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Metabolism of o-, m- and p-Fluoro-, -Bromo- and -Iodo-nitrobenzenes in the Rabbit
By H. G. BRAY, SYBIL P. JAMES AND W. V. THORPE Department of Physiology, The Medical School, University of Birmingham (Received 9 September 1957)
In a study of the metabolism of the 19 members of -melting points of the acetanilides were: o-fluoro, 750; mthe chloromononitrobenzene series (for summary fluoro, 860; p-fluoro, 152°; o-bromo, 990; m-bromo, 890; and references see Table 4 of Bray, James & Thorpe, p-bromo, 1680; o-iodo, 1080 and m-iodo, 121°. p-Iodo1957 b) it was observed that in the rabbit several acetanilide, m.p. 1840, was purchased. N-Acetyl-S-(2-nitrophenyl)- and N-acetyl-S-(4-nittocompounds were converted into mercapturic acids phenyl)-L-cysteine were prepared as described by Bray, by replacement of a relatively labile chlorine atom. James & Thorpe (1956). In that paper the m.p. of the The formation of mercapturic acid by acetyl- former was described erroneously as 156-158° instead of cysteyldebromination of 4-bromo-3:5-dichloronitro- 173-174°. 3- and 4-Nitrocatechols were prepared according benzene (Betts, Bray, James & Thorpe, 1957) to Weselsky & Benedikt (1882). and of 2-bromo-4-chloro-, 2-bromo-5-chloro- and 4-bromo-3-chloro-nitrobenzene (Bray, James & METHODS Thorpe, 1957 a) has also been noted. It was thereAnimals, diet and dosage. Doe rabbits (2-3 kg. body wt.) fore of interest to examine the metabolic fate of some other halogenonitrobenzenes with particular were maintained as described by Bray, Ryman & Thorpe (1947). Compounds were by reference to the formation of mercapturic acids. suspensions in water. Theadministeredwerestomach tube as dose levels 0-1 g./kg. body For this purpose the monohalogenonitrobenzenes weight for o-fluoronitrobenzene, 0-15 g.lkg. for o-bromowere chosen since they were more readily available and o- and m-iodo-nitrobenzene and 0-2 g./kg. for the than polyhalogenonitrobenzenes. Some prelimin- other compounds. All the fluoro and all the ortho and meta ary experimnents have been reported by Bray & compounds caused some anorexia. Occasionally the dose of m-fluoronitrobenzene was fatal. James (1957). Determination of metalites. The methods were essentiBetts et al. (1957) found that in the rabbit 2:4:5- and 2:4:6-trichloronitrobenzene yielded ally as described by Bray et al. (1956, 1957a). Recoveries metabolites in which a chlorine atom was replaced (%±5%) of the anilines added to urines were: o-fluoro, by an hydroxyl group. The present investigation 93; m-fluoro, 108; p-fluoro, 108; o-bromo, 98; m-bromo, 103; p-bromo, 103; o-iodo, 103; m-iodo, 107; p-iodo, 105. has revealed further examples of hydroxyldehaloQualitaive examination of urines. This was carried out as genation. described by Bray et al. (1956). Ether extracts B were prepared by continuous extraction with ether for 24 hr. of MATERIALS urine adjusted to pH 1 with 2w-HSO4. Extraction of the All melting points recorded are uncorrected. Elementary residual urine after hydrolysis with an equal volume of microanalyses were carried out by F. and E. Pascher, Bonn. 10N-H2SO4 gave extracts C. Extracts D were obtained Most of the halogenonitrobenzenes were purchased. from the residual urine from C adjusted to pH 7. o-Fluoronitrobenzene was prepared by decomposition of Paper chromatography. The procedure was as described o-nitrobenzenediazonium borofluoride (cf. Schiemann & by Bray, Thorpe & White (1950) except that to minimize Pillarsky, 1929; Dippy & Williams, 1934). The solid product decomposition of aminophenols and loss of nitrophenols of the reaction was extracted with ether, the solvent was papers were dried without heating. The solvents and deremoved from the extract and the residue was steam- tecting reagents used and the BF values of the compounds distilled. The distillate was extracted with ether. After are given in Tables 3 and 4. removal of ether the liquid residue from the extract was Indophenol reaction. Synthetic p-fluoroaniline reacts purified by fractional distillation. The product had b.p. 950 with the indophenol reagents to give a blue colour slowly. at 12 mm.; n24 1-5452 [Schiemann & Pillarsky (1929) give (Normally a blue colour is obtained with these reagents n172 1.5331]. o- and m-Iodonitrobenzene, m.p. 490 and 320 only from p-aminophenols.) This caused little difficulty in respectively, were prepared by the method of Ullmann interpretation of paper chromatograms, since p-fluoro(1896). aniline runs faster than p-aminophenol with which it o-Fluoro- and o- and m-iodo-aniline were prepared by might otherwise have been confused (see Table 3). The preduction of the corresponding halogenonitrobenzenes with fluoroaniline was shown to be free from p-aminophenol by Sn and HCI in ethanol. The other halogenoanilines were paper chromatograms treated with other detecting reagents. purchased. All halogenoanilines were characterized as the -The slow blue colour always appeared in the zone which corresponding acetanilides, which were prepared by treat- other detecting agents had shown to be that occupied by ment with acetic anhydride at room temperature. The p-fluoroaniline. It seems likely that a small proportion of 36 Bioch. 1958, 68
g.X Y RESULTS The average daily excretions of normal metabolites by the rabbits used were of the same order as those found previously (e. Hybs. (1957a). The ortho compounds and p-fluoronitrobenzene form relatively large amounts of mercapturic acid. 0t. . THORPE I958 o 4) p-fluoroaniline is defluorinated by the reagents used in the indophenol reaction. All the aminophenols isolated had m. The absorption spectra of the mercapturic acids were identical with those obtained from the corresponding synthetic acids. and by paper chromatography. 18olatiofn of metabolites. o4 '4 o'4 at0 i° 0y f '4o 4) 0 a d) . 0 D~~~~~~~~.5aO.4 4)0 Metabolitea i8olated or detected The metabolites isolated and the yields obtained are given in Table 2.4%) from m-bromonitrobenzene (see Table 2).i!CO 4t0i 9 io9 . C and D of the urines are given in Tables 3 and 4.and p-iodo-nitrobenzene are probably too low.0 4 :9 O 4) 0o es . significant amounts of phenolic metabolites seem to be excreted unconjugated (see Tables 3 and 4). for CBH6ONBr: N. 0e co '00~~~~~0 £ '4 O . 7. JAMIES AND W. would be higher than the true control values for the experimental period. The isolated acetanilides and mercapturic acids were shown to be identical with authentic synthetic specimens by mixed m. G. with some of the compounds. Except with p-fluoronitrobenzene. Furthermore. The metabolites detected by paper chromatography in extracts B. m * 4) oE )0 c4 " ..p. S. makes it probable that there is no significant excretion of mercapturic acid from these compounds. P. As several of the compounds caused anorexia. James & Thorpe. (1956). BRAY.p.._ . a large part of the dose was excreted as phenolic metabolites. For these reasons 'the totals accounted for' given in Table 1 for all compounds except p-bromo. Halogenoanilines were isolated as the acetanilides as described by Bray et al. The results obtained by quantitative analysis of the urines of dosed animals are summarized in Table 1. .. the values for glucosiduronic acid formed from these compounds are probably low. Except with p-bromo. V. CaJc. 4)S aS 0o 'E OD o i 3 o ES 4 v O XX 6o o 4X c4 :~~~~ :: t04) 05' * * . Bray.'s in agreement with those given in the literature except the o-aminobromophenol (Found: N. . The small values [by the modified iodometric method of Stekol (1936)] for mercapturic acid from the meta compounds. a relatively small proportion of the dose was excreted as the corresponding aniline. together with failure to detect any mercapturic acid in the urines. which were used as base lines. 7-3.6. 1953). (The toxicity of the compounds administered precluded the use of larger doses.562 H. since the values for excretion before and after the experiment.5'. Mercapturic acids and aminophenols were isolated as described by Bray et al.and piodo-nitrobenzene.) All spots from *'Fe 4)5 X . In addition to the nitro compounds listed there were faint traces of other yellow spots but these were not regularly reproducible and the amounts were too small for satisfactory identification after reduction.
This is supported by the finding of these spots on a paper chromatogranm of hydrolysed B extract. since six of the B extracts gave no naphthoresorcinol tests for glucuronic acid and two. The acetyl derivative prepared from the phenol isolated had m. to some extent be deduced on the basis of chemical tests for o-.Vol. Six phenols which were isolated had properties in agreement with those given in the literature. of M.p. Metaboites i8olated from urine of rabbits given halogenonitrobenzene8 563 Melting points given in parentheses are for the derivative isolated. and p-aminophenols as described by Bray et al. ether-soluble glucosiduronic acids of either these or m- other phenols (cf. For eight of the halogenonitrobenzenes.and mamino-phenols obtained from o-fluoronitrobenzene. Doubt therefore remains only about the m-nitro. 88° and Schutt (1885) gave m. Phillips (1930) reported 1770 for 2-acetamido-4-bromophenol. the B extract of which gave a positive naphthoresorcinol test. 1840. (1957 b). the possibility of the presence of such conjugates can be dismissed. 4-Amino-3-iodophenol m-Iodoaniline* p-Iodoaniline* 2-Amino-5-iodophenol t Berg & Newbery (1949) give 1620.1 6 N-Acetyl-S-(4-nitrophenyl)-L-cysteine 1494 3 6 2-Amino-5-bromophenol 1 o-Iodo(107) 2 0-4 o-Iodoaniline* m-Iodop-Iodo* 04 0*6 6 6 2 2 6 As monoacetyl derivative. N-Acetyl-S-(2-nitrophenyl)-L-cysteine 170 146t (121) (183) 4 1 3 4 139$ diazotizable compounds have been listed in Tables 3 and 4 except two from slow-running derivatives of o-fluoronitrobenzene (see below).or -6-bromophenoill 4 (168) p-Bromo2 0-6 p-Bromoaniline* 156 0. tests for o. § Gattermann (1894) gave 1630 and Hodurek (1897) 155°.and p-aminophenols would no longer provide evidence of structure since the conjugates of o. 1957 b). the labour of attempting the synthesis of the appropriate reference phenols did not seem justified. however. There is the possibility that some of the halogenophenols. Bray et al. To save space. in fact. Authentic synthetic specimens of the halogenonitrophenols and aminohalogenophenols were not available. which gave a positive reaction (those from p-fluoro. With each halogenonitrobenzene the products which would result from hydroxyldehalogenation were also looked for and each urine was examined for 3. 2-Amino-6-bromophenol has not been prepared. did not contain either nitro. unless positive results were obtained. This chromatogram also bore a spot corresponding to 4-amino-3-fluorophenol and one corresponding to the aminophenol formed by reduction of the 2. however.or 4-fluoro-3-nitrophenol which had been found in B extracts. The constitution of the phenols detected can. As there are 60 possible metabolites of this type and at least 26 would be required for identification by direct comparison.p. the dehalogenated phenols are entered only once in Table 3. (% of dose) administered Metabolite isolated rabbits (g. t In near agreement with Hodgson & Kershaw (1928).and 4-nitrocatechol. If the phenols conjugated. 128° for 2-amino-4-bromophenol.) 1760 10 o-Fluoro1 0*33 N-Acetyl-S-(2-nitrophenyl)-L-cysteine 1 (850) 2 m-Fluoro0-52 m-Fluoroaniline* 2 161t 6 4-Amino-2-fluorophenol 2 2 (151) p-Fluoro0*53 p-Fluoroaniline* 157 10 3 N-Acetyl-S-(4-nitrophenyl)-L-cysteine 2 o-Bromo2 0*4 o-Bromoaniline* (99) 172 12 6 N-Acetyl-S-(2-nitrophenyl)-L-cysteine 1514 2 6 4-Amino-3-bromophenol 1 (87) 2 m-Bromo0-6 m-Bromoaniline* 1 165§ 4-Amino-2-bromophenol 6 1 135 6 2-Amino-4. which were detected in the (unhydrolysed) B extracts and tentatively identified as m-nitro. 68 METABOLISM OF HALOGENONITROBENZENES Table 2. Nitrobenzene Yield derivative Dose/rabbit No.p. 11 Schlieper (1893) and Hunter & Barnes (1928) gave m.and p-iodo-nitrobenzene). it is unlikely that they were glucosiduronic acids. As spots corresponding to these two phenols were also obtained from paper chromatograms of the (hydrolysed) C or D extract. All compounds isolated were recrystallized from aqueous ethanol.or amino-halogenophenols. were.or m-amino-phenols.and p-aminophenols would react more like m-aminophenols. These spots probably represented the were so 36-2 .p.
and one p-nitrofluorophenol and the corresponding aminophenols.or o-nitro-fluorophenol was detected.and m-nitro-phenol were found in the B extracts but the m-aminophenol was not that which was formed by reduction of the m-nitrophenol. Blue develops slowly after addition of the coupling reagent. Small amounts of a rn-amino. benzene-acetic acid-water (1:1:2. From o-fiuoronitrobenzene. due to streaking.and 4-Nitrocatechol have RF values 1. Detected Colour with Rp values in in extract solvent mixture detecting reagent _ k _A AA A B c B C a d C D b Derivatives of o-fluoronitrobenzene o-Fluoroaniline x 1.5 which was not found in extracts B and D but was obtained when extract B was hydrolysed (see Results section).0 0.p. brown.0 x x 0-20 (2-Amino-5-fluorophenol) B (+) y PP 0.5 hr.05 0*70 (±) Y pP 4-Nitrophenol 0*83 0. by vol.0 0.564 H. JAMES AND W. Authentic specimens of the compounds in parentheses were not available for comparison. From m-fluoronitrobenzene. The following are the arguments for the constitution of the phenolic metabolites listed in Tables 3 and 4.or -2-fluorophenol)*t * * (+) 0. orange. 11 3.0 (+) 0*79 M 2-Nitrophenol dY 1. 20% (w/v) Na2CO3. 2N-HCI. C. B. Hydroxylation can give one o-.05 0-58 ( +) 0*17 0 0. solvent mixture C) corresponding to the nitrophenol was obtained after hydrolysis of the B extract and was therefore probably present only as an ethersoluble conjugate (see above). P.50 M (3-Amino-4. d. x indicates that the spots obtained were not satisfactory for identification. V. (+). No o-amino. G. The m-aminophenol (R.) (1. 1957 b).g. Corner & Young.0 0*90 0 60 N-Acetyl-S-(4-nitrophenyl)-L-cysteine 0-87 dY 0. by vol. + + x x (2. absent. The main phenolic metabolites were a p-aminofluorophenol and its corresponding nitrophenol (shown by reduction to the aminophenol). by vol. t Lack of a reference compound made it impossible to decide which BR values pertained to the given aminophenol. + + .and one p-nitrofluorophenol and the corresponding aminophenols are possible.0 0*28 G 4-Nitrocatecholli (±) (±+) B 4-Aminophenol P 0-01 00 0*72 B * Reduction of the nitrophenol gave the aminophenol of RF 0. presumably glucosiduronic acids. Colours of spots: B. pale.0 x 1-0 (+) (±) B4 Bn 0-02 0. 4 paper: A. present in traces. Y.5% ammonium sulphamate and 0-1% N-(l-naphthyl)ethylenediamine dihydrochloride (Bratton & Marshall.or -4-fluorophenol)*t 0. The main phenolic Table 3.. (4:3:3. 0. 0 5 % FeCl3. by vol. BRAY. 0.75 M (5-Amino-2-fluorophenol) 0. 3N-NH.0 0-89 M 0415 4 y 07 (2-Fluoro-4-nitrophenol) 0-06 0*57 + B B 0-17 0-61 4-Amino-2-fluorophenol§ 0.or -6-fluorophenol) 0-52 Bt 0-25 (+) ± dY 1. the arguments for the suggested constitutions are given in the Results section. C and D (see Methods). soln. deep..0 0-77 BP (+) Derivatives of p-fluoronitrobenzene 0-70 B p-Fluoroaniline M 0.) (1. 90°)-benzene-98 % formic acid (3:1:2. G.). -. green. magenta. Present. +.11 0-81 3-Nitrophenol PP (±) (±) x 3-Nitrocatecholl1 x 0-25 OBn G 0-17 G 0-28 . which gave spots of B.). values and detection of metabolites of fluoronitrobenzenee Solvent mixtures and times of run on Whatman no. Bn.0 Y . + (3-Fluoro-4-nitrophenol) 0*68 0-08 0*40 Y . 0.or 4-Fluoro-3-nitrophenol)* 0-50 * Y (+) (+) 0. Compounds found in extracts of hydrolysed urine (C and D) are probably excreted in combined form.CO-aq. 1954) (3 hr. D. p. THORPE I958 hydrolysis products of material.0 + x (2-Amino-4. P.0 and 0 2 respectively in solvent mixture D-and give purple and red colours respectively with w-NaOH. purple. Two o-. e. light petroleum (b.00 0 4-Nitrocatechol!l (+) (+) 3-Aminophenol PBn 0-05 0.) (1 hr.. 1939). 0 5.0 B 00 0*71 (4-Amino-3-fluorophenol) B x x (3-Amino-2. two m. agreeing with that given in the literature. benzene-acetic acid-water (2:2:1. blue.0 009 M Steam-distillate 0-46 N-Acetyl-S-(2-nitrophenyl)-L-cysteine 0-65 0. R.). S.). d. Under RF values. butanol-3N-(NH4). t Bright-yellow colour with nitrous acid.p. Detecting reagents: a. Metabolites were sought in extracts B. M. b.0 0-81 2-Aminophenol Derivatives of m-fluoronitrobenzene m-Fluoroaniline 1. § Isolated specimen of this compound had m. indophenol reaction (Bray et al.5 hr. c. one m. about 0 and 0413 (solvent mixture C) on the chromatogram of unhydrolysed B extract. yellow.1% NaNO2.
present in traces. RB valume and detection of metabolite8 of bromo.or -4-iodophenol) § (3-Amino-4. times of run. Similar arguments to those used for the fluoro compounds apply. The presence of 4-nitrocatechol is also more readily accounted for as a product of further hydroxylation of 3-nitrophenol. Metabolites were sought in extracts B. agreeing with those given in the literature. Present. Detected Colour with Rp values in solvent mixture in extract detecting reagent A o-Bromoaniline B c a b c B - C - D + N-Acetyl-S-(2-nitrophenyl)-L-cysteine (3-Bromo-4-nitrophenol) 4-Amino-3-bromophenol* (3-Amino-2. nitrofluorophenol and the corresponding aminophenols are possible.or -6-iodophenol) p-Iodoaniline N-Acetyl-S-(4-nitrophenyl)-L-cysteine x 0-0 0-80 B: 2-Amino-5-iodophenol* * * ~ ~ * Isolated specimens of these compounds had m. Blue develops slowly after addition of the coupling reagent. From the bromo. Both the aminophenols were detected but the main phenolic metabolite was the defluorinated product. due to streaking. C and D (see Methods).. +. .and 3-amino-phenol respectively. A m-nitro.or -4-bromophenol) mn-Bromoaniline (2-Bromo-4-nitrophenol) 4-Amino-2-bromophenol* (2-Amino-4. t Approximate. + - - ( -t) * + - y + B dY + . 4-aminophenol. Compounds found in extracts of hydrolysed urine (C and D) are probably excreted in combined form. B .and 3-amino-5fluoro-phenol would both have the same R.or -6-bromophenol was isolated.or -6-bromophenol) (3-Amino-5-bromophenol) p-Bromoaniline N-Acetyl-S-(4-nitrophenyl)-L-cysteine 2-Amino-5-bromophenol* (5-Amino-2-bromophenol) o-Iodoaniline N-Acetyl-S-(2-nitrophenyl)-L-cysteine (3-Iodo-4-nitrophenol) 4-Amino-3-iodophenol* (3-Amino-2. The absence of dehalogenated phenols simplified the identification. detecting reagents and colour abbreviations are as in Table 3. Under RF values. One o.and 3-amino-phenol in Table 3. y y + + + . § Lack of a reference compound made it impossible to decide which Rp values pertained to the given aminophenol. x indicates that the spots obtained were not satisfactory for identification. * _ (+) + - (+) + . Authentic specimens of the compounds in parentheses were not available for comparison. but its properties were not in satisfactory agreement with Table 4.and iodo-nitrobenzene8 Solvent mixtures.and the p-aminoFrom p-fluoronitrobeizene. ( + ).and iodo-nitrobenzene8. - dY . since it is unlikely that 3-fluoro-5-nitro. the arguments for the suggested constitutions are given in the Results section.and mamino-phenol were detected and are recorded as 3-nitro. absent. . paper. spot very elongated. . - + - + _ _ * + - - Y + _ + - ± + - * + - + dY + - . values as have 3-nitro. The compound from m-bromonitrobenzene described in Table 4 as 2-amino-4. + Derivatives of o-bromonitrobenzene 1-0 M 1-0 1-0 0-65 0-0 0-46 0-90 0-0 0-63 B 0-62 0-0 0-90 M 0-50 0-25t 0-10 Derivatives of m-bromonitrobenzene 1-0 1-0 1-0 0-31 0-22 0-50 x 0-0 0-70 x x 0-87 x 0-25t 0-80 Derivatives of p-bromonitrobenzene x 1-0 M 1-0 0-85 0-0 0-60 0-63 0-0 0-92 Bt x x M 0-05 Derivatives of o-iodonitrobenzene 1-0 M x 1-0 0-65 0-0 0-46 x 0-02 0-66 B 0-0 0-69 0-30 x M 0-0 0-06 x M 0-95 0-28 Derivatives of m-iodonitrobenzene x M 1-0 0-90 0-76 0-10 0-67 0-28 0-0 0-83 Bt Derivatives of p-iodonitrobenzene x P 1-0 0-90 0-85 0-0 0-60 - .Vol. t Bright-yellow colour with nitrous acid.g.and one mphenol. 68 METABOLISM OF HALOGENONITROBENZENES 565 metabolites were the p-nitro. -. e. + - - + _ _ ) + * -Y + + _ + _ + + B * -.or -2-iodophenol)§ m-Iodoaniline (2-Iodo-4-nitrophenol) (2-Amino-4. Traces of an o-aminophenol were found but it was not possible to distinguish between the two possible o-aminophenols.p.
All the o. hydroxylation precedes or follows reduction of the The extent of hydroxyldefluorination of o. Betts two trichloronitrobenzenes (Betts et al. m-Fluoronitrobenzene yields m-nitro. sufficient evidence mercapturic acid was shown by paper chromato.and (1952) failed to detect 3-nitrocatechol as a metap-halogenonitrobenzenes form mercapturic acids bolite of o-nitrophenol.and o-iodo-nitrobenzene. no satisfactory evidence identified from m-chloronitrobenzene. except that 3:5-dichloro-2evidence that this occurs in the monofluoronitro.and the triHydroxyldechlorination has been observed in chloronitrobenzenes (Bray et al. in traces. such as would have been expected amongst the metabolites of o-fluoronitrobenzene is from those obtained by Bray et al. Furthermore. benzenes were o-aminophenols and no o-nitroor at room temperature for 18 hr. is not not available.) forms is neglected. Although lack of reference compounds preby acetylcysteyldehalogenation. 2-Amino-6-brQmophenol is unknown.phenols may also have offset the probability of anisole by treatment with sodium methoxide revealing differences when mixtures of phenols (Holleman & Beekman. o-chloro. 1957a. There is. however.chromatograms when reference compounds were zenes.metabolites.and p-bromo. The question whether is equally stable under these conditions. The detected in urine. The mono.only m-bromonitrobenzene yields a metabolite defluorination might therefore have been ex. BRAY.major phenolic metabolites of the p-halogenonitrofluoronitrobenzene had been kept at 370 for 4 hr. 1957). however. properties of the phenols which can be so formed It was therefore concluded that meta-hydroxylation those given in the literature for 2-amino-4-bromophenol (see Table 2).or m-aminophenols can be formed by hydroxyl. the were examined in even three different solvent present work suggests that hydroxyldefluorination mixtures. o.been given the corresponding chloroanilines. (-NO2) and reduced (-NH.2:3:6-trichloronitrobenzene. althoughthedetectionof4-nitrocatecholstrengthens the probability that defluorination ofm-fluoronitroThe results in the present investigation are. fewer haloHalogens in a meta position in nitrobenzenes are genophenols have been detected than with the particularly stable (see Bunnett & Zahler.566 H. are summarized in Table 5. 1956). apart from m-fluoronitrobenzene. 2:5-dichloro-4-nitrobenzenes. The results p-fluoro-. The evidence now presented is stronger than that obtained with the vented a complete identification of the hydroxylchloro compounds.nitrophenol and. the phenolic metabolites excreted after rabbits had With both these compounds.and nitro group was studied for the monochloronitrom-fluoronitrobenzene is not so great. Smith & Williams corresponding chloro compounds. (1956) by examination of traces of defluorinated products were detected.and moccurs within the animal since no nitrophenol halogenonitrobenzenes were p-nitrophenols. These ation in a position ortho or meta to the nitrogen can compounds yielded only o. found only in traces and only as metabolites of osince 4-nitrophenol and 4-nitrocatechol were fluoro-. JAMES AND W. to a benzene occurs. from which the formation of ated halogenated metabolites.and p-aminophenols occur without displacement of fluorine. The defluorination probably predominant nitrophenols from all the o. In the monohaloation without defluorination. The absence of 3-nitrocatechol large extent.and chloronitrobenzenes (Bray et al. where nitrophenols were detected as not in the monochloronitrobenzenes. 1957). G.which does not correspond to one which was pected. phenols is of interest.and monoiodo-nitrobenzenes do not appear formed if the nitro group directed hydroxylation to undergo hydroxyldehalogenation but there is into the meta position. It is unequivocal that defluorination of phenol were identified as metabolites of 2:4. V. In general. P. The chemical similarity of the surprising. 1951) chloro compounds. S. it cannot be stated unequivocally that they can be distinguished with certainty from the relevant phenols formed by defluorination. but this may have been due and the lack of evidence for the formation of partly to the greater difficulty of interpreting paper mercapturic acids from the m-halogenonitroben. but et al. As the and there was no evidence of meta-hydroxylation. The could be detected after aqueous suspensions of p. Formation of mercapturic acid by acetylcysteyl. for the formation of mercapturic acid from mThe position of the hydroxyl group in the nitrofluoronitrobenzene.genonitrobenzenes. (1956) with the not unexpected since Robinson. DISCUSSION . since only benzenes by Bray et al.and p-chloro-nitrobenzenes.has been obtained to show a general similarity grams. With the di. however.and p-fluoronitrobenzene occurs since a p-aminophenol 2:5-dichloronitrobenzene respectively and traces of is the main phenolic metabolite excreted and only 2:3:5-trichloro-4-nitrophenol were obtained from o. m-nitrophenols were fluorination must occur in the nitro compound. b. since the mercapturic acids have been with the chlorophenol metabolites of the monoisolated from the urines of rabbits given o. hydroxyl. p-Fluoroaniline phenols were detected. they were those which would be bromo. THORPE I958 are unknown. At least some de. 1903). If the distinction between unreduced of m-fluoronitrobenzene can occur in the rabbit.
in conjunction with the fact that only traces of the products of ortho-hydroxylation of o-chloronitrobenzene were found (Bray et al. SUMMARY 1. Chem. (1949). o-. 128.and iodo-nitrobenzenes so closely resembled those from the corresponding chloro compounds (see Table 5) it seems reasonable to suppose that the origins of the various hydroxylation products are similar to those of the corresponding chloro compounds. Nitrobenzene derivative o-Fluoroo-Chloroo-Bromoo-Iodom-Fluorom-Chlorom-Bromom-Iodo. probably because the major phenolic metabolite was one formed by hydroxyldefluorination).and o-aminophenols (4amino-2-iodophenol was the only phenol of thIs type not detected). Ortho A hydroxyldehalogenation _ A_ _ Meta -A_ Para A______ 4-Nitro- Nitro + Amino Nitro Amino +or4 +2. The ortho and para compounds form mercapturic acids (over 30 % of the dose with the fluoro.and para-hydroxylation can occur before reduction. V. J. As the phenolic metabolites of the fluoro. 5. o. only a small amount of this metabolite was excreted.and p-fluoro-. . Taylor for assistance with the quantitative analyses. The aminophenols were almost certainly present in greater amounts. Betts.g. 537. +4 + Nitro ++ ++ Amino ++ ++ Nitro + - Amino catechol - - - +2. biol. G. Phenols formed by hydroxylation Phenols formed by Position of hydroxyl relative to the nitrogen . except with p-bromo. & Thorpe. 4. (3) only in the fluoronitrobenzenes is the halogen group sufficiently labile to be replaced by hydroxyl.. (1956). J.and amino-phenols excreted are approximately the same. & Newbery. The main phenolic metabolite of p-fluoronitrobenzene is p-aminophenol. It should be emphasized that this grading is based mainly on the appearance of spots on paper chromatograms and not on quantitative determinations. those present in small amounts or traces by + and compounds not detected by -. chem. James. A. 2. S. The metabolism of o-. E.. Wood and Mrs B. 3. only aminophenols were present in sufficient amount to permit isolation (see Table 2).and p-iodo-nitrobenzene has been studied in the rabbit.. G. Phenolic metabolites of the monohalogenonitrobenzene8 567 The major phenolic metabolites are indicated by + +. Bratton. We wish to thank Mr P. however. 610.and p-iodo-nitrobenzene (30 %) less than 10% of the dose is excreted as the corresponding anilines. Results for the chloronitrobenzenes are from Bray et al. G. The most conspicuous general features of the hydroxylation products of the halogenonitrobenzenes are: (1) the main phenols from the ortho compounds are p-aminophenols and those from the meta compounds are p. (1939). K. J. p.and o-. & Marshall. 68 METABOLISM OF HALOGENONITROBENZENES Table 5. m. (2) the main phenols from the p-halogenonitrobenzenes are o-amninophenols (with p-fluoronitrobenzene. particularly in the + + grading. Except with p-fluoronitrobenzene (less than 10%) about 30-60% of the dose is excreted as phenols conjugated with glucuronic and sulphuric acids. C.and m-Fluoronitrobenzene also probably undergo hydroxyldefluorination. H.(apart from products of hydroxyldefluorination).Vol. Bray. +4 + 2 + +4 - +2or4 - +2or4 + - +3 +4 2 + + pCFluorop-Chloro- + +4 + +2. W. An inferior figure indicates the position of the halogen relative to the hydroxyl group if more than one product is possible with the hydroxyl group in the given position. 642. S. All the four possible chloronitrophenols were excreted by rabbits given o-chloronitrobenzene so that in this compound ortho-. J. bromo.. REFERENCES Berg. e. Apart from hydroxyldehalogenation in the fluoro compounds the metabolic fate of the fluoro-. 66. B. but this is not surprising for the reasons given above. With the other ohalogenonitrobenzenes the products of orthohydroxylation were not detected (see Table 5). in general.. qualitatively similar to that of the corresponding chloro compounds. m. jun. (1957). bromo. J. it should not be taken that the amounts of nitro. P. Soc.r4 + +2or4 ++ + p-Bromop-Iodo- - - ++ ++ - + - ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ Para position ( + + + + +_+ + J occupied by halogen X occurred only in the nitro compounds.compounds) but the meta compounds do not. 1956). mand p-bromo.and iodo-nitrobenzenes is. S. . Biochem. meta.and o-bromo..
p. W. & Benedikt. 80. & Barnes. G. P. (1957). H. Chem. Institute of Animal Physiology. Allfrey & Mirsky (1949). & Thorpe. J. Hunter. Ge8. Chargaff. (1903). 62. 2400. (1885). Babraham. Ges. Ber. 27. 1927. J. R. & James. Bray. and (ii) methods involving the interaction of isolated deoxyribonucleic acid with proteins (Brown & Watson. Biochem. in * Present address: Department of Biochemistry. chem. S. 41. 6. i. J. H. 477. chem. chem. S. Gattermann. Biochem. 2703. Bray. J. Soc. (1957b). & Thorpe. may be regarded as a group of closely related polymeric species having significantly different compositions. 2051. W. 3035. Cambridge.. 46. J. Phillips. H. Biochem. p. S. Ge8. 483. Chem. (1929). 64. 1955). V. (1957a). Schlieper. Soc. M.. . D. 61. Lipshitz & Chargaff. S. & Thorpe. either on a concentration gradient (Crampton. W. Hodurek. The solid material was separated by centrifuging (4000 g for 30 min. Holleman. & Pillarsky. (1936).) was homogenized in a Waring Blendor for 6 min. Stekol. V. (1930). Bunnett. D. Robinson. J. 29. J. 386. 66. Hybs. JAMES AND W. Chem. R. 273. J. Ges. W. Corner. (1896). this was stored at . G. another connexion. 279. 1956) or on a time gradient (Lucy & Butler. H. (1947). R. biol. Bray. p. 1953. dt8ch. R. J. Rev. and obtained as a gel by centrifuging at 81 000g. & Williams. 1878. E. James. F. chem. Smith. J. 65.). Akad. The methods of fractionation commonly employed hitherto fall into two groups: (i) those dependent on the extraction of denatured deoxyribonucleoproteins with solutions of sodium chloride. Soc. N. (1893). R. Ber. J. chem. 0. Biochem. (1952). Univer8ity of Oxford (Received 14 January 1957) Recent investigations have show-n that the total deoxyribonucleic acid. Ullmann. 49. 50. G. Ab8tr. Unchanged nucleoproteins and the insoluble conjugate formed were removed by centrifuging. P. W. WARD* Department of Biochemi8try. J. R. The product was reprecipitated four times from M-NaCl soln. G. 271. Wet. G. Bray. 1953). J. Ge8. Biochem. Bray. (1951). The material was extracted from minced glands (obtained immediately after slaughter) by the method of Daly. The washed solid was then mixed rapidly in a Waring Blendor with M-NaCl (3 vol. Biochem. and the accompanying liberation of deoxyribonucleic acid fractions. H. P. V. W. J. Mh. 2469. Crampton & Lipshitz. J. Schuitt. S. that protamine combines with heparin yiekling an -insoluble complex conjugate. p.. T.) and washed twice with 3 vol. 26. V. (1953). J. of NaCltrisodium citrate soln. P. F. Rosenkranz & Beiser. L. 38. (1934). H. S. W. prakt. 212. (1956). E. Ox-spleen deoxyribonucleoprotein. Am8t. (1894). 113. (1928). F. J. 32.. A. (1950). Ber. After . & Beekman. James. 3. K. H. Chem. J. J. 232. P. of 0*14M-NaCl soln. H. Schiemann. G. P. L.568 H. Lipshitz & Chargaff.] Displacement Fractionation of Deoxyribonucleoproteins by Heparin and Dextran Sulphate BY P. A. (1954). F. & Thorpe. chem. Thorpe. & Zahler. G.. G. It was shown by Chargaff & Olson (1938). Fresco. Ryman. Addition of subequivalent quantities of these polysulphates to deoxyribonucleoprotein in 0-14M-sodium chloride leads to the liberation of deoxyribonucleic acid in solution. Repetition of the process thus led to the preparation of a series of deoxyribonucleic acid fractions. W. 53. THORPE I958 Bray.. (1897). dtsch.18°. 1955) or ionic-exchange reagents (Bendick. V. EXPERIMENTAL MateriaLs Calf-thymus deoxyribonucteoprotein. 327. S. & Williams. Bray. F. dt8ch. Dippy. HICHENS AND P. Weselsky. James. KENT. Z. Proc. 647. chem. Ber. 221. dt8ch. & Thorpe. Where necessary. K. V. F. J.. 607. Lipshitz & Chargaff. Soc. 266. We have now shown that isolated calf-thymus histone behaves in a simnilar way and is precipitated in 0 14M-sodium chloride by titration with heparin or with dextran sulphate. The present studies are concerned with an alternative means of fractionation of undenatured deoxyribonucleoproteins from calf-thymus gland and ox spleen. E. [Quoted from J. & Kershaw. polypeptides (Spitnik. containing 0-O1M-trisodium citrate. James. G. (1928). Biochem. F.. 1955). 1466. A. 45P. J. Fresh tissue (150 g. B. chem. founded on the stepwise displacement by strong anionic polymers of the protein moiety of the nucleoproteins. Hodgson. M. 1954a. Soc. P. & Young. chem. Biochem. chem. BRAY. 86. W. (1882). in 350 ml. V. H. (1904). L. dt8ch. S. Biochem. Ber. 67. V. b. isolated from a single biological source. F. & White. 58. A.
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