unc 615 Coden: ACPHEE ISSN 1330-0073 ACTA PHARMACEUTICA 3/2003 PUBLISHED BY — IZDAVAC CROATIAN PHARMACEUTICAL SOCIETY HRVATSKO FARMACEUTSKO DRUSTVO Acta Phar. pp. 151-240 Zagreb, September 2008,Fede ere‘Acta Pharm, §3 (2008) 175-186 sgt ter per Novel 1,2,5-oxadiazine derivatives - Synthesis and in vitro biological studies Month masaric ‘A new synthetic approneh to the 1.25-exadiazine ring SANDRA KRALIVIC! ynthetic approach to the 12.5-0xadi 8 somnkeHenc system is described, 2-Substituted oF 24-disubstituted URANKA Z0RC"| 2H-1.25-oxadiazine-3,6(4H,5H1)-dione derivatives 4 were prepared by cyclisation of hydroxamic acids 3 derived "Faculty of Pharmacy and Biachemistry ftom N-(-bencotriazolylearbony))-amino acids 1. The University of Zagreb, Zagreb, Croatia” structures of the synthesised compounds were fully characterised by IR, "H and "C NMR specteosenpy and 2 Ruadjr Bodkooié Institute ‘elemental analysis. The aim ofthis study was to evaluate Zagreb, Croats biological activity of the newly synthesised oxadiazine derivatives. Cytotosie and cytostatic activities. were tested on two cell ines (HeLa and GMK) and evaluated by MTT:test. Two human DNA viruses (adenovirus 7 and herpesvirus 1) and two human RNA viruses (coxsa- ‘kiovirus BS and echovirus 7) were used in the antiviral test, Selected biological studies indicated that 2;phenyl- 2H-L2S-oxadiarine-S6(GH.SH)-dione (4a) and. 4-ben- 2yl2-phenyl-2H-1,2,Soxadiazine36(4H,SH)-dione (Ae) statistically significantly inhibited cell growth. A minor antiviral effect was observed upon adenovirus, herpesvi sus and enterovinises, Keywords: 12,S-oxadiazine derivatives, syntness, cytoto eed Jane 12290 sic effect, cytostatic elect, antiviral activity, cytopathic Ae Ngt 25, 203, eet ‘Oxadiazines are interesting and promising heterocyclic compounds. A diversity of biological effects is associated with oxadiazines bearing heteroatoms at 1,2,4 or 1,84 po- sitions. 6H-1,2.4-oxadiazine-3,5-(2HA)-dione, the 6-oxa analogue of uracil, has been shown to significantly inhibit growth in several bacterial strains while not being highly inhibitory to mammalian cells (1), 1,3,4-Oxadiazine derivatives exhibit cardiovascular, antibacterial, plant growth regulating, miticidal and nematocidal, acricidal, insectici and anticonvullsive ac al ties (2, 3). In addition, oxadiazines are useful intermediates in the synthesis of tenidap prodrugs or f/-lactam antibiotics, in particular in the synthesis ‘of carbapenems and penems (4,5). + Cutrespondence, «nai rlovrkfyahoo.comRania a Nove 12. mnie dates ~ Syed nit lol sion, Ai har 820) 15-86 On the other hand, 1,2,5-oxadiazines are not very common heterocyclic systems. A comprehensive review of their synthesis and reactivity has been reported by Smalley (6). ‘The promising therapeutic potential of this class of compounds prompted us to synthe- sise and biologically screen several 1,2,5-oxadiazine derivatives. Our synthetic approach involves hydroxamie acids as the starting compounds, which themselves have been of ‘multifold biological and pharmacological interest (7-9). Herein we present a new synthetic approach to 1,2.5-oxadiazines derivates and their biological activity on mammalian cells. Furthermore, their antiviral activity agaist adenoviruses, herpes viruses and enteroviruses was investigated EXPERIMENTAL ‘Materials and analytical methods Melting points were determined on a Boétius Microheating Stage (Franz Kistner Nacht: KG, Germany) and were uncorrected. IR spectra were recorded on a FTIR Perkin Elmer Paragon 500 spectrometer (Perkin Elmer, UK). 'H and 8C NMR spectra wore rex corded on a Varian Gemini 300 spectrometer (Varian, USA), operating, at 300 and 755 Miz for the 1H and '8C nucle, respectively. Samples were measured in DMSO-d, solu tions at 20 °C in 5 mm NMR tubes. Chemical shifts (6) in ppm were referred to TMS. Coupling constants () in Hz, were observed through three bonds. For TLC, silica gel plates Kieselge! 60 Fas (Merck, Germany) and the following sot- Went mixtures were used: cyclohexane/ethyl acetate/methanol (31:03), eyclohe xano/ ethyl acetate (1:1), chloroform/methanol 1). Spots were visualised by shortwave UY Tight and iodine vapour. Column chromatography was performed on size gel (Kemika, Croatia), 0.063-0.200 mmm, with dichloromethane/methanol (8:1) o¢ cyclohe- xane/ethiyl acetate (1:1) as eluent “Amino acids were purchased from Kemika (Croatia). N-Phenylhydroxylamine was Prepared by reduction of nitrasoberzene with L-ascorbate (11) All solvents were of ana lytical grade purity and wore dried prior to use Syntheses N-Q-berzetrnzolyleartonyl)-amivo acids (N-Bte-antino acids, tac). ~ N-Btc-glycine (1a), N-BieTealanine (1b) and N-Bte-L- phenylalanine (Ie) were synthesised following the Procedure published previously (12). N-(T-benzotriazolylcarbony!)-amino acid chlorides (2a-e). ~ Chlorides 2 were prepared from N-Btc-amino acids 1a-¢ and thionyl chloride (13) Hydroxamie acids 3a-c. ~ To a suspension (2a) or solution (2b,¢) of 3-S minol chloride 2 In §5-80 ml. cold toluene (0 °C), a solution of 3-5 mmol N-phenylhydroxylamine (PHA) in 45-75 mL toluene and a solution of 5.5 mul N-methylmorpholine (MM) in 30 mL toluene were acided simuttaneously and dropwise. The reaction mixture was stirred al room temperature for 3-24 h. Product 3a partially precipitated from the reaction mixture, together with MM hy: Gtochloride, The precipitate was filtered off and dissolved in a water /ethy! acetate mix. 175 Mr a: Novel 12a ture. The organic lay twice with wat to give 0.525 g (35%) of 33 toluene mother liquor by Proctucts 3b and 3e with water, three times w. layer was dried over anhy pressure. The analstically (eluent: dichloromethane, and cyclohexane, Pure pr hexane mixture 1.25-Oxntiazine 3,60 acid 3a oF 3c in 130 mL acy ‘The reaction mixture was salt was filtered off and the dichloromethane and extre The organie layer was deie lunder reduced pressure. Pi (eluent: cyclohexane/ethyl Cell tines Green monkey kidney ‘noma cells (HeLa; ATCC C ina modified eagle mediun. supplemented with 10% in, Lglutamine, 10 mmo! L-1 mg mL"! streptomycin), anc MIT-test MTT-Hest was used tw (4S-dimethylthiazol-2-y1) zane crystals in live cells an Formazane crystals dissolv. abled the spectrophotometr was possible to determine | agent or if it stimulated cell (CD if specteophotometric Values measured after 24 h « values measured after 72h growth, fe, cytostatic effect values measured ater 72h cell growth (proliferative eff Acyclovir (A as da and de. Fa wes used ompa‘Phar $8000 175-186 terocyclic systems. A rorted by Smalley (6) ompted us to synthe: ir synthetic approach selves have been of awines derivates and iviral activity against Stage (Franz Kastner ceded on a FTIR Perkin NMR spectra were re- rating at 300 and 755 red in DMSO-dg solu- were referred to TMS. and the following sol- ol :1:0.3), cyclohe- sualised by short-wave formed on silica gel ranol (9:1) oF eyelohe- ryllydroxylamine was solvents were of ana- }. — N-Ble-glycine (1a), thesised following the vrides 2 were prepared ») of 3-5 mmol chloride phenylhydroxylamine rrorpholine (MM) in 30 vn mixture was stirred logether with MM by- cer/ethyl acetate mixx Mui al: Novel 12S-xadariedtivaves = Stevan init Halos Ae Pa 9) 175.186 ture. The organic layer was extracted with water, twice with cold 1% HCI solution and twice with water, then dried over anhydrous sodium sulphate, filtered and evaporated to give 0.525 g (35%) of 3a. An additional amount (0.315 g, 21%) of 3a was isolated from toluene mother liquor by an analogous extraction procedure, Products 3b and 3¢ were isolated as follows. The reaction mixture was extracted with water, three times with 1% HCI solution and two times with water, The organic layer was dried over anhydrous sodium sulphate, filtered and evaporated under reduced pressure. The analytically pure 3b was obiained by purification on silica gel column (cluent; dichloromethane/ methanol 91) and additional trituration with ethyt acetate and cyclohexane, Pure product 3e was obtained by recrystallisation with ether/eyelo- hexane mixture 1,2,5-Oxaiazine-3,6-tione derivatives (44,0). ~ To a solution of 1,6 mmol of bydroxamic acid 3a oF 3c in 130 mL acetone, 5 mL of 10% solution of sodium carbonate was added, The reaction mixture was stirred at room temperature for 2.5 h. Precipitated inorganic salt was filtered off and the mother liquor was evaporated. The residue was dissolved in dichloromethane and extracted twice with cole 1% HCI solution and twice with water, ‘The organic layer was dried over anhydrous sodium sulphate, filtered and evaporated under reduced pressure. Products da and 4c were purified by column chromatography (eluent: cyclohexane/ethyl acetate 1:1) Cell tines Green monkey kidney cells (MK; ATCC CCL-26, USA) and human cervical carci noma cells (Hela; ATCC CCL-2, USA) were used. Monolayer cell cultures were grown inva modified eagle medium (OMEM; Dalbecco minimum essential medium, Difeo, USA) supplemented with 10% inactivated foetal calf serum (F5; Gibco BRL, UK), 2 mmol L=! Leglutamine, 10 mmol Lt Hepes (pH 7.4) and antibiotics (100 U mi! penicillin, 100 mg ml streptomycin), and were incubated at 37 °C and 5% of CO, MTT-test MITE-test was used to measure the mitochondrial activity in cells (14). When added, 3-(4,5-dimethylthiazo|-2-y))-2,5-diphenyl-2H-tetvazolium bromide (MTT) formed forma- zane crystals in five cells and cells in an early apoptosis cycle (programmed cell death). Formazane crystals dissolved when dimetyl sulphoxide (DMSO) was added. This en- abled the spectrophotometric reading, at 670 nm. According, to the MTT-test results, it ‘was possible to determine if a tested compound acted as a cytotoxic agent, cytostatic agent or if it stimulated cell growth. The tested compound had a cytotoxic effect on cells (CT) if specteophotometric values after 72 h of incubation were below the initial control values measured after 24 h of incubation. Values at 72 h of incubation below the control values measured after 72 h of incubation indicated the inhibitory effect (IC) of cell growth, {e., cytostatic effect, whereas values at 72.h of incubation higher than contral values measured after 72 h of incubation meant that the tested compound stimulated cell growth (proliferative effect), Acyclovir (Ac) was used as a positive control compound at the same concentrations as da andl 4e. Fach compound was dlissolved in DMSO to produce 0.1 mol L-! solution wr‘Marts Novel 125-natvine duns Sythe ite boi so, Ae lem $82 15-1 and further diluted in DMEM to concentrations of 10 mol L- (\), 10-9 mol L=!(¢)] and 10-6 mol 1 (ey). The final concentration of DMSO in different dilations of compouinls ‘was less than 0.1% and at that concentrations it did not influence the cell growth Antiviral assay Five different viral titres (V-1 to V-5, relative titres) obtained by serial dilution (1:2 for adenovirus and herpesvirus, and 1:10 for enteroviruses) were used to test the inhibi tion of the cytopathic effect (CPE) (15). HeLa and GMK cells were seeded at 10° cells per mL on 24-well microtitre plates (Becton Dickinson, USA), Viral infection was performed on one-day-old confluent cell monolayers with addition of the tested compounds at dil- ferent concentrations, The CPE was followed by an optical microseape 24 h after infec tion for enteroviruses (coxsackievirus and echovirus) and 48 h for adenovirus and her pesvirus. The CPE of enteroviruses was observed on GMK cells, while CPE of adeno- virus and herpesvirus was observed on HeLa cells (10) Statistics ‘The results are shown as the percentage of CPE inhibition compared to CPE without compounds on each plate, The results were statistically analysed on a personal com- puter by ANOVA test and shown graphically. The criteria for statistical significance was p <0.05. The concentration values that inhibit 50% of cell growth (ICs) were calculated. using the linear regression model. RESULTS AND DISCUSSION Syntheses ‘The starting compounds for the synthesis of hydroxamic acids 3 were N-Bic-amino acids (la-e), They were prepated from the appropriate amino acids and l-benzotriazole carboxylic acid chloricle and converted into the corresponding chlorides 2 by mneans of ef ° MN . erty AY? soc, ar sive be _ “6 . a ne ase aren fr b R=Me Ap wy? © Ro Det 200, fun wpteyysostanine Cavey EE An, Se . TW But benzowrarole o Jae dae Scheme 1 | MDa Not end 3.6 acids Bae and 12.5-axadin ‘Dble 1. Reaction con CHN analysis (%) Molecular Mp. ¢ vie Time Solvent 6 formula °C) ) )Mtb: Novel L2S-naoine derientivs = Sy andr bisa sda Pr 5 (0) 7586 ‘ta ar 85006) 178185 wm semeians 903 sunjiodain oss popad Sueur "3, G38 16 ers 0c ‘S91 ‘DAE “OSI ‘SOL LI “BALE SHE cH ODS SOT a ed o “et rr SEH GY aes Lerten) cnet ele DE SEM or sc9s TOTNES ck um "969 ‘08s “FES 86 ‘SCL HFT 95rT PTZL HS HS49 “ror ‘gs0t 'eez 108) “asi “Fost “BOOL “ORLE “ex “DEDE THE GHZL az Esso CONSE © eEI~UET rez tema HYDE 69 “SSL ‘OB “968 “peol ‘Zé01 ‘SELL ‘SECT “SRT ‘ORET‘DEHL GOT SBF Eres (eESTE) “egSt Esl SL91 TULL “TELL “ORSE TSE STZ SF ans —O'NTHD Serer ste woe, «He 609 169 "E82 “F00r‘060T . ‘geet ‘9501 “TOET 'S6et “ZEEE ‘Sort ‘DEST GO FH SLES (6e'tLE) ‘yest “691 “2991 ‘ICZI SSE TE THEE OST ITH 98zsTOFNTESID —FI-Zer we wn, NoHOO cn) oN tu) pune}/"P21e eposi05 punod Gay) at (e) sisfteue NH sR sunt wang gy HOD 2ivp saanecnnp auoyp 9'g-suzmpoce-c7'T pun aE size apuneaphy Jo sep pose pun su nied woy2eey 1 gas rd to test the inbibi- seeded at 10% cells per fection was performed. cssted compounds at dif- Neaetyhnoepatine bencorazle 51), 10-3 mol Ll (e) and dilutions of compounds see the cell growth, by serial dilution (1:2 roscope 24 halter infec- for adenovirus and her- Is, while CPE of adeno- dmpared to CPE without sed on a personal com- tistical significance was th (Cag) were calculated cids 3 were N-Blcamino ids and I-benzotriazole chlorides 2 by means of ~ Ripon oH = Me =o imtit as Noel L2S-naine deinses~ Sythe nd i to lg suds, Ae Pars, 8 (208 15-18 thionyl chloride (12, 13). In the next reaction step, acyl chlorides 2 reacted with N-phe- nylhydroxylamine and gave the following hydroxamic acids: N’-hydroxy-N'-phenyl -N-Ble-glycine amide (3a), N”-hydroxy-N"-phenyH-Bic-L-alanine amide (3b), and N' -hydroxy-N =phenyl-N-Ble-L-phenylalanine amide (3c) (Scheme 1). Its worth pointing cilorides with hydroxylamine by ‘out that the analogous reactions of N-Bleamino ac drochloride in our hands failed to give the corresponding hydroxamic acids (toluene or acetonitrile were used as solvents and triethylamine or N-methylmorpholine as hydtra- xen chloride acceptors) Under basie conditions, hydroxamic acids 3 readily underwent cyclisation to afford 41,25-oxadiazine-3,6-diones (4) (Scheme 1). The substituent on C-4 depends on the starting, amino acid, while the substituent on C-2 atom depends on the hydroxylamine used in the previous reaction step. Two oxadiazine derivatives, viz. 2-phenyl-2H-1,2,5-oxadiazine “36(4HSH)-clione (4a) and 4-benzyl-2-phenyl-2H-1,2 5-oxadinzine-3,6(4H1,511)-dione (Ae), were isolated and analysed, while the corresponding L-alanine derivative (4b) was only detected by TLC, Spectral assignment and CHN analysis of the synthesised new compounds 3 and 4 confirmed their structures. IR spectra of 3 showed the absorption maxima of carbonyl ‘group bound to benzotriazole at 1731-1739 and the hydroxamic acid carbonyl group at 1658-1687 cm-I, In the IR spectra of oxadiazine derivatives, two carbonyl absorptions were visible: carbonyl at position 3 at 1747-1772 em and carbonyl at position 6 at 1688-1692 (amide 1) and 1590-1594 em (amide I), IH_NMR spectra of products 1a-¢ confirmed presence of carboxylic acid group at 13.15-12.00, NH group at 942-934, aromatic protons at 8:15-7.50 and C-2 atom at 4.50-4.04 ppm. C spectra confirmed presence of two carbonyl groups: carboxylic ac carbonyl at 173.28-172.18 and carbonyl bound to benzotriazole at 149.44-148.87 and, fi- nally, chiral C-2 atom at 54.98-41.84 ppm. IH NMR spectra of products 3a-c revealed presence of N-hydroxyl group at 11 ppm, instead of carboxy group. Hydroxamic acid carbonyl was shifted to lower ppm values (167.80-165.25 ppm) than carboxylic acid carbonyl, while carbonyl bound to benzotria- zole, aromatic C-atoms and C-2 had practically the same values as in cormpouinds Tae. 'HLNMR spectra of products 4a,c confirmed presence of NH group ai 884-872, aro matic protons at 7.56-7.27 and C-2 atom at 4.49-4.10 ppm. "SC spectra confirmed pre- sence of two carbonyl groups at 164.85-164.56 and 152.83-152.69, aromatic C-atoms at 19739-1221 and C-2 atom at 55.48-44.20 ppm Reaction conditions, yields and analytical data of the newly synthesised com- pounds are presented in Table I and NMR spectral data are given in Table IL Biological studies The synthesized oxadiazines were used in biological studies. Values obtained by the MTT-test were all below control values 72 h following incubation (Figs. 1 and 2). No growth inhibition of GMK cells was observed for compounds 4a and 4e at concentration 5, but at concentrations ¢ and cy a statistically significant growth inhibition of GMK ved, ranging from 40% lo 6% (Fig, 1). Similarly, a statistically significant ’ of HeLa cells was observed for 4a at concentration ¢; (Fig, 2), cells was obs 180 Roof as Nove Souda zine 3,6 i 1.2S:oradie a of B-amina acids Ya-c, hydroxamic acids 3a-e NMR and 3 NMIad Ph $98) 15-16 tos 2 reacted with N-phe- N’-hydroxy-N’-phenyl- sine amide (3b), and N’- © 1), Its worth pointing with hydroxylamine hy- ovamic acids (toluene or aylmorpholine as hydro vent cyclisation to afford 4 depends on the starting yeroxylamine used in the snyl-2H-1,2,5-oxadiazine- ne-3,6(417,5H)-dione (4c), derivative (4b) was only new compounds 3 and 4 stion maxima of carbonyl ic acid carbonyl group at wo carbonyl absorptions carbonyl at position 6 at carboxylic acid group at 15-7.50 and C-2 atom at yl groups: carboxylic acid feat 149.44-14887 and, fi- \ydroxyl group at 11 ppm, fted to lower ppm values sony bound to benzotria- 5.5 in compounds a-c Hi group at 884-8.72, aro- pectra confirmed pre- 2.68, aromatic C-atoms at newly synthesised com- siven in Table I ies. Values obtained by the sation (Figs. 1 and 2). No 4a and de at concentration srowth inhibition of GMK ‘ya statistically significant ‘Feoncentration ¢y (Fig. 2), ine3.6ctione derivatives 4a Sosa 1, z e 2 g 2 y S § § z z Novel 12Senlizve drives Syahid bilgi adi, Ae Phar 9) 15-186 BC NMR ‘Compound No. 5.54 ( 17262 (i), 9.44 (4), 13 1s 41880) aw 75 Ha), 27 He), 7.49 27 Ha, 7.33 (4, 2H, 7, 11, He), 723 (2 a 3.10.7 53 He), 805 (4, 1H, 5 1H, 3, Hi, 9,272, 2) 765 I 12.15 @ 1H, OH), 934 (4, 1H, 3, J = 83 He), = 8 iH, } 181tril ls Rove 12x dress = Sybil ti apes, At art 92 15-4 Mtr at Nove 2 Sadan while cell proliferation wa: growth inhibition of Hela 2). The higher growth inh GMK cells, was probably present in Hela cells (data Results of the antivical i Sad stimulated both GMK and 2 at ane | mw i a a3 885 i | ge 8 gees o Sq £ gen8 aaa lg We a 2 3 z z 2. a te ; Fig. 2. Percentage of grow : 58 & 3 2 : Hela cells determined by MAT : eS ¢ : in the absence (control) or | s a7 hat <. a Se a presence of da, 4c and acyelovi 3 Ro age eh 2 ge (6 = 104 mol LA, = 10 me z SR ERG ze 8 25 : 6) 104 mol Lan = SD @ gR aks fe 2 2o : 10° mol Lan 5, se seq $8 es |= f3 ges gz 8 Re At S28 39, 2 23 As the cytostatic effect B18 fee 8 88 anel are presented in Table om 2 a than on Hela cells, while ang 2A ge i The inhibition of CP 2 ; alltested viruses, while tre * : observed only at concentra 7 concentration cy (approxi. 2 = FR zl GE £ -()ei| #5 - 4 Sef ge Coll tir | [as ls 8 # a3 8 ux | | a Het i 1 23 1iy At Par 590 1. M tart a Noel 125 onze derives» Sot a is, At hm 88D) 15-186 while cell proliferation was observed at concentrations ¢z and cy, Statistically significant , srovth inhibition of HeLa cells was observed also for 4c, ranging from 50% to 75% (Fig Le 2), The higher growth inhibition of the tested compounds on HeLa cells, compared tt =f GMK cells, was probably due to the programmed cell death phenomena permanently ¢ present in HeLa cells (data not shown) (16). Contrary to the tested compounds, acyclovir = Stinvalated both GMK and HeLa cell yrowth at all concentrations (Figs. 1 and 2), 9, 137.10 @), 123.16 Diets — Fig, 1 Percentage of growth of GMK cells determined by MTT-tet in the absence (control) or in the presence aff, de and acyclovir (Ac) 0 J (= 104 mol L*, ep = 10 mol Lo, aaa (aaa (a ae 0° mol L-})(oneant SD, n = 12), a 300) “ he i £ 5 2 wo E Fig. 2 Percentage of growth of Cem oh i HeLa ces determined by MTTest B ect inthe absence (contol) or inthe & i precnocofaaseandecycovie(ag | = Gritmltgsiemlis, 9! : c= 10 mol L*) (mean SD, n= 12) reassert a ten : As the eytostatic effect of 4a and 4e was obsorved, ICyy values (17) were calculated and are presented in Table III. A compound 4a showed higher cytostatic effect on GMK than on HeLa cells, while 4c was more cytotoxic on Hela than on GMK ells, Results of the antiviral assay’ (percentage of CPE inhibition) are shown in Table IV The inhibition of CPE (approximately 5-20%) for 4a was observed at concentration ¢, for all tested viruses, while the inhibition of coxsackievirus CPE (approximately 5-15%) was ‘observed only at concentrations c, and ¢y, Compound 4c inhibited CPE of adenovirus at ‘concentration cy (approximately 20%) as well as CPE of herpesvirus and enteroviruses at 4 ‘Table I IC for compounds 40 and 4 ion 8 (PP) atl ine ; “IC (mol La) # e6 ea Tis tot S0 28 Hea ast att i i 183Mit Noel 125-ndiorinederentvs = Syed wt ilps sadn Ace Mar $3) 17586 concentrations ey, ¢ and ¢, (approximately 5-20%). A satisfactory inhibitory effect rang- ing from 21 to 40% of acyclovir, as expected (18), was observed on herpesvirus at all tested concentrations and virus titres, Poor CPE inhibition of da and 4e observed for all tested viruses (maximum 20%) and the lack of CPE inhibition at minor virus titres and at lower concentrations of the tested compounds was probably not an antiviral effect but more likely their inhibitory clfect upon cell growth (shown previously by the MPT:test). Thus, the lack of CPE bition at low virus titres, wl hie is usually possible to observe the inhibition of CPE for antiviral compounds (¢.¢. acyclovir), may confirm the previous statement Table 1V. Cytopathic eee (CPE) on cell culture of different vrusest ypon 4a, 4¢ and acylovie (Ac)? compared to contol (solvent DMEM) Virus/titee CPE inhibition (a) ae 4 StrivAe-CEeeee ‘Adenovires 7 Peet va o 0 » 0 oO “ s = va o 0 » 9 Oo ape v3 w 0 0 oO 0 Sie eee vt Mie ing gig < = 5 oo! Gee Oo - + Hlerpesvius 1 va oo 6 ee ee ) v2, o 0 wm ww mM wD OD va 0 0 2 © 0 0 » % » va 6 0 5 6 6 6 me Tchaviras 7 o 5 DM Wo = = mo 0 ww Ww - - go-to eta ote Heeb Fateh i oo 0 o mB tb Ss 0 m MW - = » 5 2 mM wo oe ss VAL to V5: soil dilution 12 for edonovrus 7 and Rerpesvius 1a 1:10 for echoes 7 and 10 ta Lt p= 109 wl Lh, 65 = 4 molt Mbit Noel 12 Sordi 13.5-Oxadinaine-36 propriate hydroxamic ac of Nemonosubstituted hy eneral method for const studies indicated that ox cells at tested! concenteat hibited ell growth in the centration. A minor inhit ‘compounds 4a and 4c, w. antiviral drugs could be tion should be undertake Ackuooledgemonts.-Th Ministry of Science and Tec govae for providing the « 1, PT. Berkowitz, R.A, Le bial activity of certain & 2.K/M. Khan, S. Rahat, ¥ and A, Malik, Syntbesis oxadiavine-4propanenit 3.M.J. Komnet, Synthese « ines and 2aryl3-dia 247-2049. 4. RP Robinson and K, M ives of ant-inflammto 5. M. B. Gravestock, Ppa penem Intermeites, Eur 10, 6.8K: Smalley, 1.25-0%0 (Ba. A.J. Boulton), Yor 7. RJ. Bergeron, Synthesis 597-02. 8. S.Hanessian and 5 John Beticarbonyl compound: 9. M. Whittaker, C.D. Flay matrix metalloprateinas 10. Fundamental vetogy, 21 11.5. UIsié V Vitek, D. He sobenzenes Kole 0 the 2, 12,1. Butula, B. Zore sn ¥. setzung mit Aminesaun.At Phar, $9208 15-1 vey inhibitory effect rang- ‘don herpesvirus at all viruses (maximum 20%) wer concentrations of th ore Itkely their inhibitory vs, the lack of CPE init- the inhibition of CPE for s statement 1 da, de and acyclovir (Ac! mm 0 tahun a: Nove 1.2.S-nsne drat Sys nd ie ili tu, At Phar 89 (20 175-186 CONCLUSIONS 1,3,5-Oxadinzine-3,6-diones can be successfully prepared by cyclisation of the ap- propriate hydroxamic acids. Since various methods for the preparation of a wide array of Nemonosubstituted hydroxylamines are available, this procedure constitutes @ new {general method! for construction of the 1,3,5-oxadiazine ring, system. Selected biological studies indicated that oxadiazines da and 4e had no cytotoxic effect on HeLa and GMK calls at sntrations, but they had a pronounced cytostatic effec, i, they in- hibited cell growth in the 25-75% range, depending on the compound, cel Line and con- centration. A minor inhibition of CPE on adenovirus, herpesvirus and enteroviruses, for ‘compounds 4a and de, was observed. These preliminary findings indicate that potential antiviral drugs could be found within oxadiazine derivatives. Thus, further investiga- tion should be undertaken, ested con, Ackotaledgemonts. ~ This stuly was supported by grants (Nex D006549 and 0096104) from the Ministry of Science and Technology of the Republic of Crostis. 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Ispitano je biotosko djetovanje spojeva da i de u in vitro wvjetima, Citotoksigno i citostatsko djetovanje testirano je na dvije stanigne linije (HeLa i GMK) i evaluirano MTTtestom, Antivicalni testovi provedeni su na dva humana DNA virusa (adenovitus 7 i herpesvirus 1) i dva humana RNA virusa (coxsackievirus BS i echovirus 7). Ustanov- leno je da 2-fnil-2H-1,25-oksadinzin-3,641,5H)-dion (4a) i d-benzil-2-fenil-2H-1, ~oksadicin-3,6(4H,SH)-dion (4) statistitki znagajno inhbiraju rast stanica GME i stanica HeLa, te da imaju slabi protuvirusn ueinak na fsitane viruse jute ijt 1.2.5-oksadiazin derivati,sknteza, cltotoksiga uéinak, djelovanje, ctopatski ukinak lostatski ucinak, protuvirusne Formaceutcko-biskemijpk fakultet Seewtlitta u Zagreb, Zagreb Institut Rudjor BoBkovt, Zagreb 186