Synthesis and cytotoxic evaluation of some new 4(3H)-quinazolinones on HeLa cell line

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1 Rsr in Prmutil Sins, My 2012; 7(2): Riv: Sp 2011 Apt: D 2011 Sool o Prmy & Prmutil Sins Isn Univrsity o Mil Sins riinl Artil Syntsis n ytotoxi vlution o som nw 4(3)-quinzolinons on L ll lin G.A. Kormi, M. Smsiri n F. ssnz Dprtmnt o Miinl Cmistry n Isn Prmutil Sins Rsr Cntr, Sool o Prmy n Prmutil Sin, Isn Univrsity o Mil Sins, Isn, I.R.Irn. Astrt Quinzolinon kon is prsnt in lr numr o iotiv sustns. Sin rmrkl ytotoxi tivity is ssoit wit som 4(3)-quinzolinons, in tis stuy som 4(3)-quinzolinon wr syntsiz n srn inst L lls. T syntsis ws prorm vi rtion o ntrnili i wit iroxyli nyris to prou roxyli is rivtivs. T prouts wr t in ti nyri to prou nzoxzinons. Finlly, 4(3)-quinzolinons wr syntsiz y rtion twn nzoxzinons n primry mins. T ssssmnt o t strutur o t syntsiz ompouns ws s on sptrl t (FT-IR, Mss n 1 MR). Susquntly, ytotoxi tivity o ompouns 3, 6, 9 n 13 (iniviully n in omintion wit oxoruiin) ws vlut on L ll lin usin MTT ssy. T rsults init tt t tst ompouns i not sow siniint ytotoxiity lon n in omintion wit oxoruiin (1 n 20 µm). Kywors: Quinzolinon; Syntsis; Cytotoxiity; L ll ITRDUCTI Cnr is li trtnin iss wit omplx ptonsis wi trts umn li rtly. Quinzolinon is uilin lok or svrl lklois isolt to t (1). 4(3)-Quinzolinons r us troyls (2) wit ivrs rn o ioloil tivitis..; ytotoxiity, ntimiroil, protin tyrosin kins iniitory n nti-inlmmtory proprtis (1,3). Triyli quinzolinon rivtiv, "Doxyvsinon" nturl lkloi is wll known s ytotoxi nt (4,5). Strutur tivity rltionsip stuis init tt sustitutions t 2 n 3 positions o quinzolinon rin r ily ssoit wit ytotoxi tivity o ts ompouns. Tror, ltrin t sustitutions on ts positions my rsult in nnin ytotoxi tivity o ts ompouns (6-8). In t lit o ov onsirtions, w pln to syntsiz numr o 2,3 isustitut rivtivs o quinzolinon. In ition, tir possil ytotoxi tivitis wr lso ssss. Aorin to t litrtur, usully t irst stp in t proution o quinzolinon rin is t rtion twn ntrnili i n n yl lori or ti nyri (8,9). T prout in ot rtions is n mi sustitut wit simpl lkyl roups wi upon rin losur ivs t nzoxzinon. Finlly, lkyl sustitut quinzolinons r otin rom t rtion o nzoxzinon wit irnt mins. In t urrnt stuy, ntrnili i ws rt wit yli iroxyli nyris (10). Applition o yli iroxili nyris lik suini nyri inst o simpl yl loris or ti nyri or r roxyli i t t n o t mi si in. T mi is n to t orrsponin nzoxzinon n rt wit irnt mins to iv t inl quinzolinon rivtivs. T prsn o n xtr roxyli moity on t quinzolinon si in my srv s nw sit or urtr sustitutions on quinzolinon si in or possily n xtr rin losur to prou triyli quinzolinon rivtivs. Corrsponin utor: F. ssnz, tis ppr is xtrt rom t Prm.D tsis o Tl , Fx Emil: ssnz@prm.mui..ir

2 G.A. Kormi t l. / RPS 2012; 7(2): T prsn o r roxyl roup on t quinzolinon strutur oul possily ltr t ioloil proprtis inluin ytotoxi tivitis. Bus it s n sown tt irnt iroxyli i rivtivs possss onsirl ytotoxi tivitis (11), ll iroxyli i intrmits prou in tis work wr lso vlut or tir ll toxiitis. MATERIALS AD METDS Mltin points wr trmin on n Eltro trml 9200 mltin point pprtus. 1 MR sptr wr ror on 400 Mz sptromtr Brukr (Grmny); mil sits r xprss in ppm wit rrn to TMS. Mss sptrl t wr otin on Simzu LC/MS-2010 pprtus (Jpn). Inrr (FT-IR) sptr wr ror on WQF-510 FT-IR sptromtr (Cin). Tin lyr romtorpy ws prorm on Mrk m pr-ot (0.25 mm) sili l GF254 plts (Mrk Co., Grmny); ompouns wr tt wit 254-nm UV lmp (Prkin Elmr 550s, USA). Sili l ( ms) ws mploy or routin olumn romtorpy sprtions. RPMI 1640, MTT n ntiiotis (pniillin G/ strptomyin) wr purs rom Sim (Enln), tl l srum (FCS) n trypsin-edta wr purs rom Gio (Sotln). P 4, K 2 P 4, Cl, KCl, Cl n wr purs rom Mrk (Grmny) n L ll lin ws otin rom CBI (Irn). Cmistry T nrl rtion sm or t prprtion o t trt ompouns is sown in Fi. 1. Syntsis o -nzyl-3-(3-nzyl-4-oxo-3, 4- iyroquinzolin-2-yl) propnmi (5) A mixtur o ntrnili i 1 (5.4, 0.04 mol) n suini nyri 2 (4, 0.04 mol) ws rlux or 5 in lil ti i (GAA) (20 ml). T solvnt ws vport n t rsiu ws rrystlliz in ton to yil 2-(3-roxypropnmio) nzoi i 3 s yllowis rystl. T rsultin roxyli i 3 (2, mol) ws t t ºC in ti nyri (8 ml) or 1 n tn ws onntrt usin vuum pump to or 3-(4-oxo-4-nzo[][1,3]oxzin-2-yl) propnoi i 4 s rk visos oil. Bnzoxzinon rivtiv 4 (1.84, mol) ws rlux wit nzylmin (1.48, mol) in loroorm (20 ml) or 3. T solvnt ws vport n rsultin oily mss ws issolv in tyln lyol (20 ml), (0.25 ) ws n t mixtur ws t t ºC or 3. T mixtur ws kpt ovr nit t room tmprtur. T p o rsultin mixtur ws just to 7-8 y ition o Cl 3%. T pripitt ws iltr n ws wit ol wtr n rrystlliz in isopropyl lool to or - nzyl-3-(3-nzyl-4-oxo-3,4-iyroquinzolin -2- yl) propnmi 5 s wit rystls. Syntsis o 3-(4-oxo-3, 4-iyroquinzolin- 2-yl) propnoi i (6) 3-(4-xo-4-nzo[][1,3]oxzin-2-yl) propnoi i 4 (1.84, mol) wi ws lry syntsiz n ormmi (1.133, mol) wr rlux in solut tnol (25 ml). Atr 4, t ot suspnsion ws iltr, t luis smisoli ompoun otin ws rtriz s 2,3- iyropyrrolo [2, 1-] quinzolin-1, 9-ion 7. T iltrt ws llow to ool t room tmprtur. 3-(4-oxo-3, 4-iyroquinzolin - 2-yl) propnoi i 6 ws pripitt s wit rystllin powr. Syntsis o -nzyl-4-(3-nzyl-4-oxo-3, 4- iyroquinzolin-2-yl) utnmi (11) A mixtur o ntrnili i 1 (3.5, mol) n lutri nyri 8 (4, mol) ws rlux or 10 in GAA (15 ml). T solvnt ws vport to or smisoli prout wi ws rrystlliz in loroorm to yil 2-(4-roxyutnmio) nzoi i 9 s wit rystls. T rsultin roxyli i 9 (0.5, mol) ws t t ºC in ti nyri (2 ml) or 1. Solvnt ws vport to yil 4-(4-oxo-4nzo[][1, 3] oxzin-2-yl) utnoi i 10 s visos oil. Bnzoxzinon rivtiv 10 (0.47, mol) ws rlux wit nzylmin (0.98, mol) in loroorm (5 ml) or 3. T solvnt ws vport n t rsultin 120

3 Syntsis n ytotoxi vlution o som nw... 2 in AAG C 3 C 1 C 2 8 in AAG C 9 j i C 12 in AAG C 13 C 5 i 3 Ati nyri o C C 2 4 in tnol C 7 Ati nyri 2 9 C o C j k l i Fi. 1. Gnrl rtion sm or t prprtion o trt ompouns. 121

4 G.A. Kormi t l. / RPS 2012; 7(2): oil ws issolv in tyln lyol (5 ml), (0.1 ) ws tn n t mixtur ws t t ºC or 3. T mixtur ws kpt ovr nit t room tmprtur. T p o t rsultin mixtur ws just to 7-8 y iition o Cl 3%. T rsultin mixtur ws onntrt y rotry vportor n rtiont on PTLC to iv - nzyl-4- (3- nzyl -4- oxo-3, 4-iyroquinzolin-2-yl) utnmi 11 s wit powr. Syntsis o 2-(3-roxyrylmio) nzoi i (13) In n ttmpt to prou -nzyl-3-(3- nzyl -4- oxo -3,4- iyroquinzolin -2 -yl) rylmi mixtur o ntrnili i 1 (5.4, 0.04 mol) n mli nyri 12 (3.92, 0.04 mol) ws rlux or 6 in GAA (20 ml) to yil 2-(3-roxyrylmio) nzoi i 13 s pl yllow powr. Furtr rtions i not ollow t usul prour n sir ompoun ws not isolt. Bioloil tivity L ll lin ws rown in RPMI 1640 mium (striliz usin 0.22 µm miroioloil iltrs) supplmnt wit 10% FCS, 100 U/ml pniillin G, 100 m/ml strptomyin n L-lutmin (12). T p o t mium ws tn just twn 7.3 n 7.6 usin Cl or n kpt t 4ºC or us. Clls wr ultur in n tmospr o 5% C 2 in umiii ir t 37ºC (12). L ll lin ws s in 96-wll plt t ll nsity o lls/ml n inut or 24. Tn 20 µl o vrious onntrtions (1-500 µm) o t syntsiz ompouns (3, 6, 9 n 13), DMS 1% (s ntiv ontrol) n oxoruiin 20 µm (s positiv ontrol) wr to t ultur mi n inut or 48. In sprt st o xprimnts, 22.5 µl o oxoruiin (1, 20 µm) n 22.5 µl o ompouns (100 µm) ws to wll n inut t t sm onition s mntion ov. Cytotoxiity ws trmin y rpi olorimtri ssy usin MTT. T lls wr inut t 37ºC or 3 wit MTT. Tn t mium ws rmov n t rsultin ormzn rystls wr issolv in 180 µl o DMS (13). Asorn o wll ws msur usin miroplt rr t 540 nm. All rsults wr ompr wit untrt ontrol. T rsults wr lso ompr wit oxoruiin in t omintion xprimnts. E st o xprimnts ws prorm in triplit witin tr onsutiv ys. RESULTS 2-(3-Croxypropnmio) nzoi i (3) C ; Yil 90%; m.p ºC; wit powr; FT-IR (KBr, m -1 ): 3323 (-), 3150 (C- Ar), 2939 (C- Alipti), (C), 1682 (C=); 1 MR (400 Mz-DMS): (2, s, ), (1, s, ), 8.47 (1,, j=8.4z, ), 7.97 (1,, j=8z, ), 7.57 (1, t, j=7.2z, ), 7.13 (1, t, j=7.6z, ), 2.6 (2, t, j=6.4, ), 2.54 (2, t, j=6, ); Ms (m/z, %): 236 (M-1, 100). - nzyl -3- (3- nzyl -4- oxo -3, 4- iyroquinzolin-2-yl) propnmi (5) C ; Yil 65%; m.p ºC; wit rystl; FT-IR (KBr, m -1 ): 3309(-), 3028(C- Ar), 2954 & 2885 (C- Alipti), 1682 & 1630 (C=); 1 MR (400 Mz- CDCl3): 8.23 (1,,, j=8z, j=1.2 z, ), 7.63(1,, t, j=8.4z, j=1.6z, ), (2, m, ), (10, m, ), 6.39 (1, s, ), 5.39 (2, s, ), 4.35 (2,, j=4.8z, ), 3(2, t, j=5.6, ), 2. 7 (2, t, j=5.6, i); Ms (m/z, %): 397 (M +, 31). 3- (4- xo -3, 4-iyroquinzolin -2- yl) propnoi i (6) C ; Yil 54%; m.p ºC; wit rystl; FT-IR (KBr, m -1 ): 3417 (-), 3064 (C- Ar), 2935 (C- Alipti), 1770 & 1693 (C=); 1 MR (400 Mz-DMS): 7.88 (1, s, ), 7.7 (1,, j=7.6z, ), 7.58 (1, t, j=7.4z, ), 7.5 (1, t, j=7.4z, ), 7.28 (1, s, ), 7.25 (1,, j=7.6, ), (4, m, ); Ms (m/z, %): 218 (M +, 100). 2, 3-Diyropyrrolo [2, 1-] quinzolin-1, 9-ion (7) C ; FT-IR (KBr, m -1 ): 2922 & 2852(C- Alipti), 1803 & 1711 (C=); 1 MR(400 Mz-DMS): 8.14 (1,, 122

5 Syntsis n ytotoxi vlution o som nw % Cll Survivl ompoun 3 ompoun 6 ontrols C+ C Conntrtion µm Fi. 2. Cytotoxi ts o ompouns 3, 6 on L lls. Followin xposur to irnt onntrtions o ompouns 3, 6, ll viility ws ssss usin MTT mto. Dt r prsnt s mn ± SD; P<0.05 (ompr to ntiv ontrol); n=9; C+ =Positiv ontrol (Doxoruiin 20 µm); C- =tiv ontrol (DMS 1%) % Cll Survivl ompoun 9 ompoun 13 ontrols C+ C Conntrtion µm Fi. 3. Cytotoxi ts o ompouns 9, 13 on L lls. Followin xposur to irnt onntrtions o ompouns 9, 13, ll viility ws ssss usin t MTT mto. Dt r prsnt s mn ± SD; P<0.05 (ompr to ntiv ontrol); n=9, C+ =Positiv ontrol (Doxoruiin 20 µm); C- =tiv ontrol (DMS 1%). 100 % Cll Survivl onntr on 100 onntr on 100 +ox 1 onntr on ox 20 ontrols 0 Comp.3 Comp.6 Comp.9 Comp.13 Dox1µM C+ C Compouns Fi. 4. Cytotoxi ts o ompouns 3, 6, 9 n 13 iniviully n in omintion wit oxoruiin on L lls. Followin xposur to irnt onntrtions o ompouns 3, 6, 9 n 13 n oxoruiin, ll viility ws ssss usin t MTT mto. Dt r prsnt s mn ± SD; P<0.05 (ompr to ntiv ontrol); n=9, Dox1µM = Doxoruiin 1 µm; C+ =Positiv ontrol (Doxoruiin 20 µm), C- =tiv ontrol (DMS 1%). 123

6 G.A. Kormi t l. / RPS 2012; 7(2): j=6.8 z, ), 7.88 (1, t, j=6.8z, ), (1, t, j=6.8z, ), 7.46 (1,, j=7.6z, ), (4, m, ); Ms (m/z, %): 201 (M+1, 36) 2-(4-Croxyutnmio) nzoi i (9) C ;Yil 60%; m.p ºC; wit powr; FT-IR (KBr, m -1 ): 3321 (-), 3118 (C- Ar), 2960 (C- Alipti), (C), 1703 & 1676 (C=); 1 MR (400 Mz-DMS): (1, s, ), (1, s, ), 11.1 (1, s, ), 8.46 (1,, j=8.4. z, ), 7.96 (1,, j=8z, ), 7.57 (1, t, j=7.2z, ), 7.13 (1, t, j=7.6z, ), 2.42 (2, t, j=7.2z, ), 2.29 (2, t, j=7.6z, i), 1.82 (2, m, j); Ms (m/z, %): 251 (M +, 20) - nzyl -4- (3- nzyl -4- oxo -3, 4- iyroquinzolin-2-yl) utnmi (11) C ;Yil 5%; m.p C; wit powr; FT-IR (KBr, m -1 ): 3288(-), 3064 & 3030 (C- Ar), 2951 & 2868 (C- Alipti), 1680 & 1635 (C=); 1 MR (400 Mz-CDCl3): 8.2 (1,,, j=1.2z, j=9.2z, ), 7.64 (1,, t, j=1.6z, j=8.4z, ), 7.46 (1,, j=8z, ), 7.4 (1, t, j=7.6z, ), (8, m, ), 7.11 (2,, j=6.8z, ), 6.13 (1, s, ), 5.38 (2, s, ), 4.34 (2,, j=5.6z, i), 2.76 (2, t, j=7.2z, j), 2.27 (2, t, j=6.8z, k), (2, m, l); Ms (m/z, %): 411 (M +, 76.5) 2-(3-Croxyrylmio) nzoi i (13) C ; Yil 85%; m.p C; rm powr; F-IR (KBr, m -1 ): 3072 (C- Ar), (C), 1689 (C=); 1 MR (400 Mz - DMS): (2, s, ), (1, s, ), (1,, j=8z, ), 7.99 (1,, j=6.8z, ), 7.6 (1, t, j=7.2z, ), 7.2 (1, t, j=8z, ), 6.6 (1,, j=12z, ), 6.3 (1,, j=12z, ); Ms (m/z, %): 234 (M-1, 100) Cytotoxi vlution o t prpr ompouns (3, 6, 9, n 13) on L ll lin rvl tt ts ompouns t tst onntrtions oul not ru t viility o lls siniintly s sown in Fis. 2 n 3. Comintions o ts ompouns t onntrtion o 100 µm wit oxoruiin (1 n 20 µm) oul not improv t ytotoxiity o oxoruiin itr (Fi. 4). 124 DISCUSSI Aylloris r t most rport sustrts or t irst nulopili ttk (8, 9), rri out on ntrnili i in t proution o 2-sustitut quinzolinon rivtivs. r yli iroxyli nyris (i.. suini nyri) srv s sustrt to prou 2-sustitut rivtivs vin rtiv ronyl roup tt to t n o t si in (ompouns 3, 9 n 13). T roxyli rivtivs prou sily ws pripitt u to tir ir polrity tn t strtin ntrnili i. In t nxt stp nzoxzinon ws prou in t prsn o ti nyri, trou yrtiv yliztion mnism. T rsult ltons r vry unstl (14,15) n ry or rtion wit ny nulopils vill in t mi inluin wtr n primry mins. Rtions o propr lipti mins wit nzoxzinon rsult in t proution o sir quinzolinons s t inl ompouns (ompouns 5, 6, 7 n 11). Dirt miiition o lunky roxyli n roup s wll s t proution o quinzolinon rin is lso possil wn nulopil is us in xss quntity s sn in ompouns 5 n 11. T rsults o ytotoxi vlution o ompouns 3, 6, 9 n 13 on L ll lin i not sow siniint ytotoxi tivitis vn t 500 µm onntrtion. Comintion o ts ompouns t onntrtion o 100 µm wit oxoruiin (1 n 20 µm) oul not lso improv t ytotoxiity o oxoruiin. Dspit mpl mount o inormtion rlt to t ytotoxi ts o similr quinzolinon rivtivs, t lk o tivity osrv r oul ttriut to t low soluility o t stui ompouns in RPMI mium or t ntur o t ompouns. It soul not tt som o t ompouns prou r wr not solul nou to vlut or ytotoxi tivity wit routin prours (ompouns 5, 7 n 11). CCLUSI T mil prour ppli r, sussully rsult in t syntsis o sir ompouns n numr o si

7 Syntsis n ytotoxi vlution o som nw... prouts, wi wr luit trou routin nlytil prours. Syntsiz ompouns i not sow onsirl ytotoxiity on L lls t tst onntrtions. tr ioloil vlutions inluin ytotoxiity on otr ll lins n ntimiroil tivitis sms niil u to t struturl similrity o syntsiz ompouns wit known tiv ytotoxi sustns. ACKWLEDGMET Tis rsr ws support y t Rsr Counil o Isn Univrsity o Mil Sins. REFERECES 1. Msk BS, Ar P. T mistry o rntly isolt nturlly ourrin quinzolinon lklois. Tt Ltt. 2006;62: Connolly DJ, Cusk D, Sullivn TP, Guiry PJ. Syntsis o quinzolinons n quinzolins. Tt Ltt. 2005;61: Liu JF, Wilson CJ, Y P, Spru K, Srnt K, Si Y, t l. Privil strutur s quinzolinon nturl prout-tmplt lirris: intiition o novl tuulin polymriztion iniitors. Bioor M Cm Ltt. 2006;16: Liu JF, Y P, Spru K, Srnt K, Yonns D, Blino CM, t l. ovl on-pot totl syntss o oxyvsiinon, mkinzolinon, isinioton, n tir rivtivs promot y mirowv irrition. r Ltt. 2005;7: Lmouri F, Touik, Bouzzin SM, mii M, Bourin M. Exprimntl n omputtionl stuy o ioloil tivitis o lklois isolt rom Pnum rml ss. J. Mtr. Environ. si. 2010;1: R D, Elr MC, Dion G, Mio B. Syntsis, ytotoxiity n iniitory t on tuulin polymriztion o nw 3-troylo sustitut 2-styryl quinzolinons. Eur J M Cm. 2004;39: Slvm P, Bu K, Pmrj R, prsoons L, Clrq E. Syntsis, ntivirl n ytotoxi tivitis o som novl 2-pnyl-3-isustitut quinzolin-4(3)- ons. Ar J Prm Prmol. 2008; 2: As SE, Al Gw M, Gory, Aull J. w quinzolinon rivtivs: syntsis, nti-inlmmtory n ntitumor tivitis. Cm T. 2010;2: Cnrik PM, Yki T, rsi B, Srir V, Vnuopl G, Vnktswr Ro J, t l. Syntsis linto novl 2, 4, 6-trisustitut quinzolinon rivtivs, tir ntitril n ytotoxi tivity inst TP-1, L-60 n A375 ll lins.inin J Cm. 2009;48: Mj Kn A, Anjum S, Coury MI, Att-ur- Rmn. Syntsis o iotiv suinylntrnili i str n its nlous. Worl J Cm. 2006;1: ll I, Izyor RA, Wrrn AE, Brns CR. Cytotoxiity n mo o tion o lipti iroxyli is in L1210 lympoyti lukmi lls. Antinr Rs. 1999;19: Jntov S, Cipk L, Slmnov D, orvt V, Ruko P. Inution o ytotoxiity n ssda rks y 9-romo-5morpolinottrzolo[1,5-] quinzolin in tumor lls ultur in vitro. Toxiol in Vitro. 2003;17: Kml A, Brti EV, Rmi MJ, Dstiri D, Ry JS, Viswnt A, t l. Quinzolinon link pyrrolo[2,1-][1,4]nzoizpin (PBD) onjuts: Dsin, syntsis n ioloil vlution s potntil ntinr nts. Bioor M Cm Ltt. 2010;18: Mkour MF. Rtivity o 4-3, 1-nzoxzin-4- ons towrs nitron n ron nulopili rnts: pplitions to t syntsis o nw troyls. Arkivo J. 2004;i: r AA, Rti LG, Cu, Pis VJ, Bnl A. Mirowv ssist on pot syntsis o 2, 3-isustitut Quinzolin-4-(3)-ons n tir potntil ioloil tivity. Dr Prm Cmi. 2010;2: