B Y T

TRNG I HC DC H NI

L ANH CHIN

TNG HP V TH TC DNG

SINH HC MT S DN CHT

5-(4-HYDROXYBENZYLIDEN)

THIAZOLIDIN-2,4-DION

KHA LUN TT NGHIP DC S

H Ni - 2014

B Y T

TRNG I HC DC H NI

L ANH CHIN

TNG HP V TH TC DNG

SINH HC MT S DN CHT

5-(4-HYDROXYBENZYLIDEN)

THIAZOLIDIN-2,4-DION

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. TS. Phan Th Phng Dung

2. ThS. Trn Th Lan Hng

Ni thc hin:

B mn Ha Dc

H Ni - 2014

LI CM N

Trc khi bt u vit nhng phn chnh trong kha lun ny, ti xin c

gi nhng li cm n chn thnh nht n nhng ngi trong sut thi gian qua

lun bn cnh gip ti hon thnh mt cch tt nht kha lun tt nghip.

Trc ht vi lng knh trng v bit n su sc nht ti xin by t li cm

n chn thnh n PGS.TS. Nguyn Hi Nam, TS. Phan Th Phng Dung, ThS.

Trn Th Lan Hng B mn Ha Dc trng i hc Dc H Ni. Thy, c

khng ch to nhng iu kin tt nht gip ti hon thnh kha lun m lun

c nhng hng dn chnh xc v kp thi nhng lc ti gp kh khn, lun bn

ng vin ti, to cho ti ng lc v nim tin ln.

Ti cng xin gi li cm n n cc thy gio, c gio v cc anh ch k

thut vin ca B mn Ha Dc trng i hc Dc H Ni, Khoa Ha i

hc Khoa hc t nhin H Ni, Vin khoa hc v cng ngh Vit Nam, Khoa Dc

i hc quc gia Chungbuk Hn Quc lun gip , to iu kin thun li

cho ti thc hin kha lun ny.

Ti cng xin by t nhng tnh cm thn thng n cc anh ch v cc bn

trong nhm thc nghim ti b mn Ha Dc, nhng ngi chia s vui bun,

ng hnh cng ti trong sut thi gian qua.

Cui cng ti xin gi li cm n n gia nh, ngi thn v bn b quan

tm, ng vin to ng lc cho ti i n ngy hm nay.

H Ni, thng 3, nm 2014

L Anh Chin

MC LC

T VN 1

CHNG 1. TNG QUAN 2

1.1 Tc dng sinh hc ca thiazolidindion 2

1.1.1 c tnh t bo v khng t bo ung th 2

1.1.2 Tc dng chng i tho ng 4

1.1.3 Tc dng khng khun, khng nm 6

1.1.4 Tc dng khc 8

1.2 Mt s phn ng s dng trong tng hp dn cht thiazolidindion 10

1.2.1 Cc phn ng tng hp thiazolidin-2,4-dion 10

1.2.2 Phn ng to cu ni alkyl 11

hn ng ngng t gia aldehyd thm v nhn thiazolidin-2,4-dion 11

CHNG 2. NGUYN LIU, THIT B, NI DUNG V PHNG PHP

NGHIN CU 13

2.1 Nguyn vt liu, thit b 13

2.1.1 Ha cht 13

2.1.2 Thit b, dng c 13

2.2 Ni dung nghin cu 14

2.2.1 Tng hp ha hc 14

2.2.2 Th tc dng sinh hc ca cc cht tng hp c 14

2.3 Phng php nghin cu 14

2.3.1 Tng hp ha hc v kim tra tinh khit 14

Xc nh cu trc 14

2.3.3 Th c t nh t bo invitro 15

nh gi mc ging thuc ca cc d n cht tng hp c 16

CHNG 3. THC NGHIM, KT QU V BN LUN 17

3.1 Ha hc 17

3.1.1 Tng hp ha hc 17

3.1.2 Kim tra tinh khit 25

Xc nh cu trc 26

3.2 Th hot tnh sinh hc 30

3.2.1 Th c tnh t bo cc cht c tng hp 30

nh gi mc ging thuc 31

3.3 Bn lun 31

3.3.1 Tng hp ha hc 31

3.3.2 Khng nh cu trc cc cht tng hp c 32

3.3.3 Hot tnh sinh hc 34

KT LUN V KIN NGH 35

TI LIU THAM KHO 36

PH LC 40

DANH MC CC K HIU, CC CH VIT TT

13C-NMR

13C nuclear magnetic resonance (ph cng hng t ht nhn 13C)

1H-NMR

1H nuclear magnetic resonance (ph cng hng t ht nhn 1H)

COX Cyclooxygenase

CTCT Cng thc cu to

DCM Dicloromethan

DMF N,N-dimethylformamid

DMSO Dimethlsulfoxid

EtOH Ethanol

IC50 Nng c ch hot t bo xung mt na

IR Ph hng ngoi

MeOH Methanol

MS Ph khi lng

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid

PPAR- Peroxisom proliferator activated receptor gamma

PTP 1B Protein tyrosin phosphat 1B

SAHA Suberoylanilid acid

STT S th t

SW620 T bo ung th i trng

Tonc Nhit nng chy

TEA Triethylamin

TLC Phng php sc k lp mng

TZD Thiazolidin-2,4-dion

DANH MC CC BNG

STT Tn bng Trang

Bng 3.1 Ch s l ha v hiu sut tng hp cc cht 3a-d 25

Bng 3.2 Gi tr Rf v Tonc ca cc cht 3a-d 26

Bng 3.3 Kt qu phn tch ph hng ngoi (IR) cc cht 3a-d 26

Bng 3.4 Kt qu phn tch ph khi lng (MS) cc cht 3a-d 27

Bng 3.5 Kt qu phn tch ph 1H (

1H-NMR) cc cht 3a-d 28

Bng 3.6 Kt qu phn tch ph 13

C (13

C-NMR) cc cht 3a-d 29

Bng 3.7 Tc dng khng t bo ung th i trng (SW620) 30

Bng 3.8 nh gi mc ging thuc ca cc cht 3a-d 31

DANH MC S V HNH V

STT Tn hnh Trang

Hnh 1.1 Tc dng ca TZD trn PPAR- 4

Hnh 1.2 Mt s TZD ng dng trn lm sng 5

Hnh 1.3 Cc TZD c ch PTP1B mi c nghin cu 6

Hnh 3.1 Quy trnh tng hp cc cht 17

1

T VN

Thiazolidindion v cc dn cht l nhm cc hp cht hu c d vng c

nhiu nh khoa hc trong v ngoi nc nghin cu tng hp, sng lc tc dng

sinh hc tm kim cc thuc mi. Nhiu cng trnh nghin cu chng t cc

Thiazolidindion c nhng tc dng sinh hc rt ng quan tm.

c mt s cng trnh nghin cu [7,9,12] chng minh thiazolidindion v dn

cht l cc cht c tc dng chng i tho ng thng qua c ch hot ha th th

peroxisom proliferator activated gamma (PPAR-) v c ch protein tyrosin

phosphat 1B (PTP1B), cc thiazolidindion gip c th tng tnh nhy cm vi

insulin ni sinh, tng tiu th glucose v gim ng huyt. Cho n nay mt s

thuc iu tr i tho ng type 2 da trn khung thiazolidindion c s

dng trn th trng nh Pioglitazon.

Bn cnh tc dng h ng huyt, hin nay cc cht mang khung

thiazolidindion cn c th tc dng khng t bo ung th. Nhiu nghin cu ca

cc nh khoa hc khng nh cc dn cht mang khung thiazolidindion c tc

dng c ch s pht trin ca nhiu loi t bo ung th [4,20,21].

Ngoi ra cc thiazolidindion cng th hin cc tc dng sinh hc khc nh:

khng khun, khng nm, khng vim, chng c thn kinh...

Vi mong mun ng gp mt phn lm phong ph thm cc nghin cu v dn

xut thiazolidindion, ti: Tng hp v th tc dng sinh hc mt s dn cht 5-

[4-hydroxybenzyliden]thiazolidin-2,4-dion c thc hin vi cc mc tiu nghin

cu sau:

1. Tng hp c mt s dn cht 5-[4-hydroxy benzyliden]thiazolidin-2,4-

dion.

2. Th tc dng gy c t bo ca cc dn cht tng hp c.

2

CHNG 1. TNG QUAN

1.1 Tc dng sinh hc ca thiazolidindion

Thiazolidindion (TZD) hay cn c gi l glitazon l nhm cc dn cht c cha

gc thiazolidin-2,4-dion c cu trc nh sau:

y l mt trong nhng dy cht hu c c nghin cu h thng v mt ha

hc cng nh tc dng sinh hc. Rt nhiu cng trnh nghin cu tng hp v th

tc dng sinh hc ca cc TZD cho thy chng c tc dng kh phong ph v a

dng nh chng i tho ng, khng t bo ung th, khng khun, khng nm,

khng vim

1.1.1 c tnh t bo v khng t bo ung th

Nm 1985, Eshba v Salama [28] tng hp mt lot cc dn cht ca 5-(2-oxo-

3-indolinyl)thiazolidin-2,4-dion vi v tr 1 ca vng isatin v 3 ca vng

thiazolidin l cc base Mannich v th hot tnh khng ung th ca cc cht ny.

C 5 cht c nh gi tc dng khng dng t bo ung th bch cu P388

chut. Trong hp cht c base Mannich cha dimethyl amino c ch mnh nht,

kt qu kho st 5 cht ny cng cho thy s c mt ca nhm Br ti v tr 5 vng

thm nhn isatin lm tng tc dng.

Nm 2010, Vijay Patil v cng s [21] tin hnh tng hp mt s dn cht

mang nhm TZD v th c tnh invitro trn 7 dng t bo ung th bao gm:

3

HOP62 (t bo ung th phi), PC3 (t bo ung th tuyn tin lit), MCF7 (t bo

ung th v), HEPG2 (t bo ung th gan), K562 (t bo ung th bch cu),

GURAV (t bo ung th ming) v KB (t bo ung th vm hng).

Kt qu nghin cu cho thy, c 5 cht tng hp c u c tc dng trn t bo

ung th cc mc khc nhau (trong hp cht c R l 3-

(trifloromethyl)phenyl c tc dng c ch 5/7 dng t bo ung th th nghim).

Khng cht no c tc dng trn dng t bo ung th gan.

Tip tc theo hng nghin cu ny, nm 2012 Alegaon Shankar v cng s [4]

cng tin hnh tng hp nhm hp cht c cha khung thiazolidindion v nh

gi c tnh trn cc dng t bo ung th ngi gm: HeLa (t bo ung th c t

cung), HT-29 (t bo ung th i trc trng), A549 (t bo ung th phi), MCF-7

(t bo ung th v).

Kt qu nghin cu cho thy, cc cht tng hp c c tc dng c ch t bo

ung th mc khc nhau. Trong hp cht vi nhm th l vng benzen v

methyl benzen cho gi tr IC50 t 60-100 (M). Khi vng benzen gn thm cc

nhm th halogen l brom hoc flo th c tnh trn cc dng t bo ung th th

nghim tng ln ng k vi gi tr IC50 thu c trong khong 40-50 (M). Hp

4

cht c tc dng mnh nht mang nhm th 3,4,5-trimethoxybenzen vi cc gi tr

IC50 t 30-36 (M). Ngoi ra, cc hp cht d vng cha N v O cho cc gi tr IC50

mc trung bnh t 60-90 (M).

1.1.2 Tc dng chng i tho ng

Cc TZD c tc dng chng i tho ng nh hot ha th th peroxisom

proliferator activated gamma (PPAR-) v c ch protein tyrosin phosphatase 1B

(PTP1B). y l cc tc nhn ng vai tr quan trng trong vic iu ha glucose,

triglycerid, cholesterol huyt, gip c th tng tnh nhy cm vi insulin ni sinh,

tng tiu th glucose, lm gim ng huyt [8,19,20,24,26].

Nghin cu ca Cantello Barrie (1994) [9], nghin cu ca Shubhanjali Shukla

(2012) [26], nghin cu ca Avupati Pasudeva (2012) [6] cho thy thiazolidindion

l nhm cht ch vn chn lc trn th th PPAR-. Thng qua hot ha PPAR-,

TZD iu chnh s biu hin ca mt s gen mc tiu ca PPAR- v tc ng ln

qu trnh chuyn ha lipid ca t bo. Kch thch PPAR- s kch thch d tr v s

dng acid bo, triglycerid t bo m, kch thch s dng glucose v c ch oxi ha

acid bo c, c ch s tng hp glucose gan, ngoi ra cn tng cng s oxi

ha cc LDL i thc bo. T ci thin s nhy cm vi insulin ca t bo,

gim nng glucose trong mu, gim acid bo trong mu, chng x va ng

mch v cao huyt p (xem minh ha hnh 1.1)

H nh 1.1: Tc dng ca TZD trn PPAR-

Da trn c ch ny, nhm thuc iu tr i tho ng mang khung TZD ra i.

Cho n nay, c nhiu dn cht cht mang khung TZD c tng hp v th

5

hot tnh h ng huyt. Trong s , troglitazon c a ra th trng u tin,

nhng sau b rt v c tnh trn gan. Hin nay, pioglitazone v rosiglitazone

vn ang c s dng trn lm sng. Tuy nhin, cc thuc ny vn c c tnh

nht nh trn gan, tim mch v huyt hc v cc tc dng khng mong mun ny

thng gp hn trong liu trnh iu tr km vi sulfonylureas hoc insulin

[10,13,14]. Hai trong s cc cht ch vn PPAR th h mi l Balaglitazon v

Rivoglitazon thuc nhm TZD vn ang trong giai on th nghim lm sng

[2,16].

H nh 1.2: Mt s TZD c ng dng trn lm sng

Trong mt s nghin cu gn y, cc dn cht TZD cn c chng minh c tc

dng c ch enzym PTP1B, do lm tng nhy, p ng ca insulin v leptin

ti th th, dn n lm gim nng glucose, lipid trong mu [7,25]. Mt s dn

cht ca TZD c tng hp v nghin cu tc dng trn ch PTP1B [7,12].

6

H nh 1.3: Cc TZD c ch PTP1B mi c nghin cu

Nghin cu chng minh hai dn cht ny ng thi c ch PTP1B v hot ha

PPAR-. Trn in vivo c tc dng nh l cht h ng huyt, ci thin ng k

dung np glucose. Hai hp cht ny cng c ch ng k s tng cn, chng bo ph

do c tc dng h triglicerid, cholesterol huyt. c im cu trc c bit l s c

mt ca nhm sulfonyl trn cht 4b c vai tr quan trng trong to tng tc vi c

th th PTP1B v PPAR-. Nghin cu ny m ra hng pht trin cc

thiazolidindion mi vi tc dng h ng huyt mnh ng thi c th dng nh

nhng cht iu ha lipid mu, chng bo ph v gim thiu cc bnh v tim mch.

1.1.3 Tc dng khng khun, khng nm

y l tc dng ng ch ca dy dn cht thiazolidin-2,4-dion. Nhiu cng

trnh nghin cu cho thy dy dn cht ny c tc dng khng khun mnh, v tc

dng khng nm [3,11,22,29].

Nm 1974 E.B.Akerblom [3] tng hp v th tc dng khng khun ca cc

dn cht thiazolin-2,4-dion:

Cc cht tng hp c u c xc nh nng c ch ti thiu trn 5 chng

vi khun (bao gm 2 chng Gram (+): Sta.aureus, -haem streptococcus v 3 chng

7

Gram (-): E.coli, Pseu.aeruginosa, P.vulgaris). Kt qu thu c trong nghin cu

ny cho thy c 9 cht thc s c tc dng dit khun r rt trn 5 chng vi khun

kim nh. Gi tr MIC nm trong khong t 0,2-12,5 g/ml.

Nm 2006, M.C.Unlusoy v cng s [29] tng hp dy dn cht ca benzyl-5-

(4-cloro-2-piperidin-1-yl-thiazol-5-yl-methylen)thiazolidin-2,4-dion, c cu trc

nh sau:

Kt qu th hot tnh sinh hc cho thy dy cht ny c tc dng kh mnh trn

cc chng vi khun Gram (+): Staphylococcus aureus, Bacillus subtilis, vi khun

Gram (-): E.coli, vi vng c ch t 9-14 mm so snh vi ampicillin (10 mm). Cc

cht ny hu nh khng th hin tc dng c ch trn vi nm Candida albicans.

Nm 2011, nh khoa hc n Deepak K Aneja v cng s [11] tng hp

c 24 hp cht mi l dn xut pyrazolyl-2,4-thiazolidindion v th hot tnh

khng nm, khng khun ca chng trong iu kin in vitro.

Kt qu cho thy:

8

V hot tnh khng nm: C 11 hp cht c ch trn 60% Aspergillus niger, 11

hp cht c ch trn 60% Aspergillus flavus v 5 hp cht c ch c 2 vi nm trn

60%.

V hot tnh khng khun: Tt c cc hp cht u th hin tnh khng cc vi

khun Gram (+) (Staphylococcus aureus v Bacillus subtillis) vi MIC t 32-128

g/ml, khng c hp cht no th hin tnh khng cc vi khun Gram (-)

(Escherichia coli v Pseudomonas aeruginosa).

Nm 2012, nhm nghin cu ca Prajwal L.Lobo [22] tng mt s dn cht

ca thiazolidin-2,4-dion v th hot tnh ca cc cht ny trn vi sinh vt (2 vi

khun Gram (+): Staphylococcus aureus v Bacillus subtilis v 2 vi khun Gram (-

): Escherichia coli v Pseudomonas aeruginosa, 2 vi nm: Candida albicans v

Aspergillus niger).

Nhm tc gi nhn thy tt c cc cht tng hp c u th hin kh nng c

ch trn vi khun vi vng c ch t 8-28 mm, v trn vi nm vi vng c ch t

10-27 mm. Trong dn cht cha nhm NH th hai gn vi vng benzen c nhm

NO2 v tr para th hin tc dng c ch trn cc vi khun, vi nm tt hn c.

1.1.4 Tc dng khc

Ngoi cc tc dng k trn, trong thi gian gn y, nhiu nghin cu chng

minh cc hp cht thiazolidin-2,4-dion cn c nhiu tc dng khc nh: chng

vim, chng c thn kinh

Nm 2010, Amal M. Youssef v cng s [5] tng hp mt lot cc dn cht

pyrazolyl-2,4-thiazolidindion:

9

Cc hp cht c th nghim trong iu kin in vitro tc dng gy c t bo,

khng vim v chng c thn kinh. Kt qu cho thy, cc hp cht 5c, 5e th hin

kh nng chng c thn kinh nng thp hn nng gy c t bo ca

chng, cc hp cht ny (5c, 5e) cng th hin tc dng c ch COX. Bn hp cht

(5a, 5b, 5c, 5e) c tc dng khng vim invitro c la chn th nghim in

vivo v hot tnh ny trn chut. Vi m hnh gy vim cp, kh nng khng vim

ca cc cht th nghim t 24-53%, so snh vi celecoxid (41%). Vi m hnh gy

vim bn cp (sub-acute), kh nng khng vim ca cc cht th nghim t 44-

53%, so snh vi celecoxid (54%).

Nm 2011, trong nghin cu ca L.C. Santos v cng s [23], 3 hp cht mi l

dn xut ca thiazolidin-2,4-dion c tng hp v tin hnh th hot tnh khng

vim trn chut Wistar c, so snh vi indomethacin.

10

Kt qu cho thy cc hp cht ny c kh nng chng vim tt, hp cht (4a, 4b)

c tc dng thp hn indomethacin, hp cht 4c c tc dng khng vim mnh hn

indomethacin 15%.

1.2 Mt s phn ng s dng trong tng hp dn cht thiazolidindion

1.2.1 Cc phn ng tng hp thiazolidin-2,4-dion

1.2.1.1 Phn ng ng vng ca thioure vi acid monocloacetic

Nm 1954, E. Mameli v cng s [18] tng hp vng thiazolidin-2,4-dion theo

s sau:

Phn ng xy ra qua hai giai on:

- Giai on 1: to pseudothiodohydantoin:

- Giai on 2: thy phn pseudothiohydantoin di tc dng ca HCl sinh ra

trong giai on mt:

Phng php ny c la chn s dng nhiu v cho hiu sut cao v vic thc

hin phn ng khng qu phc tp.

1.2.1.2 Phn ng ng vng ca ethyl cyanothioacetat c mt acid clohydric

Phng php ny do K.C.Joshi [15] tm ra. Mc d quy trnh phn ng kh n

gin nhng cho hiu sut thp nn t c s dng.

11

1.2.2 Phn ng to cu ni alkyl

Phn ng th SN2: xy ra gia dn xut halogen v mt tc nhn base c cp in

t t do hay giu electron nh cc alcol, phenol hay ester [1].

Phn ng alkyl ha gia nhm imid (NH) trong nhn thiazolidin-2,4-dion, nhm

amid (NH) trong nhn indolin-2,3-dion v cc alkyl halogenid u din ra theo c

ch SN2. Phn ng c bit thun li khi tin hnh trong mi trng kim.

1.2.3 Phn ng ngng t gia aldehyd thm v nhn thiazolidin-2,4-dion

Nhm methylen v tr s 5 trong nhn thiazolidin-2,4-dion c tnh linh ng, do

c kh nng tham gia phn ng ngng t vi ci aldehyd thm theo s phn

ng:

Phn ng c th tin hnh trong cc dung mi: ethanol, methanol hay acid acetic.

c tc c s dng l piperazin hoc c th l natri acetat khan. Do s linh ng

ca nhm methylen trong nhn thiazolidin-2,4-dion nn phn ng ngng t ny xy

ra d dng. Cc tc nhn xc tc c th loi b bng dung dch acid hoc lc v ra

vi nc.

C ch phn ng: phn ng xy ra theo hai giai on

- Giai on cng hp: nhm methylen v tr s 5 c cc hydro linh ng c

th d dng tch khi nguyn t cacbon khi c mt xc tc base, hnh thnh tc

Trng thi chuyn tip

12

nhn i nhn mnh. Anion va hnh thnh tn cng vo cacbon mang in tch

dng ca nhm cacbonyl trong aldehyd to thnh sn phm cng hp. Giai on

ny din ra theo s :

- Giai on ngng t: Di tc dng ca proton, mt phn t nc c loi

ra to thnh lin kt 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 ny din ra theo s sau:

Sn phm ngng t c to thnh ch c cu hnh (Z), iu ny c chng

minh qua kt qu nghin cu nhiu x tia [4,8].

13

CHNG 2. NGUYN LIU, THIT B, NI DUNG V PHNG PHP

NGHIN CU

2.1 Nguyn vt liu, thit b

2.1.1 Ha cht

Cc ha cht, dung mi s dng trong qu trnh thc nghim l loi dnh cho tng

hp c nhp t cng ty Merk hoc Sigma-Aldrich. Cc ha cht ny c s

dng trc tip khng qua tinh ch thm.

Ha cht chnh

P-hydroxybenzaldehyd 3-Bromopropanamin

Thiazolidin-2,4-dion 5-Bromoisatin

4-Nitrobenzensulfoclorid 5-Fluoroisatin

2-Bromoethanamin 2-Bromoethyl clorid

Dung mi v cc ha cht khc

Dicloromethan Piperazin

Dimethylformamid HCl

Triethylamin NaH

Methanol

2.1.2 Thit b, dng c

- Dng c thy tinh: bnh cu y trn dung tch 50 ml c nt mi, sinh hn

hi lu, pipet, bnh chit, phu thy tinh, bnh chy sc k lp mng (TLC).

- My khuy t gia nhit.

- My ct quay chn khng Buchi R-210.

- Cn phn tch, cn k thut Shimazu.

- T lnh, t sy, my siu m.

- Bn mng silicagel Merck 70-230 mesh chy sc k lp mng.

- My o nhit nng chy nhit in (Electrothermal digital) xc nh

nhit nng chy.

- My Perkin Elmer xc nh ph IR.

14

- My khi ph Agilent 6310 Ion Trap MS

- My cng hng t Bruker AV-500 ghi ph 1H-NMR, 13C-NMR.

2.2 Ni dung nghin cu

2.2.1 Tng hp ha hc

- (Z) 3-(2-(5-Floro-2,3-dioxoindol-1-yl) ethyl)-5-(4-hydroxybezyliden)thiazolidin-

2,4-dion (3a)

- (Z) 3-(2-(5-Bromo-2,3-dioxoindol-1-yl) ethyl)-5-(4-hydroxybezyliden)thiazolidin-

2,4-dion (3b)

- (Z) N-(2-(5-(4-hydroxybenzyliden)-2,4-dioxothiazolidin-3-yl) ethyl) -4-

nitrobenzensulfonamid (3c)

- (Z) N-(3-(5-(4-hydroxybenzyliden)-2,4-dioxothiazolidin-3-yl) propyl)-4-

nitrobenzensulfonamid (3d)

2.2.2 Th tc dng sinh hc ca cc cht tng hp c

Th c tnh t bo in vitro, trn dng t bo: SW620 (t bo ung th i

trng)

S b nh gi tnh ging thuc ca cc cht tng hp c.

2.3 Phng php nghin cu

2.3.1 Tng hp ha hc v kim tra tinh khit

Da trn nhng nguyn tc v phng php c bn ca ha hc hu c

tng hp cc dn cht.

Dng TLC theo di qu trnh tin trin ca phn ng.

Kim tra tinh khit ca sn phm bng TLC v o nhit nng chy.

2.3.2 Xc nh cu trc

Cc cht tng hp c xc nh cu trc bng cc loi ph sau: ph hng ngoi

( R), ph khi (MS), ph cng hng t proton (1H-NMR), ph cng hng t

carbon (13

C-NMR).

Ph hng ngoi ( R): ph hng ngoi c ghi ti Khoa ha, trng i Hc

KHTN H Ni, trn my Perkin Elmer vi k thut vin n n KBr trong vng 4000-

15

600cm-1. Cc mu rn c phn tn trong KBr sy kh vi t l 1:200 ri p

di dng film mng di p lc cao c ht chn khng loi b hi m .

Ph khi (MS) c o my khi ph Agilent 6310 Ion Trap MS ca

vin Ha hc cc hp cht t nhin Vit Nam

Ph cng hng t ht nhn (1H-NMR, 13C-NMR) c ghi trn my Bruker

AV-500 ti trung tm khoa hc v cng ngh Vit Nam dng DMS lm dung mi,

cht chun ni l tetramethylsilan (TMS).

2.3.3 Th c t nh t bo invitro

Th hot tnh khng t bo ung th in vitro c thc hin ti khoa Dc, Trng

i hc uc gia Chungbuk, Cheongju, Hn uc theo phng php MTT v gi

tr C50 c tnh trn phn mm GraphPad Prism.

Dng t bo th nghim:

- SW620: t bo ung th i trng.

Cc dng t bo ung th c s dng t Ngn hng t bo ung th ca Vin

nghin cu sinh hc v cng ngh sinh hc Hn uc (KR BB) v c nui cy

trong mi trng DMEM (Dulbecco s modified) hoc RPM b sung 10 BS

(huyt thanh bo thai b).

c tnh t bo ca cc cht c th bng phng php MTT theo cc bc sau:

c 1 hun b

Cc t bo pha logarit c tripsin ha v phn tn vo h n dch n t bo

trong mi trng DMEM hoc RPM b sung 10 BS. iu chnh nng

khong 1,5.104 n 3,5.104 t bo, sau chia u vo cc ging ca a 96 ging,

m i ging 200 l, cc a sau c 37oC trong iu kin 5 C 2.

Sau 24 gi , cc mu th c chun b trong 20 l mi trng DMEM RPM b

sung 10 BS t dung dch gc 10 mg ml trong dimethyl sulfoxid (DMS ) ri

thm 2 l mu th vo cc ging nhiu nng khc nhau. Cc a ny sau

c thm 48 gi. Tt c cc mu c chun b sau cho nng cui cng ca

DMS khng qu 0,1 .

c 2 Tin hnh th

16

Sau 48 gi , thm vo m i ging 20 l thuc nhum MTT (nng MTT 5

mg/ml trong mui m phosphat PBS). Cc a c thm 3 gi 37oC trong

iu kin 5 C 2. Tip theo m i ging c cho 100 l dung dich DMS , 5

pht cho MTT formazan c ha tan. hp thu c c bc sng 510 nm.

Gi tr IC50 l nng ca mu th m , hp th gim i 50 so vi nhm

chng (trng m tnh l ging ch thm mi trng nui cy): kt qu cui cng l

gi tr trung bnh ca 4 ln o c lp vi gi tr hp th khc nhau khng qu

5 . Gi tr C50 c tnh da trn phn mm GraphPad Prism. Trong phng php

th ny, C50 20 g ml c coi l c hot tnh.

2.3.4 nh gi mc ging thuc ca cc d n cht tng hp c.

Tnh gi tr logP ca cc dn cht tng hp c bng phn mm EP suite: uy

trnh bao gm v cu trc 2D, to Smile notation bng phn mm Chemsketch 4.0

ca ACD labs sau a Smile notation vo phm mm EP suite tnh cc gi tr

logP. nh gi mc ging thuc ca cc dn cht da trn quy tc Lipinsky [17]:

- Khi lng phn t ca cht phi nh hn 500 g/mol.

- S trung tm nhn lin kt hydro (N, ) phi nh hn 10.

- S trung tm cho lin kt hydro (NH, H) phi nh hn 5.

- Cn bng gia tnh thn nc du: logP 5.

17

CHNG 3. THC NGHIM, KT QU V BN LUN

3.1 Ha hc

Qu trnh tng hp 4 cht trong kha lun ny c tin hnh theo s :

H nh 3.1. Quy trnh tng hp cc cht

Ghi ch: i) Thiazolidin-2,4-dion, ethanol, piperazin, 80oC, 24h; ii) 2-bromoethyl clorid,

K2CO3, DMF, 80oC, 3h; iii) NaH, DMF, 80

oC, 24h; iv) 2-bromoethanamin hydrobromid

(hoc 3-bromopropanamin hydrobromid), TEA, DCM, to phng, 24h; v) NaH, DMF,

100oC, 24h.

3.1.1 Tng hp ha hc

3.1.1.1 Tng hp 5-(4-hydroxybenzyliden)thiazolidin-2,4-dion (1)

18

S phn ng:

Tin hnh

- Ha tan 244 mg p-hydroxybenzaldehyd (2 mmol) bng 5 ml EtOH khan trong

bnh cu 50ml, thm vo 292 mg thiazolidin-2,4-dion (2,5 mmol) v 215 mg

piperazin (2,5 mmol)

- Hi lu 80oC, kt hp khuy t trong 24h.

- Kim tra phn ng bng TLC vi pha ng DCM : MeOH (9 : 1).

X l

- Loi bt dung mi

- h n hp phn ng vo 10 ml HCl 5% lnh, thy xut hin ta mu vng nht.

- Lc, ra ta bng nc ct n khi dch lc trung tnh

- Sy kh 60oC trong 24h

Kt qu

- Thu c cht rn, mu vng

- Rf = 0,3 (DCM : MeOH = 9:1)

- H = 90 %

3.1.1.2 Tng hp (Z) 3-(2-(5-floro-2,3-dioxoindol-1-yl) ethyl)-5-(4-

hydroxybezyliden)thiazolidin-2,4-dion (3a)

Qu trnh tng hp cht 3a din ra theo 2 bc:

a. Tng hp 1-(2-cloroethyl)- 5-floroindolin -2,3-dion (2a)

S phn ng:

Ghi ch: ii) 2-bromoethyl clorid, K2CO3, DMF, 80oC, 3h

19

Tin hnh

- Ha tan 165 mg 5-floro isatin (1 mmol) bng 1ml DMF trong bnh cu 50ml.

Thm 207 mg K2CO3 (1,5 mmol) khan, siu m 1 pht.

- un 60oC v khuy t trong 60 pht, cho ti khi h n hp chuyn sang mu tm

nu.

- Thm 83 mg KI (0,5 mmol) vo bnh phn ng

- Chun b 2-Bromoethan clorid trong 1 ml DMF v nh t t vo h n hp phn

ng, nng nhit ln 80oC

- Tin hnh phn ng trong 3 gi, kt hp khuy t

- Kim tra phn ng bng TLC vi pha ng DCM : MeOH (9 : 1).

X l

- Lm lnh bnh phn ng, pha long h n hp phn ng bng 20 ml nc lnh

- Acid ha bng HCl 5 n pH=4, thy c ta mu nu xut hin

- Lc, ra ta bng nc ct n khi dch lc trung tnh

- Sy kh 60oC trong 24h.

Kt qu

- Thu c cht rn, mu nu

- Rf = 0,67 (DCM : MeOH = 9:1)

- H = 78 %

b. Tng hp (Z) 3-(2-(5-floro-2,3-dioxoindol-1-yl) ethyl)-5-(4-

hydroxybezyliden)thiazolidin-2,4-dion (3a)

S phn ng:

Ghi ch: iii) NaH, DMF, 80oC, 24h

20

Tin hnh:

- Ha tan 222 mg 5-(4-hydroxybenzyliden)thiazolidin-2,4-dion (1 mmol) trong 1

ml DMF trong bnh cu 50 ml.

- Thm 2 mmol NaH, un 80oC trong 1h

- Thm t t 226,5 mg 1-(2-cloroethyl)- 5-floroindolin -2,3-dion (1 mmol) ha

tan trong DMF vo bnh cu.

- Tin hnh phn ng trong 24h, kt hp khuy t

- Kim tra phn ng bng TLC vi pha ng DCM : MeOH (9 : 1)

X l:

- Lm lnh bnh phn ng

- h n hp phn ng vo 20ml HCl 5% thy c ta mu vng cam m xut

hin.

- Lc, ra ta bng nc

- Sy kh 60oC trong 24h.

- Kt tinh li 3a trong EtOH, lc ly tinh th, sy kh

Kt qu:

- Thu c 229 mg cht rn mu cam m

- Rf = 0,53 (DCM : MeOH = 9:1)

- Tonc = 221-223oC

- H = 56%

3.1.1.3 Tng hp (Z) 3-(2-(5-bromo-2,3-dioxoindol-1-yl) ethyl)-5-(4-

hydroxybezyliden)thiazolidin-2,4-dion (3b)

Qu trnh tng hp cht 3b din ra theo 2 bc:

a. Tng hp 5-bromo-1-(2-cloroethyl)indolin -2,3-dion (2b)

S phn ng:

Ghi ch: ii) 2-bromoethyl clorid, K2CO3, DMF, 80oC, 3h

21

Tin hnh:

Qu trnh tng hp cht 5-bromo-1-(2-cloroethyl)indolin-2,3-dion (2b) t 226 mg

(1 mmol) 5-bromoisatin v 2-bromoethan clorid tin hnh tng t nh qu trnh

tng hp cht (2a).

Kt qu:

- Thu c cht rn mu nu

- Rf = 0,68 (DCM : MeOH = 9:1)

- H = 64,4 %

b. Tng hp (Z) 3-(2-(5-bromo-2,3-dioxoindol-1-yl) ethyl)-5-(4-

hydroxybezyliden)thiazolidin-2,4-dion (3b)

S phn ng:

Ghi ch: iii) NaH, DMF, 80oC, 24h

Tin hnh:

- Qu trnh tng hp cht (Z)-3-(2-(5-bromo-2,3-dioxoindol-1-yl) ethyl)-5-(4-

hydroxybezyliden)thiazolidin-2,4-dion (3b) t 222 mg (1 mmol) cht (1) v

288,5 mg (1 mmol) cht (2b) tin hnh tng t nh qu trnh tng hp cht

(3a). Kt tinh li 3b trong EtOH, lc ly tinh th, sy kh.

Kt qu:

- Thu c 237 mg cht rn mu vng cam

- Rf = 0,55 (DCM : MeOH = 9:1)

- Tonc = 216-217oC

- H = 50%

3.1.1.4 Tng hp (Z) N-(2-(5-(4-hydroxybenzyliden)-2,4-dioxothiazolidin-3-yl)

ethyl)-4-nitrobenzensulfonamid (3c)

Qu trnh tng hp cht 3c din ra theo 2 bc:

22

a. Tng hp N-(2-bromoethyl)-4-nitrobenzensulfonamid (2c)

S phn ng:

Ghi ch: iv) TEA, DCM, to phng, 24h

Tin hnh:

- Ly 442 mg 4-nitrobenzensulfoclorid (2 mmol), 1,5 ml DCM v 0.5 ml DMF vo

bnh cu 50 ml, lm lnh bng nc

- Thm 450 mg 2-bromoethanamin hydrobromid (2,2 mmol)

- Thm t t 0,6 ml TEA (4,4 mmol) lc u trong h n hp nc

- Tin hnh phn ng nhit phng trong 24h

- Kim tra phn ng bng TLC vi pha ng DCM : MeOH = 40:1

X l

- Ct quay chn khng loi bt dung mi

- Acid ha bng 15 ml HCl 5%, lc u, thy c ta xut hin, ta 30 pht trong

t lnh

- Lc, ra bng nc ct n khi dch lc trung tnh, sy kh 60oC trong 24h

Kt qu

- Thu c cht rn, mu trng ng

- Rf = 0,8 (DCM : MeOH = 20:1)

- H = 86%

b. Tng hp (Z) N-(2-(5-(4-hydroxybenzyliden)-2,4-dioxothiazolidin-3-yl) ethyl)-4-

nitrobenzensulfonamid (3c)

S phn ng:

23

Ghi ch: v) NaH, DMF, 100oC, 24h.

Tin hnh:

- Ha tan 221 mg 5-(4-hydroxybenzyliden)thiazolidin-2,4-dion (1 mmol) bng 1

ml DMF trong bnh cu 50 ml

- Thm 50 mg NaH (2 mmol), hot ha trong 1h nhit phng

- Thm 309 mg N-(2-bromoethyl)-4-nitrobenzensulfonamid (1 mmol) un 100oC

trong 24h.

X l

- Thm vo bnh phn ng 2 ml MeOH, lm lnh bnh phn ng 20 pht trong

- h n hp phn ng vo 10 ml HCl 5 lm lnh, khuy u, xut hin ta.

lnh khong 30 pht.

- Lc, ra ta bng nc ct n khi dch lc trung tnh, sy kh 60oC trong 24h

- Kt tinh li sn phm trong EtOH, lc ly tinh th, sy kh

Kt qu

- Thu c 290 mg cht rn mu vng

- Rf = 0,5 (DCM : MeOH= 9:1)

- Tonc = 234-235oC

- H = 65%

3.1.1.5 Tng hp (Z) N-(2-(5-(4-hydroxybenzyliden)-2,4-dioxothiazolidin-3-yl)

propyl)-4-nitrobenzensulfonamid (3d)

Qu trnh tng hp cht 3d din ra theo 2 bc:

a. Tng hp N-(3-bromopropyl)-4-nitrobenzensulfonamid (2d)

S phn ng:

24

Ghi ch: iv) TEA, DCM, to phng, 24h

Tin hnh:

Qu trnh tng hp cht N-(3-bromopropyl)-4-nitrobenzensulfoamid (2d) t 442

mg (2mmol) 4-nitrobenzensulfoclorid v 480 mg (2,2mmol) 3-bromopropanamin

hydrobromid tin hnh tng t qu trnh tng hp cht (2c).

Kt qu:

- Thu c cht rn mu trng ng

- Rf = 0,81 (DCM : MeOH = 20:1)

- H = 80%

b. Tng hp (Z) N-(2-(5-(4-hydroxybenzyliden)-2,4-dioxothiazolidin-3-yl)

propyl)-4-nitrobenzensulfonamid (3d)

Ghi ch: v) NaH, DMF, 100oC, 24h.

Tin hnh:

- Qu trnh tng hp cht (Z) N-(2-(5-(4-hydroxybenzyliden)-2,4-

dioxothiazolidin-3-yl) propyl)-4-nitrobenzensulfonamid (3d) t 221 mg (1 mmol)

cht (1) v 323 mg, (1 mmol) N-(2-bromoethyl)-4-nitrobenzensulfonamid tin

hnh tng t qu trnh tng hp cht (3c). Kt tinh li 3d trong EtOH, lc ly

tinh th, sy kh

Kt qu:

- Thu c 277 mg cht rn mu vng

- Rf = 0,6 (DCM : MeOH = 9:1)

- Tonc = 235-237oC

- H = 60%

25

Bng 3.1: Ch s l ha v hiu sut tng hp cc cht 3a-d

Cht CTCT KLPT Mu sc Hiu sut

(%)

3a

412 Cam m 56

3b

473 Vng cam 50

3c

449 Vng 65

3d

463 Vng 60

3.1.2 Kim tra tinh khit

tinh khit ca cc cht 3a-3d c kim tra bng sc k TLC v o nhit

nng chy (Tonc) ca chng.

Sc k lp mng (TLC): TLC c tin hnh trn bn mng nhm trng

silicagel Merk 70-230 mesh, quan st vt sc k di n t ngoi bc sng

254nm. Dung mi ha tan cc cht l aceton. H dung mi l DCM : MeOH = 9:1

Kt qu thu c trn sc k u cho thy mt vt gn, r khi soi di nh sng

n t ngoi.

o nhit nng chy: cc cht sau khi c tinh ch bng cch kt tinh li

vi dung mi EtOH, c 4 cht 3a-d u c dng tinh th rn. im nng chy ca

cc dn cht c xc nh bng my o nhit nng chy nhit in

(Electrothermal digital). Nhit nng chy ca cc dn cht dao dng trong

khong 1-2oC.

Nh vy c th khng nh cc cht c tng hp c tinh khit.

26

Bng 3.2: Gi tr Rf v Tonc ca cc cht 3a-d

Cht CTCT H dung mi Rf Tonc (

oC)

3a

DCM:MeOH = 9:1 0,53 221-223

3b

DCM:MeOH = 9:1 0,55 216-217

3c

DCM:MeOH = 9:1 0,5 234-235

3d

DCM:MeOH = 9:1 0,6 235-237

3.1.3 Xc nh cu trc

khng nh cu trc ca cc cht tng hp c, tin hnh ghi ph hng ngoi

( R), ph khi lng (MS), ph cng hng t ht nhn (1H-NMR), ph cng

hng t carbon (13C-NMR) ca cc sn phm cui. Kt qu ph ca cc cht c

trnh by c th phn phn tch ph v ph lc.

3.1.3.1 Ph hng ngoi (IR)

Bng 3.3: Kt qu phn tch ph hng ngoi (IR) cc cht 3a-d

Cht CTCT S sng (cm-1) ng vi cc dao ng

O-H N-H Csp2 C=O C=C

3a

3587 3060 1730

1667

1591

1515

1485

3b

3643 1740

1670

1602

1579

1514

1472

27

3c

3535 3290 3110 1732

1677

1601

1579

1532

1514

3d

3443 3268 3100 1745

1676

1603

1582

1518

Ghi ch: : dao ng ha tr

Nhn xt: Ph IR ca cc cht 3a-d cho thy cc tn hiu c th nhn dng s

b s hin din mt s nhm chc nh C= , O-H thng qua cc gi tr ca di

hp thu, cng pic, hnh dng pic

3.1.3.2 Ph khi lng (MS)

Bng 3.4: Kt qu phn tch ph khi lng (MS) cc cht 3a-d

Cht CTCT KLPT m/z

3a

412 412,8 [M+H]+

3b

473 472,8 [M]-

3c

449 450,04[M+H]+

3d

463 461,7[M-H]-

Nhn xt: Kt qu phn tch ph bng 3.4 v ph cho thy cc ph u c

pic phn t c s khi ng bng s khi d kin vi cng mnh.

3.1.3.3 Ph cng hng t ht nhn 1H (1H-NMR)

28

Bng 3.5: Kt qu phn tch ph cng hng t ht nhn 1H (1H-NMR) cc

cht 3a-d

Cht CTCT S liu phn tch ph 1H-NMR

3a

1H-NMR (500MHz, DMSO, ppm):

3,88-3,90 (2H, t, CH2); 3,95-3,97 (2H, t,

CH2); 6,90-6,91 (2H, d, J= 9,0, H-3,5);

7,28-7,30 (1H, m, 4 ); 7,44-7,50 (3H, m,

H-2,6,6 ); 7,57-7,61 (1H, m, H-7 ); 7,76

(1H, s, CH); 10,4 (1H, s, OH)

3b

1H-NMR (500MHz, DMSO, ppm):

3,88-3,96 (4H, m, CH2); 6,90-6,91 (2H, d,

J= 8,5, H-3,5); 7,24-7,26 (1H, d, J= 8,5,

H-4 ); 7,45-7,46 (2H, d, J= 8,5, H-2,6);

7,74-7,76 (2H, d, J= 12,5, H-6 ,7 ); 7,88-

7,89 (1H, d, CH); 10,39 (1H, s, OH)

3c

1H-NMR (500MHz, DMS , ppm):

3,10-3,13 (2H, m, CH2); 3,67-3,69 (2H, t,

J= 6,0, CH2); 6,91-6,92 (2H, d, J= 8,5, H-

3,5); 7,45-7,47 (2H, d, J= 8,5, H-2,6); 7,77

(1H, s, CH); 7,97-7,99 (2H, d, J= 9,0, H-

2 ,6 ); 8,30-8,32 (1H, t, NH); 8,37-8,39

(2H, d, J= 9,0, H-3 ,5 ); 10,37 (1H, s, OH)

3d

1H-NMR (500MHz, DMS , ppm):

1,67-1,72 (2H, m, CH2); 2,81-1,72 (2H,

m, CH2); 3,60-3,63 (2H, t, CH2); 6,90-

6,92 (2H, d, J= 8,5, H-3,5); 7,45-7,47 (2H,

d, J= 9,0, H-2,6); 7,78 (1H, s, CH); 8,02-

8,04 (2H, d, J= 8,5, H-2 ,6 ); 8,07-8,09

(1H, t, NH); 8,39-8,41 (2H, d, J= 8,5, H-

3 ,5 ); 10,37 (1H, s, OH)

29

Ghi ch: : chuyn dch ha hc (ppm); s: singlet, vch n; d: doublet ,vch i; t:

triplet, vch ba; m: multiplet, a vch

Nhn xt: Kt qu phn tch ph bng 3.5 v cn c vo ph c th thy: s

lng proton, bi tn hiu, dch chuyn ph hp vi cng thc cu to ca 4

cht 3a-d.

3.1.3.4 Ph cng hng t ht nhn 13C (13C-NMR)

Bng 3.6: Kt qu phn tch ph cng hng t ht nhn 13C (13C-NMR) cc

cht 3a-d

Cht CTCT S liu phn tch ph 13C-NMR

3a

13C-NMR (125MHz, DMS , ppm): 182,28

(C3 ); 167,730 (C5 ); 165,82 (C3 ); 160,21

(C2 ); 159,51 (C6 ); 157,59 (C4); 146,48

(C9 ); 133,71 (C7); 132,64 (C2-6); 124,46

(C8 ); 123,70 (C7 ); 118,24 (C4 ); 118,19

(C6 ); 116,4 (C3-5); 116,17 (C2 ); 111,59

(C5 ); 38,57 (C6 ); 37,46 (C7 )

3b

13C-NMR (125MHz, DMS , ppm): 181,65

(C3 ); 167,73 (C5 ); 165,81 (C3 ); 160,21

(C2 ); 157,95 (C4); 149,13 (C9 ); 140,10

(C7 ); 133,74 (C7); 132,65 (C2-6); 126,96

(C1); 123,70 (C5 ); 119,05 (C4 ); 116,40 (C3-

5); 116,17 (C2 ); 115,25 (C6 ); 112,59 (C8 );

38,53 (C6 ); 37,47 (C7 )

3c

13C-NMR (125MHz, DMS , ppm): 167,52

(C5 ); 165,75 (C3 ); 160,07 (C4); 149,52 (C4 ),

146,07 (C1 ); 133,19 (C7); 132,49 (C2-6);

127,89 (C2 -6 ); 124,64 (C3 -5 ); 123,82 (C1);

116,71 (C2 ); 116,38 (C3-5); 41,39 (C6 )

3d

13C-NMR (125MHz, DMS , ppm): 167,48

(C5 ); 165,77 (C3 ); 160,12 (C4); 149,57

(C4 ); 145,89 (C1 ); 133,35 (C7); 132,57 (C2-

6); 128,09 (C2 -6 ); 124,62 (C3 -5 ); 123,87

(C1); 116,69 (C2 ); 116,40 (C3-5); 40,32 (C6 );

39,92 (C8 ); 27,4 (C7 )

Ghi ch: : chuyn dch ha hc (ppm)

30

Nhn xt: Kt qu phn tch ph bng 3.6 v ph cc cht phn ph lc

(ph lc 23-30) cho thy: S lng carbon, dch chuyn ha hc ca cc pic l

ph hp vi cng thc cu to d kin.

Nh vy, kt hp kt qu phn tch cc ph IR, MS, 1H-NMR, 13C-NMR c th

khng nh cc cht tng hp c c cng thc cu to ng nh d kin. Trn c

s 4 cht 3a-d tip tc tin hnh th hot tnh sinh hc.

3.2 Th hot tnh sinh hc

3.2.1 Th c t nh t bo cc cht c tng hp

Cc cht 3a-d c tin hnh th c tnh t bo invitro trn dng t bo ung th

i trng (SW620) ti khoa Dc, Trng i hc uc gia Chungbuk, Cheongju,

Hn uc theo phng php MTT v gi tr C50 c tnh trn phn mm

GraphPad Prism. so snh cc cht (1) v 5-bromoisatin v 5-floroisatin ng

thi cng c th hot tnh. Kt qu c trnh by bng 3.7

Bng 3.7: Tc dng khng t bo ung th i trng (SW620)

Cht CTCT

Tc dng hng t bo ung th S 2

IC50

(g/ml) 1

IC50

(M)2

3a

9,76 23,69

3b

11,04 23,34

3c

4,081 9,09

3d

10,23 22,09

1

48,84 221,00

31

5-floroisatin

21,15 128,00

5-bromoisatin

17,16 75,93

hi ch: 1n ng g/ml c ch pht trin ca t bo ung th; 2n ng (M)

c ch 50% s pht trin ca t bo ung th

3.2.2 nh gi mc ging thuc

Bng 3.8: nh gi mc ging thuc ca cc cht 3a-d

theo quy tc Lipinsky

Cht CTCT LogP KLPT S NH,

OH S N, O

3a

1.61 412 1 7

3b

2.30 473 1 7

3c

1.79 449 2 9

3d

2.28 463 2 9

Nhn xt: Tt c cc cht u tha mn c 4 yu cu ca quy tc Lipinsky v

ging thuc.

3.3 Bn lun

3.3.1 Tng hp ha hc

Phn ng tng hp 5-(4-hydroxybenzyliden)thiazolidin-2,4-dion: u trnh

ngng t gia p-hydroxybenzaldehyd vi nhm methylen ca vng thiazolidin-2,4-

dion c tin hnh trong mi trng ethanol, vi xc tc l piperazin. Vng

thiazolidin-2,4-dion c hai nhm carbonyl ht in t mnh, xc tc piperazin c

32

tnh base do lm tng kh nng phn ng ca nhm methylen v tr s 5. Phn

ng ngng t cho hiu sut cao ( 90 )

Phn ng tng hp cc cht 2a-b: phn ng ny cn tin hnh cn thn, m

bo isatin c hot ha hon ton v phn ng ht, nu khng loi ht isatin trong

sn phm, phn ng tip theo s c nhiu sn phm ph do isatin cng l mt tc

nhn nucleophin mnh.

Phn ng tng hp cc hp cht 2c-d: ch cho TEA vo t t, v TEA l mt

amin bc 3, s to mui TEA.HCl gip y phn ng i theo chiu thun. Nu cho

qu nhanh, phn ng s tng nhit NO2-C6H4-SO2(+) s d phn ng vi nhn

phenyl ca phn t khc, to ra cc sn phm ph.

3.3.2 Khng nh cu trc cc cht tng hp c

Ph hng ngoi:

- Cc cht u c nh hp th ca nhm C=O trong khong t 1850-1650 cm-

1 vi pic nhn, mnh, sc nt.

- Cc di hp th ca vng thm vi cc pic dao ng trong khong t 1500-

1450 cm-1

.

- C 4 cht 3a-d u c s hin din nh hp thu ca nhm O-H trong

khong t 3700-3200 cm-1.

- Nh vy c th s b kt lun d liu ph IR ph hp vi cng thc cu to

d kin ca 4 cht 3a-d. Tuy nhin da trn ph IR vn cha c nhiu d liu

khng nh mt cch chc chn cu trc ca cc cht.

Ph khi lng:

- Cc pic ion phn t c s khi ph hp vi s khi d kin ca cc cht 3a-d

Ph cng hng t ht nhn 1H:

- D liu ph 1H-NMR ph hp vi cng thc cu to d kin ca 4 cht 3a-

d. Trn ph ca cc cht u quan st thy:

- Cc pic ca cc proton nhn thm nm trong vng dch chuyn 5,0-9,0.

- Cc pic proton cu ni methylen, proton ca nhm chc OH, NH th hin s

lng proton, bi tn hiu, dch chuyn ph hp.

33

Ph cng hng t ht nhn 13C:

- Kt qu phn tch ph cc cht cho thy: s lng cacbon, dch chuyn

ha hc ca cc pic l ph hp vi cng thc cu to d kin. Hai cht 3a-b c 4

pic ca 4 nhm C=O, cc pic ca vng benzen, cc pic ca d vng indol v 2 pic

ca 2 nhm CH2. Hai cht 3c-d u c 2 pic ca 2 nhm C=O, cc pic ca vng

benzen, cht 3d c 3 pic ca 3 nhm CH2, cht 3c ch thy c 1 pic ca nhm CH2

do pic ny b che bi cc pic ca DMSO.

Khng nh cu hnh sn phm 1:

- Phn ng ngng t to ra sn phm c dy ni i, nh vy c th tn ti

ng phn Z v E. Tuy nhin mt s nghin cu cho thy sn phm ngng t ch

c duy nht cu hnh Z [4,8]. Thc hin nhiu x tia v da trn ph cng hng

t ht nhn proton ca cc cht c cu trc tng ng, nhn thy ch tn ti duy

nht cu hnh Z m khng c cu hnh E d cc iu kin phn ng khc nhau:

AcOH/AcONa, toluen/piperazin.

Khng nh cu trc sn phm cui:

- Phn ng SN2 tng hp cc cht 3a-d: phn ng c th xy ra theo hai

hng: to thnh sn phm O-alkyl hoc N-alkyl do c nhm OH phenol v NH

imid u tr thnh tc nhn nucleophin trong mi trng kim. Tuy nhin kt qu

phn tch ph IR ca cc cht 3a-d u thy c nh hp th ca nhm OH, trn ph

1H-NMR cc cht 3a-d u thy c s hin din ca proton nhm OH. Bn cnh

theo mt nghin cu mi c cng b gn y ca Steven R. LaPlante v cng s

[27] a ra phng php phn bit N-alkyl hay O-alkyl bng ph NMR, l

da trn s khc bit v chuyn dch ha hc trn ph 13C-NMR gia C lin kt

trc tip vi N v C lin kt trc tip vi O, kt qu thc t trn ph 13C-NMR cc

cht tng hp c cho thy chuyn dch ha hc ca C dy ni -CH2 ph hp

vi chuyn dch ha hc d kin ca sn phm N-alkyl. Nh vy c th khng

nh chc chn cc sn phm c tng hp l cc sn phm N-alkyl ng nh

d kin.

34

3.3.3 Hot t nh sinh hc

- Th c tnh t bo 4 cht 3a-d trn dng t bo ung th i trng (SW620)

cho thy IC50 ca cc cht u nh hn 20g/ml, tc c 4 cht u c hot tnh

khng t bo ung th SW620. Trong cht 3c cho IC50 thp nht (IC50 = 4,081

g/ml), cc cht 3a, 3b, 3d cho gi tr IC50 mc trung bnh (9,760-11,04 g/ml).

Do cc cht tc dng theo c ch phn t nn so snh hot tnh gia cc cht

chng ti quy i gi tr IC50 t g/ml sang M.

- C 5-(4-hydroxybenzyliden)thiazolidin-2,4-dion (cht 1) v cc dn cht

isatin u c bit l nhng cht c c tnh t bo. Khi gn cc isatin vo cht 1

c cc cht 3a, 3b c c tnh t bo mnh hn (cc cht 3a, 3b c IC50 (M)

gim 9,3-9,5 ln so vi 1 v gim t 3,25-5,4 ln so vi cc isatin ban u). Nh

vy, nhiu kh nng tc dng ca 3a v 3b l tc dng hip ng ca c hai hp

phn isatin v 5-benzyliden-thiazolidin-2,4-dion theo cc c ch khc nhau.

- Cc cht 3c, 3d cng c c tnh t bo mnh hn cht 1 (gi tr IC50 (M)

gim 24 ln cht 3c v gim 10 ln cht 3d). Nh vy hp phn 4-

nitrobenzensulfonamid gn thm cng lm tng mnh c tnh t bo ca cht to

thnh (3a, 3b). C th mt phn c tnh l do bn thn phn 4-

nitrobenzensulfonamid gy ra (vo t bo nhm nitro b kh ha to amin, sau

to nitrosoamin gy c). Kt qu th nghim cho thy cht 3c c c tnh t bo

mnh hn 2,43 ln cht 3d, so snh cht 3c v cht 3d, cht 3c c t hn mt nhm

CH2 mch ni, cht 3c c gi tr logP = 1,79 thp hn cht 3d vi logP = 2,28, nh

vy cc dn cht 4-nitrobenzensulfonamid hot tnh ca cc cht rt nhy cm vi

tnh thn du thn nc. C ch ca tc dng ny cn cn c nghin cu su hn.

- V ging thuc ca bn cht 3a-d theo quy tc Lipinsky, cc cht u

tha mn c yu cu ca quy tc, cho thy kh nng hp thu tt, thm tt qua

mng t bo, c tim nng tip tc nghin cu, pht trin nhm sng lc, tm

kim cc thuc mi.

35

KT LUN V KIN NGH

I. Kt lun

T cc kt qu nghin cu trnh by c th rt ra mt s kt lun sau:

1.1.V tng hp ha hc v khng nh cu trc

tng hp c cc cht nh d kin, cc cht u cha c cng b trong bt

k ti liu no, bao gm:

- (Z) 3-(2-(5-Floro-2,3-dioxoindol-1-yl) ethyl)-5-(4-hydroxybezyliden)thiazolidin-

2,4-dion (3a)

- (Z) 3-(2-(5-Bromo-2,3-dioxoindol-1-yl) ethyl)-5-(4-hydroxybezyliden)thiazolidin-

2,4-dion (3b)

- (Z) N-(2-(5-(4-hydroxybenzyliden)-2,4-dioxothiazolidin-3-yl) ethyl) -4-

nitrobenzensulfonamid (3c)

- (Z) N-(3-(5-(4-hydroxybenzyliden)-2,4-dioxothiazolidin-3-yl) propyl)-4-

nitrobenzensulfonamid (3d)

khng nh cu trc cc cht tng hp c nh phn tch cc d liu

ph IR, MS, 1H-NMR, 13C-NMR.

1.2.V hot tnh sinh hc

Bc u th hot tnh c ch t bo ung th ca cc cht tng hp c.

Kt qu cho thy c 4 cht tng hp c u c hot tnh c ch trung bnh vi

dng t bo ung th i trng SW620, trong cht 3c c hot tnh mnh nht

(IC50=9,09 M).

II. Kin ngh

Vi cc kt qu t c, chng ti kin ngh:

Tin hnh th hot tnh khng t bo ung th in vitro ca cc cht tng hp

c trn cc dng t bo ung th khc.

Tip tc nghin cu, pht trin nhng cht mi l dn cht ca 5-(4-

hydroxybezyliden)thiazolidin-2,4-dion cha khung p-nitrobenzensulfonamid v cc

nhn thm khc trong iu tr ung th.

36

TI LIU THAM KHO

Ting Vit Nam:

1. B Y t (2008), Ha hu c, NXB Y hc, tp I

Ting nc ngoi:

2. Agrawal R., Jain P. and Dikshit S. N. (2012), Balaglitazone: A Second

Generation Peroxisome Proliferator-Activated Receptor (PPAR) Gamma ()

Agonist, Mini Rev Med Chem, 2012 Feb, 12(2), 87-97.

3. Aherblom E.B. (1974), Synthesis and structure Activity relationship of a

series of antibacterial active 5-(5-nitro-2-furfulidene) thiazolones, J.Med-Chem,

17(6), 609-614.

4. Alegaon Shankar G., Alagawadi Kallanagouda R. (2012), New thiazolidine-

2,4-diones as antimicrobial and cytotoxic agent, Medicinal Chemistry Research,

21(10), 3214-3223.

5. Amal M. Youssef, M. Sydney White, Erika B. Villanueva, Ibrahim M. El-

Ashmawy, Andis Klegeris (2010), Synthesis and biological evaluation of novel

pyrazolyl-2,4-thiazolidinediones as anti-inammatory and neuroprotective agents,

Bioorganic & Medicinal Chemistry, 18, 20192028.

6. Avupati Vasudeva Rao, Yejella Rajendra Prasad, Akula Annapurna, et al.

(2012), Synthesis, characterization and biological evaluation of some novel 2, 4-

thiazolidinediones as potential cytotoxic, antimicrobial and antihyperglycemic

agents, Bioorganic & medicinal chemistry letters, 6442-6450.

7. Bhattarai Bharat Raj, Kafle Bhooshan, Hwang Ji-Sun, et al. (2009),

Thiazolidinedione derivatives as PTP1B inhibitors with antihyperglycemic and

antiobesity effects, Bioorganic & medicinal chemistry letters, 19(21), 6161-6165.

8. Bruno G., Costantino L., Curinga C., et al. (2002), Synthesis and aldose

reductase inhibitory activity of 5-arylidene-2, 4-thiazolidinediones, Bioorganic &

medicinal chemistry, 10(4), 1077-1084.

9. Cantello Barrie CC., Cawthorne Michael A., Cottam Graham P., et al.

(1994), [[. omega.-(Heterocyclylamino) alkoxy] benzyl]-2, 4-thiazolidinediones as

37

potent antihyperglycemic agents, Journal of medicinal chemistry, 37(23), 3977-

3985.

10. Chien-Jung Lu, Yu Sun, Chih-Hsin Muo, Rong-Chi Chen, Pei-Chun Chen,

Chung Y. Hsu (2013), Risk of Stroke with Thiazolidinediones: A Ten-Year

Nationwide Population-Based Cohort Study, Cerebrovasc Dis 2013, 36, 145151.

11. Deepak K Aneja, Poonam Lohan, Sanjiv Arora, Chetan Sharma, Kamal R

Aneja and Om Prakash (2011), Synthesis of new pyrazolyl-2, 4-thiazolidinediones

as antibacterial and antifungal agents, Aneja et al. Organic and Medicinal

Chemistry Letters 2011, 1-15.

12. Douty Brent, Wayland Brian, Ala Paul J., et al. (2008), Isothiazolidinone

inhibitors of PTP1B containing imidazoles and imidazolines, Bioorganic &

medicinal chemistry letters, 18(1), 66-71.

13. orman L.M., Simmons, D.A.; Diamond, R.H. (2000), Hepatic failure in a

patient taking rosiglitazone, Ann. Intern. Med. 2000, 132, 118-121.

14. Hastings K.L., D.A.B.T, P.H., Thiazolidinedione Hepatotoxicity: The

PPAR Paradigm, Office of new Drugs, Center for Drug Evaluation and Research,

U.S.Food and Drug Administration

15. Joshi K.C. (1968), 2,4-thiazolidindione, Indian J.Chem, 5(4), pp. 139-141

16. Koffarnus RL, Wargo KA, Phillippe HM, Rivoglitazone: a new

thiazolidinedione for the treatment of type 2 diabetes mellitus, Ann Pharmacother,

2013 Jun, 47(6), 877-85.

17. Lipinski C.A., et al., (2001), Experimental and computational approaches to

estimate solubility and permeability in drug discovery and development

settings, Advanced Drug Delivery Reviews , 46(3-26), 3-26.

18. Mameli E. Zorgi L. (1954), Amidine derivaties of thiazolidin, Il Farmaco,

9, 691-704.

19. Mauricio J. Reginato and Mitchell A. Lazar, Mechanisms by which

Thiazolidinediones Enhance nsulin Action, TEM Vol. 10, No. 1, 1999

20. Neha Saraf, Promod K. Sharma, Sambhu C. Mondal, Vipin K. Garg and

Avnesh K. Singh (2011), Role of PPARg2 transcription factor in

38

thiazolidinedione-induced insulin sensitization, Royal Pharmaceutical Society

2012 Journal of Pharmacy and Pharmacology, 64, 161171.

21. Patil Vijay, Tilekar Kalpana, Mehendale-Munj Sonali, et al. (2010),

Synthesis and primary cytotoxicity evaluation of new 5-benzylidene-2,4-

thiazolidinedione derivatives, European journal of medicinal chemistry, 45(10),

4539-4544.

22. Prajwal L. Lobo, Boja Poojary, Manjunatha K, Prathibha A, N. Suchetha

Kumari (2012), Novel thiazolidine-2,4-dione mannich bases: Synthesis,

characterization and antimicrobial activity, Der Pharma Chemica, 4(3), 867-871.

23. Santos L.C., Uchoa F.T., Canas A.R.P.A., Sousa I.A., Moura R.O., Lima

M.C.A., Galdino S.L., Pitta I.R., Barbe J. (2011), Synthesis and anti-inflammatory

activity of new thiazolidine-2,4-diones, 4-thioxothiazolidinones and 2-

thioxoimidazolidinones, Heterocyclic Communications, Volume 11, issue 2.

24. Schoonjans Kristina, Staels Bart, Auwerx Johan (1996), Role of the

peroxisome proliferator-activated receptor (PPAR) in mediating the effects of

fibrates and fatty acids on gene expression, Journal of lipid research, 37(5), 907-

925.

25. Seely B Lynn, Staubs Patricia A., Reichart Donna R., et al. (1996), Protein

tyrosine phosphatase 1B interacts with the activated insulin receptor, Diabetes,

45(10), 1379-1385.

26. Shubhanjali Shukla, Pankaj Kumar, Nirupam Das, N.S.Hari Narayana

Moorthy, Sushant Kumar Shrivastava, Piyush Trivedi and Radhey Shyam

Srivastava (2012), Synthesis, Characterization, Biological evaluation and docking

of Coumarin couple Thiazolidinedione derivatives and t bioisosteres as PPAR

agonists, Medicinal Chemistry, 8, 834-845.

27. Steven R. LaPlante, Franois Bilodeau, Norman Aubry, James R. Gillard,

Jeff Meara, Ren Coulombe (2013), N- versus O-alkylation: Utilizing NMR

methods to establish reliable primary structure determinations for drug discovery,

Bioorganic & Medicinal Chemistry Letters, 23, 46634668.

39

28. Surendra Nath Pandeya, Sivacumar Smitha, Mayank Jyoti, Sesaiah Krishnan

Sridhar (2005), Biological activities of isatin and its derivatives, Acta Pharm, 55,

2746.

29. Unlusoy M.C., Dundar O.B., Atlantar N., Ertan R. (2006), Synthesis and

antimicrobial activity of some new 3-substituted benzyl-5-(4-chloro-2-piperidin-1-

yl-thiazole-5-yl-methylene)thiazoidine-2,4dione derivaties, Turk.J.Chem, 30, 355-

360.

40

PH LC

Ph lc 1 Ph khi MS ca cht 3a

Ph lc 2 Ph khi MS ca cht 3b

Ph lc 3 Ph khi MS ca cht 3c

Ph lc 4 Ph khi MS ca cht 3d

Ph lc 5 Ph hng ngoi IR ca cht 3a

Ph lc 6 Ph hng ngoi IR ca cht 3b

Ph lc 7 Ph hng ngoi IR ca cht 3c

Ph lc 8 Ph hng ngoi IR ca cht 3d

Ph lc 9 Ph H1-NMR ca cht 3a

Ph lc 10 Ph H1-NMR m rng ca cht 3a

Ph lc 11 Ph H1-NMR ca cht 3b

Ph lc 12 Ph H1-NMR m rng ca cht 3b

Ph lc 13 Ph H1-NMR ca cht 3c

Ph lc 14 Ph H1-NMR m rng ca cht 3c

Ph lc 15 Ph H1-NMR ca cht 3d

Ph lc 16 Ph H1-NMR m rng ca cht 3d

Ph lc 17 Ph H1-NMR m rng ca cht 3d

Ph lc 18 Ph C13

-NMR ca cht 3a

Ph lc 19 Ph C13

-NMR m rng ca cht 3a

Ph lc 20 Ph C13

-NMR ca cht 3b

Ph lc 21 Ph C13

-NMR m rng ca cht 3b

Ph lc 22 Ph C13

-NMR ca cht 3c

Ph lc 23 Ph C13

-NMR m rng ca cht 3c

Ph lc 24 Ph C13

-NMR ca cht 3d

Ph lc 25 Ph C13

-NMR m rng ca cht 3d

Ph lc 26 Ph C13

-NMR m rng ca cht 3d

41

Ph lc 1. Ph khi MS ca cht 3a

42

Ph lc 2. Ph khi MS ca cht 3b

43

Ph lc 3. Ph khi MS ca cht 3c

44

Ph lc 4. Ph khi MS ca cht 3d

45

Ten may : GX-PerkinElmer-USA Nguoi do: Nguyen Thi Son DT: 0912140352

Mail: sonhuco@yahoo.comResolution: 4cm-1

BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN

14BDate: 5/3/2013

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 500 .0

0 .0

10

20

30

40

50

60

70

80

90

99.3

cm-1

%T

3587

3444

3060

1730

1667

1614

1591

1515

1485

1433

1371

1341

1281

1238

1176

1156

1132

1096

1046

887

832

798

745

716

611

533

Ph lc 5. Ph hng ngoi IR ca cht 3a

46

Ten may : GX-PerkinElmer-USA Nguoi do: Nguyen Thi Son DT: 0912140352

Mail: sonhuco@yahoo.comResolution: 4cm-1

BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN

MAU 14DDate: 3/13/2013

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 .0

0 .0

10

20

30

40

50

60

70

80

90

101 .5

cm-1

%T

3643

3176

2925

2852

1740

1670

1602

1579

1514

1472

1442

1396

1373

1338

1285

1250

1177

1152

1126

1097

1043

905

867

832

741

706

689

Ph lc 6. Ph hng ngoi IR ca cht 3b

47

Ten may : GX-PerkinElmer-USA Resolution: 4cm-1

BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN

Nguoi do: Phan Thi Tuyet MaiTen mau: 9CDate: 4/1/2013

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 .0

50.0

52

54

56

58

60

62

64

66

68

70

72

74

76

78

80

82

84

86

88

90

92

94

96

97.4

cm-1

%T

3535

3290

3110

1732

1677

1601

1579

1532

1514

1439

1385

1356

1305

1279

1203

1160

1120

1098

1037

1010

944

905

859

830

740

687

636

Ph lc 7. Ph hng ngoi IR ca cht 3c

48

Ten may : GX-PerkinElmer-USA Nguoi do: Nguyen Thi Son DT: 0912140352

Mail: sonhuco@yahoo.comResolution: 4cm-1

BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN

DUNG-12CDate: 5/3/2013

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 500 .0

0 .0

10

20

30

40

50

60

70

80

90

97.1

cm-1

%T

3443

3268

3100

2942

1745

1676

1603

1582

1518

1434

1385

1352

1313

1288

1218

1168

1120

1074

905

858

827

741

687

669

559

5312862

Ph lc 8. Ph hng ngoi IR ca cht 3d

49

Ph lc 9. Ph 1H-NMR ca cht 3a

50

Ph lc 10. Ph 1H-NMR m rng ca cht 3a

51

Ph lc 11. Ph 1H-NMR ca cht 3b

52

Ph lc 12. Ph 1H-NMR m rng ca cht 3b

53

Ph lc 13. Ph 1H-NMR ca cht 3c

54

Ph lc 14. Ph 1H-NMR m rng ca cht 3c

55

Ph lc 15. Ph 1H-NMR ca cht 3d

56

Ph lc 16. Ph 1H-NMR m rng ca cht 3d

57

Ph lc 17. Ph 1H-NMR m rng ca cht 3d

58

Ph lc 18. Ph 13C-NMR ca cht 3a

59

Ph lc 19. Ph 13C-NMR m rng ca cht 3a

60

Ph lc 20. Ph 13C-NMR ca cht 3b

61

Ph lc 21. Ph 13

C-NMR m rng ca cht 3b

62

Ph lc 22. Ph 13C-NMR ca cht 3c

63

Ph lc 23. Ph 13C-NMR m rng ca cht 3c

64

Ph lc 24. Ph 13C-NMR ca cht 3d

65

Ph lc 25. Ph 13C-NMR m rng ca cht 3d

66

Ph lc 26. Ph 13C-NMR m rng ca cht 3d