Tổng hợp và thử hoạt tính kháng tế bào ung thư của 5 -(4''-bromobenzyliden) hydantoin và một số dẫn chất base mannich.pdf

B Y T

TRNG I HC DC H NI

NGUYN TIN HIP

TNG HP V TH HOT TNH

KHNG T BO UNG TH CA 5-(4-

BROMOBEZYLIDEN)HYDANTOIN V

MT S DN CHT BASE MANNICH

KHA LUN TT NGHIP DC S

H NI - 2014

B Y T

TRNG I HC DC H NI

NGUYN TIN HIP

TNG HP V TH HOT TNH

KHNG T BO UNG TH CA 5-(4-

BROMOBEZYLIDEN)HYDANTOIN V

MT S DN CHT BASE MANNICH

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. TS. V Trn Anh

2. PGS.TS inh Th Thanh Hi

Ni thc hin: B mn Ha hu c

H NI 2014

LI CM N

Li u tin, ti xin by t lng knh trng v bit n su sc ti TS.V

Trn Anh, PGS.TS inh Th Thanh Hi, PGS. TS Nguyn Quang t l

ngi thy tn tnh hng dn, to mi iu kin thun li v ng vin ti

hon thnh kha lun ny.

Ti cng xin chn thnh cm n s gip nhit tnh ca : Ths ng V

Lng (phng NMR-Vin Ha Hc v Cng Ngh Vit Nam), Nguyn Th

Sn (b mn Ha vt liu- Khoa Ha Trng i hc Khoa hc T nhin),

TS. Trn Th Nh Hng (Vin Ha hc cc hp cht thin nhin) v ton th

cc thy c gio, cn b k thut vin ca B mn Ha hu c, cc thy c

gio trong trng, cc phng ban, th vin-Trng i hc Dc H Ni.

Cui cng ti, xin cm n gia nh v bn b lun gip v ng vin

ti trong sut qu trnh hc tp v nghin cu va qua.

H Ni, Ngy 13 thng 5 nm 2014

Sinh vin

Nguyn Tin Hip

MC LC

DANH MC CC K HIU, CH VIT TT

DANH MC CC BNG, BIU

DANH MC CC HNH V, TH

T VN

CHNG 1.TNG QUAN

1.1 TC DNG SINH HC CA CC DN CHT HYDANTOIN ..... 2

1.1.1 Tc dng chng co git ........................................................................... 2

1.1.2 Tc dng khng khun, khng nm ........................................................ 3

1.1.3 Tc dng chng ung th .......................................................................... 4

1.2 CC PHNG PHP TNG HP 5-(4'-

BROMOBENZYLIDEN)HYDANTOIN V CC BASE MANNICH .9

1.2.1 Phn ng tng hp 5-arylidenhydantoin .............................................. .10

1.2.2 Phn ng tng hp dn cht base Mannich.13

CHNG 2. NGUYN VT LIU, TRANG THIT B V PHNG

PHP NGHIN CU

2.1 NGUYN VT LIU, HA CHT, DUNG MI ............................ 15

2.2 THIT B TH NGHIM .................................................................... 16

2.3 PHNG PHP NGHIN CU ........................................................ 16

2.3.1 Phng php tng hp 5-(4-bromobenzyliden)hydantoin v dn cht

base Mannich. .................................................................................................. 16

2.3.2 Phng php xc nh cu trc ........................................................... 17

2.2.3 Phng php th tc dng gy c t bo ung th. ............................. 17

CHNG 3. THC NGHIM V KT QU

3.1 TNG HP HA HC ........................................................................ 20

3.1.1 Tng hp 5-(4-bromobenzyliden)hydantoin (H0). .............................. 21

3.1.2 Tng hp cc dn cht base Mannich ca 5-(4-

bromobenzyliden)hydantoin (H1-H 6). ............................................................ 22

3.2 XC NH CU TRC CA CC CHT TNG HP C ... 27

3.2.1 Ph hng ngoi ...................................................................................... 27

3.2.2 Ph cng hng t ht nhn ................................................................. 27

3.2.3 Ph khi lng ...................................................................................... 27

3.3 TH HOT TNH GY C T BO UNG TH ........................ 27

CHNG 4. BN LUN

4.1 V TNG HP HA HC. ................................................................ 36

4.2 V XC NH CU TRC CA CC CHT TNG HP C.

4.2.1. V ph hng ngoi (IR)......................................................................... 36

4.2.2 V ph cng hng t ht nhn 1H-NMR ............................................ 37

4.2.3 V ph cng hng t ht nhn 13C-NMR ........................................... 37

KT LUN V KIN NGH

TI LIU THAM KHO

DANH MC PH LC

DANH MC CC K HIU, CH VIT TT

13C- NMR : Ph cng hng t ht nhn carbon 13

1H- NMR : Ph cng hng t ht nhn proton

A549 : T bo ung th phi ngi

Ac2O : Anhydrid acetic

AcOH : Acid acetic

DMF : Dimethylformamid

EGFR : Epidermal growth factor receptor

(th th ca yu t tng trng biu m)

EtOH : Ethanol

ESI : Phng php phun m in t

Hep- G2 : T bo ung th gan ngi

HMBC : Ph tng tc d ht nhn qua nhiu lin kt

(Heteronuclear Multiple Bond Connectivity)

HSQC : Ph tng tc d ht nhn qua mt lin kt

(Heteronuclear Single-Quantum coherence)

IC50 : Nng c ch 50%

IR : Ph hng ngoi

MCF7 : T bo ung th biu m v ngi

MeOH : Methanol

MS : Khi ph

(Mass spectrometry)

PC3 : T bo ung th tuyn tin lit

DANH MC CC BNG, BIU

BNG TRANG

Bng 3.1 kt qu tng hp 5-(4-bromobenzyliden)hydantoin v cc dn

cht base Mannich (H1-H6)

29

Bng 3.2 S liu ph IR v ph 1H-NMR ca cc cht tng hp c

(H0-H6)

30

Bng 3.3 S liu ph 13C-NMR ca cc cht tng hp c (H0-H6) 31

Bng 3.4 S liu ph ESI_MS ca cht H0 v cc dn cht base Mannich

(H1-H6)

32

Bng 3.5 Kt qu th tc dng ca cc cht tng hp c trn mt s

dng t bo ung th ngi (theo phng php SRB)

34

Bng 3.6 Kt qu xc nh gi tr IC50 (g/ml) ca mt s cht tng hp

c trn dng t bo Hep G2 (theo phng php SRB).

35

Bng 4.1 Cc tng tc 1H-13C ca 5-(4-bromobenzyliden)hydantoin 42

Bng 4.2 Cc tng tc 1H-13C ca 5-(4-bromobenzylden)-3-

pyrrolidinomethylhydantoin (H6)

45

DANH MC CC HNH V, TH

HNH

TRANG

Hnh 4.1.1. Ph tng tc HSQC ca cht H0

40

Hnh 4.2.1.Ph tng tc HMBC ca cht H0

41

Hnh 4.6.1. Ph HSQC ca hp cht H6

43

Hnh 4.6.2 Ph HMBC ca hp cht H6

44

1

T VN

Trong cuc chin chng li bnh tt, con ngi khng ngng tm ti, nghin

cu pht hin ngy cng nhiu loi thuc mi c hiu qu trong phng v

iu tr bnh. Thuc mi c tm ra c nhiu ngun gc khc nhau, trong

cc thuc c to ra bng phng php tng hp v bn tng hp ng vai

tr quan trng.

Trong lnh vc tng hp thuc, nhanh chng tao ra cc thuc mi, cc nh

nghin cu thng da trn cu trc ca cc cht ang c dng lm thuc

hoc cc cht c hot tnh sinh hc c trin vng to ra cc cht mi d

an c tc dng tt hn, t c tnh hn v c hiu qu hn trong iu tr

Cc dn cht ca hydantoin l dy cht c quan tm nghin cu v tng

hp ha hc, tc dng sinh hc v ng dng rng ri lm thuc. Mt s dn

cht hydantoin c dng rng ri trong lm sng nh phenytoin (thuc

chng ng kinh), nitrofurantoin (thuc khng khun), dantrium (thuc gin

c vn), nilutamid (thuc iu tr ung th tuyn tin lit di cn) [19].

Trong nhng nm qua, tm thy nhiu dn cht 5-arylidenhydantoin thin

nhin v tng hp c hot tnh khng khun, khng nm, v c tc dng

chng ung th rt ng quan tm [1], [5], [7], [8], [15], [20].

tip tc gp phn nghin cu, tm kim cc dn cht 5-arylidenhydantoin

c tim nng v hot tnh chng ung th, chng ti thc hin kha lun vi

cc mc tiu sau.

1. Tng hp 5-(4-bromobenzyliden)hydantoin v mt s dn cht base

Mannich.

2. Th sng lc hot tnh gy c t bo trn mt s dng t bo ung th

ngi ca cc cht tng hp c.

2

CHNG 1.TNG QUAN

1.1 TC DNG SINH HC CA CC DN CHT HYDANTOIN

Hydantoin (imidazolidin-2,4-dion) l hp cht hu c c A.Bayer

pht hin nm 1861[29] bng phn ng hydro phn (hydrogenolysis) allantoin

( cht c pht hin trong ti dch niu (the allantoin fluid) ca b ci),

l ngun gc tn hydantoin. Sau , nm 1864, A.Bayer [30] tng hp c

hydantoin vi nguyn liu u l bromoacetylure. Tuy nhin cu trc vng

ca hydantoin ch c xc dnh bi Strecker vo nm 1873 [31].

NH

NH

O

O

4

12

35

Hydantoin

Bn thn hydantoin khng th hin tc dng sinh hc, tuy nhin nhiu cng

trnh nghin cu tng hp v th tc dng sinh hc cho thy cc dn cht ca

hydantoin c tc dng phong ph v a dng nh thuc chng ng kinh,

khng khun, chng ung th [1], [3], [4], [8], [11],.... Cc dn xut th v tr

N1, N3, c bit th 2 ln v tr C5 c nghin cu v cho thy tc dng

sinh hc ng ch

1.1.1 Tc dng chng co git

Dn cht hydantoin c dng nhiu nht trong iu tr l 5,5-

diphenylhydantoin (phenytoin) (1), bit dc l Dilantin (bit dc ca

Parke-Davis), Epanutin (bit dc ca Pfizer) [3],[9].

NH

HN

O

O

1

3

Phenytoin c tc dng chng co git, thng c s dng trong iu tr bnh

ng kinh [3],[9]. Tuy nhin phenytoin cn c mt s tc dng ph nh gim

bch cu, gim tiu cu, bun nn, pht ban,... v th c nhiu nghin cu

nhm tng hp cc dn cht ca hydantoin c t tc dng ph hn nhng vn

duy tr c tc dng chng co git. V d, cc dn cht ca phenytoin c

tng hp gm ethotoin (3-ethyl -5-phenylhydantoin), mephenytoin (5-ethyl-3-

methyl-5-phenylhydantoin), 5-methyl-5-phenylhydantoin, v 5-pentyl-5-

phenylhydantoin . Cc cht c tng hp u cha khung hydantoin, cha

cc nhm th c kh nng to lin kt hydro (nhm C=O, nhm NH) v

phn khng k nc nh nhn thm lin kt vi C-5 [27].

1.1.2 Tc dng khng khun, khng nm

Nitrofurantoin (2) l dn cht hydantoin c dng iu tr nhim khun

ng tit niu t nm 1959 v cho n nay vn c s dng trong iu tr

[12].

OO2N

N N

NH

O

O

2

Mt dn cht ca nitrofurantoin th N-3 vi nhm hydroxymethyl l

Nifurtoinol [3-hydroxymethyl-1-(5-nitrofurfurylidenamino)hydantoin] (3)

cng c tc dng nh Nitrofurantoin v c p dng trong iu tr [17].

O

N

O

O

O2N

OH

3

Mt s dn cht base Mannich N-3 ca Nitrofurantoin (4) c tng hp

v th nghim c tc dng khng khun, khng nm tt, trong c cht

ang c dng lm thuc tng t nh Nitrofurantoin nhng bnh nhn d

4

dung np hn vi bit dc l Nifurfolin (1-(5nitro-2-furfurylidenamino)-3-

morpholinomethylhydantoin) (5) [17]

OO2N

N N

N

O

O (CH2)nNHR

NHR

-N(CH3)2

-N

-N O4

OO2NN N

N

O

O CH2 N O

5

Dn cht khc cng c tc dng khng khun, hin c s dng trong iu

tr nhim khun ng tit niu l Furazidin (1-{[3-(5-nitro-2-

furyl)allyliden]amino}hydantoin (6) [17].

HNN

O

O

NO

NO2

6

1.1.3 Tc dng chng ung th

Mt s dn cht hydantoin c ng dng trong iu tr ung th nh

l Nilutamid (7).

O2N

F

F

F N

NH

CH3CH3

O

O

7

Cht ny c tng hp t nm 1992, c ng dng trong iu tr ung th

tuyn tin lit di cn t nm 1996 [9],[17].

5

Mt s hp cht thin nhin c cu trc 5-arylidenhydantoin c phn

lp v tm thy c hot tnh c ch t bo ung th nh cc cht aplysinopsin

(8) v spongiacidin (9) t bt bin [16].

NH

N

N

CH3

O

O CH3

8

HN

HN

O

Br

R

NH

HN

O

O

9

Nhiu dy cht 5-arylidenhydantoin c tng hp v th nghim hot

tnh chng ung th nc ngoi cng nh Vit Nam.

Nm 2003, cc tc gi inh Th Thanh Hi, Nguyn Quang t, L Mai

Hng [6] cng nghin cu hot tnh khng hai dng t bo ung th

ngi (t bo ung th biu m (KB) v t bo ung th mng t cung (FL))

ca dy cht 5-(5-nitro-2-furfurliden)hydantoin v cc dn cht base

Mannich ca n. Kt qu l tm c hai cht l 5-(5-nitro-2-

furfuryliden)imidazolidin-2,4-dion (10) v 3-piperidinomethyl-5-(5-nitro-2-

furfuryliden)imidazolidin-2,4-dion (11) c hot tnh khng t bo ung th rt

mnh (gi tr IC50 l 0,2-1,04 (g/ml) i vi dng t bo FL v 0,6-1,15

(g/ml) i vi dng t bo KB).

NH

HN

O

O

O

NO2

10

NCH2N

NH

O

O

ONO2

11

Nm 2006, C.Carmi v cng s [13] nghin cu tng hp mt s dn

cht 1-phenyl-5-benzylidenhydantoin v th hot tnh c ch EGFR (receptor

ca yu t tng trng biu m) v chng tng sinh ca ung th biu m

ngi A431. Kt qu nghin cu ca tc gi Carmi cho thy mt s cht tng

6

hp c c tc dng c ch qu trnh phosphoryl ha ca EGFR, c tc dng

c ch s pht trin v tng sinh ca t bo ung th biu m A431 khi th

nng 20M. Cht c tc dng mnh nht l (E)-5-p-hydroxybenzyliden-1-

phenethylhydantoin (12) vi IC50=27M trn invivo.

Nm 2009, V.Zuliani v C.Carmi tip tc hng nghin cu cc dncht

5-benzylidenhydantoin c hot tnh c ch EGFR v hot tnh chng ung th,

tng hp v th nghim hot tnh chng tng sinh dng t bo ung th

phi A549 ca mt s cht [28]. Kt qu cho thy mt s dn cht 5-

benzyliden-1-phenethylhydantoin c hot tnh c ch tng sinh t bo nng

20M. cht c tc dng mnh nht (E)-5-p-hydroxybenzyliden-1-

phenethylhydantoin (12).

HO NH

N

OO

12

Cc tc gi gi thit c ch tc dng kp ca dy cht ny l do lin kt i

lin hp ngoi vng to cho nhn hydantoin c kh nng tc dng l

tng tc vi v tr hot ng ca EGFR v alkyl ha cc cht i nhn sinh

hc. Gi thuyt ny c ng h bi hot tnh gy c t bo ung th bit

ca nhiu hp cht thin nhin v tng hp c cha nhm carbonyl ,-khng

no [14].

Nm 2009, M.Mudit, M.Khanfar cng b cng trnh nghin cu v

pht minh, thit k v tng hp cc dn cht 5-benzylidenhydantoin c hot

tnh chng ung th di cn ly cm hng t hp cht thin nhin bin [21].

T loi bt bin Hemimycale Arabica (Mycalidae) bin Hng Hi, cc tc

gi phn lp c 3 cht bit l (Z)-5-(4-hydroxybenzyliden)hydantoin

7

(13), 5-(4-hydroxybenzyl)hydantoin (14), (Z)-5-(6-bromo-1H-indol-3-

yl)methylenhydantoin (15).

NHHN

O

O

HO

13

NHHN

O

O

HO

NHHN

O

O

HO

13

NHHN

O

O

HO

NHHN

O

O

HO

14

15

HNNH

O

O

NH

Br

Tip cc tc gi tng hp 39 dn cht 5-benzylidenhydantoin, trong

c 8 cht mi. Th nghim hot tnh chng ung th cho thy cht (13) t

nhin v cht tng hp (Z)-5-(4-ethylthio)benzylidenhydantoin (16) c hot

tnh mnh c ch s tng trng v s xm ln ca t bo ung th tuyn tin

lit trn invitro. V mi lin quan cu trc tc dng ca dy cht ny, cc tc

gi c nhn xt l yu t lp th nh hng n tc dng nhiu hn yu t

in t v cc cht c nhm th v tr para ca nhm benzyliden m c cha

cc nguyn t oxy, nit hoc lu hunh c hot tnh cao. Cc hp cht ny c

tnh cht nhn hydro, v th c th c tng tc lin kt hydro v tr para

NHHN

O

O

S

16

Nm 2010, tc gi V Trn Anh [1] nghin cu tng hp v th hot

tnh ca cc dn xut hydantoin 1725, 17(a-d)-25(a-d). Kt qu tng

hp c 43 dn cht ca hydantoin trong c 9 dn xut ca 5-

arylidenhydantoin v 34 dn cht base Mannich ca cc dn xut 5-

arylidenhydantoin. Tc gi th hot tnh khng t bo ung th ca 43 cht

tng hp c i vi 2 dng t bo ung th ngi l t bo ung th gan

Hep-G2 v t bo ung th phi LU. Kt qu cho thy trong s 43 cht tng

8

hp c c 4 dn cht 5-arylidenhydantoin v 9 dn cht base Mannich ca

chng c hot tnh khng t bo ung th gan ngi Hep-G2

(IC50=0,385g/ml). Cc dn cht base Mannich u c c im cu to

chung l cha nhm ht in t (-Cl, -NO2) trong hp phn aryliden.

CHNH

NHO

OR1 234

56

1'

2'3'

4'

5' 6'

17 25

R= H; 2' -OH; 4'-N(CH3)2; 4'-OH; 4'-F

2' -Cl; 4'-Cl; 3'-NO2; 4' -NO2

CHNH

NO

OR1 234

56

1'

2'3'

4'

5' 6'

CH2 NR1R2

17(a-d) - 25(a-d)

NR1

R2

N O (a) NR1

R2

N N-CH3(c)

NR1

R2

N (b) NR1

R2

NH CH3 (d)

Nm 2012, tc gi Th Thu Hng [7] tng hp v th hat tnh khng

t bo ung th ca 15 dn cht 5-halogenoarylidenhydantoin v base Mannich

18(a-d)-20(a-d). Kt qu cho thy 11 cht c hot tnh khng t bo ung th

i trng HTC116 vi IC50=10,17-19,23g/ml.

9

1 234

5

6

1'

2'3'

4'

5' 6'

R = F (18) ; Cl (19) ; Br (20).

18(a-d) - 20(a-d)

NR1

R2

N O (a) NR1

R2

N N (c)

NR1

R2

N (b) NR1

R2

NH (d)CH3

CH3

CHNH

NHO

O

R

1 234

5

6

1'

2'3'

4'

5' 6'

CHNH

NO

O

R

CH2NR1R2

Nm 2013, tc gi Trnh Vn Ninh [8] tng hp v th hot tnh khng

t bo ung th ca 13 dn cht 3-aryl-5-arylidenhydantoin . Kt qu cho thy

c 2 cht c hot tnh c ch t bo ung th i trng HTC116 vi IC50

=10,99 g/ml v IC50=15,25 /ml, v c 2 cht c hot tnh c ch t bo ung

th tuyn tin lit PC3 vi IC50=4,04 g/ml v IC50=2,01 g/ml.

O2N N

NH

O

OR

R = H (21) ; 4'-F (22) ; 3'-Cl(23) ; 4'-Cl (24) ; 2',6'-Cl2 (25) ; 2'-Br (26) ; 3'-Br (27) 4'-Br (28) ; 3'-NO2 (29) ; 2'-OH (30) ; 4'-OH (31).

(21-31)

1.2 CC PHNG PHP TNG HP 5-(4-

BROMOBENZYLIDEN)HYDANTOIN V CC BASE MANNICH

Hydantoin (imidazolidin-2,4-dion) c cng thc cu to nh sau:

10

NH

NH

O

O

4

12

35

T cu trc phn t hydantoin cho thy nhiu kh nng phn ng chuyn ha

to thnh nhiu dn cht khc nhau ca hydantoin.

1.2.1 Phn ng tng hp 5-arylidenhydantoin

S phn ng:

NH

NH

O

O

RCHO 1 234

5

NH

NH

O

O

12

345HC

OH

RNH

NH

O

O

12

34

5R+

R = phenyl, phenyl th, furyl, pyryl, pyridyl, quinolyl, hoc indolyl

Nm 1911, Wheeler v Hoffman l nhng ngi u tin nhn thy

hydantoin c th ngng t vi aldehyd thm to thnh dn cht c ni i gn

C-5 ca hydantoin [26]. Phn ng c thc hin trong acid acetic bng,

vi natri acetat khan, anhydrid acetic. Cc aldehyd thm c s dng l

benzaldehyd, 2-hydroxybenzaldehyd, furfural, vanillin,

NH

NH

O

O

Ar-CHO 1 234

5

NH

NH

O

O

12

345Ar+

-H2O

AcOH, AcONa

Phng php ca Wheeler v Hoffman c s dng tng hp nhiu dn

cht th C-5 ca hydantoin [26]. Mt nhc im ca phng php trn l

phi s dng mt lng ln acid acetic v natri acetat.

Nm 1982, tc gi A.Kleeman v cng s [16] tin hnh phn ng

ngng t hydantoin vi mt s aldehyd thm vi xc tc mui amoni ca cc

acid carboxylic trong acid acetic bng. Kt qu thu c so snh vi phng

php s dng xc tc natri acetat ca Wheeler v Hoffman cho hiu sut tt

hn hn. V d: hiu sut tng hp 5-benzylidenhydantoin t benzaldehyd v

11

hydantoin theo phng php Kleeman l 95% so vi 46% theo phng php

Wheeler v Hoffman.

Nm 1993, Marton, J. [20] s dng xc tc c tnh base mnh l

ethanolamin tin hnh phn ng ngng t aldehyd thm vi hydantoin

trong dung mi l hn hp nc:ethanol (1:1), phn ng c tin hnh

nhit hi lu trong 4 gi. Tc gi s dng mt s aldehyd thm (p-

clorobenzaldehyd, p-nitrobenzaldehyd, p-hydroxybenzaldehyd,), hiu sut

phn ng 44-89%. Nm 2004, tc gi Thenmozhiyal v cng s [24] s

dng xc tc ny trong iu ch cc phenylmethylenhydantoin v iu ch

c 54 dn cht.

S phn ng:

rt ngn thi gian phn ng, hin nay cc nh ha hc s dng vi sng

trong tng hp hu c. Nm 2006, tc gi Lamiri [17] s dng vi sng

trong tng hp 5-arylidenhydantoin. Hn hp natri acetat, oxyd nhm c

cho vo hn hp hydantoin v aldehyd trong dung mi CH2Cl2. Khuy hn

hp phn ng nhit phng, sau ct loi CH2Cl2, dng vi sng tn s

2405Hz, cng sut 1250W tc ng vo trong 2 pht.

RCHONH

NH

+O

O

Natri acetat/oxyd nhm

vi sng, 2 pht NH

NH

O

O

H

R

O H

R

+NH

NHO

O

NaHCO3/H2O-EtOH

Ethanolammin,t0 NH

NH

O

O

R

R = Alkyl, alkoxy (-OR), -NR, -OH, -X, -CN, -CF3, -NO2

12

Phn ng c hiu sut tng i cao (74-96%), vi cc aldehyd l

benzaldehyd, 0-clorobenzaldehyd, m-clorobenzaldehyd, p-

clorobenzaldehyd,....

V c ch phn ng ngng t

Phn ng ngng t gia hydantoin v aldehyd thm l phn ng ngng

t kiu aldol xy ra theo c ch sau [2]:

- Giai on cng hp:

Nhm methylen v tr 5 ca hydantoin rt hot ng, nguyn t H y d

dng tch khi carbon (khi c xc tc base) v anion (II) c to thnh l

mt tc nhn i nhn mnh:

NH

NH

O

O

+ B HCNH

NH

O

O

+ BH

Anion (II) tn cng vo carbon mang in tch dng phn C(+) ca nhm

carbonyl ca aldehyd to sn phm cng hp (IV):

HCNH

NH

O

O

Ar C

O

H(+)

(-)

+ NH

NH

O

O

H

CHAr

O

(III) (II) (IV)

Ion alcolat (IV) to thnh li ly mt proton ca BH, tr li xc tc B(-) :

NH

NH

O

O

H

HCAr

O

(IV)

BH NH

NH

O

O

H

HCAr

OH

(V)

+ B+

13

- Giai on ngng t loi nc:

1.2.2 Phn ng tng hp dn cht base Mannich

a) nh ngha phn ng Mannich.

Phn ng Mannich l phn ng aminomethyl ha cc hp cht hu c c

nguyn t H linh ng bng tc dng ca formaldehyd (hoc cc aldehyd

khc) v cc amin bc 1 hoc amin bc 2 (hoc NH3) to ra dn cht c nhm

th aminomethyl gi l base Mannich [2]. iu kin cn thit xy ra phn

ng l tnh i nhn ca amin phi mnh hn tnh i nhn ca hp cht cha

hydro linh ng. Phn ng thng c tin hnh trong mi trng acid [2],

[25].

S phn ng:

C H + HCHO + NH

R1

R2

H+

H2OC C N

R1

R2

Phn ng xy ra d dng trong dung mi alcol nhit khng cao (

14

- Giai on 2: Cation aminomethyl phn ng vi hp cht c hydro linh

ng.

+ Trng hp hp cht c hydro linh ng khng c kh nng to enol ha

(nh trng hp hydro linh ng cc lin kt N-H, S-H, Se-H, P-H hoc

lin kt C-H nhn indol, pyrol ) th cation aminomethyl tn cng vo

nguyn t mang hydro linh ng to sn phm base Mannich:

+N

R1

R2

CH2+

+ CHN

R1

R2

CH2 CH+

+Trng hp hp cht c hydro linh ng c kh nng to enol (hydro linh

ng nm bn cnh nhm carbonyl ca phn t) th cation aminomethyl l tc

nhn i in t tn cng vo lin kt i C=C ca dng enol to ra hp cht

cha nhm aminomethyl (base Mannich):

C

O

C H

H

C

O

C

H

N

R2

R1

H2C

+

C

OH

C

N

R2

R1H2C

- HN

R2

R1

H2CC

O

C

Base Mannich

15

CHNG 2. NGUYN VT LIU, TRANG THIT B V PHNG

PHP NGHIN CU

2.1 NGUYN VT LIU, HA CHT, DUNG MI

STT Tn nguyn liu Ngun gc TCCL

1 Hydantoin Merck 98%

2 4-bromobenzaldehyd Merck 100%

3 Morpholin Merck 98%

4 Piperidin Merck 98%

5 N-methylpiperazin Merck 98%

6 P-toluidin Merck 98%

7 Pyrrolidin Merck 98%

8 P-anisidin Merck 98%

11 Cloroform Trung Quc 99%

12 Dimethylformamid Trung Quc 99.5%

13 Ethyl acetat Trung Quc 99.5%

14 Formaldehyd Trung Quc 37-40%

15 Methanol Trung Quc 99.5%

16 Natri bicarbonat khan Trung Quc 99.5%

17 Ethanol tuyt i c Giang (VN) 99.5%

16

2.2 THIT B TH NGHIM

- Dng c thy tinh (bnh cu 3 c dung tch 500ml, sinh hn, phu nh

git, ng ong, cc thy tinh cc loi, bnh lc ht, phu Buchner )

- Cn k thut in t Shimadzu (Nht).

- Bm ht chn khng DIVAC.1.21 (M).

- Bm ht chn khng VRL mode 200-7.0 (M).

- My ct quay Buchi R-210 (Thy S).

- T sy Memmert (c).

- T sy chn khng Shellab (c).

- My khuy t IKA-RCT (c).

- My o nhit nng chy EZ-Melt (M).

- Ph hng ngoi (IR) ghi trn my Perkin Elmer, phng Ha Vt liu,

Khoa Ha hc, Trng i hc Khoa hc T Nhin, i hc Quc Gia

H Ni

- Ph cng hng t ht nhn (1H-NMR, 13C-NMR, HQSC, HMBC) ghi

trn my AVANCE, Bruker AV500 ti Vin ha hc, Vin khoa hc v

cng ngh Vit Nam.

- Ph khi lng (MS) ghi theo phng php phun m in t (ESI-MS)

trn my LC-MSD-Trap-SL, ti Vin Ha hc, Vin Hn lm Khoa hc v

Cng ngh Vit Nam.

2.3 PHNG PHP NGHIN CU

2.3.1 Phng php tng hp 5-(4-bromobenzyliden)hydantoin v dn

cht base Mannich.

- S dng cc phng php thc nghim trong ha hu c tng hp cc

cht d kin.

- Dng phng php kt tinh li tinh ch sn phm thu c.

- Dng SKLM theo di phn ng v s b xc nh tinh khit.

17

2.3.2 Phng php xc nh cu trc

Cc phng php ph (IR, 1H-NMR, 13C-NMR, HQSC, HMBC, MS)

c s dng khng nh cu trc cc sn phm tng hp c. Cc s

liu ph ca cc cht tng hp c i chiu vi ti liu.

2.3.3 Phng php th tc dng gy c t bo ung th.

Th tc dng gy c t bo ung th ca cc cht tng hp c theo

phng php: SRB [18], [22]

Nguyn tc:

y l phng php th nghim in vitro o s tng sinh v sng st ca t

bo. T bo ung th c nui cy trong a 96 ging..

- Hp cht Sulphorhodamin B (SRB) bin i thnh aminoxanthin c mu

hng, c cc acid amin cn thit trong t bo sng hp thu. Cng nhiu t

bo sng, s hp thu cng nhiu. Cc t bo sng, sau c c nh. nh

lng cc cht mu c t bo hp thu bng my quang ph k bc sng

495-515 nm. T tnh c s lng t bo sng st. Phng php ny hin

ang c p dng ti Vin nghin cu ung th quc gia M (National

Cancer Institution - NCI).

Dng t bo th nghim:

3 dng t bo:

- Dng t bo MCF-7 (t bo ung th v)

- Dng t bo Hep-G2 (t bo ung th gan)

- Dng t bo PC3 (t bo ung th tuyn tin lit)

Cht chun dng tnh

S dng cht chun c kh nng dit t bo: Ellipithine, Vinblastine hoc

Taxol pha trong DMSO

18

Tin hnh th hot tnh

a/ Chun b t bo

- T bo ung th c duy tr iu kin tiu chun. Sau khi t bo c

hot ha pht trin n phase log s c s dng cho th test vi cc cht

th chun b sn 4 - 10 thang nng khc nhau, lp li ba ln trn phin

vi lng 96 ging i vi phng php SRB.

- Mu th nghim bao gm: t bo + mi trng nui cy + mu th c

trong t m CO2/370C t bo tip tc pht trin.

- a nui cy t 48 gi-72 gi (37oC, 5% CO2) cho php cht th pht

huy tc dng.

b/ Quy trnh th nghim c tnh t bo theo phng php SRB [18],[22]

- T bo sau khi c t 48 gi-72 gi c c nh bng TCA 40C

trong 1 gi, sau ra sch bng nc v kh t nhin.

- Thm 50 l dung dch sulphorhodanin B (SRB) vo mi ging ca a

nui cy, nhum nhit phng trong 30 pht.

- Ra sch dung dch sulphorhodanin B (SRB) d bng dung dch acid

acetic 1% 5 ln.

- Hon tr sulphorhodanin B c nhum vo t bo. Lc k ha tan

hon ton.

- c mt quang bc sng 492-515nm. Mt quang s phn nh s

lng t bo sng st.

d. Tnh kt qu.

* Tnh t l t bo sng st (gi tr CS)

Gi tr CS: l kh nng sng st ca t bo nng no ca cht th

tnh theo % so vi i chng. Da trn kt qu o c ca chng OD (ngy

19

0), DMSO 10% v so snh vi gi tr OD khi trn mu tm gi tr CS (%)

theo cng thc sau:

CS% =

Gi tr CS% sau khi tnh theo cng thc trn c a vo tnh ton

Excel tm ra % trung bnh lch tiu chun ca php th c lp li 3

ln theo cng thc ca Ducan nh sau.

lch tiu chun = ( xi - x )

2

n - 1

* Tnh gi tr IC50

Cc mu c biu hin hot tnh (CS

20

CHNG 3. THC NGHIM V KT QU

3.1 TNG HP HA HC

Trong kha lun ny, chng ti tin hnh tng hp 5-(4-

bromobenzyliden)hydantoin v mt s dn cht base Mannich ca chng, bao

gm cc cng vic sau:

- Tng hp 5-(4-bromobenzyliden)hydantoin bng phn ng ngng t

aldehyd thm 4-bromobenzaldehyd vi hydantoin theo phn ng:

Br CHO +NH

NH

O

O

BrHC

NH

NH

O

O

1 234

51'

2'3'

4'

5' 6'

1 2

34

51'

2'3'

4'

5' 6'

H0

6

NaOAc/AcOH

- Tng hp mt s dn cht base Mannich ca 5-(4-

bromobenzyliden)hydantoin bng cch cho 5-(4-bromobenzyliden)hydantoin

tc dng vi fomaldehyd v cc amin bc 1, bc 2 theo s phn ng sau:

BrHC

NH

NH

O

O

12

34

51'

2'3'

4'

5' 6'

+ HCHO + NHR1

R2Br

HC

NH

N

O

O

12

34

51'

2'3'

4'

6'

CH2 NR1R2

H0 H1-H6

6 6

5'

EtOH/DMF

NR1

R2

N O

NR1

R2

N (H2)

(H1)

N

R1

R2

HN (H3)

NR1

R2

N N (H4)

OCH3

CH3

NR1

R2

N (H6)

N

R1

R2

HN CH3 (H5)

hi lu

hi lu

21

3.1.1 Tng hp 5-(4-bromobenzyliden)hydantoin (H0).

Trn c s l thuyt v phn ng ngng t aldehyd thm vi hydantoin

c trnh by phn tng quan, chng ti tin hnh phn ng theo

phng php phn ng c thc hin trong acid acetic bng vi natri acetat

khan theo quy trnh ca tc gi Wheeler v Hoffman [26].

Cng thc:

BrHC

NH

NH

O

O

12

34

51'

2'3'

4'

5' 6'

6

CTPT: C10H7BrN2O2; KLPT: 266,08

Quy trnh tng hp.

Cho vo bnh cu 9,25g (0,05 mol) 4-bromobenzaldehyd ; 5,00g (0,05

mol) hydantoin; 16,4g (0,1 mol) natri acetat khan v 52,5 ml acid acetic bng.

Khuy u cho hn hp tan hon ton v un nng hn hp, sau hn hp

phn ng c un hi lu v duy tr nhit phn ng 1300-1350. Phn

ng kt thc sau 4 gi. Theo di phn ng bng SKLM vi h dung mi (A)

dicloromethan:methanol (12:1), h dung mi (B) dicloromethan:methanol

(14:1), hoc h dung mi (D) cloroform:methanol (9:1). ngui, hn hp

ra cc c m, ngm lnh 5 pht sau hn hp nhit phng trong 30

pht. Lc ht kit,ra ta nhiu ln bng nc ct cho n khi ht acid acetic

(th bng giy qu). Ra tip vi ln bng cn lnh. Sy kh ta thu c.

Kt tinh trong dung mi EtOH/DMF (15:4).

Khi lng sn phm 3,56g tinh th mu trng. Hiu sut: 26,7%

Nhit nng chy: 295-2970C

Rf = 0,74 (TLC, silicagel 60 F254, h dung mi CH2Cl2 : CH3OH (12:1))

22

Rf = 0,66 (TLC, silicagel 60 F254, h dung mi CH2Cl2 : CH3OH (14:1))

Rf = 0,65 (TLC, silicagel 60 F254, h dung mi CHCl3 : CH3OH (9:1))

Ph IR (KBr, max(cm-1

)): xem ph lc 1.1, bng 3.2

Ph 1H-NMR (500MHz, DMSO-d6), (ppm): xem ph lc 1.2, bng 3.2

Ph 13C-NMR (125 MHz, DMSO-d6), (ppm): xem ph lc 1.3, bng 3.3

ESI-MS(m/z, %): xem ph lc 1.4, bng 3.4.

3.1.2 Tng hp cc dn cht base Mannich ca 5-(4-

bromobenzyliden)hydantoin (H1-H 6).

Trong phn tng quan, chng ti trnh by v phn ng Mannich ca

hydantoin v cc dn cht th C-5. Cn c trn cc kt qu nghin cu

cng b, chng ti la chn iu kin tin hnh phn ng Mannich ca 5-

(4-bromobenzyliden)hydantoin l nhit 850C vi t l s mol 5-(4-

bromobenzyliden)hydantoin : formaldehyd : amin l 1:1:1. Chng ti tin

hnh tng hp vi cc amin (morpholin, piperidin, pyrrolidin, N-

methylpyperazin, p-toluidin, p-anisidin).

Qui trnh tng hp chung.

Cho vo bnh phn ng 0,0025 mol cht H0, thm 15ml ethanol, khuy

nhit phng cho phn tn u trong 15 pht. Cho tip 0,21ml formol

(0,0025 mol HCHO) v 0,0025 mol amin, 3-4 git CH3COOH, khuy nhit

phng trong vng mt gi. Sau un cch thy nng dn nhit ti

850C th thm tip DMF vo t t hn hp tan hon ton. Sau , tip tc

khuy v gi nhit phn ng 850C. Theo di phn ng bng sc k lp

mng vi h dung mi A: CH2Cl2 : CH3OH (12:1) vi cc cht H1 , H4. H

dung mi B: CH2Cl2 : CH3OH (14 :1) vi cc cht H2, H3. H dung mi C:

H2O : AcOH : MeOH (5:1:4) vi cht H5, h dung mi D : CHCl3 : CH3OH

23

(9:1) vi cht H6, bn mng silicagel 60 F254, pht hin vt n t ngoi

bc sng 254nm. Sau 5 gi phn ng t ti u. hn hp phn ng ra

cc c m ngui n nhit phng, qua m. Sau co thnh cc

tht k to ta tt. Lc ht, ra bng ethanol lnh thu c sn phm th.

Kt tinh li bng dung mi thch hp, sy kh 600C trong t sy chn khng

trong 3h thu c sn phm.

Sau y l cc trng hp c th .

1) 5-(4-bromobenzyliden)-3-morpholinomethylhydantoin (H1)

BrHC

NH

N

O

O

12

34

51'

2'3'

4'

5' 6'

6

CH2 N O


T 0,67g (0,0025 mol) cht (H0); 0,21 ml formol (0,0025 mol HCHO) v

0,22g (0,0025 mol) morpholin, thi gian phn ng l 5 gi. Sn phm c

kt tinh li trong dung mi EtOH/DMF (40:11).

Sn phm thu c l 0,71g tinh th mu vng , hiu sut 77,8%

Nhit nng chy: 232-2330C, Rf = 0,78 (H dung mi A ).


)): xem ph lc 2.1, bng 3.2


Ph 13C-NMR (125MHz, DMSO-d6), (ppm): xem ph lc 2.3, bng 3.3


2) 5-(4-bromobenzyliden)-3-piperidinomethylhydantoin (H2).

Br CH N

H

N

O

O

12

34

51'

2'3'

4'

5' 6'

6CH2 N


24

T 0,67g (0,0025 mol) cht (H0); 0,21 ml formol (0,0025 mol) HCHO v

0,21g (0,0025 mol) piperidin, thi gian phn ng l 5 gi. Sn phm c kt

tinh li trong dung mi EtOH/DMF (5 : 1).

Sn phm thu c l 0,74g tinh th mu vng, hiu sut 81,5%.

Nhit nng chy 220-2230C. Rf = 0,6 (H dung mi B )


)): xem ph lc 3.1, bng 3.2




3) 5-(4-bromobenzyliden)-3-(4-methoxyphenylamino)methylhydantoin

(H3).

BrHC

NH

N

O

O

12

34

51'

2'3'

4'

6'6

CH2 NH OCH31''

2'' 3''

4''

5''6''

5'



0,31g (0,0025 mol) p-anisidin, thi gian phn ng 5 gi. Sn phm c kt

tinh li trong dung mi EtOH/DMF (7:1).


Nhit nng chy: 234-2370C, Rf = 0,85 (H dung mi B).


)): xem ph lc 4.1, bng 3.2



25


4) 5-(4-bromobenzyliden)-3-(N-methylpiperazino)methylhydantoin (H4).

Br CH N

H

N

O

OCH2 N N CH3

12

34

56

1'

2'3'

4'

5' 6'

1''

2'' 3''

4''

5''6''

CTPT: C16H20BrN4O2 ; KLPT: 378,25.


0,25g (0,0025 mol) N-methylpiperazin, thi gian phn ng l 5 gi. Sn phm

c kt tinh li trong dung mi EtOH/DMF (20:0,4).

Sn phm thu c c khi lng l 0,605g tinh th mu trng, hiu sut

64,0%

Nhit nng chy 238-2400C, Rf = 0,76 (H dung mi A).

Ph IR (KBr, max (cm-1

)): ph lc 5.1, bng 3.2

Ph 1H-NMR (500MHz, DMSO-d6), (ppm): ph lc 5.2, bng 3.2

Ph 13C-NMR (125MHz, DMSO-d6), (ppm): ph lc 5.3, bng 3.3


5) 5-(4-bromobenzyliden)-3-(4-methylphenylamino)methylhydantoin (H5).

BrHC

NH

N

O

O

12

34

51'

2'3'

4'

5' 6'

6

CH2 NH CH31''

2'' 3''

4''

5''6''



0,27g (0,0025 mol) p-Toluidin, thi gian phn ng l 5 gi. Sn phm c

kt tinh li trong dung mi EtOH/DMF (50 : 21).

26

Sn phm thu c l 0,84 tinh th mu vng chanh, hiu sut 87,2%.

Nhit nng chy: 243-2450C, Rf = 0,92 (h dung mi C)


)): xem ph lc 6.1, bng 3.2



ESI-MS(m/z,%): xem ph lc 6.4, bng 3.4.

5) 5-(4-bromobenzyliden)-3-pyrrolidinomethylhydantoin (H6).

BrHC

NH

N

O

O

12

34

51'

2'3'

4'

5' 6'

6

CH2 N1''

2''3''

4''

5''



0,18g (0,0025 mol) pyrrolidin, thi gian phn ng 5 gi. Sn phm c kt

tinh li trong dung mi EtOH/DMF (20:3).


Nhit nng chy: 224-2270C, Rf = 0,74 (h dung mi D)

Rf = 0,56 (TLC, silicagel 60 F254, h dung mi CHCl3 : MeOH (9:0,5))


)): xem ph lc 7.1, bng 3.2



ESI-MS(m/z,%): xem ph lc 7.4, bng 3.4.

27

3.1 XC NH CU TRC CA CC CHT TNG HP C

3.2.1 Ph hng ngoi

Ph hng ngoi c ghi trn my Perkin Elmer ti phng Ha vt liu,

Khoa ha hc, i hc Khoa hc t nhin, i hc Quc gia H Ni. 7 ph

c ghi cc ph lc 1.17.1. Kt qu phn tch s liu ph hng ngoi

c ghi bng 3.2

3.2.2 Ph cng hng t ht nhn

Ph cng hng t ht nhn proton, 13C v ph tng tc hai chiu (2D-

NMR) c ghi trn my ph AVANCE Spectrometer AV500 (BRUKER,

c) trong dung mi DMSO-d6, cht chun ni TMS ti Vin Ha hc -

Vin Hn lm Khoa hc v Cng ngh Vit Nam. 7 ph c ghi cc

ph lc 1.27.2 v 1.37.3. Kt qu phn tch s liu ph cng hng t ht

nhn proton v 13C c ghi bng 3.2 v 3.3.

3.2.3 Ph khi lng

Cc cht tng hp c ghi ph khi lng theo phng php phun m

in t (ESI-MS) trn my LC-MSD-Trap-SL, ti Vin Ha hc, Vin Hn

lm Khoa hc v Cng ngh Vit Nam. 7 ph c ghi cc ph lc

1.67.6

3.2 TH HOT TNH GY C T BO UNG TH

Th hot tnh gy c t bo trn mt s dng t bo ung th ngi c

tin hnh ti Phng Sinh hc thc nghim-Vin Ha hc cc hp cht thin

nhin (Vin Hn lm Khoa hc v Cng ngh Vit Nam).

Kt qu th hot tnh gy c t bo ung th

Ti Phng sinh hc thc nghim- Vin Ha hc cc hp cht thin nhin

(Vin Hn Lm Khoa Hc v Cng Ngh Vit Nam) 7 cht do chng ti tng

hp (H0-H6) c th hot tnh gy c t bo trn dng t bo ung th

28

biu m v MCF-7, dng t bo ung th tuyn tin lit PC3 v dng t bo

ung th gan ngi Hep-G2. Kt qu c trnh by bng 3.5, bng 3.6.

Kt qu cho thy c 5 cht l cht H1 (IC50= 4,54 g/ml), H2 (IC50= 2,89

g/ml ), H3 (IC50= 3,73 g/ml), H4 (IC50= 2,84 g/ml), H6 (IC50= 1,59 g/ml)

c hot tnh c ch t bo ung th gan Hep-G2. Cc cht tng hp c

khng c tc dng vi dng t bo MCF7 v PC3 th nghim.

Bng 3.1: kt qu tng hp 5-(4-bromobenzyliden)hydantoin v cc dn cht base Mannich

STT Cng thc K hiu

cht

M

Thi gian phn ng

(gi)

Dung mi kt tinh li

Rf

Nhit nng chy

(0C)

Hiu sut (%)

1 Br CH N

H

NH

O

O

12

34

56

1'

2'3'

4'

5' 6'

H0 266,08 5 EtOH:DMF

(15:4)

0,74A

0,66B

0,65D

295-297 26,7

2 1 234

5

61'

2'3'

4'

5' 6'

1''

2'' 3''

4''

5''6''Br CH N

H

N

O

OCH2 N O

H1 365,21 5

EtOH:DMF

(40:11) 0,78

A 232-233 77,8

3 1 234

5

61'

2'3'

4'

5' 6'

Br CH N

H

N

O

OCH2

1''

2'' 3''

4''

5''6''

N

H2 363,24 5

EtOH:DMF

(5:1) 0,60

B 220-223 81,5

4 1 234

5

61'

2'3'

4'

5' 6'

Br CH N

H

N

O

OCH2 NH OCH3

1''

2'' 3''

4''

5''6''

H3 401,24 5

EtOH:DMF

(7:1) 0,85

B 234-237 61,8

5 Br CH N

H

N

O

OCH2 N N CH3

12

34

56

1'

2'3'

4'

5' 6'

1''

2'' 3''

4''

5''6''

H4 378,25 5

EtOH:DMF

(50:1) 0,76

A 238-240 64,0

6 1 2

345

6

1'

2'3'

4'

5' 6'

1''

2'' 3''

4''

5''6''

NHN

O

O

BrCH2 NH CH3

H5 385,24 5

EtOH:DMF

(50:21) 0,92

C 243-245 87,2

7 N

HN

O

O

BrCH2 N

H6 349,21 5

EtOH:DMF

(20:23) 0,74

D 224-227 74,4

Bng 3.2: S liu ph IR v ph 1H-NMR ca cc cht tng hp c (H0-H6)

STT CTCT

K hiu

NR1R2

IR (KBr, cm-1) 1H-NMR (DMSO-d6,ppt, J, Hz)

C=C C2=O

C4=O CH2 H6(s) CH2(s) Gi tr khc

1 Br CH N

H

NH

O

O

12

34

56

1'

2'3'

4'

5' 6'

H0 1664 1767

1735 6,377

11,276 (s;1H;N3-H);

10,586 (s;1H;N1H); 7,567 (m;4H;H2,3,5,6)

2 1

234

5

61'

2'3'

4'

5' 6'

1''

2'' 3''

4''

5''6''Br CH N

H

N

O

OCH2 N O

H1 N O

1655 1768

1713 2950 6,511 4,355

7,597 (m;4H;H2,3,5,6); 3,541 (m,4H;H3,5);

2,50 (m;4H;H2,6).

3 1

234

5

61'

2'3'

4'

5' 6'

Br CH N

H

N

O

OCH2

1''

2'' 3''

4''

5''6''

N

H2 N

1662 1767

1716 2940 6,487 4,344

7,578 (m;4H;H2,3,5,6); 2,503 (m;4H;H2,6);

1,467 (m;4H; H3,5); 1,312 (m;2H;H4).

4 1

234

5

61'

2'3'

4'

5' 6'

Br CH N

H

N

O

OCH2 NH OCH3

1''

2'' 3''

4''

5''6''

H3 NH OCH3

1659 1771

1717 2961 6,38 4,878

10,829 (s;1H;N1H); 7,588 (m;4H;H2,3,5,6);

6,793 (m;2H;H3,5); 6,713 (m;2H;H2,6); 6,160

(m;1H;NH); 3,660 (s;3H;OCH3).

5 Br CH N

H

N

O

OCH2 N N CH3

12

34

56

1'

2'3'

4'

5' 6'

1''

2'' 3''

4''

5''6''

H4 N N CH3 1665

1767

1718 2946 6,483 4,358

7,57(m, 4H, H2,3,5,6); 2,507(m,8H,H2,3,5,6);

2,126(s,3H,CH3).

6 12

345

6

1'

2'3'

4'

5' 6'

1''

2'' 3''

4''

5''6''

NHN

O

O

BrCH2 NH CH3

H5 NH CH3

1662

1762

1715 2919 6,506 4,89

10,831(s;1H;N1H); 7,584 (m;4H;H2,3,5,6);

6,901

(m;2H;H3,5);6,745(m;2H;H2,6);6,336(m;1H;NH);2,174 (s;3H;CH3).

7 NHN

O

O

BrCH2 N

H6 N

1660 1767

1713 2971 6,436 4,466

7,567(m;4H;H2,3,5,6); 2,507 (m;4H;H2,5);

1,63 (m;4H;H3,4)

Bng 3.3: S liu ph 13C-NMR ca cc cht tng hp c (H0-H6)

STT CTCT K

hiu NR1R2

13C-NMR (DMSO-d6, ppm)

C6 C4=O C2=O Cc gi tr khc

1 Br CH N

H

NH

O

O

12

34

56

1'

2'3'

4'

5' 6'

H0 106,808 165,384 155,624 121,456 (C4); 132,244 (C1); 131,605(C3,5); 131,211(C2,6);

128,54 (C5)

2 1

234

5

61'

2'3'

4'

5' 6'

1''

2'' 3''

4''

5''6''Br CH N

H

N

O

OCH2 N O

H1 N O

108,113 164,06 155,762

132,066 (C1); 131,636 (C3,5); 131,333 (C2,6); 126,998

(C5); 121,726 (C4); 65,972 (C3,5); 59,878 (NCH2N);

50,313 (C2,6).

3 1

234

5

61'

2'3'

4'

5' 6'

Br CH N

H

N

O

OCH2

1''

2'' 3''

4''

5''6''

N

H2 N

107,853 165,06 155,965

132,142 (C1); 131,464 (C3,5); 131,196 (C2,6); 127,172

(C5); 121,726 (C4); 60,709 (NCH2N); 51,168 (C2,6);

25,41(C3,5); 23,323(C4)

4 1

234

5

61'

2'3'

4'

5' 6'

Br CH N

H

N

O

OCH2 NH OCH3

1''

2'' 3''

4''

5''6''

H3 NH OCH3

108,303 164,072 155,125

151,081 (C4); 139,765 (C1);131,982 (C1); 131,607 (C3,5);

131,372 (C2,6); 126,885 (C5); 121,786(C4); 114,356

(C3,5); 113,775 (C2,6); 55,205 (OCH3); 48,547 (NCH2N).

5 Br CH N

H

N

O

OCH2 N N CH3

12

34

56

1'

2'3'

4'

5' 6'

1''

2'' 3''

4''

5''6''

H4 N N CH3 106,43 165,369 155,693 135,139(C1); 133,648(C4); 129,017(C5); 128,25(C2,6);

61,469(NCH2N); 54,205 (C3,5); 49,970 (C2,6); 40,090

(CH3).

6 1 2

345

6

1'

2'3'

4'

5' 6'

1''

2'' 3''

4''

5''6''

NHN

O

O

BrCH2 NH CH3

H5 NH CH3

108,326 164,039 155,087

143,558 (C1); 131,985 (C1); 131,615 (C3,5); 131,27

(C3,5); 129,34 (C2,6); 126,879 (C5); 125,732 (C4);

121,794 (C4); 112,72 (C2,6); 48,009 (NCH2N); 20,01

(CH3).

7 NHN

O

O

BrCH2 N

H6 N

107,921 164,995 155,911

132,12 (C1); 131,604 (C3,5); 131,205 (C2,6); 127,17 (C5);

121,667 (C4); 55,532 (NCH2N); 49,818 (C2,5); 23,344

(C3,4).

32

Bng 3.4: S liu ph ESI-MS ca cht H0 v cc dn cht base Mannich

(H1-H6)

STT K

hiu Cng thc

phn t

KLPT

(M)

Tnh

[M-H]-

m/z (%)

Ion khc

m/z (%)

1 H0 C10H7BrN2O2 266,08 264,93 (100)

2 H1 C15H16BrN3O3 365,21 363,96 (100)

M CH2 N O : 264,90 (20%)

CH2 N O : 100 (0,02%)

3 H2 C16H18BrN3O2 363,24 361,91 (2) M CH2 N : 264,90 (95%)

4 H3 C18H16BrN3O3 401,24 399,95 (100)

M CH2 NH OCH3 : 264,90 (70%)

CH2 NH OCH3 : 136(0,05%)

5 H4 C16H20BrN4O2 378,25 377,05 (34)

M CH2 N N-CH3 : 264,90 (88%)

CH2 N N-CH3 : 113 (0,02%)

6 H5 C18H16BrN3O2 385,24 383,95 (100)

M CH2 NH CH3 : 264,98 (41%)

CH2 NH CH3 : 120,0 (0,1%)

7 H6 C15H16BrN3O2 349,21 M CH2 N : 264,9 (100%)

33

Bng 3.5: Kt qu th tc dng ca cc cht tng hp c trn mt s

dng t bo ung th ngi (theo phng php SRB)

STT K hiu

mu

Nng

mu

(g/ml)

Dng t bo

Cell survival (%) Kt lun

Hep-G2 MCF7 PC3

DMSO 100,00,0 100,00,0 100,00,0

Chng

(+) 5 0,20,03 1,50,2 2,50,08 Dng tnh

1 H0 5 62,31,1 78,50,7 86,30,5 m tnh

2 H1 5 44,20,5 73,70,9 81,71,2 Dng tnh vi

dng Hep G2

3 H2 5 25,30,6 78,91,4 96,81,1 Dng tnh vi

dng Hep G2

4 H3 5 34,81,4 88,10,7 92,50,8 Dng tnh vi

dng Hep G2

5 H4 5 24,80,7 86,80,5 91,21,3 Dng tnh vi

dng Hep G2

6 H5 5 54,70,9 85,21,5 91,40,5 m tnh

7 H6 5 9,81,2 71,70,5 92,31,4 Dng tnh vi

dng Hep G2

34

Bng 3.6. Kt qu xc nh gi tr IC50 (g/ml) ca mt s cht tng hp

c trn dng t bo Hep G2 (theo phng php SRB).

Stt K hiu mu

Dng t bo

Gi tr IC50 (g/ml) Kt lun

Hep-G2 MCF7 PC3

Chng (+) 0,24 0,31 0,26 Dng tnh

1 H1 4,54 >5 >5 Dng tnh vi

dng Hep G2

2 H2 2,89 >5 >5 Dng tnh vi

dng Hep G2

3 H3 3,73 >5 >5 Dng tnh vi

dng Hep G2

4 H4 2,84 >5 >5 Dng tnh vi

dng Hep-G2

5 H6 1,59 >5 >5 Dng tnh vi

dng Hep G2

35

CHNG 4. BN LUN

4.1 V TNG HP HA HC.

- Phn ng ngng t 4-bromobenzaldehyd vi hydantoin c thc hin

trong dung mi l acid acetic bng v xc tc l natri acetat khan, hiu sut

26,7%.

- V thi gian phn ng ngng t: kt qu thc nghim cho thy khng c

s khc bit v thi gian phn ng tng hp cc halogenoarylidenhydantoin

nh cc cng trnh cng b [1], [7],[8]

- Cc phn ng tng hp cc dn cht base Mannich ca 5-(4-

bromobenzyliden)hydantoin c thc hin trong hn hp dung mi

EtOH/DMF (17:1) v xc tc acid acetic c hiu sut kh cao (61,8-87,2%).

- Thc hin phn ng Mannich chng ti la chn 4 amin bc 2 v 2 amin

bc 1c lc base mnh l N-methylpiperazin, morpholin, piperidin, p-toluidin,

pyrrolidin ,p-anisidin v vi cc amin ny phn ng xy ra kh d dng v l

cc hp phn cu to ca cc base Mannich c th c hot tnh sinh hc cao

trong cc cng trnh cng b [1], [11], [13], [14].

4.2 V XC NH CU TRC CA CC CHT TNG HP C.

4.2.1. V ph hng ngoi (IR)

7 cht tng hp c ghi ph hng ngoi cc ph lc 1.17.1. Qua

nghin cu ph ca cc cht, chng ti nhn bit cc di hp th c trng

ca cc nhm chc v lin kt ca cc cht tng hp c [10]. Kt qu bin

gii ph c trnh by bng 3.2

Sau y l mt s nhn xt chung:

36

- Ph hng ngoi ca 7 cht tng hp c u xut hin 2 di hp th

ca cc nhm C=O (17711713 cm-1) v di hp th ca lin kt C=C ca

nhn benzen c to thnh do phn ng ngng t nm trong vng hp th

16651655 cm-1.

- Ph IR ca cht H0 c di hp th ca NH amid v NH imid ca nhn

hydantoin. cht H0 ch xut hin mt di hp th tng ng vi dao ng

ca nhm NH (31373053 cm-1) c l do hai di hp th NH xen ph nhau.

Trong khi cc dn cht ca base Mannich (H1-H6) khng c di hp th

NH amid chng t H ca N-3(NH imid) c thay th bi phn ng

Mannich v xut hin bng sng hp th nm trong 29712909cm-1 c trng

cho dao ng ha tr ca lin kt CH2, iu ny cho thy phn ng Mannich

xy ra v tng hp c cc dn cht nh d kin.

- Ph IR ca cht H0 v H(1-6) xut hin di hp th c trng cho lin kt

Carom-Br (12801072cm-1

).

- Cc s liu trng vi cc ti liu tham kho c. Cc kt qu s b c

th khng nh cc sn phm to thnh c cu trc nh d kin.

V xc nh ng phn hnh hc ca cc dn cht to thnh da

trn ph IR.

Trn ph IR ca cc cht tng hp bng 3.2 cho thy xut hin di hp

th c trng cho lin kt i ethylenic (C(5)=C(6)) c to thnh do phn

ng ngng t nm trong vng t 16651655cm-1 . Theo tc gi Tan v cng

s [26] ng phn E c cu trc phng nn lm m rng h lin hp gia aryl

v vng hydantoin, t lm gim tn s dao ng ca lin kt C=C v C=O.

Trong khi ng phn Z c dng xon lm cho h lin hp gp tr ngi. V

th di dao ng ha tr vng tn s cao c trng cho lin kt C=C ca

ng phn Z. Nghin cu ca Tan v cng s [23] cho thy di hp th nm

trong vng 16751660cm-1 ng vi dao ng ha tr ca lin kt C=C ca

37

ng phn Z, nm trong vng16401630cm-1 ng vi dao ng ha tr C=C

ca ng phn E. iu ny cng ph hp vi kt qu thu c ca tc gi V

Trn Anh[1], Th Thu Hng [7], Trnh Vn Ninh [8] (cc dao ng ha tr

ca lin kt C=C ca cc cht tng hp c xut hin trong vng

16661653cm-1, y l cc ng phn Z). Kt qu ghi ph hng ngoi ca H0

v base Mannich cho thy cc dao ng ha tr ca lin kt C=C ca cc cht

xut hin trong vng 16651655 chng t cc cht ca chng ti tng hp l

cc ng phn Z.

4.2.2 V ph cng hng t ht nhn 1H-NMR

Ph cng hng t proton cho php nhn bit dng proton v s lng

proton tng dng ca 7 cht c ghi ph qua s liu v dch chuyn ha

hc (), bi v cng ca cc pic c trnh by bng 3.2 . Sau y l

bin gii v mi lin quan cu trc phn t v ph 1H-NMR ca cc cht tng

hp c.

- Ph 1H-NMR ca cht H0 cho thy tn hiu ca proton H-N1 10,586

ppm v H-N3 xut hin 11,276 ppm, trong khi cc dn cht base

Mannich H(1-6) khng c tn hiu cng hng ca proton H-N3, ch c tn hiu

ca proton H-N1 (10,82910,831 ppm).

- Ph 1H-NMR ca 7 cht u cho thy tn hiu ca proton ethylenic (H-

6) to thnh t phn ng ngng t hydantoin v aldehyd thm (Ho l

6,377ppm v H(1-6) l 6,4786,511ppm).

- Ph 1H-NMR ca H(1-6) tng hp c cn cho thy s xut hin tn

hiu ca proton nhm >N-CH2-N< (4,3444,883ppm).

Ngoi ra cn cc dng proton khc v s proton tng dng ca 7 cht ghi

ph c nhn bit vi cc s liu ghi bng 3.2. Ph 1H-NMR cho thy

cc tn hiu c trng ca cc proton trong phn t ca cc cht to thnh ph

hp vi cu trc d kin.

38

V xc nh ng phn hnh hc ca cc dn cht to thnh da

trn ph 1H-NMR.

Cc hp cht ca chng ti c xut hin lin kt i lin kt vi C-5 ca

hydantoin, v th c th c ng phn hnh hc Z v E do s cn quay xung

quanh lin kt i C=C C-5. Hai ng phn Z v E u c th phn bit vi

nhau da trn dch chuyn ha hc ca proton ethylenic (H-6) v proton

orthophenyl trn phn benzyliden (H2 v H6).

Do nh hng ca hiu ng anisotrop ca nhm carbonyl C4=O vi proton

ethylenic =CH gy hiu ng gim chn dng ng phn Z so vi ng phn

E nn proton ethylenic ca dng ng phn Z s cng hng trng yu

hn v c dch chuyn ha hc ln hn.

Nhn xt trn ph hp vi cc nghin cu tin hnh ca tc gi Tan, S.F

v cng s, tc gi Khanfar v cng s, tc gi Zuliani v cng s [28]. Theo

nghin cu ca cc tc gi trn chuyn dch ha hc ca proton ethylenic

vng trng thp (H 6,47,00 ppm) s tng ng vi ng phn Z, vng

trng cao (H 6,206,30 ppm) s tng ng vi ng phn E.

Cht H0 c dch chuyn ha hc ca proton ethylenic t 6,377ppm tng

ng vi ng phn Z. Vy cc cht H0 chng ti tng hp c c cu hnh

Z.

Hiu ng anisotrop ca nhm carbonyl C-4 cng lm cho cc proton

orthophenyl trn hp phn benzyliden ca ng phn E b gim che chn hn

so vi proton tng ng ng phn Z. Theo Tan v cng s [23] chuyn

dch ha hc ca proton H0 (H2, H6) nm trong vng 7,527,65 ppm i vi

ng phn Z v trong khong 7,907,98 ppm i vi ng phn E ca mt s

dn cht 5-arylidenhydantoin c nhm th v tr 4 l Cl, -CH3, -OCH3 v

39

H hoc CH3 v tr 1. Cht H0 ca chng ti tng hp c c dch

chuyn proton H0(H2, H6) l 7,5487,589 ppm chng t cht H0 tng hp

c c cu hnh l ng phn Z:

12

34

5

NH

NH

O

OCH 6

Br

1'2'

3'

4' 5'

6'

12

34

5

NH

NH

O

OC

H

6

Br

1'

2'

3'4'

5'

6'

ng phn Z ng phn E

Nh vy nhm th N-3 khng gy nh hng lm thay i cu hnh ca

cc base Mannich so vi aryliden ban u.

4.2.3 V ph cng hng t ht nhn 13C-NMR

7 cht tng hp c c ghi ph 13C-NMR. Kt qu phn tch ph

c trnh by bng 3.3 cho thy cc cht c ghi ph c s carbon v

dch chuyn ha hc ca cc v tr carbon ph hp vi cng thc d kin.

- Ph 13C-NMR ca 7 cht (H0 v H(1-6)) u cho thy tn hiu cng

hng ca carbon nhm C=O (amid) l C-4 (165,384 ppm v

164,039165,06 ppm), ca nhm C=O (imid) l C-2 (155, 624 ppm v

155,087155,965 ppm) v nhm ethylenic C-6 (106,808 ppm v

107,853108,326 ppm).

- thy r tng tc gia proton v carbon C-13 cc hp cht 4-

bromobenzylidenhydantoin v cc dn cht base Mannich chng ti tin

hnh ghi ph 2D-NMR (HSQC v HMBC) ca cc hp cht (H0, H3, H5, H6),

sau y l bin gii cc cht.

40

+ Vi cht H0

-Trn ph HSQC cho thy tng tc gn gia proton ca ethylenic (H-6) c

gi tr bng 6,377ppm vi nguyn t carbon ethylenic (C-6) c gi tr

bng 106,808

-Vng phenyl xut hin nhm th C4, xut hin cc tng tc gn gia

proton H-2 v H-6 ( 7,567 ppm) vi nguyn t C-2 v C-6 ( 131,211

ppm), gia cc proton H-3 v H-5 ( 7,567 ppm) vi nguyn t C-3 v C-5

( 131,605 ppm) (xem hnh 4.1.1).

- Trn ph HMBC ca cht H0 tng hp c cho thy proton ethylenic

(H-6) c tng tc 3J-HMBC vi C-4 (carbonyl amid), C-2 v C-6 ca vng

benzen.

Hnh 4.1.1. Ph tng tc HSQC ca cht H0

BrHC

NH

NH

O

O

12

34

51'

2'3'

4'

5' 6'

6

41

CH

NH

NH

O

O

1 234

56

1'2'

3'4'

5'

6'

Br

+ Trn ph HMBC ca H0 cho thy proton ca H-2,6 c tng tc 3J-

HMBC vi C-4 ca vng benzen v C-6 (carbonethylenic). Proton H-3,5 c

tng tc 3J-HMBC vi C-3,C-5, C-1 ca vng benzen (xem hnh 4.1.2).

CH

NH

NH

O

O

1 234

56

1'2'

3'4'

5'

6'

Br

H

H

Hnh 4.1.2.Ph tng tc HMBC ca cht H0

42

Bng 4.1: Cc tng tc 1H-13C ca 4-bromobenzylidenhydantoin (H0)

+ Vi cc dn cht base mannich.

- Trn ph HSQC ca cc cht H(3,5,6) u thy xut hin tng tc gn

gia cc proton ca nhm >N-CH2-N< nm trong vng 4,4664,890 ppm vi

nguyn t carbon ca nhom >N-CH2-N< nm trong vng 48,00955,532

ppm.

+ Trn ph HSQC ca hp cht H6 (hnh 4.6.1) cho thy tng tc gn gia

cc proton vng 7,5657,588 ppm vi cc nguyn t carbon trong vng

131,194131,623 ppm ca nhn thm ca hp phn benzylidenhydantoin.

hp phn amin l pyrrolidin xut hin cc tng tc gn gia cc proton H-2

Proton Carbon lin kt trc tip

Carbon cch 3 lin kt

V tr (ppm) V tr (ppm) V tr (ppm)

H-6 6,377 C-6 106,808 C-2,6 131,211

H-2 7,567 C-2 131,211

C-4

C-6

C-6

121,456

131,211

106,808

H-6 7,567 C-6 131,211

C-4

C-2

C-6

121,456

131,211

106,808

H-3 7.567 C-3 131,605 C-5

C-1

131,605

132,244

H-5 7,567 C-5 131,605 C-3 131,605

43

v H-5 c 2,507 ppm vi C-2 v C-5 trong vng 49,818ppm. Tng

tc ca proton H-3 v H-4 c 1,63ppm vi C-3 v C4 trong vng

23,344 ppm.

Vi cht H6, ph HMBC (hnh 4.6.2) cho thy proton ca H-2 v H-5 c

tng tc 3J-HMBC vi carbon ca nhm >N-CH2-N

44

Bng 4.2: Cc tng tc 1H-13C ca 5-(4-bromobenzyliden)-3-

pyrrolidinomethylhydantoin (H6).

Proton Carbon lin kt trc tip

Carbon cch 3 lin kt

V tr (ppm) V tr (ppm) V tr (ppm)

H-6 6,436 C-6 107,921

C-2

C6

C-4

131,205

131,205

164,995

H-2 7,567 C-2 131,205

C-6

C-6

C-4

107,921

131,205

121,667

Hnh 4.6.2 Ph HMBC ca hp cht H6

45

H-3 7,567 C-3 131,604 C-1

C5

132,12

131,604

H-5 7,567 C-5 131,604 C-1

C-3

132,12

131,604

H-6 7,567 C-6 131,205

C-6

C-2

C-4

107,921

131,205

121,667

H-2 2,507 C-2 49,818

C-5

C-4

C(CH2)

49,818

23,344

55,532

H-3 1,63 C-3 23,344 C-5 49,818

H-4 1,63 C-4 23,344 C-2 49,818

H-5 2,507 C-5 49,818

C-2

C-3

C(CH2)

49,818

23,344

55,532

H(CH2) 4,4661 C(CH2) 55,532

C-2

C-4

C-2

C-5

155,911

164,995

49,818

49,818

4.2.4 V Ph MS.

7 cht tng hp c ghi ph khi lng (ph lc 1.4-7.4). T kt qu

trnh by cc ph lc c th nhn thy c 6 cht ghi ph u c pic phn

t vi s khi ng bng khi lng phn t tnh theo cng thc d kin l

46

H0, H1, H2 ,H3, H4, H5, cn H6 khng c pic phn t vi s khi c trng

m ch c cc pic ion mnh. Ngoi ra trn ph 7 cht th c 5 cht c cc

pic ca cc ion mnh c trng ph hp vi s phn mnh l H1, H3, H4,

H5, H6 c ghi bng 3.4. Cc cht cha nguyn t Brom trong phn t u

c pic ng v i km pic phn t v pic ca ion mnh cha brom vi s khi

ln hn 2, t l cng (1:1) ca pic ph hp vi t l ng v 81Br/79Br l

98,1% [10].

4.3 V tc dng gy c t bo ung th

Kt qu cho thy:

- 5 cht H1, H2, H3, H4, H6 c tc dng c ch dng t bo ung th Hep-

G2 vi IC50 ln lt l 4,54; 2,89; 3,73; 2,84 v 1,59 g/ml.

- 2 cht H0 v H5 khng c hot tnh vi 3 dng t bo th nghim.

Kt qu cho thy cc base Mannich ca 5-(4-bromobenzyliden)hydantoin

c th mang li hot tnh c ch t bo ung th.

c nhiu cng trnh nghin cu tng hp v th hot tnh khng t bo

ung th ca dn cht 5-arylidenhydantoin v cc base Mannich c cng

b [1], [5]. Kt qu nghin cu ca tc gi V Trn Anh [1] cho thy trong 43

cht tng hp c c 13 cht c hot tnh c ch t bo ung th gan ngi

Hep-G2 (IC50= 0,385g/ml) trong cht c hot tnh mnh nht l 5-(4-

clorobezyliden)-3-(N-methylpiperazino)methyl hydantoin (IC50= 0,38g/ml).

Tc gi Thu Hng [7] tng hp v th hot tnh gy c t bo ung

th ca 15 cht 5-(3-halogenoaryliden)hydantoin v base Mannich. Kt qu

cho thy c 11 cht c hot tnh c ch t bo ung th i trng HTC116 vi

IC50= 10,1719,23g/ml.

Tc gi Trnh Vn Ninh [8] tng hp v th hot tnh gy c t bo ung

th ca 13 cht ca mt s dn cht 3-aryl-5-arylidenhydantoin. Kt qu cho

47

thy c 2 cht c hot tnh c ch t bo ung th i trng HTC116 v 2 cht

c hot tnh c ch t bo ung th tuyn tin lit PC3.

So snh kt qu th hot tnh gy c t bo ung th ca cc dn cht base

Mannich ca 5-(4-bromobenzyliden)hydantoin vi cc dn cht 5-

arylidenhydantoin, cc dn cht 5-(3-halogenaryliden)hydantoin v 3-aryl-5-

arylidenhydantoin cng b [1],[7],[8]. Th thy cc base Mannich c hot

tnh tng ng vi dng t bo ung th gan Hep-G2 v khng c hot tnh

vi cc dng t bo ung th tuyn tin lit PC3 v ung th biu m v MCF7

m tc gi V Trn Anh [1], Thu Hng [7], Trnh Vn Ninh [8] cng

b . iu ny cng khng nh thm rng cc nhm th ht in t trong vng

aryl gp phn mang li hot tnh khng t bo ung th gan ngi Hep-G2

theo tc gi V Trn Anh [1].

48

KT LUN V KIN NGH

1. KT LUN

T nhng kt qu nghin cu trnh by trn y, chng ti rt ra mt

s kt lun nh sau:

1.a) tng hp c cht 5-(4-bromobenzyliden)hydantoin (H0) v 6

dn cht base Mannich, trong tt c u cha thy cng b trong cc ti

liu tham kho c.

1.b) Tt c cc cht tng hp c u c kim tra tinh khit bng

sc k lp mng v o nhit nng chy. Cu trc cc cht tng hp c

xc nh bng phn tch ph hng ngoi, ph cng hng t proton (1H-

NMR), ph cng hng t ht nhn (13C-NMR), ph khi lng (MS) v c 4

cht (H0, H3, H5, H6) c tin hnh o ph 2 chiu (HMBC,HSQC). Kt qu

thu c cho php chng ti kt lun cc cht tng hp c c cu trc ng

nh d kin.

2) th hot tnh gy c t bo ung th ca 7 cht tng hp c i

vi 3 dng t bo ung th ngi l t bo ung th biu m v MCF7, t bo

ung th gan Hep-G2, t bo ung th tuyn tin lit PC3. Kt qu cho thy c

5 cht l cc cht H1 (IC50= 4,54 g/ml), H2 (IC50= 2,89 g/ml ), H3 (IC50=

3,73 g/ml),H4 (IC50=2,86 g/ml), H6 (IC50= 1,59 g/ml) c hot tnh c ch

t bo ung th gan Hep-G2. C 7 cht u khng c hot tnh c ch t bo

ung th tuyn tin lit PC3 v t bo ung th biu m v MCF7.

2. KIN NGH

T cc kt qu nghin cu trn y chng ti xin c mt s xut nh

sau:

- Tip tc th sng lc hot tnh gy c t bo ca cc cht tng hp

c trn cc dng t bo ung th khc nh: ung th d dy, ung th ty,

49

- Tip tc tng hp v sng lc hot tnh gy c ca cc dn cht 5-

benzylidenhydantoin v cc dn cht base Mannich khc nhm tm kim cc

cht c trin vng.

TI LIU THAM KHO

Ting Vit

1. V Trn Anh (2010), Tng hp mt s dn cht ca hydantoin v thm

d tc dng khng khun, khng nm v chng ung th, Lun n tin s

Dc hc, Trng i hc Dc H Ni.

2. Phan nh Chu (2003), Cc qu trnh c bn tng hp ha dc hu

c, NXB Khoa hc v k thut.

3. B mn dc l, Trng i hc Dc H Ni (2005), Dc l hc

lm sng, NXB Y hc, tr 289-300.

4. Nguyn B c (2003), Ha cht iu tr bnh ung th, NXB Y hc,

393-394.

5. inh Th Thanh Hi, Nguyn Quang t, Nguyn Vit H (2002),

Tng hp v th tc dng khng khun, khng nm ca 5-(5-nitro-2-

furfuryliden)-imidazolidin-2,4-dion v dn cht, Tp ch dc hc, 7,

14-15.

6. inh Th Thanh Hi, Nguyn Quang t, L Mai Hng (2003),

Nghin cu hot tnh gy c t bo ca mt s dn cht ca 5-

nitrofurfural , Tp ch Dc hc, 2003, 1, 18-20.

7. Th Thu Hng (2012), Tng hp v th hot tnh khng t bo ung

th ca mt s dn cht 5-halogenarylidenhydantoin, Lun vn thc s

Dc hc, Trng i hc Dc H Ni.

8. Trnh Vn Ninh (2013), Tng hp v th hot tnh sinh hc ca mt s

dn cht 3-aryl-5-arylidenhydantoin, Lun vn thc s Dc hc,

Trng i hc Dc H Ni.

9. Phm Thip, V Ngc Thy (2008), Thuc bit dc v cch s dng,

NXB Y hc.

10. Nguyn nh Triu (1999), Cc phng php vt l ng dng trong

ha hc, NXB i hc Quc gia H Ni.

Ting Anh

11. Advendano, C. and Menendez, J.C (1983), Hydantoin, Kirk-Othmer

Encyclopedia of Chemical Technology, Vol. 12, 3rd

Ed., New York,

John Wiley, 672-700.

12. Arya, S.C.; Agarwal, N. ; Agarwal, S. (2006), Nitrofurantoin:an

effective and ignored antimicrobial, International Joural of

Antimicrobial Agents, 27(4), 354-355.

13. Carmi, C. et al. (2006), 5-Benzylidene-hydantoins as new EGFR

inhibitors with antiproliferative activity, Bioorganic and Medical

Chemistry Letters, 16, 4021-4025.

14. Dimmock, J.R.; Taylor, W.G. (1974), Synthesis and physical

properties of substituted 4-dimethylaminomethyl-1-phenyl-1-nonen-3-

ones possessing antitumor properties, Journal of Pharmaceutical

Sciences, 63(1), 69-74.

15. Kazlauskas, R.; Murphy, P.T. et al. (1977), Aplysinopsin, a new

trytophan derivative from a sponge, Tetrahedron Letters, 18(1), 61-64.

16. Kleeman, A. (1982), Process for the production of 5-

arylidenhydantoin, US patent 4.305.072.

17. Lamiri, M. et al. (2006), Microwave-Assisted Solvent-Free

Regiospecific Synthesis of 5-Alkylidene and 5-Arylidenehydantoins

Synthetic Communications, 36, 1575-1584.

18. Likhitwitayawid, K.; Angerhofer, C.K. et al. (1993), Cytotoxic and

antimalarial Bisbenzylisoquinole alkaloids from Stephania erecta,

Journal of Natural products, 56 (1), 30-38.

19. Martindale (2005), The complete drug reference, 34ed, Pharmaceutical

Press.

20. Marton, J. ; Enisz, J. (1993), Preparation and Fungicidal Activity of 5-

Substituted Hydantoins and their 2-Thio Analogs, J.Agric.Food

Chem., 41, 148-152.

21. Mudit, M. ; Khanfar, M. et al. (2009), Discovery, design, and

synthesis of anti-metastatic lead phenylmethylene hydantoins inspired

by marine natural products, Bioorg. Med. Chem., 17(4), 1731-1738.

22. Skehan, P.; Storeng, R. (1990), New colorimetric cytotoxicity assay

for anticancer-drug screening, Journal of the National Cancer Insitute,

8(13), 1107-1112.

23. Tan, S.F.; Ang, K.P and Fong, Y.F (1986), (Z) and (E) -5-

arylmethyenehydantoins : Spectroscopic properties and Configuration

Assignment, J.Chem.Soc.Perkin Trans. II, 1941-1944

24. Thenmozhiyal, J.C. et al. (2004), Anticonvulsant Activity of

Phenylmethylidenehydantoins: A Structure-Activity relationship

study, J.Med. Chem., 47, 1527-1535.

25. Tramontini, M. (1973), Advances in the chemistry of Mannich bases,

Synthesis, 12, 703-775.

26. Wheeler, H.L et al. (1911), On Hydantoins: A synthesis of

phenylalanine and of tyrosine, Am. J. Chem., 45, 368-383.

27. Wong, P.T.H (1989), Pharmacological Profiles of 5-

phenylenehydantoin and its methoxy substituted derivaties, Japhan J.

Pharmacol., 49, 309-315.

28. Zuliani, V.; Carmi, C. et al. (2009), 5-Benzylidene-hydantoins:

Synthesis and antiproliferative activity on A549 lung cancer cell line,

European Journal of Medicinal Chemistry, 44(9), 3471-3479.

Ting c.

29. Baeyer, A. (1861), Vorlufige notiz ber das Hydantoin, Justus

Liebigs Ann. Chem., 117, 2, 178-180.

30. Baeyer, A. (1864), Mittheilungen aus dem organischen Laboratorium

des Gewerbeinstitutes in Berlin: Untersuchungen ber die

Harnsuregruppe, Justus Liebigs Ann. Chem., 130, 2, 129-175.

31. Stecker A. (1873), Ueber Uroxansure und die chemische constitution

der Harnsure, Justus Liebigs Ann. Chem., 155, 2, 177-185.

DANH MC CC PH LC

Ph lc 1 (1.1-1.6) Cc ph ca hp cht H0







Ph lc 8 Cc Phiu tr li kt qu th hot tnh

gy c t bo ung th

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

Mail: [email protected]: 4cm-1

BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN

Date: 5/7/2014

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

100.0

cm-1

%T

3487

3226

1795

1767

1735

1664

1583

1487

1428

1383

1353

1308

1277

1253

1198

1094

1073

1007

884

878

817

751

736

712

651

3271

Ph lc 1.1: Ph IR ca hp cht H0

Ph lc 1.2: Ph 1H-NMR ca cht H0

BrHC

NH

NH

O

O

Ph lc 1.3: Ph 13C-NMR ca cht H0

BrHC

NH

NH

O

O

Ph lc 1.4: Ph HSQC ca cht H0

BrHC

NH

NH

O

O

Ph lc 1.5 : Ph HMBC ca cht H0

BrHC

NH

NH

O

O

Ph lc 1.6: Ph MS ca cht H0

BrHC

NH

NH

O

O

Ph lc

1.1: Ph

IR ca

hp cht

H1




MAU H1Date: 4/21/2014

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

3453

3293

2837

1768

1713

1655

1584

1487

1431

1335

1280

1154

1113

1073

1004

887 817

767

720

677

29092950

Br CH

NH

N

O

OCH2 N O

Ph lc 2.1: Ph IR hp cht H1


BrHC

NH

N

O

OCH2 N O




Mau H2Date: 4/21/2014

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

102.3

cm-1

%T

3301

2940

2915

2849

2808

2762

1767

1716

1662

1584 1487

1435

1413

1393

1329

1278

1264

1178

1162

1120

1072

1007

1002

986

880

838

820

778

762

718

662

3095


Br CH

NH

N

O

OCH2 N


Br CH N

H

N

O

OCH2 N




MAU H3Date: 4/21/2014

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

100.0

cm-1

%T

3384

3306

3036

2961

2833

1771

1717

1659

1583

1524

1489

1444

1423

1363

1350

1315

1253

1171

1098

1074

1028

1006

874

821

763

722

668

633


NH

N

O

OCH2NH OCH3

HCBr


Br CHNH

N

O

OCH2 NH OCH3

Ph lc 4.5: Ph HMBC ca cht H3

Br CHNH

N

O

OCH2 NH OCH3


Br CHNH

N

O

OCH2 NH OCH3




MAU H4Date: 4/21/2014

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

100.0

cm-1

%T

3228

2946

2817

1718

1665

1584

1489

1436

1377

1339

1288

1254

1176

1144

1076

1007

875

816

763

727

670

644

3064

1767

1739


Br CH N

H

N

O

OCH2 N N CH3

Ph lc 5.2: Ph 1H-NMR ca hp cht H4

Ph lc 5.3: ph 13C-NMR ca hp cht H4

Br CH N

H

N

O

OCH2 N N CH3

Phc lc 5.4: Ph MS ca cht H4

Br CH N

HO

OCH2 N N CH33




MAU H5Date: 4/21/2014

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

100.8

cm-1

%T

3410

3366

3317

3018

2919

1762

1715

1662

1618

1585

1524

1486

1454

1419

1359

1290

1257

1172

1132

1095

1072

1006

873

817

764

718

669

650

637


BrHC

N

O

OCH2 NH CH3


BrHC

NH

N

O

OCH2 NH CH3




MAU H6Date: 4/21/2014

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.0

0.0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100.0

cm-1

%T

3280

2940

2821

1767

1713

1660

1585

1489

1435

1416

1384

1364

1333

1307

1257 1154

1072

1006

993

877

834

815

785

765

730

704

666

2971

3214


BrHC

NH

N

O

OCH2 N


BrHC

NH

N

O

OCH2 N

VIN HN LM KHOA HC V CNG NGH VIT NAM

VIN HO HC CC HP CHT THIN NHIN

Phng Sinh hc thc nghim

Vietnamese Academy of Science and Technology, Hanoi, Vietnam

(VAST)

Institute of Natural Products Chemistry (INPC)

The Bioassay Lab.

18 Hong quc Vit- Cu Giy- H Ni- Vit Nam

Tel: 844-8361899; Fax: 844-7564390; Email: [email protected]

S: /TLKQ

PHIU TR LI KT QU

HOT TNH GY C T BO (CYTOTOXICITY ASSAY)

Ngi gi mu: Trn Anh

a ch: B mn Ha Hu c i hc Dc H Ni

S lng mu: 35

Loi mu: Mu tinh

Ngy nhn mu: 17/04/2014 Ngy tr li kt qu: 06/05/2014

PHNG PHP PHN TCH

Theo phng php ca Skehan & CS (1990) v Likhiwitayawuid & CS

(1993) hin ang c p dng ti Vin nghin cu ung th Quc gia ca

M (NCI) v trng i hc Dc, i hc Tng hp Illinois, Chicago, M.

Dng t bo

- MCF 7: Human Breast adenocarcinoma (Ung th biu m v) - PC 3: Prostate cancer (Ung th tuyn tin lit) - Hep G2: Human Hepatocellular carcinoma (Ung th gan ngi)

Mi trng nui cy t bo: DMEM (Dulbeccos Modified Eagle Medium) hoc MEME (Minimum Essential Medium with Eagles salt)

c b sung L- glutamine, Sodium piruvat, NaHCO3, PSF (Penixillin-

Streptomycin sulfate- Fungizone); NAA (Non-Essential Amino Acids); 10%

BCS (Bovine Calf Serum)

Tripsin-EDTA 0,05%; DMSO (Dimethyl Sulfoside); TCA (Trichloro Acetic

acid); Tris Base; PBS (Phosphate Buffered Saline); SRB (Sulfo Rhodamine

B); Acid Acetic.

Cc dng c dng 1 ln: Bnh nui cy t bo, phin vi lng 96 ging,

pipet pasteur, cc u tup cho micropipet

Cht chun chng dng tnh:

Dng cht chun c kh nng dit t bo: Ellipithine, Vinblastine hoc

Taxol pha trong DMSO

- c trn my ELISA bc sng 495-515nm

Tnh kt qu:

Gi tr CS (Cell Survival): l kh nng sng st ca t bo nng

no ca cht th tnh theo % so vi i chng. Da trn kt qu o

c ca chng OD (ngy 0), DMSO 10% v so snh vi gi tr OD khi

trn mu tm gi tr CS (%) theo cng thc:

OD (mu) OD (ngy 0)

CS% = x 100

OD (DMSO) OD (ngy 0)

Gi tr CS% sau khi tnh theo cng thc trn, c a vo tnh ton

Excel tm ra % trung bnh lch tiu chun ca php th c lp li

3 ln theo cng thc ca Ducan nh sau: lch tiu chun

(xi - x )2

=

n - 1

Cc mu c biu hin hot tnh (CS < 50%) s c chn ra th nghim

tip tm gi tr IC50

Gi tr IC50 : dng gi tr CS ca 10 thang nng , da vo

chng trnh Table curve theo thang ga tr logarit ca ng cong pht

trin t bo v nng cht th tnh gi tr IC50. Cng thc:

1/y=a+blnX

Trong Y: nng cht th; X: Gi tr CS (%)

KT QU THC NGHIM

STT K hiu

mu

Nng mu

(g/ml)

Dng t bo


Hep-G2 MCF7 PC3

DMSO 100,00,0 100,00,0 100,00,0

Chng (+) 5 0,20,03 1,50,2 2,50,08 Dng tnh

1 AN1 5 77,630,7 82,30,4 84,70,6 m tnh

2 AN2 5 60,10,5 78,41,2 79,51,4 m tnh

3 AN3 5 49,30,9 69,30,7 85,31,2 Dng tnh vi dng Hep G2

4 AN4 5 83,71,2 73,21,3 91,50,4 m tnh

5 AN5 5 52,80,4 82,30,4 82,60,7 m tnh

6 H0 5 62,31,1 78,50,7 86,30,5 m tnh

7 H1 5 44,20,5 73,70,9 81,71,2 Dng tnh vi dng Hep G2




11 H5 5 54,70,9 85,21,5 91,40,5 m tnh

12 H6 5 9,81,2 71,70,5 92,31,4 Dng tnh vi

dng Hep G2

13 T1 5 90,10,7 92,40,9 86,30,7 m tnh

14 T2 5 47,10,7 81,61,3 84,21,1 Dng tnh vi dng Hep G2

15 T3 5 62,10,4 94,31,2 96,51,6 m tnh

16 T4 5 83,30,9 80,40,6 99,20,8 m tnh

17 T5 5 99,21,5 82,61,1 65,30,6 m tnh

18 T6 5 71,50,8 80,20,9 71,20,9 m tnh

19 T7 5 20,50,6 73,10,6 90,51,3 Dng tnh vi dng Hep G2

20 T8 5 65,30,9 71,21,1 95,61,1 m tnh

21 T9 5 71,21,2 86,30,4 91,20,7 m tnh

22 T10 5 93,20,5 82,51,3 64,20,4 m tnh

23 T11 5 64,60,0 76,50,9 92,50,9 m tnh

24 T12 5 48,30,6 75,80,9 85,71,2

Dng tnh vi dng Hep G2

25 T13 5 81,90,8 86,40,5 93,81,6 m tnh

STT K hiu mu

Dng t bo

Gi tr IC50 (g/ml) Kt lun

Hep-G2 MCF7 PC3

Chng (+) 0,24 0,31 0,26 Dng tnh

1 AN3 4,95 >5 >5 Dng tnh vi dng

Hep G2

2 H1 4,54 >5 >5 Dng tnh vi dng

Hep G2


Hep G2


Hep G2


Hep G2


Hep G2

7 T2 4,66 >5 >5 Dng tnh vi dng

Hep G2

8 T7 3,22 >5 >5 Dng tnh vi dng

Hep G2

9 T12 4,72 >5 >5 Dng tnh vi dng

Hep G2

STT

K hiu

mu

Nng mu

(g/ml)

Dng t bo PC 3


DMSO 100,00,0

Chng

(+) 5 2,50,08 Dng tnh

1 II 5 90,61,6 m tnh

2 IIa 5 84,70,9 m tnh

3 IIb 5 95,31,2 m tnh

4 IIc 5 91,70,6 m tnh

5 IId 5 78,30,7 m tnh

6 III 5 96,40,9 m tnh

7 IIIa 5 94,71,2 m tnh

8 IIIb 5 98,30,6 m tnh

9 IIIc 5 75,11,1 m tnh

10 IIId 5 82,60,4 m tnh

Xc nhn ca Vin Ph trch phng Ngi c kt qu

TS. Hu Ngh Trn Th Hng H

Documents

Tổng hợp và thử hoạt tính kháng tế bào ung thư của 5 -(4''-bromobenzyliden) hydantoin và một số dẫn chất base mannich.pdf