B Y T TRNG I HC DC H NI NGUYN QUC THNG NGHIN CU TNG HP V TH HOT TNH SINH HC CA MT S DN CHT THIAZOLIDIN-2,4-DION KHA LUN TT NGHIP DC S H NI - 2013 B Y T TRNG I HC DC H NI NGUYN QUC THNG NGHIN CU TNG HP V TH HOT TNH SINH HC CA MT S DN CHT THIAZOLIDIN-2,4-DION KHA LUN TT NGHIP DC S Ngi hng dn: 1.TS. Phan Th Phng Dung 2.DS. Lm Th Ha Ni thc hin: B mn Ha Dc Trng i hc Dc H Ni H NI - 2013 LI CM Nu tin vi tt c s knh trng, ti xin by t lng bit n su sc nthy gioPGS.TS.NguynHiNamvcgioTS.PhanThPhngDung,cc thyctrctiphngdnvchbotntnhgiptihonthnhkhalun ny. Ti xin gi li cm n n DS. Lm Th Ha cng cc thy c, cc anh ch kthutvinbmnHaDcTrngihcDcHNigipvto iu kin tt nht cho ti trong thi gian thc hin kha lun. Ti cng xin gi li cm n n cc thy c gio Trng i hc Dc H Ni tn tnh dy d, ch bo ti trong sut 5 nm hc va qua. TixincmnsgipcacccnbphngPhntchhucVin Hp cht thin nhin - Vin Hn lm Khoa hc v Cng ngh Vit Namv Phng Ha vt liu Khoa Ha Trng i hc Khoa hc t nhin i hc Quc gia H Ni. Cuicngtixingilicmnngianhcbitlbmti,ngi thn v bn b quan tm, ng vin to ng lc cho trong cuc sng v hc tp. H Ni, ngy 21 thng 5 nm 2013 Sinh vin Nguyn Quc Thng MC LC CHNGTNG QUN ................................................................................... 2 Tc dng sinh hc ca thiazolidindion ........................................................... 2 c tnh t bo ......................................................................................... 2 1.1.2. Tc dng c ch enzym histon deacetylase .............................................. 5 3 Mt s tc dng khc ca thiazolidindion .............................................. 12 2 Mt s phn ng s dng trong tng hp dn chtthiazolidindion ............. 14 2Phng php tng hp thiazolidin-2,4-dion ............................................ 14 22 Phn ng th SN2 .................................................................................. 15 23 Phn ng ngng t ................................................................................ 15 CHNG 2 NGUYN LIU, THIT B, NI DUNG V PHNG PHP NGHIN CU ...................................................................................................... 17 2 Nguyn vt liu, thit b ............................................................................... 17 22 Thit b, dng c .......................................................................................... 17 23 Ni dung nghin cu ................................................................................... 19 24 Phng php nghin cu ............................................................................. 19 24 Tng hp ha hc v kim tra tinh khit ........................................... 19 242 Xc nh cu trc .................................................................................. 20 243 Th tc dng c tnh t bo in vitro ..................................................... 20 244 nh gi mc gingthuc ca cc dn cht tng hp c ............. 21 3 Ha hc ....................................................................................................... 22 3 Tng hp ha hc.................................................................................. 22 32 Kim tra tinh khit............................................................................ 32 33 Xc nh cu trc .................................................................................. 33 32 Th tc dng khng t bo ung th ca cc cht c tng hp .................. 38 33 Bn lun ...................................................................................................... 39 33 Tng hp ha hc.................................................................................. 39 332 Tc dng khng t bo ung th .............................................................. 41 KT LUN V KIN NGH ............................................................................... 43 TI LIU THM KHO PH LC DANH MC CC K HIU, CC CH VIT TT: 13C-NMRPh cng hng t ht nhn 13C (13C nuclear magnetic resonance) 1H-NMRPh cng hng t ht nhn proton (1H nuclear magnetic resonance) CTCTCng thc cu to CTPTCng thc phn t DCMDicloromethan DMFN,N-dimethylformamid DMSODimethyl sunfoxid EtOHEthanol HATHiston acetyltranferase HDACHiston deacetylase IRPh hng ngoi (Infrared spectroscopy) HepG2T bo ung th gan MeOHMethanol MS Ph khi lng (Mass spectroscopy) PPAR-Peroxisom proliferator activated receptor gamma MTTThuc nhum 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromid PTP 1BProtein tyrosin phosphat 1B SAHAAcid suberoylanilid STTS th t SWL68T bo gan bnh thng SW620T bo ung th i trng TZDThiazolidin-2,4-dion TLCSc k lp mng (Thin Layer Chromatography) Rf H s lu gi (Retention factor) DANH MC CC BNG STTTn bngTrang Bng 1.1Kt qu th c tnh t bo ca cc hp cht TZD3 Bng 1.2Gi tr IC50 ca cc TZD (M) v gi tr logP4 Bng 3.1 Hiu sut v cc ch s ha l ca cc sn phm c tng hp 32 Bng 3.2Gi tr Rf v tonc ca cc sn phm cui 4a-4d33 Bng 3.3Kt qu phn tch ph khi lng ca cc cht trung gian34 Bng 3.4Kt qu phn tch ph hng ngoi ca cc cht34 Bng 3.5 Kt qu phn tch ph khi lng ca cc cht sn phm cui 35 Bng 3.6Kt qu phn tch ph 1H-NMR ca cc sn phm cui36 Bng 3.7Kt qu phn tch ph 13C-NMR ca cc sn phm cui37 Bng 3.8Tc dng khng t bo ung th i trng (SW62)39 Bng 3.9nh gi ging thuc ca dy cht c tng hp42 DANH MC S V HNH V HNH V STTTn hnhTrang Hnh 1.1Vai tr cn bng ng gia HDC v HT i vi s phin m 6 Hnh 1.2Cu to trung tm hot ng ca HDAC7 Hnh 1.3Cu trc mt s hot cht c ch HDAC8 Hnh 1.4Cu trc chung ca mt s dy cht nghin cu cng b8 Hnh 1.5Cu trc ca SH v trung tm hot ng ca HDAC9 Hnh 1.6Cu trc thit k v hp cht c ch HDC10 Hnh 1.7M hnh phn t I v II to lin kt chelat vi ion Zn2+ trong protein 10 Hnh 1.8c tnh t bo ti cc nng 11 Hnh 1.9c tnh t bo theo thi gian 12 Hnh 1.10Kt qu c ch HDC ca cc cht sau 8h12 Hnh 1.11Tc dng ca TZD trn PPR-13 Hnh 1.12Mt s TZD c ch PTP1B mi c nghin cu14 S `STTTn s Trang 3.1Quy trnh tng hp cc cht22 3.2Tng hp thiazolidin-2,4-dion22 3.3Tng hp dy cht 2a-d23 3.4Tng hp dy cht 3a-d26 3.5Tng hp dy cht 4a-d29 1 T VN Thiazolidindion (TZD)v cc dn cht c bit nc tc dng chng ithongnhhothaththperoxisomproliferatoractivatedreceptor gammavcchproteintyrosinphosphat1B[13,14].Mtsthuciutri tho ng tp 2 da trnkhung TZDvn ang c s dng trn th trng nh Pioglitazon [2, 17]. Bn cnh , cc hp cht TZD cn c nghin cu khai thc cc tc dng khcnhtcdngkhngkhun[6],khngnm[7,8],chngoxihatbo[18]. cbittrongthigiangny,ccnghincuvdnchtcaTZDthng hng ti hot tnhkhng trn cc dng t bo ung th: ung th phi, ung thv, ungthganvmtsdngtbokhc[12,26,28].Cctcgithitkcng thcmicaTZD,trongnhmTZDngvaitrcchenzymhiston deacetylase (mt enzym quan trng trong qu trnh hnh thnh cc t bo ungth) [26]. Kt qu thu c rt kh quan khi so snh vi mt s cht chng ung th hin hnh.T cc kt qu nghin cu trn, nhm mc ch tm kim thm cc dn cht thiazolidindion trin vng, chng ti thc hin ti Nghin cu tng hp v th tcdngsinhhccamtsdnchtthiazolidin-2,4-dionviccmctiu sau: 1. TnghpN-(2-(4-((2,4-dioxothiazolidin-5-yliden)methyl)phenoxy)ethyl) benzensulfonamid v mt s dn cht. 2.Th c tnh t bo ca cc cht tng hp c. 2 CHNG 1. TNG QUAN 1.1. Tc dng sinh hc ca thiazolidindion Thiazolidindioncncgilglitazonlnhmccdnchtcchagc thiazolidin-2,4-dion: CcTZDcchngminhcnhiutcdngsinhhcngchnh: tc dngkhngkhun [6,7], chng nm [7,8], chng oxy ha [18], gy c t bo, chngungth[12,26,28],hothaththPPAR-,cchproteinPTP1B [8,12,14]. Hinnay,hngnghincuvhottnhcaTZDangcquantm nhiu l c tnh t bo, c ch HDAC, hot ha PPAR- v c ch PTP1B. 1.1.1.c tnh t bo Nm2,VPatilcngcngstinhnhtnghpmtsdncht mangnhmTZD(I)vthctnhinvitrotrn7dngtboungthbaogm: Hop62(ungthphi),PC3(ungthtuyntinlit),MCF7(ungthv),HepG2 (ungthgan),K562(ungthbchcu),Gurav(ungthming)vKB(ungth vm hng). Nng cc cht th nghim trong khong t 10-4 ti 10-7 M, tin hnh song song vi 2 mu th, mt mu khng s dng cht c ch v mt mu s dng chtcchldoxorubicin[29].Sau48gi,nhgimcphttrincami dngtbo,xcnhccnngtikhnngphttrincatbobcch 50% (GI50). Tnh gi tr logarit nng ti GI50, cc gi tr m th hin cho s nhy cmcaccdngtbo,cchpchtcgitrlogGI50-4,0-4,53>-4,0-6,72-4,53>-4,0>-4,0 I b >-4,0>-4,0>-4,0-6,73>-4,0>-4,0>-4,0 I c -5,65>-4,0>-4,0-4,51-5,65-6,71>-4,0 I d >-4,0-4,53>-4,0-5,64-4,53>-4,0>-4,0 I e -6,72-6,71>-4,0>-4,0-5,60-6,73-5,65 Doxorubicin-5,59-6,8830 4d-Cl-3,3,8312,36 Ghi ch: *nng (g/ mL) c ch 50% s pht trin ca t bo ung th Nhn xt: C 4 cht u c tc dng c ch pht trin t bo ung th Cc cht 4a, 4b, 4c c ch yu, gi tr IC50 > 3 gmL Cht 4d c tc dng c ch mnh hn 3 cht cn li, gi tr IC50 =2,36 gmL 3.3. n lun 3.3.1. Tng hp ha hc Phnngtnghpvngthiazolidin-2,4-dionthchintheophngphp caE.Mamelivcngs[3,19].Phnngdinrathunlivthiusutcao (72%). Phn ng SN2 gia cc dn cht halogen v cc alcol hay phenol to lin kt ether[1].Ccdnxutbromtrongdyphnngviccphenolkhkhn,phn ng xy ra khng hon ton. S dng xc tc l K2CO3 chuyn nhm phenol thnh phenolat,nhmtngkhnngphnng.ldehydchatantrongdungmil methanol sau thm K2CO3 dng bt, gia nhit v khuy trn trong 3-60 pht nhm hot ha aldehyd. Sau thm t t 4-hydroxy bezaldehyd vo, hi lu trong 24 gi. Phn ng xy ra khng hon ton, lun lun cn d 4-hydroxybenzaldehyd vphiloibtrongqutrnhtinhch.4-hydroxybenzaldehydckhnngphn 40 ng vi thiazolidin-2,4-dion phn ng tip theo, nh hng n s tinh khit ca sn phm cui. Qutrnhngngtgiaccaldehydvinhmmethylencavng thiazolidin-2,4-dionctinhnhtrongdungmiethanol,vixctcl piperazin.Vngthiazolidin-2,4-dionchainhmcarbonylhtintmnh,xc tcpiperazinctnhbasedolmtngkhnngphnngcanhmmethylen trong vng. Phn ng ngng t cho hiu sut cao (> 90%). C ch phn ng: phn ng xy ra theo hai giai on -Giaioncnghp:nhmmethylenvtrs5ccchydrolinhngcth d dng tch khi nguyn t cacbon khi c mt xc tc base, hnh thnh tc nhn i nhnmnh.Anionvahnhthnhtncngvocacbonmangintchdngca nhm cacbonyl trong aldehyd to thnh sn phm cng hp. Giai on din ra theo s : -Giai on ngng t: Ditcdngcaproton,mtphntnc cloiratothnhlinkt i, qu trnh xy ra d dng do sn phm to thnh c mc nng lng thp nh h ni i lin hp. Giai on din ra theo s sau: 41 Phnngngngttorasnphmcdynii,nhvycthtnti ng phn Z v E. Tuy nhin mt s nghin cu cho thy sn phm ngng t ch c duy nht cu hnh Z. Thc hin nhiu x tia X v da trn ph cng hng t ht nhnprotoncaccchtccutrctngng,nhnthychtntiduynht cuhnhZmkhngccuhnhEdcciukinphnngkhcnhau: AcOH/AcONa, toluen/piperazin [6,13]. 3.3.2. Tc dng khng t bo ung th Kt qu th tc dng ca cc dy cht c tng hp trn dng t bo ung th i trng (SW62) cho kt qu khng tt, t l % t bo pht trin mc cao ( 4a,4b,4cut8-85%).Tuynhindnchtmangnhmthclolichothy kh nng c ch t bo mnh hn nhiu so vi cc dn cht cn li, gi tr thu c l -33,83%. Gi tr IC50 cao i vi ba cht 4a, 4b v 4c (IC50> 3 gmL), cht 4dchogitrIC50mctrungbnh(IC50=12,36gmL).Cthnhnthydn xut ca nhm th clo cho hot tnh tt hn cc dn cht khng c nhmth, nhm th methyl v methoxy. V ging thuc ca cc cht trn theo quy tc Lipinsky, cc cht u tha mncyucucaquytc.Dodkhngthhincctnhtbott nhngcutrcccchungcadychtctnghpcngchothykhnng 42 thm tt quamng t bo, t c th tip tc nghin cu tng hp vi ccnhm dn cht khc c th cho hot tnh sinh hc tt hn. ng 3.9: nh gi ging thuc ca dy cht c tng hp ChtCTCTlogPMWS NH, OH S N,OS in t inh ng 4aR=H2,32405252 4bR=CH3 2,87419252 4cR=OCH3 2,40435252 4dR=Cl2,97439252 43 KT LUN V KIN NGH I.Kt un 1.tnghpcccchtnhdkin,ccchtuchaccngbtrong bt k ti liu c tham kho no, bao gm: -N-(2-(3-((2,4-dioxothiazolidin-5-yliden)methyl)phenoxy)ethyl)benzen sulfonamid. -N-(2-(3-((2,4-dioxothiazolidin-5-yliden)methyl)phenoxy)ethyl)-4-methylbenzen sulfonamid. -N-(2-(3-((2,4-dioxothiazolidin-5-yliden)methyl)phenoxy)ethyl)-4-methoxy benzensulfonamid. -4-chloro-N-(2-(3-((2,4-dioxothiazolidin-5-yliden)methyl)phenoxy)ethyl) benzensulfonamid. Cu trc ca cc cht trung gian v sn phm c xc nh bng cch phn tch cc ph MS, IR, 1H-NMR, 13C-NMR. 2.Bc u th c hot tnh c ch t bo ung th i trng ca cc cht tng hp c. Kt qu cho thy: -Tc dng c ch t bo ung th: c bn cht u th hin tc dng c ch dng t boungthSW62,cbacht(4a,4bv4c)thhintcdngcchkmvi IC50 mc cao, cht 4d c tc dng tt hn vi IC50 mc trung bnh. II.Kin ngh Vi kt qu th hot tnh t bo thu c chng ti kin ngh nh sau: -Tinhnhthhottnhcaccchttnghpctrnmtschtcdng khccpngvinhmthiazolidin-2,4-dioncaratrongccnghin cu: HDAC, PPAR-, PTPB. -Tinhnhtnghpccdnchtthiazolidin-2,4-dionviccnhmthkhc nhau. 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Taxonomy,fermentation,isolation,physico-chemicalandbiologicalproperties,and antitumor activity", J Antibiot (Tokyo). 47(3), p. 301-310 Ph c Ph lc 1:Ph khi ng ca cht3a Ph lc 2:Ph khi ng ca cht 3b Ph lc 3:Ph khi ng ca cht 3c Ph lc 4:Ph khi ng ca cht 3d Ph lc 5:Ph khi ng ca cht4a Ph lc 6:Ph khi ng ca cht4b Ph lc 7:Ph khi ng ca cht4c Ph lc 8:Ph khi ng ca cht4d Ph lc 9:Ph hng ngoi ca cht4a Ph lc 10:Ph hng ngoi ca cht4b Ph lc 11:Ph hng ngoi ca cht4c Ph lc 12:Ph hng ngoi ca cht4d Ph lc 13:Ph H1-NMR ca cht4a Ph lc 14:Ph H1-NMR m rng ca cht4a Ph lc 15:Ph H1-NMR ca cht4b Ph lc 16:Ph H1-NMR m rng ca cht4b Ph lc 17:Ph H1-NMR ca cht4c Ph lc 18:Ph H1-NMR m rng ca cht4c Ph lc 19:Ph H1-NMR m rng ca cht4c Ph lc 20:Ph H1-NMRca cht4d Ph lc 21:Ph H1-NMR m rng ca cht4d Ph lc 22:Ph H1-NMR m rng ca cht4d Ph lc 23:Ph C13-NMR ca cht4a Ph lc 24:Ph C13-NMR m rng ca cht4a Ph lc 25:Ph C13-NMR ca cht4b Ph lc 26:Ph C13-NMR m rng ca cht4b Ph lc27:Ph C13-NMR ca cht4c Ph lc 28:Ph C13-NMR m rng ca cht4c Ph lc 29:Ph C13-NMR ca cht4d Ph lc 30:Ph C13-NMR m rng ca cht4d Ph lc 1: ph khi ng MS cht 3a Ph lc 2: ph khi ng MS cht 3b Ph lc 3: ph khi ng MS cht 3c Ph lc 4: ph khi ng MS cht 3d Ph lc 5: ph khi ng MS cht 4a Ph lc 6: ph khi ng MS cht 4b Ph lc 7: ph khi ng MS cht 4c Ph lc 8: ph khi ng MS cht 4d Ph lc 9: ph hng ngoi IR cht 4a Ph lc 10: ph hng ngoi IR cht 4b Ph lc 11: ph hng ngoi IR cht 4c Ph lc 12: ph hng ngoi IR cht 4d Ph lc 13: ph 1H-NMR cht 4a Ph lc 14: ph 1H-NMR m rng cht 4a Ph lc 15: ph 1H-NMR cht 4b Ph lc 16: ph 1H-NMR m rng cht 4b Ph lc 17: ph 1H-NMR m rng cht 4b Ph lc 18: ph 1H-NMR cht 4c Ph lc 19: ph 1H-NMR m rng cht 4c Ph lc 20: ph 1H-NMR cht 4d Ph lc 21: ph 1H-NMR m rng cht 4d Ph lc 22: ph 1H-NMR m rng cht 4d Ph lc 23: ph 13C-NMR cht 4a Ph lc 24: ph 13C-NMR m rng cht 4a Ph lc 25: ph 13C-NMR cht 4b Ph lc 26: ph 13C-NMR m rng cht 4b Ph lc 27: ph 13C-NMR cht 4c Ph lc 28: ph 13C-NMR m rng cht 4c Ph lc 29: ph 13C-NMR cht 4d Ph lc 30: ph 13C-NMR m rng cht 4d