tổng hợp và thử hoạt tính sinh học của 3-(4-nitrophenyl) - hydantoin và một số...

B Y T

TRNG I HC DC H NI

L TRNG T

TNG HP V TH HOT TNH SINH HC CA 3-(4-NITROPHENYL)-

HYDANTOIN V MT S DN CHT

KHA LUN TT NGHIP DC S

H NI-2013

B Y T

TRNG I HC DC H NI

L TRNG T

TNG HP V TH HOT TNH SINH HC CA 3-(4-NITROPHENYL)-

HYDANTOIN V MT S DN CHT

KHA LUN TT NGHIP DC S

Ngi hng dn:

ThS. Hong Thu Trang DS.Trnh Vn Ninh

Ni thc hin:

B mn Ha Hu C

H NI-2013

LI CM N Li u tin, ti xin by t lng knh trng v bit n su sc ti ThS

Hong Thu Trang, DS.Trnh Vn Ninh, PGS.TS Nguyn Quang t,

TS.V Trn Anh 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 cm n s gip nhit tnh ca : Ths ng V Lng

(Phng NMR- Vin ha hc- Vin Khoa hc v Cng ngh Vit Nam), Ths.

Phm Th Nguyt Hng (Khoa Dc l-Sinh ha, Vin Dc liu Vit

Nam), TS. Nguyn Th Sn (B mn Ha vt liu, khoa Ha, trng i hc

Khoa hc T nhin, i hc Quc gia H Ni) 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.

Mc d c nhiu c gng nhng do hn ch v thi gian, kin thc

cng nh ti liu tham kho nn kha lun ca ti khng th trnh khi nhng

sai st, khim khuyt trong ni dung v hnh thc, ti rt mong nhn c s

gp ca cc thy c kha lun c hon thin hn.

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 1 thng 5 nm 2013

Sinh vin

L Trng t

DANH MC CC K HIU, CH VIT TT A549 : T bo ung th phi ngi 13C NMR : Ph cng hng t ht nhn carbon 13

AcOH : Acid acetic

DMF : Dimethylformamid

DMSO : Dimethylsulfoxid

EGFR : Epidermal growth factor receptor, th th ca yu t tng trng biu m

EtOH : Ethanol

EtOAc : Ethyl acetat

HCT116 : T bo ung th i trng ngi.

Hep G2 : T bo ung th gan ngi

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

(Heteronuclear Multiple Bond Connectivity) 1H NMR : Ph cng hng t ht nhn proton

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

(Heteronuclear Single-Quantum coherence)

IC50 : Nng c ch 50%

IR : Ph hng ngoi

LU : T bo ung th phi

MCF7 : T bo ung th biu m v ngi

MeOH : Methanol

MS : Khi ph ( Mass spectrometry)

DANH MC CC BNG

Bng Trang

3.1: Kt qu tng hp ca cc dn cht 3-aryl-5-

arylidenhydantoin tng hp c I-V 27

3.2: S liu ph IR ca cc cht tng hp c 28

3.3: S liu ph 1H-NMR ca cc cht tng hp c 29

3.4: S liu ph 13C-NMR ca cc cht tng hp c 30

3.5: Kt qu th tc dng khng cc dng t bo ung th

ngi ca cc cht tng hp c

31

3.7: Gi tr IC50 (g/ml) ca cc dn cht hydantoin trn Hep-G2 38

3.8: Gi tr IC50 (g/ml) ca cc dn cht hydantoin trn HTC116 39

DANH MC CC PH LC

Ph lc 1 (1.2-1.3) : Cc ph ca hp cht A

Ph lc 2 (2.1-2.6) : Cc ph ca hp cht B

Ph lc 3 (3.1-3.6) : Cc ph ca hp cht I

Ph lc 4 (4.1-4.6) : Cc ph ca hp cht II

Ph lc 5 (5.1-5.6) : Cc ph ca hp cht III

Ph lc 6 (6.1-6.6) : Cc ph ca hp cht IV

Ph lc 7 (7.1-7.6) : Cc ph ca hp cht V

Ph lc 8 : Phiu tr li kt qu th hot tnh khng t bo

ung th

Trang

CC CH V K HIU VIT TT

DANH MC CC BNG

DANH MC CC HNH V S

T VN 1

CHNG 1. TNG QUAN 2

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 c ch t bo ung th 4

1.2 CC PHNG PHP TNG HP HYDANTOIN V DN

CHT

8

1.2.1 Phn ng tng hp hydantoin v hydantoin th 8

1.2.1.1 Phn ng ng vng ca cc -aminoacid vi cc cyanat 8

1.2.1.1 Phng php tng hp t -aminoacid v isocyanat hu c 8

1.2.1.3 Phn ng ng vng ca cc -dicarbonyl vi ure hoc dn xut ca

ure

9

1.2.1.4 Phn ng Bucherer-Bergs 9

1.1.1.5 Phn ng ca cc cyanoacetamid vi kim hypohalid 10

1.2.2 Phn ng tng hp dn cht 5-arylidenhydantoin 10

1.2.2.1 Phn ng ngng t vi aldehyd 10

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

PHP NGHIN CU

14

2.1 NGUYN VT LIU, HA CHT, DUNG MI 14

2.2 THIT B TH NGHIM 15

2.3 NI DUNG NGHIN CU 15

2.4 PHNG PHP NGHIN CU 15

2.4.1 Phng php tng hp cc dn cht 3-aryl-5-arylidenhydantoin 15

2.4.2 Phng php xc nh cu trc 16

2.4.3 Phng php th tc dng khng t bo ung th 16

CHNG 3. THC NGHIM V KT QU NGHIN CU 17

3.1 TNG HP HA HC 17

3.1.1 Tng hp 3-(4-nitrophenyl)hydantoin 17

3.1.1.1 Tng hp N-(4-nitrophenylamino)carbonyl)glycin 18

3.1.1.2 Tng hp 3-(4-nitrophenyl)hydantoin 19

3.1.2 Tng hp cc dn cht 3-aryl-5-arylidenhydantoin (I-V) 19

3.2 XC NH CU TRC CA CC CHT TNG HP C 23

3.2.1 Ph hng ngoi 23

3.2.2 Ph cng hng t ht nhn 1H v 13C 24

3.2.3 Ph khi lng 24

3.3 TH NGHIM HOT TNH KHNG CC DNG T BO UNG

TH NGI

24

3.4 BN LUN 33

3.4.1 V TNG HP HA HC 33

3.4.2 V XC NH CU TRC CA CC CHT TNG HP

C

35

3.4.2.1 V ph hng ngoi (IR) 35

3.4.2.2 V ph cng hng t ht nhn 1H-NMR 36

3.4.2.3 V ph cng hng t ht nhn 13C-NMR 36

3.4.2.4 V ph MS 37

3.4.3 V TC DNG KHNG T BO UNG TH 37

CHNG 4. KT LUN V KIN NGH 40

4.1 KT LUN 40

4.2 KIN NGH 40

TI LIU THAM KHO

DANH MC PH LC

PH LC

1

T VN

Trong tt c cc lnh vc, vic tm ra nhng phng php mi nng

cao hiu qu s dng lun l vn c quan tm hng u. Cng nh vy,

trong lnh vc Dc phm, cc nh khoa hc lun n lc khng ngng nhm

tm ra nhng dn cht mi c tim nng tc dng sinh hc c th nng cao

cng tc phng v cha bnh.

Hin nay, c nhiu phng php c p dng nhng phng php

tng hp v bn tng hp vn gi vai tr quan trng v khng th thiu. Cc

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

thuc hoc cc cht c tc dng dc l, c trin vng to ra cc cht mi

d on c tc dng tt hn, t c tnh hn v c hiu qu cao hn trong iu

tr.

Vi nhng tim nng v tc dng sinh hc, cc dn cht ca hydatoin

c quan tm nghin cu v mang li nhng hiu qu nht nh. C th k

n mt s dn cht c s dng rng ri trong y hc 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).

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

c tim nng v hot tnh sinh hc, chng ti thc hin kha lun vi cc mc

tiu sau:

1.Tng hp 3-(4-nitrophenyl)hydatoin v mt s dn cht.

2.Th tc dng chng ung th ca cc hp 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 [11] bng phn ng hydro phn (hydrogenolysis)

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

ci), l ngun gc ca tn hydantoin. Sau , nm 1864, A.Bayer [11]

tng hp c hydatoin vi nguyn liu ban u l bromoacetylure. Tuy

nhin cu trc vng ca hydantoin ch c xc nh bi Strecker vo nm

1873 [8]. Cho n nay, nhiu dn cht ca hydantoin c tng hp v

mt s cht c s dng lm thuc.

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 kh phong ph v a dng nh ng chng

ng kinh, khng khun, chng ung th [1], [2], [3], [6], [12], [14], [15]

Cc dn xut th v tr C1, C3, c bit th 2 ln v tr C5 c nghin

cu v cho thy nhiu 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-

diphenylhydatoin (phenytoin) (1), bit dc l Dilantin (bit dc ca Parke-

Davis), Epanutin (bit dc ca Pfizer).

NH

HN O

O (1)

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

bnh ng kinh [2], [6], [19]. Tuy nhin phenytoin cn c mt s tc dng

3

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

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 [24].

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

[9], [16].

ON N

NHO2N

O

O (II)

Nm 1993, tc gi Janos Marton v cng s [20] tng hp v th tc

dng khng khun,khng nm ca dn cht 5-arylidenehydantoin (3 a-h) cho

thy chng c tc dng tt. Cc cht tng hp c tc dng vi chng nm gy

bnh trn la m, u nh Erysiphegraminis f. sp. Stritici, Uromyces

appendicutalus, Botrytis nicerea.

HN

HN OO

R1

R2

R3 R4

R1=R2=R3=R4=H (a) R1=OH, R2=R3=R4=H (e)

R3=OCH3, R1=R2=R4=H (b) R2=OCH3, R3=OH, R1=R4=H (f)

R1=OCH3, R2=R3=R4=H (c) R2=OH, R3=OCH3, R1=R4=H (g) R2=R3=OCH3,R1=R4=H (d) R1=OH, R2=OCH3, R3=R4=H (h)

3 a-h

4

1.1.3 Tc dng c ch t bo ung th Mt dn cht hydantoin c ng dng trong iu tr ung th l

Nilutamid (4)

FFF

O2N

N

NHO

O

CH3CH3

(4)

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

tuyn tin lit di cn nm 1996 [3], [6], [19]. Nilutamid c ch nh trong

iu tr ung th tuyn tin lit c di cn, dng phi hp vi phu thut ct b

tinh hon.

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

(5) v spongiacidin (6) t bt bin [15], [17].

NH

N

NO

O

CH3

CH3HN

OHN

NH

HN

R

BrO

O 5 6

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

Hng [4] 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)

Kt qu l tm c hai cht l 5-(5-nitro-2-furfuryliden)imidazolidin-

2,4-dion(8) v 3-piperidinomethyl-5-(5-nitro-2furfuryliden)imidazolidin-

2,4-dion (9) c hot tnh khng t bo ung th rt mnh (gi tr IC50 l 0,2-

5

1,04 (g/ml)) i vi dng t bo FL v 0,6-1,15 (g/ml) i vi dng t bo

KB.

N

NH

ONO2

CH2N

O

ONH

HN O

O ONO2

7 8

Nm 2006, C.Carmi v cng s [12] 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

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 (10) vi IC50= 27M trn invivo.

Nm 2009, V.Zualiani v C.Carmi tip tc hng nghin cu cc dn

cht 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 [33]. 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 (9).

NH

N

O OHO

9

6

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 hai 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 [23].

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

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

(10), (R)-5-(4-hydroxybenzyl)hydantoin (11), (Z)-5-(6-bromo-1H-indol-3-

yl)methylen)-hydantoin (12).

HNNH

OHO

O

HNNH

OHO

O

10 11

HN

NH

NH

O

O

Br

12

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 8

t nhin v cht tng hp (Z)-5-(4-ethylthio)benzylidenhydantoin (13) c

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

7

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 benzlydien m c cha

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.

HNNH

O

O

S

13

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

tnh ca cc dn xut ca hydantoin (14). 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 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.

NH

NO

OCH

CH2-NR1R2

R

1'

2'3'

4'

5' 6'

1234

56

(14)

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

4-F; 2-Cl; 4-Cl; 3-NO2; 4-NO2.

NR1

R2

N O (a)

N (b)NR1

R2

N N (c)CH3

HN (d)CH3

NR1

R2

NR1

R2

8

1.2 CC PHNG PHP TNG HP HYDANTOIN V DN CHT

Hin nay nhiu dn cht ca hydantoin ngoi vic c ng dng lm

thuc cn c s dng lm nguyn liu trong tng hp ha hc, trong ha

phn tch, trong nng nghip v nhiu ng dng khc. Do tim nng ng dng

a dng ca cc dn cht hydantoin, nn t cui th k 19 n nay c nhiu

phng php tng hp hydantoin v dn cht c cng b trong cc ti liu

khc nhau.

1.2.1 Phn ng tng hp hydantoin v hydantoin th

1.2.1.1 Phn ng ng vng ca cc -aminoacid vi cc cyanat.

y l phng php c in tng hp hydantoin th C-5 hoc N-1,

s dng phn ng gia -aminoacid v kalicyanat, c tc gi Urech tin

hnh u tin vo nm 1873. Phn ng c tin hnh trong dung dch nc

1000C [8], [21].

S phn ng:

RHC COOHNHR'

KCN RHC COOKNCONH2R'

NR'HN

O

O R

H2O

1000C

HCl

25%

Mui kali ca acid hydantoic

Phng php tng hp ny cho hiu sut cao nu i t nguyn liu l

mui hydroclorid ca ester ethylglycin.

1.2.1.2 Phng php tng hp t -aminoacid v isocyanat hu c.

y l phng php m rng ca phng php Urech [34], c s

dng tng hp dn cht hydantoin th N-3. Akyl isocyanat v aryl

isocyanat c s dng rng ri trong tng hp hydantoin t -amino acid

[13].

9

R CH COOHR'NCO N NH

O

O

HN

OC

NH CHR

COOH- H2O

NH2

R'

R'

R 1.2.1.3 Phn ng ng vng ca cc -dicarbonyl vi ure hoc dn xut ca

ure

Ty thuc vo iu kin phn ng v t l cc cht tham gia phn ng c

th thu c sn phm trung gian l dihydroxyimidazolidon hoc glycouril.

Khi un nng cht trung gian vi acid hydrochloric, hydantoin s to

thnh [8], [21].

S phn ng:

NH2

NH2O O

OHN NH

O

OHOH

NH2CONH2

NH

HN

HN

NH

OO

dihydroxyinidazolidinGlycouril

HN NH

O

O

HCl

-NH 2

CONH

2

-H 2O

HCl-H2 O

1.2.1.4 Phn ng Bucherer-Bergs

Phn ng Bucherer-Bergs tng hp cc dn cht hydantoin th C-5 t

hp cht carbonyl vi KCN hoc t cyanohydrins tng ng v amoni

carbonat [8], [29].

S phn ng: R

CR'

OKCN R

CCN

R' OH

RC

CNR' NH2

RC

CNR' NHCOOH

CO2NH3

+ NH3

RC

CNR' NHCONH2

HN NH

RR'

O

NHH2O

HN NH

RR'

O

O

10

1.2.1.5 Phn ng ca cc cyanoacetamid vi kim hypohalid [8], [19].

S phn ng:

C CNR'

RCONH2

C CNR'

RN C O HN

HNO

O

R'R

NaOBr

Sn phm ca phn ng l cc sn phm th hai ln v tr C-5.

1.2.2 Phn ng tng hp dn cht 5-arylidenhydantoin Hydantoin (imidazolidin-2,4-dion) c cng thc cu to nh sau:

NH

NH

O

O

T cu trc phn t hydantoin cho thy nhn hydantoin c nhm

methylen hot ng (C-5) c kh nng ngng t vi cc aldehyd thm to

ra dn cht 5-arylidenhydantoin.

1.2.2.1 Phn ng ngng t vi aldehyd S phn ng:

NH

NH

O

O

NH

NH

O

OCHR

OH

1

4 3NH

NH

O

O154 3

2RRCHO 5 2

R = phenyl, phenyl th, furyl, pyryl, quinolyl

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

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

gn C-5 ca hydantoin [29]. 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,

11

NH

NH

NH

NH

O

O

O

OArAr CHO

-H2O

AcOH, AcONa

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

dn cht th C-5 ca hydantoin [26], [31].

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 [27] s

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

c 54 dn cht.

S phn ng: O H

R

NH

NH

O

O NH

NHO

OR

NaHCO3/ Nuoc-EtOH

Ethanolamin, t0

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

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

sng trong tng hp hu c. Nm 2006, tc gi Lamiri [18] 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 2045Hz, cng sut 1250W tc ng vo trong 2 pht.

NH

NH

O

NHH

R

O

ORCHOnatri acetat/oxyd nhm

vi sng, 2 pht

O

12

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

benzaldehyd, o-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 nh sau [25], [28]

- 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

HC

O

O

NH

NH

O

O

+ + BHB

(I) (II)

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

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

NH

NH

HC

O

O

NH

NH

O

OHC

H

Ar

O

Ar C

O

H(+)

+

(-) (III) (II) (IV)

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

NH

NH

O

OHC

H

Ar

O

NH

NH

O

OHC

H

Ar

OH

+BH+ B

(IV) (V)

13

- Giai on ngng t loi nc:

Xc tc base (B-) ly nguyn t H linh ng v tr s 5 v tch anion

hydroxyd to thnh sn phm tng ng.

NH

NH

O

O

H

HCOH

Ar + B

NH

NH

O

OCH2

Ar+ BH + OH

14

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 Hm Lng

1 4-Nitrophenylisocyanat Merck 98%

2 Benzaldehyd Merck 97%

3 4- Clorobenzaldehyd Merck 100%

4 4- Nitrobenzaldehyd Merck 100%

5 3-Nitrobenzaldehyd Merck 98%

6 4-Fluorobenzaldehyd Merck 98%

7 Glycin Merck 98%

8 1,4-Dioxan Merck 98%

9 Aceton Trung Quc 99.5%

10 Acid acetic Trung Quc 99.5%

11 Cloroform Trung Quc 99%

12 Ethylacetat Trung Quc 99.5%

13 Dimethylformamid Trung Quc 37- 40%

14 Methanol Trung Quc 99.5%

15 Natri acetat Trung Quc 82.03%

16 Ethanol tuyt i c Giang (VN) 99.5%

17 Bn mng Silicagel Merck Silicagel 60 F254

18 Kali hydroxyd Merck 85%

19 Acid hydrocloric Trung Quc 36.46%

15

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 Bchi 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, B mn ha vt

liu,Khoa ha hc,HKHTN, H Quc gia H Ni.

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

AVANCE, Bruker AV500 ti Vin ha hc, Vin khoa hc v cng

ngh Vit Nam.

- Ph khi lng (LC/MS) ghi trn my LS-MSD-Trap-SL, ti Vin

ha hc, Vin khoa hc v cng ngh Vit Nam.

2.3 NI DUNG NGHIN CU - Tng hp v xc nh cu trc cc dn cht ca 3-aryl-5-

arylidenhydantoin.

- Th hot tnh khng mt s dng t bo ung th ca cc cht tng

hp c.

2.4 PHNG PHP NGHIN CU 2.4.1 Phng php tng hp cc 3-aryl-5-arylidenhydantoin

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

cc cht d kin.

16

- 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.

2.4.2 Phng php xc nh cu trc Cc phng php ph (IR, 1H-NMR, 13C-NMR, MS) c s dng

khng nh cu trc ca cc sn phm tng hp c. Cc s liu ph ca cc

cht tng hp c i chiu vi ti liu.

2.4.3 Phng php th tc dng khng t bo ung th Nguyn tc:

- p dng phng php MTT c bin son bi tc gi Mosmann

(1983) nh gi c tnh trn t bo [22]. Nguyn tc ca phng php

nh sau: MTT l th nghim in vitro o s tng sinh v sng st ca t

bo. T bo c nui cy trong mt a 96 ging, y bng. Hp cht MTT

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, c mu vng

c thm vo mi ging v t bo c 37oC, 5% CO2. Mu vng ny b

bin i thnh formazan tm trong ty th ca nhng t bo sng. Kh nng

hp th ca dung dch c mu ny c th c nh lng bng my quang

ph k bc sng 540 600nm. S bin i mu ch xy ra khi enzyme

reductase trong ty th l hot ng, v do s chuyn i c th lin quan

trc tip n s lng t bo sng st. Chng ti s dng phng php ny

nghin cu c tnh ca thuc i vi t bo.

17

CHNG 3. THC NGHIM V KT QU NGHIN CU

3.1 TNG HP HA HC Trong kha lun ny chng ti tin hnh thc hin tng hp 1 s dn cht

3-aryl-5-arylidenhydantoin bao gm cc cng vic sau:

- Tng hp dn cht 3-(4-nitrophenyl)hydantoin bng phn ng ng

vng gia glycin v 4-nitrophenyl isocyanat:

H2C COOH + O2N N=C=ONH2

1.KOH/H2O

-H2O HCl 25%,t0

O2N NH

NH

OH

O

O

O2N N

NHO

O

(A)

(B)

2.HCl

- Tng hp cc dn cht 3-aryl-5-arylidenhydantion bng phn ng

ngng t sn phm trn vi cc aldehyd thm.

RCHO O2N N

NHO

O+ O2N N

NHO

O

R

(B)

R = -H (I); -4-Cl (II); -4-NO2 (III); -3-NO2 (IV); -4-F (V)

3.1.1 Tng hp 3-(4-nitrophenyl) hydantoin

Gm 2 giai on:

- Tng hp N-((4-nitrophenylamino)carbonyl)glycin (A)

- ng vng to dn cht 3-(4-nitrophenyl)hydantoin (B)

3.1.1.1 Tng hp N-((4-nitrophenylamino)carbonyl)glycin (A)

Cng thc:

18

O2N NH

NH

OH O

O

CTPT: C9H11N3O4,KLPT: 225,20

Tin hnh:

Ha tan 0,68g (0,0103mol) KOH vo 8ml H2O, sau lm lnh n 100C.

Cho t t 0,75g (0,01mol) glycin vo hn hp trn, khuy cho tan ht,

thm 4ml 1,4-dioxan.

Sau thm t t 1,8g (0,011mol) 4-nitrophenyl isocyanat, trong qu

trnh cho ha cht vn lm lnh 100C.

Hn hp phn ng c khuy u trong 24h, duy tr nhit thng.

Theo di phn ng bng SKLM vi dung mi khai trin l MeOH.

Thm 70ml H2O vo hn hp khuy u, sau lc qua giy lc thu c

dch lc.

Acid ha dch lc bng HCl (2N), lc qua giy lc thu c ta, ra ta

bng nc v sy kh.

Khi lng sn phm: 1,42g cht rn mu vng.

Hiu sut: 55,69%.

Nhit nng chy: 209-2100C.

Rf = 0,55 (TLC, silicagel 60 F254, dung mi khai trin MeOH).

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

3,817 (2H,d,J=5,5,CH2); 6,675(1H,t,J=6,N1-H); 7,621( 2H,d,J=9,5; H2,6);

8,137(2H,d,J=9,5, H3,5); 9,602(1H,s,N3-H); 12,650( 1H,s,OH).

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

8,518(s;1H;N1-H); 8,324(d;2H;H3,5); 7,728(d;2H;H2,6); 4,104(s;2H;H5)

19

3.1.1.2 Tng hp 3-(4-nitrophenyl) hydantoin (B)

Cng thc:

O2N N

NHO

O

CTPT: C9H7N3O4, KLPT: 221,17

Tin hnh:

1,05g (0,0044mol) cht A c phn tn trong acid hn hp 9ml dung

dch HCl 25%.

un hi lu trong vng 4 gi.

Theo di phn ng bng SKLM vi h dung mi khai trin l

EtOAc/MeOH (4:1).

Sau hn hp phn ng c lm lnh v lc, ht kit v ra ta nhiu

ln cho ti khi ht acid (th bng giy qu).

Sau ra vi ln bng cn lnh. Sy kh ta thu c. Kt tinh li trong

dung h dung mi EtOH:DMF (5:1).

Khi lng sn phm: 0,7g cht rn mu trng.

Hiu sut: 67,88%.

Nhit nng chy: 249,3-252,80C (ph hp vi ti liu tham kho [13]).

Rf = 0,43 (TLC, silicagel 60 F254, h dung mi EtOAc/MeOH (4:1).

Ph IR (KBr), max (cm-1) : xem ph lc 2.1.

Ph 1H-NMR(500MHz, DMSO-d6), (ppm): xem ph lc 2.2.

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

3.1.2 Tng hp cc dn cht 3-aryl-5-arylidenhydantoin

Quy trnh tng hp chung

20

Cho vo bnh cu 0,03 mol aldehyd thm; 0,66g (0,03 mol) cht B; 0,74

(0,09 mol) natri acetat v 5ml acid acetic. Khuy u cho hn hp tan hon

ton, un nng hn hp, duy tr nhit phn ng 150-1600C. Xc nh

thi gian phn ng l 6 gi. 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 li trong dung mi

thch hp. Theo di phn ng bng SKLM vi h dung mi khai trin

cloroform : methanol (9: 0,5). Sau y l kt qu c th:

1. 5-benzyliden-3-(4-nitrophenyl)hydantoin (cht I) Cng thc

O2N N

NHO

O

CTPT: C16H11N3O4; KLPT: 309, 28

Tin hnh:

0,32g benzaldehyd (0,003mol) 0,66g cht B (0,003 mol)- 0,74g natri

acetat (0,009 mol) 5ml acid acetic. un hi lu trong vng 6 ting, kt tinh

li trong hn hp dung mi EtOH:DMF (5:1).

Sn phm thu c l 0,56g cht kt tinh mu vng.

Hiu sut 54,88%.


Rf = 0,75 (TLC, silicagel 60 F254, h dung mi CHCl3 : MeOH (9:0,5)).

Ph LC-MS: [M-H]- (tnh ton: 309,28 [C16H11N3O4], tm thy 308,0)

(xem ph lc 3.4).



21


2. 5-(4-clorobenzyliden)-3-(4-nitrophenyl)hydantoin (cht II) Cng thc:

O2N N

NHO

O

Cl

CTPT:C16H10ClN3O4; KLPT: 343,72

Tin hnh:

0,42g 4-clorobenzaldehyd (0,003mol) 0,66g cht B (0,003 mol) - 0,74g

natri acetat (0,009 mol) 5ml acid acetic. un hi lu trong vng 6 ting, kt

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

Sn phm thu c l 0,61g cht kt tinh mu vng nht.

Hiu sut 59,45%.



Ph LC-MS: [M-H]- (tnh ton: 343,72 [C16H10ClN3O4], tm thy 342,0)

(xem ph lc 4.4).




3. 5-(4-nitrobenzyliden)-3-(4-nitrophenyl)hydantoin (cht III) Cng thc

O2N N

NHO

O

NO2

CTPT:C16H10N4O6, KLPT: 354,27

Tin hnh

22

0,45g 4-nitrobenzaldehyd (0,003 mol) 0,66g cht B (0,003 mol)- 0,74g



Sn phm thu c l 0,60g cht kt tinh mu vng m.

Hiu sut 56,82%.

Nhit nng chy 264-2650C.



(xem ph lc 5.4).

Ph IR (KBr), max (cm-1): xem ph lc 5.1.

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

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

4. 5-(3-nitrobenzyliden)-3-(4-nitrophenyl)hydantoin (cht IV) Cng thc:

O2N N

NHO

O NO2

CTPT:C16H10N4O6, KLPT: 354,27

Tin hnh:

0,45g 3-nitrobenzaldehyd (0,003 mol) 0,66g cht B (0,003 mol)- 0,74g



Sn phm thu c l 0,79g kt tinh mu trng.

Hiu sut 74,81%.

Nhit nng chy 245-2460C.


23


(xem ph lc 6.4).

Ph IR (KBr), max (cm-1): xem ph lc 6.1.

Ph 1H-NMR (500MHz, DMSO-d6), (ppm): xem ph lc 6.2.

Ph 13C-NMR (125MHz, DMSO-d6), (ppm): xem ph lc 6.3.

5. 5-(4-fluorobenzyliden)- 3-(4-nitrophenyl)hydantoin (cht V) Cng thc:

O2N N

NHO

O

F

CTPT:C16H10FN3O4; KLPT: 327,27

Tin hnh:

0,372g 4-fluorobenzaldehyd (0,003mol) 0,66g cht B (0,003 mol)-

0,74g natri acetat (0,009 mol) 5ml acid acetic. un hi lu trong vng 6

ting, kt tinh li trong hn hp dung mi EtOH:DMF (5:1).

Sn phm thu c l 0,58g tinh th mu vng nht.

Hiu sut 56,50%.



Ph LC-MS: [M-H]- (tnh ton: 327,27 [C16H10FN3O4], tm thy 326,0)

(xem ph lc 7.4).




3.2 XC NH CU TRC CA CC CHT TNG HP C

3.2.1 Ph hng ngoi

24

Ph hng ngoi c ghi trn my Perkin Elmer ti B mn ha vt liu,

Khoa ha hc,HKHTN, H Quc gia H Ni. 5 ph c ghi cc ph

lc 2.1 7.1. 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 c ghi trn my ph AVANCE

Spectrometer AV500 (BRUKER, c) trong dung mi DMSO-d6, cht

chun ni TMS ti Vin ha hc - Vin Khoa hc v Cng ngh Vit Nam.

By ph c ghi cc ph lc 1.2 7.2, 1.3 7.3. Kt qu phn tch s

liu ph cng hng t ht nhn proton v 13C c ghi bng 3.3, 3.4.

3.2.3 Ph khi lng

Ph khi lng (LC/MS) ghi trn my LS-MSD-Trap-SL, ti Vin

ha hc, Vin khoa hc v cng ngh Vit Nam. 5 ph c ghi cc

ph lc 3.4 7.4. Kt qu ph MS c ghi bng 3.1.

3.3 TH NGHIM HOT TNH KHNG CC DNG T BO UNG

TH NGI

Ti khoa Dc l- Sinh ha (Vin dc liu), 5 cht do chng ti tng

hp c th hot tnh khng cc dng t bo ung th ngi theo phng

php s dng thuc nhum MTT Gi tr IC50 c tnh bng phn tch hi

quy khng tuyn tnh ca ng cong p ng liu tng ng, s dng phn

mm Exell [32]. Kt qu c trnh by bng 3.5.

Cch tin hnh:

- Nui cy t bo: Cc dng t bo c lu gi trong nit lng, nh

thc v duy tr trong mi trng DMEM (Dulbecco's Modified Eagle

Medium) c b sung huyt thanh b ti 10%, dung dch khng sinh v

khng nm 1% (penicillin 50,000 units/L v streptomycin 50 mg/L). T

bo c nui cy cho pht trin ti mc khong 70%, thay mi trng

sch, t bo ny c dng lm th nghim.

25

- Mu th nghim: Ha mu th nghim vo dung dch DMSO 100% sao

cho nng gc ca cc mu l 2 mg/ml. Tip theo pha thuc nghin

cu thnh thang nng gm 5 nng 100, 50, 25, 12.5, v 6.25

g/ml. Nng ca thuc th c dng theo tiu chun sng lc

thuc chng ung th c ngun gc dc liu ca tc gi Teicher 1997

[10]. Ellepticine c s dng lm chng dng, vi nng th ln

lt l 50; 25; 12,5; 5,25; v 3,125 M. Dung dch DMSO 0,1% c

s dng lm chng m.

- Quy trnh nui cy:

1. Cho khong 2X104 t bo/ml mi trng nui cy (i vi cc dng

t bo MCF-7, Hep-G2, A549) v 4X104 t bo/ml mi trng nui

cy (i vi dng t bo HTC116). 2 ging trng lm chng

trng (blank control).

2. Cc t bo c nui trong mi trng 37oC v 5% CO2 cho php

t bo gn vo y mi ging ca a nui cy. Gi t bo trong 24

gi t bo n nh.

3. Sau 24 gi nui cy, thm 20 l thuc th v thuc chun cc

nng khc nhau vo mi ging. Mi nng c lp li 2

ging. Lc nh a nui cy thuc c tan hon ton trong mi

trng.

4. a nui cy t 48 gi (37oC, 5% CO2) cho php thuc pht huy

tc dng. Quan st t bo hng ngy bng knh hin vi.

5. Chun b dung dch MTT nng 5mg/ml.

6. Thm 20 l dung dch MTT vo mi ging ca a nui cy. Lc

nh cho MTT khuch tn u trong mi trng nui cy.

7. 37oC trong 3 gi MTT c chuyn ha.

8. Loi b mi trng trong cc ging ca a nui cy.

26

9. Hon tr formazan (sn phm chuyn ha MTT) bng 100 l

DMSO. Lc k formazan c th tan hon ton.

10. c mt quang bc sng 540 nm. Mt quang s phn nh

s lng t bo sng st.

- % t bo sng sau khi x l thuc so vi chng trng c tnh theo

cng thc nh sau:

% t bo sng = [OD540 ca t bo c x l] / [OD540 ca t bo

khng c x l thuc] x 100%.

- Cch tnh gi tr IC50: Gi tr IC50 c tnh bng phn tch hi quy

khng tuyn tnh ca ng cong p ng liu tng ng, s dng phn

mm Exell.

T bo: gm 4 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 A549 (t bo cung th phi).

- Dng t bo HTC116 (t bo ung th i trng).

Ha cht:

- MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)

ca hng in vitrogen.

- Adriamycin ca hng Sigma.

- DMSO ca hng sigma.

- V cc ha cht khc.

My mc thit b: my c ELISA ca hng Thermo Labsystems, knh

hin vi soi ngc, v cc my mc khc.

Cht chun dng tnh: Adriamycin c s dng lm chng dng.

27

Bng 3.1: Kt qu tng hp cc cht

STT

K

hiu

cht

M

(.v.C)

Thi gian

phn ng

(gi)

Dung mi kt

tinh li

Thc nghim

Nhit nng

chy (0C) Hiu sut (%) [M-H]-

1 I 309, 28 6 EtOH:DMF (5:1) 288-289 54,88% 308,0

2 II 343,72 6 EtOH:DMF (5:1) 271-272 59.45% 342

3 III 354,27 6 EtOH:DMF (5:1) 264-265 56,82% 353

4 IV 354,27 6 EtOH:DMF (5:1) 245-246 74,81% 353

5 V 327,27 6 EtOH:DMF (5:1) 262-263 56,50% 326

6 B 221,17 4 EtOH:DMF (5:1) 249-2520C 67,88%

28

Bng 3.2 Kt qu ph IR ca cc cht tng hp c

STT K hiu cht IR (KBr, cm

-1)

NH CHarom C2=O C4=O NO2 Caryl-X

1 I 3221 2929-2856 1752 1715 1523-1339

2 II 3238 2919-2850 1758 1723 1524-1338 1093(X-Cl))

3 III 3445 3195-3122 1773 1734 1516-1342

4 IV 3195 3087 1769 1725 1528-1345

5 V 3227 3099 1764 1726 1537-1345 1237(X-F)

6 B 3244 3116-3086 1787 1740 1525-1347

29

Bng 3.3 S liu ph 1H NMR ca cc cht tng hp c

Cng thc cu to chung:

O2N N

NHO

O

123 4

5

1"

2"3"

4"

5" 6"

(B) :

O2N N

NHO

O

123 4

5

R

1'

2' 3'

4'

5'6'

1"

2"3"

4"

5" 6"

(I-V)

STT K hiu cht 1H-NMR (DMSO-d6, ppm)

1 B 8,518(s;1H;N1-H); 8,320(d;2H;H3,5,J=9); 7,724(d;2H;H2,6,J=9); 4,104(s;2H;H5)

2 I 11,227(s;1H;N1-H); 8,391(d;2H;H3,5, J=9,5); 7,830(d;2H;H2,6, J=9,5); 7,719(d;2H;H2,6, J=7,5);

7,451(t;2H;H3,5,J=7.5); 7,383(t;1H;H4,J=7,5); 6,695(s;1H;CH)

3 II 11,278(s;1H;N1-H); 8,368(d;2H;H3,5,J=9); 7,812(d;2H;H2,6,J=9); 7,715(d;2H;H2,6,J=8,5);

7,487(d;2H;H3,5,J=8,5); 6,683(s;1H;CH)

4 III 11,538(s;1H;N1-H); 8,393(d;2H;H3,5,J=7,5); 8,240(d;2H;H3,5,J=9); 7,929(d;2H;H2,6,J=9);

7,835(d;2H;H2,6,J=7); 6,774(s;1H;CH)

5 IV 11,530(s;1H;N1-H); 8,471(s;1H; H2); 8,403(d;2H;H3,5,J=9,5); 8,178(d,1H,H4,J=8); 8,090(d,1H,H6,J=8); 7,824(d,2H,H2,6,J=9,5); 7,709(t,1H,H5,J=8), 6,836(s;1H;CH)

6 V 811,245(s;1H;N1-H); 8,379(d;2H;H3,5,J=9); 7,833(d;2H;H2,6,J=9); 7,773(t;2H;H2,6,J=5,5);

7,274(t;2H;H3,5,J=9); 6,712(CH)

30

Bng 3.4 S liu ph 13C NMR ca cc cht tng hp c

Cng thc cu to chung

O2N N

NHO

O

123 4

5

1"

2"3"

4"

5" 6"

(B) :

O2N N

NHO

O

123 4

5

R

1'

2' 3'

4'

5'6'

1"

2"3"

4"

5" 6"

(I-V)

STT K hiu

cht 13C-NMR (DMSO-d6, ppm)

1 B 170,705(C-4); 155,573(C-2); 145,716(C-4); 138,171(C-1); 126,594(C-2;6);124,016(C-3;5); 46,053(C5)

2 I 162,78(C-4); 153,317(C-2); 146,009(C-4); 137,712(C-1); 132,655(C-1); 129,622( C-2,6); 128,867(C-

3,4,5); 126,947(C-2,6); 126,023( C-5); 124,128(C-3,5);110,684(CH)

3 II 162,658(C-4); 153,285(C-2); 146,033(C-4); 137,624(C-1); 133,321(C-1); 131,602(C-4); 131,242 ( C-2,6);

128,803(C-3,5); 126,896(C-2,6); 126,524( C-5); 124,110(C-3,5);109,218(CH)

4 III 162,555(C-4); 153,450(C-2); 146,528(C-4); 146,139(C-4); 139,589 (C-1); 137,465(C-1);130,379( C-2,6);

129,010 ( C-5); 126,954(C-2,6); 124,165(C-3,5); 123,799(C-3,5); 107,423 (CH)

5 IV 162,532(C-4); 153,495(C-2); 148,225(C-3); 146,084(C-4); 137,553(C-1); 135,407( C-6); 134,535(C-1);

130,200(C-5); 128,389(C-5); 126,974(C-2,6); 124,168(C-3,5); 123,887(C-2); 122,962(C-4)

6 V 162,748(C-4); 161,103(C-4); 153,340(C-2); 146,015(C-4); 137,685(C-1); 131.921(C-2); 131,854(C-6);

129,268(C-1); 126,939(C-2,6); 125,809(C-5); 124,135(C-3,5);115,968(C-3); 115,797(C-5); 109,620(CH)

31

Bng 3.5: Tc dng ca cc mu th nng 100 g/ml trn mt s dng t bo ung th

STT K hiu

mu

Dng t bo % sng st Kt lun

A549 MCF-7 Hep-G2 HTC116

1 DMSO 100 100 100 100

2 Adriamycin 29,2 16,8 26,9 17,5 Dng tnh

3 I 74,9 108,7 70,8 56,3 m tnh

4 II 85,0 116,7 80,3 51,4 m tnh

5 III 87,6 178,5 87,7 52,0 m tnh

6 IV 87,9 158,4 79,9 46,3 Dng tnh trn 1 dng HTC116

7 V 68,3 270,3 67,3 44,3 Dng tnh trn 1 dng HTC116

8 B 79,8 81,4 82,2 52,5 m tnh

32

3.4 BN LUN

3.4.1. V tng hp ha hc

3.4.1.1 V phn ng cng hp to 3-(4- nitrophenyl)hydantoin (B)

Phn ng gm c 2 giai on:

Cng hp i nhn.

ng vng.

C ch phn ng c th gii thch nh sau:

Phn ng cng hp i nhn: glycin phn ng vi kali hydroxyd to mui glycinat a nhm NH2 v dng t do l tc nhn i

nhn.

Giai on ng vng (HCl 25%, t0) tng t cc phn ng ng vng khc.

H2C COOK

NH2

H2C COOH

NH2

+ KOH

H2C COOK

NH2

+ O2N N=C=O N C O

N

CH2KOOC

O2N

HH

O2NHN C

NHCH2

KOOC

OO2N NH C

NH

CH2COH

O

OHClO2N N

NHO

O-H2O

HCl/t0

3.4.1.2 V phn ng ngng t:

33

Da trn cc ti liu tham kho c [1], [5], [20], [21], [27], [30],

[35] phn ng ngng t aldehyd thm vi hydantoin c th xy ra vi cc

iu kin sau:

- Xc tc natri acetat trong acid acetic c, t0= 130-1350C.

- Xc tc amonicarbonat trong acid acetic bng.

- Xc tc ethanolamin trong ethanol, t0=900C.

Chng ti tin hnh kho st thm d phn ng ngng t ca aldehyd

thm vi 3-(4-nitrophenyl)hydantoin vi cc iu kin.

- Xc tc natri acetat trong acid acetic c, t0= 130-1350C.

- Xc tc ethanolamin trong ethanol, t0=900C.

Cc kt qu kho st ca chng ti nhng iu kin trn u khng thu

c sn phm ngng t mong mun.

Da trn quy trnh tng hp 3-aryl-5-arylidenhydantoin ca Lima nm

1992 [35], vi iu kin phn ng ngng t ca 3-arylhydantoin v aldehyd

thm nhit 145-1550C, chng ti tin hnh phn ng vi xc tc natri

acetat trong acid acetic c nhng nng nhit ln 150-1600C th thu c

sn phm ngng t mong mun.

Nh vy iu kin nhit l iu kin quan trng t c kt qu

ca phn ng ngng t cc aldehyd thm vi 3-arylhydantoin.

Kt qu thc nghim trn cho thy kh nng phn ng ca nhm CH2

dn cht 3-(4-nitro phenyl)hydantoin km hn so vi cc hydantoin khng

th. Kt qu ny c th gii thch nh sau: do nhm 4-nitrophenyl ht in t

lm lch orbital v cc orbital p C ca nhm C4=O do lm gim kh

nng siu lin hp ca cc lin kt C-H ca C5 v tr so vi C4=O, do

lm gim tnh linh ng ca hydro trong nhm CH2 ny. V vy kh nng

tch proton trong giai on cng hp ca c ch phn ng ngng t xy ra

kh khn hn

34

NH

N N

O

O

O

H

H

O

12

34

5

3.4.2 V xc nh cu trc ca cc cht tng hp c

3.4.2.1 V ph hng ngoi (IR)

6 cht tng hp c c ghi ph hng ngoi. Qua nghin cu ph

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

nhm chc v lin kt ca cc cht tng hp c [7].

- Ph hng ngoi ca 6 cht (B, I-V) u c xut hin cc di hp th

mnh tng ng vi dao ng ho tr ca nhm carbonyl C2=O v C4=O, nm

trong vng t 1787-1752 cm-1 v 1740-1715cm-1

.

- Trn ph ca ca 6 cht (B, I-V) u c xut hin mt di hp th

tng ng vi dao ng ho tr ca nhm NH (imid) ca vng hydantoin,

nm trong vng t 3245-3195 cm-1.

- Trn ph ca 6 cht (B, I-V) u c xut hin di hp th tng

ng vi dao ng ho tr ca nhm CHarom , nm trong vng t 3195-2850

cm-1

.

- Trn ph IR ca cc cht (B, I-V) u xut hin hai di hp th mnh

c trng cho dao ng ho tr lin kt v phn lin kt ca nhm NO2 , nm

trong vng t 1537-1516cm-1 v 1347-1338 cm-1

.

- Trn ph IR ca cht II xut hin di hp th s sng 1093 cm-1

c

trng cho dao ng ho tr ca Cl lin kt trc tip vi nhn thm Caryl-Cl

v tr para c mt trong cu trc ca cht ny.

- Trn ph IR ca cht V xut hin di hp th s sng 1237cm-1

c

35

trng cho dao ng ho tr ca F lin kt trc tip vi nhn thm Caryl-F v

tr para c mt trong cu trc ca cc cht ny.

Cc s liu trn trng vi ti liu tham kho c [7]. y l cc tn hiu

quan trng cho php s b khng nh s to thnh sn phm ca phn ng

iu ch dn cht th 3-arylhydantoin v phn ng ngng t gia hydantoin

v cc aldehyd thm (xem bng 3.2, ph lc 1.1-6.1).

3.4.2.2 V ph cng hng t ht nhn (1H-NMR)

Ph1 H-NMR cho thy cc tn hiu c trng ca cc proton v lng

proton tng dng ca 6 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 quan h cu trc phn t v ph 1H-NMR ca cc cht tng

hp c. S tn hiu proton xut hin trong ph 1H-NMR bng vi s

nguyn t hydro c mt trong cu trc d kin.

- Ph 1H-NMR cho thy tn hiu ca proton H-N1 (tn hiu t, cng

yu) xut hin trong vng 8,518-11,588 ppm (xembng 3.3).

- Ph 1H-NMR ca 5 cht I-V u cho thy tn hiu proton ethylenic (H-

6) to thnh t phn ng ngng t ca 3-arylhydantoin v aldehyd thm trong

vng 6,683-6,836 ppm

- Trn ph 1H-NMR ca cc cht cn xut hin cc tn hiu c trng

ca proton nhn thm hp phn aldehyd. (Bng 3.3, ph lc 1.2-6.2).

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

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

c trnh by bng 3.4 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.

- Nguyn t carbon nhm C=O (amid) l C-4 c tn hiu cng hng

trong vng t 162,532-170,705 ppm .

36

- Nguyn t carbon nhm C=O (imid) l C-2 c tn hiu cng hng

trong vng t 153,285-155,573 ppm .

- Nguyn t carbon ethylenic l C-5 c tn hiu cng hng trong vng

125,809-128,389 ppm

- Ph 13C-NMR ca cc cht cho thy s carbon v dch chuyn

ha hc c trng ca carbon nhn thm hp phn aldehyd. (Bng 3.4, ph

lc 1.3-6.3).

3.4.2.4 Ph khi lng (MS)

khng nh thm v cu trc ca cc 3-aryl-5-arylidenhydantoin

chng ti tin hnh ghi ph MS (LC-MS) ca cc hp cht ny. T cc kt

qu ghi ph thu c c th nhn thy 6 cht tng hp c u xut hin pic

phn t c cng phn t ln, khi lng ca ion phn t ph hp vi

khi lng phn t d on ca cht d kin.

3.4.3 V tc dng khng t bo ung th.

Kt qu th cho thy

C 2 cht IV, V c tc dng c ch dng t bo HTC116 nng 100g/ml ( % dng t bo sng st ln lt l 46,3%; 44,3%).

Ring cht V c xc nh IC50= 10,99g/ml i vi HTC116

c coi l c hot tnh khng HTC116.

Cht B khng c hot tnh vi 4 dng t bo. T kt qu trn cho thy hp cht 5-aryliden c th mang li hot tnh

khng t bo ung th khi c thm nhm th ht in t nh m-NO2, p-F.

c nhiu cng trnh nghin cu v tng hp v th tc dung khng t

bo ung th ca dn cht 5-arylidenhydantoin c cng b [1], [5]. Theo

tc gi Trn Anh [1] cho thy trong s 43 cht tng hp c c 13 cht (VI,

VIa, VIb, VII, VIIb, VIIc, VIId, VIII, VIIIa, VIIIb, VIIId, IX, IXa) c

37

hot tnh khng t bo ung th ngi Hep-G2 (IC50=0,385g/ml) trong

cht c hot tnh mnh nht l cht VIIc (IC50=0,38 g/ml).

Bng 3.6 Gi tr IC50 (g/ml) ca cc dn cht hydantoin trn Hep-G2 [1]

K

hiu

Tn cht

Gi tr IC50

(g/ml) trn

Hep-G2

VI 5-(2-clorobenzyliden)hydantoin 2,72

VIa 5-(2-clorobenzyliden)-3-morpholinomethylhydantoin 4,22

VIb 5-(2-clorobenzyliden)-3- piperidinomethylhydantoin 4,31

VII 5-(4-clorobenzyliden)hydantoin 4,69

VIIb 5-(4-clorobenzyliden)-3- piperidinomethylhydantoin 1,53

VIIc 5-(4-clorobenzyliden)-3-(N-methylpiperazino)methylhydantoin 0,38

VIId 5-(4-clorobenzyliden)-3-(4-

methylphenyl)aminomethylhydantoin

2,89

VIII 5-(3-nitrobenzyliden)hydantoin (VIII) 5

VIIIa 5-(3-nitrobenzyliden)-3-morpholinomethylhydantoin 4,31

VIIIb 5-(3-nitrobenzyliden)-3- piperidinomethylhydantoin 3,71

VIIId 3-(4-methylphenyl)aminomethyl-5-(3-

nitrobenzyliden)hydantoin

1,99

IX 5-(4-nitrobenzyliden)hydantoin 3,70

IXa 3-morpholinomethyl-5-(4-nitrobenzyliden)hydantoin 2,77

Tc gi Th Thu Hng [5] tng hp v th hot tnh khng t bo ung th

ca 15 cht 5-halogenoarylidenhydantoin v base Mannich, kt qu cho thy c 11

cht (I(a-c),II, II(a-d),IIIa, IIIb, IIId) c hot tnh khng t bo ung th i trng

HCT116 vi IC50=10,1719,23g/ml.

38

Bng 3.7: Gi tr IC50 (g/ml) ca cc dn cht hydantoin trn HTC116 [5]

K hiu

Tn cht

Gi tr IC50 (g/ml) trn

HTC116 Ia 5-(3-fluorobenzyliden)-3-morpholinomethyl hydantoin 16,63 Ib 5-(3-fluorobenzyliden)-3-morpholinomethylhydantoin 17.31 Ic 5-(3-fluorobenzyliden)-3- piperidinomethylhydantoin 18,22 II 5-(3-clorobenzyliden)hydantoin 10.17 IIa 5-(3-clorobenzyliden)-3-morpholinomethylhydantoin 16,38 IIb 5-(3-clorobenzyliden)-3- piperidinomethylhydantoin 13,42 IIc 5-(3-clorobenzyliden)-3-(N-

methylpiperazino)methylhydantoin 17,96

IId 5-(3-clorobenzyliden)-3-(4-methylphenyl)aminomethylhydantoin 11,13

IIIa 5-(3-bromobenzyliden)-3-morpholinomethylhydantoin 16,37 IIIb 5-(3-bromobenzyliden)-3- piperidinomethylhydantoin 19.23 IIId 3-(4-methylphenyl)aminomethyl-5-(3-

bromobenzyliden)hydantoin 13,77

So snh kt qu th hot tnh khng t bo ung th ca cc dn cht 3-

aryl-5-arylidenhydantoin vi cc dn cht 5-arylidenhydantoin cng b [1]

th thy cc dn cht 3-aryl-5-aryliden hot tnh km hn i vi dng t bo

ung th gan [1]v km hn cc dn cht 3-halogenoarylidenhydantoin tc

dng i vi dng t bo HTC116. iu ny chng t nhm 3-aryl khng

mang li kt qu thun li v hot tnh khng t bo ung th gan v HTC116.

Khi tin hnh th nghim hot tnh khng t bo ung th, nhn thy cc cht

tng hp c c kh nng ha tan rt km nn c th hn ch tc dng trn

t bo ung th th nghim.

39

CHNG 4. KT LUN V KIN NGH 4.1 KT LUN

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

s kt lun nh sau:

a) tng hp c 5 dn cht 3-aryl-5-arylidenhydantoin (I-V) cha

thy cng b trong cc ti liu tham kho c

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 (13C-NMR), ph khi lng (MS).

Kt qu thu c cho php chng ti kt lun cc cht tng hp c c cu

trc ng nh d kin.

c) th tc dng khng t bo ung th ca 6 cht tng hp c i

vi 4 dng t bo ung th ngi l t bo ung th phi A549, t bo ung th

gan HepG2, t bo ung th i trng HCT116 v t bo ung th biu m v

MCF7. Kt qu cho thy c 2 cht IV v V hot tnh khng t bo ung th

i trng HCT116. C 6 cht u khng c tnh khng t bo ung th gan

Hep-G2,t bo ung th phi A549 v t bo ung th biu m v MCF7.

4.2 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 khng t bo ung th ca cc cht tng

hp c trn cc dng t bo khc nh : ung th tin lit tuyn, ung th d

dy

Tip tc tng hp v sng lc hot tnh chng ung th ca cc dn cht 3-aryl-5-arylidenhydantoin khc nhm tm kim cc cht c trin vng trong dy cht ny.

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

sng, NXB Y hc, tr 289-300.

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

tr.393-394.

4. 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.

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

ung th ca mt s dn cht 5-halogenoarylidenhydantoin, Lun vn

thc s Dc Hc,trng i hc dc H Ni

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

dng, NXB Y hc.

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

ha hc, NXB i hc Quc gia H Ni

Ting Anh

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

Encyclopedia of Chemical Technology, Vol. 12, 3rd Ed., New York,

John Wiley, 672-700

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

effective and ignored antimicrobial, International Joural of

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

10. Beverly A. Teicher.(1997) Anticancer drug development guide:

Preclinical screening, clinical trials, and approval. Humana Press,

Totowa, New Jersey, 1997

11. (a) A. Baeyer, Justus Liebigs Ann. Chem., 119, 126 (1861). (b) A.

Baeyer, Jusrus Liebigs Ann. Chem., 117,178 (1861). (c) A. Baeyer,

Justus LiebigsAnn. Chem., 130,129 (1864).

12. Carmi, C. and al. (2006), 5-Benzylidene-hydantoins as new EGFR

inhibitors with antiproliferative activity, Bioorganic and Medical

Chemistry Letters,16, 4021-4025.

13. Dakin, H.D (1942), "On Lysine and ornithine ", J. Biol. Chem., 146,

237 240.

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

properties of substituted 4-dimethylaminomethyl-1-phenyl-1-nonene-

3-ones possessing antitumor properties, Journal of Pharmaceutical

Sciences, 63(1), 69-74

15. Inaba, K.; Sato, H. and al. (1998), Spongiacidins A-D, New

Bromopyrrole Alkaloids from Hymeniacidon Sponge, J. Nat. Prod.,

61(5), 693-695.

16. Kashanian, J.;Hakiamian, P. and al. (2008), Nitrofurantoin as a

first-line in uncomplicated UTIs, The Journal of Urology,179 (4),

84

17. Kazlauskas, R.; Murphy, P.T and al. (1977), Aplysinopsin, a new

tryptophan derivative from a sponge, Tetrahedron Letters, 18 (1),

61-64.

18. Lamiri, M. and al. (2006), Microwave-Assisted Solvent-Free

Regiospecific Synthesis of 5- alkylidene and 5- arylidenhydatoin,

Synthetic Communications,36,1575-1584.

19. Martidale (2005), The complete drug refrence, 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. Meusel, M.; Gtschow, M. (2004), Recent developments in

hydantoin chemistry, Organic Preparations and Procedures

International : The New Journal for Organic Synthesis, 36(5), 391-

443.

22. Mosmann T. Rapid colorimetric assay for cellular growth and

survival: application to proliferation and cytotoxicity assays. J

Immunol Methods: 65, 55-63, 1983.

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

synthesis of anti-metastatic lead phenylmethylene hydantoins inspired

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

24. Poupaert, J. H.; Vandervorst, D. and al. (1984), Structure- Activity

Relationships of Phenytoin like Anticonvulsant Drugs, J. Med.

Chem., 27, 76-78.

25. Smith, M.B. (2007), Marchs advanced organic chemistry, Reaction,

Mechanism, and Structure, 6th edition, Wiley-interscience, John

Wiley and sons, Inc., publication.

26. Szymanska, E.; Kononowicz, K.K. (2002), Antimycobacterial

activity of 5-arylidene aromatic derivates of hydantoin, Il Farmaco,

57, 355-362.

27. Thenmozhiyal, J.C. and al. (2004), Anticonvulsant Activity of

Phenylmethylenehydantoins: A Structure- Activity relationship

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

28. Tietze, L.F.; Beifuss, U. (1991), Comprehensive Organic Synthesis,

Trost, B.M., Ed, Pergamon: Elmsford, NY, 1991, Vol.2, 341-394.

29. Ware. E. (1950), Bucherer-Berg reaction, Chem.Rev.46, 422

30. Wheeler, H.L and al. (1911), On hydantoins : A synthesis of

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

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

phenylenehydantoin and its methoxy substituted derivaties, Japhan

J. Pharmacol., 49, 309-315.

32. Zhao Ming, Peng Shiqi (2009), Pharmacetical bioassays: Methods

and applications, John Wiley and sons, Inc

33. Zuliani, V.; Carmi, C. and 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

34. Urech, F. (1873), Ueber Lacturaminsure und Lactylharnstoff,

Justus Liebigs Ann Chem, 165, 1, 99-103.

Ting Php

35. De Lima, J.G. (1992) Thiazolidinediones et imidazolidinediones

substitues: synthse, stucture, tude pharmacologique et relations

quantitatives structure-activit, These grade de Docteur, de

IUniversite Joseph-Fourier GRENOBLE 1

PH LC 8.PHIU TR LI KT QU TH HOT TNH KHNG T BO UNG TH

Ch thch:

K hiu Cht

D1 I

D2 II

D3 III

D4 IV

D5 V

Ph lc 1.2 Ph 1H ca cht A

O2N NH

NH

OH O

O

Ph lc 1.3 Ph 13C ca cht A

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

TRAN ANH-MAU BDate: 4/2/2013

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.00.0

10

20

30

40

50

60

70

80

90

101.3

cm-1

%T

3244

31163086

1787

1740

16131595

1525

1503

14471407

1377

1347

1325

1246

1179

11101093

1055

854

755744

722685

652

O2N N

NHO

O

123 4

5

Ph lc 2.1 ph IR ca cht B

Ph lc 2.2 Ph 1H ca cht B

O2N N

NHO

O

Ph lc 2.3 Ph 13C ca cht B

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


Nguoi do: Phan Thi Tuyet MaiTen mau: D1Date: 4/1/2013

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.080.0

82

84

86

88

90

92

94

96

98

100

102

104104.8

cm-1

%T

34383221

29292856

1752

1715

1654

1595

15231497

1455

14001339

13111265

12391185

11201107

949928

889876

853

804

768

743730

685

661

NO2 N

NHO

O

123 4

5

Ph lc 3.1 ph IR ca cht I

Ph lc 3.2 Ph 1H ca cht I

Ph lc 3.3 Ph 13C ca cht I

Ph lc 3.4 ph MS ca cht I



Nguoi do: Phan Thi Tuyet MaiTen mau: O2Date: 4/1/2013

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.00.0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

99.2

cm-1

%T

3446

3238

3128

29192850

1758

1723

1659

1608

1590

1524

1494

13951338

1313

1238

11821122

1093

1011

973953

930

914884

853

822

792

772

756

731

712

685652

NO2 N

NHO

O

123 4

5 Cl

Ph lc 4.1 ph IR ca cht II

Ph lc 4.2 Ph 1H ca cht II

Ph lc 4.3 Ph 13C ca cht II

Ph lc 4.4 ph MS ca cht II




4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.00.0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100103.1

cm-1

%T

3445

31953122

1773

1734

1667

1596

15161394

1342

1237

1191

1128927

889

856

829

761743

684

NO2 N

NHO

O

123 4

5 NO2

Ph lc 5.1 ph IR ca cht III

Ph lc 5.2 Ph 1H ca cht III

Ph lc 5.3 Ph 13C ca cht III

Ph lc 5.4 ph MS ca cht III




4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.020.0

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

101.6

cm-1

%T

31933086

1769

1725

1669

1597

1528

1499

1406

1345

12401191

1122

1106

950911

890

854

828807

759733

707

675

658

NO2 N

NHO

O

123 4

5

NO2

Ph lc 6.1 ph IR ca cht IV

Ph lc 6.2 Ph 1H ca cht IV

Ph lc 6.3 Ph 13C ca cht IV

Ph lc 6.4 ph MS ca cht IV

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

Mail: [email protected]: 4cm-1


TRAN ANH-D5Date: 4/2/2013

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.00.0

10

20

30

40

50

60

70

80

90

100

106.4

cm-1

%T

3227

3099

1764

1726

1663

1613

15971537

15151499

14121345

1313

1237

11911164

11191105

899

852828

803

755727

657

NO2 N

NHO

O

123 4

5 F

Ph lc 7.1 ph IR ca cht V

Ph lc 7.2 Ph 1H ca cht V

Ph lc 7.3 Ph 13C ca cht V

Ph lc 7.4 ph MS ca cht V

Nguyn tc:- p dng phng php MTT c bin son bi tc gi Mosmann (1983) nh gi c tnh trn t bo [22]. Nguyn tc ca phng php nh sau: MTT l th nghim in vitro o s tng sinh v sng st ca t bo. T bo c nui cy trong m...CTPT: C9H11N3O4,KLPT: 225,208,518(s;1H;N1-H); 8,324(d;2H;H3,5); 7,728(d;2H;H2,6); 4,104(s;2H;H5)3.1.1.2 Tng hp 3-(4-nitrophenyl) hydantoin (B)CTPT: C9H7N3O4, KLPT: 221,17Ph IR (KBr), max (cm-1) : xem ph lc 2.1.Ph 1H-NMR(500MHz, DMSO-d6), (ppm): xem ph lc 2.2.3.1.2 Tng hp cc dn cht 3-aryl-5-arylidenhydantoinCTPT: C16H11N3O4; KLPT: 309, 28Tin hnh:Hiu sut 54,88%.Ph IR (KBr), max (cm-1) : xem ph lc 3.1.Ph 1H-NMR(500MHz, DMSO-d6), (ppm): xem ph lc 3.2.CTPT:C16H10ClN3O4; KLPT: 343,72Hiu sut 59,45%.Ph IR (KBr), max (cm-1) : xem ph lc 4.1.Ph 1H-NMR(500MHz, DMSO-d6), (ppm): xem ph lc 4.2.Ph 13 C-NMR (125MHz, DMSO-d6), (ppm) : xem ph lc 4.3.Cng thcCTPT:C16H10N4O6, KLPT: 354,27Tin hnhHiu sut 56,82%.Ph IR (KBr), max (cm-1): xem ph lc 5.1.Ph 1H-NMR (500MHz, DMSO-d6), (ppm): xem ph lc 5.2.CTPT:C16H10N4O6, KLPT: 354,27Tin hnh:Hiu sut 74,81%.Ph IR (KBr), max (cm-1): xem ph lc 6.1.Ph 1H-NMR (500MHz, DMSO-d6), (ppm): xem ph lc 6.2.CTPT:C16H10FN3O4; KLPT: 327,27Hiu sut 56,50%.Ph IR (KBr), max (cm-1) : xem ph lc 7.1.Ph 1H-NMR(500MHz, DMSO-d6), (ppm): xem ph lc 7.2.3.2.1 Ph hng ngoi3.2.3 Ph khi lngNui cy t bo: Cc dng t bo c lu gi trong nit lng, nh thc v duy tr trong mi trng DMEM (Dulbecco's Modified Eagle Medium) c b sung huyt thanh b ti 10%, dung dch khng sinh v khng nm 1% (penicillin 50,000 units/L v strepto...Mu th nghim: Ha mu th nghim vo dung dch DMSO 100% sao cho nng gc ca cc mu l 2 mg/ml. Tip theo pha thuc nghin cu thnh thang nng gm 5 nng 100, 50, 25, 12.5, v 6.25 g/ml. Nng ca thuc th c dng theo tiu chun s...- Quy trnh nui cy:T bo: gm 4 dng t bo:Ha cht:Bng 3.3 S liu ph 1H NMR ca cc cht tng hp cCng thc cu to chung:Cng thc cu to chung

tổng hợp và thử hoạt tính sinh học của 3-(4-nitrophenyl) - hydantoin và một số...

Documents