Tong hop va thu* hoat tinh khang te bao ung thu*
cua mot so dan chat 5-halogenoisatin
Nguyen Le Hien', Nguyen Quang Dat' Tran Viet Hung' Bo mon Hoa Hiru ca - Trumg Dgi hoc Duac Hd Noi
Dat van de
Cac d i n chit isatin la mpt day c h i t hifu co da dygc quan tam nghien ciru ve hoa hpe va tac dung sinh hpe. Nhiiu edng trinh nghien eifu da cdng bo cho thiy cac d i n c h i t cua Isatin cd hoat tinh sinh hpc phong phu da dang nhy eae tae dung: khang virus, khang lao, khang khuin, khang n i m , chong ung thy, ifc c h i monoaminooxydase '^-^'•''-''°\
Trong cac thdng bao trydc day [7-10] chiing tdi da trinh bay k i t qua nghien eifu tong hgp va thCf hoat tinh sinh hpe cua mdt sd d i n c h i t isatin. Oe tiip tuc va phat trien hydng nghien ciru v i day chit nay, trong bai bao nay, chiing tdi trinh bay k i t qua tong hgp va thy hoat tinh khang te bao ung thy cua 5-fluoroisatin, 5-cloroisatin va cac d i n chit cua chung.
Dieu kien va thiPC nghiem
D i i u kien SKLM dygc thyc hien tren ban mdng silicagel Kieselgel 6OF254 (Merck). Diem chay dygc ghi tren may Electrothermal digital.
Pho IR dygc ghi tren may Perkin Elmer. Pho UV dygc ghi tren may Gary I E UV -Visible spectrophoto-meter varian. Pho MS dygc ghi tren cac may HP - 989B - MS, LC-MSD-Trap - SL va tren may Autospee Premier. Pho ' H - N M R va '^C-NMR dygc ghi tren may Avance 500 MHz
<Bruker).
Qui trinh chung tdng h o p hai c h i t (1) va (2) Theo tai lieu tham khao '^l
Qui trinh chung tong h a p hai c h i t ngu'ng tu (3) va (4)
Cho vao binh c i u 0,05mol c h i t 1 (hoac ehit 2), 0,15mol natri acetat khan, 0,05mol thiazolidin- 2,4-dion va 100ml acid acetic bang. Dun hdi lyu
each cat. Hdn hgp sdi d 117 - 121 0 thi b i t d i u tinh gid. Theo ddi qua trinh tien trien phan ifng bing SKLM vdi he dung mdi CHCl3:MeOH (13:1) de xac d|nh thdi gian phan ifng toi y u . De ngudi, cho k i t tinh sau dd dem lpc hiit chan khdng, rya bing nyde da d i n khi h i t acid. S i y khd. K i t tinh lai trong ethanol : DMF (1: 1). K i t qua tong hgp dygc ghi d bang 1.
Qui trinh chung tdng h a p cac base Mannich
Cho vao binh c i u 0,002mol c h i t 3 (hoac c h i t 4), 20ml ethanol, khuiy trdn d i u . Cho t i i p 0,0045mol hgp phin amin, 0,006mol formaldehyd (0,5ml formol). Khuiy d i u 0 nhiet dp phdng trong 2 gid ddi vdi c h i t (5, 6, 9, 10) va khuiy 0 nhiet dp phdng trong 1 gid d i u , d 50°C trong vdng 1 gid sau ddi vdi c h i t (7, 8). Theo ddi phan ifng bing SKLM vdi he dung mdi CHCl3:MeOH (13:1) de xac dinh thdi gian phan ifng toi yu. De k i t tua, Ipe hiit chan khdng; rCra tua bing ethanol lanh. S i y khd. K i t tinh lai trong ethanol. K i t qua tong hgp dygc ghi 0 bang 1.
Phu'O'ng phap thiF hoat tinh khang t l bao ung thu"
Phyong phap thy nghiem hoat tinh khang t i bao ung thy dygc t i i n hanh theo md hinh thy nghiem cua Vien nghien cifu ung thy Qude gia cua My (NCI) theo phyong phap cua Likhiwitayaw/uid '®' va dygc thyc hien tai phdng sinh hpc thyc nghiem (Vien hda hpc cae hgp chit thien nhien - Vien khoa hpe va Cdng nghe Viet Nam).
Chiing tdi da tiin hanh thCf kha nang gay dpe t i bao cua 6 c h i t ( 1 , 2, 3, 4, 6, 7) doi vdi cac ddng te bao ung thy gan ngydi (Hep-2) va ddng t i bao ung thy mang tim ngydi (RD).
Ket qua va ban luan
Tdng hop hda hoc: Cac d i n c h i t 5-halogenoisatin d y g c tong hgp theo s o do phan irng sau day:
-r CbCCH(OH); - H;N-0H.HC1
C H = N - O H
70 - 80'C
(3) X = F (4) X = C1
( 5 - 8 )
Trong 36 (5) x = F . — N ;
( 6 ) X = F . — N ;
(7) X = CI.—N-;
( 9 - 1 0 )
o
• — N O
(8) X = C1 .—N'C - — N >
(9) X - F . Ar = <, ^—CH,
( 1 0 ) X - F . A r - <, ^ V
Tong hgp 5-fluoroisatin (1) va 5-eloroisatin (2) bing phan irng Sandmayer nam 1919 gdm 2 byde: Byde 1: Cho p-fluoroanilin/p-cloroanilin phan ih-ng vdi cloralhydrat va hydroxylamin trong mdi tOfdng acid thu dygc p-fluoroisonitroso acetanilid va p-cloroisonitroso acetanilid vdi hieu suit 90,6%
va 83,1%. Byoe 2: Ddng vdng eac isonitrosoacetanilid dydi tac dung cua H2SO4 dac g 80°C thu d y g c 5-fluoroisatin va 5- eloroisatin vdi hieu s u i t 73.5% va 68%.
Ngyng tu 5-fluoroisatin va 5-cloroisatin vdi thiazolidin-2.4-dion trong dung mdi acid acetic bang va xuc tac natri acetat khan, thu dygc hai c h i t (3) va (4). Thyc hien phan ifng Mannich ddi
vdi hai chat (3) va (4) da thu d y g c 4 d i n chat base dlMannich (5 ^ 8) va d i n c h i t base monoMannich (9, 10). Cac phan ifng Mannich dygc thifc hien trong dung mdi ethanol, d nhiet dp thich hgp (nhiet dp phdng vdi 4 c h i t 5,6,9,10 va nhiet dp 50°C vdi 2 c h i t (7, 8). Qua trinh phan ifng dygc theo doi b i n g s i c ky Idp mdng de xac d|nh thdi gian phan irng thieh hgp. K i t qua tong hgp 10 ehit (1 •;• 10) vdi cac sd lieu ghi d" bang 1.
Op tinh khiit cua cae c h i t tong hgp dygc da dygc kiem tra bing SKLM. C i u triic cua 10 c h i t (1 •;- 10) dygc xac nhan qua phan tieh pho IR, MS. H ' - N M R , ' ^ C - N M R . Sd lieu phan tieh pho dygc ghi d cac bang 2, 3, 4.
Pho IR cua 10 ehit (1+10) d i u cd dai h i p thu eua cae nhdm >C=0 (1763-1736em"'' va 1709-1676 cm'''), lien k i t C=C cua nhan benzen (1627-1607 cm"\ 1593-1542 cm"'; 1489-1464cm"''). Pho IR cua 6 chit (1, 3, 5, 8, 9, 10) cd dai h i p thu cua lien k i t Carom-F (1193-1165cm"'). Pho IR cua 4 ehit (2, 4, 7, 8) ed dai h i p thu cua lien k i t Carom-CI (1066-1042 cm"'). Pho IR cua cac chit (1, 2, 3, 4) cd dai h i p thu cua NH amid va NH imid eua nhan isatin va nhan thiazolidin-2,4-dion (3217-3179 cm"''), trong khi dd cac d i n chit base di-Mannich (5, 8, 7, 8) khdng ed ea hai dai h i p thu nay va xuit hien dai hip thu eua nhdm >CH2 (2965-2934 em"'' va 2861- 2850em"'). Pho IR eua hai d i n chit base mono- Mannich (9, 10) cd dai h i p thu -NH- d' hgp phin amin thom (3360-3349cm"'') va -NH- amid (3210- 3150em"'') va dai h i p thu cua nhdm >CH2 ( 2959- 2947em"' va 2864-2852 cm"^).
Pho khoi lygng cCia 10 chit tong hgp d i u cd pic phan tif phil hgp vdi edng thifc chit dy kien va tren pho do MS cd cae pic cua eae ion manh dae tryng phil hgp vdi so do phan manh. Bon chit (2, 4, 7, 8) cd chifa nguyen tCf 01 trong phan tCf deu cd cac pic dong vj di kem pic phan tCf va eac pic eua ion manh chifa Cl vdi so khoi Id'n hon 2 va ty le cydng dp (3:1) cua hai pic tyong irng phii hgp vdi ty le ddng vi ^''CI/^^CI trong ty nhien la 32,5%.
Pho ' ' H - N M R cua 8 c h i t tong hgp dygc (1 -s- 4, 6, 8, 9, 10) cd d i y du tin hieu eua eae proton cd
trong phan ty. Hai ehit 5-fluoroisatin (1) va 5- eloroisatin (2) ed pie ed 5 la 11,02ppm (ehit 1) va 11,12ppm (chit 2) cua proton >NH amid d vdng isatin. Hai ehit ngyng tu (3) va (4) cd eae cap pie cd 5 la 12,84ppm (ehit 3) va 12,86ppm (ehit 4) eua proton >NH imid d' vdng thiazolidin-2,4-dion va 11,16ppm (ehit 3) va 11,26ppm (ehit 4) cua proton >NH- amid d' vdng isatin. Hai c h i t (6,8) khdng cd cac pic cCia proton NH- amid d vdng isatin v^ proton >NH imid cua vdng thiazolidin-2,4- dion da neu tren, dong thdi ed eac pic cua 2 proton cua nhdm -CH2-N(3) la 4,62ppm (ehit 6 va chit 8) va eua 2 proton cua nhdm -CH2-N(r) la 4,50ppm (chit 6 va c h i t 8). Sd lieu pho ^H-NMR neu tren cua 2 chit (6 va 8) da gdp phin xac nhan e i u triic base di-Mannich cua 2 c h i t nay diing nhy dy kiin. Hai chit (9,10) cd pic eua proton >NH amid d vdng isatin vdi 5 la 11,20ppm (ehit 9) va 11,23ppm (chit 10) va khdng ed pie cua proton NH-imid d vdng thiazolidin-2,4-dion, dong thdi cd pic cua 2 proton eua nhdm CH2-N(3) vdi 5 la 5,10 ppm (ehit 9 va ehit 10) va cd pic eua proton cua NH-amin thom vd'i 5 la 8,56 ppm (chit 9) va 8,58 ppm (chit 10). Sd lieu pho ^H-NMR neu tren da gdp phin chifng minh e i u triic base mono- Mannich cua 2 c h i t 9, 10. Pho ^^C-NMR cua 3 chit dai dien (3, 4, 6) eho thiy 3 ehit nay ed so carbon va dp dieh ehuyen hoa hpe 5 eua eae vi tri carbon phu hgp vdi cdng thirc dy kiin.
Bang
C h i t
1 2 3 4
5 6 7 8 9 10
1 : Ket qua tdng hgp cic
CTPT
C8H4O2NF C8H4O2NCI C11H5O3N2FS C11H5O3N2CIS C21H23O5N4FS C23H27O3N4FS C21H23O5N4CIS C23H27O3N4CIS C19H14O3N3FS C18H11O3N3F2S
KLPT
165 181,5
264 280,5
462 458 478,5 474,5 383 387
Chit (1 - 10) Thd'i gian phan Lpng
(gio-) 1 1 6 6 2 2 2 2 2 2
Nhiet dp phan y n g
t°C) 80 80 1 1 7 - 1 2 1 1 1 7 - 1 2 1
25 25 50 50 25 25
Dung moi tinh lai EtOH EtOH
k§t
EtOH : DMF(1:1) EtOH : DMF
EtOH EtOH EtOH EtOH EtOH EtOH
(1:1)
Diem chay
("O
226 - 228 252 - 254 327 - 329 366 - 367 1 9 6 - 1 9 8 . 1 6 4 - 165
1 9 4 - 195 1 7 9 - 180 1 1 5 - 118 1 6 6 - 1 6 8
Hieu suat
(%)
73,5 68,0 81,4 59,9 60,8 66,3 70,0 80,0 66,6 74,9
Bang 2: Sd lieu phd UV, IR va MS cua cac chit tdng hgp dwgc Pho UV Pho IR (KBr), cm"^
I'JOIf
Chat Xmax ( n m
v N H V C H 2 , C H 3 V C = 0 v C ^ ^ Carom v C a r o m - F v C a r o m - C I
Pho khoi (M^)
1 206;245;294;429 3212 1737 1618 1193 165 1489
2 212;248;295;425 3202 1763 1619 1061 181 1709 1472
3 261;279;366 3217 1763 1620 1189 264 1694 1589
4 270:280:368 3179 1742 1617 1066 281 1676 1542
5 261;280;373 2965 1742 1618 1165 463 2859 1681 1594
6 280:371 2934 1740 1616 1176 458 2850 1682 1468
7 271:281:368 2957 1740 1607 1050 481 2861 1682 1464
8 209:281 ;372 2937 1743 1609 1042 477
• 2851 1682 1457
9 244;280;368 3349 2974 1736 1614 1192 382 3150 2864 1681 1590
10 236:261 ;280;368 3360 2959 1739 1627 1193 386 3210 2852 1670 1593
Bang 3: Sd lieu phd ^H-NMR vi ^^C-NMR ciia mot sd chit tdng hgp dwgc
Chat ^H-NMR (DMSO-de), 5(ppm)
1 11,02(s, I H , H-1); 7,42(m, I H , H-7); 7,37(m, I H , H-4); 6,90(q, I H , H-6) 2 11,12 (s, I H , H-1); 7,60 (q, I H , H-7); 7,53 (d, I H , H-4); 6,91 (d, I H , H-6)
3 12,84 (s, I H , H-3); 11,16 (s, I H , H-1'); 8,49 (q, I H , H-7'); 7,20 (m, I H , H-4'); 6,90 (q, I H , H-6') 4 12,86 (s, I H , H-3); 11,26 (s, I H , H-l'); 8,71 (s, I H , H-7'); 7,38 (m, I H , H-4'); 6,90 (q, I H , H-6') g 8,74 (q, 1H, H-7'); 7,22 (m, 1H, H-4'); 6,91 (q, 1 H,H-6'); 4,62 (s, 2H, CH2-N(3)); 4,50 (s, 2H, CH2-
N(i ));2,49 (m, 8H, H-2",6",2"', 6'"); 1,45 (m, 8H, H-3",5",3"',5"'); 1,32 (m, 4H, H-4",4"')
„ 8,99 (q, I H , H-7'); 7,44 (m, I H , H-4'); 7,21 (q, I H , H-6'), 4,62 (s, 2H, CH2-N(3));4,50 (s, 2H, CH2-N(r));
2,50 (m, 8H, H-2",6",2"',6"'); 1,46 (m, 8H, H-3",5",3"', 5"'); 1,34 (m, 4H, H-4", 4"')
g 11,20(d, I H , H-1'); 8,56 (m, I H , HN-Ar); 7,94 (s, I H , H-7'); 7,26 (m, I H , H-4'); 6,90 (m, I H , H- 6'); 6 , 5 2 - 6 , 7 3 (m, 4H, Ar-H); 5,10 (d, 2H, CH2-N); 2,12 (m, 3H, CH3)
10 11,23 (d, I H , H-1'); 8,58 (d, I H , HN-Ar); 8,50 (d, I H , H-7'); 7,24 (m, I H , H-4'); 7,05 (m, I H , H- 6'); 6,81 - 6 , 9 9 (m, 4H, Ar-H); 5,10 (d, 2H, CH2-N)
Chat ^'C-NMR (DMSO-ds), 8(ppm)
3 170, 53 (C-4); 168,22 (C-2); 167,45 (C-2'); 158,37 (C-5'); 156,51 (C-3'); 140,04 (C-7'a); 133,94
(C-5); 120,47 (C-3'a); 118,60(0-7'); 114,31 (C-6'); 111,06 (C-4') ^ ^ . 170, 47 (C-4); 167,98 (C-2); 167,38 (C-2'); 142,32 (C-7'a); 134,18 (C-5'); 131,60 (C-7'); 127,10
(C-6'); 125,79 (C-3'); 125,06(0-5); 121,07 (C-3'a); 111,69(0-4')
167,97 (C-2); 162,28 (C-2'); 158,74 (C-5'); 156,87 (C-3'); 140,71 (C-7'a); 120,31 (C-7'); 130,96 6 (C-6'); 111,16 (C-4'); 64,25 (N(3)-CH2); 62,45 (N'dj-CHz): 51,36 (C2-,6); 51,10 (Cj-.e-); 25,43
(C3-,5-); 25,26 (C3-,5-); 23,18 (C4); 22,19 (C4O
Kit qua thu" hoat tinh khang t§ bao ung thu" (4) cd t^c dung khang ca hai ddng t i bao ung thy K i t qua thy hoat tinh khang t i bao ung thy thy nghiem. Cae ehit (2, 3, 6) d i u ed tac dung dygc trinh bay d bang 4. K i t qua cho t h i y c h i t khang ddng te bao ung thy gan.
Bang 4: Kit qua thOhoat tinh khing ti bio ung thw ngwoi
Chat
Chifng (+) 1 2 3 4 6 7
Gia trj IC50 (ng/ml) Dong t l bao
0,25
> 5 4,88 4,31 2,18 4,95
> 5 Hep-2
Ket luan Dong t l bao RD
0,20
> 5
> 5
> 5 1,79
> 5
> 5
Dyong tinh Am tinh
Dyong tinh vol 1 dong t l bao Dyong tinh voi 1 dong te bao Dyong tinh voi 2 dong t l bao Dyong tinh vd'i 1 dong t l bao
Am tinh
Nhan xet v§ tuxyng quan ciu true - tac dung Tif k i t qua thCf nghiem cho thiy c i u tao khung phan ty va cae nhdm t h i ed anh hydng Idn doi vdi tac dung khang ddng te bao ung thy 0 ngydi cua cac d i n chit 5- halogenoisatin tong hgp dygc. Chit (1) khdng ed tae dung vdi ea hai ddng t i bao, chit (2) cd tac dung tren ddng t i bao ung thy gan (Hep-2). Nhy vay, nhdm t h i -01 da gdp phin mang lai hoat tinh nay d ehit (2).
Mat khae, vdng thiazolidin-2,4-dion trong phan tCf cSe ehit ngyng tu ed anh hydng dang ke d i n hoat tinh khang t i bao ung thy so vdi c h i t ban d i u (1,2): vdi sy ed mat eua vdng nay, c h i t (3) da cd tae dung khang ddng te bao ung thy gan, chit (4) cd tac dung khang ca hai ddng te bao thy nghiem va tae dung vd'i ddng te bao ung thy gan manh hon h i n c h i t (2). Trong khi dd, cac nhdm t h i base Mannich (morpholinomethyl va piperidinomethyl) trong phan tCf eae ehit (6, 7) lai lam giam hoat tinh khang te bao ung thy so vd'i chit ban d i u la hai chat ngyng tu (3, 4)..
Ket luan
Da tong hgp d y g c 10 d i n c h i t 5- halogenoisatin, trong dd cd 8 c h i t ( 3 ^ 1 0 ) chya thiy edng bo trong eae tai lieu ma ehiing tdi tham khao dygc.
K i t qua thy tac dung khang te bao ung thy eho thay cd 4 c h i t ed tac dung khang t i bao ung
thy 0 ngydi, trong dd ehit (4) cd tac dung khang ea hai ddng t i bao ung thy gan (Hep-2) va ung thy mang tim (RD). Cae chat (2, 3, 6) cd tac dung khSng ddng t i bao ung thy gan (Hep-2).
Lai cam an
Odng trinh nay la k i t qua eua de tai nghien eifu CO ban do Bd Khoa hpe va Cdng nghe tai trg. Cac tac gia xin chan thanh cam on!
Summary
5-Fluoroisatin (1) and 5-cloroisatin (2) have been prepared by Sandmayer reaction in 1919.
By condensation of (1) and (2) with thiazolldin- 2,4-dion, two compounds (3, 4) were obtained.
These hvo compounds (3, 4) underwent Mannich reaction to give six Mannich base derivatives (5 •;•
10). The structures of the synthesized compounds were confirmed by IR, UV, ^H-NMR,
^^C-NMR and MS. On biological activity screening one compounds (4) exhibited a high cytotoxic activity on Hep-2 and RD cells, the other three compounds (2, 3, 6) exhibited a high cytotoxic activity on Hep-2 cells.
Keywords: Isatin, 5-halogenoisatin, Mannich bases, cytotoxic activity.
