethyl(l24% increased life-span). I S . The 2-thiomethyl-IPA possibly because of its very low water solubility is inactive in this system.0 -% increased life-span (over control) 100 122 135 112 125 128 Toxic 116 Toxic Toxic Inactive 124 115 125 125 Inactive Inactive Inactive 108 Inactive 117 Toxic Cyclohexyl Cyclopropylmethyl Tetrahydro furfuryl Geranyl Farnesyl a4-Pyridoxyl trans-4-Hydroxy-methyl-2-butenyl (zeatin riboside) frans-3Chloro-2-butenyl 3 11 8 6 6 6 200 100 100 100 100 75 100 200 11 5 8 3 ethyladenosines are inactive in vitro. The sparingly soluble thiomethyl-IPA is slightly active while the cis and trans chlorobutenyl analogs are quite active.3 9. Val. as was found previously in the case of ethoxyethyladenosine which was active in vivo.2 x 10" None at 2 X lo-' (suspension) 6X 1. The corresponding adenine bases are relatively inactive. Table IV.2 x 10-5 None at (suspension) None at 1. paralleling their high cytokinin activity. The compds were also tested against mouse mammary carcinoma cells (TA-3) in culture. The isopentenyl analog (IPA)" is more active in this cell line than in sarcoma 180. trans-Zeatin riboside.N 6-Substituted Adenosines JournalofMedicinaI Chemistry.3 x 2. is inactive in this tumor system. The adenosine nucleosides are far more active than the corresponding bases as obsd before' in this tumor system.6 9.0 10. N6-2-propoxyethyl (1 28% increased life-span). a very potent cytokinin and mi§See also the case of Nb-2-ethoxyethyladenosine (reference 2).9 X l o v 4 No.2 189 Table V. The satd chain compds have lower effectiveness than their unsatd analogs paralleling their cytokinin effects. Zeatin riboside.5 x 10-5 7x 1.

as the ancillary acid binder. To 50 ml of abs EtOH was added 1. 373 mmoles) was dissolved in 250 ml of anhyd Et. and the amine was collected as the fraction which distd at 83-81": izZ5D1..H. N6-Cyclohexyladenosine(IV).O and sucked dry.41 g (14 mmoles) of Et.. is virtually inactive in the case of this tumor as in the case of the in vitro tumor cell tests. Uv spectra were obtained on a Cary Model 14 recording spectrophotometer. Since it was noted above that 2-hydroxy-N6-IPA had sharply curtailed cytokinin activity vs. when chromatog controls indicated that 6-chloropurine riboside was no longer present in the mixt.N or CaCO. 1. It was filtered and dried (0.) C. Inz. 1. The solids were crystd from MeOH-H. Y.H.00 g (7 mmoles) of 6-chloro-9-~-D-ribofuranosyl-9H-purine (6chloropurine riboside). (C. The material was recrystd from MeOH: yield. 1.SO. was filtered. The amines employed are identified under each prepn. was filtered. r l Z S D 1.4 g (14 mmoles) of CaCO. (adding 2 ml of HOAc to the first CHCI. mp 125". stirring contd for I hr and the mixt allowed to stand for 2 h r .4% of the theoretical values. 5. N.S§ and 1. 95% EtOH). 1. 2.and farnesyladenosines are too insoluble at levels needed to be active. IS. The suspension was stirred 18 hr allowing the temp to rise to room temp.).^_.O in 3 hr. whereupon cyclohexylamine hydrochloride pptd in cryst form.O. with stirring for 5 hr when chromatog controls showed the absence of 6-chloropurine riboside. and fractionation.. l 4 boiling point determined by Siwoloboff's method. Where analyses are indicated only by symbols of the elements.4196comparing with the same lit. Whatman paper No. a'-Pyridoxyladenosine contains 2 OH groups in the side chain and is inactive. was evapd.N5~J C. 95% EtOH). 2.Anal. H.00 g (7 mmoles) of 6-chloropurine riboside.O (26.5" (c 0.46 g (21 mmoles) of n-2-butoxyethylamine* * was refluxed and stirred as above for 18 hr after which the reaction chromatog controls indicated the absence of 6-chloropurine riboside. (2) high cytokinin activity is parallelled by E.5 mmoles) of 6-chloropurine riboside. as above. washed with chilled EtOH.73 g (13 mmoles) of 6-chloropurine riboside.415 g of Et. The use of CaCO. N.N.5'. (Eastman Kodak Co.4 g (14 mmoles) of CaCO. The hot reaction mixt was filtered through Celite and the product crystd from the filtrate on cooling. mp 108".'. [a]2sD --59" (c 0.. and the mixt was refluxed 6 hr.__.O and dried.O. Scott. gave the same results.O..455 g (4. Recrystn from MeOH yielded _______. N6-CyclopropyImethyladenosine (V). On adding Et. SSThis amine was generously donated by Hoffman-I. filtered.N. and dried: yield.4587. (a) Cyclopropylmethylamine.O and dried (Na.54 g (21 mmoles) of distilled n-BuNH.25 mmoles) of 6-chloropurine riboside.O. and the ppt was washed with Et. 95% EtOH).9 ml. This compd cannot be made vin the N'-quaternization procedure' since adenosine does not react with iodocyclohexane in DMF nor in N. N.O.39 g (14 mmoles) of cyclohexylamine (nzSD 1.@ and 0. The reaction mixt was filtered and evapd to dryness.N (14 mmoles). (C.No.17 g of LAH (373 mmoles) in 200 ml of anhyd Et.. W-Tetrahydrofurfuryladenosine (VI). 2.45'70. N.39. Anal.O..4" (c 0. the analytical results obtained for those elements are within +0.N. through the courtesy of I k .312. The material was recrystd from H. The prepn of this compd by the nucleophilic substitution method using either Et. and dried: yield. N6-Farnesyladenosine(VIII). l was (6-chloropurine riboused. er aLI4 The b p w a s detd b y Siwoloboff's method. mp 171-172". H.. mp 185".) C.'NH.).98 g (81%).SO. Optical rotation was measured on a Jasco Model ORD-UV5 optical rotatory dispersion recorder. 0.2 of their vol and cooled. The crystals were filtered.) 126. An alternate method of isolation is to take up the evapd filtered material (after removal of cyclohexylamine hydrochloride) in 100 ml of H. portion to take up any amine).O to yield 80% silky needles melting at 176": (CY]~'D -64. The reaction mixt was refluxed under N. N. Experimental Section Mps were detd on a Mel-Temp mp apparatus and are not cor.7%).50 g (20. The combined CHCI.. The Ca salts were removed by filtration of the hot soln.. yielding the same results. 1. H. (b) N6-Cyclopropylmethyladenosine (V). and the mixt was refluxed under N. the filtrate and wash were evapd to about 0. washed with cold EtOH.) and 1.- crobial inhibitor. (C. It was recrystd from EtOH-Et.5Ns0. N6-n-2-Propoxyethyladenosine (11). It was cooled. and the product was filtered off and washed with chilled EtOH then Et. which crystd on cooling. basifying.00 g (7 mmoles) of 6-chloropurine riboside. Anal.95% EtOH). The reaction mixt was evapd and redissolved in hot abs EtOH. 1.O (14:1). EtOH).644 g (2.) C.4207 $$Supplied b y Aldrich Chemical Co. N. To summarize it may be said that in Nbsubstituted adenosine analogs with side chains containing 4-7 C atoms: (1) cytokinin activity is optimal when a double bond is present in the side chain..0 g (3. .aHoche. with stirring 6 hr when chromatog controls showed the absence of 6-chloropurine riboside. 1. Plainview.) C. and washed with chilled MeOH-H. 3.O and dried.. and 2.$$ and 1. as in the preceding prepn. 1.41 g (14 mmoles) of Et. Vol. On cooling to room temp the product crystd out. Inc.Anal.N. A mixt of 100 ml of abs EtOH.0" (c 0.# and 1. 2. The white suspension was filtered. N6-Geranyladenosme (VII).90 g (38.___.75 g (38.H. The material had bp 1 5 2 O (750 mm) and nZSD1. and the residue was crystd from EtOH-Et.O filtrates were sepd and dried (Na. **Supplied by K . The reaction mixt was stirred and refluxed for 17 hr when chromatog controls indicated the absence of 6-chloropurine riboside. This amine has been made by Roberts and MazuP by the redn of cyclopropyl cyanide with Na in abs EtOH followed by isolation of the hydrochloride. fractions were washed with H. H..7 mmoles) of Et. trans-3-Chloro2-butenyladenosine shows slight activity at 100 mg/kg per day and reduction of dosage to 7 5 mg/kg per day renders it inactive. H. The mixt was filtered hot to remove Ca salts and the product deposited from the filtrate on cooling. mp 208") and analyzed correctly for C. and 2. (3) tumor cell growth inhibition follows cytokinin activity with an exception that the presence of an OH group in the side chain diminishes antitumor effects. E.N.H.H.7 mmoles) of the cyclopropylmethylamine. N.. (C. hydroxylation may effect an unexpected behavior. The Et.N.04 g (74%). 1.O to the mother liquor the product crystd out.__I.06 g (84%). To 100 ml of abs EtOH was added 2.. To 100 ml of abs EtOH was added 2.O and then dried: yield.492 moles) was added dropwise. Geranyl. Cyclopropyl cyanide?? (25 g. yield. It was filtered and washed with cold EtOH followed by Et. values of Harder. [cyIzSD --59. H. The redn of the nitrile could also be effected with LAH in Et. presents some difficulty in isolation of the desired product. 2.15 g (87%).306 95% EtOH). ??Supplied by Aldrich Chemical Co.00 g (7 mmoles) of 6-chloropurine riboside. To 100 ml of abs EtOH were added 2.84 g(9270). was refluxed with stirring 18 hr when chromatog controls indicated absence of 6-chloropurine riboside. After filtration the material was crystd from EtOH and dried: yield.4 g (93%). I. Use of CaCO. mp 148". To 30 ml of abs EtOH were added 0.N.O was evapd. The crystals were filtered off and washed with cold EtOH then with Et.- . and 1.N. To 100 ml of abs EtOH was added 3. The reaction mixt was refluxed with stirring for 18 hr after which chromatog controls indicated no further presence of 6-chloropurine riboside in the reaction.& K Laboratories. 1.I90 Journal of Medicinal Chemistry.342. Anal.O and ext 3 times with 100-ml portions of CHC1. The soln was added dropwise to a stirred and cooled suspension of 14. The combined Et. and washed with Et. et al.26 mmoles) of farnesylamine.AnaZ. (Cl.N-dimethylacetamide. The reaction mixt was refluxed with stirring for 4 hr at which time chromatog controls indicated the absence of 6chloropurine riboside. W.. The material had a bp 1 2 9 O ( 7 5 0 mm) and nZsD 1.44 g (14 mmoles) of n-2-propoxyethylamine.H.) C.50 g (2. Since no product crystd out on cooling. n Z 5 D1. H. N.414 g (14 mmoles) of tetrahydrofurfurylamine. 1972.4250. values of IXarder. [ a J Z 5 D C.4127 comparing with the same lit. coli growth inhibition..H. mp 176".). [aIZsD--55" (c 0. [alzsD -59.76 g.Q. To the stirred and cooled suspension.. cold H.07 g (7 mmoles) of geranylamine. as an ancillary condensing agent practically abolished reactivity. yield.216. that of IPA. 2 Fleysher A mixt of 100 ml of abs EtOH. Anal.O.O and dried: yield. N6-n-2-Butoxyethyladenosine (111). C. Aldrich Chemical Co. 3. The product. The CHC1.90 g (84%). ether extn of the amine. #Supplied by K tk K Laboratories Inc.0 g (7 mmoles) of 6-chloropurine riboside.318.5" (C 0. mp --56" (c 0. It appears that the introduction of an OH group in the side chain of IPA sharply lowers antitumor activity. N'-n-Butyladenosine (I).O.50 mmoles) of Et.40 g (14 mmoles) of CaCO. HC1. The solvent systems used for descending chromatog are given in Table I.N. 6-Chloro-9p-D-ribofuranosyl-9H-purine side) was purchased from K & K Laboratories.217 95% EtOH). [aIzSD-71.O.H. (C.

30. On refluxing a white ppt deposited initially which later dissolved leaving an opalescence in the reaction mass.5%). 12. Bloch. 921 (1966). and 6. R. Shaw. Alexander Bloch. The microbial assays were carried out according to techniques previously published.Biochemistry. 130. ibid. MEDI 54..Science. H. Each animal was injected ip with 1 x lo6 cells of leukemia L 1210. Schmitz. The control cultures increased by 10. R. (9) W. Science. 1272 (1969). to Dr.. Lowry. coli (K-12) was grown in the synth medium of Gray and Tatum.. I. 1114 (1964). Chem SOC. H.O. Chem Soc.I9and by protein detns. and the ppt was washed with EtOH and dried at room temp: yield. (17) C. Skoog. J. Armstrong. D. Stasiuk. Tatum.8. Roberts and R. 97. N. mp 249".H. 11. Lummis. G.. (16) A.95% EtOH). and (T-436) from the American Cancer Society. With further refluxing a white solid deposited out of soh.886 (1966). The compds were administered in a homogenized 1% CMC emulsion. H. Biochemistry. Joumal of Medicinal Chemistry. for help with the mouse study... 9. N. Y.... Pfeil. M. and Dr.. 6. J. J. The cells were exposed to the compds for 6 days during which time there were 3 changes of medium. Szweykowska. Gemer. Sept 1970. 961 (1968). 166. (6) S. (C. and R. 88. Chem. J. Bloch and C. H. SOC. Hecht. Phytochemistry. Morton. E.90 g (93. Med.t o 15-fold. Ass.. 15. Amer. Grady. 192. Assay of Cytokinin Activity. Robins. No. 1056 (1969). M. 132 (1959). Female DBA. E. Amer. (C. 265 (1951). Bloch. Carraway. Hecht. Harder.' Results are given Table 111. (22) V. J. Hamzi. Rosenbrough. [aIZ5D -63. Skoog. The cytokinin effect of the new compds was assayed with the callus derived from the pith from apical stems of Wisconsin 38 tobacco plants using the method and media of Murashige and Skoog.H. Hakala and Miss A. A. Fan. This mammalian cell line (mouse mammary adenocarcinoma TA-3) was grown in RPMI 1640mediumzl supplemented with 10% horse serum. M. (13) A. Med. Mazur. Results are given in Table IV. to Dr. Anal. and C. Randall. Y. J. Chem.06 g (60. All the concns of the compds were tested in 5 tubes each. N. and C."Laboratory Technique in Organic Chemistry. Chem. 2 191 assays.86 g (10 mmoles) of 6chloropurine riboside. Skoog.1169 (1967). H. H. Fujji. Microbial Assay Procedure. S. N. Schmitz. G . Eagle. the reaction mass was filtered hot. Nichol. Schmitz.C . 6.6' (c 0. A. M. I. 73. Skoog. [a]"D -51.H. Chem Ber. F. and R. (14) U. Robins. 539 (1968).. U. or Li. Sci. The test system consisted of tube cultures (12 X 75 mm) inoculated with 5 X l o 4 cells in 1 ml of medium. 305 (1956).. Survival time was noted on each animal (Table VI). E. Franklin. (11) R.1" (c 0. The medium was replaced with fresh medium on the second day by centrifuging the cells for 10 min at 500 rpm and by aspirating off the supernatant. 9. Chem. J.N6-Substituted Adenosines 0. R. 199. N. Soc. N. A. J. Hall. Abstracts of the 160th National Meeting of the American Chemical Society. M. Eagle. J. . M. A.3345 (1966). Gerald Grindey. and J. Leonard. J. W. 3071 (1969). (7) S. T. (4) G . L.00 g (12. Loeppky. A. (10) S. Med. A. Leonard. Isr. H. D./HA mice (6-8 weeks old) (18-20 g) were obtd from the RPMI breeding colony.. M. Sahai Srivastava for help with the cytokinin . Physwl. (3) M. R. Anal. 3345 (1966).Biochemistry. 6. (5) F. supplemented with another milliliter of medium containing the compd and then incubated at 36" in upright position. J. J.N. Vol. J.. Hecht.35% EtOH). L.. Hakala. Public Health Service. (8) T. 1938. Hall. 2509 (195 1).. Phytochemistry. N. New York.. Chicago. Fleysher.NSO. (b) Carcinoma TA-3 Cells. Proc. (a) Sarcoma-180 Cells. H. and R. and Mr. 3. Chem. To 100 ml of abs EtOH was added 2. 161." 1st ed. Hall. Thedford.CO. Burrows. 1972. 91. and treated with the drug once daily for 6 consecutive days starting the day after tumor inoculation. (21) G. Thedford. (2) M. T. Miss Ginger Dutschmann.510 (1964). Ill. Science. The compds were added in aq soln (or as a suspension if the solubility was low) to cultures of S-180 cells grown as monolayers in T-15 flasks in Eagle's'' medium containing 5% horse serum. E. Halgeson. Skoog.I6 E.). as ancillary condensing aEntsresulted in poor reactivity. B. Maue. Occolowitz. Y.. Effects on the Growth of Sarcoma-180 and Carcinoma TA-3 Cells in Vitro." (Table V).0 mmoles) of Et. Plantarum 15. 5 .5 mmoles) of pyridoxamine dihydrochloride (Sigma Chemical Co. Leonard. A. Use of CaCO. (19) J. Coutsogeorgopoulos.) C. 404 (1944). H. M. J. (15) J. Skoog. and K. M. Oyama and H. K. V. R. Fleysher. 20. Fleysher. 9.s 19 (1967). Amer. Moore. Mulhern for cell culture determinations. Wilson. J. A. Smallwood. J.Exp. S. and R. N.447 g (42%): mp 98".'. (12) M. Thedford. Bloch. Boyle. Hecht. M. After 20 hr at reflux temp. J. U. 1173 (1970).155. Murashige and F. This work was supported in part by research grants (CA-11047) from the National Cancer Institute. ibid.2614 (1966). L. Biol. Cancer Res. and F. Biol. W. H. L. 1837 (1967). Effect of NWubstituted Adenosines on Leukemia L 1210. and D. McGraw Hill Book Co. and R. Leonard. Zenner. Hall. L. R. Med.717 (1968). H. and F. Gray and E. J. The quantity of cells was estimated by the crystal violet method of Grady. T. N. and R. P. Nat. Robert Maue for the microbial assay procedures. 1907 (1970). References (1) M. Q.Proc.646. (18) H. Leonard. The growth of the controls as estd by protein assayzzwas 12to 15-fold in 3 days (Table V). T. H. S. Y. H. 3. R. N. and F. Smith. The compds were added to the medium at lO-'-lO-' M.. p 51. J. M. Acknowledgment.O) C. in vivo. (20) 0. A. and H. Hakala. T. et a1. J. Fleysher. Acad. N6-Pyxidoxyladeno~ine(a4-pyridoxyl-N 6-adenosine) ( I X ) . The author wishes to express his thanks t o Dr. J.

1021/jm00272a016 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. Agrawal.doi.W.org on April 25. Sartorelli J. 2009 More About This Article The permalink http://dx. 1972. 15 (2). Randall Wheaton. Cushley. Med. Methylated .Potential antitumor agents. Lipsky.-(N)-heterocyclic carboxaldehyde thiosemicarbazones Krishna C. 1155 Sixteenth Street N.. Seymour R.org/10. Chem. 5. DC 20036 .acs.alpha. Robert J.1021/jm00272a016 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. Washington. J.. and Alan C. 192-195• DOI: 10.

however.192 Journal of Medicinal Chemistry.' Blockade of the formation of RNA and protein also occurs. the identification of these compounds is under investigation. Connecticut 06510. one of the most active members of this series. A similar mechanism of action appears to be operative with both 3-hydroxy-2-formylpyridine thiosemicarbazone and 5-hydroxy-2-formylpyridine thiosemicarbazone . Minn. No. 15.6 The structural specificity of the formyl thiosemicarbazone side chain was found to be critical. but these pathways are considerably less susceptible to drug induced inhibition.* J.3. Minneapolis.4a Substitution of these groups (Le. Received August 16. Dehydrogenation was carried out in good yield. was considerably more efficacious than the parent compound on the L1210 lymphoma. 1969. however. The syntheses of Me-substituted derivatives of isoquinoline.^ Investigation of the structure-activity relationships required for antineoplastic activity4 have indicated that certain of the overall molecular dimensions of 1-formylisoquinoline thiosemicarbazone (IQ-1). 5-. Lipsky. OH and OAc) in the 4 position of the isoquinoline ring resulted in compounds which were both less active and less toxic than the parent compound against sarcoma 180 ascites cells in mice. 1972. the isoquinoline and pyridine.4dihydroisoquinoline with Pd at 200" produced in part unknown compounds which were found to have no aromatic protons in nmr. 2 Agrawal.and pyridinecarboxaldehyde thiosemicarbazones were initiated from corresponding dimethylsubstituted heterocyclic ring systems. Division of Health Science Resources. ' \ 4 c n 3 S . 1971 Several monomethylated derivatives of a-(N)-heterocyclic carboxaldehyde thiosemicarbazones were prepared to define the molecular dimensions compatible with antineoplastic activity. Lutidines were obtained commercially and dimethylisoquinolines were synthesized utilizing the Bischler-Napieralski reaction.3-dimethyl-3. Thus. A variety of thiosemicarbazones of a-(N)-heterocyclic carboxaldehydes have been shown to be potent inhibitors of transplanted rodent neoplasms. et al. $Section of Physical Sciences. New Haven. U..2 and DNA viruses of the Herpes fa mil^.4-dimethyl-3. the sodium salt of the 4-OH derivative. Vol. Chemistry. S.and 1.' spontaneous lymphomas of dogs.and 6-methylsemicarbazones of 3-. have been synthesized and biologically evaluated. Methylated a-(N)-Heterocyclic Carboxaldehyde Thiosemicarbazones? Krishna C. These were the thioand of 3-.* Seymour R. 4-. dine ring system with relatively great therapeutic indices as antineoplastic agents.* Robert J.& Sodium salts of several other OH derivatives of both isoquinoline and pyridine ring systems have also been prepared as a means of solubilizing for parenteral administration these extremely insoluble compounds. Considerable difficulties were encountered in the dehydrogenation with Pd of 3. 5. Agrawal. Direct oxidation of 1. 4-.' could be modified with the retention of high biological activity.4-dihydroisoquinolines when prepared according to a published procedure. Sartorelli Department of Pharmacology and Section of Physical Sciences.\yH:Nk k q h 6 ~ ~ 5 CH-NNHCNHz .' The investigations indicate that in mammalian cells the primary site of action of these agents is the biosynthesis of DNA. Randall Wheaton. Potential Antitumor Agents. These compounds were therefore synthesized by rearrangement of their N-oxides with Ac2O '* Scheme I CH3 CH3 1 2 I AczO 2 dil HCi OH I CHO CHZOH cn3 3 5. Tests for tumor-inhibitory potency indicate that in general the pyridine derivatives were better inhibitors of the growth of the L1210 lymphoma than were the isoquinolines. Public Health Service.4-dimethylisoquinolines (1 and 7) with SeOz resulted in poor yields of the respective 1carboxaldehydes.. and Grant T-23 from the American Cancer Society and the Biotechnology Resources Branch of the National Institutes of Health (FR 00356). Dehydrogenation of 1. and 5-methylisoquinoline-l-carboxaldehyde pyridine-2-carboxaldehyde. by heating the dihydroisoquinolines with Ph2S2 and removing the formed thiophenol by distillation. Yale University School of Medicine. since modifications made at the various positions of the side chain resulted in either a decrease or a complete loss of antitumor activity.. To this end a number of monosubstituted alkylated derivatives of the two most active heterocyclic ring systems in this series. this work was supported by Grant CA-02817 from the National Cancer Institute. in many instances such solubilization conferred greater therapeutic gain. Cushley.4-dihydroisoquinoline or 1. with the location of the metabolic lesion being the conversion of ribonucleotides to deoxyribonucleotide forms. and Alan C.9 two thiosemicarbazone derivatives of the pyri?Presented in part before the Division of Medicinal Chemistry at the 157th National Meeting of the American Chemical Society.4b The biochemical basis for the growth-inhibitory activity of IQ-1 has also been studied in our laboratory. Me substituents at the other positions of these ring systems did not significantly increase the carcinostatic potency of the parent compounds. substituents such as 5-OH and 5-OAc were found to confer therapeutic indices to the resultant derivatives that were greater than for the parent compound. lo It was deemed desirable to further define the molecular dimensions compatible with the biological activity of this relatively new class of antineoplastic agents. April. Introduction of a 6-Me group in the pyridine ring and of an analogous 3-Me substituent in the isoquinoline structure resulted in compounds with no antineoplastic activity indicating an apparent intolerance to substitution at the a' position to the heterocyclic N atom for inhibitory action.

Introduction of a 3-Me group (18) did not result in therapeutic improvement over PT. Biological Results and Discussion. Similarly. The reaction of 8 with Ac20 contrasts with the attempted rearrangement of 4-methylisoquinoline N-oxide by Robison and Robison12 who could not isolate a discrete chemical material from the polymeric mass which was produced. 2 193 Scheme II (Schemes . The findings indicate that. As an example.9 Hz.1-hydroxymethyBsoquinoline (12) and then purified by alumina column chromatography. The 5-Me group in this compound was a triplet.0 ~ i d e .5-dimethylisoquinoline (15) to the corresponding 1-carboxaldehyde (16) was achieved in fair yield with Se02 (Scheme 111). The antitumor activity of methylated a-(N)-heterocyclic carboxaldehyde thiosemicarbazones against the L12 10 lymphoma in mice is given in Table I.follows to produce the anhydro base 10 which undergoes an intramolecular rearrangement. in I1 the Me is a doublet showing a 5-bond long range coupling. unless C-8 or the ring N contains substituents (e. 19. All peaks assigned to replaceable protons were confirmed by addition of CF3C02H. 3-methyl-1-hydroxymethylisoquinoline (3) and 1. J l . the Me-substituted derivatives 6.H-3 = 1. Compd Scheme III were prepared by previously published procedure^'^ utilizing the rearrangement of corresponding lutidine N-oxides with Ac~O. the chemical shift for a 1-Me group occurs between 6 2. Oxidation of 3 with Mn02 followed by reaction with thiosemicarbazide gave the desired thiosemicarbazone 6.3% loss in body weight. An interesting long-range coupling in isomers I and I1 of 3.a .3-dimethyl-4-hydroxyisoquinoline (4). IQ-1. Compd 12 was then oxidized with Mn02 to the corresponding carboxaldehyde 13 which on reaction with thiosemicarbazide yielded the desired product 14.& some useful empirical results have been obtained which can be used to characterize compds in the isoquinoline series. 14. A minor isomer. I Figure 1. On the other hand. _ ~ I and 11). the rearrangement presumably proceeds by an intramolecular mechanism through intermediates 9 and 10 in a manner analogous to that proposed for 2-picoline N .. with a 4-Me substituent. Nmr Studies. Abstraction of a proton from 9 by AcO.No. the pyridine derivatives were better inhibitors of the L1210 lymphoma than the isoquinoline series.26. was not only found to be equally active with PT as a tumor inhibitor.+3 = 1. Methylated pyridine-2-carboxaldehydes..4-dimethylisoquinoline N-oxide (8) with Ac20 (Scheme 11) produced mainly 4-methyl. and 17 increased the average survival time to a lesser extent. this compound was relatively toxic as shown by a 14. the precursors of the synthesis of thiosemicarbazones. but was also much less toxic causing only 4. the 1-Me substituent (6 2.3-dimethylisoquinoline Noxide (2) with Ac20. From the relatively abundant number of compds investigated in the present study. Positive identification of this trace material was not accomplished. N + 0).1-acetoxymethylisoquinoline (1 1). differentiation of the various isomers from nmr results has been possible. doubled the average life-span of tumor-bearing mice as compared to untreated controls. The rearrangement of l-methylisoquinoline N-oxide has been reviously reported and a possible mechanism proposed. possibly l-methyl4-acetoxymethylisoquinoline. However. Compound 11 was hydrolyzed with dil HCl to yield 4-methyl.4-dihydroisoquinolines(Figure 1) has been used to establish the cyclized nature of the compounds.81 and 2. but still increased the average survival time to 18. In the pyridine series 2-formylpyridine thiosemicarbazone (PT) was the most active agent causing more than a 2-fold increase in the average survival time. in general. In the case of the 5-Me compounds. doubly bonded electronegative substituents on the C directly bonded to C-1 cause a paramagnetic shift of the proton resonance at C-8 into the region 6 9. Selective oxidation of the 1-Me group in 1.42 to an octet which is the AB subspectrum of the ABMXBspin system. IS.4% loss in body weight at the optimal dose level of 5 mg/kg administered twice daily.13-9.Anitumor 7Rwsemicarbazones Journal ofhfedicinal Chemistry. with a Me group in position 1. This C-8 proton multiplet lies to lower field than the other ring protons. Vol. . only in 16 was a splitting resolved between the 5-Me group and the protons at C-4 and C-6. were isolated after acid hydrolysis of the mixture of esters obtained by heating 1. Treatment of 1. l Nucleophilic ~ attack of the Noxide O on Ac20 results in a formation of intermediate 9. 1972. due to coupling with the single proton on &-3. however. Although the parent compound. as well as previously. Nmr spectral parameters for the compds prepared were in accord with the structures proposed.2 days. J = 0.7 Hz. Intro- 16 on reaction with thiosemicarbazide produced the desired derivative (17). Two compounds.J1-a. In the case of 9.& A similar mechanism would be expected to be operative in the transformation reactions of 1 (Scheme I). Double irradiation of the 1-Me resonance lines in I reduced the multiplet structure at 6 3.5 Hz. due to long-range coupling to the two H-3's (dark lines).g.was present as a contaminant. Compd 20 having a 5-Me substituent was relatively nontoxic as shown by a minimal loss in body weight.32) is a triplet.92. In I.Such longrange coupling must involve the heterocyclic N atom.

2.).).). The procedure employed was similar to the prepn of 1. S 20 5 224-22Sd EtOH C8H1CDN4S C.68 (s. N. l5 This difference might be explained by the extreme water insolubility of 17 which leads to poor uptake of the compound by neoplastic cells.6 17 20 + 8. Orange. S 21 6e aRecrystn was not necessary.0) and 3-methyl-1hydroxymethylisoquinoline (3)was extd with Et. 8. % Crystn solvent Formula Analyses Isoquinolines 207-208 6 3 87 THF-cyclohexane C I Z H l 2N4S C. where(4) pptd..H. was heated to 225" in an oil bath permitting the by-product C. The residue was acidified with 10%HC1. Chemotherapy Research Laboratory. mp 150-151" . A similar result was obtained in the isoquinoline series where a 3-Me substituent. 1.). The residual oil was distd at 100-105" (0.46 g of NaOAc and added slowly to 4. days Compound daily dose.48 (s. 1 H. y.O and dried. H. Experimental Section Melting points were determined with a Thomas-Hoover capillary melting point apparatus and are uncor.). The mixt was then extd with Et20. 15.7 8.4% of the theoretical value.3-Dimethylisoquinoline (1).8 14 20 +11. the corresponding amides by published procedures" according to the Bischler-Napieralski reaction. Rearrangement of 1.O. (C.SH to distill off. and concd.O was removed leaving a dark oil which was extd with Et.14b dReported mp 220. 3-CH. 4-CH3). 3 H. 1 H. Dimethyl-3.. N.H Table 11.61 (s.14a eThis derivative was generously supplied by MI.6 18.27 g) and 12.O.84 (s. and the residual oil was distd at 90-93" (0. The mixt was concd to give a dark oil which distd at 130-150" (0. This derivative (17) has been reported to be equal in activity to 14 as an inhibitor of the target enzyme ribonucleoside diphosphate reductase in vitro. The ppt was upon 4-hydroxy-1. Drugs were administered ip in fine suspension beginning 24 hr after tumor implantation and treatment was continued twice daily at 12-hr intervals for 4 consecutive days.. 4-CH. 2.5 13. Palo Alto.). Mount Zion Hospital and Medical Center. duction of a 6-Me group in PT (21) resulted in a compound with only minimal biological activity.4-Dimethylisoquinolinine (7). Determination of antineoplastic activity was based upon the prolongation of survival time afforded by the drug treatmert. tan-colored crystals: yield 58%.77 (s. which may be considered to be analogous to the 6-Me group of pyridine series. et al. No.2 mm) to yield 4. bAverage wt change from onset to termination of drug treatment. solvent was removed. The thiosemicarbazones were tested for antineoplastic activity in mice bearing the L1210 lymphoma. The AcOH was removed by flash evapn. H.5°.O.NO) C. Conn. CH. l-CH.0.O for 2 h r . 3 H.NO) C.4 21. Recrystn from EtOAc contg a small amt of MeOH yielded 0. mp 73-76". Chemical shifts (6) are given in ppm downfield from TMS. H.86 (s.1 ml) was mixed with 0. H.. 2.26 g (37%) as colorless crystals: mp 103-104". French. Anal. H. 2 was then extd with Et .4-Dimethylisoquinoline N-Oxide.85 (s. 2 Table I. 1-CH. H-3).35 mm) yielding 4.89 (s.5 mm). 2.3-dimethylisoquinoline filtered. The soln was basified with NaOH s o h and extd with Et. The alk layer was neutralized to pH 7. Compd 2 (4.2 'Administered twice daily for 4 consecutive days at 12-hr intervals beginning 24 hr after tumor transplantation. S 14 4 214-21 5 70 EtOH-H.4dihydroisoquinolines were synthesized by cyclizing §Where analyses are indicated only b y symbols of the elements. 1.0 Hz. 3 H. 8. nmr 6 2. 2. Effect of Me-Substituted a-(N)-Heterocyclic Carboxaldehyde Thiosemicarbazones on the Survival Time of Mice Bearing the L1210 Lymphoma Maximum effective Average A Average survival.5 21 40 + 1.74 g of 1 preheated at 80".39 (broad s. H. Compd 17 which contains a 5-Me group in the isoquinoline ring also showed no antineoplastic activity.4 19 5 . dd. nmr 6 2.). H. 19 72.4 g (88%) of an oily mixt of esters. The residual solid was recrystd from petr ether to give 1. The Et.5 17. 1 H.194 Journal of Medicinal Chemistry. 3 H.S.O 1 ' 2H1.). Complete details of the screening procedure have been described earlier. 3-CH.28 g of this mixt of esters 100 ml of 10% HCl was added and heated for 1 hr at 100". 1 H. The mixt was heated for 4 hr with stirring and then allowed to remain at room temp overnight. A 40% s o h of AcO.50 (s. Antitumor Screening... resulted in a compd (6) with much lower antineoplastic activity. 1-CH. H. H-3). (s.6 The ascites tumor was transplanted by inoculating BDF.O. mg/kgn weight. Only those resonance signals necessary for differentiating the various compds are described.O was removed and then the oil was distd at 167-170" (0. analytical results obtained for those elements are within 0. N.). Woodside.0 with 10%HCI.5 10. d.5 g) was heated at 110" in 1.6 6 20 + 8. 3 H.4. N. Compd 11 (0.0. The crude 12 was .14 g (61%). The pale yellow oil crystd upon cooling: yield 1.NO) C. H-3). 3-CH3).:" mp 92-93" (reported 97-98").H.4-DimethylisoquinolineN-oxide (8) was synthesized according to the ptocedure of Robison and Robison" for the synthesis of isoquinoline N-oxide. filtered. and the aq layer was basified with NaOH soln.86 (broad s. 1-CH.40 (d. 5.05 (s.O for 2 hr. nmr 6 2.8 g) was heated at 100" in 20 ml of 10%HC1 for 1 hr. This compd was synthesized according to Spath. 1. Methylated a-(N)-Heterocyclic Carboxaldehyde Thiosemicarbazones Me Substitution Compound position Mp. Anal. bReported mp 209°. "C dec Yield.. (C. Frederic A. 7. l-CH. 3 H. Vol. S 17 5 233-235 83 a C I Z H l 2N4S C. the impurities were extd with Et.95 g (89%) of pure colorless product: nmr S 2. 3 H. OH). N. The soln was made alk with NaOH soln (pH 11.. Spectra were obtd in DMSO-d.. (C. 5-CH.O. etc).0 8.Excess Ac. mice ip with approximately 4 X lo6 tumor cells. 3.3-Dimethylisoquinoline N-Oxide. 3 H. Calif. Several recrystns from petr ether raised the mp to 76-77".55 (s. H.14a 'Reported mp 194-196°.nmr 6 2.s-Dimethylisoquinoline (15).N4S C. S Pyridines 18 3 214-2 16& EtOH C8H10N4S C.H. S 19 4 193-195' EtOH-KO C8H10N4S C.60 (s. N.3 mm) to yield 7. and the compd was distd at 153-155" (0.01 g) washeated at 120" in 40 ml of Ac.0 IQ-1 20 . Elemental analyses8 were performed by the Schwarzkopf Microanalytical Laboratory. singlet. Compd 8 (1. and concd to give 4methyl-1-hydroxymethylisoquinofine (12).9 16.50 (s. washed with H. %b None + 5.SO. Agrawal.H. ~. CH. 2 H. 1. To 4. Recrystn from EtOAc and cyclohexane (Norit A) gave pale. doublet. et al. 1.).3 20. m. and flash evapd.3-dimethyl-3. N.07 g (78%) of pure material which crystd on standing: mp 50-51". dried (NaZS04 ).2 20 15 .). 3 H. Anal. J = 7. 3 H. The Et.). H.4 PT 5 -14.71 g of Ph.6 12.H.).76 (s.NO) C. multiplet. 1-CH. Anal.4-dihydroisoquinoline (9. The 4 methyl-1-acetoxymethylisoquinoline (1 1) was possibly contaminated with the 4-acetoxymethyl isomer and was purified after acid hydrolysis. 3 H. (6.5 ml of Ac.3 g (80%) of pure colorless compd: nmr 6 2. 2.). Rearrangement of 1. 3 H. dried (NaJO.6 18 15 -10. (C.7 g (20%) of pure material: mp 169170'. N. An equimolar mixture of 1.nmr 6 2.4 mm) to give 7. doublet of doublets.. Nmr spectra were detd with a Bruker HFX-3 spectrometer operating at 90 MHz and fitted with an H-P frequency counter. filtered.O layer was dried (Na.and the Baron Consulting Co. 3 H.3-Dimethylisoquinoline N-Oxide (2).10 (broad s.

7 Hz. Fed.. Biochem. J. (CliHJO) N.and 6-isoprenyl-2. Vol. Agrawal and A. Gwardyak for excellent assistance.228 (1971). Inhibition of the succinoxidase system was relatively nonspecific in respect t o the side chain. Miss Lynn A. (11) E. N. nmr 6 10. A.NO) N. 758 (1957).Pardini. 9. Sartorelli. G. 8. . Coenzyme Q has a widespread distribution in biological systems4 including the malarial parasite. mp 108-109".nmr 6 10..Coenzyme Q-Enzyme Inhibitors chromatogd on 6 0 g of silica gel (100-200 mesh) and eluted with EtOAc (500 ml). J.H. A. Catlin. Sartorelli. Med.. Brockman. 2 H. (15) A. No.18 (dd. C. oxidizing 12 with MnO. J. Jill Corby Morton. Gann. James C. Crystn from hexane (Norit) yielded colorless fibrous material: 0. Chem. A. M. Acknowledgment.). The mixt was refluxed for 2. 13. 134 (1930). 2 3 2 (1955). (C.. J = 5. 2 195 References (1) (a) R.3dimethoxy-5-hydroxy-1. K. Agrawal. Res. 3 H.O). Agrawal. Pharm.)... which were also succinoxidase inhibitor^. Stinson. (C. and Karl Folkers" Stanford Research Institute. 9 . Bon Tempo.. 2. K. These findings link one kind of inhibition of mitochondrial electron transport at the coenzyme Q loci to chemotherapy of malaria. (3) W. Compd 15 (0. 57. Jr. 2. was added slowly. A. Thomson. Cancer Res. 4-Methylisoquinoline-1-carboxaldehyde ( 13) was synthesized by the same procedure as 5. washed (H. Sartorelli.22 g (2 mmoles) of SeO. Exp. and S. 1 H. ( 5 ) F. Commun. Austin.. Preliminary structure-activity investigations demonstrated that mitochondrial succinoxidase activity was inhibited by various antimalarial naphthoquinone analogs. Chem..81 (d. The acid layer was filtered and made alk with NaHCO. Kuntara.^ have been synthesized. J. 1638 (1966).K. and E. 5. and dried. Booth. 7 0 0 (1968). CoQs is the dominant CoQ of Plasmodium A series of benzoquinones structurally related to known antimalarial naphthoquinones. Kuroiwa. C. 12. L. ibid. Agrawal. F. ibid. Amer. Pharmacob.25 g (72%). ibid.Anal.71 (s. Anal. Blanz.05 (d. (CllH1. Spath. and the activi t y was restored b y coenzyme Q and its derivative^. McMurray. Pharm. nmr 6 2. CHO). Bell.56 (s. 5 8 5 (1966). Martello. 27. Joseph C. 6-Alkyl. C.NO)C. and the resulting ppt was collected. C. and A. 5 1 (1967). 1 1 .4-benzoquinones were found to inhibit mitochondrial NADHoxidase and succinoxidase systems from beef heart. A. 1 H.38 g. were 'Author t o whom correspondence should be addressed at the University of Texas. 25. Mitochondrial reconstruction'Y2 and spectrophotometric investigations on the kinetics of coenzyme Q turnover during electron transport3 are responsible for the view that coenzyme Q participates in the primary electron transport sequence. Mathes and W. 15. and A. B. Biol. 192 (1967). J. Booth.^ Next. French and E. C. Agrawal. Chem. E. (b) F. The University o f Texas at Austin. 1972. A. C. The most effective group in the 5 position was OH for the 6-phytyl derivatives.771 (1969). Texas 78712. 31. 3 . (14) (a) W. S. W.H.. CH. The 5-Me0 derivative was essentially noninhibitory. and the C. (8) E. 8. and W. E. Agrawal.68 (s. 80. 16. and H.167 (1956).'-' Mammalian succinoxidase and NADH-oxidase systems have been extensively studied and may be considered representative of the coenzyme Q electron transport sequences. was removed to yield 5. H-3). C. C. Sei. C. 2. 4-CH3). 3 H. (6) K. 2-wcyclohexyloctyl-3-hydroxy-1. (b) E. 2 . Cancer Res. ReceivedAugust 9. and in diminishing degree. Moore. C. Sartorelli. R..69 (t. Traynelis and R. Exp. the inhibitory action of antimalarial agents was reversed by coenzyme Q. 2 9 . Heidker..4-benzoquinones were similarly evaluated.3-dimethoxy-6-phytyl-l.431 (1970)." These benzoquinones also have structural resemblance to coenzyme Q. M. Brockman.6590 (1958).1948 (1968). B. dioxane was removed.609 (1970). Cushley. Journal of Medicinal Chemistry. Inhibition of the NADH-oxidase system was greatest when the hydroxyquinone possessed a side chain of from 16 and 17 C. C. C.5 hr and filtered. C. (b) K. Jr. Recrystn from petr ether gave colorless needles: 0.." Again.J. 90.. Sidwell. Skipper. They represent potential antagonists of coenzyme Q function in mitochondrial electron transport. A. hence.21 (s. 9 0 8 (1970). 26 (1967). R. Jr.. 1971 5-Substituted 2. California 94025. they also represent potential antimalarial activity. 2 mmoles) was dissolved in 25 ml of dioxane and 0. Specificity of Inhibition of Coenzyme Q-Enzyme Systems by Lipoidal Benzoquinone Derivatives? Ronald S. Soc.. K. Med. 3-Methylisoquinoline-1-carboxaldehyde (5). 4 4 9 2 (1970). Sartorelli. Fed. Bull. (c) K. mp 71-72'. 2. 20. 63B. 3 H.3119 (1971). C. 31. 3 H. Sartorelli. 1 4 1 . C. Furukawa and Y ..5 and 0. C. shown to inhibit beef heart mitochondrial succinoxidase systems. 4-CH. S-CH. Chem. and 0. ibid.. The thiosemicarbazones were prepd by treating alcoholic solns of the corresponding carboxaldehydes with an aq soln of thiosemicarbazide contg a few drops of dil AcOH.8 Hz. ibid. (13) V. (10) (a) F. W. Booth. C. J. CHO). J. and A.0 mmoles) of MnO. Blanz. Anal. 5-Methylisoquinoline-1-carboxaldehyde (16). H. (2) W. (b) A. F. Blanz. Berger.). The 5 4 1 and 5-Br derivatives were less inhibitory than the 5-OH derivatives. chloroquine and a new napthoquinone antimalarial. Ber. A. Biochemistry. C. The authors wish to thank Mrs. C. (7) (a) A. and the residue was extd with dil HCl. CHO). Sartorelli. Sartorelli.235 (1971). Moore. French and E. 3443 (1958). Creasey. W. J. J. Arnett. Jr. Robison. 1 H . and A. Menlo Park.27 g (72%). Thiosemicarbazones. French and E.2 mmoles) was dissolved in 20 ml of C.22 (s. Compd 3 (0.Biochem. Soc. K.Biophys.. R. 1 H. J. These investigations demonstrated that the function of coenzyme Q in the '-' . Recrystn from petr ether yielded colorless crystals in 70% yield: mp 59-60". CancerRes. Shaddix.35 g (4. Robison and B. was added..28 (s. The succinoxidase system was generally more sensitive to most of the benzoquinones tested than was the NADH-oxidase system.%NO) N.. C. Zedeck. Sartorelli. Moore. Ber. . 9 . French. 80. nmr 6 10.. Soc. 3-CH. (4) (a) K. and E.H. Med.). and A.Proc. Sartorelli. M. "Coenzyme Q.4-naphthoquinone. and Miss Annette F. (9) (a) B. C. Proc. 133. BioL. The solvent was removed and the residue was recrystd from hexane to give colorless crystals: 0. Relevant data concerning these compds are listed in Table 11. J. and A. The mixt was refluxed for 4 hr and fitered. (12) M.. Moore. C. J. Blanz. Amer.27 g (42%). J = 5. mp 87-88".1454 (1965). Separate sites for the function of coenzyme Q in the succinoxidase and NADH-oxidase systems i n beef heartl2'l3 and yeast13>14 mitochondria have been implicated by studies on the structural specificity of coenzyme Q. 26. Anal. and Institute for Biomedical Research. Pharmacologist. (b) S. Sauermilch. (b) F.. H4). Agrawal and A. Sartorelli.32 g. 1 H.

W. Heidker. 1972. Joseph C. DC 20036 . Med.. 195-197• DOI: 10.org/10. 1155 Sixteenth Street N. Catlin.. Pardini. Washington. Chem. 2009 More About This Article The permalink http://dx.acs. 15 (2).org on April 25. James C.doi.1021/jm00272a017 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. and Karl Folkers J.1021/jm00272a017 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.Specificity of inhibition of coenzyme Q-enzyme systems by lipoidal benzoquinone derivatives Ronald S.

BioL. W. 1971 5-Substituted 2. 2 195 References (1) (a) R. 3-Methylisoquinoline-1-carboxaldehyde (5). ibid. H-3).. (8) E. Spath. (2) W..235 (1971). 6-Alkyl. McMurray.%NO) N. Amer. 1 H.. Chem. 3443 (1958). French and E. Agrawal. and A. Sartorelli. (13) V. Agrawal and A. French. Brockman. Sartorelli..68 (s. Inhibition of the NADH-oxidase system was greatest when the hydroxyquinone possessed a side chain of from 16 and 17 C. CHO).nmr 6 10. 2-wcyclohexyloctyl-3-hydroxy-1. The authors wish to thank Mrs. Sartorelli. The 5-Me0 derivative was essentially noninhibitory. 57.K. Moore.H. B. (b) S.1948 (1968).. Proc. and A.O). Soc.). They represent potential antagonists of coenzyme Q function in mitochondrial electron transport. 2. dioxane was removed. J = 5. Pharm. (12) M. and in diminishing degree. Mitochondrial reconstruction'Y2 and spectrophotometric investigations on the kinetics of coenzyme Q turnover during electron transport3 are responsible for the view that coenzyme Q participates in the primary electron transport sequence. R. mp 71-72'. A. CancerRes. mp 87-88". J. M. Sauermilch. Skipper. (15) A. 2 H. 133. C. These findings link one kind of inhibition of mitochondrial electron transport at the coenzyme Q loci to chemotherapy of malaria.22 (s. J.. 4-CH. CHO). C. 758 (1957). Soc. J = 5.05 (d. Commun. (CliHJO) N.7 Hz.. Pharm. Brockman. J. C. Sartorelli. 31. 4 4 9 2 (1970). Blanz.35 g (4." These benzoquinones also have structural resemblance to coenzyme Q. nmr 6 10. 1 H.. Sei. 2. (4) (a) K. 20. J. and Miss Annette F. and Institute for Biomedical Research. ibid.. A. Blanz. CHO). (b) F. Compd 3 (0. Heidker. Sartorelli. Fed.8 Hz. Fed. K. 1972.38 g. 3 H.Anal. B. The succinoxidase system was generally more sensitive to most of the benzoquinones tested than was the NADH-oxidase system. Jr. . Journal of Medicinal Chemistry. 3 H.25 g (72%). shown to inhibit beef heart mitochondrial succinoxidase systems. 90. R.3-dimethoxy-6-phytyl-l. nmr 6 2.5 and 0. 1 H . C.0 mmoles) of MnO. Cushley. 7 0 0 (1968). ibid. Catlin.167 (1956).^ have been synthesized. 5 8 5 (1966). Preliminary structure-activity investigations demonstrated that mitochondrial succinoxidase activity was inhibited by various antimalarial naphthoquinone analogs. Furukawa and Y .. Berger.. Bell. Med.27 g (72%). C. and A. Sartorelli. Blanz. 1 H. (b) E. (7) (a) A. 8. 16. W. and 0. chloroquine and a new napthoquinone antimalarial. M. (b) K.1454 (1965). Martello. Robison. Ber. Soc. Stinson. C. Arnett. Jr. the inhibitory action of antimalarial agents was reversed by coenzyme Q. C. Relevant data concerning these compds are listed in Table 11. C. L. and S. Austin. ReceivedAugust 9. Chem.28 (s. C.. Agrawal. Agrawal. 1638 (1966). C. H. J. E. A. Agrawal.'-' Mammalian succinoxidase and NADH-oxidase systems have been extensively studied and may be considered representative of the coenzyme Q electron transport sequences. Booth. No. Compd 15 (0. 63B. and the activi t y was restored b y coenzyme Q and its derivative^. Moore.32 g. C. was added slowly. Creasey.. oxidizing 12 with MnO. and W. Anal.18 (dd. and H. Biol. K. 2 .81 (d.. and A. S. was removed to yield 5.. Bull. 2..2 mmoles) was dissolved in 20 ml of C. Bon Tempo. 4-CH3). 4-Methylisoquinoline-1-carboxaldehyde ( 13) was synthesized by the same procedure as 5. C. 192 (1967). 1 4 1 . The most effective group in the 5 position was OH for the 6-phytyl derivatives. Moore.). Anal. 9 . Anal. The solvent was removed and the residue was recrystd from hexane to give colorless crystals: 0.228 (1971).56 (s.Biophys. Exp. (3) W. C. nmr 6 10. ibid. Zedeck. (b) A. James C. and the C.J. (14) (a) W. 134 (1930). C. Biochemistry.4-naphthoquinone. Med. Chem. (9) (a) B. Exp.and 6-isoprenyl-2. and E. Recrystn from petr ether yielded colorless crystals in 70% yield: mp 59-60". ibid.. washed (H. and the residue was extd with dil HCl. J. C. J. C.Proc. J. Gann. French and E. Chem. Gwardyak for excellent assistance.21 (s.431 (1970). and the resulting ppt was collected.NO) N.Biochem. A.. Texas 78712. These investigations demonstrated that the function of coenzyme Q in the '-' . and A. M. 3 . were 'Author to whom correspondence should be addressed at the University of Texas. Sartorelli. Amer.^ Next. Jr. 8. 2 mmoles) was dissolved in 25 ml of dioxane and 0. Agrawal and A. C. R. Booth. Med.69 (t. 3 H.27 g (42%). (c) K. J. .4-benzoquinones were found to inhibit mitochondrial NADHoxidase and succinoxidase systems from beef heart.3119 (1971). A. C. 27. 9 . 1 H. 12. Pharmacologist. J. Crystn from hexane (Norit) yielded colorless fibrous material: 0. A.). Sidwell. Cancer Res. Shaddix. H4).). C. Cancer Res. hence. 2 9 . A. Specificity of Inhibition of Coenzyme Q-Enzyme Systems by Lipoidal Benzoquinone Derivatives? Ronald S. The 5 4 1 and 5-Br derivatives were less inhibitory than the 5-OH derivatives." Again. (10) (a) F.. 31. Kuroiwa. Thiosemicarbazones. Blanz. C.71 (s. Vol. Sartorelli. Sartorelli. The mixt was refluxed for 2. The acid layer was filtered and made alk with NaHCO. Agrawal. Traynelis and R.3dimethoxy-5-hydroxy-1. 3 H.H. Ber.. Moore.22 g (2 mmoles) of SeO. J. Jill Corby Morton. (C. Recrystn from petr ether gave colorless needles: 0. Robison and B. K. 3-CH. (11) E. 25. (b) F. Acknowledgment. 5-Methylisoquinoline-1-carboxaldehyde (16).771 (1969).. California 94025. 80. was added. Separate sites for the function of coenzyme Q in the succinoxidase and NADH-oxidase systems i n beef heartl2'l3 and yeast13>14 mitochondria have been implicated by studies on the structural specificity of coenzyme Q. Jr. and E.Coenzyme Q-Enzyme Inhibitors chromatogd on 6 0 g of silica gel (100-200 mesh) and eluted with EtOAc (500 ml). which were also succinoxidase inhibitor^. N. Sartorelli. The thiosemicarbazones were prepd by treating alcoholic solns of the corresponding carboxaldehydes with an aq soln of thiosemicarbazide contg a few drops of dil AcOH. 5 1 (1967). 26 (1967). CH. A. 15. Coenzyme Q has a widespread distribution in biological systems4 including the malarial parasite. Menlo Park.NO)C. and A. 5. 80. F. 9 0 8 (1970). ( 5 ) F. "Coenzyme Q. French and E. 9. Kuntara. (C. The mixt was refluxed for 4 hr and fitered. Miss Lynn A. F. and Karl Folkers" Stanford Research Institute. Inhibition of the succinoxidase system was relatively nonspecific in respect t o the side chain. mp 108-109". Joseph C. Biochem.Pardini. and dried.. G. C.H.609 (1970). (6) K. W.4-benzoquinones were similarly evaluated. Pharmacob. 2 3 2 (1955). K. Sartorelli. 13. (CllH1.6590 (1958). 2. E. CoQs is the dominant CoQ of Plasmodium A series of benzoquinones structurally related to known antimalarial naphthoquinones. The University o f Texas at Austin. 1 1 . Res.5 hr and filtered. S-CH.. J. C.. 26. they also represent potential antimalarial activity. Booth. Mathes and W. Thomson.

CH=C(CH.4-benzoquinones.450 lratom of 0 consumed per min per mg of protein..).250 to 0.1 ml of EtOH per 3 ml of reaction mixt. Voi.CH.300 to 0.)CH. and 5-bromo-6-phytyl-substituted quinones. Coenzyme Q and the various compds were added in EtOH. of protein) of the coenzyme Q.)CH.4-benzoquinones Inhibition of succinoxidase Inhibition of NADH-oxidase E and Ra L non-Eb E and Ra L non-Eb R ~~~~~ ~~ ~ c H 3 0 CH. and the data are in Table I. Table 11. Similar results were obtained with the unextracted succinoxidase system. supplemented (100 nmoles/flask) systems and the extd control was defined as 100% activity. although the magnitude of the inhibitions was less.The flasks also contd 100 nmoles of added coenzyme Q. but the magnitude of the inhibitions was less.(CH. Asolectin was added to serve as a carrier for the quinones.’ Portions of the lyophilized HBHM were extd with pentane’ to remove the coenzyme Q.H (phytyl) (CH.49. and 5-methoxy-6-phytyl derivatives inhibited 96. The 5-OH. succinoxidase system has little specificity for the various isoprenologs. Inhibition of Mitochondrial Succinoxidase and NADH-oxidase by 6-Substituted 2..5-Br. The differences in Tables I and I1 for inhibition of the extracted and reconstructed NADH-oxidase and succinoxidase systems between the specific activity (patoms of 0 consumed/min per mg The effect of various 5-substituted 2. Consequently. respectively.O 0~ R 0 H (CH. 14H(tetraprenyl) (CH.H (solanesyl) . Inhibition of Mitochondrial Succinoxidase and NADH-oxidase by 6-Substituted 2. and 2% of the controls. unextd NADH-oxidase. 24.CHzCHCHz).H (dihydrophytyl) (CHz)i&& (CHz).CH=C(CH. 52.3-Dimethoxy-6-phytyl-l .400 to 0. 5-C1.O H 3 0 ~ ~ H z C H = C ~ C H 3 ~ C H z ( C H ~ C H z C ).C(CH. No.7% 0 41 100 15 9 23 62 39 56 16 100 91 83 89 100 90 86 45 32 85 69 78 19 65 76 54 CH.0 mg per flask.. (w-cyclohexyloctyl) 96 92 100 67 76 62 49 65 41 39 ‘E and R = extracted and reconstructed.3-methoxy-6-phytyl1.H HCHz OH 0 c l Br OCH.)HCH.). The extracted-reconstructed succinoxidase system was depressed to 17. and 5-methoxy-6-phytyl derivatives. 1972.5-1.H (famesyl) (CH. on beef heart mitochondrial succinoxidase and NADHoxidase activities.. and 0. 5-C1. The compd (100 nmoles) was tested in the presence of 100 nmoles of coenzyme Q.0. Each value in Table I and in Table I1 represents the average of from 2 to 6 assays.CH=C(CH. b L non-E = lyophilized and nonextracted. (CH.3-Dimethoxy-5-hydroxy-l. The coenzyme Q deficient mitochondria were reconstructed by adding coenzyme Q and asolectin (a soybean phospholipid).4-benzoquinones on mitochondrial electron transport systems was determined.3-dimethoxy-5-hydroxy-6-alkyl-1. respectively. The lyophilized unextracted NADH-oxidase system was similarly affected. particularly the analogous 5-Me0-.to obtain the data in Tables I and 11.CH=C(CH. The activities of the lyophilized unextd and the extd-reconstructed mitochondrial succinoxidase and NADH-oxidase enzyme systems were detd manometrically in the absence and presence of the various benzoquinones to be tested. and unextd succinoxidase ranged from 0. and the data are herein described. These data demonstrate that the most significant structural change for inhibition of coenzyme Q-enzyme systems is the replacement of the 5-Me group of Table I. 96 52 24 2 62 36 6 8 83 51 21 0 78 25 24 0 aE and R = extracted and reconstructed.CH=C(CH&CH.H (geranyl) (CH. Results and Discussion Experimental Section “Heavy beef heart mitochondria” (HBHM) were i~olated’~ and lyophilized as described.CH.).)CH. 5-C1-. respectively.C. bL non-E = lyophilized and nonextracted . et 01. and the concn of EtOH was maintained in the flasks at 0. and 100%of the controls in the presence of the 5-OH.650. The per cent activities in Tables I and I1 for the lyophilized and unextd NADH-oxidase and succinoxidase systems were detd from the specific activity of the unextd control which was defmed as 100%. 15. The per cent activity of the assay systems (specific activity for the compd minus the specific activity of the extd control) was calcd from the uninhibited specific activity representing 100%.)CH.). This investigation was designed to evaluate the inhibition of various 2.0. but the activity of coenzyme Q in the NADHoxidase system is directly proportional to the length of the isoprenoid side chain..79. extd-reconstructed succinoxidase.4-benzoquinones R Inhibition of NADH-oxidase E and Ra L non-Eb Inhibition of succinoxidase E and Ra L non-Eb 7% c CH.350 to 0. 2 Folkers. The specific activities of the uninhibited extd-reconstructed NADH-oxidase.196 Journal of Medicinal Chemistry.. This different organic structural specificity for the succinoxidase and NADH-oxidase systems will surely influence the capacity of various coenzyme Q analogs to inhibit each of the enzyme systems.H.350.450.). an investigation of the structure-inhibition relationships was conducted on the recently synthesized benzoquinones. The mitochondrial protein was detd by the biuret methodI6 and was maintained at 0.5-Br.

References (1) R. Lunan. is situated in two different molecular environments. 168. Geiman. Heidker. However. 6-phytyl. J. 42. Lenaz. J. and K. and K.Ball. and 6-farnesyl derivatives inhibited the activity of the extracted-reconstructed succinoxidase to 0-25% of that of the controls. Chem. G. and K. Folkers. Similar structure-inhibition relationships were observed in the experiments conducted with preparations of lyophilized HBHM which was not extracted with pentane to remove CoQlo. Biochem... Schnell. 6-heptadecyl. 47. P. 3. for this fellowship.Methods Enzymol. 2 197 coenzyme Q with 5-OH. E. (14) A. Lunan. 110) . 257 (1947). et d . Blair. (13) G. Jr. Castelli. the 6-geranyl derivative is too hydrophilic to be functional in electron transfer in the hydrophobic environment associated with the activity of coenzyme Qlo. D. D. Colloq. ' that the inhibition by hydroxybenzoquinones of a mitochondrial NADH-oxidase system was reversed by coenzyme Qz . S. (11) J. 6-cyclohexyloctyl (C 14). J. V. Harris of Woodside.Lenaz. This inte retation is consistent with the findings of Castelli.78 (1967). Dr. Castelli.405 (1967). The 5-C1 derivative was a more effective inhibitor than the 5-Br derivative. E.'Soc.P. Folkers. G. Biochem. (2) L. C. and 6-heptadecyl (Cl. A. F. (9) . In the lyophilized and unextracted HBHM. K. Skelton. Biophys. (17) A. Littarm. G.4benzoquinones on the activities (HBHM) of NADH-oxidase and succinoxidase was assessed. Commun. 90. (3) M. Mitochondria. (8) J. P. S. 142..519 (1966).. Lester and S. those 5-hydroxybenzoquinones containing the 6-tetraprenyl (CZO). These relative inhibitions could also be influenced by a greater affinity for coenzyme Q in the NADH-oxidase than the succinoxidase system. The 6-tetraprenyl. Apparently. 6-phytyl (CZO).3572(1968). and 6-heptadecyl (C groups inhibited the phytyl ( extracted-reconstructed mitochondrial NADH-oxidase sysand 0% of the controls.. and K. Szarkowska.. Chem. W. J.G. but those compounds containing the 6-farnesyl (CIS). Folkers. W. and 6-solanesyl (C45) groups depressed the enzyme activity to 25-65% of the controls. (15) P. The 5-Me0 derivative was essentially noninhibitory in all forms of the enzyme systems.Coenzyme Q-Enzyme Inhibitors Journal ofhfedicinal Chemism.). 13. Crane. G . Pardini held the Stanley E.). Folkers. K. D. an alkyl side chain of 17 or 20 C atoms was more effective for the inhibition of the NADH4 or 1 9 C atoms. Skelton.Folken. 6-solanesyl. and K.. Castelli.F. A. J. G .539 (1968).ll (1967).5334 (1968). Fleischer. .806 (1971). Biophys. tem to 0. (7) F. J. 10. R. Rietz. 602 (1970). S. ibid. Folkers. C. He and Dr.. Quinones. Pardini. Biophys. (12) G. 14. oxidase system than were side chains of 1 Branching or degree of unsaturation of the side chain appeared to be of less structural significance than the general length of the side chain.-. 183 (1965). Anfinsen. The 5-OH substituted quinone was the most effective inhibitor of all 4 of the CoQ-enzyme systems. S. (4) F. E. ibid. and Q. On the basis of these data.. S. (5) P. Siddiqui. Med. C.3-dimethoxy-S-hydroxy-1. .407 (1971). ) . Jr. Vol. (6) F. The observed greater sensitivity and the lower structural specificity of the succinoxidase as compared to the NADHoxidase system for the various h droxybenzoquinones also support the previous con~lusion'~'~ that coenzyme Q has separate locations in complexes I and I1 and. Harris Postdoctoral Fellowship in Biomedical Research. Kroger. 1972. Rietz. Geiman.. Schnell. M. Biochim. and K. J.. 6-dihydrophytyl.4. No. 1284 (1969). The finding that the extracted-reconstructed mitochondrial enzyme systems are more sensitive to the analogs of coenzyme Q than are their respective unextracted preparations implies that the site of inhibition of these hydroxybenzoquinones is at the region of coenzyme Q in the electron transport chain. Calif. . since the inhibitor and coenzyme Q were both present during reconstruction at equal concentration. L.Klingenberg and A. S . Int. J. 6-nonadecyl (C 19). the succinoxidase system appeared to be more sensitive to the various antimetabolites of coenzyme Q than was the NADH-oxidase system. The specificity for the length of the alkyl side chain which was observed in the studies in NADH-oxidase inhibition was not apparent in the succinoxidase inhibition. Folkers. Bertoli. 6-phytyl (C. 1966. Bertoli. the most effective inhibitors were the 6-tetraprenyl (C. G. and K. The other derivatives caused less than 50%inhibition.D.Catlin. Since the derivative with the 5-OH group was the most effective inhibitor of these 5-substituted derivatives. Karl Folkers express their gratitude to Mr. Acknowledgments. 113. Daves. 2. Med. (16) E. Biophys. L. K. Biophys. Daves. The 6-geranyl analog inhibited the enzyme activity to 50% of that of the controls. and K. 34..) derivatives which inhibited the enzyme activity to 35-40%. Biochem. Layne. The greater sensitivity of the succinoxidase system to inhibition may be explained by a greater affinity of the CoQ-site associated with succinoxidase for the hydroxybenzoquinones. and the data are in Table 11.358 (1961). Biochem. Littarru. With the exception of the 6-phytyl and the 6-dihydrophytyl analogs. Res. These data show that the extracted-reconstructed mitochondrial NADH-oxidase activity was not inhibited by the 5-hydroxybenzoquinone containing a 6-geranyl (Clo) side chain. M. Q. Biochem. Ronald S. 8. Skelton. 120. 90. Chem.47 (1957). the effect of various 6-alkyl-substituted 2.200 (1968). Acta. the extracted-reconstructed succinoxidase system was more sensitive to the various 5-hydroxybenzoquinone analogs than was the comparable NADH-oxidase system. Biol. Again. Commun. V. 15. S. Amer.. Vitaminforsch.. 37... Siddiqui. Pardini. ibid. Folkers. 6-cyclohexyloctyl.8. A. Res. Biochemistry. F. 6-nonadecyl.. Arch.1026 (1971). therefore. Skelton. Chem.B. . J. respectively. Lenaz. Heidker. Skelton. Biochem. Cooper. Folkers. Arch. C. Folkers. R. Lenaz. 6-dihydroC .. and K. and 0. B..

1155 Sixteenth Street N.doi. DC 20036 . 1972.org on April 25. Da Re.. 2009 More About This Article The permalink http://dx. Primofiore J. Med.-Adrenergic blocking agents of the chromone and xanthone groups P. P. P.W.beta.org/10.1021/jm00272a018 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. Borraccini. and G. 198-199• DOI: 10. Chem. Washington. A.acs.. 15 (2).1021/jm00272a018 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.. Valenti.

H. R' = tert-C4H9 and in the 2 position for 19 and 20. Borraccini. prepared by standard methods.. (c) Contraction Rates of Isolated Right Atrial Strips. IS. ' HCl CHOH 269-270c C22H26C1N03 Crystn solvent: 'ligroin. As an alternative. respectively. C1. H. H. 2 Notes 0-Adrenergic Blocking Agents of the Chromone and Xanthone Groups P. 12.H. H. 13. The synthesis of these compds was carried out from the corresponding Ac derivatives. R' = i-C3H7 6.2C1NO. Vol.' quinoline. H.H. In Table I all the chromone and flavone derivatives prepared are shown.~ benzofuran. C1. R' = tertIC4H9 12. 19. Chromone and Flavone Derivatives Compdd R R' analogs are described in detail in the Experimental Section for illustrative purposes. . CMeOH-Et20. 6 .8) derivatives.4.H.. Compd 4 is therefore a selective P-adrenergic blocking I__.6. C6H5 I . H. Valenti. 19.H. Figure 1 reports cumulative log concn response curves for the agonist (isoproterenol) in the presence of various concns of the antagonist (4).20) on the basis of the results obtained among the CNS stimulants of the same The basic chain was located.H. N NH-i-C3H. ' HCI co 220-223' 1 6H20CM03 C. down to 2 X IO-'M. 10 pg/ml of 4. C1. We have also prepared 2 analogous xanthone derivatives (19. C1.g. On the basis of the ECzo values.6. and 12 melted respectively (ligroin) at 105-107".____ _ I _ _ -_______-- X MP.198 Journal of Medicinal Chemistry. C1. Br C. HC1 233-235' 1 6H22C1N03 C. R = C6HS. while the xanthone Table I.' ~. H. H. l2 The norepinephrine-induced lipid mobilization in rats is competitively blocked by equimolecular doses of 4. the activity of the former group decreases in the order 4 > 19 > 10. 146-148". 1 2 3 4 CH. b e n ~ o d i o x a n e . propranolol is approximately 10 times more active. R = CH3. amination. Primofiore Institute of General Chemishy.R' = tert-CJI. dThe bases corresponding to compds 4. Received March 23. N NH-CC3H7 HCI CHOH 10 C6H5 co 257-259' C22H24C1N03 C. 10. CH3 CH3 CH3 CH3 CH. The same doses of 4 cause a slight and transient hypotensive response of the order of -5 and . C1. N NH-tert-C. and 20 have been evaluated for their fl-adrenergic blocking activity in a number of tests at the Institute of Pharmacology of the University of Padua. bEtOAc. P.* P." Iv administration of 1 and 5 mg/kg of 4 causes a 100%and 87% reduction of the hypotensive response to 0. H. 6 H co 154-156' C18HL403 C. Compds 4. and G. R = C6H. University of Pisa. C1. by bromination.C1NO. (d) Lengthening of the Refractory Period of the Isolated Guinea Pig Auricles. Da Re. the reductive amination of the glyoxalyl derivatives (obtained by SeOz oxidation of the same starting materials) according to Fodor and Kovacs" gave unsatisfactory results.001 pg/ml of isoproterenol is reduced to about half and completely abolished with doses of 5 and. 10.No.H. and 12 R' 4.'6 An average 20% (ECZo)reduction in the maximal driven rate (MDR) at which the isolated guinea pig auricles respond to electrical stimulation is produced by 10 pg/ml of 4. We now report the preparation of some new &adrenergic blocking derivatives of the chromone and flavone group. The N-isopropyl derivatives were significantly more active than the tert-Bu analogs. ( f ) Blood Pressure in Anesthetized Dogs. R' = i-C3H7 10.. A. H Br co 143-146b 13H1 lBr03 C. 157-159". (b) Isolated Short-circuited Frog Skin.indole. in position 6 for 4. and reduction of the final amino ketone intermediates. was the most active in all tests employed. 20. (a) Lipomobilizing Activity. according to the synthetic possibilities. The chronotropic effect of 0.14 The increase of the short-circuited current in isolated frog skin is antagonized (60%) by 4 at 10-6M. HC1 11 C6H5 C. N CHOH NH-CC.6 dibenzofuran." The isoproterenol reduction of the contraction of the isolated guinea pig tracheal chain caused by carbachol (0. N NH-i-C. I pg/kg of iv isoproterenol. N 12 C6H5 NH-tert-C. N NH-tert-C. H. 5 C. with which we wish to illustrate further the versatility of the benzo-y-pyrone molecule as a carrier moiety in medicinal research.3-dimethylchromone (4). HC1 co 137-139' c 2 lH2ZClN0 3 9 C6H5 202-204' c21H24CM03 C. In this group.. HC1 co 200-202' c l. N NH-tert-C.. . R = CH3. I971 The search for new P-adrenergic blocking drugs with enhanced therapeutic properties is still very active' and there has recently been an increasing interest in heterocyclic (e. "C--___ ____-_Formula Analyses RfCHZX&t3 H co 136-138' I 3H1203 C.2 and. 6. 121-122". respectively. Br 8 C6H5 Br CO C. 1972. 10.10 mm. (e) Isolated Guinea Pig Tracheal Chain.H. Italy. HC1 CHOH 244-247' c132. C1. R' = i-CJr.5 pg/ml) is antagonized by very small doses of 4.[1hydroxy-2-isopropylaminoethyl] -2. etc. 561 00 Pisa. H 7 2 16-2 17 C18H13Br03 C.

Ordinate: per cent of maximal release. N] . Br.) C. and the mixt was kept at room temp with stirring for 4 hr. J. Pharm. and dried. Noseda. H. (C. Exp. washed (dil NaOH-H. F. 1972. N. of PhH. (7) R. dissolved (NaHCO.. J. (12) G. Director of the Institute of Pharmacology.) C.) C. B. H. 2. The general methods of synthesis described are illustrative of those of analogous compounds. the collected oil solidified on cooling.. A mixt of 7. C1.H. C1..O). V. .5 1. The soln was filtered from the catalyst and evapd to dryness. was obtd.). On crystg from ligroin. A. Pharmacodyn.5 hr.O. H.) C.[ l-Hydroxy-2-isopropylaminoethyl] xanthone Hydrochloride (19). and 45 g of PhCOONa was heated in an oil bath at 180-190" for 7-8 hr. Brit. 3 g of the corresponding amino ketone hydrochloride (18). gave 6 g of white cryst solid. H.H.. gave the amino ketone as the HCl salt. 169 (1970). C. J... Carpenedo. 1970.. C1. Chem. mp 199-200". 30 g of BzCl.. and L. Fitzgerald.. using a Biichi apparatus. for his kind permission t o report some biological data. Binon. (2) G. 95 g of AlC1. Zerahn.OJ C. The data reported seem to confirm the pharmacological potentialities of the benzo-y-pyrone molecule also in this field of medicinal chemistry. uptake ceased. S. [Anal. 2 199 90 " "1 M 1 I.. Chem. Mitchell. H. gave 15 g of yellow solid.Notes Journal of Medicinal Chemistry. (16) F. N. 1045 (1949). 2-tert-Butylaminoacety~anthone Hydrochloride (18). Blank.v.O).. mp 136-138". 23. a soln of 3. ActaPhysiol. Chodnekar. Pharmacol. Howe. Arzneimittelforsch. Abstr... Pharmacol.butylaminoethyl]xanthoneHydrochloride (20). Med. Med. The residue was distd at 175-180' (4-5 mm). Borland. after 2 crystns from EtOAc. 82119r (1967).O. N]. Cima. (14) G.. 5. Marchetti.O. 13. Pharmacol. Fassina. (C.0) iv in albino mice] is 2. S. Wandestrick. Duncan.285. treated with HC1 gas.O gave 2. and R. M.O.4% of the theoretical values. (C. P.H. was obtained. Arzneim. (C. Ther.ClNO. Vol. J. (C. C. Arch. 13. mp 108-1 10" (ligroin) [Anal. Goldenberg.577 (1965).H. 20. mp 224-226" [Anal. 166.A. Pharmacol. was added. Howard.511 (1969). (11) G. and J. The sepd solid was collected. 2-Isopmpylaminoacetylxanthone Hydrochloride (17). starting from 7. (17) P.base.. (C.O and the sepd solid was collected. J. Arzneim. Da Re. Chem.O. N]..) C.281 (1967). The reaction mixt was kept on a steam Acknowledgment...O).O>. Cl. Anal. Shanks. 3. 6-Acetyl-2. in 2 hr. In comparison with propranolol.1 g of 17 in 40 ml of MeOH was hydrogenated over 10%Pd/C until H. Pharmacodyn. The PhH layer. No.H. mp 194-196".O. References (1) M. on crystg from EtOHH. (C.. Luduena. Ussig and K. Chemother. Patil. 292 (1969). Anal. 32. H.5 g of white product. A mixt of 15 g of 2-propionyl-4acetylphenol(13). H. Pharm. N.) C.. The reaction mixt was taken up in H. H.) C. (C. Where analyses are indicated only by symbols of the elements. To a soln of 17 g of 16 in 1. and L.3 g of 14 was added in small portions. Arch. Int. was obtd. bath for 1. and M.-%O).. Lum.O. N] . agent of the propranolol type. layer was dried and filtered. Valenti. H.5 hr and then poured into ice H.-Forsch.3-dimethylchromone (1). 0 M. Ther.O. 36. The authors are indebted to Professor R. 23 g of white product (mp 64-65') was obtd. A. (8) W. 4 has a potency ratio of 0. The residue. and V..5-242. Amer. 43 (1968). and R. On crystg from ligroin.o 10 "1 10 ' . (6) R. mp 210-211". F.H9Br0. Fortschr. mp 112-114' (ligroin) [Anal. K. H. 2-Propionyl-4-ace~lphenol(l3).2 x 10-6M. To a soh of 4...718 (1968). Mancini. L. Fodor and 0.CINO. gave 3 g of white solid. 2-Bromoacetylxanthone (16). In a similar manner. and dried.O was heated in an oil bath at 170-180' for 7 hr. H.I8 with membrane activity (test d) and devoid of intrinsic sympathomimetic activity (test f). Charlier.. H. Anal.[ I-Hydroxy-2-tert. G.H. 18. Da Re.. 6 g of anhyd NaOAc. N. (13) H. mp 189-193" dec (MeOH-Et. Mancini.7 g of the corresponding amino alcohol hydrochloride as a white solid. and 0. 663 (1968). washed (H. was added in 1 hr.. University of Padua. 6-Acetyl-3-methylflavone (7). Kovacs.. Rao. J. Hepworth.368 (1969). 2-Carboxy-4'-acetyldiphenyl Ether (14). (18) J. 1965).308 (1968). H.3-dimethylchromone. 3. 15. and the solvent was evapd. 4 g of NaOH. washed (NaOH-H.H. Toth. 71. Anal. 8 g of yellow product. and G. and the sepd product was isolated by filtration. French Patent 3697 (Dec 27. Sagramora.NO.240 (1968). ( 5 ) R. Santi. V. 25. Anal. 107.. The fused mixt was taken up in H. (10) P. Pharmacol. Chem. C. E. Pharmacol. white cryst solid. S. a slight excess of i-PrNH. Scand. Removal of the solvent left a residue which on crystg from ligroin. Clin. mp 185-186".ClN0. and extd with CHCl...6 10 7 10 6 ID Io q 10 -* I Figure 1.O. Brit. A mixt of 10 g of 2-propionyl-4-acetylpheno1(13). The CHC1. Sei. H.. The soln was washed (H. B. white cryst solid. mp 230-233" (MeOH-Et .. H. 18. Fiandini. On crystg from EtOH. and 1 0 ml of Ac. Ther.2 x 10-SM)of 6-[l-hydroxy-2-isopropylaminoethyl] -2. (15) J.H. Biel and B.-Forsch. J. J.) C. mp 154-156". (C. Anal. J.) C. Cumulative log concentration-response curves for isoproterenol in the presence of various concns (a. (3) R. (C. Chodnekar. 10. Turner.8H. Turner. analytical results obtained for those elements were within +0. Experimental Section All melting points were detd in open glass capillaries. . was obtd...O) and dried. Hill and P.527 (1970).8 g of 0-chlorobenzoic acid. 6 g of p-hydroxyacetophenone.1 (test d).3 g of 2-bromoacetylxanthone (16). washed (NaHC0. ). After removal of the solvent the residue was treated with ice and HCl and extd with CHCl. (9) P. The mixt was poured into ice water and acidified with dil HCl. D.. P. On crystallg the crude product from MeOH-Et. K.8 g of 15 in 300 ml of CHCl. To a soln of 25 ml of o-hydroxypropiophenone and 25 ml of AcCl in 100 ml of CSd. Anal. and dried. 3. and the mixt was kept at 50-60 for 1hr. (C. but its LDS0 [223 mg/kg (205.. Merlo. Cima. The crude product.H. in 75 ml of CHCl. L. Brit.JI. K.base.. Soc.. 58. (4) J. Abscissa: molar concns of isoproterenol. The residue. and 100 g of 85% %PO. on crystg from MeOH-Et. 10.5 times lower. S.) C. and the solvent was evapd. Haley. and R.NO. (C. with stirring.5 g of white solid.ClNO. W. was added. J. and are uncor. 160. With the same procedure 3 g of rert-butylaminoacetylxanthone hydrochloride (18) gave 1. Chim. 46 (1966). W. The reaction mixt was transferred t o a separatory funnel. Fassina. 2-Acetylxanthone (15). Inf. Hill and P. To a soh of polyphosphoric acid (from 100 g of P.335 (1956). 9. 8 g of white solid.. 110 (1951). J. Ghouri and T.O). Howe. A soln of 4.) C. and repptd with dil HC1.2 X lO-'M.5 g of Cu powder was heated in an oil bath at 200" for 0..2 g of Br. Anal. 2.

1972.acs.org on April 25. 15 (2).W. 2009 More About This Article The permalink http://dx. 1155 Sixteenth Street N. Replacement of methylene by polar groupings Eugene L. 200-201• DOI: 10. DC 20036 . Stogryn J. Chem..1021/jm00272a019 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society.Synthesis of trimethoprim variations.org/10..doi. Med.1021/jm00272a019 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. Washington.

Biological Tests.4. Replacement of CHp by Polar Groupings? Eugene L. The direct condensation of 3. Received February 22. I971 The antimalarial efficacy of 5-ary1-2. 20.4-diaminopyrimidines. Esso Research and Engineering Company.§ The target structures did not affect the mean survival time of Plasmodium berghei infected mice sufficiently. mp 65.1 3.5-trimethoxyphenol.4. A mixt of 16.2 2. - - CH=N CH.4diamino-5-(3. hN. Experimental Section 2.O gave 14. gallinaceum) failed to improve the survival time of the test animals beyond day 1. E.85.S. X MP. 15.3 g of 3. 3. giving good and 2.S04 salt.5-195 THF H. Cl 4a 4a with NH. The mixt was added to 140 ml of 3% NaOH and extd with Et.O $Intramolecular cyclization of 5-ureido and -thioureidopyrimidines with expulsion of amines. Displacement of both C1 required high temp and pressure amination techniques. 2 Notes Synthesis of Trimethoprim Variations.7 g of the phenoxyacetonitrile and 11.4.01%) failed to produce a response.5-trimethoxyphenoxy)pyrimidine (1). New Jersey. fH.2 g of ethyl formate. days NHl 1 2 3 4 5 6 7 8 9 10 0 N=N~ CONH~ NHCOg co. 901 from the Army Research Program on Malaria. bMice were treated 3 days postinfection sc with a single dose of the compd being screened. found 21.NH NHCONHg NHCSNH 204-205 270-272 140-142 226-228 222-224 185-185. i H O A ~ salt. &my Medicinal Research and Development Command under Contract No.5-triaminopyrimidineto yield 7 was intolerably slow and isolation of product difficult. to be classified as active. Several of these syntheses were atypical and merit comment. the synthetic techniques detailed in the Experimental Section. p 339 and ref 4.5 169-172 193.2 g of K. Treatment of this oil in 1 1. of Et. comparatively few studies have dealt with this structural parameter.4.C03 in 60 ml of dry Me.CO was refluxed overnight. Although aminolysis of chloropyrimidines' are generally high temp processes. At a drug concn of 0.4. An amine exchange between this Schiff's base occurred readily. This is Contribution No.5 0.-EtOH over extended time periods failed to effect complete amination of 4a. are known synthetic entries into purines.7 0 0.5-trimethoxybenzaldehyde with 2. 1972. For discussion of purines see ref 3.1%. Stogryn Government Research Laboratory.3 g (88%) of 3.O. and 12. Spectral data of the reaction product suggested that under these conditions. 53. as a function of the electronic demands of aryl substituents. DADA1 7-68-C8 1 0 3 . No.4-Diamino-5-(3. at the maximum dose levels (640 mg/kg).33. berghei (see ref 6). 1 produced 100%abnormal -. CAST at 640 mg/kg. EtOH tThis work was supported by the U. the next lower dose level (0.4. Evapn of the Et. ghlonohydrate. "C Recrystn solvent OMe Formula Analysisa Antimalarial act. Neutralization of the aq phase and extn with Et.4.O gave 10.5-trimethoxyaniline. at a dose level of 320 mg/kg.8N607S C14H19NS0S C14H1$505 14H16N405 7N503 C18H27N507 C14H19N503 C14H20N605 14H I sN6O 3 ' i i e 0. 6.H16N404 C13H. . L Rane. Linden.5-66' (PhH-hexane). calcd. was defined by the extensive studies of Hitchings' and coworkers.6 g of chloroacetonitrile.NH~~~ CH. found 53.200 Journal of Medicinal Chemistry.2 0.O was added.5-trimethoxybenzyl)pyrimidine] congeners appeared particularly pertinent in light of its recently reported activity against drugresistant strains of malaria.5 g of a viscous oil.$ Purification by sublimation or recrystallization failed. iDecompd on heating. appear to undergo facile thermal alteration.5trimethoxyphenoxyacetonitrile. However. Compds 2. obtained virtually quantitatively.*Vc A ST.4. To a slurry of NaH (3.5-trimethoxybenzaldehyde i-PrNH2.3 0.77. The target compounds listed in Table I were prepared by Table I.O HlO HlO EtOH PhH PhH 1 ' . was a competing nucleophile. Vol. calcd.' We wish to describe structural variations of trimethoprim in which the CH2 group that connects the aryl and pyrimidine rings has been replaced by polar functionality. #The rodent and avian activity screens were performed by Dr.5 0. the known promotional influence of carbonyl functionality in nucleophilic displacements presented the possibility of obtaining 4 by treating oocyst and 100%suppression of oocyst in the mosquito (Aedes aegypti) screen.4% of the theoreticai value.5-triaminopyrimidine yields of 7. Gerberg. Although their work also indicated an activity dependence on the nature of the functionality uniting the aryl and pyrimidine rings. Reaction of 4a with refluxing NH. Compds 9 and 10.7 g of 50%NaH) in 130 ml of PhH was added a soln of 16. After stirring at ambient temp overnight 100 ml of H.6 a m e r e analyses are indicated by symbols of elements analytical results were obtained for those elements within ?0.4.42. Attempts to sublime either 9 or 10 resulted in the isolation of 3. and 6-9 in the avian activity screen (P. No. Acidification of the aq phase yielded 3 g of 3. These synthetic deficiencies were alleviated by an initial preparation of the and Schiff s base from 3. The change in survival time (AST) is an indication of activity against P. eNot evaluated in the rodent screen. The significance of this screen is described in ref 5 . dC.4. with destruction of 9 and 10. An examination of this parameter in trimethoprim [2.5-trimethoxyphenol. The mosquito activity screen was conducted b y Dr.-EtOH at atm pressure.

Evapn of solvent left 48 g of a white solid. Perrotta.5-trimethoxybenzylideneamino)pyrimidine (6).6 g of 3. "The Pyrimidines. National Cancer Institute. soln pptd 3 g of 2 as a yellow solid. Anal. 26.4. 2. After 5 min sufficient NaHCO.4-Diamino-5-pyrimidinecarbox-3.4.5-trimethoxybenzamido)pyrimidine Monohydrate (3). Chem. 5 " (MeOH-H. The University of Texas at Houston. P.. (2) D. bp 145" (0.5 mm).N (1.5-trimethoxybenzylamino)pyrimidine Acetate Salt (7).5-triaminopyrimidine7 (1 g) and 1.4 g). Houston..N. A mixt of 5 g of 4a. and G.232 (1967). C. filtered. 2. Heterocycl. mp 43-43.4.N.9 g of a dark viscous oil which gradually solidified. (c) P. Russell.2 Of the 5-triazenoimidazoles. poured into a large vol of Et.4-Diaminopyrimidinyl)] -3-(3. and G. 112 (1957). and L. No.4-Diaminopyrimidiny1)]-3-(3. 2. 8.HCl.4-Dihydroxy5-pyrimidinecarboxylic acid was converted into the titled compound by the procedure Gershon* used to prep the analogous 6-pyrimidinecarbonyl chloride. 2. 2.5-tnmethoxyanilide (4a).rldichloro-5-pyrimidinecarbonyl chloride in 50 ml of THF. After 15 min the CHC1. A soln of 40 g of 3. R.62 g) and 2... Brown. Gerberg.J. J. maintg the temp below 5". U. Richard.Cl. Gerns.4-Diamino-5-( 3. Arnold.NO.7 g of guanidine hydrochloride and 5.4-diaminopyrimidine in 110 ml of H. 15.5-triaminopyrimidine was refluxed for 20 hr and then allowed to stand at room temp for 2 days.2 g. To a soln of 2 g of 2. Hitchings. cooled. yield 2 g.4. Med. pp 153-154.5-trimethoxybenzoyl chloride in 250 ml of PhMe.3-dimethyl-1-triazeno)imidazole-4-carboxamide (DIC) in particular.4. Falco.4diamino-S-hydroxypyrimidine hydrochlorideQ and 3. evoln ceased.N.Notes Journal ofMedicina1 Chemistry.4.3-dimethyltriazeneP Ying-Tsung Lin.* Department of Developmental Therapeutics.4.. 3507 (1962). The red-brown solids. Soc. and recrystd. Received May 20.CO eluate. Amer.. 50% yield.4.5-trimethoxybenzylideneisopropylamine and 8. Appl.O soln of 7 was made basic with NH. 1.5-trimethoxybenzoyl chloride in 15 ml of THF.4. Anderson Hospital and Tumor Institute. T. ibid.9 g of the above described methylated aldehydonitrile in 125 ml of MeOH was refluxed for 18 hr. J. The MeOH was evapd and the residue extd with hot THF. N. the precursor of the purine base.5-Trimethoxyphenyl Isothiocyanate. Chem.431 (1967). DuBreuil.4-Diamino-5-( 3. An H.4.1 mm). D. To a cold soln of 2. it is entirely possible that as a derivative of 5aminoimidazole-4-carboxamide (AIC).4. Anal. S.NO. and 66 ml of liquid NH. were filtered and extd with 500 ml of boiling 1N H.J.8 g. 1-[5-(2.1 g of 3. H. However. Stirring was contd until the characteristic acyl halide ir absorption was absent. and J. Ass...O.6 Nevertheless. This product solidified on standing.. 1. A. The mixt was stirred for 20 min and concd. mp 63" (hexane). O and EtOH. bp 120-140" (0. Chem. H. filtered. Org.54rimethoxyphenyl isocyanate. Hitchings. 27. 73.N in 20 ml of H. (C. Amer. formed after standing overnight. filtered. 2. The distillate solidified on standing. (5) E.N in 20 ml of THF-H. and recrystd.4-Diamino-5-(3. B. H.S) C. and the contents were filtered and recrystd. (C. yield 2 g.5-trimethoxyphenyl)urea Monohydrate (9).4.O. B. Chem. 2 201 with 5. (3) D. Hitchings.4. Srikrishna Vadlamudi.OH.O was added 1. To a slurry of NaN. 10. The product. (9) R.H. mp 7 6 5 7 7 . National Institutes of Health. H. The mixt was then heated on a steam bath until N.The ~ mechanism of the antitumor action of DIC remains obscure.4. An EtOH soln.SO.$rlO.OH. H.3-dimethyltriazene these compounds have not been (la-If). Russell and G. yield 3 g.4. The residue from the THF soh was chromatogd through base-treated silica gel. yield 4 g.H. (6) T. Y. On cooling the %SO.108 (1966).1 g of Et. and G. (d) F.O). 2.4-Diamino-5-(3. J. T.. Med.5 g of Et. (C.5-trimethoxybenzylamino)pyrimidine (8).4.JI.. Compounds. is clinically useful for the induction of temporary remission in malignant m e l a n ~ m a . Powdered 3.5-trimethoxyaniline in 300 ml of CHCl. 1962.' To extend these observations and. layer was sepd and dried over CaCl.7 g of 3. To a cold soln of 1 g of 2. B. Low pressure hydrogenation of 2. to elucidate the structural requirements for antitumor activity in the triazenes and specifically t o ascertain whether an imidazole ring with a carboxamide moiety ortho to the triazeno side chain is indispensable in DIC.54rimethoxyaniline in 250 ml of 1N HCl at 0" was added 8 g of NaNO. References (1) (a) E. 1-[5-(2. The pptd solid was filtered and recrystd. Poole. 5-(3. C1. Osdene. A.4. Hul1.4-Diamino-S-(3.. was heated in an autoclave for 8 hr at 180". (C. 73. (b) E.) C. Falco.36 g of 3. After 20 min the reaction mixt was concd.5-trimethoxybenzoyl chloride in 20 ml of THF. (7) D.. H.4. Public Health Service. New York.. After 1 hr stirring the ppt was filtered and washed thoroughly with H. 3. S.1 g of 2.4.' An excellent and comprehensive review of this subject has recently appeared.350 ml. Preparation and Antitumor Activity of Derivatives of 1 -Phenyl-3. 2. above all. we have synthesized 6 derivatives of 1-phenyl-3. Robins. Ti Li Loo. (C. J. (about 35 g) was added to raise the pH to 8.O at 0".4 g of 3. ibid. 3763 (1951). J.476 (1968). Except for ?Supported in part by Contracts PH 43-66-11 5 6 and PH 43-681283 with Chemotherapy. and repeatedly washed with Et. and distd (14..5 g in 125 ml of PhMe was added 23.O.5-trimethoxyphenyl)thiourea (10).) C. A number of 5-triazenoimidazoles are active against experimental tumors.) N. Vol.5 g of 6 in 140 ml of HOAc over PtO.5-trimethoxyphenyl isothiocyanate (2 g) was added to a soh of 2. occurred in 15 min. K.84 g of 3. 203.O. Bethesda. (4) R.4. To CSCI.3 g of 2. This soln was added to 11 g of 2. 3758 (1951).8 g of 3.4.4. it has been proposed that the triazenes may act as alkylating agents through the in vivo generation of carbonium ions. filtered. Et. Med. D.4. Inc.O and EtOH. Hitchings. was found in the second Me. Anal. Chem. 73. N.5 -trimethoxybenzoy loxy )pyrim idine ( 5 ) . Chem. 19. 3." Interscience. The bomb was cooled. 2033 (1956).O was added 30.4. H. The residue was washed with cold dil NaHCO.4. M. (8) H. and recrystd from PhH-hexane. Anal. .4-Dichloro-S-pyrimidinecarbonyl Chloride.5-triaminopyrimidinein 40 ml of 75% EtOH.. P. 3753 (1951). J. The pptd yellow solid was filtered and recrystd. Bethesda.. N.5-Trimethoxybenzylideneisopmpylamine. A.4-Dichloro-5-pyrimidinecarbox-3. 1972.5-trimethoxyphenylazo)pyrimidine Sulfate (2).5-triaminopyrimidine in 150 ml of 50% EtOH was added 2. Microbiological Associates.95 g of 3. 19 71 2. It was cooled.5".O washed. Soc. Mosquito News. Brown. Rane. Texas 2. poured into a large vol of H.5-trimethoxyanilide Monohydrate (4).. Maryland. Martin and J.O. S.3 mm).O) N. 3.8 g of product. L. gave 8. mp 196-198". J.5-Trimethoxyphenyl Isocyanate. Anal.4-Diamino-5-(3.3 g of CH. 9. mp 110-1 11" (MeOH). After standing ovemight the reaction was filtered and the THF evapd. S.. Gershon. A s o h of guanidine (from 9. bp 90-91" (0.. (25 g) suspended in 180 ml of ice H.' Antitumor activity has been observed in several derivatives of phenyltriazene. in 40 ml of H.4 g of NaOMe) and 8. in 200 ml of THF was refluxed for 5 hr. Distn gave 24. Russell.O was added 1. yield.. 359 (1966). To a soh of 20. The reaction mixt was filtered. contg 15. 13 ml of NH. Cl. After several hours stirring at room temp the ppt was filtered and washed d t h H . on the basis of studies of the biotransformation of DIC4>' and the carcinogenesis of phenyltriazenes. Add1 quants of 6 could be obtd by concn of the filtrate.5-trimethoxyaniline in 100 ml of THF was added to a cold soln of 3.4. 5-triazenoimidazole may somehow-interfere with imidazole and purine metabolism. Maryland and Abraham Goldin National Cancer Institute. National Institutes of Health. H.4.H.5-trimethoxybenzaldehyde and 35 ml of i-PrNH.

acs.org/10.3-dimethyltriazene Ying-Tsung Lin.1021/jm00272a020 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. 1972. Ti Li Loo. and Abraham Goldin J. 15 (2). Med. 2009 More About This Article The permalink http://dx.Preparation and antitumor activity of derivatives of 1-phenyl-3.1021/jm00272a020 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society.org on April 25. 201-203• DOI: 10. 1155 Sixteenth Street N.W. Srikrishna Vadlamudi.. Washington.doi. DC 20036 . Chem..

This product solidified on standing..54rimethoxyaniline in 250 ml of 1N HCl at 0" was added 8 g of NaNO. ibid. we have synthesized 6 derivatives of 1-phenyl-3.O was added 1. (d) F. Gerberg.3-dimethyltriazeneP Ying-Tsung Lin..4.O.5-triaminopyrimidine was refluxed for 20 hr and then allowed to stand at room temp for 2 days. Gershon. H.4. above all.1 g of Et..4.. Chem.4. Med. layer was sepd and dried over CaCl. P. 19 71 2.4-Diamino-5-( 3. filtered.350 ml.. in 40 ml of H. J.N in 20 ml of THF-H. the precursor of the purine base.O).4-Dihydroxy5-pyrimidinecarboxylic acid was converted into the titled compound by the procedure Gershon* used to prep the analogous 6-pyrimidinecarbonyl chloride. After 20 min the reaction mixt was concd. T. Russell and G. yield 2 g. 73. A. Brown. The residue from the THF soh was chromatogd through base-treated silica gel.4.O was added 30.. 3758 (1951). gave 8. J. yield 3 g. (5) E.5-trimethoxybenzylamino)pyrimidine Acetate Salt (7). The mixt was stirred for 20 min and concd. After 1 hr stirring the ppt was filtered and washed thoroughly with H. (about 35 g) was added to raise the pH to 8.4-Dichloro-5-pyrimidinecarbox-3.N.95 g of 3. ibid. Chem. Add1 quants of 6 could be obtd by concn of the filtrate.232 (1967). (6) T..1 g of 2. Hitchings.5 mm). Falco. Hitchings. is clinically useful for the induction of temporary remission in malignant m e l a n ~ m a . The pptd yellow solid was filtered and recrystd. Anal. Maryland.O was added 1. Hul1.4. Anal.3 mm).' Antitumor activity has been observed in several derivatives of phenyltriazene. 19. (8) H. To a cold soln of 1 g of 2.5-trimethoxyphenyl)urea Monohydrate (9).5-tnmethoxyanilide (4a). was found in the second Me. Brown.4.5-trimethoxybenzylideneamino)pyrimidine (6).2 g. filtered. it is entirely possible that as a derivative of 5aminoimidazole-4-carboxamide (AIC).N.O and EtOH.5-triaminopyrimidine7 (1 g) and 1. An H. 3753 (1951).The ~ mechanism of the antitumor action of DIC remains obscure.O.4-Dichloro-S-pyrimidinecarbonyl Chloride. was heated in an autoclave for 8 hr at 180".4.8 g. and G. 112 (1957).4-Diamino-S-(3.. mp 63" (hexane). 2. Stirring was contd until the characteristic acyl halide ir absorption was absent.36 g of 3. 26.N in 20 ml of H. 3. Heterocycl. N. 2. S. J. mp 110-1 11" (MeOH). Org.O. H.4 g of NaOMe) and 8. 1972. B.O.J.6 g of 3. Russell. Perrotta.5-Trimethoxybenzylideneisopmpylamine. After standing ovemight the reaction was filtered and the THF evapd. were filtered and extd with 500 ml of boiling 1N H. (b) E. The red-brown solids. yield. (C. Anderson Hospital and Tumor Institute. The bomb was cooled. Received May 20.4-Diamino-5-(3. 1. 50% yield. evoln ceased.O. T. H. National Cancer Institute.O washed.4-Diaminopyrimidiny1)]-3-(3.Cl.4. Evapn of solvent left 48 g of a white solid. B.5-trimethoxybenzamido)pyrimidine Monohydrate (3).5-triaminopyrimidine in 150 ml of 50% EtOH was added 2. A soln of 40 g and 35 ml of i-PrNH.8 g of 3.6 Nevertheless. H. Amer. New York. U.. On cooling the %SO.108 (1966).4. J. To a slurry of NaN. DuBreuil. 15.5 g of 6 in 140 ml of HOAc over PtO. Russell. P. R. (4) R. The product.5-Trimethoxyphenyl Isocyanate. A number of 5-triazenoimidazoles are active against experimental tumors.5-trimethoxybenzoyl chloride in 20 ml of THF. 359 (1966). soln pptd 3 g of 2 as a yellow solid.) C. To a soln of 2 g of 2. mp 43-43. After 15 min the CHC1. 8. 73.5-trimethoxyaniline in 300 ml of CHCl. Rane. 2.1 mm).5-trimethoxybenzylideneisopropylamine and 8.5 g of Et. The MeOH was evapd and the residue extd with hot THF.4. The distillate solidified on standing. (25 g) suspended in 180 ml of ice H. D. pp 153-154.4-Diaminopyrimidinyl)] -3-(3.4. K.84 g of 3. occurred in 15 min.5 g in 125 ml of PhMe was added 23.$rlO.4 g of 3. Ti Li Loo.2 Of the 5-triazenoimidazoles. formed after standing overnight. (C. (C. N. Anal.H.4.4-Diamino-5-pyrimidinecarbox-3.5 -trimethoxybenzoy loxy )pyrim idine ( 5 ) . J.JI.. 3763 (1951).4. on the basis of studies of the biotransformation of DIC4>' and the carcinogenesis of phenyltriazenes. 3507 (1962).N.3 g of CH. 2. Poole. Cl. mp 196-198". yield 2 g. 2 201 with 5. 203. No. Richard.62 g) and 2.. and recrystd. 5-(3.9 g of the above described methylated aldehydonitrile in 125 ml of MeOH was refluxed for 18 hr. H. Mosquito News.' An excellent and comprehensive review of this subject has recently appeared. However. and G.5-trimethoxyphenylazo)pyrimidine Sulfate (2)." Interscience.H. and recrystd from PhH-hexane. Inc. Low pressure hydrogenation of 2. Ass. The mixt was then heated on a steam bath until N. 2.rldichloro-5-pyrimidinecarbonyl chloride in 50 ml of THF.5". (2) D.54rimethoxyphenyl isocyanate.. The University of Texas at Houston. Gerns.431 (1967). in 200 ml of 3.H.4. it has been proposed that the triazenes may act as alkylating agents through the in vivo generation of carbonium ions. mp 7 6 5 7 7 . Soc. Chem. bp 120-140" (0. After 5 min sufficient NaHCO. H. Med. Bethesda. N. and repeatedly washed with Et. poured into a large vol of H. to elucidate the structural requirements for antitumor activity in the triazenes and specifically t o ascertain whether an imidazole ring with a carboxamide moiety ortho to the triazeno side chain is indispensable in DIC. and recrystd.5-trimethoxyphenyl isothiocyanate (2 g) was added to a soh of 2.9 g of a dark viscous oil which gradually solidified.O) N. and the contents were filtered and recrystd. Med. and G. Bethesda. A mixt of 5 g of 4a. The reaction mixt was filtered. S.. bp 90-91" (0.4-Diamino-5-( 3.4. Texas 2.7 g of guanidine hydrochloride and 5. A. maintg the temp below 5".. A.. 73.5-trimethoxyphenyl)thiourea (10). cooled. D.CO eluate.4-diaminopyrimidine in 110 ml of H. . L.8 g of product. poured into a large vol of Et. yield 4 g.4. National Institutes of Health. J.1 g of 3. A s o h of guanidine (from 9.5-Trimethoxyphenyl Isothiocyanate. "The Pyrimidines. References (1) (a) E.J.. Arnold.Notes Journal ofMedicina1 Chemistry.S) C. The residue was washed with cold dil NaHCO. Houston. 1-[5-(2. The pptd solid was filtered and recrystd. Y.5-trimethoxybenzoyl chloride in 15 ml of THF. 5 " (MeOH-H.7 g of 3. filtered.5-trimethoxyaniline in 100 ml of THF was added to a cold soln of 3.OH. 1-[5-(2. Srikrishna Vadlamudi. Appl. (c) P. To a cold soln of 2..4-Diamino-5-(3.. B. Chem. (C. 2. contg 15. Amer. Chem.476 (1968).4.N (1.HCl.3 g of 2. Soc. H. 3. J.4. C. It was cooled.O soln of 7 was made basic with NH.NO. H.5-triaminopyrimidinein 40 ml of 75% EtOH.OH. (C. and J. 3. Et.NO. O and EtOH.5-trimethoxyanilide Monohydrate (4). C1. To a soh of 20. 9. Distn gave 24. Chem. An EtOH soln. After several hours stirring at room temp the ppt was filtered and washed d t h H . Microbiological Associates.. To CSCI. Robins.4-Diamino-5-(3. and 66 ml of liquid NH. (9) R. 1962.5-trimethoxybenzoyl chloride in 250 ml of PhMe. (3) D. Public Health Service.. S.5-trimethoxybenzaldehyde of THF was refluxed for 5 hr. 2. Compounds. 1. Falco. Powdered 3.. filtered. Vol. Preparation and Antitumor Activity of Derivatives of 1 -Phenyl-3.4. This soln was added to 11 g of 2.4. Osdene.O at 0".SO.4.4. Maryland and Abraham Goldin National Cancer Institute. 13 ml of NH. National Institutes of Health.) C. 2.4. 5-triazenoimidazole may somehow-interfere with imidazole and purine metabolism. (7) D. Anal. Except for ?Supported in part by Contracts PH 43-66-11 5 6 and PH 43-681283 with Chemotherapy. S. Martin and J. 27. 10. and distd (14. M.5-trimethoxybenzylamino)pyrimidine (8).' To extend these observations and.4.4 g). Anal. and L.3-dimethyltriazene these compounds have not been (la-If).4diamino-S-hydroxypyrimidine hydrochlorideQ and 3. bp 145" (0.3-dimethyl-1-triazeno)imidazole-4-carboxamide (DIC) in particular.4. 2033 (1956).* Department of Developmental Therapeutics. Hitchings.4.) N. Hitchings.

3-dimethyl-l -triazeno)benzoic acid and ethyl chloroformate. respectively. After stirring at room temp for 30 min. A second crop of the crude product was obtd by extg the filtrate with PhH followed by concn. the orientation of either the CONH2 or the COzCH3 substituent with respect to the dimethyltriazeno side chain is not critical. Vol. 10 mmoles) and ethyl chloroformate (1 ml. 10.3-dimethyltriazene derivatives with that of DIC. 10. ZN40 ZN4' CQH12N40 I$.3-dimethyltriazene with no annular substituent does not increase the survival of mice bearing L1210 leukemia (sensitive to methotrexate)8 would suggest that the carboxamide moiety is necessary for antileukemic activity. and the pyrazole ring are all equivalent therapeutically in the triazenes. The solid formed was removed by filtration. and para) show comparable antileukemic activity.5 (0) 8 13 13 11 (3) (3) (3) (0) 25 10 5 0 15 (2) 12. with the addn of a few drops of EtOH)."'" it appears that the imidazole ring.39 ml.3-benzotriazin-4(3H)one instead of the required diazo intermediate.5 (0) 0 0 30 20 5 2 10.p described previously. In fact. The light brown crude phenyltriazene derivative was collected by filtration and dried over P. mice inoculated ip with l o 5 L1210 leukemic ascites cells. ILS of 20%or greater is suggestive of reproducible activity. accompanied by thorough agitation.5 ( 1 ) 1 1 (0) 0 5 20 5 10 10 11 11 10 10 (2) (0) (0) (0) (0) 0 10 10 0 0 7 14 11 11 10 (6) (3) (0) (0) (0) 0 40 10 10 0 50 25 30 0 10. mC.3%for C.3 mmoles. R = OCH3.O. Microanalyses were performed by Dr. (9 g. (ortho. However.O).5 (5) 12 (3) 10. (0. In Table I we compare the anti- dicates that the CONHz group in the phenyltriazenes may be replaced with C02CH3 with no adverse effect. dild with 20 ml of anhyd Et. The fact that l-phenyl-3. R = NHz.. Number in parenthesis represents the average body loss in grams on the 6th day.OH (2.~ because the diazotization of o-benzamide directly afforded 1. If.5 (2) 0 10. with MezNH in Na2C03 ~ o l n However. yields of the crude product were 94-100%.3-Dimethyl-l-triazeno)benzamide.5 (4) 35 14 (2) 40 1 3 (2) 12. N. the above cold s o h contg the diazo compound was carefully added to Me. 8 mice per group and 46 untreated controls.No. An analytical sample was further recrystd from PhH-Et. Antitumor Activities.5 (2) 10 (2) 5 0 11.2. 1 la. pd.O and p-dioxane) was added Et. Finally.3dimethyl-1-triazeno)benzoic acid (2 g. The arom amine. However. %. From these results. Ammonolysis of the ethyl benzoyl carbonate which was not isolated gave la.O. 0-(3. %crease in life span.N (1. the stirring was contd at room temp for an add1 2-3 hr. After standing Table I. leukemic activity of the 1-phenyl-3. R = NH.O instead.5 (0) 1 1 (0) 10. 5 . because these phenyltriazenes and DIC are equally active and because the pyrazole analog of DIC likewise displays antileukemic property. they are nevertheless as effective as DIC in increasing the survival time of the leukemic mice. IS.8 g. it is clear that although the tolerated dose range is lower for the Ph analogs.5 (3) 25 13 (1) 30 11.5 (5) 12. the temp being kept between 5 and 15". "C Molecular formula" 'QH1 la 56 127-13 1 lb 73 145-146 IC 78 176-178 le 70 43-45 If 80 103-104 aAll compounds analyzed within *0. days.5 (2) 5 15 (2) 50 14 (1) 40 1 3 (1) 30 13. at least one of these.O.5 (0) 30 30 10 40 40 35 =BDF.5 (3) 11 (2) 11 (2) 25 10 10 2 6 (6) 1 3 (4) 12 (0) 10. 2 Notes N=NN(CHJ. the mixt was made ammoniacal with concd NH.5 (3) 14 (2) 12 (0) 12.5 (4) 5 13. The determination of the activity of each compound against a variety of transplantable tumors is currently in progress. Further. Median survival time of untreated controls: 10 days. Comparison of the Antitumor Activity of Phenyl Analogs of DIC with DIC Itself in Mouse Leukemia L1210a Dose. m.b mg/kg 833 5 00 300 180 108 65 180 108 65 108 65 39 23 DIC la MSTC I L S ~ MST 12 11 11 10 11 10 12 11 11 (3) (2) (2) (2) (1) (0) (0) (0) (0) ILS lb MST ILS IC MST Id ILS MST ILS le MST ILS If MST ILS Treatment Day 1 only 20 10 10 0 10 0 20 10 10 12. The combined crude product was recrystd from PhH. At the end of the mixing. Yield.NH (40%in H.5 (4) 5 10. The crude compound was dissolved in abs MeOH and treated with activated charcoal. f.O. Alford and associates of the National Institute of Arthritis and Metabolic Diseases. l e was recrystd from anhyd Et.13 mole) dissolved in 25 ml of H. - SMp's were detd with a Fisher-Jones apparatus.202 Journal of Medicinal Chemistry.R=OCHj.1 ml.35 mmoles in 30 ml of an equivol mixt of anhyd Et. la. They were readily prepared by treating the corresponding diazotized methyl aminobenzoates and m-and p-aminobenzamide.3dimethyltriazene.5 (3) 10 (0) 10 (0) 0 5 25 0 0 2 10. l a was synthesized by a modified procedure. bTreatment ip CMedian survival time.N. our present work in- at 0-5" for 20 min.O to ppt the purified product. . The clarified MeOH soln was dild with H. in vacuo. and 9 10.. 15. to whom we wish to express our gratitude. may have a more favorable therapeutic index than DIC. Compound % MP. I9 72. Experimental Section$ Derivatives of l-Phenyl-3. meta. William C. Table 11.O ( 4 : l by vol. 0. as all 3 derivatives. R = OCHj.ob. Oe. Diazotization was achieved by the slow addn. National Institutes of Health. H. R = NH2. An ethyl benzoyl carbonate was prepared from o-(3.13 mole) was added to a mixt of 100 g of crushed ice and 33 ml of concd HCl with vigorous stirring at 0-5". To a soln of 0-(3.14 mole) and ice (75 g) with stirring.5 (0) Treatment Days 1-9 6 (3) 0 6 (3) 0 9.5 (1) 35 30 15 12. 0.5 (0) 13 (0) 10 (2) 14 (1) 14 (1) 13. the Ph ring. . 15 mmoles) while stirring was contd for another 3. 3N302 Cldr.5 (0) 15 40 20 25 11 (2) 10 (1) 10 (1) 10 0 0 Treatment Days 1.5 hr. of NaNO.

M. 60. H. ( 3 ) J. A number of studies on the pharmacological action of vitamin B6 and its derivatives have been reported. H. Chemistry. CLit. Issacs. Exp. by the applkation of this method using pyridoxal with several 3-hydroxyphenethylaminesand hista- R XIII.2. Cheng. G. 54. and G .545 (1969). C.6. prepared by the usual method. Although the structure of the above products could be thought to be that of Schiff bases. bLit.N.H. Shedy.O. W. Pharmaceutical Institute.4-Tetrahydro-6-hydroxy4-pyridoxylisoquinolines and 4-Pyridoxyl4. " R' f l \ N H 1 111. Stock. L. R = OH. Vol. Koizumi. R. Sot.3 We had found a method of synthesizing the various 1. R = OAc ..2. fNot analyzed. R = H. Tokyo. Okui. Biol. respectively. C.Aobayama.N.5H10d 'The free base was prepd as usual.1533 (1970). Biochem. Treatment of VII-XI with dil HCl led . this was ruled out by the following evidence.398 (1956).. and J. R = OH. Synthesis and Activity of Pyridoxal Derivatives (Studies on the Syntheses of Heterocyclic Compounds. 0. Soc.. 12. Sci. 15. mg (%) Mp. Sendai. Med. Burchenal.Notes Joumal of Medicinal Chemistry. dDried over PzOs at 120" (1 mm) for 24 hr. Beal. Japan R R ' \ <H T N C O C H 3 K. 4-pyridoxyl-4. J. XIV 241-243 MeOHb C&mNlO. Sei.Noell and C. Montgomery. J.:N~O. and their Ac derivatives. "C Recrystn solvent Formulau c.10 mp 252-253" dec. We wish to report the synthesis of 1. R' = H xv Table 1. Med. Pharm.3. 2 203 References (1) Y. R = OH. Received May 12.S . K. H. Skibba. eC.l (1969). and H. Hey. J.5-c]pyridine No. R' = OMe VI1 Biochemical Studies on Drugs and the Central Nervous System. Kametani. Loo. N anal. Rep. dec.* M. 1. ( 5 ) G.6. mg Amine. R = H. 18. Elks and D.H.3. R' = OMe It is well known that vitamin B6 has significant action on the CNS. A.2. in press.sH.. Hengy. PharmacoL. A.o. Chem.2043 (1970). Bryan. ODell.Od XI 500 198-200 Pale yellow needles Cl. (6) R.. D.3. Biochem.. A.NzO. "C Appearance VI I VI11 IX Formulae 252-254b Colorless plates C13H1SN.. R' = OAc XIV. A. Pharmacol. C. Laster. Proc.. Y. 92 420 836 1050 410 I1 IIIa-HCl IV V-HC1 VIa-HCI 150 470 845 1020 160 (82) 400 (53) 810 (51) 630 (33) 120 (16) N anal.. M.O. J. CHO H I R 11.J.H . R = H: R' = OH IX. and that it is of importance in the metabolism of brain cells.f 270-273 Colorless needles C16H18N20S X 243-245 Pale brown needles C. Beyer. Org. Ono Research Laboratories. L. Thurman.. R' = O H X. mg Yield. Krauth. bColorless prisms. K.. (8) J.Shealy. Shealy. B. A. Their activity on the CNS has also been examined.4-tetrahydro-l-pyridoxylisoquinoline derivatives (VIII-XI). 438l) T. Nishii. R = OH. Dagg. Cancer Chemother. (11) Y.7-tetrahydro-3H-imidazo[4. R' = OH IV. 19. ibid. (4) J.5. Phamacol. Jr. von Hodenberg. Ramirez. 441 (1943).F. H.Ob XI11 264-206 MeOH-EtiOC C. R' = OH V. Products from Pyridoxal and 3-Hydroxyphenethylamines and with Histamine Starting materials Product Compd Pyridoxal. Chugai Pharmaceutical Co. L. and M. mine. MP. W. 4tetrahydroisoquinoline derivatives by cyclization of the corresponding carbonyl compounds with 3-hydroxyphenethylamine derivatives 111-VI without acid4-' as a catalyst. W. Pharm. J.. Japan. 1971 VIII.F. Talley..554 (1971). Table 11.HIJVIO.2 .. Ltd. The Properties of Acetyl Derivatives of 1. Montgomery. J. Reussmann. Exp.Luce.7-tetrahydro-3H-imidazo [4. (9) J. R' = H VI. (7) Y. 'C.119 (1970).. F. C. 1972. R' = OH XI. XI1 181-183 MeOH-El. Ther. 91. No. 3-Hydroxyphenethylamine derivatives (IIIVI). F. were cyclized with pyridoxal to give the cyclized compounds listed in Table I. 27.674 (1962). 59. Tohoku University.E.. and C..ll mp 242-244" dec. (10) C. and W. R = H. T. D. and R. Chem. CColorless needles. Shealy and C.01f 243-24F Colorless needles C. (2) Y.H.2150 (1962).Housholder and T. . R = H. Chem.5-c]pyridine (VII)...

W. Nishii.1021/jm00272a021 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.1021/jm00272a021 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. Med.org on April 25. 1972. 2009 More About This Article The permalink http://dx. Chem.org/10. and M. Ono J. 1. 203-204• DOI: 10. K.doi. Okui. DC 20036 ..Synthesis of heterocyclic compounds. Koizumi. Kametani. Biochemical studies on drugs and the central nervous system. Synthesis and activity of pyridoxal derivatives T. 438. Washington.. Y. M. 15 (2).acs.

398 (1956).2. (9) J.. A. VI I VI11 IX 92 420 836 1050 410 I1 IIIa-HCl IV V-HC1 VIa-HCI Table 11. R = H: R' = OH IX. Burchenal. CHO H I R 11. CColorless needles.3.H.2. XI1 181-183 MeOH-El.6. Received May 12.J. ibid.545 (1969).5H10d 'The free base was prepd as usual..* M. 4-pyridoxyl-4. The Properties of Acetyl Derivatives of 1.10 mp 252-253" dec. prepared by the usual method. Cheng.H.. Ramirez. (8) J. this was ruled out by the following evidence.3 We had found a method of synthesizing the various 1. . and their Ac derivatives. J. 27. Sei..ll mp 242-244" dec. Biol.. Chem.6. 1971 VIII. " R' f l \ N H 1 111. R = H. XIV 241-243 MeOHb C&mNlO. Thurman. dec.. ( 3 ) J. R = OAc .. von Hodenberg. 91.3. Shealy.. Chugai Pharmaceutical Co. Kametani. mg Yield. Proc. ODell. 2 203 References (1) Y. 1. Treatment of VII-XI with dil HCl led . A. M. Soc. R. bColorless prisms.7-tetrahydro-3H-imidazo [4.4-Tetrahydro-6-hydroxy4-pyridoxylisoquinolines and 4-Pyridoxyl4. K. 0. Nishii. C. R = OH. Chem. Elks and D. 54.H. R = H. and M. H.. Phamacol. and G . (11) Y. J. R' = OMe VI1 Biochemical Studies on Drugs and the Central Nervous System.F.Notes Joumal of Medicinal Chemistry. G.l (1969). Although the structure of the above products could be thought to be that of Schiff bases. F. Montgomery. R' = H xv Table 1. J. R' = OH V. Loo. 441 (1943). H. Pharm. Tokyo. 15. 60. "C Recrystn solvent Formulau c. H. were cyclized with pyridoxal to give the cyclized compounds listed in Table I.. R' = O H X.E.119 (1970). 19. 'C..f 845 810 (51) 270-273 Colorless needles C16H18N20S X 1020 630 (33) 243-245 Pale brown needles C.S ..2150 (1962). Bryan. N anal. Jr.Luce.F. Synthesis and Activity of Pyridoxal Derivatives (Studies on the Syntheses of Heterocyclic Compounds. Chemistry. Ono Research Laboratories. "C Product Appearance Formulae 150 160 (82) 252-254b Colorless plates C13H1SN. Med. Biochem.H . mg Amine. mine.2. R' = H VI.. and J.Shealy. Exp. Stock. respectively. H. Krauth. Exp. Org. Vol. Cancer Chemother..2 . mg (%) Mp. (4) J. R = OH.01f 470 400 (53) 243-24F Colorless needles C. Beyer. A. Pharm.o. R' = OAc XIV. Shealy and C. A. and H. R' = OMe It is well known that vitamin B6 has significant action on the CNS. J. Shedy. 18. Laster. 12. Products from Pyridoxal and 3-Hydroxyphenethylamines and with Histamine Compd Starting materials Pyridoxal. No.4-tetrahydro-l-pyridoxylisoquinoline derivatives (VIII-XI). 1972. C. Rep.N.HIJVIO. ( 5 ) G. and that it is of importance in the metabolism of brain cells.5-c]pyridine (VII). fNot analyzed. D. Skibba. Sendai. and R. Koizumi. MP. (2) Y. R' = OH XI.O. in press. Chem. F. C. R = OH. L. and C. 438l) T.N. 59.Aobayama. 3-Hydroxyphenethylamine derivatives (IIIVI).. (10) C.554 (1971).Housholder and T.2043 (1970). Montgomery. J. Ther. 4tetrahydroisoquinoline derivatives by cyclization of the corresponding carbonyl compounds with 3-hydroxyphenethylamine derivatives 111-VI without acid4-' as a catalyst. Sci. Japan. Their activity on the CNS has also been examined. L.7-tetrahydro-3H-imidazo[4. W. A number of studies on the pharmacological action of vitamin B6 and its derivatives have been reported. Okui. L.NzO.674 (1962). Pharmacol. Med. A. D. N anal. Dagg. Hengy..O. Beal. (6) R. CLit. R' = OH IV. PharmacoL. C. B. by the applkation of this method using pyridoxal with several 3-hydroxyphenethylaminesand hista- R XIII.Ob XI11 264-206 MeOH-EtiOC C. Issacs. Japan R R ' \ <H T N C O C H 3 K. bLit. Hey.. Sot.Od XI 500 120 (16) 198-200 Pale yellow needles Cl. Talley. K. Pharmaceutical Institute. R = H. We wish to report the synthesis of 1. Reussmann.1533 (1970). dDried over PzOs at 120" (1 mm) for 24 hr. Ltd.5-c]pyridine No. R = OH.. T. W. (7) Y..:N~O.3. J.5.sH.Noell and C. W. Y. M. and W. eC. Tohoku University. R = H. Biochem.

Chem. 5445 (1970). and M. To male mice (ddy strain. and pnitrodiphenylamine was prepared. Antimalarial Compounds. T. and X. Kametani. 88. Since the compd was insol in all the solvents. Aoyama. Poland. Pharmacological Activities of Pyridoxal Derivativesa ~ Notes Compds Aminophylline VI1 VI11 1X Behavioral observation Pain response Flexor reflex Barbiturate potentiating action Relative activity Judgement Traction test 30 min TTb FTC 0 - 90 min TTb FTc - 45 min 1. Vol.414 (1952 ) . A mixt of 100 mg (0.1235 (1971). M.7-tetrahydro-3H-imidazo[ 43cjpyridine (VII). and K. Amer. Warsaw 10. Folkers. I971 Acknowledgments. E. Piotrowska. X and XI have the same analgetic effects as aminophylline.5 mmole) of 4-pyridoxyl-4.59)..0 0.60 (1961). 134. Soc.1 1 .. Chugai Pharmaceutical Co. 1253 (19. Kametani.’ and of tetrahydroisoquinoline derivatives of 280 nm..Kuryinyk. which was dissolved in 5 ml of 5% NaOH. which showed the absorption band attributable to NHC=O at around 1650 cm-’ in the ir (KBr).9 X No 0. S. Pharmacological Tests (Table 111). 23-28 g)] .. bTT = tranquilizing tendency. Soc. The compounds so obtained were tested for analgetic effect. Hayasaka. S.13 0 0 1.4-7 The compounds. 4 1 . the lack of absorption due to C=N was observed in the ir spectrum. Van Marren.9 a[Each number shows the mean value for 5 mice (ddy strain. In the present work we tried to investigate to what extent the nitro and sulfo group were responsible for the antimalarial activity of pnitrodiphenyl sulfone derivatives. The authors are indebted to the Psychopharmacology Division of Research Laboratories. and. and K. 70.O gave 120 mg (61%) of colorless prisms. Chem. A. Hiragi.9mmole) of pyridoxal. were found to have slightly more analgetic activity than aminophylline.Heyl. and N. recrystn of which from MeOH-Et. Harris.8 Compounds VI1 and VIII were identical with the authentic sample prepared by Heyl and his coworkers.36 0 0 0. The starting amines I-VI were prepared according to the l i t e r a t ~ r e ~ and ’ ~ subsequently caused to react ?The financial support of this work from the World Health Organization is gratefully acknowledged. the sepd crystals were collected by filtration. M. p 373.9 0. VIII. H. J. Fukumoto. D.. Exp.Dereymaeker. which are derivatives of histamine. Strahlentherapie. and analgetic action were tested by the methods of Kuhn and coworkers.. A mixt of 150 mg (0. Experimental Section? Cyclization of Pyridoxal ( 1 ) with Amines (Table I).9 mmole) of histamine. (2) P..46 0 0. 2 ml of Ac.23 0 .?. used as a control. J. Koch. Technical University (Politechnika). and K. Ther. J. 19 72.0 2 XIV No 1. 26. Folkers. Elsevier Publishing Co. Liverpool School of Tropical Medicine. and T. proton of XV was resonant at r 1 there appeared no proton at a lower field than r 2. tractive action.3669 (1948). Continuing our research on potential antimalarial agents. Amsterdam.Luz. Ltd. After administration. Pharm.1285 (1968). Ishimaru. Kigasawa. Harris. 2 Table 111. tMelting points were determined on a Yanagimoto microappara. T. and hypnotic action using mice as described later in the Experimental Section. Barbiturate potentiating action. Tetrahedron. W. 16. Pharmacol..204 Journal of Medicinal Chemistry. Pharmacology.” Co~rvosier’~ and Burn. followed by neutralization with 5% HCI.333 (1967). England H. Yukugaku Zasshi. Chem. VII. D. traction. K.A. 1123 (1968). Angelino. Guanidine Derivatives of Diphenyl Sulfone and Related Compounds W. K. B. Burn. The results are listed in Table 111. T.91 0 1 . H. d . I S . Exp. H. W. Moreover.”>’’ These facts are consistent with the cyclic structures presented here. Chem. London.89 (1960). H. J. 14.8 0.” Garanttini and Ghetti. A. Ed. general behavior was observed for 90 min and classified into 43 types. - - to recovery of the starting material (VII-XI).82 0. 23-28 g). Ahrens.63 1. Peters. Mason.O. 6 j : XI No 1.. the uv spectrum showed the maximum characteristic of pyridoxal derivatives at 320 nm. Yagi.90 - 0 0 0 0 ++ +++ ++ ++ +++ - 1.6. purification was done by repptn. Pharmacol. Soc.o 0. Liverpool L3 SQA. Evap of the solvent gave a pale yellow powder. 1 1.07 - 1 . Takano. p-nit rodiphenyl ether. S. S.7 tendency. 6 XI1 No 1.953 (1968). Satoh. “Psychotropic Drugs.8 XI11 No 1.Kametani. Chem. “Biological Standardization. Bull.Med.Heyl. pnitrodiphenylmethane. References (1) T. Sugahara.7 Analgetic action 90” Judgement 1.” Oxford University Press. and S. J. 17.07 1 .No. Med. Kigasawa. 12. F. for the pharmacological data presented. After filtration. Shibuva.25 1. 16.Kametani. Yagi. although the H. 92 mg (0. F.1 in our product. and T. E.’‘ respectively. K. ibid. were acetylated in the usual way to afford the Ac derivatives. (5) T.. 74. Courvosier..L.2 a series of biguanide and amidineurea derivatives of phalodiphenyl sulfones. Compounds VI1 and XII. Received August 31. 112 (1968). tus (MP-S2) and uncorrected.Kametani. mp 181-183”. H. Hiiragi.5 0.2 Chemistry. S. 113. 1950. Fukumoto. J. . and H. 0 1. G. Acetylation of Pyridoxal Derivatives (Table 11). C. and the excess reagents were distd off to leave the Ac derivative XU. CFT = fallen No No - 0. ibid. (3) R.82 0 0. Luz. Urba6ski Institute of Organic Chemistry and Technology. . Serafin. 100 mg/kg of each compd suspended in 1% arabic gum was administered per os. (4) T. and 1 ml of pyridine was heated on a water bath for 3 hr.5. Hibino.Kuhn and E. IS. and 5 ml of EtOH was heated on a water bath for 7 hr. * .

and T.. Guanidine derivatives of diphenyl sulfone and related compounds W. Urbanski J. B. 2009 More About This Article The permalink http://dx. 12. 1155 Sixteenth Street N. Serafin..doi.W. 1972. 204-206• DOI: 10. DC 20036 .org/10. Piotrowska.acs. Med. H. Washington. Peters. Chem. 15 (2).1021/jm00272a022 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society.org on April 25.1021/jm00272a022 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.Antimalarial compounds.

?.5 mmole) of 4-pyridoxyl-4..A. Hayasaka. Piotrowska. and T. References (1) T. W.23 0 .4-7 The compounds. 112 (1968).L. Courvosier. J. Angelino. H. (3) R.9 1 .Heyl. . F. purification was done by repptn.9 0.. Continuing our research on potential antimalarial agents. and the excess reagents were distd off to leave the Ac derivative XU. England H.90 - 0 0 0 0 ++ +++ ++ ++ +++ - 0 X No 0. M. traction. and. Acetylation of Pyridoxal Derivatives (Table 11). - to recovery of the starting material (VII-XI). and X. Exp. Vol. J. S. Mason.. p 373.. the lack of absorption due to C=N was observed in the ir spectrum.8 1. and 5 ml of EtOH was heated on a water bath for 7 hr. K. Yagi.414 (1952 ) . A. 74.2 a series of biguanide and amidineurea derivatives of phalodiphenyl sulfones. Exp.’ and of tetrahydroisoquinoline derivatives of 280 nm. Sugahara.Kuryinyk. Experimental Section? Cyclization of Pyridoxal ( 1 ) with Amines (Table I). After filtration. Soc.5 0..13 0 0 2 XIV No 1. Chugai Pharmaceutical Co. Chem.. Pharmacological Tests (Table 111). London. tractive action. Burn. Kigasawa.O. Ahrens. Harris.Luz.1 in our product. 6 1. Yukugaku Zasshi. 6 1 .46 0 0.91 0 j : XI No 1.9mmole) of pyridoxal. Tetrahedron. IS. and M. 26. 92 mg (0. S. were found to have slightly more analgetic activity than aminophylline. 17. A mixt of 100 mg (0. 1253 (19. Hibino. although the H. Barbiturate potentiating action. were acetylated in the usual way to afford the Ac derivatives. d . Pharmacological Activities of Pyridoxal Derivativesa ~ Notes Compds Aminophylline VI1 VI11 1X Behavioral observation Pain response Flexor reflex Barbiturate potentiating action Relative activity Judgement Traction test 30 min TTb FTC 0 - 90 min TTb FTc - 45 min 1. Serafin. proton of XV was resonant at r 1 there appeared no proton at a lower field than r 2. 4 1 . E.. and N. Yagi.7 Analgetic action 90” Judgement 1. tMelting points were determined on a Yanagimoto microappara.2 Chemistry. and T. A mixt of 150 mg (0. Ltd. Guanidine Derivatives of Diphenyl Sulfone and Related Compounds W. The starting amines I-VI were prepared according to the l i t e r a t ~ r e ~ and ’ ~ subsequently caused to react ?The financial support of this work from the World Health Organization is gratefully acknowledged. Antimalarial Compounds.8 Compounds VI1 and VIII were identical with the authentic sample prepared by Heyl and his coworkers. Ed.1 * . (5) T. Pharmacol. J.07 XI1 No 1.953 (1968). ibid.Kuhn and E. Fukumoto. 23-28 g). Aoyama. CFT = fallen No No - 0. 16. The results are listed in Table 111. Poland.7-tetrahydro-3H-imidazo[ 43cjpyridine (VII).Kametani. H. Strahlentherapie. 134. The authors are indebted to the Psychopharmacology Division of Research Laboratories. Evap of the solvent gave a pale yellow powder.59). Pharmacol. recrystn of which from MeOH-Et..O gave 120 mg (61%) of colorless prisms.Med. 70. Shibuva. which was dissolved in 5 ml of 5% NaOH. Compounds VI1 and XII. 23-28 g)] . X and XI have the same analgetic effects as aminophylline. J. and hypnotic action using mice as described later in the Experimental Section. K. S. Chem.82 0. Chem. Bull. Takano. C.o 0. ibid. T. Urba6ski Institute of Organic Chemistry and Technology.63 1. Koch. 88. (2) P. which showed the absorption band attributable to NHC=O at around 1650 cm-’ in the ir (KBr). Liverpool School of Tropical Medicine. 113.0 0. for the pharmacological data presented. S. Pharmacology. Amer. H.204 Journal of Medicinal Chemistry. followed by neutralization with 5% HCI. Folkers. Fukumoto. Luz. Chem. Peters. and S.5. 1 1. Elsevier Publishing Co.. Kigasawa. D.1235 (1971). T. used as a control. Warsaw 10. 0 XI11 No 1. E. After administration. 12..333 (1967).8 0. . B.Dereymaeker. Chem.60 (1961). Hiragi. M. and K. A. 2 Table 111.” Co~rvosier’~ and Burn.Kametani. Soc. Since the compd was insol in all the solvents.3669 (1948). T. - 1 . I S . Liverpool L3 SQA. I971 Acknowledgments.. Folkers. and analgetic action were tested by the methods of Kuhn and coworkers. In the present work we tried to investigate to what extent the nitro and sulfo group were responsible for the antimalarial activity of pnitrodiphenyl sulfone derivatives.No. the uv spectrum showed the maximum characteristic of pyridoxal derivatives at 320 nm.9 mmole) of histamine. Kametani. and K. Amsterdam.89 (1960). Pharm. VIII.36 0 0 a[Each number shows the mean value for 5 mice (ddy strain. The compounds so obtained were tested for analgetic effect. Hiiragi. Harris. F. the sepd crystals were collected by filtration.07 - 1. Received August 31. and 1 ml of pyridine was heated on a water bath for 3 hr. tus (MP-S2) and uncorrected. (4) T. Satoh. 1950. Soc. bTT = tranquilizing tendency. Technical University (Politechnika). p-nit rodiphenyl ether. “Psychotropic Drugs. 5445 (1970).” Oxford University Press. H. G.7 tendency. J. and H.9 1 . S. 1123 (1968). 100 mg/kg of each compd suspended in 1% arabic gum was administered per os. and K.Kametani.25 1. Med. 14.6. Kametani. 16.1285 (1968).’‘ respectively. Van Marren.”>’’ These facts are consistent with the cyclic structures presented here. Moreover. 2 ml of Ac. K. 19 72.” Garanttini and Ghetti. VII. Ishimaru. general behavior was observed for 90 min and classified into 43 types. which are derivatives of histamine. To male mice (ddy strain. “Biological Standardization. H.Heyl. pnitrodiphenylmethane. mp 181-183”. D.82 0. and pnitrodiphenylamine was prepared. W. Ther. J.0 0.

Compounds VII-IX and XII-XIV showed some antimalarial action. that produced no deaths. T.0 5. Toxicity.8 Inactive Inactive 17 17 75 100 100 85.0 7. The crude product upon washing with acetone crystd from 50% EtOH: mp 201-202". Amidineureas XIII-XVII were obtained from biguanides VII-XI. Antimalarial Activity. g 100 100 100 < 100 > 300 450 100 660 100 830 100 > 300 > 300 > 100 200 200 30 100 93.5 5 . Wt. ml Reaction time. 1 g (48%).2 0.5 7.3 g of VI.0 g of cyanoguanidine in 10 ml of pyridine for 4 hr. Grzelecka in preparation of the samples $Tests were carried out by the method of Peters. K.O 6. Warsaw (acute and subacute oral tests) and the Liverpool School of Tropical Medicine (subacute sc tests). $Tests were carried out partly at the Institute of Drugs.8 58.2 m1/20 g of body wt (Table I).0 25 .o 1 .8 3. min Yield Amidineurea MP. The LD. XI and XV-XVII were significantly less toxic sc while X was the most toxic. The biguanide XI was prepared by refluxing 2.1 71. No.9 73. Animals received 4 consecutive daily doses of drug in 0.Notes Table I. on heating in dil HCl (Table 111). cyanoguanidine was added.5 300 41. 1912. probably due to better oral absorption than VII-IX.O 50. XI or XV-XVII. The technical assistance of Mrs.. .7 89."C g % I1 111 IV v 7.1 1. For details see Experimental Section. No activity was detected at the maximum doses tested in X.5 30.0 2. ml EtOH. VIII.0 6.. Experimental Section All analytical data of the new compounds were in agreement with the calcd ones for the expected structures which were also confirmed by ir spectra. was calcd graphically by Litchfield and Wilcoxon's method as modified by Roth. 2 205 sc LDo/da PO Antimalarial activity (parasitaemia relative to controls) mg/kg per day sc 1 3 10 VI1 1166 VI11 2500 I X X XI X I 1 X I 1 1 XIV XV XVI XVII Inactive 'The highest dose.0 60 60 60 120 VI1 VI11 I x X 178-179 175-176 180-181 174-175 3.' at the Liverpool School of Tropical Medicine. that produced no deaths in groups of 10 or 15 mice.2 ml of soln or suspension sc from day 0 through day +3. Antimalarial Activity.0 20. NO. 5 10 10 20 20 20 15 15 120 90 15 0 xv X I 1 1 XIV XVI XVII 190-191 187-188 215-217 206-208 >260 1. XI1 being the most active.O 5 . Table 11. J. min Yield BG Mp.0 7. XI1 X I 1 1 XIV I X X xv 0 XI XVI XVII 2 Biguanides VII-X were prepared as follows.7 % 52 60 20 15 37 Recrystn solvent 60% EtOH 60% EtOH 80% EtOH 80% EtOH DMF with cyanoguanidine in alcoholic HC1. PO Journal of Medicinal Chemistry.0 51. 1 .o 1 . Haworth of the World Health Organization for their kind interest and aid.0 1 . 15. mg/kg (mice) No. g 35% alc HC1. Amidineureas XII-XVII were prepared by hydrolysis of the corresponding biguanides. g 10% HC1. Cyanoguanidine. Biguanide No. NH NH Amine I I1 I11 IV V VI II II AU = -NHCONHCNH. 1.O 10.8 100 55.yield. SO2 SO2 Y F c1 Br NO NO. LD.5 6. Wt. Subacute toxicity was assessed by de$ the highest dose administered on 4 consecutive days orally (as above) or sc (in a 4% soln of Tween 80).4 55.1 0 9. Amine No.0 5 . Lepes and Dr.2 0.6 18.7 83. and the mixt was refluxed. dried.7-0.$ The antimalarial activity of VIIXVII was tested against Plasmodium berghei in mice sc (Table I).$ All tests were carried out in white mice (CFl line) infected ip on day 0 with donor blood contg approximately lo' parasitised red blood cells. The resulting hydrochloride was made alk with 5% NaOH. "C g VI1 V I 1 1 I X X XI 2. The soln was poured into H. The percentage of parasitised red blood cells was counted in treated groups of animals on day +4 and compared with the percentage in saline-treated controls. Vol.* Acute toxicity of VII.5 h a c tive Inactive VII-IX and XII-XIV showed a similar level of subacute toxicity sc but the latter were more toxic orally. ___ Acknowledgments. ml Reaction time. and boiled with PhMe to remove the unreacted starting amine (Table 11).8m1/20 g of body wt.7 77. p-Nitro-p'-biguanidodiphenylamine (XI) was prepared from VI and cyanoguanidine in the presence of pyridine hydrochloride.5 7. and 1. and XII-XIV was tested by oral administration and subacute toxicity of VIIXVII (4 daily doses) by oral and sc administration (Table I).O 7. administered on 4 consecutive days. The amine was dissolved in alc HCl.$ Acute toxicity on oral administration was tested in white mice (Porton breed) in groups of 20. In no case was complete activity obtained at the L D 0 p or below. respectively.2 0. The animals were observed for 7 days. NH AU II BG V I 1 VI11 X SO. Toxicity.2g of pyridine hydrochloride. Scheme 1 BG = -NHCNHCNH. Toxicity. We wish to express our gratitude to Dr.The compds were administered by gavage in a 5% suspension of aq gum arabic in a vol of 0.9 52.3 83.1 40 35 65 65 Recrystn solvent 40% EtOH 40% EtOH 50% EtOH MeOH + EtOH Table 111.O and made alk with 5% NaOH. Doses (sc) were contained in a vol of 0.

206

Journal ofMedicina1 Chemistry, 1972, Vol. 15, No. 2 Table I. Enzyme Activity of Analogs Compound R, R, R,

hrotes

for biological screening and of Mrs. J. Portus and Mr. B. Robinson for antimalarial screening is also gratefully acknowledged. References
(1) B. Serafin and M. H. Aldridge, J. Med. Chem., submitted for publication (part 11). (2) B. Serafin, T. Urbahski, D. C. Warhurst, ibid., 12,33 (1969). (3) C.Richter and W. Frey, Swiss Patent No. 278939 (1952). (4) L. C. Raiford and J. C. Colbert, J. Amer. Chem. Soc., 48,2660 (1926). ( 5 ) L. H. Litvinenko and K. Levchenko, Zh. Obshch. Khim., 29, 1970,3079 (1959). (6) F.Ullman and K. Dahmen,Ber., 41, 3753 (1908). (7) W. Peters, Exp. Parasitol., 17, 80 (1965).

PDH activity, %

1 Pyridoxine Me 2 w-Methylpyridoxine' Et 3 &Rando$ i-PI 4 C1 anal08 Me 5 n - R analog VI11 n-Pr 'Data for these PDH activity estimates Marshall.*

OH H 100 OH €I 95 OH 1 1 25 OH e1 22 OH H 5 were obtd from Melius and

Synthesis and Enzymological Activity of 3-Hydroxy-2-n-propy1-4,S-pyridinedimethanol
Paul Melius* and Joseph L. Greene, Jr.
Department of Chemistry, Auburn University, Auburn, Alabama. Received July 15, 1971

The conversion of 3-hydroxy-2-ethy1-4,5-pyridinedimethan01 and 3-hydroxy-2-isopropyl-4,5-pyridinedimethanol to their corresponding aldehydes by yeast pyridoxine dehydrogenase was described by Melius and Marshall.' Also 3-hydroxy-2-methyl-6-chloro-4,5-pyridinedime than01 was found to be oxidized by the enzyme with an activity of the order of that for the i-Pr analog. The rate of reaction for the Me and Et analogs was about 4 times that for the i-Pr analog and the hydroxychloro compound. In the present report the synthesis and yeast pyridoxine dehydrogenase action on 3-hydroxy-2-n-propyl-4,5-pyridinedimethanol (VIII) is described. The synthesis of VI11 involved an initial condensation of ethyl-n-butyropyruvate and cyanoacetamide to form 4carbethoxy-3-cyano-6-n-propyl-2-pyridone (I).2 I was then carried through a sequence of reactions involving n i t r a t i ~ n , ~ chlorination, reduction with reduction with Pd-H2,' hydrolysis with HCl,3 diazotization, and reduction with NaBh,6 to give finally VIII. Thus a modification of the reduction of the NOz group was utilized here, in which SnC12 was used in place of Fe which had been utilized in the preparation of the i-Pr analog. The pyridoxine dehydrogenase enzyme used here was a Acknowledgment. We wish to thank Miss Barbara K. preparation described by Morino and Sakamoto.2 The Newton for her technical assistance. assay of enzymatic activity toward VI11 is given in Table I and compared with the activities of the compds prepared and studied by Melius and Marshall.' References Experimental Section?
4Carbethoxy-3-cyano-6-n-propyl-2-pyridone (I) was prepd refluxing a soln of the Na salt of ethyl n-butyr~pyruvate~ (208g; 1.0 mole) and cyanoacetamide (92g; 1.1 moles) in abs EtOH (1400 ml) for 3 hr. After standing at room temp overnight, the reaction mixt was chilled and treated with an ice cold soln made up by dilg concd HCl(200 ml) to 1200 ml with ice and H,O. The crude
?Melting points are corrected and were determined in a Mel-Temp apparatus (Laboratory Devices, Cambridge, Mass.) Microanalyses were by Galbraith Laboratories, Knoxville, Tenn.

product thus pptd was washed thoroughly with H,O before being crystd from aq EtOH (3000ml; (60:40)EtOH-H,O) to give 152 g (65%) of I, mp 146-148'. 4Carbethoxy-3-cyano-6-n-propylJ-nitro-2-pyridone (11). Compd I (23.5g; 0.1mole) was nitrated with HN0,-Ac,O, essentially as described by W ~ e s t to , ~give, after recrystn from 50% aq EtOH, 18.5 g (66.3%) of 11, mp 163-164". 4Carbethoxy-2-chloro-S-nitro-6-n-propylnicotinonitrile (111). Compd I1 (27.9g; 0.1 mole) and PCl, (22.9g; 0.11 mole) were mixed and heated together at 125 +5" for 2 hr. POCl, was removed in vacuo before the residue was triturated with crushed ice until solidification was complete. Recrystn of the crude product from 8 5 0 ' . abs EtOH gave 21.2 g (71%) of 111, mp 4 5-Amino-2-chloro-4-carbethoxy-6-n-pmpylnicotinonitr~e (IV). Compd 111 (29.8g; 0.1 mole), suspended in Et,O was treated with a freshly filtered s o h of SnCl, (78g) in concd HCl(l65 ml) in a manner analagous to that described by Greene and Montgomery.4 The crude product was recrystd from abs EtOH (625 ml) to give 23 g (86%) of IV, mp 168-169". 5-Ammo-4-carbethoxy-6-n-propylnicotinonitrile (V). Hydrogenation of IV (26.8g; 0.1 mole) over 5% Pd-BaCOS3 and work-up of the reaction mixt gave 10.5 g (45%) of V after recrystn from abs EtOH, mp 132-133'. 3-Amin0-2-n-propylpyridine-4,S-dicarboxylic Acid (VI). Hydrolysis of V (15.6g; 0.067 mole) with concd HCl' gave 9.0 g (60%) of the dicarboxylic acid VI, mp 215-216' dec. 3-Hydroxy-2-n-propylpyridine-4,5dicarboxytic Acid (VII). Compd VI (8.3 g; 0.037mole) was diazotized at 80" in aq s o h to give 4.1 g (49.4%) of VIII, mp 230-232". 3-Hydroxy-2-n-propyl-4,5-pyridinedimethanol Hydrochloride (VIII). Reduction of the 2 CO,H groups of VI1 (2.25g; 0.01 mole) with NaBH,-AlCl, as described by Blackwood6 gave the n-Pr analog of pyridoxine hydrochloride (0.94g; 41%); mp 2102 1 2 ' dec.Anal. (C,,H,,NO;HCl) C, H, N. Enzymatic assays were carriedout as described by Melius and Marshall. l The yeast pyridoxine dehydrogenase (PDH) activity was measured by the reaction of phenylhydrazine with the aldehyde produced from the pyridoxine and its analogs.'

(1) P. Melius and D. L. Marshall, J. Med. Chem., 10,1157 (1967). (2) Y. Morino and Y.Sakamoto, J. Biochem. (Tokyo), 48,733 (1960). (3) H.M. Wuest, J. A. Bigot, Th. J. DeBoer, B. van der Wal, and J. P. Wibaut, Reel. Trav. Chim. Pays-Bas, 78,226 (1958). (4) J. L. Greene, Jr., and J. A. Montgomery, J. Med. Chem., 6, 294 (1963). (5) C.S. Marvel and E. E. Dreger in "Organic Synthesis," Collect. Vol. I, H. Gilman, Ed., Wiley, New York, N. Y., 1946,p 238. (6) R. K.Blackwood, G . B. Hess, C. E. Larrabee, and F. J. Pilgram, J. Amer. Chem. Soc., 80,6244(1958). (7) H. Wada and E. E. Snell, J. Biol. Chem., 236, 2089 (1961).

Synthesis and enzymological activity of 3-hydroxy-2-propyl-4,5-pyridinedimethanol
Paul Melius, and Joseph L. Greene Jr.
J. Med. Chem., 1972, 15 (2), 206-206• DOI: 10.1021/jm00272a023 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.acs.org on April 25, 2009

More About This Article
The permalink http://dx.doi.org/10.1021/jm00272a023 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036

206

Journal ofMedicina1 Chemistry, 1972, Vol. 15, No. 2

hrotes

for biological screening and of Mrs. J. Portus and Mr. B. Robinson for antimalarial screening is also gratefully acknowledged. References
(1) B. Serafin and M. H. Aldridge, J. Med. Chem., submitted for publication (part 11). (2) B. Serafin, T. Urbahski, D. C. Warhurst, ibid., 12,33 (1969). (3) C.Richter and W. Frey, Swiss Patent No. 278939 (1952). (4) L. C. Raiford and J. C. Colbert, J. Amer. Chem. Soc., 48,2660 (1926). ( 5 ) L. H. Litvinenko and K. Levchenko, Zh. Obshch. Khim., 29, 1970,3079 (1959). (6) F.Ullman and K. Dahmen,Ber., 41, 3753 (1908). (7) W. Peters, Exp. Parasitol., 17, 80 (1965).

Table I. Enzyme Activity of Analogs Compound R, R, R, PDH activity, %

1 Pyridoxine Me 2 w-Methylpyridoxine' Et 3 &Rando$ i-PI 4 C1 anal08 Me 5 n - R analog VI11 n-Pr 'Data for these PDH activity estimates Marshall.*

OH H 100 OH €I 95 OH 1 1 25 OH e1 22 OH H 5 were obtd from Melius and

Synthesis and Enzymological Activity of 3-Hydroxy-2-n-propy1-4,S-pyridinedimethanol
Paul Melius* and Joseph L. Greene, Jr.
Department of Chemistry, Auburn University, Auburn, Alabama. Received July 15, 1971

The conversion of 3-hydroxy-2-ethy1-4,5-pyridinedimethan01 and 3-hydroxy-2-isopropyl-4,5-pyridinedimethanol to their corresponding aldehydes by yeast pyridoxine dehydrogenase was described by Melius and Marshall.' Also 3-hydroxy-2-methyl-6-chloro-4,5-pyridinedime than01 was found to be oxidized by the enzyme with an activity of the order of that for the i-Pr analog. The rate of reaction for the Me and Et analogs was about 4 times that for the i-Pr analog and the hydroxychloro compound. In the present report the synthesis and yeast pyridoxine dehydrogenase ac(VIII) tion on 3-hydroxy-2-n-propyl-4,5-pyridinedimethanol is described. The synthesis of VI11 involved an initial condensation of ethyl-n-butyropyruvate and cyanoacetamide to form 4carbethoxy-3-cyano-6-n-propyl-2-pyridone (I).2 I was then carried through a sequence of reactions involving n i t r a t i ~ n , ~ chlorination, reduction with reduction with Pd-H2,' hydrolysis with HCl,3 diazotization, and reduction with NaBh,6 to give finally VIII. Thus a modification of the reduction of the NOz group was utilized here, in which SnC12 was used in place of Fe which had been utilized in the preparation of the i-Pr analog. The pyridoxine dehydrogenase enzyme used here was a Acknowledgment. We wish to thank Miss Barbara K. preparation described by Morino and Sakamoto.2 The Newton for her technical assistance. assay of enzymatic activity toward VI11 is given in Table I and compared with the activities of the compds prepared References and studied by Melius and Marshall.' Experimental Section?
4Carbethoxy-3-cyano-6-n-propyl-2-pyridone (I) was prepd refluxing a soln of the Na salt of ethyl n-butyr~pyruvate~ (208g; 1.0 mole) and cyanoacetamide (92g; 1.1 moles) in abs EtOH (1400 ml) for 3 hr. After standing at room temp overnight, the reaction mixt was chilled and treated with an ice cold soln made up by dilg concd HCl(200 ml) to 1200 ml with ice and H,O. The crude
?Melting points are corrected and were determined in a Mel-Temp apparatus (Laboratory Devices, Cambridge, Mass.) Microanalyses were by Galbraith Laboratories, Knoxville, Tenn.

product thus pptd was washed thoroughly with H,O before being crystd from aq EtOH (3000ml; (60:40)EtOH-H,O) to give 152 g (65%) of I, mp 146-148'. 4Carbethoxy-3-cyano-6-n-propylJ-nitro-2-pyridone (11). Compd I (23.5g; 0.1mole) was nitrated with HN0,-Ac,O, essentially as described by W ~ e s t to , ~give, after recrystn from 50% aq EtOH, 18.5 g (66.3%) of 11, mp 163-164". 4Carbethoxy-2-chloro-S-nitro-6-n-propylnicotinonitrile (111). Compd I1 (27.9g; 0.1 mole) and PCl, (22.9g; 0.11 mole) were mixed and heated together at 125 +5" for 2 hr. POCl, was removed in vacuo before the residue was triturated with crushed ice until solidification was complete. Recrystn of the crude product from 8 5 0 ' . abs EtOH gave 21.2 g (71%) of 111, mp 4 5-Amino-2-chloro-4-carbethoxy-6-n-pmpylnicotinonitr~e (IV). Compd 111 (29.8g; 0.1 mole), suspended in Et,O was treated with a freshly filtered s o h of SnCl, (78g) in concd HCl(l65 ml) in a manner analagous to that described by Greene and Montgomery.4 The crude product was recrystd from abs EtOH (625 ml) to give 23 g (86%) of IV, mp 168-169". 5-Ammo-4-carbethoxy-6-n-propylnicotinonitrile (V). Hydrogenation of IV (26.8g; 0.1 mole) over 5% Pd-BaCOS3 and work-up of the reaction mixt gave 10.5 g (45%) of V after recrystn from abs EtOH, mp 132-133'. 3-Amin0-2-n-propylpyridine-4,S-dicarboxylic Acid (VI). Hydrolysis of V (15.6g; 0.067 mole) with concd HCl' gave 9.0 g (60%) of the dicarboxylic acid VI, mp 215-216' dec. 3-Hydroxy-2-n-propylpyridine-4,5dicarboxytic Acid (VII). Compd VI (8.3 g; 0.037mole) was diazotized at 80" in aq s o h to give 4.1 g (49.4%) of VIII, mp 230-232". 3-Hydroxy-2-n-propyl-4,5-pyridinedimethanol Hydrochloride (VIII). Reduction of the 2 CO,H groups of VI1 (2.25g; 0.01 mole) with NaBH,-AlCl, as described by Blackwood6 gave the n-Pr analog of pyridoxine hydrochloride (0.94g; 41%); mp 2102 1 2 ' dec.Anal. (C,,H,,NO;HCl) C, H, N. Enzymatic assays were carriedout as described by Melius and Marshall. l The yeast pyridoxine dehydrogenase (PDH) activity was measured by the reaction of phenylhydrazine with the aldehyde produced from the pyridoxine and its analogs.'

(1) P. Melius and D. L. Marshall, J. Med. Chem., 10,1157 (1967). (2) Y. Morino and Y.Sakamoto, J. Biochem. (Tokyo), 48,733 (1960). (3) H.M. Wuest, J. A. Bigot, Th. J. DeBoer, B. van der Wal, and J. P. Wibaut, Reel. Trav. Chim. Pays-Bas, 78,226 (1958). (4) J. L. Greene, Jr., and J. A. Montgomery, J. Med. Chem., 6, 294 (1963). (5) C.S. Marvel and E. E. Dreger in "Organic Synthesis," Collect. Vol. I, H. Gilman, Ed., Wiley, New York, N. Y., 1946,p 238. (6) R. K.Blackwood, G . B. Hess, C. E. Larrabee, and F. J. Pilgram, J. Amer. Chem. Soc., 80,6244(1958). (7) H. Wada and E. E. Snell, J. Biol. Chem., 236, 2089 (1961).

6-bis(trifluoromethyl)-9-phenanthrenemethanol R. 15 (2). 1972. 2009 More About This Article The permalink http://dx. Washington. 207-208• DOI: 10.1021/jm00272a024 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.. E.Antimalarial activity and conformation of erythro.-(2piperidyl)-3.org/10.acs.doi.W. Chem. 1155 Sixteenth Street N. Med. DC 20036 .alpha.1021/jm00272a024 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society..and threo-. Olsen J.org on April 25.

dF.~ mixture of IIA and IIB. in which nonbonded inter[3. giving an estimate of the rotamer populations. by examination of the nmr DMSO are consistent with the importance assigned to intraspectra of 111 and IV.' thus producing a racemic mixture of erythro and threo amino alcohols.70 5. Received June 10.6-bis(trifluorome thyl)-9-phenanthryl]hexahydro-3Hoxazolo[3. JAB Values for Epimeric (~-(2-~per~dyl)-3.52 4. CAn average of 6 runs. Solvent DMSSd. one would expect an increase in intermolecular H bonding" [OH---O=C(CH.O.2 Hz. 1971 Table I.68e 'Spectra were obtd at indicated temp on a Varian Model DP-60 high-resolution spectrometer at a concn of 10%. B.6 'Spectra obtd on a Varian Model DP-60 hi h resolution spectrometer in CDCl. IC should be the preferred rotamer. as the final reaction step.bonding (OH-N). intramolecular The difference in chemical shift between the two C3 CH2 H bonding must significantly influence the conformational of 1-substituted-hexahydro-3H-oxazolo [3. values accurate t o an estimated k0. The erythro (I) of intramolecular H bonding and has a high degree of nonand threo (11) amino alcohols can lie in 3 possible staggered bonded interaction.HC1 II.6 and 10. et al. Upon conversion to the TsOH salt. concn of the mother liquors gave a residue containing roughly equal amounts of the diastereomers.95 0. The increase in JAB observed in This is Contribution No. COH resonance eliminated by addn of D.41 C6D6 50 2.O.8 and 10S9and 2. and C) with the vicinal coupling constant.43 0.96 h z (comand the geminal coupling constant for the same 2 protons pared with the CDCIBvalue).6-Bis(trifluoromethyl)-9-phenanthryl. Sargent3 reported the isolation of both piperidinemethanol epimers from the catalytic reduction of 2-piperidyl 6-methoxy-4The coupling constants for the HC1 salts of I and I1 (Table quinolyl ketone. lesser steric interactions. respectively.09 4.91 50 2. 15. in Me&O and can be employed to assign configuration to the amino alcohol precursors. 2-Pyridyl 3. The increase in JAB of 1. 2 207 Antimalarial Activity and Conformation of ery throand threo-cw-(2-Piperidyl)-3. Previous piperidinemethanol preparations were accomplished either by a different synthetic scheme? usually yielding only one isolated diasteromeric form.65c 7.6-bis(trifluoromethyl)-9-phenanthryl ketone was prepared by the procedure of Nodiff. e3. California 95813. I1 + IV). the pure gauche and trans coupling constant values have been suggested as 2. The amino alcohols were treated with methanolic CH20 and converted to the corresponding 1. The hydrochloride of the major epimer (I) was sparingly MeOH soluble and was obtained in pure form after 2 evaporative recrystallizations. lophurae in ducks and found to be of comparable a~tivity. data6 for the racemic epimers of 1-phenylhexahydro-3H-oxazolo. It is expected conducted since it is possible their pharmacological activities the population of IIC would be minimal.92 0. TFA Acetone -d CDCl. The data for 111 and OH---O=S(CH&] with a decrease in intramolecular H and IV are shown in Table I.4a] pyridine are included.4u]pyridines preferences. E. The nmr data indicates that I1 exists as --__a mixture of IIA and IIB. the increased importance of steric factors in these solvents. dConfign of phenyl 2-piperidylmethanols has also been established by Dudas and Weisz' and by Kovar. since it is incapable are related to conformational preferences.Notes Journal ofhfedicinal Chemistry. S.09 6. at room temp at 10% concn." TheJAB values for I and I1 and their HCl salts in solvents of differing polarity are shown in Table 11. for comparative purposes.GemC A convenient route to antimalarials containing a 2-piperidinemethanol group involves. the JAB values An nmr study of I and I1 and their protonated forms was did not show as marked a solvent dependence.H.).98 50 4.39 and 1. Sacramento.70e 6. Table 11.4a]pyridire derivative (I + 111. Vol. a catalytic hydrogenation of a 2-pyridyl ketone. JAB.6 Thus.78 4. In the threo amino alcohol.66c 60 2.64 4. we have assigned the configuration of molecular H bonding as in these solvents. There thus appears to be limited data available as to the effect of conformation on the antimalarial activity of piperidinemethanols..43 -1.3 -1. 1972. JH.7 --2. actions enhance intramolecular H bonding. eExchangeable protons replaced with D by shaking overnight with D. relative to TMS. Phenyls e (111) Phenyls e (IV) Cisd Transd 3. g I i 6 values.55 100 4. the minor epimer (11) could be isolated in pure form after 2 recrystallizations from MeOH.6-Bis(trifluoromethy1)-9-phenanthyl lar models the latter shows severe nonbonded interactions.6-bis(trifluoro~ethyl)-9-phenanthrenemethanol and Their HCl Saltsn JAR. The materials were tested as SN2157 and SN8279 11) indicate the preferred protonated rotamers are IA and a against P . bAn average of 6-10 runs.21 4. conformations (A.4-a]pyridinesU Ar Configuration Of HaHb Chemical shift c3 protonsb *H.HCI 7.5 -2. No. Army Medical Research and Development Command under Contract DA-49-193-MD-2891. Ha Hb These data suggest that in nonpolar solvents the erythro I 111 epimer resides almost entirely in the gauche forms IA and I1 IV IB. respectively. Nmr Data for l-Aryl-hexahydro-3H-oxazolo[ 3.CCO.31 2. with IIA favored as it has the ?This work was supported by the U. Hzb Temn O C I I1 I.3 Hz.6-bis( trifluoromethy1)-9phenanthrenemethanolt R.17 4. But in view of JABvalues. In similar compounds. with IA favored over IB since on inspection of molecuAI = 3. It is of interest to compare the JAB .69 6. et al.. $H. Olsen Aerojet Solid Propulsion Company.[3.92 0. 947 from the Army Research Program on MezCO and DMSO (compared to CDC13) is consistent with Malaria.$ or on a scale insufficient to permit isolation and characterization of the isomer formed in lesser amounts.58 6.' and hydrogenated at atm pressure to give about an 85: 15 mixture (analysis via nmr and tlc) of epimeric piperidinemethanols. Based on these steric interactions alone.H. I and I1 as erythro and threo.

E."J. ?This investigation was supported b y NIH Grant NS 08738. W. Czech. N. H. and M. (10) M. and P. A. F. The solids were dissolved in MeOH (Darco) M. Elemental anal.O. Matsuura.A.6-bis(trifluoromethyl)-9N-Demethylation of Morphine and Structurally phenanthryl ketone. (6) T..0g (63%) of I11 as white flakes. removed a yellow impurity to leave 11.E. Darco was added and. Recrystn (MeOH-H. 68.2710 (1946).. 68. and the product was recrystd (EtOH. Blaha. J. Levy. the reflux was contd overnight. and J. The mother liquors from the above recryst were concd to dryness. Chapman and R. Chem. Portoghese. P. Norton. Ber. H.. and dried. Rapport.NOF. T.2697 (1946). Kovar. treated with dil K. the minimum dosage showing activity was 20 and 160 mg/kg for the threo and erythro compds. 68. mp 284-285" (44.. M. Leo Rane at the University of Miami. Lutz. K. _IV. H. ibid. Anal. Benson. J. Osdene. Edwards.459 (1967). D. (8) J. 28. A. Koepfli. C1. Koepfli. 68. Recrystn (2x. M. Heterocycl. J a y . 14..Munk. (12) T. 0. ibid. R. (11) 0.No. E. Lutz. Thus. ibid. was passed through a mixt of 115 g (0.9 g (25 mmobs) of TsOH . Burger.. The test results. 68..$JOF.HCl.A. Nodiff. Tyagi. Amer.Ainley and H. 19411945. ibid. ibid. Buchman and D.. P. _I ?H IA IB IC IIA IIB IIC Acknowledgment. were correct (*0. R.R. Experimental Section# dl-erythro-ol-(2-Piperidyl)-3.2g. Darco) gave an analytical material. ibid. Patel. H. and cooled to ppt I1 . 4.. K. 1 0 .27 14 (1946). Heterocycl. (C. 5. J. T. Tenn.412 (1960). Buchman. (9) P. Refluxing with CCl. Add1 (C%O). (C. Chem. Minneapolis. G. M. TsOH. Sargent. J. E.6-Bis(trifluoromethyl)-9-phenan~l] hexahydro-3H-o~azolo[3.. Commun. pptg a mass of white crystals.Brown. 68. H.. giving 5 cures out of 5 infected mice at dosages of 40 mg/kg for the threo epimers and 5 cures at 80 mg/kg for the erythro compds. R.. W.S) C. A. 125. Similarly. E. Seyfried. of Minnesota. A. furnished to us through the Walter Reed Army Institute of Research. Franklin.. Bergstrom.6-bis(trifluoromethyl)-9-phenanthrenemethanol were tested12 for antimalarial activity as their HCl and TsOH salts and as their oxazolopyridine derivatives against Plasmodium berghei in mice and P.H. Newton. Anal.46]pyridine.ly2The most common of these modifications is replacement of the Me group attached to the basic N with some other substituent. et al. Y. (Engelhard 85%). R. . protonation of I1 should increase the population of IIA with a concomitant decrease in IIB. ibid. and L.60 R.. Chem. and J. and A. Abdel-Monem a n d P. The solid was dissolved in CHCl. mp 269-270".0g (84%) of IV as a white powder. Dudas and I. B. Meyers. 95%). Koepfli. Senear. respectively. 3.6-Bis(trifluoromethyl)-9-phenanthryl coupling constants observed in DMSO for the free bases and their protonated forms. A mixt of 4. Sargent. This would enhance the population of IA at the expense of IB and IC. Chem. B.E.O and satd with HCl g a s to ppt 11. Howton. Instruments employed were: Beckman IR-9infiared spectrophotometer..J. Rane. J. Med. and K. berghei. Inc. F. J. Buchman.gallinaceum in chicks by Dr. Chem. 111. Vol.[ 3.1813 (1946). Ar = 3. 86. 94. Ser. (2) A. Portoghese* and again concd to 10% the original vol to give 96. D. The mixt of I and 11 (15. W.3%) of Department of Medicinal Chemistry. Soc. 2705 (1938). 68. C. 4. Senear. Chem.. R. Soc. Amer. Chem. (1946). Collect. Sargent. C. mp 181-182'. E.. (4) F. Chen.1057 (1967). J... W.. R. Jacobs. and eluted with CHCl.9 g (1 1 mmoles) of I.6-bis(trifluoromethyl)-9-phenan- threnemethanol Hydrochloride (I HCI). the bulk of the solvated quaternary N in I . IS. Knoxville. Meyers. 431 (1967).275mole) of 2-pyridyl3. after filRelated Compounds with Chloroformate Esterst tration. H.. Varian Model DP-60high-resolution nmr spectrophotometer and Beckman DK-2uv spectrophotometer. (C. Y. related compounds have been performed and the compounds tested in an effort to analyze the relationship between structure and analgetic activity. Seymour. (3) H.921 (1971). Med. Minnesota 55455. and R.J-L.0 g.2718 (1946). E.. B.5 g of crude product.Mead. HCl. due to the increased steric requirements of the solvated ammonium group. Winstein. The author wishes to thank Roy Putnam for the nmr spectra.. T. F. The dl-erythro and dl-threo isomers of a-(2-piperidyl)-3.H. 68. I. College of Pharmacy. Proc. and F. J. L. A scmilar reaction of I1 gave 4. 2605 (1946).. Against P..Kondo. Anal. R. 10. I1 was dissolved in anhyd Et. the filtrate was evapd in vacuo to 10% the original vol. B. Soc. Meilahn. 2 Notes for 5 min. HCl is significantly increased by H bonding with DMSO. F. Chem. The mixt was cooled and filtered.NOF. S. Henderson..NO. A. Chem.O). 1946.CO. Chem. E. and 40 ml of concd HCl for 16 hr. C1. 2 ml of (CH. Crabb and R. J. of MeOH. B.Senear.H.2721 (1946). To attempt a general correlation of epimer conformation with antimalarial activity on the above limited data is premature.2697 (1946). Ann Arbor. H. N...3480(1968). L. Biological Data. N. (C. King. B.0g (82. A.E.) C. Evidently.. show these materials to be highly active against P.. F. Weisz. Russell. H. refluxed Hundreds of modifications of morphine and structurally §The synthesis and activity of I ' HC1 has been previously reported b y Nodiff. 68.. University I .H. 19 72. (7) A. F. Anal.3%) and were performed b y Galbraith Laboratories. Mich. soln. (C. I971 methanol Hydrochloride (I1 HCI). Howton. soln. S. Sargent. M..§ No toxic deaths were reported up to dosages of 640 mg/kg. MeOH. Boykin.. Med. and 50 ml of MeOH was refluxed 8 hr. mp 331-332" dec.0 g of PtO. Darco) to give 3. Tanabe. HC1 as a white powder. HCl) C . dl-threo-or-(2-Piperidyl)-3.6-bis(~uorome~yl)-9-ph~n~~eneReceived August 5.2 1.2199 (1963). C.418 (1966).. H. Linden. 1256 (1964). H. ibid.) C. on protonation. and J . ibid.Wiselogle. Mead. Seneker. s o h (2 ml) was added. J.A.F. E. and J. F. R.. N. I1 ' TsOH (50 g. H. Roy. Mead.2688 (1946). 68.R. "A Survey of Antimalarial Drugs. et al.O) gave 4. 33.A. mp 167-168'. HC1) C. galZinaceum.2708 (1946). Seibert. T. Org.JVOF. References (1) D. ibid. S. N. et al.208 Journal of Medicinal Chemistry. King. (5) E.S.Anal. S. 68. J. poured onto a silica gel H column (75 g). and D. B. 35 mmoles) in 200 ml of MeOH was treated with 4.' #All melting point (uncorr) were taken 01% a Buchi apparatus.1 mole) was neutralized by stirring overnight with dil aq NaOH soh.

N-Demethylation of morphine and structurally related compounds with chloroformate esters M.. 1972.W.1021/jm00272a025 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. Portoghese J.. 15 (2). 1155 Sixteenth Street N. 2009 More About This Article The permalink http://dx. Abdel-Monem. 208-210• DOI: 10.org/10. and P. Chem. S.org on April 25. DC 20036 . M. Washington.doi.acs.1021/jm00272a025 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. Med.

68. 0. 2705 (1938). . Seibert. and J. 19 72.ly2The most common of these modifications is replacement of the Me group attached to the basic N with some other substituent. Darco) to give 3.$JOF. Chem.H.27 14 (1946). J. Edwards. (C.6-Bis(trifluoromethyl)-9-phenan~l] hexahydro-3H-o~azolo[3. furnished to us through the Walter Reed Army Institute of Research. Amer. W. F.46]pyridine. E. T. R. G. Biological Data. giving 5 cures out of 5 infected mice at dosages of 40 mg/kg for the threo epimers and 5 cures at 80 mg/kg for the erythro compds. (C. Osdene.O. (C.. Soc. Tanabe. B. ibid. Chem. I971 methanol Hydrochloride (I1 HCI). Crabb and R. Seneker. Senear. Roy.[ 3. of Minnesota. E. Soc.. (3) H. 14. on protonation. ibid. Org. J.921 (1971). Levy. R. and eluted with CHCl.) C. due to the increased steric requirements of the solvated ammonium group. Chem. 86. 68. Recrystn (2x. IS. H. N.. refluxed Hundreds of modifications of morphine and structurally §The synthesis and activity of I ' HC1 has been previously reported b y Nodiff. Anal. Knoxville. 68. Buchman. were correct (*0. Meilahn. Mead. Soc.J-L.1813 (1946).R. poured onto a silica gel H column (75 g). and J . the bulk of the solvated quaternary N in I . R. HCl) C . Burger. Howton. show these materials to be highly active against P. M. (6) T. Chen. and cooled to ppt I1 .Anal. L.Brown. F. F.S. Ann Arbor.S) C. Bergstrom.2g.0 g.. Elemental anal. Refluxing with CCl. Blaha. R. 68.2697 (1946). (12) T. K. R. Proc.JVOF. 3.A. 431 (1967). Rane. Chem. the reflux was contd overnight.. (4) F. 28. Chem. E. S.HCl. D.0g (82. mp 269-270".6-bis(~uorome~yl)-9-ph~n~~eneReceived August 5. Patel. 94. 68. after filRelated Compounds with Chloroformate Esterst tration.R.. and L. H.0g (84%) of IV as a white powder. Med. M. Buchman and D. Anal. Meyers. F. (9) P.2708 (1946). J. F. J. Mead. Anal. ibid. Med. Dudas and I. L.) C. 2 ml of (CH. 10.gallinaceum in chicks by Dr. E. ibid. B. The solid was dissolved in CHCl. and M.NOF. Russell. A. (1946). (8) J. of MeOH.. 4... Mich.A. 2605 (1946). (C. J. 1 0 . N. R.. Heterocycl. C. H. mp 181-182'. University I .. 35 mmoles) in 200 ml of MeOH was treated with 4.2688 (1946). Linden.. A scmilar reaction of I1 gave 4. Vol.275mole) of 2-pyridyl3. References (1) D. Similarly.Kondo. Koepfli.6-bis(trifluoromethyl)-9N-Demethylation of Morphine and Structurally phenanthryl ketone..459 (1967). Tyagi. Ber. ibid. K. J a y . Lutz. (5) E.. mp 167-168'. B. Sargent.418 (1966). H. Newton. 1946.. Meyers. 33.. ibid. A. Varian Model DP-60high-resolution nmr spectrophotometer and Beckman DK-2uv spectrophotometer.6-bis(trifluoromethyl)-9-phenanthrenemethanol Hydrochloride (I HCI). Darco was added and. Leo Rane at the University of Miami.. Anal. Franklin. and J. HC1) C. P. Instruments employed were: Beckman IR-9infiared spectrophotometer. and A. T. S. E. and D. A mixt of 4. R. Koepfli. (C.§ No toxic deaths were reported up to dosages of 640 mg/kg.. Amer. C. T. B. The mixt of I and 11 (15. The dl-erythro and dl-threo isomers of a-(2-piperidyl)-3. Seymour.. Chem. E. E. Henderson. treated with dil K. W. Med.. Portoghese. The test results. M. Sargent. s o h (2 ml) was added. College of Pharmacy.Wiselogle.NO.. J. pptg a mass of white crystals. Winstein. (7) A. King. Chem. ibid. 68. 95%).0g (63%) of I11 as white flakes.O) gave 4.2199 (1963). Jacobs.2 1.. A.CO.Ainley and H. R. A. soln. W.2710 (1946). respectively. B.No. Against P.F. Czech. Minneapolis. Boykin. F. Minnesota 55455. Add1 (C%O). removed a yellow impurity to leave 11. Chem. Matsuura. and J. Chem. H. was passed through a mixt of 115 g (0. (2) A. et al.9 g (25 mmobs) of TsOH .6-bis(trifluoromethyl)-9-phenanthrenemethanol were tested12 for antimalarial activity as their HCl and TsOH salts and as their oxazolopyridine derivatives against Plasmodium berghei in mice and P... (11) 0. 2 Notes ?H IA IB IC for 5 min. H.O and satd with HCl g a s to ppt 11. and 50 ml of MeOH was refluxed 8 hr. 68.E. N. 68. 1256 (1964).3%) and were performed b y Galbraith Laboratories. J. J.NOF.O). Howton. and the product was recrystd (EtOH. Lutz.A. J.3%) of Department of Medicinal Chemistry. 4. Abdel-Monem and P.Senear.Mead. Kovar.9 g (1 1 mmoles) of I. I1 ' TsOH (50 g. _IV. and F. J. Y. Sargent. Thus. Collect.. The mixt was cooled and filtered. C1. I1 was dissolved in anhyd Et. Chem. berghei.6-Bis(trifluoromethyl)-9-phenanthryl coupling constants observed in DMSO for the free bases and their protonated forms. This would enhance the population of IA at the expense of IB and IC.412 (1960). HC1 as a white powder. 68.. Heterocycl. Experimental Section# dl-erythro-ol-(2-Piperidyl)-3. Norton.Munk. dl-threo-or-(2-Piperidyl)-3. Buchman. E. B. Ar = 3. HCl. soln. et al. (10) M.3480(1968). related compounds have been performed and the compounds tested in an effort to analyze the relationship between structure and analgetic activity. _I IIA IIB IIC Acknowledgment. C1. Benson. 5.. T. D. S. Seyfried. ibid. 68. ibid. Recrystn (MeOH-H. Rapport. The author wishes to thank Roy Putnam for the nmr spectra. Commun. I. Ser. 111. To attempt a general correlation of epimer conformation with antimalarial activity on the above limited data is premature. H. Chapman and R. H.E. M. The solids were dissolved in MeOH (Darco) M. W. Tenn.1 mole) was neutralized by stirring overnight with dil aq NaOH soh."J. C. F. ibid.0 g of PtO.. and dried. the minimum dosage showing activity was 20 and 160 mg/kg for the threo and erythro compds.' #All melting point (uncorr) were taken 01% a Buchi apparatus. and R. N..2697 (1946).H. 125. the filtrate was evapd in vacuo to 10% the original vol. The mother liquors from the above recryst were concd to dryness. and P.2721 (1946). 68. J.E. et al. Senear. and K. Portoghese* and again concd to 10% the original vol to give 96. Koepfli. and 40 ml of concd HCl for 16 hr. Inc. mp 284-285" (44. Nodiff. protonation of I1 should increase the population of IIA with a concomitant decrease in IIB. P.208 Journal of Medicinal Chemistry.60 R. S. H.1057 (1967).. mp 331-332" dec.. Darco) gave an analytical material.2718 (1946). N. H. HCl is significantly increased by H bonding with DMSO. 19411945. Sargent. (Engelhard 85%). MeOH.H. Evidently. Y. TsOH.. H. ?This investigation was supported b y NIH Grant NS 08738.. King. B.H. Weisz.. galZinaceum. A. "A Survey of Antimalarial Drugs.A.J.. A.5 g of crude product.

The mixt was refluxed under N.O. The solvent was removed in vaeuo to afford 0. the EtOH was removed in vacuo. The carbamate was obtained in crystalline form by selectively hydrolyzing the phenyl chloroformate contaminant with aq K2C03.H.001 mole) of 2 in a mixt of EtOH (80 ml) and 50% KOH (20 ml) was refluxed under N.O layer was extd with 1N HC1. (C]&$JO*HCl) C.-i-PrOH (3:l). No.. Removal of E t 2 0 afforded 0. R = Me. The mixt was treated with 2 N HCl(50 ml) and sodium potassium tartrate (6 g) and refluxed for 4 hr. Structure 9 was corroborated by its reconversion to 8 with LAH.5%. structures related to morphine. The Et. Demethylation of 8 also was carried out with ethyl chloroformate. The Et. whereas compounds such as morphine. Basification of the aq soln afforded 0. and extd with CHC1. Treatment of morphine (1) with phenyl chloroformate in the presence of KHC03 in boiling CHC13 and subsequent treatment of the product with a mild base afforded.2 g) in THF (150 ml).O) afforded 2. 74. A stirred suspension of 2. (+)-3-Methoxymorphinan Hydrochloride (10 HC1). for 24 hr. adjusted to pH 8. corydine. of this product (1 1) with HBr-HOAc afforded 3-hydroxymorphinan (12). The mixt was maintd at room temp for 24 hr and the MeOH removed in vacuo. phase was sepd and concd in vacuo.. (C.5. Codeine (4) was demethylated with ethyl chloroformate in a two-phase system containing aq KOH and CHC13. R = Me. was removed in vacuo.. HCl: yield. R' = R" = COOEt 6. (40 ml).8 Gadamer and Knoch' studied the effect of ethyl chloroformate on a variety of cyclic tertiary amines and observed that bulbocapnine.0 g (0. H.) C. solution or KBr disk. 70-325 mesh) and eluted with Et. R' = COOPh 10. and extd with Et. A THF soln (100 ml) contg 0. possibly because esterification of the 3. It is noteworthy that the reaction of morphine with phenyl chloroformate was unsuccessful in the absence of base. The acid phase was filtered. R = Me. and the residue was suspended in 1N HCl (50 ml) and extd with Et.O (1:4) to afford 0.or 6-OH generates HCl which may protonate the basic N and hence diminish its reactivity. mp 307"dec (reported 305' dec).O soln was concd under reduced pressure and the oily residue was dissolved in MeOH (60 ml) and treated with 2% aq K. for 4 hr. R' = H.O.8 g (0.6 g of KOH and 10.8 g (0. ' ~ Experimental Section! Cleavage of tertiary amines with chloroformate esters was first reported in 191 1. Anal. Mich. E.NO. and the residue was crystd from 50 ml of MeOH-H. Thus. University of Minnesota. the MeOH was removed and the residue was dild with H. H. in high yield. based on its spectral properties.0 g of KHCO. have successfully prepared 'H-labeled morphine b y employing a modification of our procedure. RoR Ron 8. R" = Me 2.5-255'. R = Me. and refluxed for 30 min.7 g (yield. of Pharmacology.O ext contd some unreacted 2 as shown by tlc. cooled. Crystn (MeOH-H.O. for 24 hr. SA. R = M e : R ' = H 11. (C. This assignment was confirmed further by LAH reduction of 2 to morphine and by its hydrolytic conversion to normorphine (3) with KOH. they suffer from the disadvantage of the toxicity of reagents employed and/or the low yields of demethylated product.5 g (0. [3H]LAH reduction of the carbamate intermediate obtained from the demethylation step would afford a 3Hlabeled Me group. Vol.. The nmr spectra were obtd with a Varian A-60D spectrometer (CDCl. (+)-3-Methoxy-l7-carbophenoxymorphinan (9).O. After the mixt was acidified with concd HCl. stirred suspension of 0. 1972. treated with an aq soln (100 ml) contg 5.002 mole) of 9 was added slowly to a cooled. R = R' = H. (250 ml) was treated with 11. H. One approach to this problem which seemed worthwhile exploring involved demethylation of analgetics with a chloroformate ester' followed by hydrolysis of the resulting carbamate to the secondary amine as outlined below.4 g (0. The soh was treated with concd HCl(20 ml). N. recrystd 10. N. Normorphine (3).8 g of 9.O was dried (MgSO. Takemori and J. (30 ml) was maintd at 25' for 24 hr. a nonbasic crystalline material which was assigned structure 2. R = R' = H 1. TMS).45 g of crude 10.15 mole) of KHCO.002 mole) of 2 was added slowly to a cooled.C1. LAH Reduction of Carbophenoxynormorphe (2). and tropine were not cleaved. R = Me. Hydrolysis of 9 with KOH gave the desired secondary amine (10). An EtOH soln (80 ml) contg 2.) C. A THF soln (50 ml) contgO. R = R' = M e 9.14 We wish to describe the utilization of this reaction as a means of conveniently demethylating. Several examples of this reaction have been published since. LAH Reduction of 9. Merck AG. Although there are several methods reported"' for the demethylation of tertiary amines.H. R = R' = H. The acid phase was adjusted to pH 8. R" =Me 5. and stirred under N. heroin.CO. for 20 hr. basified. R' = R" = H Reaction of 3-methoxy-N-methylmorphinan (8) with phenyl chloroformate at room temp afforded carbamate ester 9. R = Me: R ' = COOEt 12. The exts were concd in vacuo. R" = COOEt 7. mp 100-102". Mass spectra were obtd with a Hitachi Perkin-Elmer RMU-CD mass spectrometer.. Anal. Dept. in CHCI. and laudanosine were converted to the corresponding ethyl carbamates..'@l3 and in this regard. for 24 hr. N.12 g of 2: mp 127-130' (yield 91%). The soln was dild with HzO (20 ml) and the EtOH was partially removed under reduced pressure.2 g of LAH in THF (100 ml). The mixt was refluxed under N.CI. HCl (MeOH-EtOAc). The spectral characteristics of the neutral intermediate appear to be in harmony with 5 . The aq suspension was extd with Et. mass spec (70 eV) m/e 391. A soln of 3. The CH. 15. The residue was dissolved in MeOH (150 ml).5%. This was concd in vacuo to remove THF and extd with Et.O (100 ml) and the CHCI. HCl. !Melting points were detd in open capillary tubes using a ThomasHoover melting point apparatus and are uncorrected.I7 yield.0088 mole) of morphine (1) and 15 g (0. It is noteworthy that the present procedure also can be employed as a convenient and relatively inexpensive method for introducing radioactivity into a N-Me group. . R = R ' = R " = H 4. Wang.5 g (0. The ethyl carbonate group of 5 could be hydrolyzed selectively to 6 which then was cleaved to norcodeine (7) under more vigorous conditions.01 1 mole) of 8 and 2.077 mole) of phenyl chloroformate and refluxed for 60 hr.) and treated with ethanolic HC1 to give 1. The i r spectra were obtd with a Perkin-Elmer 237B spectrophotometer in CHCI.013 mole) of phenyl chloroformate in CH.45 g of morphine: mp 252254" (reported 254-256")16 (yield 76%). stirred suspension of LAH (0.Notes Journal of Medicinal Chemistry. R" = COOPh 3. codeine. phenyl chloroformate was found to be superior to both benzyl and ethyl chloroformate in the cleavage of tertiary amines.$ A 14C label may be incorporated by demethylating with [ ''C]C1COOR followed by r e d ~ c t i o n .O was removed and the oily residue was chromatogd on a column of 45 g of silica gel (E. The Et.NO." 3 . and the solvent was removed in vacuo to afford 3.-i-PrOH (3:l).R' = H . A soln of 0.O.86 g of unreacted 8.5 and extd with a mixt of CHC1. Hydrolysis N-Carbophenoxynormorphine(2).O and extd (Et.007 mole) of 9 was treated with 50% aq KOH (20 ml) and the reaction mixt was refluxed under N.O). . Fractions 6-12 (50 ml each) were pooled. 65%) of 9. 2 209 the secondary amine serves as an important intermediate in the synthesis of such compounds. The aq acid soln was filtered. The Et. mp 253. The mixt was treated with H. and the residue was extd with Et.12 g of 3: mp 277' dec (reported 276-277"). treated with EtOAc (15 ml).5 g (0. after purification. Garden City.O and the Et. Microanalyses were performed b y M-H-W Laboratories. Anal. 43.0 g (0.

(4) 0.O. (12) M. filtered. and Sergio Boveri Research Laboratories. Academic Press. (17) Reference 16.O and 1N NaOH altemately.O was extd with 1N HC1. was removed in vacuo to afford an oil (3..012 mole) of codeine (4) in CHCl. After addn of H. and KOH s o h was added when necessary to maintain a pH > 10.30.. J. all the others with a Buchi apparatus. and 38.31.CO. After refluxing under N. Five successive 1-ml portions of ethyl chloroformate were added to the reaction mixt at 1-hr intervals. E. After cooling again to 7-8'. The aq acid soln was basified and extd with Et. ~ x COOH y I R y~yCOOCOOC.O.. S. 251 (1936). Nador. and basified to afford 1.0 g of the oil in 95% EtOH (80 ml) was treated with 50% aq KOH (20 ml) and refluxed under N. p 749. and 50 ml of dioxane was stirred at room temp until a clear soln resulted. RIY\YCONHRz I11 0 I1 I 1 IV References (1) P.C0. McMahon in "Advances in Tracer Methodology. deStevens. Chem.. Grussner. Bracco Industria Chimica. (10) E. The aq acid soln was basified and extd with Et. In preliminary CNS screening the majority of the compounds were found to be without hypnotic or sedative activity. Ber. Fischer.O. 2 Notes cooled.O (20 ml) and the EtOH was removed under reduced pressure.(10 ml) for 2 hr. J. but all the compounds showed too low a therapeutic index. A.. U.O Synthesis of 4(3H)-Pteridinones Ernst Felder. Rahway. and the dried Et. the CHCl. Org.O afforded 2. (9) J. A mixt of 5. keeping the temp at 11-12'. 1972.. and recrystd. Ed.0 g (0. 3-Aminopyrazinecarboxamides(1-20). New York. 5 5 . This was treated with a mixt of MeOH (90 ml) and 10% aq K. M. L. B. p 702. I . Vol. Sei. Merck and Co. 4.. (11) J. Stecher. The residue was acidified with 6 N HCl and extd with Et. G. and. Compds 23. Speyer and L. Where analyses are indicated only by symbols of the elements.. 25 ml of ortho ester.. mp 309311" dec (reported mp 309" dec). For 23 the reaction was carried out in anhyd HCO. The residue was triturated with 20 ml of EtOH. New York.. ibid. 7.5 g) which was dissolved in a mixt of glacial AcOH (10 ml) and 48% HBr (10 ml). hotplate test).O ext was treated with ethanolic HC1 to afford 0. J. in contrast t o the 4(3H)-pteridinones (IV).30. (8) F. Gadamer and F. Ed. Recrystn solvents and physical data are given in Table I. S . (+)-3-Hydroxymorphinan (12).O. layer was sepd and extd with 1N HCl.2 g) whose ir spectrum included characteristic absorptions at 1690 cm-' (C=O of N-C0. The synthesis of the title compounds involved the intermediate 3-aminopyrazinecarboxamides. Campbell.O. the mixt was filtered and concd in vacuo t o remove THF. McMahon.04 mole of the appropriate amine hydrochloride was added. Knoch. 34.7 g (yield.. which were obtained. Diels and E. Abdel-Monem and T. 0. Litchfield and Wilcoxon) being 1250 (1042-1500).O afforded an oil (2.' via the mixed anhydride (11)6 and reaction of the latter with appropriate amines (RzNHz). 221 1 (1951).. filtered. Received May 24. from 3-aminopyrazinoic acid (I). and 38 some analgetic activity was observed at 150-250 mg/kg (mouse. No." G. € W. The soln was dild with H. Chem. phenylbenzoquinone test) and 150-500 mg/kg (mouse. 56. N. Chem. Helv. (18) 0. Milan.H. which is helpful for their identification by chromatography. The residue was suspended in 1 N HCl(50 ml) and extd with Et. 259. (15) R.." 7. The solvent was removed on a rotatory evaporator under reduced pressure and the residue was triturated for 30 min with 50 ml of H.210 Journal ofMedicinal Chemistry. D. 15 (1967).. Amer. Bayer and Co. washed with Et. Pkarm. Plenum Press. 4(3H)-Pteridinones (2147). the reaction mixt was poured in ice water (300 ml). Kraiso and L. Org. German Patent 255. Ber. Chim.824 (1967). 3104 (1955).8 g (0.342. Soc.. Recrystn solvents and physical data are given in Table 11. A CHCl. A.01 mole of 111.4% of the theoretical value. R. Jr. (16) Merck Index. Patent 3. The reaction mixt was stirred for 24 hr and the CHC1. respectively. (5) A. 69B. Pohland and H.H-Ac. Marion. Ed. The 2-phase reaction mixt was shaken for 24 hr. At the end of 24 hr. Y. Soc. N. J. Rothchild.O. H.reveal a characteristic fluorescence under uv light. extd with Et. for 24 hr. S. (13) G. Pkarm.O. Pharm. p 747.. Inc.942 (1911). Flynn. Arch. Sullivan. 47. 43%) of 7: mp 183-185" (reported mp 185"). and recrystd. 1220 (1070-1391). and for 24 in 1: 1 anhyd HC0. 852 (1930). dried. (3) H.* Davide Pit&.. 22. A soln of 3.O was removed under reduced pressure to give 0.51 g (yield. 0. mp 260-263" [reported for (-)-3-hydroxymorphinan. and 31 showed a slight sedative activity at 300 mg/kg (mouse) and with 30. and are uncorrected. and R.. 865 (1966).976 (1967). Y. 1968. (19) Reference 16. Chapter 4 (1953). mp 260-262'1 .Ig sedative activities. dried. Italy.(2 g). Zajic. and the reaction was allowed to proceed at room temp for 3 hr. and E. A mixt of 0. The Et. Removal of Et. 135 (1921). I * Norcodeine (7). Acta.Murphy. 1259 (1957). Schnider and A.04 mole) of 3-aminopyrazinoic acid. in good yield.4 g (0. (14) J. J. 1965. Spath.56 g (0." Vol.5 g of an oily uncrystallizable material. Removal of Et. 8th Ed. Portoghese. mouse. React. 7. N. then removed in vacuo.0015 mole) of 7 was treated with ethyl chloroformate (20 ml) and anhyd K. 1968. Hobson and J. (Weinheim). treated with EtOAc (10 ml). for 6 hr. and the Et. Uv and ir spectra were measured for some typical compds and were as expected. This soln was cooled to 7-8" and 4 ml (0. R . D. 1971 The sedative-hypnotic activity of some 4(3H)-quinazolones' prompted us to synthesize a number of the isosteric 4(3H)-pteridinones and to investigate their hypnotic and . 78%) of 8 * HC1. and 20-30 ml of Ac.04 mole) of Bu. p 29. The CHC1.Et) and 1740 cm-' (C=O of 06-C02Et). The amides (III). Tetrahedron Lett. Hageman.O.1 g (31%) of 12. 69B. and 1750 (1400-2187) for 23. the LDS0(mg/kg.H. extd with Et. described in Table I.31.. The existing literature gives only a few examples of preparation of 3-alkyl or 3-aryl substituted 4(3H)-pteridinone~~-~ and no data at all on their pharmacology. 15. analytical results obtained for those elements were within +0. (7) E. Walther. A soln of 2. 71.O was refluxed for 5 hr and then concd on a rotatory evaporator at room temp in vacuo. The 3-aminopyrazinecarboxamides(111) could be cyclized to the desired 4(3H)-pteridinones (IV) by condensation with an ortho ester R3C(OC2H5)3in AczO solution. Soine.N. (2) "Analgetics. soln (25 ml) contg 4.2043 (1914). after evapn of the solvent. D.HC1. Sei. and refluxed for 30 min. (6) E.. McCluskey.04 mole) of EtOCOCl was added dropwise. 57 (1971). J. (50 ml) was treated with ethyl chloroformate (5 ml) and 15% aq KOH (50 ml). concd in vacuo to remove MeOH. 575 (483-684). Experimental Section The melting points of all but four compds 21-47 were taken with a Mettler FP-1 apparatus...

1155 Sixteenth Street N. 2009 More About This Article The permalink http://dx.. 1972. Washington.org/10..W. and Sergio Boveri J.doi. Chem. 210-211• DOI: 10.acs. Med.Synthesis of 4(3H)-pteridinones Ernst Felder. DC 20036 . 15 (2). Davide Pitre.1021/jm00272a026 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.1021/jm00272a026 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society.org on April 25.

04 mole of the appropriate amine hydrochloride was added. Pharm. Chem. React. analytical results obtained for those elements were within +0.C0. I . Rothchild. Pkarm.824 (1967). Vol. phenylbenzoquinone test) and 150-500 mg/kg (mouse. 4(3H)-Pteridinones (2147).. Org. Ber. (10) E. 15 (1967). Abdel-Monem and T. Hageman. Flynn.5 g) which was dissolved in a mixt of glacial AcOH (10 ml) and 48% HBr (10 ml). was removed in vacuo to afford an oil (3.O. after evapn of the solvent. which were obtained. 43%) of 7: mp 183-185" (reported mp 185"). Removal of Et.. Portoghese. (2) "Analgetics.04 mole) of 3-aminopyrazinoic acid. 8th Ed.O ext was treated with ethanolic HC1 to afford 0. (4) 0. and for 24 in 1: 1 anhyd HC0. McMahon in "Advances in Tracer Methodology. 15. 1965. The aq acid soln was basified and extd with Et. Gadamer and F. and recrystd. M.H.O. The solvent was removed on a rotatory evaporator under reduced pressure and the residue was triturated for 30 min with 50 ml of H. B.O was extd with 1N HC1.976 (1967). 3104 (1955).H. 5 5 . J. The synthesis of the title compounds involved the intermediate 3-aminopyrazinecarboxamides. (15) R. The residue was triturated with 20 ml of EtOH. Chem. 575 (483-684). (16) Merck Index.. Kraiso and L. 0. Ed. I * Norcodeine (7). treated with EtOAc (10 ml).012 mole) of codeine (4) in CHCl. Pohland and H.942 (1911). layer was sepd and extd with 1N HCl. N. Helv. This was treated with a mixt of MeOH (90 ml) and 10% aq K.Ig sedative activities. Bracco Industria Chimica. J.O and 1N NaOH altemately. p 749. (3) H. Chim. and 1750 (1400-2187) for 23. Diels and E.0 g (0. (18) 0. 2 Notes cooled..8 g (0. ibid. At the end of 24 hr.342.. and Sergio Boveri Research Laboratories.04 mole) of EtOCOCl was added dropwise.O was refluxed for 5 hr and then concd on a rotatory evaporator at room temp in vacuo.1 g (31%) of 12. Jr. 135 (1921). Ber. A mixt of 0. Acta. Nador. concd in vacuo to remove MeOH. Soine. extd with Et... Compds 23. the reaction mixt was poured in ice water (300 ml). and basified to afford 1. Chapter 4 (1953). D. (12) M. N. and 31 showed a slight sedative activity at 300 mg/kg (mouse) and with 30. The residue was suspended in 1 N HCl(50 ml) and extd with Et.O. 4. New York. E.O afforded an oil (2. 34. Arch. hotplate test).Murphy. The amides (III). and R. The Et. 47. L.4% of the theoretical value. filtered.0015 mole) of 7 was treated with ethyl chloroformate (20 ml) and anhyd K." Vol. The 3-aminopyrazinecarboxamides(111) could be cyclized to the desired 4(3H)-pteridinones (IV) by condensation with an ortho ester R3C(OC2H5)3in AczO solution.51 g (yield. then removed in vacuo. S. R .O. 259. (13) G. Amer. J. Marion. 7. and. S. Received May 24. The aq acid soln was basified and extd with Et. McCluskey. N. 852 (1930)." G. 1220 (1070-1391). soln (25 ml) contg 4. Soc. (Weinheim). Stecher.N. The residue was acidified with 6 N HCl and extd with Et. which is helpful for their identification by chromatography. Fischer. The CHC1..reveal a characteristic fluorescence under uv light. RIY\YCONHRz I11 0 I1 I 1 IV References (1) P. After addn of H. 56. p 29. for 6 hr.31. Knoch. (5) A. U..CO.Et) and 1740 cm-' (C=O of 06-C02Et). Grussner. in good yield. The soln was dild with H..4 g (0.2043 (1914). Schnider and A. filtered. Y. Zajic. A. (14) J. Bayer and Co. p 702.' via the mixed anhydride (11)6 and reaction of the latter with appropriate amines (RzNHz). for 24 hr. the mixt was filtered and concd in vacuo t o remove THF. After refluxing under N. D. (19) Reference 16. and 38. Academic Press. Soc. p 747. Sullivan. (17) Reference 16.5 g of an oily uncrystallizable material..(2 g). and recrystd. deStevens.O afforded 2. ~ x COOH y I R y~yCOOCOOC. New York. 1971 The sedative-hypnotic activity of some 4(3H)-quinazolones' prompted us to synthesize a number of the isosteric 4(3H)-pteridinones and to investigate their hypnotic and .O. respectively. mouse.2 g) whose ir spectrum included characteristic absorptions at 1690 cm-' (C=O of N-C0.. keeping the temp at 11-12'. all the others with a Buchi apparatus.H-Ac. Hobson and J. Five successive 1-ml portions of ethyl chloroformate were added to the reaction mixt at 1-hr intervals. dried. washed with Et.31. but all the compounds showed too low a therapeutic index. 25 ml of ortho ester. and the reaction was allowed to proceed at room temp for 3 hr. Org. H. and 38 some analgetic activity was observed at 150-250 mg/kg (mouse.(10 ml) for 2 hr." 7.30. 1259 (1957).O was removed under reduced pressure to give 0. € W. 1968. Campbell.. 69B. 251 (1936). S . Walther. Litchfield and Wilcoxon) being 1250 (1042-1500). For 2 3 the reaction was carried out in anhyd HCO. Tetrahedron Lett. Speyer and L. A soln of 2. (9) J.O. 0.. (+)-3-Hydroxymorphinan (12)..56 g (0. The reaction mixt was stirred for 24 hr and the CHC1. and E. This soln was cooled to 7-8" and 4 ml (0. and the dried Et. Ed.. Spath. J. and refluxed for 3 0 min.210 Journal ofMedicinal Chemistry. 1972. Ed. After cooling again to 7-8'. Milan. A CHCl.. Experimental Section The melting points of all but four compds 21-47 were taken with a Mettler FP-1 apparatus. The existing literature gives only a few examples of preparation of 3-alkyl or 3-aryl substituted 4(3H)-pteridinone~~-~ and no data at all on their pharmacology. and 20-30 ml of Ac. McMahon. In preliminary CNS screening the majority of the compounds were found to be without hypnotic or sedative activity.0 g of the oil in 95% EtOH (80 ml) was treated with 50% aq KOH (20 ml) and refluxed under N. (50 ml) was treated with ethyl chloroformate (5 ml) and 15% aq KOH (50 ml). described in Table I.O (20 ml) and the EtOH was removed under reduced pressure.O. A mixt of 5. 69B.. German Patent 255. Rahway.O Synthesis of 4(3H)-Pteridinones Ernst Felder. J. A.04 mole) of Bu. the LDS0(mg/kg. Uv and ir spectra were measured for some typical compds and were as expected. R. the CHCl. mp 260-262'1 .HC1. (11) J.01 mole of 111. extd with Et.. Inc. dried. D. Plenum Press. Merck and Co.O. Sei.. Italy. 78%) of 8 * HC1. mp 309311" dec (reported mp 309" dec). Chem. and KOH s o h was added when necessary to maintain a pH > 10. J. and are uncorrected. 71. mp 260-263" [reported for (-)-3-hydroxymorphinan. Where analyses are indicated only by symbols of the elements. Recrystn solvents and physical data are given in Table 11.7 g (yield. 22. (7) E.30. and the Et. in contrast t o the 4(3H)-pteridinones (IV). Y. Removal of Et. Pkarm. and 50 ml of dioxane was stirred at room temp until a clear soln resulted. from 3-aminopyrazinoic acid (I). 3-Aminopyrazinecarboxamides(1-20). 7. The 2-phase reaction mixt was shaken for 24 hr. Recrystn solvents and physical data are given in Table I. Sei.. A soln of 3. 221 1 (1951). Patent 3. G. (8) F..* Davide Pit&. No. 865 (1966). 57 (1971). (6) E. 1968.

(3) J. Saxena.5 -204 200.O l H l 6N40 C10H14N40 1 ' CI lHl.H9NS0 C. 40. (6) D.. (5) S.O * HCl * H.H.O 14H 1 E N 4 0 Cl. C. .C6H5 H H H H H Br H H H H H H H H H H H H H H C7H6N40 'SH1 O N '.5 188. Cyclo-C6Hll o-CH.5 177. Bicking. p.(1955).N. Chem.H6-P EtOH EtOH EtOH EtOH-i-Pr.C6H4 m-CH3C6H4 PCH3C6H4 o-(i-Pr)C6H4 p-(i-h)C6H4 O-EtOC6H4 m-EtOC..H9BrN40 Cl I B I N 8 134 134 73 107-108 105 136 112 158 154 125 134-135 104-105 139-140 134 180 136 190-191 155-156 157 176 33% EtOH EtOH EtOH-H.35.364 (1966). Jr. H H H H H H H H H H H H H H H H H H Br Br C6H8N40 C8H. Med.5 179. (2) E. C13H10N40 13H10N40 C14H11N402 C. 64. o-CH3C6H4 m-CH3C6H4 p-CH3C6H4 C6H5 o-(i-Pr)C6H4 p-(i-Pr)C6H4 O-EtOC6H4 m-EtOC6H4 p-EtOC..'Albert and D.. ibid.5-296.. Rosati. Ber. SOC. bCalcd.H14N402 1 ' 3H14N402 C15H18N402 C19H18N402 C. of these Research Laboratories. C2H5 C2H5 C2H5 CH.Hl8N. G. Jones. C17H1$r1402 C21H18N401 294. R.5 MeCN EtOH C6H6-P CHCl -Et 2 0 MeCN EtOH MeCN THF n-Bu.67.. . CCalcd. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 CH3 CH(CH3). Boveri. 62. R.5-207 166. Vol. Taylor. 3-Aminopyrazinecarboxamides(111) No.5 201-204.5-190. Chem. Grabitz. L. dBiichi apparatus. 2 211 Formula MP. Jr. Res. Cyclo-C5H9 Cyclo-C..5-172..5-1 82. Acknowledgment. R. S. S. C.5 164. ZN4O 6N40 C16H1340 C15H14N402 1 5H14N401 ClSH14N40.1651 (1952). found. J. Brown. found. Chem.5-167 203-204. p.. 62. I . SOC. Chem.899 (1967). N. Cb l6 l6 Cyclo-C. H.82. Pit&.15. P..5 -147d >299 202-205. Gujral.5 117-121.5-150. o-CH3C6H4 m-CH3C6H4 PC2H30C6H4 o-CH3C6H4 H CH(CH3)z Cyclo-C.74. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 31 38 39 40 41 42 43 44 45 46 41 CHI CH(CH3).5 160-163 165. B. "C Crystn solventa Anal. 99. 4(3H)-Pteridinones (IV) No.O c 1. Sonn.N.4850 (1907).637.(i-Pr)C6H4 pEtOC.5-180.74 (1953).)@C& CH CH -CH-CHl 3-Py C6H5 H H H H H H CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH. J.H.5 148d 196-198.H.5 aP = petr ether. N.HI 8 4 0 CiaHiiN40 C11H12N40 14H1 6N40 16N40 I 3H14N40.. 63.5 -24 3. . No.O C6H61P C.5 240.5 226-227.H.. Hi N H.5 dec -195d 147. We thank Dr.5-167.BrN40 C7H6N40 CIOHl l N 4 0 C12H14N40 C13H16N40 C14H11N40 aN40 IsH.H.Indian Med. J.5 239-24 1.. Journal of Medicinal Chemistry.5 204.O 33% EtOH 50% EtOH 50% EtOH 50% EtOH EtOH 95% EtOH 95% EtOH 65% EtOH 65% EtOH 65% EtOH EtOH n-BuOH 95% EtOH Dioxane-%O EtOH 95% EtOH 95% EtOH T a b l e 11.EtOC 6H4 p-C4H90C6H4 C6H5(CH.5 -144d 155-159. and E.H.BrN. References (1) M.H9CIN40 C10H9N50 leH. and E. 15. for the pharmacological screening. Cragoe. 43. and R. J. T i w a r i .O MeOEtOH EtOH EtOH EtOH EtOH C6H6-P EtOH C6H6-P EtOH EtOH EtOH EtOH-Et aO C. I9 72. J. Amer.5 235-236. 10. R3 Rl Formula MP.Notes Table I. "C Crystn solvent Anal. (4) A. J. pEtOC6H4 H H Br H H H H C21H 1. Gabriel and A.H.O C16H16N402 C21H18N402 198.H.

org/10.org on April 25.1021/jm00272a027 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society.W.acs. Chem. 1972. Brown. 15 (2).doi.. 2009 More About This Article The permalink http://dx. 212-212• DOI: 10. 1155 Sixteenth Street N. Med.Hepatocarcinogenicity of the trimethyl homologs of 4-dimethylaminoazobenzene Ellis V. DC 20036 .1021/jm00272a027 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.. Washington. and Alice Kruegel J.

2 Notes scopic examinations were made whenever an animal died or at the end of the experiment. 15. Much of the doubt as to the pathogenicity of Pityrosporum species has arisen from the difficulties encountered in artificial culture. vitamin A palmitate.06%. Walling. Young male rats of the Sprague-Dawley strain. C. C.5 55 C. Osborne and Mendel salt mixt. Norman J.NMe. The C.~ bp 224-228”. Received May 26.4’. 40 g. Chem.4’. (8) C. Mesidine (60 g) was dissolved in a mixt of 113 ml of concd HC1 and 376 ml of H.6‘-trifluoro deriva t i ~ e It . Miller. Cancer Res. Brentford. 3-Aminopseudocumeneg was produced by Fe-AcOH reduction of 3-nitropseudocumene which in turn was produced by the hypophosphorus acid reduction of the diazonium salt from 3-nitro-6-aminopseudocumene.212 Journal ofMedicinal Chemistry. 269 (1951). from Charles Bowman Co. 67. A Hall. A group received DAB at the 0. Yamada. Jones. “C 2’. Vol. H.. In spite of this limited pathogenicity or perhaps limited virulence. was obtd from 5-nitrohemimellitene. Amer. patterned after the “low protein. Brown and A.O and diazotized at 0” using 30. Kropf.6’-TrimethyLDAB 104. 5-Aminopseudocumene. Tinea versicolor was formerly attributed to the presence of Malassazia furfur. Definite to strong carcinogenic activity has been shown by some monosubstituted and disubstituted derivatives of 4-dimethylaminoazobenzene (DAB). K. CancerRes. 264 ml of H. Dolinsky. Fisher and C. and 109 g of NaOAc was added. 120 g.). Amer. Huender. C. Chem. 75. were distributed as equally as possible in initial body weight into groups of 10 animals each. Chem. Lexington.’2 2’. Leicester. Experimental Section All melting points were detd on a Fisher-Johns apparatus and are uncorrected. Sapp. N analyses were performed in this department on an F and M Model 185 analyzer by MI. Ritchie..5‘-Trimethyl-DAB 101..Jap. 3’.H21N.6 g of NaNO.O. Agr. Ugelow.5 mg. J. 50 g. City of Leicester Polytechnic. 1.5’-Trimethyl-DAB 131. J.4’. Me~idine. A. Chem. Each group was fed a diet.. 19 71 Results DAB gave tumor incidences of 6/ 10 at 4 months and 9/ 10 at 6 months. low riboflavin” diet of Miller‘ to which had been added one of the azo compounds at a level of 0. Pays-Bas. 552 ml of 95% EtOH.. Developments in the cultural technique’*’ have much reduced the problems of assessment of this class of compound. Biological Properties.O. N - Yield“ recryst % Pityrosporuni ovale and orbiculare.~ seemed of interest to synthesize and test for rat hepatocarcinogenic activity all of the trimethyl homologs of DAB with Me groups in the primed positions only. E. University of Kentucky College of Medicine. Trimethvl4-dimethvlaminoazobenzenes ~~ Compd Formulab . Brown* and Alice Kruegel Department of Chemistry.. J. 770 g. 27. One-half hr after the final addn. 2436 (1956). for the microscopic evaluation of the tumors. Department of Pathology. Trav.~mp 29-27”. Daryl Sharp. Soc. This sensitivity makes assessment of antipityrosporum agents difficult and earlier reports of measurements suspect. was prepared from 5-nit~ohemimellitene~ by reduction (Fe-AcOH).06% level while the control group received only the basal diet. H. 1972.3’. J. Med.Mp. 42. since isolation and maintainance in vitro of these lipophilic organisms are markedly influenced by variations in the constituents of the media. Offic. Laparotomies were performed at the indicated times and micro- Some Aliphatic Amines as Antipityrosporum Agents Harold B x o n . Acknowledgment. bAll compds were analyzed for C. 3‘.709 (1959).. Fujima.H. 97. Soc. Recent work in pyrrolidine chemistry has shown that ethyl AT-alkyl-4-hydroxy- . (10) H.9 g of the dye as bright orange needles which were recrystd from abs EtOH. Miller and E. was prepd from nitromesitylene6 by reduction with Sn-HCl. A.’ 6-aminopse~documene~ was prepd from 6-nitropseudocumene. None of the other trimethyl-DAB homologs produced tumors at 9 months at which time the experiment was terminated.. Yates. Hamdan.4% of the theoretical values.. SOC.5‘-Trimethyl-DAB 145-146. England and John K. 11. England. budding yeasts o f the family Cyptococcacae’ are not usually regarded as pathogens. Nat. Y .5‘-DAB gave 9/10 at 4 months and 10/10 at 6 months. ovale on the scalp. The first orange band was eluted. (9) K. (7) M. 2635 (1953). M. Purvis. and A. 1 (1915). 0. Justus LiebigsAnn.663 (1961). mp 62-63’. Where analyses are indicated only by symbols of the elements analytical results obtained for those elements were within +0. T. (3) E.. Sato. 442 (1968). 163 (1875). Daniel L. 11. Trimethylmilines.3’. Chem. ~is claimed that P.4’-TrimethyLDAB 147-151 26 C17H21N3 2‘. Chem. 179.5-134 8 C17H21N3 “All samples recrystd from 95% EtOH after chromatog on alumina from toluene-heptane. Cerelose. (5) A. The composition of the basal diet per kilogram was as follows: crude casein.IN.5-106” (see Table I). in 150 ml of H.. Chim.5-1 06 17 C17H21N3 2’. V. Chem. Soc. and the s o h was stirred for 24 hr. References (1) J. The authors are indebted to Dr. No.6’-TrimethyLDAB. 57. ovale and P. Kentucky 40506. Landenburg.339 (1953). 2’. I9 71 Table I.” mp 75-78’.5-103 25 C’. Weiss and Dr. Van Abbe2 has discussed the possible relationship between dandruff and the presence of P.. Ber.5-133 57 C17H21N3 2’. orbiculare are only pathogenic in susceptible persons. 3’.” and 5-aminohemimellitene. D. Ass. R.). 1-3 The only active trisubstituted-DAB tested has been the 2’. approximately 8 weeks old and weighing 150-200 g.4’. 41. 1234 (1968). (4) J. furfur and P. (11) F. and R. 34. Extn with PhH and evapn of PhH left a semisolid material which was submitted to column chromatog over alumina in toluene-heptane soh.. E..4’.H.3 but more recent work has shown that M. 4-Amin0hemimellitene. these yeasts do seem to be implicated in the causation of skin disorders and their suppression is clinically desirable. riboflavin. (12) A. orbiculare are probably the same organisms in different phases of g r ~ w t h It . 89. and the solvent was removed to give 19.6’-Trimethyl-DAB 132. Yoneyama. Received August I I . Sugden* School of Pharmacy. Miller. All the rats were kept individually in screen-bottomed cages and were offered food and water ad libitum. €1. Reel. Vitab (rice bran concentrate.4’. H. J. Hock and H. These are all new compounds and can be prepared by the diazotization of the proper trimethylanilines followed by coupling with PhNMez. L. Hepatocarcinogenicity of the Trimethyl Homologs of 4-Dimethylaminazobenzene Ellis V. The new azo compounds are listed in Table 1. A. 1700 (1935). 20 g. (6) J. T. and M. (2) E.4’.500 IU. J. J. Beringer and I. Bull. Miller. CancerInst. University of Kentucky. V. Beecham Products (U. Van AbbB.5-Trimethyl-DAB appears to be about as hepatocarcinogenic as DAB.1546 (1910). mp 104.Aduan. W. was prepd from 5-nitropseud~cumene~ in the same way. corn oil. Chem. a s o h of 54 g of C. Brown.3’.

. Sugden J.. 15 (2).1021/jm00272a028 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. 2009 More About This Article The permalink http://dx.Aliphatic amines as antipityrosporum agents Harold Dixon. and John K. 1972.1021/jm00272a028 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society.org on April 25.acs. Chem.doi.org/10. 1155 Sixteenth Street N.W. 212-214• DOI: 10. DC 20036 . Van Abbe. Norman J. Washington. Med.

Hepatocarcinogenicity of the Trimethyl Homologs of 4-Dimethylaminazobenzene Ellis V. 3’.4’-TrimethyLDAB 147-151 26 C17H21N3 2‘. from Charles Bowman Co. 3-Aminopseudocumeneg was produced by Fe-AcOH reduction of 3-nitropseudocumene which in turn was produced by the hypophosphorus acid reduction of the diazonium salt from 3-nitro-6-aminopseudocumene. Fisher and C. 1.NMe. a s o h of 54 g of C. was prepd from 5-nitropseud~cumene~ in the same way. Weiss and Dr.Jap. Med.. vitamin A palmitate. 27. 120 g. orbiculare are only pathogenic in susceptible persons. 11. Leicester. 2 Notes scopic examinations were made whenever an animal died or at the end of the experiment. Kentucky 40506. Experimental Section All melting points were detd on a Fisher-Johns apparatus and are uncorrected. Vol.. 552 ml of 95% EtOH.’2 2’. This sensitivity makes assessment of antipityrosporum agents difficult and earlier reports of measurements suspect.4’. Chem.. Osborne and Mendel salt mixt.H21N. V. 1700 (1935). (3) E. 2635 (1953). (10) H. Norman J. (11) F. H. riboflavin. ~is claimed that P. Definite to strong carcinogenic activity has been shown by some monosubstituted and disubstituted derivatives of 4-dimethylaminoazobenzene (DAB). T. Cerelose. and R. A. Agr. E. H. Amer. A. Landenburg. 2’. since isolation and maintainance in vitro of these lipophilic organisms are markedly influenced by variations in the constituents of the media. Much of the doubt as to the pathogenicity of Pityrosporum species has arisen from the difficulties encountered in artificial culture. Hock and H. J. 20 g. 19 71 Results DAB gave tumor incidences of 6/ 10 at 4 months and 9/ 10 at 6 months. Chim.O and diazotized at 0” using 30. 11. 269 (1951). and M. Received May 26. (2) E.06% level while the control group received only the basal diet. Soc. 179.. No. approximately 8 weeks old and weighing 150-200 g. furfur and P.5-106” (see Table I). 34. bAll compds were analyzed for C.O. ovale and P. University of Kentucky College of Medicine.5-1 06 17 C17H21N3 2’. Van AbbB.). Beecham Products (U. 1972. mp 62-63’.6 g of NaNO.” and 5-aminohemimellitene. J. Hamdan. Chem. (12) A.5-Trimethyl-DAB appears to be about as hepatocarcinogenic as DAB. (5) A. E. for the microscopic evaluation of the tumors. 67. The new azo compounds are listed in Table 1. Chem. Justus LiebigsAnn. I9 71 Table I.5 mg.5‘-Trimethyl-DAB 145-146. Ugelow.5-103 25 C’. The composition of the basal diet per kilogram was as follows: crude casein.1546 (1910). T. Pays-Bas.. Vitab (rice bran concentrate. City of Leicester Polytechnic.212 Journal ofMedicinal Chemistry. Sato.H. Yates. (9) K. Laparotomies were performed at the indicated times and micro- Some Aliphatic Amines as Antipityrosporum Agents Harold B x o n . 1-3 The only active trisubstituted-DAB tested has been the 2’. R. Y . Me~idine.’ 6-aminopse~documene~ was prepd from 6-nitropseudocumene.O.4% of the theoretical values. L. patterned after the “low protein.3’. Department of Pathology.5’-Trimethyl-DAB 131. Sapp. These are all new compounds and can be prepared by the diazotization of the proper trimethylanilines followed by coupling with PhNMez.IN. 15.~ bp 224-228”. Reel. 5-Aminopseudocumene. Kropf. Beringer and I. was obtd from 5-nitrohemimellitene. were distributed as equally as possible in initial body weight into groups of 10 animals each.663 (1961). Brown.6‘-trifluoro deriva t i ~ e It . Trimethvl4-dimethvlaminoazobenzenes Yield“ Compd Formulab recryst % . Cancer Res. Huender.4’. University of Kentucky. N ~~ Pityrosporuni ovale and orbiculare. In spite of this limited pathogenicity or perhaps limited virulence. 57.. 42. References (1) J. Acknowledgment. was prepared from 5-nit~ohemimellitene~ by reduction (Fe-AcOH).3 but more recent work has shown that M. A group received DAB at the 0. Van Abbe2 has discussed the possible relationship between dandruff and the presence of P. was prepd from nitromesitylene6 by reduction with Sn-HCl. 770 g. Chem.. Offic.). Miller. Developments in the cultural technique’*’ have much reduced the problems of assessment of this class of compound. Ass. €1.. Where analyses are indicated only by symbols of the elements analytical results obtained for those elements were within +0. 1 (1915).339 (1953).709 (1959).H.3’. Jones.. Amer. 3’.3’. None of the other trimethyl-DAB homologs produced tumors at 9 months at which time the experiment was terminated.4’. Fujima. CancerRes. N analyses were performed in this department on an F and M Model 185 analyzer by Mr. mp 104. Miller. One-half hr after the final addn. All the rats were kept individually in screen-bottomed cages and were offered food and water ad libitum. (6) J. (4) J. Tinea versicolor was formerly attributed to the presence of Malassazia furfur. Dolinsky. Yamada. M. CancerInst. orbiculare are probably the same organisms in different phases of g r ~ w t h It . Chem. and the solvent was removed to give 19. Nat. and A. The authors are indebted to Dr. C. H. V. Recent work in pyrrolidine chemistry has shown that ethyl AT-alkyl-4-hydroxy- . Miller. The first orange band was eluted.4’. Lexington.500 IU. J. 89.6’-TrimethyLDAB 104. 75. 97. Each group was fed a diet. corn oil.~ seemed of interest to synthesize and test for rat hepatocarcinogenic activity all of the trimethyl homologs of DAB with Me groups in the primed positions only. 4-Amin0hemimellitene. (7) M.5-134 8 C17H21N3 “All samples recrystd from 95% EtOH after chromatog on alumina from toluene-heptane.” mp 75-78’. K. C. England and John K. Brentford. Trimethylmilines. Purvis.. 41. 3‘. low riboflavin” diet of Miller‘ to which had been added one of the azo compounds at a level of 0. 0. and the s o h was stirred for 24 hr. Mesidine (60 g) was dissolved in a mixt of 113 ml of concd HC1 and 376 ml of H. Chem.5‘-Trimethyl-DAB 101. C.. Walling. Young male rats of the Sprague-Dawley strain. Ber..4’. 163 (1875). Daryl Sharp.. Chem. J. England. 264 ml of H.Mp.06%. Brown and A. A. Trav.6’-TrimethyLDAB. Miller and E. W. and 109 g of NaOAc was added. Extn with PhH and evapn of PhH left a semisolid material which was submitted to column chromatog over alumina in toluene-heptane soh. The C. 2436 (1956). Chem. 442 (1968).9 g of the dye as bright orange needles which were recrystd from abs EtOH. Bull. 50 g. Biological Properties. Soc.6’-Trimethyl-DAB 132. Brown* and Alice Kruegel Department of Chemistry. Soc. in 150 ml of H..4’.5‘-DAB gave 9/10 at 4 months and 10/10 at 6 months. Received August I I . (8) C.~mp 29-27”. Daniel L. Ritchie. J.5 55 C.4’. J. J. D. Yoneyama. these yeasts do seem to be implicated in the causation of skin disorders and their suppression is clinically desirable. ovale on the scalp. budding yeasts o f the family Cyptococcacae’ are not usually regarded as pathogens. 40 g.Aduan. “C 2’. 1234 (1968).5-133 57 C17H21N3 2’. Sugden* School of Pharmacy. SOC. A Hall.

05 mole) and catalyzed by Triton B s o h (0.Notes Table I JoumalofMedicinal Chemistry. orbiculure but for studies with P.8) 220 (0. The presence of a secondary amino group was found to be essential for the activity of amines 1 in both assays. ovale. and X = CN having minimal activity of the groups tested. influences the activity of amines 1 against P. Examination of Table I shows that amines (1) have modest activity against P. ir. H. 0. (C.1 mole) were heated in abs EtOH (100 ml) for 18 hr.1 mole).' The final concn of compd tested in agar was within the range 1000-8 . Y. yield. orbiculare (Table I) there is no indication that the length of the aliphatic chain R is critical for inhibitory action.~ work is to investigate the antipityrosporum activity of smaller molecules than those described before' and to indicate some structure-activity relationship with regard to P.0 g (26%). except under condns of primary isolation. 0. ovule. H. 6. N-Hexadecyl-3-aminopropionamide was prepd from hexadecylamine (12. yield.8) 231-232 245 -24 7 Yield. much less active.5 16 125 62. bp 60-80" (see Table I).NO. The technique for detg the MIC involved making serial dilns of the compd in agar and surface inoculating the growth medium with test organisms. 13. with X = CONH. Microbiological Testing. P. Apparently optimal activity is found when the side chain. mp 3940". With regard to P.05 mole) and acrylamide (3.1 g. X = C02Et showing maximal inhibition. Vol. In the case of P. and anhyd Na. ovale and P. Anal. N.05 ml) in the usual manner: mp 7880".1 mole) and recrystd from petr ether: bp 40-60". 0. Formulaa Form Base HC1 Salt Base Base Acetyl deriv of 4 HBr salt Acetyl deriv of 6 Acetyl deriv of 1 HBr salt HBr salt Base Base Base HCl salt HBr salt % P.ovale of amines 1 on malt extract Tween agar medium was observed in those compounds where R was Clo or more. orbiculare is a much less vigorous organism in artificial culture and more exacting in its growth requirements than is P. "C 134-136 (0. ovule. The selectivity of 1. Experimental Section Uv. 0. 0. with X = CN having an intermediate level of activity amongst the groups tested.2 g.No.) C.1 mole). 0. ovule in the malt extract Tween agar medium assay (1 1 and 9 being less active than 6 ) ..3 g. The nature of the functional group. H.1 mole) and ethyl or methyl acrylate. 0.8 g (81%). orbiculare.5>1000 >lo00 500 >lo00 >lo00 31 31 64 128 64 128 Zinc pyriihGne as a control aAll compds analyzed for C. being the least active. ovule there is some correlation between the tests on malt extract Tween agar medium (without added fatty acids or bile salts) and the suspension assay technique. since acetylation abolishes activity (7 and 8). 2 213 P. X = C02Et being the most active and X = CONH. is (CH.O) C. N.).5 500 31 31 31 125 16 strains 14 64 125 >lo00 500 >loo0 125 >loo0 >lo00 48 56 26 81 55 50 50 55 16 30 45 64 63 25 80 128 125 >lo00 500 >loo0 125 >loo0 >lo00 125 256 256b 62. Primary amines (0. needed for P.orbiculare. Alkyl N-Substituted-3-aminopropionates (1).5 g.C03 (10.2 g.5 g (50%). n-Dodecylamine (18.5 500 31 31 31 62..the ox bile and glyceryl monooleate were omitted since these appeared to inactivate the test materials and were not essential for the growth of P. 0. bPartial inhibition at 128 pg/ml.O (20 ml) by a conventional method: mp 64-65".1 mole) and acrylonitrile (5. H. and the product was isolated in theusual way: bp 180-190" (0. yield.6 g. and ethyl chloroacetate (12. After dispersing the compd in 2% Tween 40.N. X = C02Me being slightly less active. they are.7 mm). 12. Ethyl N-Dodecylaminoacetate.. yield. in contrast with the results of tests on P. Minimum Inhibitory Concentration. or ethyl methacrylate (0. X.5>1000 >loo0 16 8 8 5-oxo-3-pyrroline-3-carboxylates have moderate activity The object of the present against P. (CI6H. The latter observation adds some weight to these yeasts being regarded as separate species. ovale and P. N. 1972. and nmr spectra were measured for all compds and were as expected. Acetamido-N-hexadecylamino-3-propionitrile was prepd from N-hexadecyl-3-aminopropionitrile (12. and the product was isolated by the method described by Sugden6 and characterized as the base or converted into an appropriate salt.H4. IS. was significant.5) 176 (0. ovule. two-fold dilns were prepd in measured amt of liquefied culture medium. N-Hexadecyl-3-aminopropionitrile was prepd by the method of Caldo' from hexadecylamine (24. orbicul~re. Optimal activity against P. orbiculare. N. Anal. R X Y Mp or bp (mm).08 mole) in Ac. orbiculare No. The nature of the side chain Y had considerable influence on the activity of the compounds against P. The nature of the functional group.1 g. and 14 does tend to justify the classification of these yeasts as distinct species. orbiculare. ovule strains 1 2 3 8 >loo0 >loo0 > l o 0 0 125 125 125 500 500 500 500 500 500 >loo0 >loo0 >loo0 31 31 31 >loo0 > l o o 0 > l o o 0 500 500 500 125 62.0 g.N.8) 179-180 78-80 38-40 64-65 171-172 3-5 200 (11) 135-136 182-184 180 (0. ovule and P.0) C. 4. The secondary amino group appears to be essential. however. (CI9H4.5-0. Melting points were taken on a Biichi apparatus and are uncorrected. but possibly less effect on activity when tested by the suspension assay.0 g (22%). 500 1000 31 62. X. The growth medium was Dixon's formula' without modification for tests against P.1 mole) were heated under reflux in abs EtOH (100 ml) for 1 hr. Acetyl derivatives were prepd by conventional methods and recrystd from petr ether.Anal. 10. 6 being more active than 9 and much more active than 11.

101g. cryst hydrochloride. min. In view of the very broad pharmacological utility of substituted 2-phenylethylamines. Ph. mp 185. A taxonomic study. The inoculated plates together with the appropriate organism controls were incubated for 3 days at 37" in the case of P. pp. then chilled in ice.'? The dried Et.. Amer. Nystrom.* and Roland K. Synthesis of 2-Fluoro-9-0-D-rib0furanosylpurine ( 2-Fluoronebularine) Masajiro Kawana. C o m e t . 257 (1960).O. I9 71 The antibiotic nebularine (9-p-D -ribofuranosylpurine) has shown tuberculostatic. Hawaii 96822. MIC's were detd by . The org phase was washed with %0. 1 hr).5-186.66 mole) of PhCHO.536 mole) of the hydrochloride by stirring with concd aq NaOH. and heated for an add1 20 min.SO. Org.^ The mode of action has been proposed to be in the purine biosynthetic pathway!.O.' in 200 ml of PhH was added through the condenser at such a rate as to maintain reflux (15 min). Activity of the compds was assessed on the growth observed. C.O. (7) C.D. (2) N. K." North Holland Publishing Co. University of London. and P. Ph. mp 212-214". The medium was poured into petri dishes and left to harden ovemight at room temp. Lucier. 753 (1962). J. which was washed thoroughly with ice-cold 20% EtOH-Et. 28..O followed by 250 ml of 20% of aq NaOH. Received August 6. Syn. The resulting voluminous. The filtrate was mixed with one-third its vol of abs EtOH and 60 ml of concd HCl was added slowly with continuous swirling and ice cooling.72 (1964). E. the mixt was stirred for 90 min and then added to 300 ml of H. 44. alkyl-. No. then. Honolulu. After cooling in ice. was added portionwise. The yield was 83 g (77%). 19 71 from anisyl alcohol. we wish to contribute a synthetic procedure which. After chilling t o Oo. pg/ml. Chem. and discarded. Smiley and C. (5) J. The aq DMSO layer was extd with two 100-ml portions of Et. H. and the resulting soln was evacd at the aspirator for 30 min.. J. The hydrochloride may be recrystd from Et. University of London. Robert J. Chim. Harris. orbiculare. 84. New Compounds A Rapid.2 and anticancer activit^.O. A. Robins ICN Nucleic Acid Research Institute. identified by mass spectroscopy [ m / e 122. p-Anisyl alcohol (100 g. A. 1972. Vol. (Milan). Lodder..214 Journal of Medicinal Chemistry. and halogen-substituted phenethylamines.O. Invest. Each test compd (0. Two Et. 1166-1186. Hogan. and eliminates the need for isolation of intermediates and other time-consuming operations. The mixt was heated under reflux until no more H. Arnold. ~ ~ ~ .65 mole) of Me. After addn was complete.O exts of the basic aq phase were added to the amine layer which sepd.O to remove unreacted PhCHO and made strongly basic with 50% aq NaOH. identified by mass spectroscopy [ m / e 121. granular ppt of NaCl and LiCl and aluminate was removed by filtration.. A sample was inoculated with P.' with icawater cooling to maintain the temp at 35-40". and treated dropwise with 25 ml of H. an attached Dean-Stark trap was removed and a soln of 82 g (0.1 ml of standard suspension contg lo6 organisms/ml) and stored at room temp for 1 hr.725 mole) was shaken with 500 ml of concd HCl for 2 NaHCO. Suspension Technique. 1970.O and dried (MgSO.O and dried. 44. Soc. Convenient Preparative Procedure for Phenethylamines Edgar F. 1370. Received August 16. it is also applicable without substantive modification to other ring alkoxy-. M. (3) T.O. 15.5% then added over 40 min t o a stirred slurry of 49 g (1. cooled slightly. ovule (0. (6) J.. 77.5'. and the whole was washed once with H. which were combined with the product layer. 1964. A dry flask was charged with ca.observing the lowest concn which inhibited growth under the prescribed condns. Although based entirely on standard synthetic methods. 389 (196 1). Thesis.^. The overall yield was 75% tLAH alone and other metal hydride reagents are unsatisfactory for the reduction of benzylic nitriles to amines. "The Yeasts. because of its versatility and convenience. 165 (M+)]. 30. 151 (M+)].O and treated with 50 ml of concd HCl with swirling and cooling to yield the white. Chem. Dermafol.609 (1964). generated from 100 g (0.O through the condenser to replenish losses and facilitate stirring.. 2 New Compounds for 3 days.02 ml of standard suspension). washed well with Et. (4) R. ' ' We wish to report the synthesis of 2-fluoronebularine (2a). (2) R. The 2-phase mixt was heated for 90 min on the steam bath. with periodic addn of Et. Van Abbb. may find considerable use.J. the aq layer was washed twice with Et. Burke. F. Caldo. This material was dissolved in 500 ml of 20% abs EtOH-Et. without cooling.). N-Methyl-p-methoxyphenylethylamine Hydrochloride. California 92664. S . J. J. treated with 200 ml of H. Sternberg and F. 1hr). 0.0 mole) of NaCN in 400 ml of DMSO.6 mole) of anhyd AlC1. and the small upper phase sepd. 36. Ind. Irvine.' antimitotic. (3) J.999 (1961). Kiefer Department of Chemistry. Arch. ovule and up to 5 days in the case of P. The surface of the agar was then inoculated with the test organisms (0. the overall scheme is specifically tailored to the properties of the benzylic intermediates involved. 600 ml of abs Et.6 mole) of LAH. Korosec.O was present in the condensate (ea. Amsterdam. p-Me thoxyphenethylamine.1 g) was dissolved or suspended in Tween 40 (2 ml) and the vol made up to 100 ml with sterile dist H. Experimental Section 4-MethoxyphenylethylamineHydrochloride. University of Hawaii. and H. The mixt was stirred for 2 hr. The procedure is described for the pmethoxy derivative. was treated with 100 ml of PhH and 70 g (0. the cryst amine hydrochloride was collected.O soln of crude p-methoxyphenylacetonitrile was added at such a rate as to maintain gentle reflux without extemal heat (cu. 15. Synthesis of the title compound 2a was accomplished by removal of the benzylthio group from 6-benzylthio-2-fluoronebularine (1)8 with Raney Ni. A mildly exothermic reaction began at once. The test samples and appropriate controls were plated out on petri dishes of Dixon's medium' and incubated at 37" References (1) J. the cooling bath was removed. J. References (1) R. SOC.O and chilled in ice as 80 g (0.' It has limited usefulness because of its high t o ~ i c i t y .D. Dermafol. followed by 23 g (0. Rousseau. leaving 90 g (102%) of crude N-methyl-p-methoxyphenethylamine. 44.2544 (1955). Sugden.OEtOH or i-PrOH. D. . Thesis. Org. 25. 121. Chem.. Keddie.

2009 More About This Article The permalink http://dx. 1155 Sixteenth Street N. convenient preparative procedure for phenethylamines Edgar F.1021/jm00272a029 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society..org on April 25.1021/jm00272a029 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. Chem.Rapid. Med.W. 214-214• DOI: 10. 15 (2). DC 20036 .doi. 1972.acs. Washington.org/10. Kiefer J..

The filtrate was mixed with one-third its vol of abs EtOH and 60 ml of concd HCl was added slowly with continuous swirling and ice cooling. K.999 (1961).725 mole) was shaken with 500 ml of concd HCl for 2 min. and heated for an add1 20 min. and the resulting soln was evacd at the aspirator for 30 min..'? The dried Et. New Compounds A Rapid.O exts of the basic aq phase were added to the amine layer which sepd. the aq layer was washed twice with Et. After chilling t o Oo. Nystrom. 2 New Compounds pg/ml. Experimental Section 4-MethoxyphenylethylamineHydrochloride. identified by mass spectroscopy [ m / e 121. then chilled in ice. generated from 100 g (0. The mixt was stirred for 2 hr. and H. (3) T. Korosec. with periodic addn of Et. The overall yield was 75% tLAH alone and other metal hydride reagents are unsatisfactory for the reduction of benzylic nitriles to amines. (4) R. References (1) J.. and halogen-substituted phenethylamines. Keddie.' with icawater cooling to maintain the temp at 35-40".observing the lowest concn which inhibited growth under the prescribed condns.5'. Received August 6. Activity of the compds was assessed on the growth observed. University of London. 151 (M+)]. we wish to contribute a synthetic procedure which. Amsterdam. The hydrochloride may be recrystd from Et. Ind. Ph. Rousseau. the overall scheme is specifically tailored to the properties of the benzylic intermediates involved. and P. 1 hr). Thesis. A sample was inoculated with P. (2) R. A taxonomic study. Org. granular ppt of NaCl and LiCl and aluminate was removed by filtration. Chim. J. Hogan. J. 121.D.' in 200 ml of PhH was added through the condenser at such a rate as to maintain reflux (15 min). 44. Received August 16. The aq DMSO layer was extd with two 100-ml portions of Et.0 mole) of NaCN in 400 ml of DMSO. Burke.O and treated with 50 ml of concd HCl with swirling and cooling to yield the white. E. D. Org. S . A mildly exothermic reaction began at once. Arch.' It has limited usefulness because of its high t o ~ i c i t y . 15. Irvine. Arnold. Synthesis of the title compound 2a was accomplished by removal of the benzylthio group from 6-benzylthio-2-fluoronebularine (1)8 with Raney Ni.2544 (1955). 36. (5) J.72 (1964). Two Et. J. C o m e t . Convenient Preparative Procedure for Phenethylamines Edgar F. 1166-1186. The medium was poured into petri dishes and left to harden ovemight at room temp. Amer. The resulting voluminous. Invest. Vol. 30. treated with 200 ml of H. Sternberg and F.' antimitotic.O.1 ml of standard suspension contg lo6 organisms/ml) and stored at room temp for 1 hr. ' ' We wish to report the synthesis of 2-fluoronebularine (2a). from anisyl alcohol. Robins ICN Nucleic Acid Research Institute. Although based entirely on standard synthetic methods. and the whole was washed once with H. The yield was 83 g (77%). Chem. ~ ~ ~ . identified by mass spectroscopy [ m / e 122. Lodder. M. without cooling.O. leaving 90 g (102%) of crude N-methyl-p-methoxyphenethylamine. which were combined with the product layer. 257 (1960). The surface of the agar was then inoculated with the test organisms (0. 600 ml of abs Et. Hawaii 96822. the cooling bath was removed. and the small upper phase sepd..1 g) was dissolved or suspended in Tween 40 (2 ml) and the vol made up to 100 ml with sterile dist H. The mixt was heated under reflux until no more H.214 Journal of Medicinal Chemistry.66 mole) of PhCHO. may find considerable use.O. The inoculated plates together with the appropriate organism controls were incubated for 3 days at 37" in the case of P.SO. mp 185.. ..O followed by 250 ml of 20% of aq NaOH.O to remove unreacted PhCHO and made strongly basic with 50% aq NaOH. cryst hydrochloride. Honolulu.02 ml of standard suspension). the cryst amine hydrochloride was collected. and treated dropwise with 25 ml of H. Caldo. and eliminates the need for isolation of intermediates and other time-consuming operations.6 mole) of anhyd AlC1. The procedure is described for the pmethoxy derivative. 1972.O and chilled in ice as 80 g (0.5-186. Dermafol. 753 (1962). N-Methyl-p-methoxyphenylethylamine Hydrochloride.5% NaHCO. J. University of London. MIC's were detd by . California 92664. A. 77. C.O through the condenser to replenish losses and facilitate stirring. 15. Chem. was added portionwise. 44. SOC. "The Yeasts.J. (7) C.6 mole) of LAH. then added over 40 min t o a stirred slurry of 49 g (1. References (1) R. mp 212-214".O and dried.D. Kiefer Department of Chemistry.. Syn. 389 (196 1). After addn was complete. washed well with Et. (2) N.O soln of crude p-methoxyphenylacetonitrile was added at such a rate as to maintain gentle reflux without extemal heat (cu." North Holland Publishing Co.. (Milan).* and Roland K.^ The mode of action has been proposed to be in the purine biosynthetic pathway!.).536 mole) of the hydrochloride by stirring with concd aq NaOH. it is also applicable without substantive modification to other ring alkoxy-. Each test compd (0. an attached Dean-Stark trap was removed and a soln of 82 g (0. A. 28.O. Dermafol. which was washed thoroughly with ice-cold 20% EtOH-Et. ovule (0. followed by 23 g (0. (3) J. Soc.84. The org phase was washed with %0. Synthesis of 2-Fluoro-9-0-D-rib0furanosylpurine ( 2-Fluoronebularine) Masajiro Kawana. Van Abbb. 1964. University of Hawaii. After cooling in ice.OEtOH or i-PrOH. the mixt was stirred for 90 min and then added to 300 ml of H. was treated with 100 ml of PhH and 70 g (0. 1970. cooled slightly. because of its versatility and convenience. A dry flask was charged with ca.609 (1964). No. alkyl-. p-Anisyl alcohol (100 g. p-Me thoxyphenethylamine. Harris. ovule and up to 5 days in the case of P.O.^.. 1hr). pp. Suspension Technique. then. F. The test samples and appropriate controls were plated out on petri dishes of Dixon's medium' and incubated at 37" for 3 days. and discarded. Robert J. Lucier. Ph.65 mole) of Me. I9 71 The antibiotic nebularine (9-p-D -ribofuranosylpurine) has shown tuberculostatic. 44.O and dried (MgSO. 1370.O. Sugden. Smiley and C. J.O was present in the condensate (ea. The 2-phase mixt was heated for 90 min on the steam bath. This material was dissolved in 500 ml of 20% abs EtOH-Et. (6) J. orbiculare. 101g. Thesis.2 and anticancer activit^. 0. Chem. 165 (M+)]. H. 19 71 In view of the very broad pharmacological utility of substituted 2-phenylethylamines. 25.

-D-ribofuranosylpurine (2-fluoronebularine) Masajiro Kawana.Synthesis of 2-fluoro-9-.doi.acs. 214-215• DOI: 10. Chem.W. 1155 Sixteenth Street N.org on April 25. Med. DC 20036 . 2009 More About This Article The permalink http://dx. Rousseau. 15 (2).1021/jm00272a030 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs. Robert J... 1972. Washington.beta. Robins J. and Roland K.org/10.1021/jm00272a030 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society.

followed by 23 g (0. identified by mass spectroscopy [ m / e 121.5'. the cryst amine hydrochloride was collected. A. pg/ml. and heated for an add1 20 min.O soln of crude p-methoxyphenylacetonitrile was added at such a rate as to maintain gentle reflux without extemal heat (cu. identified by mass spectroscopy [ m / e 122. Chem. Robins ICN Nucleic Acid Research Institute. S . because of its versatility and convenience. Hogan. 15. The inoculated plates together with the appropriate organism controls were incubated for 3 days at 37" in the case of P. 84. we wish to contribute a synthetic procedure which. and the resulting soln was evacd at the aspirator for 30 min. J. 600 ml of abs Et. MIC's were detd by . Synthesis of the title compound 2a was accomplished by removal of the benzylthio group from 6-benzylthio-2-fluoronebularine (1)8 with Raney Ni. The medium was poured into petri dishes and left to harden ovemight at room temp. Lodder. pp. 77. This material was dissolved in 500 ml of 20% abs EtOH-Et. References (1) R.1 g) was dissolved or suspended in Tween 40 (2 ml) and the vol made up to 100 ml with sterile dist H. Syn. A dry flask was charged with ca.O exts of the basic aq phase were added to the amine layer which sepd.. it is also applicable without substantive modification to other ring alkoxy-." North Holland Publishing Co. 19 71 from anisyl alcohol. (Milan).O.2 and anticancer activit^.66 mole) of PhCHO. Received August 16. A sample was inoculated with P.* and Roland K. 15. Chim. The procedure is described for the pmethoxy derivative. Experimental Section 4-MethoxyphenylethylamineHydrochloride. (6) J. ' ' We wish to report the synthesis of 2-fluoronebularine (2a). and treated dropwise with 25 ml of H. and the small upper phase sepd. generated from 100 g (0. The yield was 83 g (77%).^ The mode of action has been proposed to be in the purine biosynthetic pathway!. was added portionwise. Nystrom. Org. Soc. (3) J. granular ppt of NaCl and LiCl and aluminate was removed by filtration.' It has limited usefulness because of its high t o ~ i c i t y . 1hr). In view of the very broad pharmacological utility of substituted 2-phenylethylamines. with periodic addn of Et. (3) T. cryst hydrochloride. the aq layer was washed twice with Et. N-Methyl-p-methoxyphenylethylamine Hydrochloride. M. J. min. 753 (1962). The surface of the agar was then inoculated with the test organisms (0. Convenient Preparative Procedure for Phenethylamines Edgar F.O and chilled in ice as 80 g (0. 36. washed well with Et. Smiley and C. and the whole was washed once with H. Korosec.O.999 (1961). then chilled in ice. Honolulu. A mildly exothermic reaction began at once. "The Yeasts.' with icawater cooling to maintain the temp at 35-40". 257 (1960). The org phase was washed with %0.. D.214 Journal of Medicinal Chemistry. The overall yield was 75% tLAH alone and other metal hydride reagents are unsatisfactory for the reduction of benzylic nitriles to amines.O to remove unreacted PhCHO and made strongly basic with 50% aq NaOH. 1970. 30. 25. and eliminates the need for isolation of intermediates and other time-consuming operations. Chem. and P.J. Lucier. After chilling t o Oo. may find considerable use. Kiefer Department of Chemistry. Irvine. 44. Ph.observing the lowest concn which inhibited growth under the prescribed condns. and discarded. After addn was complete. Chem.. Invest. Amsterdam. Dermafol. and H. H.O. (2) R. SOC. mp 185. Hawaii 96822. 101g.' antimitotic. Caldo.O and dried (MgSO. ovule (0. Rousseau. Two Et. Keddie. University of Hawaii. Thesis. orbiculare. .SO. an attached Dean-Stark trap was removed and a soln of 82 g (0. F. the cooling bath was removed. No. Ph.D. Thesis. 28.'? The dried Et. Each test compd (0. which was washed thoroughly with ice-cold 20% EtOH-Et. The resulting voluminous. 1370. The test samples and appropriate controls were plated out on petri dishes of Dixon's medium' and incubated at 37" References (1) J. 1972. California 92664. and halogen-substituted phenethylamines. Amer.536 mole) of the hydrochloride by stirring with concd aq NaOH. K. J. 151 (M+)]. A. p-Me thoxyphenethylamine. New Compounds A Rapid. 1 hr).. ~ ~ ~ .O.O through the condenser to replenish losses and facilitate stirring. The aq DMSO layer was extd with two 100-ml portions of Et. Ind. 389 (196 1). I9 71 The antibiotic nebularine (9-p-D -ribofuranosylpurine) has shown tuberculostatic. which were combined with the product layer. Vol.O was present in the condensate (ea. Activity of the compds was assessed on the growth observed. A taxonomic study. ovule and up to 5 days in the case of P. J. The filtrate was mixed with one-third its vol of abs EtOH and 60 ml of concd HCl was added slowly with continuous swirling and ice cooling. the mixt was stirred for 90 min and then added to 300 ml of H. Dermafol.5-186. E.2544 (1955). then. 165 (M+)]. was treated with 100 ml of PhH and 70 g (0.' in 200 ml of PhH was added through the condenser at such a rate as to maintain reflux (15 min). 44. cooled slightly. (2) N.72 (1964). alkyl-. Sugden. The 2-phase mixt was heated for 90 min on the steam bath. C. Sternberg and F. J... 44. Arnold. University of London. Received August 6.O and treated with 50 ml of concd HCl with swirling and cooling to yield the white. 1964.5% then added over 40 min t o a stirred slurry of 49 g (1. leaving 90 g (102%) of crude N-methyl-p-methoxyphenethylamine. After cooling in ice.609 (1964). The mixt was stirred for 2 hr. the overall scheme is specifically tailored to the properties of the benzylic intermediates involved. (5) J.1 ml of standard suspension contg lo6 organisms/ml) and stored at room temp for 1 hr.O followed by 250 ml of 20% of aq NaOH. mp 212-214".02 ml of standard suspension). University of London.D.).725 mole) was shaken with 500 ml of concd HCl for 2 NaHCO. Harris. Org. Suspension Technique.O. Arch.. Synthesis of 2-Fluoro-9-0-D-rib0furanosylpurine ( 2-Fluoronebularine) Masajiro Kawana. Van Abbb. Robert J.^. Burke. 121. treated with 200 ml of H.6 mole) of anhyd AlC1. 1166-1186. p-Anisyl alcohol (100 g. The mixt was heated under reflux until no more H. 2 New Compounds for 3 days.65 mole) of Me.O.0 mole) of NaCN in 400 ml of DMSO..O and dried. without cooling.6 mole) of LAH. (4) R. (7) C. 0. C o m e t .OEtOH or i-PrOH. The hydrochloride may be recrystd from Et. Although based entirely on standard synthetic methods.

8 Hz.O.h z F H 264 (80bO). [a]"D -3. Brown and V. colorless foam. washed with a little MeCN and thoroughly with H..07g (27%) of analytically 264 nm pure 2b as a lass. Evapn of the solvents gave a column with EtOAc-EtOH (955.. H 0). and W. and the soln was extd with two 150-ml portions of CH. R. To a stirred s o h of 50 ml of 48-50% HBF. O in 30 ml of EtOH was added a suspension of 14 g of Raney Ni in 60 ml of EtOH. 1 undissolved material was removed by fitration with charcoal and the filtrate was evapd to dryness in vacuo to give 590 mg (55%) of crude 2a as a foam. Calcd for C. Hamor. and W.O. Experimental Section? 2-Fluoro-9-(p-D-ribofuranosyl)purine (2a). Clark.2 Hz. 4 X 30 cm) using EtOAc-heptane (7:3.03mole) of NaNO. University of Southern California. Paris. W.3256 (1957).01 mole) at -20 to of 9-(2. H.New Compounds SCH. Tipson. Anal. 99-loo%.. P.2 Hz. 3 ' ( c 1.343 (1960). 1 H.O over a period of 1 at the same temp for another 15 min and 50 ml of EtOH (precooled 2 0 ' ) was then added. Magrath.3752 (1965).S) C. The catalyst was removed by filtration with a Celite pad and washed thoroughly with boiling EtOH. R = COCH. Shapiro. Amer. J I t . The mixt was neutralized with ea. Weliky. A. Aures. W.Cl. and then dried (MgSO.O.O gave 8. gave I. HI. Luning. D. W. K. Where analyses are indicated only b y symbols of the elements.. Acta Chem. G. P. M. (4) M. I1 (13 g.5-146O. I.. Y.N... 19. Microanalyses were performed by Heterocyclic Chemical Corp. N.). 1 H. Zorbach and R. 2HC1) C. (9) W.13 (d. The mixt was refluxed for 40 min with stirring. . ReceivedAugust 27.3 g (0. H. Chem. Truant and H. 26 below ml of concd NH. Margolis. Amer. Where analyses are indicated only b y symbols of the elements. N. JH F = 1. H.40/0 of the theoretical values. Tor- rance.377 (1959). W. N.1037 (1971).214mole) ofN-hydroxyphthalimide in 250 ml of MeCN and 43.927 (1956)..83 ( s . Elemental anal. The bined filtrate and washings were concd to ea. 5. and S.. He).Taylor and S. Calif. The com0 ml in vacuo. Abstr. G. Chem. 87. Biesele.nmr (DMSO-d. 9-(2. Keasling. 1971 Recent reports showing the title compound (I) to possess potent in vitro and in vivo inhibition of specific histidine decarboxylase' and to markedly lower rat brain histamine.) 6 6. J.FN.p 179. in 150 ml of anhyd EtOH. The resulting ppt was removed by filtration and washed with 50 ml of cold EtOH. 205 (1970). The exts were washed with. W. To a s o h of 34. 14.. D. k&il 265 (72001.3. D'Amato. which was crystd from EtOAc contg a small amt of MeOH: mp 144.9 g (73%) of crude 2b as a gummy material.H. J. Clark.H.1019 (1953).428mole) of Et.N. Basel.. Anal. Slautterback. 9. 14572 (1965). H. 172. Chem.H.uv Aky: 263 nm (E 8000). A.7 ppm. 0 min. 'i H.1 g (0. "Synthetic Procedures in Nucleic Acid Chemistry.OS. the cryst ppt was filtered. D. Y. to yield 24 g (43%) of cryst product. Bomstein. Los Angeles. . Fed. E. 62. Fractions 26-35 contd 960 mg (24%) of 2b. F. After cooling. (7) M. Ir spectra (Nujol mull) were measured on a Perkin-Elmer infracord 137 spectrometer.. New York. 73. D. was added 3.. (2) J. D . ethanolic HC1 was added to the filtrate. 15. G. nmr for F (DMSO-d. Chem. Williams.v/v). analytical results obtained for those elements were within *0. 8..9 g (0. Vol. successively.. Chem. Experimental Section? N-(4-Thiazolylmethoxy)phthalimide(11).. S. Med. and M.'J. C.225 (1953).M. and H. Soc. J. Scand. G. H. Garmaise.. M.2y3 prompt me to report its synthesis. HI. I Journal ofMedicina1 Chemistry. hkyil 264 (7500). M. Soc:.H. G. Anal. Brown. 4-[(Aminooxy)methyl]thiazoleDihydrochloride (I). (8) J. (C. Harrisonville. Lofgren and B. and dried.Gerster and R.391 (1955). B. Schuman. The reaction mixt was stirred in 4 ml of H..which was contaminated with a trace of impurity. This product was chromatogd on a silica gel column (130g. By literature methodsF6 alkylation of N-hydroxyphthalimide with 4-chloromethylthiazole. J.4% of the theoretical values. M. S.COOH as an external standard. 8. 50 ml at 30-35" in vacuo. 2 215 HO OH RO OR 1 2 a. Menon. and the cryst solid was filtered off and dried. J. : C. 50 ml of 1% NaHCO. Science. 4th International Pharmacology Congress. Fox. 8. Best. The F nmr spectra were run with 1%CF.64 g (4 mmoles) of 1 H .88. Recrystn from EtOH-Et.05 mole) was refluxed for 2 hr with 2 . (5) R. 7.5-tri-O-acetyl-p-D-ribofuranosyl)-2-aminopurine9 -25". 4-[(Aminooxy)methyl] thiazole Dihydrochloride Glenn H. J.00 (d.H~. Fractions 36-67 were combined and evapn of the solvents gave 1. H.5" (c 2. Aures.O. Biol.8 ppm. Hamor Department of Biomedicinal Chemistry. * = 4." Interscience Publishers. Veldkamp. T. N. HR).4 Hz.9.O) 6 6. 50 ml of H.3. analytical results obtained for those elements were within +0. (1951). Absorption bands were as expected.9 g (0. H. J. J. W. and S.[ a ] " D 3 2 . No. Winzler. Howes.FN.5 g (83%) of white solid. P.455 (1965). Martin. Laws. 38..). mp 158159"(EtOH).OH to pH 6 below 1 5 ' . J . Wells. Cafid for C. Biol.). nmr (DMSO-d.06 (4JH F = 1. Drain.O. Caldwell and S. California 9000 7. B. Brown. Gordon.214mole) of 4-chloromethylthiazole hydrochloride' and refluxed for 6 hr. (6) A. and H. nmr for F (DMSO-dI. Can. K. 220.5-Tr1-O-acetyl-~-D-ribofuranosyl)-2-fluoropur~e (2b). After cooling and removal by filtration of pptd phthalhydrazide..) -27. at 4 ' occurred with concommitant displacement of the F at and formation of 9-p-D-ribofuranosyl-2-aminopurine.3 g (0.83 (s. F.N was added 36. J. D.Chem. 1969. G. Fed. Williams. Soc. British Patent 984. Anal. Proc. To a boiling soln of 1.87 (1955). Pharmacologist. 204. B. 12. I.). 1972. (3) G. Gordon and G. J.CHCl. The combined filtrate and washings were concd to ea. and 250-ml fractions were collected. The product was chromatogd on a silica gel v/v). CancerRes.. Amer.p 244.). School of Pharmacy. L. 5 g (0. L. Gelblum. 1 H.2935 References (1) N. and two 50-ml portions of H. Amer.C. ?Melting points were taken on a Thomas-Hoover capillary melting point apparatus and are uncorr.33 (d. Snyder. Exp. 0. followed by hydrazinolysis. (C.O. References D. J. and G. Cancer (Philadelphia).R=H b. S. 79. N. 1 H.: C. McKay.D. Evapn of the solvents gave 2. Biol.305 (1965).) -24. Chem. Robiqs. H. Burr. Attempts to remove the Ac blocking groups of 2b with EtOHNH. E. Mo. 1968. Soc. To this mixt was added a soln of 2. J. ?Melting points were detd with a Fisher-Johns app and are uncorr. were performed b y Elek Microanalytical Laboratories. uv (E 7300). 1 H. Chem.). R. K. mp 1761 7 7 ' dec. 8.05 mole) of hydrazine hydrate..

4-[(Aminoxy)methyl]thiazole dihydrochloride Glenn H. 1972.doi. Hamor J.1021/jm00272a031 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. 15 (2).org/10. Med. 215-215• DOI: 10. 2009 More About This Article The permalink http://dx. Washington. Chem.org on April 25. 1155 Sixteenth Street N.. DC 20036 .acs.W..1021/jm00272a031 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.

p 244. J. 50 ml at 30-35" in vacuo. Biol. ReceivedAugust 27.07g (27%) of analytically 264 nm pure 2b as a lass. Cafid for C. Amer. Amer. 12.O. Robiqs.225 (1953).OS.05 mole) was refluxed for 2 hr with 2 . washed with a little MeCN and thoroughly with H.4 Hz. (3) G. Mo. Microanalyses were performed by Heterocyclic Chemical Corp. The F nmr spectra were run with 1%CF. and W.5 g (83%) of white solid. 3 ' ( c 1.O. School of Pharmacy. were performed b y Elek Microanalytical Laboratories. and H. H. Absorption bands were as expected. W.H.. Clark.nmr (DMSO-d. McKay. H. mp 158159"(EtOH). and then dried (MgSO..927 (1956). HI. No. Science.3. Soc. D.O gave 8. Best. J. 1969. The bined filtrate and washings were concd to ea.9 g (0. The product was chromatogd on a silica gel v/v). N.) -27.R=H b. Howes. Basel. To a s o h of 34. G. J.83 ( s .3752 (1965).5-146O. Soc.Cl. G.H. CancerRes. Experimental Section? 2-Fluoro-9-(p-D-ribofuranosyl)purine (2a). This product was chromatogd on a silica gel column (130g. 8. Torrance.06 (4JH F = 1.uv Aky: 263 nm (E 8000).). and W.. mp 1761 7 7 ' dec.3 g (0. N. F.455 (1965).1037 (1971). K. the cryst ppt was filtered.3. 4 X 30 cm) using EtOAc-heptane (7:3. New York. Proc. He). (4) M. J. P.OH to pH 6 below 1 5 ' . J. J. Schuman. Pharmacologist. and S.N was added 36. H.H~.Taylor and S.01 mole) at -20 to of 9-(2. B..). Brown and V. P. Fractions 26-35 contd 960 mg (24%) of 2b. Winzler. Menon. I1 (13 g. (2) J.) -24.. and G. 14572 (1965). Fox.O. H. 73. D. 1972. and 250-ml fractions were collected.M. * = 4. Med. W. nmr (DMSO-d. A.9. W. B. 2HC1) C. O in 30 ml of EtOH was added a suspension of 14 g of Raney Ni in 60 ml of EtOH.64 g (4 mmoles) of 1 H .. N.88. J. G. HR). T.3256 (1957). By literature methodsF6 alkylation of N-hydroxyphthalimide with 4-chloromethylthiazole.. H. ethanolic HC1 was added to the filtrate. Anal. and the soln was extd with two 150-ml portions of CH. (C. 8. Luning. Evapn of the solvents gave 2. and S. Slautterback. Anal.. R = COCH. To this mixt was added a soln of 2. S. G. (6) A.O. J. Where analyses are indicated only b y symbols of the elements.3 g (0. Chem.5" (c 2...9 g (73%) of crude 2b as a gummy material. Ir spectra (Nujol mull) were measured on a Perkin-Elmer infracord 137 spectrometer.305 (1965). Gelblum. H 0). Evapn of the solvents gave a column with EtOAc-EtOH (955.05 mole) of hydrazine hydrate. S.4% of the theoretical values. M. 1 H. G. 'i H. Elemental anal. Wells.New Compounds SCH. D . G. 9-(2.H. J I t . L.Gerster and R. k&il 265 (72001. Veldkamp. J. and M. which was crystd from EtOAc contg a small amt of MeOH: mp 144. 19. British Patent 984.. (7) M. 7. N. W. B. Fed. 8. Chem. Martin. (5) R. The combined filtrate and washings were concd to ea. Hamor. 1 H. 204. The resulting ppt was removed by filtration and washed with 50 ml of cold EtOH. Lofgren and B.D. I Journal ofMedicina1 Chemistry. To a boiling soln of 1.. 4-[(Aminooxy )methyl]thiazole Dihydrochloride Glenn H. 1971 Recent reports showing the title compound (I) to possess potent in vitro and in vivo inhibition of specific histidine decarboxylase' and to markedly lower rat brain histamine. 87.2y3 prompt me to report its synthesis. H. analytical results obtained for those elements were within +0. Attempts to remove the Ac blocking groups of 2b with EtOH' occurred with concommitant displacement of the F at NH. (1951). K. Snyder.COOH as an external standard. M. 9. Tipson. analytical results obtained for those elements were within *0. in 150 ml of anhyd EtOH.9 g (0. 172. D. P.87 (1955). After cooling and removal by filtration of pptd phthalhydrazide. 1 H. : C. Chem. S. Chem.: C. Calcd for C.. Y.N. Brown. 38. Gordon and G. Clark. I. 50 ml of H. 99-loo%. Williams. R. Biol.. Soc. E.2 Hz. K.214mole) of 4-chloromethylthiazole hydrochloride' and refluxed for 6 hr. Experimental Section? N-(4-Thiazolylmethoxy)phthalimide(11). Brown.CHCl.H. University of Southern California. Scand. 4-[(Aminooxy)methyl]thiazoleDihydrochloride (I). H.. The catalyst was removed by filtration with a Celite pad and washed thoroughly with boiling EtOH. J. Weliky. 0. F. .).83 (s. 15. Chem. . at 4 and formation of 9-p-D-ribofuranosyl-2-aminopurine.5-tri-O-acetyl-p-D-ribofuranosyl)-2-aminopurine9 -25". Harrisonville. C. Gordon. Amer. (9) W. D. ?Melting points were detd with a Fisher-Johns app and are uncorr. D.h z F H 264 (80bO). Aures. Recrystn from EtOH-Et. J.. Anal.13 (d. R. 1968. J . Calif. After cooling. hkyil 264 (7500).C. H. J. 2 215 HO OH RO OR 1 2 a. M. 62.O. Abstr. 79. Keasling.. successively. The reaction mixt was stirred at the same temp for another 15 min and 50 ml of EtOH (precooled below 2 0 ' ) was then added. A. Acta Chem. 5 g (0. Fractions 36-67 were combined and evapn of the solvents gave 1..8 ppm. 5. 1 H. Biesele. Drain. Chem." Interscience Publishers..391 (1955).2935 References (1) N.FN.S) C.7 ppm. HI. M.). To a stirred s o h of 50 ml of 48-50% HBF. gave I.. Hamor Department of Biomedicinal Chemistry. The mixt was neutralized with ea. Laws. 4th International Pharmacology Congress. 205 (1970).377 (1959). References D.) 6 6. Truant and H. Anal. nmr for F (DMSO-d. Williams.1019 (1953). Burr. Zorbach and R. (8) J.v/v). and H. Bomstein. N. D'Amato. JH F = 1.Chem. (C.2 Hz.N. 50 ml of 1% NaHCO. and dried. E. The exts were washed with.1 g (0.p 179.[ a ] " D 3 2 . Y. [a]"D -3. in 4 ml of H. Magrath. 14. Can. ?Melting points were taken on a Thomas-Hoover capillary melting point apparatus and are uncorr. Cancer (Philadelphia). nmr for F (DMSO-dI.). W. Biol. L.33 (d.O) 6 6.8 Hz. Exp. 1 H. I. Shapiro.which was contaminated with a trace of impurity.FN. The com0 ml in vacuo. 8.O.O over a period of 1 0 min. and the cryst solid was filtered off and dried. Soc:.214mole) ofN-hydroxyphthalimide in 250 ml of MeCN and 43.03mole) of NaNO.00 (d. 1 undissolved material was removed by fitration with charcoal and the filtrate was evapd to dryness in vacuo to give 590 mg (55%) of crude 2a as a foam..). 26 ml of concd NH. Paris.40/0 of the theoretical values.428mole) of Et. "Synthetic Procedures in Nucleic Acid Chemistry. and two 50-ml portions of H. to yield 24 g (43%) of cryst product. Fed.5-Tr1-O-acetyl-~-D-ribofuranosyl)-2-fluoropur~e (2b). W. Caldwell and S. Chem.. Los Angeles. followed by hydrazinolysis.343 (1960). California 9000 7. was added 3. uv (E 7300). Where analyses are indicated only b y symbols of the elements. Vol. Amer. The mixt was refluxed for 40 min with stirring. 220.'J. Aures. colorless foam. Garmaise. Margolis.

1155 Sixteenth Street N.1021/jm00272a900 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society. 216-216• DOI: 10.org on April 25.1021/jm00272a900 • Publication Date (Web): 09 January 2004 Downloaded from http://pubs.. 1972.acs.Contractile Proteins and Muscle Alfred Burger J. Chem. Washington. 2009 More About This Article The permalink http://dx.org/10.. Med. 15 (2).W. DC 20036 .doi.

eti . Holloway). N. New York. steroid metabolism (P. centers on the follov. actin. It is hoped that the publishing companies in the near future will take a serious look at the best manner in which to provide texts and reference books at a reasonable price and also consider a new mechanism by which publication can be achieved in a shorter period of time. Virginia University of Virginia Charlottesville. and the withdrawal syndrome. M. and regression analysis aided by MO calculations now looks only like another tool of limited applicability which will save some time in routine molecular modification. contains a review of linear free energy and MO hypotheses in drug design. The authors are all expert. and these condensed reviews are followed by careful compilations of the effects of narcotic analgetics upon these processes and the enzymes involved. though not as closely interrelated as the first. The 6 major topics reviewed in this volume are CNS agents. The decomposition of ATP is tied to myosin and muscle work. it will prove to be an important guide-post for several years to come. J. antiarrhythmic. we know the major metabolites of several of the drugs.00. It contains a succession of segments of thin and thick filaments. These difficulties are multiplied in the attempts to explain tolerance. CNS location. 1971. physiological-chemical aspects of fatty acid oxidation (R. to be sure. Arizona University of Virginia Charlottesville. 25. takes over and encounters all the vicissitudes of such studies. ix + 300 pp. Essentially the book can be divided into two major parts. 16. the subtitle of the volume. Each major biosynthetic pathway (carbohydrates. with 14 other contributors.50 Muscle transduces chemical energy into mechanical work. 1972. $28. dependence. The thin filaments appear to make contact with the headpiece of myosin which bends out from the thick filaments. New York. Szent-Gyorgyi’s early observations on the chemistry of muscle contraction to the present. Bressler). and myosin are discussed in individual chapters. Virginia Alfred Burger Narcotic Drugs. Biochemical Pharmacology. pharmacodynamic agents (antihypertensives. The other major division. Marcel Dekker. etc. Biochemists will profit from reading about the chemical changes in contracting muscle and about muscle as a fibrous protein system. biochemical pharmacology. Metabolites of alkaloidal and synthetic potent analgetics have been identified. myotonias. lipids. Cain and 6 section editors. Y. 1971. Wakil). $9. xi + 613 pp. We have a dim outline of some three-dimensional regions of the analgetic receptors. Itanahan Department of Biochemistry Universily of Arizona Tucson. The area of muscular disorders. They represent authoritative. and quite up-todate. This is one of the few books recently published on lipids to which the term “significant” can be applied. Topics in Chemistry. tropomysin A and B. the clinically used and a few major experimental compounds and offer historical views of the intellectual processes that led t o their development. 16 X 23.5 X 23. Lands). Flagellin. with 12 contributors. 1970. and critical examination is accorded to these reactions. For the anatomist. stereochemically oriented. Hubscher). but it has not yet been possible to single out any specific enzyme system. and we PO’: sess compounds that can counteract heroin dependence or at least modify withdrawal therapeutically. The annual feature. This book will do much to make available the literature and expert judgment of the background and status in this field.50. It is obvious that the potent analgetics disrupt many physiological functions. Edited by C . Undoubtedly. their methods of chemical analysis. should now open up to causative cures. $42. 1971. there were no real breakthrough3 reported during 1970. dystrophies. glycogenoses.. However. N.S Y 2 7 h cm. the first of which encompasses fa-tty acid metabolism (S. and metabolic disease and endocrine function drugs. . No. phospholipid metabolism (E. These chapters are well developed and provide an in-depth description of progress in these fields to 1969. Virginia Alfred Burger Annual Reports in Medicinal Chemistry. bacterial lipids (W. Wakil. platelet aggregation inhibitors. ACh. Drug metabolism and adenylcyclase and CAMPanalogs are covered in Topics in Biology. The selection of the topics reflects the general activity in medicinal research. others almost free from dependence liability. K. as well as lesser articles on pharmaceutic subjects. University of Virginia Alfred Biirger Charlottesville. Where are the new drugs?” Many major companies now have promising novel drugs in clinical trial. Lennarz). Edited by Salih J. Lipid Metabolism. This is a timely and well-written book which is a formidable addi. and structure-activity relationships among these drugs. Hill and W. Perhaps the only de. biosynthesis of polyisoprenoid quinones and related compounds (R. No other exciting ideas have appeared on the horizon. Edited by Doris H. But from Chapter 4 on. chemotherapeutics. Donald J. Academic Press.00. The cycle is concluded by disengagement of the filaments and elongation of the shortened fibriles. tion to the literature of lipid biochemistry. paper-back. Samuelson). The evolution of muscles may date back to ciliary and flagellar apparatus which are treated as suitable objects of study. The many conflicting hypotheses concerning these phenorena are reviewed skillfully but the authors have been careful not to propose experimentally unwarranted conclusions.8 cm. fatty acid metabolism of plants (P. Editor Cain now publically avows the position this reviewer has taken for several years when he writes in the preface “. and some virtually free from side-effects at therapeutic doses. catecholamines. a reversal of the present pessimistic trend may be expected around 1976.). and glyceride metabolism (G. Y. proteins. Plenum Press. Y. and a chapter on the uterus as a model for medical understanding and therapy. R Stumpf). E. gastrointestinal. chapters on the functional morphology and the development of muscle and the excitable membrane will be of value. New York. this book is an account of the history of muscle research from A. J is presented to the reader as background information. which at $28. . The chapter on analysis contains all modern spectroscopic methods used in the analysis of minute quantities of foreign substances in body fluids and tissues and should be useful far beyond the scope of this specific class of drugs. tracting factor is the cost of the book. The high hopes statistical and quantitative methods have held out are beginning to fade. New York. If nothing happens to them on the way to the FDA. E. xxii + 506 pp. $28. The introductory chapters of this book deal with the structure of narcotic analgetics. or interference with a metabolite bjosynthesis as a decisive step. S. well documented. and up-todate factual reviews of . 49 contributors. 2 Book Reviews Book Reviews Contractile Roteins and Muscle. We have today analgetics thousands times more potent than morphine. W.5 cm. Medicinal chemists will welcome an outline of muscle diseases (atrophies. This may well characterize the whole area of Medicinal Chemistry at present. N. We are at the verge of recognizing the role of individual proteins in these changes. in spite of the herculean amount of work that has been done on these questions. Clouet. Wakil has done an excellent job in assembling a group of highly respected scientists to write on various facets of lipid metabolism and has provided a wellintegrated book on topics ranging from fatty acid metabolism to the biosynthesis of polyisoprenoid quinones. treatable mostly only by methods which suppress symptoms. ing important topics: prostaglandins (B. in their respective areas of interest and this is certainly indicated by depth of understanding and coverage in each chapter. All the reviews are well edited. and these sections shorten while the unengaged filaments slide. myasthenias. This book is a capable review o f all these facets and is full of suggestions for future researches.50 will rule out many graduate students and postdoctorates and even some of the less affluent professors. particularly in the industry. xii + 606 pp. Academic Press. but the explanation of the action of the drugs on tissues and enzymes is almost as hazy and inconclusive as it was 50 years ago.216 Journal of Medicinal Chemistry. But the intrinsic mode of action of analgetics still eludes us. with 29 contributors. 1970. 16. and diuretic drugs). N. the intellectual progress of drug discovery and design appears to be in a stalemate. IS.3 X 17. Y. Bentley ). Vol.5 cm. Edited by Koloman Laki.

acs.Narcotic Drugs.. Med.1021/jm00272a902 • Publication Date (Web): 09 January 2004 Downloaded from http://pubs. 1972. 216-216• DOI: 10. 15 (2). Washington.. Chem. 1155 Sixteenth Street N.doi. Biochemical Pharmacology Alfred Burger J.org/10. DC 20036 .W.org on April 25. 2009 More About This Article The permalink http://dx.1021/jm00272a902 provides access to: • • Links to articles and content related to this article Copyright permission to reproduce figures and/or text from this article Journal of Medicinal Chemistry is published by the American Chemical Society.

5 cm. Biochemists will profit from reading about the chemical changes in contracting muscle and about muscle as a fibrous protein system. the subtitle of the volume. We have today analgetics thousands times more potent than morphine.5 cm. and up-todate factual reviews of . Edited by Doris H. 1971.50. Itanahan Department of Biochemistry Universily of Arizona Tucson. Academic Press. ing important topics: prostaglandins (B.216 Journal of Medicinal Chemistry. it will prove to be an important guide-post for several years to come. N. xii + 606 pp. dystrophies. Plenum Press. Wakil has done an excellent job in assembling a group of highly respected scientists to write on various facets of lipid metabolism and has provided a wellintegrated book on topics ranging from fatty acid metabolism to the biosynthesis of polyisoprenoid quinones. S.8 cm. Academic Press. Wakil). No. Y. This may well characterize the whole area of Medicinal Chemistry at present. Wakil. 16. 1970. N. The authors are all expert. Undoubtedly. The many conflicting hypotheses concerning these phenorena are reviewed skillfully but the authors have been careful not to propose experimentally unwarranted conclusions. paper-back. . and the withdrawal syndrome. Arizona University of Virginia Charlottesville. pharmacodynamic agents (antihypertensives.S Y 2 7 h cm. myotonias.00. we know the major metabolites of several of the drugs. $28. Y. antiarrhythmic. and critical examination is accorded to these reactions. Holloway). which at $28. xxii + 506 pp. But from Chapter 4 on. the intellectual progress of drug discovery and design appears to be in a stalemate. myasthenias. tracting factor is the cost of the book. ACh. However. For the anatomist. Metabolites of alkaloidal and synthetic potent analgetics have been identified. steroid metabolism (P. Edited by Salih J. Editor Cain now publically avows the position this reviewer has taken for several years when he writes in the preface “. N. The annual feature. and glyceride metabolism (G. biosynthesis of polyisoprenoid quinones and related compounds (R. Y. in their respective areas of interest and this is certainly indicated by depth of understanding and coverage in each chapter. K. The area of muscular disorders.50 Muscle transduces chemical energy into mechanical work. 16. We are at the verge of recognizing the role of individual proteins in these changes. 49 contributors. ix + 300 pp. fatty acid metabolism of plants (P. and these condensed reviews are followed by careful compilations of the effects of narcotic analgetics upon these processes and the enzymes involved. The high hopes statistical and quantitative methods have held out are beginning to fade. biochemical pharmacology. N. Szent-Gyorgyi’s early observations on the chemistry of muscle contraction to the present. This book is a capable review o f all these facets and is full of suggestions for future researches. Essentially the book can be divided into two major parts. They represent authoritative. Vol. The thin filaments appear to make contact with the headpiece of myosin which bends out from the thick filaments. proteins.3 X 17. Lipid Metabolism. chemotherapeutics. The cycle is concluded by disengagement of the filaments and elongation of the shortened fibriles. But the intrinsic mode of action of analgetics still eludes us. 25. chapters on the functional morphology and the development of muscle and the excitable membrane will be of value. there were no real breakthrough3 reported during 1970. to be sure. These difficulties are multiplied in the attempts to explain tolerance. The decomposition of ATP is tied to myosin and muscle work. and a chapter on the uterus as a model for medical understanding and therapy. . and regression analysis aided by MO calculations now looks only like another tool of limited applicability which will save some time in routine molecular modification. but the explanation of the action of the drugs on tissues and enzymes is almost as hazy and inconclusive as it was 50 years ago.. their methods of chemical analysis. though not as closely interrelated as the first. These chapters are well developed and provide an in-depth description of progress in these fields to 1969. takes over and encounters all the vicissitudes of such studies. the first of which encompasses fa-tty acid metabolism (S. All the reviews are well edited. or interference with a metabolite bjosynthesis as a decisive step. bacterial lipids (W.00. etc.50 will rule out many graduate students and postdoctorates and even some of the less affluent professors. Topics in Chemistry. New York. gastrointestinal. CNS location. If nothing happens to them on the way to the FDA.). Perhaps the only de. and quite up-todate. Virginia Alfred Burger Annual Reports in Medicinal Chemistry. 1970. in spite of the herculean amount of work that has been done on these questions. Drug metabolism and adenylcyclase and CAMPanalogs are covered in Topics in Biology. W. University of Virginia Alfred Biirger Charlottesville. contains a review of linear free energy and MO hypotheses in drug design. others almost free from dependence liability. 1971. J is presented to the reader as background information. and some virtually free from side-effects at therapeutic doses. 2 Book Reviews Book Reviews Contractile Roteins and Muscle. Bressler). The evolution of muscles may date back to ciliary and flagellar apparatus which are treated as suitable objects of study. and we PO’: sess compounds that can counteract heroin dependence or at least modify withdrawal therapeutically. The 6 major topics reviewed in this volume are CNS agents. tropomysin A and B. Where are the new drugs?” Many major companies now have promising novel drugs in clinical trial. Medicinal chemists will welcome an outline of muscle diseases (atrophies. platelet aggregation inhibitors. with 12 contributors.5 X 23. and diuretic drugs). $42. but it has not yet been possible to single out any specific enzyme system. Lands). Clouet. Edited by Koloman Laki. J. The introductory chapters of this book deal with the structure of narcotic analgetics. phospholipid metabolism (E. and myosin are discussed in individual chapters. New York. The selection of the topics reflects the general activity in medicinal research. centers on the follov. Hubscher). Y. Edited by C . 1971. Virginia University of Virginia Charlottesville. should now open up to causative cures. Each major biosynthetic pathway (carbohydrates. Flagellin. R Stumpf). $9. the clinically used and a few major experimental compounds and offer historical views of the intellectual processes that led t o their development. eti . tion to the literature of lipid biochemistry. Lennarz). $28. It contains a succession of segments of thin and thick filaments. Cain and 6 section editors. treatable mostly only by methods which suppress symptoms. physiological-chemical aspects of fatty acid oxidation (R. Samuelson). dependence. M. 1972. glycogenoses. 16 X 23. This is a timely and well-written book which is a formidable addi. and structure-activity relationships among these drugs. Donald J. actin. xi + 613 pp. stereochemically oriented. The other major division. particularly in the industry. Marcel Dekker. It is obvious that the potent analgetics disrupt many physiological functions. with 29 contributors. New York. this book is an account of the history of muscle research from A. lipids. Biochemical Pharmacology. It is hoped that the publishing companies in the near future will take a serious look at the best manner in which to provide texts and reference books at a reasonable price and also consider a new mechanism by which publication can be achieved in a shorter period of time. Virginia Alfred Burger Narcotic Drugs. New York. This book will do much to make available the literature and expert judgment of the background and status in this field. and metabolic disease and endocrine function drugs. This is one of the few books recently published on lipids to which the term “significant” can be applied. and these sections shorten while the unengaged filaments slide. IS. E. a reversal of the present pessimistic trend may be expected around 1976. Bentley ). E. catecholamines. No other exciting ideas have appeared on the horizon. Hill and W. We have a dim outline of some three-dimensional regions of the analgetic receptors. The chapter on analysis contains all modern spectroscopic methods used in the analysis of minute quantities of foreign substances in body fluids and tissues and should be useful far beyond the scope of this specific class of drugs. with 14 other contributors. well documented. as well as lesser articles on pharmaceutic subjects.