Tổng Hợp Và Thử Hoạt Tính Sinh Học Của Một Số Dẫn Chất 5-Aryliden-2-Thiohydantoin

B GIO DC V O TO
B Y T
TRNG I HC DC H NI
TRN C LAI
TNG HP V TH HOT TNH

SINH HC CA MT S DN CHT
5-ARYLIDEN-2-THIOHYDANTOIN
LUN VN THC S DC HC
H NI 2014
B GIO DC V O TO
B Y T
TRNG I HC DC H NI
TRN C LAI
TNG HP V TH HOT TNH

SINH HC CA MT S DN CHT
5-ARYLIDEN-2-THIOHYDANTOIN
LUN VN THC S DC HC
CHUYN NGNH: CNG NGH DC PHM V BO
CH
M S: 60720402
Ngi hng dn khoa hc:
TS. V Trn Anh

PGS. TS. Nguyn Quang t
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 v PGS.TS Nguyn Quang t ht lng hng dn, ch bo,
to mi iu kin thun li v ng vin ti hon thnh lun vn ny. Ti xin
chn thnh cm n PGS.TS. inh Thanh Hi v ton th cc thy c gio,
k thut vin ca B mn Ha hu c ng vin gip ti trong thi
gian nghin cu ti b mn.
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), TS
Phan Th Nguyt Hng (Vin Dc liu), TS. Trn Th Nh Hng (Vin
Ha hc cc hp cht thin nhin), TS. Nguyn Th Sn (B mn Ha vt
liu, khoa Ha, trng i hc Khoa hc T nhin, i hc Quc gia H
Ni), PGS.TS L Mai Hng (Vin Ha hc cc hp cht thin nhin-Vin
Hn lm khoa hc Vit Nam) v 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 lun vn 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 lun vn 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
thng
Hc Vin
Trn c Lai
nm 2014
MC LC
Trang
DANH MC CC K HIU, CH VIT TT
DANH MC CC BNG
DANH MC CC HNH V, TH
T VN
CHNG 1. TNG QUAN
1.1 Tc dng sinh hc ca cc dn cht 2-thiohydantoin
1.1.1 Tc dng c ch t bo ung th
1.1.2 Tc dng khng khun, khng nm
1.1.3 Cc tc dng khc
1.2. Cc phng php tng hp 2-thiohydantoin v dn cht
1.2.1 Phn ng tng hp 2-thiohydantoin
1.2.2 Phn ng tng hp dn cht 5-aryliden-2-thiohydantoin
11
CHNG 2. NGUYN VT LIU, TRANG THIT B V
15
PHNG PHP NGHIN CU

2.1 Nguyn vt liu, ha cht, dung mi
15
2.2 Thit b th nghim
15
2.3 Phng php nghin cu
16
2.3.1 Phng php tng hp 2-thiohydantoin, cc 5-aryliden-2-
16
thiohydantoin.
2.3.2 Phng php xc nh cu trc
16
2.3.3 Phng php th hot tnh sinh hc
16
2.3.3.1 Th hot tnh gy c t bo ung th
16
2.3.3.2 Th tc dng khng khun, khng nm
20
CHNG 3. THC NGHIM V KT QU NGHIN CU
23
3.1 Tng hp ha hc
23
3.1.1 Tng hp 2-thiohydantoin
23
3.1.2 Tng hp mt s dn cht 5-aryliden-2-thiohydantoin
25
3.2 Xc nh cu trc ca cc cht tng hp c
35
3.2.1 Ph hng ngoi
35
3.2.2 Ph cng hng t ht nhn
35
3.2.3 Ph khi lng
36
3.3 Th hot tnh sinh hc
36
3.3.1 Th hot tnh gy c t bo ung th
36
3.3.2 Th tc dng khng khun, khng nm
37
CHNG 4. BN LUN
50
4.1 V tng hp ha hc
50
4.1.1 V phn ng tng hp 2-thiohydantoin
50
4.1.2 V phn ng ngng t to 5-aryliden-2-thiohydantoin
51
4.2 V xc nh cu trc ca cc cht tng hp c
54
4.2.1 V ph hng ngoi (IR)
55
4.2.2 V ph cng hng t ht nhn1H-NMR
56
4.2.3 V xc nh ng phn hnh hc ca dn cht 5-aryliden-2-
57
thiohydantoin.
4.2.4 V ph cng hng t ht nhn13C-NMR
58
4.2.5 V ph MS
61
4.3 V hot tnh sinh hc

4.3.1 V hot tnh gy c t bo ung th
62
4.3.2 V tc dng khng khun, khng nm
64
KT LUN V KIN NGH
65
TI LIU THAM KHO

DANH MC CC PH LC
PH LC
DANH MC CC K HIU, CH VIT TT

13
1
C-NMR
H-NMR
: Ph cng hng t ht nhn carbon 13

: 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
HCT116
: T bo ung th i trng 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
: Khiph
(Mass spectrometry)
PC3
: T bo ung th tuyn tin lit
DANH MC CC BNG
Bng
3.1 Kt qu tng hp 2-thiohydantoin (BT) v cc dn cht 5aryliden-2-thiohydantoin (I-XIII)
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
S liu ph 1H-NMR ca A, BM, BT

S liu ph IR ca cc dn cht 5-aryliden-2-thiohydantoin (IXIII)
S liu ph 1H-NMR ca cc dn cht 5-aryliden-2-thiohydantoin
(I-XIII)
S liu ph13C-NMR ca cc dn cht 5-aryliden-2-thiohydantoin
(I-XIII)
S liu ph ESI-MS ca cc dn cht 5-aryliden-2-thiohydantoin
(I-XIII).
Kt qu th hot tnh gy c t bo trn 3 dng t bo ung th
th nghim ca cc cht tng hp c (theo phng php SRB)
Kt qu xc nh gi tr IC50 (g/ml) ca mt s cht tng hp
c trn 3 dng t bo ung th th nghim ca cc cht tng
hp c (theo phng php SRB)
Kt qu th tc dng ca cc cht tng hp c nng 100
g/ml trn mt s dng t bo ung th ngi (theo phng php
MTT)
3.11
Kt qu xc nh gi tr IC50 (g/ml) ca mt s cht tng hp

c trn mt s dng t bo (theo phng php MTT)
Kt qu th hot tnh khng khun, khng nm ca cc cht tng
4.1
hp c
Cc tng tc 1H-13C ca cht V
3.10
Trang
38
40
41
42
43
44
45
46
47
48
49
61
DANH MC CC HNH V, TH
Trang
Hnh
4.1
: Ph HSQC ca hp cht V
59
4.2
: Ph HMBC ca hp cht V
61
DANH MC PH LC
Ph lc 1
: Ph 1H-NMR ca hp cht A
Ph lc 2 (2.1- 2.2)
: Ph 1H-NMR 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- 5.4)
: Cc ph ca hp cht III
Ph lc 6 (6.1- 6.4)
: Cc ph ca hp cht IV
Ph lc 7 (7.1- 7.6)
: Cc ph ca hp cht V
Ph lc 8 (8.1 8.4)
: Cc ph ca hp cht VI
Ph lc 9 (9.1- 9.4)
: Cc ph ca hp cht VII
Ph lc 10 (10.1- 10.4)
: Cc ph ca hp cht VIII
Ph lc 11 (11.1- 11.4)
: Cc ph ca hp cht IX
Ph lc 12 (12.1- 12.4)
: Cc ph ca hp cht X
Ph lc 13 (13.1- 13.4)
: Cc ph ca hp cht XI
Ph lc 14 (14.1- 14.6)
: Cc ph ca hp cht XII
Ph lc 15 (15.1- 15.4)
: Cc ph ca hp cht XIII
Ph lc 16
: Phiu tr li kt qu th hot tnh gy c

t bo (cytotoxicity assay)
Ph lc 17
: Phiu tr li kt qu th hot tnh khng vi

sinh vt kim nh (antimicrobial assay)
T VN
Thuc s dng trong cng tc phng v cha bnh c nhiu ngun gc
khc nhau. Bn cnh thuc c ngun gc thin nhin v vi sinh, thuc c
to ra bng con ng tng hp v bn tng hp ng vai tr quan trng.
Tng hp ha dc l mt chuyn ngnh ha hc quan trng, v ang
c quan tm nghin cu v pht trin nhm to ra cc thuc c hiu qu
trong cng tc phng v cha bnh.
Trong lnh vc tng hp thuc, nhanh chng to 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 on c tc dng tt hn, t c tnh hn v c hiu qu hn trong iu tr.
Cc dn cht 2-thiohydantoin l dy cht c quan tm nghin cu
v tng hp ho hc, tc dng sinh hc v ng dng lm thuc. Trong
nhiu dn cht ca 2-thiohydantoin c tc dng chng ung th, khng khun,
khng nm, khng virus v c ch aldose reductase, ng quan tm [6], [7],
[16], [20], [21], [23], [35], [39].
Cc thnh tu nghin cu tng hp v hot tnh sinh hc ca cc dn
cht 2-thiohydantoin nu trn cho thy vic nghin cu tng hp v th hot
tnh sinh hc ca cc dn cht 2-thiohydantoin l rt cn thit v c nhiu
trin vng . gp phn nghin cu v ho hc v hot tnh sinh hc ca cc
dn cht 2-thiohydantoin, chng ti thc hin ti ny vi cc mc tiu sau:
1.
Tng hp mt s dn cht 5-aryliden-2-thiohydantoin.
2.
Th sng lc hot tnh gy c t bo trn mt s dng t bo ung

th v tc dng khng khun, khng nm ca cc cht tng hp
c.
CHNG 1.TNG QUAN

1.1 Tc dng sinh hc ca cc dn cht 2-thiohydantoin.
2-thiohydantoin l hp cht d vng c to thnh nh phn ng ngng
t gia mt amino acid vi mt dn cht ca thiocyanat. Bn thn 2thiohydantoin khng th hin tc dng sinh hc, tuy nhin cc dn cht ca 2thiohydantoin c tc dng sinh hc c bit n trong thi gian di.
Nhiu cng trnh nghin cu tng hp v th tc dng sinh hc cho thy cc
dn cht ca 2-thiohydantoin c tc dng a dng nh: khng t bo ung th
[8], [9], [39], [40], khng t bin gen [31], khng virus [14], [17], khng
virus HIV [20], khng khun [21], [23], c ch enzyme aldose reductase [35]
Ngoi ra 2-thiohydantoin cn c s dng nhiu trong cc lnh vc khc
nh sn xut thuc tr su [36], thuc nhum [36]
1.1.1 Tc dng c ch t bo ung th.
Merbaron l mt dn cht ca 2-thiobarbituric, c tc dng gy c t
bo vi t bo ung th mu v mt s t bo ung th khc.
O
HN
S
H
N
N
H
Merbaron
Da trn s tng t v cu trc ca 2-thiohydantoin, Blanc v Cussac

[39] tin hnh tng hp mt s dn cht ca 2-thiohydantoin (1a-d), th
tc dng gy tng min dch v gy c t bo. Kt qu th nghim cho thy
cc dn cht tng hp c 1(a-d) c tc dng gy tng min dch yu, v c
tc dng trn cc t bo ung th th nghim.
O
R
N
S
R'
N
R''
1
R= CH3,
R= C6H5,
R= CH3,
R= C6H5,
R= CH2COOH,
R= CH2COOH,
R= CH2COOC2H5,
R= CH2COOC2H5,
R= H
R= H
R= H
R= H
(a)
(b)
(c)
(d)
Nm 2010, mt s dn cht ca 2-thiohydantoin (2, 3, 4, 5, 6, 7, 8)

c tc gi Al-Obaid v cc cng s [8] nghin cu th nghim invitro v
tc dng chng ung th trn 60 dng t bo ngi 5 nng khc nhau. Kt
qu thu c ng ch l cht 2, 7 c tc dng khng t bo ung th trn
dng t bo ung th bch cu, cc cht 3, 4, 5, 6, 8 c tc dng ngn cn s
phn bo v km hm s pht trin t bo th nghim.
O
Br
5-(5-bromo-thiophen-2-ylmethylen)-2NH
CH
N
H
thioxo-imidazolidin-4-on
Acid 2-[5-(5-oxo-2-thioxo-imidazolidinNH
CH
S
O
N
H
4-ylidenemethyl)-thiophen-2-ylsulfanyl]benzoic
OH
3
O
5-(4-oxo-4H-1,9-dithia-
O
NH
CH
S
N
H
cyclopenta[b]naphthalen-2-ylmethylen)2-thioxo-imidazolidin-4-on
5-(5-bromo-thiophen-2-ylmethylen)-3-
O
Br
CH
N
H
N
S
O morpholin-4-ylmethyl-2-thioxo-
imidazolidin-4-on
O
Br
3-acetoxy-6-acetoxymethyl-2-[4-(5-
NH
CH
N
bromo-thiophen-2-ylmethylen)-5-oxo-
4,5-dihydro-1H-imidazol-2-ylsulfanyl]-
AcO
tetrahydro-pyran-4-yl acetat
O
OAc
OAc
6
O
N
CH
S
O
Ph
H
AcO
2-{5-[5-oxo-1-phenyl-2-(3,4,5-
triacetoxy-6-acetoxymethyl-tetrahydroS
pyran-2-ylsulfanyl)-1,5-dihydro-
AcO
OH
Acid
imidazol-4-ylidenemethyl]-thiophen-2-
O
OAc
ylsulfanyl}-benzoic
OAc
7
O
Br
3,4,5-triacetoxy-6-[4-(5-bromo-thiophenN
CH
N
N
S
AcO
O
H
OAc
AcO
O 2-ylmethylene)-1-morpholin-4-ylmethyl-
5-oxo-4,5-dihydro-1H-imidazol-2ylsulfanyl]-tetrahydro-pyran-2-ylmethyl
acetat
OAc
8
Nguyn nhn chnh ca ung th khng thuc l do nng qu cao ca

bm tng thuc ph thuc ATP khin cho gim nng thuc, trong c
nng qu cao ca cht vn chuyn a thuc ABC (v d P-glycoprotein).
Nhiu nhm chc ha hc c kh nng c ch loi bm tng thuc (loi c
cha P-gp), bao gm 3 th h iu bin P-gp. Cc nghin cu cho thy cht 5(3-methoxy-benzyliden)-3-{3-[4-(2-methoxy-phenyl)-piperazin-1-yl]propyl}-imidazolidin-2,4-dion
(9),
5-(biphenyl-4-yl)methylen-2-thioxo4
imidazolidin-4-on (10) c tc dng c ch bm tng thuc c yu t P-gp

[33].
H3C
H3C
H
N
H
N
S
O
O
N
H
10
9
Tc gi Spengler v Hadzlik [16] tin hnh tng hp mt s dn cht

ca 5-aryliden-2-(thio)hydantoin v th tc dng c ch bm tng thuc ca
cc dn cht ny. Cht 5-(biphenyl-4-yl)methylen-2-thioxo-imidazolidin-4-on
(10) c tc dng c ch cht iu bin P-Glycoprotein trong t bo ung th
tng t verapamil.
Da trn , tc gi Hadzlik v cng s [16] tip tc tng hp dn cht
ca 5-aryliden-2-(thio)hydantoin theo s chuyn ha:
O
H
N
S
N
H
H
N
ArCHO
S
Ar
N
H
H
N
CH3I
SCH3
Ar
N
11b
11a
HO
HN
HO
H
N
N
Ar
N
11c
Cc tc gi th tc dng c ch bm tng thuc ca cc dn cht

tng hp c trn cc t bo lympho T khng thuc ca chut c gen vn
chuyn thuc ABCB1. Kt qu cho thy mt s dn cht c tc dng yu trn
bm tng thuc t bo ung th th nghim, tc dng yu hn cht 4. Hp

cht c tc dng mnh nht c 2 nhn thm.
Theo cc tc gi ny, phn nhn thm khng phn cc l yu t thun li
cho tc dng c ch bm tng thuc trn t bo ung th, cn nhm hydroxyl
l nhm th gy gim tc dng.
c bit, nhng nghin cu gn y cho thy khung benzyl ca
(thio)hydantoin c kh nng gn ln cc cht vn chuyn protein. Cc cng
trnh nghin cu ca mt s tc gi cho thy tc dng chng ung th ca cc
dn cht (thio)hydantoin rt ng quan tm [11], [16], [38]. Dn cht
(thio)hydantoin c tc dng c ch qu trnh phosphoryl ha t ng v bit
ha mt s t bo ung th do yu t EGFR (receptor ca yu t tng trng
biu m) [11], [38].
1.1.2 Tc dng khng khun, khng nm.
Tc gi Herba [17] tin hnh tng hp mt s dn cht ca
thiohydantoin v th tc dng khng khun, khng nm trn cc chng vi
khun: Staphylococcus aureus (RCMB 000108) v Bacillus subtilis (RCMB
000109) (Gram dng), Pseudomonas aeruginosa (RCMB 000103) v
Escherichia coli (RCMB 000106) (Gram m). Cc cht tng hp c th tc
dng khng nm trn cc chng Aspergillus fumigates (RCMB 002006),
Geotrichum candidum (RCMB 005008), Candida albicans (RCMB 005003),
Syncephalastrum racemosum (RCMB 005004). Kt qu cho thy cc cht
tng hp c u c tc dng khng khun, khng nm. Cht 5-oxo-2thioxo-1-(2-p-tolyl-ethylideneamino)-imidazolidin-4-yl acetat (12) c tc
dng tt nht trn chng vi khun v nm th nghim. Cht 1-acetyl-3-[(ptolylethyliden)amino]-2-thiohydantoin (13) v cht 5-thiophen-2-yliden-3-[(ptolylethyliden)amino]-2-thiohydantoin (14) c tc dng trung bnh. Cht 1acetyl-3-[(p-tolylethyliden)amino]-5-thiophen-2-yliden-2-thihydantoin
(15)
c tc dng tt trn chng vi khun v tc dng trung bnh trn chng nm

th nghim.
N
O
N
H
CH3
N
O
S
N
O
12
CH3
N
O
CH3
N
O
N
S
13
N
H
N
S
N
O
15
14
Nm 2010, tc gi Abd E.l v cng s [6] tng hp mt s dn cht

ca thiohydantoin v th hot tnh trn 4 dng vi khun l: Bacillus subtilis,
Staphylococcus aureus, Escherichia coli, Pseudomonas species v 1 dng vi
nm l Candida albicans. Kt qu thu c cho thy cc hp cht 16, 17, 18
tc dng tt trn dng vi khun Pseudomonas sp; cc hp cht 16, 17, 18 tc
dng yu trn dng Escherichia coli; 18, 19 tc dng rt tt trn vi khun
Staphylococcus aureus; 16, 17, 19, 20 tc dng rt tt trn dng vi nm
Candida albicans.
S
Br
COCH3
2-[(3-acetyl-5-oxo-2-thioxoimidazolidin-1-ylimino)-methyl]-4-
OCOCH3 O
bromo-phenyl acetat
16
S
Br
N
OH
5-acetyl-3-[(5-bromo-2-hydroxy-
NH
benzyliden)-amino]-2-thioxo-
COCH3
imidazolidin-4-on
17
S
Br
COCH3
4-bromo-2-[(3,4-diacetyl-4-bromo-5-
Br
oxo-2-thioxo-imidazolidin-1-
COCH3
OCOCH3 O
ylimino)-methyl]-phenyl acetat
18
H2NHN
Br
N
H
OH H NHN
2
4-bromo-2-[(2,5-dihydrazino-
NH
imidazolidin-1-ylamino)-methyl]phenol
19
S
Br
N
OH
3-[(5-bromo-2-hydroxy-benzyliden)-
NH
amino]-5-thiophen-2-ylmethylen-2-
thioxo-imidazolidin-4-on
20
1.1.3 Cc tc dng khc.

Trong US patent 6.826.924 tc gi Pfahl [28] tng hp cc dn cht
ca 2-thioxo-4-imidazolidindion c tc dng trong iu tr cc bnh lin quan
n chuyn ha lipid v carbonhydrat nh i tho ng typ 2, bit ha t
bo m m, bnh Hodgkin, bnh bch cu, ung th v, ung th tin lit
tuyn
R5
R4
R1
Ar
Ar
HN
R2
R3
NH
S
21
Cc dn cht th v tr 5 ca 2-deoxyuridin bi cc d vng 5 cnh

c nghin cu th tc dng khng virus [14], trong c dn cht 5-(5bromothien-2-yl)-2-deoxyuridin v 5-(5-clorothien-2-yl)-2-deoxyuridin (21)
c tc dng c ch s nhn ln ca virus Herpes typ 1. Tc gi El-Barbary
[14] s dng 5-methylen-2-thiohydantoin l d vng 5 cnh tin hnh
phn ng vi 2-deoxyuridin nhm to ra cc cht mi nghin cu tc
dng khng virus theo s sau:
S
R
N
HN
O
R
N
HN
O
O 1.(Me3Si)2NH/(NH4)2SO4
2.CF3SO3Me3, MeCN
HN
O
piperidin/EtOH
N
CHO
N
TolO
TolO
N
H
O
HN
HN
O
R= Ph
OTol
OTol
Mt s dn cht tng hp c tc dng gy c trn t bo MT4 ca virus

HIV-1, khng c tc dng ng k trn virus Herpes typ 1.
1.2 Cc phng php tng hp 2-thiohydantoin v dn cht.
1.2.1 Phn ng tng hp 2-thiohydantoin.
1.2.1.1 Phn ng ca cc -amino acid v thioure.
Phn ng:
O
O
R
S
OH
NH2
170-2200C
+
H2N
NH2
- H2O, NH3
R
HN
NH
S
y l phn ng cho hiu sut cao trong tng hp 2-thiohydantoin [22],

[36], l s ngng t trc tip gia -amino acid v thioure trong iu kin
170-220oC m khng cn dung mi no. Phn ng ny c thc hin
trong bnh phn ng, un hi lu di 4 iu kin khc nhau:
+ s dng bnh o.
+ un cch du.
+ trong l phn ng khng g kn, c trang b khuy t v un nng
bng du.
+ thit b un cch acid nhit khng i.
Phn ng ny cho hiu sut khc nhau ng vi cc amino acid khc nhau
v iu kin phn ng khc nhau. Hiu sut cao nht khi s dng iu kin
un cch du, dao ng t 70% n gn nh 100%.
1.2.1.2 Phn ng ca cc -amino acid vi ammonium thiocyanat.
Nm 1913, Treat Johson v Water Scott [19] tm ra phn ng gia amino acid vi amonium thiocyanat trong mi trng anhydrid acetic khan.
Sn phm trung gian thu c l acetylthiohydantoin, sn phm trung gian
ny b thy phn trong mi trng acid nhit cao s sinh ra 2thiohydantoin.
S phn ng:
H
N
R
OH
H2N
NH4SCN, Ac2O
H+
CH3
- CH3COOH
H
N
N
H
Trong bi bo ny, tc gi thc hin phn ng tng hp thiohydantoin

v dn cht t 7 acid khc nhau l: acid aceticphtalamic, acid pmethoxyhippuric,
acid
carbethoxyaminoacetic,
acid
m-nitrohippuric,
acetyldibromphenylalanin, benzoylalanin, acid phenylsulfonaminoacetic.

Nm 2006, Samuel Reyes v cng s [29] tin hnh tng theo phng
php trn v thu c 2-thiohydantoin. Nm 2011, Sandrine Gosling v cng
s [15] cng xy dng qui trnh tng hp 2-thiohydantoin theo s nh
trn v chng ti thc hin theo qui trnh ny.
1.2.1.3 Phn ng ca glycin ethyl ester thiocyanat vi acid amin.
10
Glycin ethyl ester thiocyanat kt hp vi acid amin, un hi lu trong

cloroform trong 4 gi c dn cht th v tr 3 ca uredoacetic ethyl ester,
sau un hi lu trong ethanol/acid hydrochloric (nng 10N, t l 1:1)
thu c 2-thiohydantoin c nhm th v tr 3 nh mong mun [27].
Phn ng:
O
NCS
O
H2N
O
a
H
N
H
N
N R
HN
a: un hi lu 4 gi trong CHCl3
b: un hi lu 3 gi trong EtOH/HCl 10N t l 1:1
1.2.2 Phn ng tng hp dn cht 5-aryliden-2-thiohydantoin.
Cng thc cu to 2-thiohydantoin (2-thioxoimidazolidin-4-on) nh sau:
O
NH
HN
T cu trc phn t 2-thiohydantoin cho thy nhiu kh nng phn ng

chuyn ha to thnh nhiu dn cht khc nhau ca 2-thiohydantoin [12],
[23], [36] Cc phn ng thc hin theo hng chnh sau:
- Phn ng th vo v tr nit N-1 v N-3.
- Phn ng ca nhm methylen hot ng C-5.
- Phn ng ca nhm C=O v C=S.
Da vo kh nng hot ng ca nhm methylen C-5 c th tng hp
cc dn cht 5-aryliden-2-thiohydantoin bng phn ng vi aldehyd thm.
OH
O
RCHO +
HN
NH
C
H
HN
NH
S
11
H2 O
C
H
HN
NH
S
Phn ng ngng t c th thc hin cc iu kin khc nhau: di iu kin

phn ng vi aldehyd thm, xc tc natri acetat, acid acetic bng [21], [35],
[40]; ngng t vi cc aldehyd thm, xc tc ethanolamin/EtOH [23], [26],
xc tc piperidin/EtOH [12], [14], di iu kin vi sng [27]
V c ch phn ng Knoevenagel [2], [24]
Phn ng ngng t cc aldehyd thm v mt s hp cht d vng cha
nhm aldehyd vi 2-thiohydantoin xy ra theo c ch phn ng Knoevenagel
tng t nh vi cc hp cht c nhm methylen hot ng khc.
Vi xc tc ethanolamin l amin bc 1, c th trnh by c ch phn ng
ngng t aldehyd thm vi 2-thiohydantoin theo c ch Knoevenagel theo
cc bc sau:
- Aldehyd thm ngng t vi ethanolamin to arylidenimin:
Ar
C
H
O + H2N CH2CH2OH
Ar
H
C
CH2CH2OH
Ar
C
H
- H2O
CH2CH2OH
OH H
- Xc tc base (ethanolamin) ly i 1 hydro ca nhm methylen hot

ng ca 2-thiohydantoin to carbanion. Anion ny tn cng vo imin to
thnh hp cht trung gian -aminocarbonyl.
HOH2CH2C
NH2
H
H
HN
O
- HO(CH2)2NH3
N R
HN
S
O
CH
N
HN
CH2CH2OH
NH
NH
S
Ar
H
C
HO(H2C)2 N
O
+ H2O
HN
NH
HN
S
H
Ar
NH
S
H
Ar
- OH
HO(H2C)2
H
C
HN
NH
O
NH
S
- Hp cht trung gian tch amin to thnh hp cht 5-aryliden-2thiohydantoin.

12
H
C
Ar
HN
NH
HO(H2C)2
NH
Ar
- 2 HO(CH2)2NH2
C
H
NH
HN
H2N CH2CH2OH
Vi xc tc natri acetat trong dung mi acid acetic

Phn ng ngng t ca aldehyd thm vi 2-thiohydantoin l phn ng
ngng t kiu aldol xy ra theo c ch sau [24]:
- Giai on cng hp:
Nhm methylen v tr 5 ca 2-thiohydantoin 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:
O
NH
+
HC
N
H
NH
+
BH
S
N
H
(II)
Anion (II) tn cng vo carbon mang in tch dng phn C(+) ca

nhm carbonyl ca aldehyd to sn phm cng hp (IV):
O
O
Ar C H
NH
HC
N
H
(III)
(II)
NH
H
S
Ar
N
CH H
O
(IV)
Ion alcolat (IV) to thnh li ly mt proton ca BH, tr li xc tc B(-) :

O
NH
H
Ar HC N
S
H
OH
NH
H
Ar HC N
S +
BH
(V)
(IV)
- Giai on ngng t loi nc:

O
O
H
Ar HC
HO
NH
N
H
(V)
+H
S
H
Ar CH
NH
N
H
OH2
13
NH
S
-H
- H2O
Ar C
H
N
H
Nm 1911, Wheeler v Hoffman [34] 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. 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,
O
Ar-CHO
4
5 2
1
N
H
AcOH, AcONa
NH
3
O
Ar
- H2O
NH
4 3
5 2
1
N
H
Phng php ca Wheeler v Hoffman c s dng tng hp nhiu

dn cht th C-5 ca hydantoin. Mt nhc im ca phng php trn l
phi s dng mt lng ln acid acetic v natri acetat.
Nm 1992, Chazeau V., v cng s [40] xy dng qui trnh tng hp
mt s dn cht 5-aryliden-2-thiohydantoin da trn nghin cu ca Wheeler
v Hoffman.
Nm 1993, Jnos Marton [23] s dng xc tc c tnh base mnh l
ethanolamin tin hnh phn ng ngng t aldehyd thm vi thiohydantoin
trong dung mi l ethanol, phn ng tin hnh nhit hi lu trong 30
pht. Tc gi s dng mt s aldehyd thm (p-clorobenzaldehyd, pnitrobenzaldehyd, p-hydroxybenzaldehyd,...), hiu sut phn ng 44-89%.
S phn ng:
O
NH
EtOH , H2N(CH2)2OH
+
S
N
H
un hi l-u
R = H, -OH, -OCH3, -N(CH3)2, -Cl, -NO2
14
NH
S
NH2
CHNG 2.NGUYN VT LIU, TRANG THIT B V PHNG

PHP NGHIN CU
2.1
Nguyn vt liu, ha cht, dung mi

Cc ha cht, dung mi dng trong qu trnh thc nghim l loi dng
trong tng hp c nhp t cng ty Merck, Sigma-Aldrich, Trung Quc.

Cc ha cht ny c s dng trc tip khng qua tinh ch thm. Bao gm:
Cc benzaldehyd:
- Benzaldehyd
- 4- Bromobenzaldehyd
- 4- Fluorobenzaldehyd
- 3- Nitrobenzaldehyd
- 2- Clorobenzaldehyd
- 4- Nitrobenzaldehyd
-2-hydroxybenzaldehyd
- 4-hydroxybenzaldehyd
-4-methoxybenzaldehyd
Amoni thiocyanat
Cc ha cht v dung mi khc: DMF, aceton, acid acetic bng,
ethanolamin, cloroform, dicloromethan, ethylacetat, methanol, natri acetat,

ethanol tuyt i, HCl, nc ct.
2.2
Thit b th nghim
- Dng c thy tinh (bnh cu 3 c dung tch 100, 250, 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).
15
- 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, ti 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, HSQC, HMBC) 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 v
LC/MS/MS-Xevo TQMS ti Vin Ha hc, Vin Khoa hc v Cng
ngh Vit Nam.
2.3
Phng php nghin cu
2.3.1 Phng
php
tng
hp
2-thiohydantoin,
cc
5-aryliden-2-
thiohydantoin
-
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.
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 ca 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 hot tnh sinh hc.
2.3.3.1 Th hot tnh gy c t bo ung th
Th tc dng gy c t bo ung th ca cc cht tng hp c theo 2
phng php:
16
- Phng php MTT [25], [37]

- Phng php SRB [18], [30].
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.
- Phng php MTT: Hp cht MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5
diphenyltetrazolium bromid, 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 540 600 nm. S
bin i mu ch xy ra khi enzym 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 cc cht tng
hp c i vi 2 dng t bo ung th ngi: t bo ung th phi A549 v t
bo ung th i trng HCT116.
- Phng php SRB: 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. Chng ti s dng phng php ny nghin cu c tnh ca cht tng
hp c i vi 3 dng t bo ung th ngi: t vo ung th tuyn tin lit
PC 3, t bo ung th gan Hep-G2, t bo ung th biu m v MCF7. 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:
5 dng t bo:
17
- 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 ung th phi)
- Dng t bo HCT116 (t bo ung th i trng)
- 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:
- Phng php MTT: Adriamycin ca hng Sigma.
- Phng php SRB: Ellipticin ca hng Sigma.
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 v lp li
hai ln i vi phng php MTT.
- 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 MTT [25],
[37]
- Chun b dung dch MTT nng 5mg/ml.
- Thm 20 l dung dch MTT vo mi ging ca a nui cy. Lc nh
cho MTTkhuch tn u trong mi trng nui cy.
- 37oC trong 3 gi MTT c chuyn ha.
- Loi b mi trng trong cc ging ca a nui cy.
18
- Hon tr formazan (sn phm chuyn ha MTT) bng 100 l DMSO.

Lc k formazan c th tan hon ton.
- c mt quang bc sng 540 nm. Mt quang s phn nh s
lng t bo sng st.
c/ Quy trnh th nghim c tnh t bo theo phng php SRB [18],[30].
- 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 492-515 nm. 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
0), DMSO 10% v so snh vi gi tr OD khi trn mu tm gi tr CS (%)
theo cng thc sau:
OD (mu) OD (ngy 0)
X 100
CS% =
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.
19
lch tiu chun s =
( xi - x )2
n-1
* Tnh gi tr IC50
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 gi tr logarit ca ng cong pht trin t bo v
nng cht th tnh gi tr IC50.s
Cng thc 1/Y = a + blnX
Trong Y: nng cht th; X: gi tr CS (%).
2.3.3.2 Th tc dng khng khun, khng nm
* Nguyn tc:
Th sng lc hot tnh khng khun, khng nm c tin hnh theo
phng php ca Berghe V.A v Vlietlinck [10], thc hin trn cc phin vi
lng 96 ging, theo 2 bc sau y:
Bc 1: Sng lc s b tm cht c hot tnh.
Bc 2: Tm nng c ch ti thiu (MIC) ca cht c hot tnh.
* Chng dng tnh:
+ Streptomycin cho vi khun Gr(-)
+ Penicillin cho vi khun Gr(+)
+ Nystatin cho nm si v nm men.
Khng sinh pha trong DMSO10% vi nng thch hp:
Streptomycin: 4mM; Penicillin: 50mM; Nystatin: 4mM
* Chng m tnh:
Vi sinh vt kim nh khng trn khng sinh v cht th
20
* Cc chng vi sinh vt kim nh bao gm:

- Vi khun Gr(-): Escherichia coli (ATCC 25922)
Pseudomonas aeruginosa (ATCC 25923)
- Vi khun Gr(+): Bacillus subtillis (ATCC 27212)
Staphylococcus aureus (ATCC 12222)
- Nm si:
Aspergillus niger (439)

Fusarium oxysporum (M42)
- Nm men:
Candida albicans (ATCC 7754)

Saccharomyces cerevisiae (SH 20)
* Tin hnh:
- Bc 1: Sng lc s b tm cht c hot tnh.
+ Chun b vi sinh vt:
. Mi trng duy tr v bo tn ging: Sabouraud Dextrose Broth
(SDB)-Sigma cho nm men v nm mc, Trypcase Soya Broth (TSB)-Sigma
cho vi khun.
. Mi trng th nghim: Mycophil cho vi nm, Eugon Broth cho vi
khun.
+ Chun b mu th: Cht th c ho tan trong DMSO nguyn cht.
+ T dung dch gc pha long thnh 4 - 10 thang nng ri nh sang
phin vi lng 96 ging.
+ Nh vo mi ging c sn dung dch VSV hot ho v c
pha long bng mi trng dinh dng cho ti nng tng ng 0,5 n
v McLand (khong 108vsv/ml).
+ i chng dng:
Dy 1: Mi trng
21
Dy 2: Khng sinh (Penicillin) + vi khun Gr(+);

Khng sinh (Streptomycin) + vi khun Gr(-);
Khng sinh (Nystatin) + vi nm.
+ i chng m: Ch c vi nm, vi khun kim nh.
+ cc phin th nghim trong t m 370C/24h i vi vi khun v
300C/48h i vi vi nm. Sau c kt qu.
+ Kt qu dng tnh l nng m khng c vi sinh vt pht
trin. Khi nui cy li nng ny trn mi trng thch a kim tra, c
gi tr CFU<5.
- Bc 2: Tm nng c ch ti thiu (MIC) ca cht c hot tnh.
Cc mu c hot tnh c sng lc ban u c pha long theo
cc thang nng thp dn, t (5 - 10) thang nng tnh gi tr nng
ti thiu m VSV b c ch pht trin gn nh hon ton.
Mu cht tinh khit c MIC 50 g/ml l c hot tnh.
22
CHNG 3. THC NGHIM V KT QU NGHIN CU

3.1 Tng hp ha hc
Trong lun vn ny chng ti tin hnh tng hp mt s dn cht 5aryliden-2-thiohydantion gm cc cng vic sau:
- Tng
hp
2-thiohydantoin
bng
phn
ng
gia
glycin
amonithiocyanat trong anhydrid acetic theo quy trnh ca Sandrine Gosling

[15] v Jnos Marton [23].
S phn ng:
OH
H2N
NH4SCN, Ac2O
H
N
S
N
1000C, 30 pht
HCl 10%
S
un hi l-u, 1 gi
N
H
O
1-acetyl-2-thiohydantoin (A)
Glycin
H
N
2-thiohydantoin (BT)
- Tng hp dn cht mt s dn cht 5-aryliden-2-thiohydantion (I-XIII)

bng phn ng ngng t 2-thiohydantoin (BT) vi mt s aldehyd thm theo
s phn ng sau:
EtOH, H2N(CH2)2OH
un hi l-u
ph-ng php A
3'
2'
1'
4'
5'
6'
O
CHO +
4 NH
51 3
2
N
H
BT
3'
1'
4'
5'
AcOH, AcONa
un hi l-u
2'
CH
4 NH
5 3
12
N
H
6'
I-XIII
ph-ng php B
R=H; 4'-F; 2'-Cl; 3'-Cl 4'-Cl;
(R=H (I); 4'-F (II); 2'-Cl (III); 3'-Cl (IV); 4'-Cl (V)
2'-Br; 3'-Br; 4'-Br; 3'-NO2;
2'-Br (VI); 3'-Br (VII); 4'-Br (VIII); 3'-NO2 (IX);
4'-NO2; 2'-OH; 4'-OH; 4'-OCH3
4'-NO2(X); 2'-OH(XI); 4'-OH (XII); 4'-OCH3 (XIII)
Trong : Phng php A : cht I , II , III , VI , VIII, X , XI.

Phng php B: cht IV ,V , VII , IX, XII, XIII.
3.1.1 Tng hp 2-thiohydantoin (BT)
23
Gm 2 giai on:
- Tng hp 1-acetyl-2-thiohydantoin (A)
- Thy phn cht A to 2-thiohydantoin (BT)
3.1.1.1 Tng hp 1-acetyl-2-thiohydantoin (A)
Cng thc:
H
N
4 3
2
51
CH3
CTPT: C5H6N2O2S, KLPT: 158,18

Tin hnh:
Cho vo bnh cu 46,00g (0,613mol) glycin, 46,60g (0,613mol) amoni
thiocyanat, thm 174 ml anhydrid acetic, un cch du v khuy u hn hp
phn ng. Theo di phn ng bng SKLM vi dung mi khai trin l
Hexan/EtOAc (7:3). Phn ng c tin hnh 1000C trong 30 pht. Kt
thc phn ng, hn hp c rt vo 500 g nc , lnh trong 4 gi. Cht
rn mu vng xut hin c lc, ra bng nc lnh 3 ln, mi ln 150ml.
Sy kh ta thu c.
Khi lng sn phm: 59,27g. Hiu sut: 61,1%
Nhit nng chy: 172-173OC (ti liu [23]: t oC = 176o C ; ti liu [29]:
t oC = 173 - 174o C ).
Rf = 0,31 (TLC, silicagel 60 F254, dung mi khai trin Hexan/EtOAc (7:3))

(ph hp vi ti liu tham kho [15]).
1
H-NMR (500 MHz, DMSO-d6), (ppm): xem ph lc 1, bng 3.2
12,558 (s,1H,N3-H); 4,401 (s,2H,H5), 2,677 (s,3H,CH3).

3.1.1.2 Tng hp 2-thiohydantoin (BT)
Cng thc:
24
H
N
3
4
51 2
N
H
CTPT: C3H4N2OS, KLPT: 116,14

Tin hnh:
59,27g (0,375mol) cht A c phn tn trong 294ml HCl 10%. un hi
lu trong vng 1 gi. Theo di phn ng bng SKLM vi h dung mi khai
trin l CH2Cl2/MeOH (14:1). Kt thc phn ng cho than hot vo hn hp
phn ng v lc nng. Dch lc c lnh qua m, thu c kt ta, lc
ta, ra nc n ht acid (th bng giy qu). Sy kh ta thu c. Kt tinh
li trong nc.
Khi lng sn phm: 19,12g cht rn mu cam. Hiu sut: 44,0 %
Nhit nng chy: 229-2300C (ti liu [23], t oC = 230 - 232o C ; Catalogue
Sigma Aldrich, t oC = 229 - 231o C ).
Rf = 0,68 (TLC, silicagel 60 F254, h dung mi CH2Cl2/MeOH (14:1))
1
H-NMR (500 MHz, DMSO-d6), (ppm): xem ph lc 2.2, bng 3.2

11,624 (s;1H;N3-H); 9,813 (d;1H; N1-H); 4,070 (s;2H;H5).
3.1.2 Tng hp mt s dn cht 5-aryliden-2-thiohydantion (I-XIII).

Da trn nhng ti liu tham kho, chng ti tng hp mt s dn cht
5-aryliden-2-thiohydantion (I-XIII) theo 2 phng php:
- Phng php A: dng xc tc ethanolamin trong dung mi ethanol
theo qui trnh ca tc gi Jnos Marton v cng s [23].
Quy trnh tng hp chung : Cho vo bnh cu phn ng 1,16g
(0,010mol) cht BT (2-thiohydantoin), 10ml EtOH, khuy u thu c hn
hp (a). Ha tan 0,010 mol aldehyd thm bng 10ml EtOH trong cc c m,
sau thm 0,9ml ethanolamin thu c hn hp (b). Thm t t tng git
(b) vo hn hp (a) trong bnh phn ng, sau thm 20ml EtOH, khuy u
25
nhit phng 15 pht. un hi lu hn hp phn ng trong 30 pht. Theo

di phn ng bng SKLM vi h dung mi khai trin l CH2Cl2 : MeOH
(14:1). ngui, hn hp phn ng ra cc c m, kt tinh trong t lnh
qua m. Cht rn c lc ht kit, ra ta bng EtOH lnh, ra tip bng
nc nng, sau ra li bng EtOH lnh. Sy kh sn phm th thu c.
Kt tinh li sn phm th bng dung mi thch hp.
Chng ti s dng phng php A tng hp cc 5-aryliden-2thiohydantoin i t cc aldehyd l benzaldehyd, 4-fluorobenzaldehyd, 2clorobenzaldehyd,
2-bromobenzaldehyd,
4-bromobenzaldehyd,
4-
nitrobenzaldehyd, 2-hydroxybenzaldehyd
- Phng php B: dng xc tc natri acetat trong dung mi acid acetic
theo qui trnh ca tc gi Chazeau V., v cng s [40].
Quy trnh tng hp chung : Cho vo bnh cu phn ng 1,16g
(0,01mol) cht BT, 0,01mol aldehyd thm, 3,77g (0,046mol) natri acetat
khan v 22ml acid acetic bng. Khuy u cho hn hp tan hon ton, un hi
lu hn hp phn ng trong 2 gi. Theo di phn ng bng SKLM vi h
dung mi khai trin CH2Cl2 : MeOH (14:1). Phn ng kt thc, ngui,
hn hp ra cc c m, ngm lnh 15 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 sn
phm th thu c. Kt tinh li sn phm th bng dung mi thch hp.
Chng ti s dng phng php B tng hp cc 5-aryliden-2thiohydantoin i t cc aldehyd l 3-clorobenzaldehyd, 4-clorobenzaldehyd,
3-bromobenzaldehyd,
3-nitrobenzaldehyd,
4-hydroxybenzaldehyd,
methoxybenzaldehyd.
Sau y l cc kt qu c th:
3.1.2.1 Tng hp 5-benzyliden-2-thiohydantoin (I)
26
4-
Cng thc:
3'
2'
1'
4'
CH
N
H
6'
5'
3NH
1 2
4
5
CTPT: C10H8N2OS; KLPT: 204,25

Tin hnh: (Phng php A) T 1,16g (0,010 mol) 2-thiohydantoin,
1,06g (0,01 mol) benzaldehyd, hi lu trong 30 pht. Kt tinh li trong dung
mi EtOH:DMF ( 17:1).
Khi lng sn phm: 0,66 g tinh th hnh kim mu vng. Hiu sut: 32,3%.
Nhit nng chy: 263,4-264,30C. (ti liu [23] : t oC = 268 - 269o C ).
Rf = 0,93 (TLC, silicagel 60 F254, h dung mi CH2Cl2 : MeOH (14:1))
Rf = 0,65 (TLC, silicagel 60 F254, h dung mi Hexan : Aceton (3:2))
IR (KBr, max (cm-1)): xem ph lc 3.1, bng 3.3
1
13
C-NMR (125 MHz, DMSO-d6), (ppm): xem ph lc 3.3, bng 3.5
ESI - MS, (m/z, %): xem ph lc 3.4, bng 3.6

3.1.2.2 Tng hp 5-(4-fluorobenzyliden)-2-thiohydantoin (II)
Cng thc:
3'
2'
1'
4'
5'
CH
3NH
1 2
4
5
N
H
6'
CTPT: C10H7FN2OS, KLPT: 222,24

1,24g (0,01 mol) 4-fluorobenzaldehyd, hi lu trong 30 pht. Kt tinh li
trong dung mi EtOH:DMF ( 10:1).
Khi lng sn phm: 1,82g cht kt tinh mu vng nht, hiu sut 78,4 %.
Nhit nng chy: 277-279 oC.
27

1
13

3.1.2.3 Tng hp 5-(2-clorobenzyliden)-2-thiohydantoin (III)
Cng thc:
Cl
3'
4'
5'
2'
1'
O
CH
3NH
1 2
4
5
N
H
6'
CTPT: C10H7ClN2OS, KLPT: 238,69

1,41g (0,01 mol) 2-clorobenzaldehyd, hi lu trong 30 pht. Kt tinh li
Khi lng sn phm: 1,51g cht kt tinh mu vng, hiu sut 63,3 %.
Nhit nng chy: : 273-274,5oC.
1
13

3.1.2.4 Tng hp 5-(3-clorobenzyliden)-2-thiohydantoin (IV)
Cng thc:
28
Cl
3'
2'
1'
4'
5'
CH
3NH
1 2
4
5
N
H
6'

Tin hnh: (Phng php B) 1,16g (0,01mol) 2-thiohydantoin, 1,41g
(0,01mol) 3-clorobenzaldehyd, 3,77g (0,046mol) natri acetat khan v 22ml
acid acetic bng, hi lu trong 2 gi. Kt tinh li trong dung mi EtOH/DMF
(4:1).
Khi lng sn phm: 1,42 g cht rn mu vng. Hiu sut: 59,0 %
Nhit nng chy: 235,5-236,6oC.
1
13

3.1.2.5 Tng hp 5-(4-clorobenzyliden)-2-thiohydantoin (V)
Cng thc:
3'
Cl
2'
1'
4'
5'
CH
3NH
1 2
4
5
N
H
6'

(0,01mol) 4-clorobenzaldehyd, 3,77g (0,046mol) natri acetat khan v 22ml
acid acetic bng, hi lu trong 2 gi. Kt tinh li trong dung mi EtOH/DMF
(4:1).
Khi lng sn phm: 1,72g cht rn mu vng. Hiu sut: 79,7 %
29
Nhit nng chy: 281-282oC (ti liu [23]: t oC = 281 - 282o C ).

1
13

3.1.2.6 Tng hp 5-(2-bromobenzyliden)-2-thiohydantoin (VI)
Cng thc:
3'
2'
Br
4'
1'
5'
O
CH
3NH
1 2
4
5
N
H
6'
CTPT: C10H7BrN2OS, KLPT: 283,14

1,85 g (0,01 mol) 2-bromobenzaldehyd, hi lu trong 30 pht. Kt tinh li
Khi lng sn phm: 1,59g cht kt tinh mu vng nht, hiu sut 56,1 %.
Nhit nng chy: 261-263oC.
1
13

3.1.2.7 Tng hp 5-(3-bromobenzyliden)-2-thiohydantoin (VII)
Cng thc:
30
Br
3'
2'
1'
4'
5'
CH
3NH
1 2
4
5
N
H
6'

(0,01 mol) 3-bromobenzaldehyd, 3,77g (0,0460mol) natri acetat khan v 22ml
acid acetic bng, hi lu trong 2 gi. Kt tinh li trong dung mi EtOH.
Nhit nng chy: 216-217oC.
1
13

3.1.2.8 Tng hp 5-(4-bromobenzyliden)-2-thiohydantoin (VIII)
Cng thc:
3'
Br
2'
1'
4'
5'
CH
3NH
1 2
4
5
N
H
6'

1,85 g (0,01 mol) 4-bromobenzaldehyd, hi lu trong 30 pht. Kt tinh li
Nhit nng chy: 279,8-282oC.
31

1
13

3.1.2.9 Tng hp 5-(3-nitrobenzyliden)-2-thiohydantoin (IX)
Cng thc:
O2N
3'
2'
1'
4'
5'
CH
3NH
1 2
4
5
N
H
6'

(0,010mol) 3-nitrobenzaldehyd, 3,77g (0,0460mol) natri acetat khan v 22ml
acid acetic bng, hi lu trong 2 gi. Kt tinh li trong dung mi EtOH.
Khi lng sn phm: 1,1 g cht kt tinh mu vng; hiu sut 50,0 %.
Nhit nng chy: 249-252 oC.
1
13

3.1.2.10 Tng hp 5-(4-nitrobenzyliden)-2-thiohydantoin (X)
Cng thc:
3'
O2N
4'
2'
1'
5'
CH
3NH
1 2
4
5
N
H
6'

32

1,51g (0,01 mol) 4-nitrobenzaldehyd, hi lu trong 30 pht. Kt tinh li trong
dung mi EtOH:DMF ( 10:1).
Khi lng sn phm: 1,94g cht kt tinh mu vng nht, bng xp; hiu sut
77,9 %.
Nhit nng chy: 282,5-284oC ( ti liu [23]: t oC = 294 - 296o C ).
1
13

3.1.2.11 Tng hp 5-(2-hydroxybenzyliden)-2-thiohydantoin (XI)
Cng thc:
OH
3' 2'
1'
4'
5'
CH
O
3NH
1 2
4
5
N
H
6'
CTPT: C10H8N2O2S, KLPT: 220.25

1,22g (0,01 mol) 2-hydroxybenzaldehyd, hi lu trong 30 pht. Kt tinh li
trong dung mi EtOH:DMF (10:1).
Khi lng sn phm: 1,25 g cht kt tinh mu cam; hiu sut 56,8 %.
Nhit nng chy: 241-242 oC (ti liu [23]: t oC = 272 - 274o C )
1

33
13

3.1.2.12 Tng hp 5-(4-hydroxybenzyliden)-2-thiohydantoin (XII)
Cng thc:
O
3' 2'
HO
1'
4'
5'
CH
3NH
1 2
4
5
N
H
6'

(0,010mol) 4-hydroxybenzaldehyd, 3,77g (0,0460mol) natri acetat khan v
22ml acid acetic bng, hi lu trong 2 gi. Kt tinh li trong dung mi
EtOH:DMF= (16:1).
Khi lng sn phm: 1,5 g cht kt tinh mu vng cam; hiu sut 68,1 %.
Nhit nng chy: 284-287 oC (ti liu [23]: t oC = 303 - 304o C )
1
13

3.1.2.13 Tng hp 5-(4-methoxybenzyliden)-2-thiohydantoin (XIII)
Cng thc:
O
3' 2'
H3CO
1'
4'
5'
6'
CH
3NH
1 2
4
5
N
H
34
Tin hnh: (Phng php B) 1,16g (0,010mol) 2-thiohydantoin, 1,36 g

(0,010mol) 4-methoxybenzaldehyd, 3,77g (0,0460mol) natri acetat khan v
22ml acid acetic bng, hi lu trong 2 gi. Kt tinh li trong dung mi
EtOH:DMF= (40:1).
Khi lng sn phm: 1,04 g cht kt tinh mu vng cam; hiu sut 44,4 %.
Nhit nng chy: 264-265oC ( ti liu [23]: t oC = 264 - 265o C )
1
13

3.2 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 vi k thut vin nn
vi KBr ti B mn Ha vt liu, Khoa ha hc, i hc Khoa hc t nhin,
i hc Quc gia H Ni. 13 ph c ghi cc ph lc 3.1 15.1. Kt
qu phn tch s liu ph hng ngoi c ghi bng 3.3
3.2.2 Ph cng hng t ht nhn
Ph cng hng t ht nhn proton, 13C v ph tng tc hai chiu (2DNMR) 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. 15 ph 1H c ghi cc ph
lc 1 2, 3.2 15.2, 13 ph 13C c ghi cc ph lc 3.3 15.3, 4 ph
tng tc 2 chiu c ghi cc ph lc: 3.4, 3.5, 4.4, 4.5, 7.4, 7.5, 14.4,
14.5. Kt qu phn tch s liu ph cng hng t ht nhn proton v
c ghi bng 3.4, 3.5.
35
13
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 v my LC/MS/MS-Xevo
TQMS, ti Vin Ha hc, Vin Hn lm Khoa hc v Cng ngh Vit Nam.
13 ph c ghi cc ph lc 3.6, 4.6, 5.4, 6.4, 7.6, 8.4 13.4, 14.6, 15.4.
Kt qu phn tch ph MS ca 13 cht (I-XIII) c trnh by bng 3.6.
3.3. Th nghim hot tnh sinh hc
3.3.1 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 Khoa Dc l-Sinh ha (Vin Dc liu) v 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 Khoa hc v Cng ngh Vit Nam) 13 cht do chng ti tng hp (IXIII) c th hot tnh gy c t bo trn cc dng t bo t bo ung th
gan Hep-G2, t bo ung th biu m v MCF-7, t bo ung th tin lit tuyn
PC3 theo phng php SRB. Kt qu c trnh by bng 3.7, 3.8.
Kt qu cho thy c 3 cht l cht V (IC50= 4,66 g/ml), cht VII (IC50=
3,22 g/ml) v cht XI (IC50= 4,72 g/ml) c hot tnh c ch t bo ung th
gan Hep-G2.
Ti khoa Dc l-Sinh ha (Vin Dc liu), 11 cht do chng ti tng
hp (I-X, XII) c th hot tnh gy c t bo trn cc dng t bo ung
th ngi theo phng php MTT. Cc dng t bo ung th c s dng l:
t bo ung th i trng HCT116, t bo ung th phi A549. Kt qu c
trnh by cc bng 3.9 v 3.10
36
Kt qu cho thy c 3 cht l: cht III (IC50= 24,4 g/ml), cht V (IC50=
27,08 g/ml), v cht VII (IC50= 26,36 g/ml) c hot tnh c ch yu trn t
bo ung th i trng HCT116.
3.3.2 Th tc dng khng khun, khng nm
Ti phng Sinh hc thc nghim - Vin Ho hc cc hp cht thin
nhin (Vin Khoa hc v cng ngh Vit Nam), 11 cht (I-X, XII) do chng
ti tng hp c th hot tnh khng khun, khng nm theo phng
php ca Berghe V.A v Vlietlink [10]. Kt qu c trnh by bng 3.11
Kt qu cho thy c 10 cht (I, III-X, XII) c hot tnh khng chng vi
khun B.subtilis (MIC=25-50g/ml).
37
Bng 3.1: Kt qu tng hp 2-thiohydantoin (BT) v cc dn cht 5-aryliden-2-thiohydantoin (I-XIII)

STT
K
hiu
cht
Cngthc
H
N
BT
S
N
H
T.gian
p.ng (h)
Dung mi
kt tinh li
Nhit
nng chy
(oC )
Rf
Hiu
sut
(%)
116,14
H2O
229-230
0,68*
44,0
204.25
0,5(A)
EtOH:DMF
(17:1)
263,4264,3
0,93*
0,65**
32,3
222,24
0,5(A)
EtOH:DMF
(10:1)
277-279
0,84*
0,57**
78,4
238,69
0,5(A)
EtOH:DMF
(10:1)
273-274,5
0,94*
0,64**
63,3
238,69
2(B)
EtOH:DMF
(4:1)
235,5-236,6
0,91*
0,61**
59,0
238,69
2(B)
EtOH:DMF
(17:1)
281-282
0,92*
0,74**
79,7
283,14
0,5(A)
EtOH:DMF
(5:1)
261-263
0,86*
0,63**
56,1
HN
NH
O
S
II
HN
F
O
S
Cl
HN
III
NH
NH
O
S
Cl
HN
IV
NH
O
S
HN
NH
Cl
Br
VI
HN
NH
O
S
Br
HN
VII
NH
O
VIII
HN
2(B)
EtOH
216-217
0,82*
0,60**
50,5
283,14
0,5(A)
EtOH:DMF
(5:1)
279,8-282
0,88*
0,65**
69,9
249,25
2(B)
EtOH
249-252
0,71*
0,50**
50,0
249,25
0,5(A)
EtOH:DMF
(10:1)
282,5-284
0,82*
0,58**
77,9
220,25
2(A)
EtOH
241-242
0,84*
0,47**
56,8
220,25
2(B)
EtOH:DMF
(16:1)
284-287
053*
0,39**
68,1
234,05
2(B)
264-265
092*
0,88**
44,4
S
NH
Br
283,14
O
S
O2N
10
HN
IX
NH
O
11
HN
NH
O2N
S
OH
12
XI
HN
XII
NH
O
13
HN
HO
S
NH
O
14
XIII
HN
H3CO
S
NH
EtOH:DMF
(40:1)
Ghi ch: *H dung mi: CH2Cl2 : MeOH (14:1); **H dung mi: Hexan:Aceton (3:2).
O
(A): phng php A; (B): phng php B
Bng 3.2: S liu ph 1H-NMR ca A, BM, BT
STT
K hiu
cht
Cng thc
N3H
O
BM*
BT**
H5
Gi tr khc
12,55
4,40
2,67 (s,3H,CH3)
CH3
H
N
4 3
2
5 1
N1H
H
N
4 3
2
5 1
H-NMR (DMSO-d6, ppm, J, Hz)
N
H
11,63
9,82
4,07
11,62
9,81
4,07
Ghi ch: BM*: Sn phm 2-thiohydantoin ca hng Sigma Aldrich

BT**: Cht 2-thiohydantoin tng hp c
Bng 3.3: S liu ph IR ca cc dn cht 5-aryliden-2-thiohydantoin (I-XIII)

R
3'
2'
1'
4'
5'
CH
4 3 NH
1 2
N
H
6'
I-XIII
Ph IR (KBr), cm-1
K hiu
cht
NH
C=S
C=O
C=C
CHarom
3220
1477
1723
1642
767-682
II
4-F
3153
1485
1729
1653
850-683
1235 (X=F)
III
2-Cl
3128
1521
1729
1666
874-757
1093 (X=Cl)
IV
3-Cl
3151
1503
1721
1648
768-681
1095 (X=Cl)
V
VI
VII
VIII
IX
X
XI
XII
XIII
4-Cl
2-Br
3-Br
4-Br
3-NO2
4-NO2
2-OH
4-OH
4-OCH3
3220
3145
3183
3226
3148
3212
3206
3170
3142
1482
1522
1499
1477
1486
1482
1484
1499
1484
1713
1730
1723
1727
1724
1745
1730
1719
1722
1647
1664
1646
1645
1647
1655
1657
1648
1643
872-671
756-663
895-837
822-625
814-632
874-759
882-677
879-686
1096 (X=Cl)
1027 (X=Br)
1073 (X=Br)
1078 (X=Br)
Carom -NO2
Caryl X
Caryl OH
1486;1385
1522;1336
1360;1225
1365;1252
1370
Bng 3.4: S liu ph 1H-NMR ca cc dn cht 5-aryliden-2-thiohydantoin (I-XIII)

3'
2'
1'
4'
5'
CH
4 3 NH
1 2
N
H
6'
I-XIII
K
hiu
cht
I
II
H
4-F
III
2-Cl
IV
VI
3-Cl
4-Cl
2-Br
VII
3-Br
VIII
4-Br
3-NO2
IX
X
XI
XII
XIII
4-NO2
2-OH
4-OH
4-OCH3
H-NMR (DMSO-d6), (ppm), J(Hz)
12,40 (1H, s, N3-H); 12,16 (1H, s, N1-H); 7,74 (2H, d, H2,6, J=7,); 7,39 (3H, m, H3,4,5); 6,48 (1H, s, -CH=)
12,38 (1H, s, N3-H); 12,17 (1H, s, N1-H); 7,82 (2H, dd, H2,6, J=8,5, JH-F=5,5); 7,27 (2H, t, H3,5, J =9);
6,49 (1H, s, -CH=)
12,47 (1H, s, N3-H); 12,27 (1H, s, N1-H); 7,81 (1 H, m, H4); 7,55 (1H, m, H5);7,42 (2H, dd, H36, J =8);
6,58 (1H, s, -CH=)
12,35 (2H,N1-H,N3-H); 7,84 (1H,s, H2); 7,65 (1H,m, H6); 7,42 (2H,m, H4,5), 6,45 (1H, s, -CH=)
7,76 (2H, d, H2,6 ,J=8,5); 7,47 (2H, d, H3,5, J =9); 6,47 (1H, s, -CH=)
12,47 (1H, s, N3-H); 12,27 (1H, s, N1-H); 7,78 (1 H, d, H3, J=7,5); 7,72 (1H, d, H6, J=8); 7,44 (1H, t, H4, J=7,5); 7,31
(1H, t, H5, J=7,5); 6,54 (1H, s, -CH=)
12,35 (2H,N1-H,N3-H); 7,96 (1H,s, H2); 7,70 (1H,d, H6, J=8); 7,55 (1H,d, H4, J=7,5); 7,35 (1H, t, H5, J=8, J=7,5);
6,43 (1H, s, -CH=)
12,19 (2H, s, N3-H, N1-H); 7,69 (2H, d, H3,5, J=8,5); 7,61 (2H, d, H2,6, J=8,5); 6,45 (1H, s, -CH=)
12,47 (1H, s, N3-H); 12,38 (1H, s, N1-H); 8,45 (1 H, s, H2); 8,18 (1H, m, H4,); 8,10 (1H, d, H6, J=8); 7,68 (1H, t, H5,
J=8); 6,61 (1H, s, -CH=)
12,56 (1H, s, N3-H); 12,41 (1H, s, N1-H); 8,22 (2H, t, H3,5, J=9); 7,97 (2H, d, H2,6, J =9); 6,56 (1H, s, -CH=)
12,27 (1H, s, N3-H); 11,82 (1H, s, N1-H); 10,30 (1H, s, -OH); 7,68 (1H, dd, H6, J=7,5;J=2); 7,21 (1H, m, H4); 6,89
(1H, d, H3,J=7,5); 6,84 (1H, t, H5,J=7,5); 6,72 (1H, s, -CH=)
7,66 (2H, d, H2,6, J=8,5); 6,80 (2H, d, H3,5, J=8,5,); 6,36 (1H, s, -CH=)
12,25 (1H, s, N3-H); 12,03 (1H, s, N1-H); 7,73 (2 H, dd, H2,6, J=8; 2); 6,98 (2H, d, H3,5, J=8,5; 3); 6,46 (1H, s, -CH=)
Bng 3.5: S liu ph 13C-NMR ca cc dn cht 5-aryliden-2-thiohydantoin (I-XIII)

R
3'
2'
1'
4'
5'
CH
4 3 NH
1 2
N
H
6'
I-XIII
K
hiu
cht
I
II
III
H
4-F
2-Cl
IV
3-Cl
V
VI
4-Cl
2-Br
VII
3-Br
VIII
IX
4-Br
3-NO2
X
XI
4-NO2
2-OH
XII
4-OH
XIII
4-OCH3
13
C-NMR (DMSO-d6), (ppm)
179,27 (-C=S); 165,77 (-C=O); 130,16 (C2,6); 129,22 (C1); 128,77 (C3,4,5); 127,72 (C5); 111,54 (-CH=)
179,27 (-C=S); 165,70 (-C=O); 161,33 (C4); 132,42 (C2,6); 128,93(C1); 127,50 (C5) 115,86 (C3,5); 110,38 (-CH=)
179,69 (-C=S); 165,47 (-C=O); 133,36 (C2); 130,90 (C1); 130,47 (C6); 130,29 (C3); 129,87 (C4); 129,55 (C5); 127,47
(C5); 105,87 (-CH=)
179,61 (-C=S); 165,68 (-C=O); 134,46 (C1); 133,67 (C3); 130,38 (C5); 128,99 (C4); 128,87 (C5); 128,75 (C2,6);
109,44 (-CH=)
179,38 (-C=S); 165,68 (-C=O); ; 133,68 (C4); 131,76 (C2,6); 131,28 (C1); 128,74 (C3,5); 128,18 (C5); 109,93 (-CH=)
179,69 (-C=S); 165,47 (-C=O); 132,80 (C2); 132,02 (C1); 131,07 (C6); 130,66 (C3); 129,85 (C4); 128,03 (C2); 123,18
(C4); 108,55 (-CH=)
179,62 (-C=S); 165,69 (-C=O); 134,75 (C1); 134,75 (C4); 131,63 (C5); 130,61 (C2); 129,24 (C5); 128,83 (C3); 122,23
(C6); 109,36 (-CH=)
179,63 (-C=S); 166,22 (-C=O); 132,21 (C1); 131,91 (C3,5); 131,58 (C2,6); 131,21 (C5) 122,18 (C4); 109,58 (-CH=)
179,86 (-C=S); 165,48 (-C=O); 148,13 (C3); 135,88 (C1); 134,11 (C6); 129,98 (C5); 129,81 (C5); 124,26 (C2); 123,15
(C4); 108,46 (-CH=)
180,56 (-C=S); 166,12 (-C=O); 147,16 (C4); 139,69 (C1); 131,40 (C5); 130,79 (C2,6); 124,15 (C3,5); 108,50 (-CH=)
178,28 (-C=S); 165,71 (-C=O); 156,27 (C2); 130,98 (C4); 130,45 (C6); 126,88 (C5); 119,46 (C1); 119,41 (C5); 115,55
(C3); 107,25 (-CH=)
178,43 (-C=S); 166,50 (-C=O); 158,69 (C4); 132,24 (C2,6); 123,74 (C1,5); 115,72 (C3,5); 112,34 (-CH=)
178,57 (-C=S); 165,77 (-C=O); 160,23 (C4); 132,11 (C2,6); 125,85 (C5); 124,85 (C1); 114,36 (C3,5); 112,11 (-CH=)
Bng 3.6: S liu ph ESI-MS ca cc dn cht 5-aryliden-2-thiohydantoin (I-XIII)
C10H8N2OS
203, 0 (100)
C10H7FN2OS
222,24
220,90 (100)
K hiu cht
Cng thc phn t
II
III
[M-H]m/z (%)
M
tnh ton
204
STT
C10H7ClN2OS
IV
C10H7ClN2OS
C10H7ClN2OS
238,20 (35Cl)
240,20 (37Cl)
238,20 (35Cl)
240,20 (37Cl)
238,20 (35Cl)
240,20 (37Cl)
282,14 (79Br)
284,14 (81Br)
282,14 (79Br)
284,14 (81Br)
282,14 (79Br)
284,14 (81Br)
280,86 (79Br) (97,65)

282,88 (81Br) (100)
280,89 (79Br) (92,66)
282,88 (81Br) (100)
280,86 (79Br) (81)
282,88 (81Br) (100)
236,90 (35Cl) (100)

236,90 (35Cl) (100)
238,98 (37Cl) (38,21)
236,90 (35Cl) (100)
VI
C10H7BrN2OS
VII
C10H7BrN2OS
VIII
C10H7BrN2OS
IX
C10H7N3O3S
249,25
247,99 (100)
10
C10H7N3O3S
249,25
247,90 (100)
11
XI
C10H8N2O2S
220,25
218,98 (100)
12
XII
C10H8N2O2S
220,25
219,0 (100)
13
XIII
C11H10N2O2S
234,05
233,0 (100)
Bng 3.7: Kt qu th hot tnh gy c t bo trn 3 dng t bo ung th th nghim(theo phng php SRB)
R
3'
2'
1'
4'
5'
CH
4 3 NH
1 2
N
H
6'
I-XIII
K hiu
mu
Nng
mu
(g/ml)
DMSO
Ellipticin
Hep-G2
Dng t bo
Cell survival (%)
MCF7
100,00,0
100,00,0
0,20,03
1,50,2
Kt lun
PC3
100,00,0
2,50,08
Dng tnh
90,10,7
92,40,9
86,30,7
m tnh
II
4-F
71,50,8
80,20,9
71,20,9
m tnh
III
2-Cl
62,10,4
94,31,2
96,51,6
IV
3-Cl
65,30,9
71,21,1
95,61,1
m tnh
m tnh
4-Cl
81,61,3
84,21,1
Dng tnh vi dng Hep G2
VI
2-Br
47,10,7
64,60,0
76,50,9
92,50,9
m tnh
VII
3-Br
73,10,6
90,51,3
VIII
4-Br
20,50,6
71,21,2
86,30,4
91,20,7

m tnh
IX
3-NO2
93,20,5
82,51,3
64,20,4
m tnh
4-NO2
83,30,9
80,40,6
99,20,8
m tnh
XI
XII
2-OH
75,80,9
85,71,26
4-OH
48,30,6
99,21,5
82,61,1
65,30,
m tnh
4-OCH3
81,90,8
86,40,5
93,81,6
m tnh
XIII
Bng 3.8: Kt qu xc nh gi tr IC50 (g/ml) ca mt s cht tng

hp c trn 3 dng t bo ung th th nghim ca cc cht tng
hp c (theo phng php SRB)
R
3'
2'
1'
4'
5'
CH
4 3 NH
1 2
N
H
6'
I-XIII
Dng t bo
K hiu
mu
Ellipticin
V
VII
XI
4-Cl
3-Br
2-OH
Gi tr IC50 (g/ml)
Kt lun
Hep-G2
MCF7
PC3
0,24
0,31
0,26
Dng tnh
>5
Dng tnh
vi dng Hep
G2
>5
Dng tnh
vi dng Hep
G2
>5
Dng tnh
vi dng Hep
G2
>5
4,66
>5
3,22
>5
4,72
46
Bng 3.9: Kt qu th tc dng ca cc cht tng hp c nng

100 g/ml trn mt s dng t bo ung th ngi (theo phng
php MTT)
R
3'
2'
1'
4'
5'
CH
4 3 NH
1 2
N
H
6'
I-XIII
K hiu mu
DMSO
Adriamycin*
Dng t bo, % sng

st
A549
HCT116
100
29,2
100
17,5
Kt Lun
53,0
51,2
m tnh
II
4-F
39,9
34,0
Dng tnh vi A549,

HCT116
III
2-Cl
30,6
29,7
Dng tnh trn dng

A549, HCT116
IV
3-Cl
66,5
78,6
m tnh
4-Cl
53,1
31,6
VI
2-Br
42,8
27,5
VII
3-Br
81,1
48,8
VIII
4-Br
56,1
35,1
IX
3-NO2
79,7
35,5
4-NO2
62,4
28,7
XII
4-OH
74,6
53,6
Ghi ch:
Dng tnh trn dng

HCT116
Dng tnh vi A549,
HCT116
Dng tnh vi
HCT116
Dng tnh trn
HCT116
Dng tnh vi
HCT116
Dng tnh vi
HCT116
Adriamycin*: Adriamycin nng 50g/g
47
m tnh
Bng 3.10: Kt qu xc nh gi tr IC50 (g/ml) ca mt s cht tng

hp c trn dng t bo HCT116 (theo phng php MTT)
R
2'
3'
1'
4'
5'
CH
4 3 NH
1 2
N
H
6'
I-XIII
STT
K hiu
mu
Dng t bo HCT116 (Gi tr

R
IC50/g/ml)
HCT116
Adriamycin
0,6
II
4-F
43,22
III
2-Cl
24,4
4-Cl
27,08
VII
3-Br
26,36
VIII
4-Br
41,26
IX
3-NO2
52,01
48
Bng 3.11: Kt qu th hot tnh khng khun, khng nm ca cc cht tng hp c.

R
3'
2'
1'
4'
5'
CH
4 3 NH
1 2
N
H
6'
I-XIII
K hiu
mu
I (T1)
Nng
mu
(g/
ml)
50
II (T6)
4-F
III (T3)
Vi khun Gr(-)
E.coli
Nng c ch ti thiu (MIC: g/ml)

Vi khun Gr(+)
Nm mc
P.aeruginosa B.subtillis
S.aureus
A.niger
Nm men
F.oxysporum S.cerevisiae C. albicans
(-)
(-)
50
(-)
(-)
(-)
(-)
(-)
50
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
2-Cl
50
(-)
(-)
50
(-)
(-)
(-)
(-)
(-)
IV (T8)
3-Cl
50
(-)
(-)
25
(-)
(-)
(-)
(-)
(-)
V (T2)
4-Cl
50
(-)
(-)
50
(-)
(-)
(-)
(-)
(-)
VI ( T11)
2-Br
50
(-)
(-)
50
(-)
(-)
(-)
(-)
(-)
VII (T7)
3-Br
50
(-)
(-)
50
(-)
(-)
(-)
(-)
(-)
VIII (T9)
4-Br
50
(-)
(-)
50
(-)
(-)
(-)
(-)
(-)
IX (T10)
3-NO2
50
(-)
(-)
25
(-)
(-)
(-)
(-)
(-)
X (T4)
4-NO2
50
(-)
(-)
50
(-)
(-)
(-)
(-)
(-)
XII (T5)
4-OH
50
(-)
(-)
50
(-)
(-)
(-)
(-)
(-)
CHNG 4. BN LUN
4.1.
V tng hp ha hc
4.1.1. V phn ng tng hp 2-thiohydantoin (BT)

S phn ng:
OH
H2N
O
H
N
NH4SCN, Ac2O
HCl 10%
H
N
S
1000C, 30 pht
N
H
un hi lu, 1 gi
O
BT
Phn ng gm c 2 giai on:

- Tng hp 1-acetyl-2-thiohydantoin (A)
- Thy phn cht A to 2-thiohydantoin (BT)
C ch phn ng c th c gii thch nh sau:
- Bc 1: Anhydrid acetic tc dng vi glycin to N-acetylglycin:
O
H3 N
H3 C
O
O
CH3
H3C
N
H
OH +
CH3COOH
- Bc 2: Acid acetic c to ra t bc 1 phn ng vi amoni

thiocyanat to ra acid thiocyanic, cht ny ng phn ha to ra acid
isothiocyanic:
CH3COOH +
NH4SCN
HS C N +
CH3COONH4
S C NH
acid isothiocyanic
- Bc 3: N-acetylglycin cng hp i nhn vo acid isothiocyanic, tip

xy ra phn ng loi nc ng vng to thnh 1-acetyl-2-thiohydantoin
(A):
50
S
HN
HN C S
HOOC
HOOC
NH
H2N
NH
C
N
HOOC
Ac
Ac
Ac
- H2O
S
HN
O
N Ac
(A)
- Bc 4: Thy phn 1-acetyl-2-thiohydantoin (A) to ra 2-thiohydantoin
(BT):
S
HN
N Ac
+ H2O
- AcOH
HN
NH
BT
Tng hp 2-thiohydantoin theo quy trnh trn i t nhng nguyn liu r

tin, d kim. Do , t tng hp c 2-thiohydantoin gip chng ti c th
ch ng nguyn liu u v gim chi ph nghin cu.
4.1.2. V phn ng ngng t to 5-aryliden-2-thiohydantoin (I-XIII)
* Tng hp dn cht 5-aryliden-2-thiohydantoin (I-XIII) c tin hnh
theo 2 phng php:
- Phng php A (s dng xc tc ethanolamin, dung mi ethanol, nhit
phn ng l 900C): Chng ti s dng phng php A iu ch cc
cht I, II, III, VI, VIII, X, XI. Chng ti la chn phng php tng hp ny
v dung mi v cht xc tc s dng u r tin, d kim v iu kin phn
ng nh nhng, t c hiu sut kh cao (50-75%). Phng php ny cng
c mt s tc gi s dng trong nhng nm gn y [1], [3], [4], [23].
Vi cc cht IV, V, VII, IX, XII, XIII chng ti cng tin hnh tng
hp theo phng php A do cc u im nh trn. Tuy nhin sn phm thu
51
c cha sch. Kt qu sc k cho thy sn phm thu c c 2 vt trn sc

k . Kt qu ghi ph 1H-NMR cng cho thy cht cha sch. Chng ti gi
thit c sn phm ph I l sn phm cng hp kiu aldol ca 2-thiohydantoin
vi aldehyd thm c to thnh theo phn ng:
H
N
R
CHO +
NH
H
N
H
C
NH
OH
I'
R = 3'-Cl (IV); 4'-Cl (V); 3'-Br (VII);
3'-NO2(IX); 4'-OH (XII); 4'-OCH3 (XIII)
R = 3'-Cl (IV); 4'-Cl (V); 3'-Br (VII);

3'-NO2(IX); 4'-OH (XII); 4'-OCH3 (XIII)
Sn phm ny c th c to thnh 1 phn do phn ng din ra 900C

cha loi nc hon ton to thnh hp cht 5-aryliden-2-thiohydantoin
(I).
- Phng php B (s dng xc tc natri acetat khan, acid acetic bng,
nhit phn ng l 130-1350C). Chng ti s dng phng php B iu
ch cc cht IV, V, VII, IX, XII, XIII. Kt qu sc k cho thy c 1 vt trn
sc k . Kt qu ghi ph 1H-NMR v 13C-NMR cng cho thy cht sch.
Nh vy, s dng phng php B thu c cht tinh khit hn c th do phn
ng tin hnh nhit cao (130-1350C) cho php phn ng loi nc xy ra
hon ton.
R
H
C
OH
H
N
S
NH
H
C
H
N
NH
- H2O
I'
Nm 2010 nhm tc gi tin hnh phn ng ngng t hydantoin vi

cc aldehyd thm theo phng php A vi thi gian phn ng l 4-5 gi [1].
So snh 2 phn ng ngng t ta thy: Phn ng ngng t 2-thiohydantoin vi
cc aldehyd thm xy ra d dng hn phn ng ngng t hydantoin vi
52
aldehyd thm th hin thi gian phn ng ngn hn (30 pht so vi 4-5
gi).
Gii thch kh nng phn ng ngng t ca 2-thiohydantoin vi aldehyd
thm: chng ti cho rng c th v kh nng phn ng d hn ca 2thiohydantoin so vi hydantoin khi phn ng ngng t vi aldehyd l do
nhm C2=S (thiohydantoin) phn cc km hn nhiu so vi C2=O
(hydantoin) nn khng nh hng n cc orbital ca C5 v th hiu ng
siu lin hp ca CH2 C-5 vi C4=O xy ra bnh thng. Trong khi
nhm C2=O (hydantoin) phn cc mnh ht in t ca lin kt CH2 C-5
v pha C2 lm lch orbital ca C5 do lm hiu ng siu lin hp ca
CH2 C5 vi C4=O gim i nn kh nng phn ng ca CH2 km hn
* V t l s mol cc cht tham gia phn ng ngng t phng php B
c nhiu cng trnh nghin cu v phn ng ngng t ca hydantoin
v cc aldehyd thm vi t l s mol khc nhau v cc cht tham gia phn ng
hydantoin : aldehyd thm : natri acetat l 1:1:2, 1:1:3 v 1:1:4,6 [34], [38],
[40], [41]. Khi p dng cc t l ny vo phn ng ngng t gia 2thiohydantoin vi clorobenzaldehyd, chng ti nhn thy rng t l 1:1:4,6
cho hiu xut cao hn so vi t l 1:1:2 v 1:1:3. T chng ti chn t l
ny trong trng hp cc cht IV, V, VII, IX, XII, XIII.
* V vic la chn nhm th trn hp phn aldehyd thm.
Vic la chn cc aldehyd thm c cc nhm th khc nhau da trn thng s
v hiu ng in t (hng s th Hammett ) v hng s th thn du () ca
nhm th v y l hai thng s c bn lin quan n nh hng ca nhm th
n tnh cht l ho v hot tnh sinh hc ca cht nghin cu. Chng ti
la chn cc nhm th i in ca 2 loi nhm th vi cc tnh cht khc
nhau v hiu ng in t v tnh thn du, thn nc nh sau:
+ , + : cc nhm th ht in t v thn du (-F, -Cl, -Br).
53
+ , - : cc nhm th ht in t v thn nc (-NO2).

- , + : cc nhm th y in t v thn du [-OCH3].
- , - : cc nhm th y in t v thn nc (-OH).
T cht I - X, chng ti la chn cc aldehyd thm l cc hp cht cha cc
nhm ht in t (nhm th cloro cc cht III-V; nhm th bromo cc
cht VI-VIII; nhm th fluoro hp cht II, nhm th -NO2 cc cht IX,
X). Theo tc gi V Trn Anh [1], y l nhng nhm th gp phn mang
li hot tnh khng khun, khng nm, hot tnh gy c t bo ca cc dn
cht 5-arylidenhydantoin v dn cht base Mannich ca chng. C ch tc
dng gi thit tng t nh nu ca tc gi V Trn Anh [1], [13] l : cc
nhm th ht in t (Cl, NO2) lm phn cc mnh lin kt i C=C ca
nhm carbonyl ,-khng no
(6) (5) (4)

C C C O
ca phn t th nghim dn ti kh
nng tham gia phn ng cng Michael ca chng vi cc cht i nhn sinh
hc ca t bo ung th vi h qu c ch s pht trin ca t bo ung th ny
[10]. Mt khc, lin kt i lin hp ngoi vng cn to cho hp cht c kh
nng tc dng l tng tc vi v tr hot ng ca EGFR (th th ca yu t
tng trng biu m). l c ch tc dng kp ca dy cht ny do cc nh
nghin cu V.Zuliani v C.Carmi nu ra rt gn y, vo nm 2009 khi
nghin cu tc dng chng ung th ca mt s dn cht 5-arylidenhydantoin
[38].
T cht XI-XIII, chng ti la chn cc aldehyd thm cha cc nhm y
in t (-OH vi cht XI, XII, -OCH3 vi cht XIII). Trong ti liu chng ti
tham kho c ca cc tc gi Carmi [11], Zuliani [38], dn cht 5-(4hydroxybenzyliden)hydantoin th hin hot tnh trn t bo ung th tin lit
tuyn PC3.
4.2.
V xc nh cu trc ca cc cht tng hp c
54
4.2.1. V ph hng ngoi (IR)

13 dn cht 5-aryliden-2-thiohydantoin (I-XIII) tng hp c c
ghi ph IR. Qua phn tch ph , chng ti nhn bit c cc di hp th
c trng ca cc nhm chc v lin kt ca cc cht tng hp c [5]. Cc
s liu ph c trng ca tng cht c ghi bng 3.3.
Sau y l cc bin gii ph hng ngoi ca cc cht tng hp c.
- Ph hng ngoi ca cc cht u xut hin mt di hp th tng ng
vi dao ng ha tr ca nhm NH nm trong vng t 32203128 cm-1
- Ph hng ngoi ca cc cht u c xut hin mt di hp th mnh
tng ng vi dao ng ho tr ca nhm carbonyl, nm trong vng t
-1
17451713cm .
- Ph hng ngoi ca cc cht u c xut hin mt di hp th mnh
tng ng vi dao ng ho tr ca nhm >C=S, nm trong vng t
-1
15221477cm .
- Trn ph IR ca 13 cht (I-XIII) u xut hin di hp th c trng
cho lin kt i ethylenic (C(5)=C(6)) c to thnh do phn ng ngng t
-1
nm trong vng t 16661642cm
- Trn ph IR ca 13 cht (I-XIII) u xut hin di hp th c trng

cho dao ng bin dng C-H ngoi mt phng ca nhn thm nm trong
-1
vng t 895625cm .
- Trn ph IR ca 3 cht III-V
xut hin di hp th s sng
-1
10961093 cm c trng cho dao ng ho tr ca lin kt C-Cl c mt trong

cu trc ca cht ny.
- Ph IR ca II xut hin di hp th c trng cho lin kt Carom-F (1235
cm-1)
55
- Ph IR ca cht 3 cht VI-VIII xut hin di hp th c trng cho lin

kt Carom-Br (10781027cm-1).
- Ph IR ca 2 cht IX, X xut hin di hp th s sng
-1
-1
15221486cm ng vi dao ng ho tr bt i xng, 13851336 cm ng

vi dao ng ho tr i xng ca nhm NO2 c mt trong cu trc ca cc
cht ny.
-
Ph IR ca 2 cht XI, XII xut hin di hp th s sng

-1
-1
13651353cm v 12521256cm c trng cho dao ng bin dng ca

nhm OH c mt trong cu trc ca cc cht ny.
-1
- Ph IR ca cht XIII xut hin di hp th s sng 1370cm c

trng cho dao ng bin dng ca nhm CH3 lin kt =C-OCH3 c mt
trong cu trc ca cht ny.
y l cc tn hiu quan trng cho php s b khng nh s to thnh sn
phm ngng t 5-aryliden-2-thiohydantoin (I-XIII).
4.2.2. V ph cng hng t ht nhn 1H-NMR
Ph 1H-NMR cho thy cc tn hiu c trng ca cc proton trong phn
t ca cc cht to thnh [5]. 15 ph 1H-NMR c ghi cc ph lc 1 2,
3.2 15.2. Kt qu phn tch s liu ph cng hng t ht nhn proton c
ghi bng 3.2 v 3.4.
- V xc nh cu trc ca hp cht A: Ph 1H-NMR ch thy tn hiu
ca proton H-N3, H-5 v CH3 chng t v tr N1 b th (xem ph lc 1).
- So snh ph 1H-NMR ca BT v BM: cc tn hiu ca proton H-N3, HN1, H-5 ghi c tng ng nhau (xem bng 3.2). Nh vy, cht tng hp
c BT ng nht vi cht mu BM.
- V xc nh cu trc ca hp cht BT: Ph 1H-NMR cho thy tn hiu
ca proton H-N1, H-N3 xut hin 9,813 ppm v 11,624ppm v H-5 c
56
=4,07ppm chng t nhm th v tr N1 ca hp cht A mt i (xem ph

lc 2.2).
- V xc nh cu trc ca cc hp cht I-XIII :
+ Vi cc cht I-XIII, 1H-NMR cho thy tn hiu ca proton H-N1, HN3 v khng thy H-5 chng t c nhm th v tr C-5.
+ Proton ethylenic (H-6) to thnh t phn ng ngng t 2-thiohydantoin
(BT) v cc aldehyd thm c tn hiu cng hng dng singlet nm trong
vng t 6,36 6,61 ppm.
+ Trn ph 1H-NMR ca cc cht I-XIII cn xut hin cc
tn hiu c trng ca proton nhn thm hp phn 5-aryliden.
4.2.3. V xc nh ng phn hnh hc ca dn cht 5-aryliden-2thiohydantoin (I-XIII) da trn ph 1H-NMR.
Cc hp cht tng hp ca chng ti c xut hin lin kt i lin kt
vi C-5 ca 2-thiohydantoin, 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 c th
phn bit vi nhau da trn chuyn dch ho hc ca proton ethylenic (H6) v proton ortho phenyl trn phn benzyliden (H2 v H6).
O
H
H6'
6
C
NH
3
2
4
5
1
N
H
1'
H4'
H3'
O
S
H2'
H5'
H2'
H6'
H5'
6
C
H
H4'
4
5
3
2
1
N
H
NH
H3'
ng phn Z
ng phn E
Do nhm carbonyl C4=O gn proton ethylenic H-6 ca dng ng phn
Z, nn hiu ng anisotropic C4=O gim chn proton H-6 dng ng phn Z
so vi ng phn E, v th proton H-6 ca dng ng phn Z s cng hng
trng yu hn v c chuyn dch ho hc ln hn.
57
Nhn xt trn ph hp vi cc nghin cu tin hnh ca tc gi

Tan,S.F v cng s [32], tc gi Zuliani v cng s [38]. Theo nghin cu ca
cc tc gi trn chuyn dch ho hc ca proton H-6 vng trng thp
(H 6,40 7,00 ppm) s tng ng vi ng phn Z, vng trng cao (H
6,20 6,30 ppm) s tng ng vi ng phn E.
13 cht (I-XIII) c chuyn dch ho hc ca proton H-6 t 6,36
6,61 ppm tng ng l ng phn Z. Vy cc cht (I-XIII) chng ti tng
hp c c cu hnh Z.
4.2.4. V ph cng hng t ht nhn 13C-NMR
Khi xt ph
13
C-NMR ca cc hp cht 5-aryliden-2-thiohydantoin
chng ti nhn thy tn hiu trn ph tng ng ph hp vi s lng

cng nh chuyn dch ho hc ca nguyn t carbon[5].
Kt qu phn tch s liu ph c ghi bng 3.5.
- Nguyn t carbon nhm C=O (C-4) c tn hiu cng hng trong
vng t 165,47-166,5 ppm.
- Nguyn t carbon nhm C=S (C-2) c tn hiu cng hng trong
vng t 178,28-180,56 ppm.
- Tn hiu ca nguyn t C-6 (carbon ethylenic) nm trong vng t
108,46-112,34 ppm.
- Tn hiu ca nguyn t C ca vng phenyl nm trong vng t 114,36161,33 ppm.
* thy r tng tc t gia proton v carbon C-13 cc hp cht 5aryliden-2-thiohydantoin (I-XIII) chng ti tin hnh ghi ph 2D-NMR
(HSQC v HMBC) ca mt s hp cht I, II, V, XII, , sau y l bin gii
cc tn hiu trn ph tng tc 2 chiu ca cht V:
58
- Trn ph HSQC ca cc cht ghi ph cho thy tng tc gn gia

proton ca ethylenic (H-6) c nm trong vng t 6,36 6,69 ppm vi
nguyn t carbon ethylenic (C-6) c nm trong vng t 108,46 112,34
ppm (xem hnh 4.1)
- Vi cht V, vng phenyl ca hp phn 5-aryliden c nhm th Cl v
tr para, khi xut hin tng tc gia proton H-2 v H-6 ( 7,76 ppm) vi
nguyn t C-2 v C-6 ( 131,76 ppm), gia proton H-3 v H-5 ( 7,47
ppm) vi nguyn t C-3 v C-5 ( 128,74 ppm) (xem hnh 4.1).
- Trn ph HMBC ca cc cht tng hp c cho thy proton
ethylenic (H-6) c tng tc 3J-HMBC vi C-4 , C-2 v C-6 ca vng
benzen.
O
4
H
6
C
6'
1
N
H
3
2
NH
2'
3'
5'
4'
Hnh 4.1 Ph tng tc HSQC ca cht V
59
- Trong cc hp cht 5-aryliden-2-thiohydantoin chng ti tng hp

c hp phn aldehyd thm, v tr 5 v 6 khng c nhm th. Trn ph
HMBC ca cc cht tng hp c cho thy proton H-5 c tng tc 3JHMBC vi C-1 v C-3 ca vng benzen, proton H-6 c tng tc 3JHMBC vi carbon C-6 (carbon ethylenic), C-2 v C-4 ca nhn thm.
O
4
H
6
C
1'
H 6'
1
N
H
3
2
NH
2'
5'
3'
H
4'
- Ph HMBC ca cht V cho thy xut hin cc tng tc xa gia cc

proton v carbon trong nhn thm cng tng t nh vi cht I. Nhng cht
V, v tr 4 c nhm th cloro nn khng cn proton H-4, khng xut hin
tng tc xa gia proton H-4 vi C-2 v C-6 (xem hnh 4.2).
3'
Cl
2'
4'
1'
5'
6'
60
CH
4 3NH
5
1 2
N
H
Hnh 4.2 Ph tng tc HMBC ca cht V

Bng 4.1: Cc tng tc 1H-13C ca cht V
Proton
Carbon lin kt trc
Carbon cch 3 lin
tip
kt
V tr
(ppm)
V tr
(ppm)
H-6
6,47
C-6
109,93
H-3,5
7,47
C-3,5
128,74
H-2,6
7,76
C-2, 6
131,76
4.2.5. V ph MS
61
V tr
(ppm)
C-2,6
131,76
C-4
165,68
C-1
131,28
C-5,3
128,74
C-4
133,68
C-6,2
131,76
khng nh thm v cu trc ca cc hp cht 5-benzyliden-2thiohydantoin (I-XIII), chng ti tin hnh ghi ph MS (ESI-MS) ca cc
hp cht ny. T kt qu phn tch ph c trnh by bng 3.6, nhn thy
13 cht (I-XIII) c pic ion phn t [M-H]-. Cc cht cha nguyn t Clo
trong phn t (III-V) u c pic ng v i km pic phn t vi s khi ln
hn 2, v t l cng (1:3) ca 2 pic tng ng ph hp vi t l ng v
37
Cl/35Cl trong t nhin l 32,5%. 3 cht (VI-VIII) c cha nguyn t Brom
trong phn t u c pic ng v i km pic phn t vi s khi ln hn 2, t

l cng (1:1) ca hai pic ph hp vi t l ng v 81Br/79Br l 98,1%.
4.3.
V hot tnh sinh hc
4.3.1. V hot tnh gy c t bo ung th

Kt qu cho thy c 3 cht l cht V (IC50= 4,66 g/ml), cht VII (IC50=
3,22 g/ml) v cht XI (IC50= 4,72 g/ml) c hot tnh c ch t bo ung th
gan Hep-G2.
So snh kt qu th hot tnh gy c t bo ung th cc cht chng ti tng
hp c (I-XIII) vi cc dn cht 5-arylidenhydantoin, cc dn cht 5-(3halogenoaryliden)hydantoin v 3-aryl-5-arylidenhydantoin cng b [1],
[3], [4] cho thy cc dn cht 5-arylidenhydantoin v 5-aryliden-2thiohydantoin u c tc dng ch yu trn dng t bo gan Hep-G2. y l
kt qu ng lu cho thy cc dn cht thuc nhm ny c tc dng nh
hng trn dng t bo ung th gan Hep-G2. C ch tc dng gi thit l :
lin kt i C=C ca nhm carbonyl ,-khng no
(6) (5) (4)

C C C O
ca phn t
th nghim dn ti kh nng tham gia phn ng cng Michael ca chng vi

cc cht i nhn sinh hc ca t bo ung th vi h qu c ch s pht trin
ca t bo ung th ny [13]. Mt khc, lin kt i lin hp ngoi vng cn
to cho hp cht c kh nng tc dng l tng tc vi v tr hot ng ca
EGFR (th th ca yu t tng trng biu m). l c ch tc dng kp
62
ca dy cht ny do cc nh nghin cu V.Zuliani v C.Carmi nu ra rt gn

y, vo nm 2009 khi nghin cu tc dng chng ung th ca mt s dn
cht 5-arylidenhydantoin [38].
C th do lin kt i lin hp vng ngoi to cho cc hp cht 5arylidenhydantoin v 5-arylidenthiohydantoin c tng tc vi v tr hot
ng trn EGFR c th ca t bo ung th gan Hep-G2 nn dn n tc
dng nh hng trn t bo ung th gan Hep-G2 ca cc dy cht 5arylidenhydantoin, 3-aryl-5-arylidenhydantoin v 5-aryliden-2-thiohydantoin.
- V mi lin quan cu trc tc dng gy c t bo ung th ca dy cht 5aryliden-2-thiohydantoin (I-XIII), c th nhn xt rng hp phn 5-aryliden
c th gp phn mang li hot tnh gy c t bo ung th khi cha nhm th
ht in t nh Cl (cht V), -Br (cht VII) v nhm y in t -OH (cht
XI). iu ny cho thy tnh cht in t ca nhm th khng nh hng quyt
nh n hot tnh gy c t bo ca dy cht ny. Mt khc, c th nhn
thy v tr ca cc nhm th trn hp phn 5-aryliden c nh hng n hot
tnh c ch t bo ung th gan Hep-G2 ca dy cht ny, th hin kt qu
nh sau:
- Cht V ( Cl v tr 4) c hot tnh, trong khi hai cht ng phn l
cht III (Cl v tr 2) v cht IV ( Cl v tr 3)li khng c hot
tnh.
- Cht VII ( Br v tr 3) c hot tnh, trong khi 2 cht ng phn
l cht VI (Br v tr 2) v cht VIII ( Br v tr 4) li khng c
hot tnh.
- Cht XI (OH v tr 2) c hot tnh, trong kh cht ng phn l
cht XII (OH v tr 4) li khng c hot tnh.
Bn cnh hot tnh c ch t bo ung th Hep-G2 ca 3 cht (V, VII, XI) kt
qu th nghim cn cho thy kt qu ng ch l 3 cht gm: Cht III
63
(IC50=24,40g/ml), cht V (IC50=27,08g/ml), cht VII (IC50=26,36g/ml) c

hot tnh c ch yu trn t bo ung th i trng HCT 116.
4.3.2. V tc dng khng khun, khng nm
T kt qu th sng lc hot tnh khng khun, khng nm ca 11 dn cht 5aryliden-2-thiohydantoin tng hp c (ghi bng 3.11), chng ti c mt
s nhn xt sau y:
- Trong s 11 cht tng hp c c 10 cht (I, III-X, XII) c hot tnh
khng chng vi khun B.subtilis (MIC=25-50g/ml).
- C 11 cht tng hp c u khng c hot tnh khng 1 chng
Gram (+) l S.aureus, 2 chng vi khun Gram (-) (E.coli, P.aeruginosa),
khng c hot tnh khng cc chng nm F.oxysporum v C.albicans.
Cc kt qu nu trn cho thy cc cht ca chng ti tng hp c c hot
tnh khng khun v khng nm khng ng k.
64
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 13 dn cht 5-aryliden-2-thiohydantoin (I-XIII),
trong s c 7 cht (II, III, IV, VI, VII, VIII, IX) 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 (1HNMR), ph cng hng t ht nhn 13C (13C-NMR), ph khi lng (MS) v
4 cht (I, II, V, XII) 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. a) th hot tnh gy c t bo ung th ca 13 cht (I-XIII) tng
hp c i vi 5 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, t bo
ung th phi A549, t bo ung th i trng HCT116. Kt qu cho thy c 3
cht l cht V (IC50= 4,66g/ml), cht VII (IC50= 3,22 g/ml),
cht XI
(IC50= 4,72 g/ml) c hot tnh c ch t bo ung th gan Hep-G2; c 3 cht

l: cht III (IC50=24,40g/ml), cht V (IC50=27,08g/ml), cht VII
(IC50=26,36 g/ml) c hot tnh c ch yu t bo ung th i trng HCT116.
C 13 cht (I-XIII) u khng c hot tnh gy c t bo ung th tuyn tin
lit PC3 v t bo ung th biu m v MCF7.
b) th tc dng khng khun, khng nm ca 11 cht (I-X, XII).
Kt qu cho thy c 10 cht (I, III-X, XII) c hot tnh khng chng vi khun
B.Subtilis (MIC=25-50g/ml)
65
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 ung th ca cc cht tng
hp c trn cc dng t bo khc nh : ung th ty, ung th d dy
- Tip tc tng hp v sng lc hot tnh gy c t bo ung th ca cc
dn cht 5-aryliden-2-thiohydantoin khc nhm tm kim cc cht khc c
trin vng trong dy cht ny.
66
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IUniversite Joseph-Fourier GRENOBLE 1.
DANH MC PH LC
Ph lc 1
: Ph 1H-NMR ca hp cht A
Ph lc 2 (2.1- 2.2)
: Ph 1H-NMR 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- 5.4)
: Cc ph ca hp cht III
Ph lc 6 (6.1- 6.4)
: Cc ph ca hp cht IV
Ph lc 7 (7.1- 7.6)
: Cc ph ca hp cht V
Ph lc 8 (8.1 8.4)
: Cc ph ca hp cht VI
Ph lc 9 (9.1- 9.4)
: Cc ph ca hp cht VII
Ph lc 10 (10.1- 10.4)
: Cc ph ca hp cht VIII
Ph lc 11 (11.1- 11.4)
: Cc ph ca hp cht IX
Ph lc 12 (12.1- 12.4)
: Cc ph ca hp cht X
Ph lc 13 (13.1- 13.4)
: Cc ph ca hp cht XI
Ph lc 14 (14.1- 14.6)
: Cc ph ca hp cht XII
Ph lc 15 (15.1- 15.4)
: Cc ph ca hp cht XIII
Ph lc 16
: Phiu tr li kt qu th hot tnh gy c

t bo (cytotoxicity assay)
Ph lc 17
: Phiu tr li kt qu th hot tnh khng vi

sinh vt kim nh (antimicrobial assay)
H
N
4 3
2
51
CH3
Ph
Ph lc 1: Ph 1H-NMR ca hp cht A
H
N
3
4
51 2
N
H
Ph l
c 2.1: Ph 1H-NMR ca hp cht BM
mu
H
N
3
4
51 2
N
H
Ph
lc 2.2: Ph 1H-NMR ca hp cht BT
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN

Ten may: GX-PerkinElmer-USA Resolution: 4cm-1 Date: 4/1/2013
Nguoi do: Phan Thi Tuyet Mai
Ten mau: T1
98.8
95
90
827
85
985
888
923
80
964
75
70
65
1764
1251
60
706
55
1091
767
50
1657
%T
1155
45
660
2813
40
1364
1348
1186
1172
682
35
1642
30
25
HN
3220
1446
1497
NH
20
15
1477
10
1723
5
0.0
4000.0
3600
3200
2800
2400
2000
1800
cm-1
1600
Ph lc 3.1 : Ph IR ca cht I
1400
1200
1000
800
600.0
S
HN
NH
O
Ph
lc 3.2 : Ph 1H-NMR ca cht I
S
HN
NH
O
Ph
Ph lc 3.3 : Ph 13C-NMR ca cht I
S
HN
NH
O
Ph lc 3.4 : Ph HSQC ca cht I
S
HN
NH
O
Ph lc 3.5 : Ph HMBC ca cht I
S
HN
NH
H
O
Ph lc 3.6 : Ph MS ca cht I

Ten mau: T6
99.9
95
3428
90
85
80
1012
992
75
714
2534
663
882
70
622
895
751
853
65
1766
2901
60
2821
55
%T
50
1271
682
966
3208
45
3056
1311
40
785
3153
830
35
1343
30
25
HN
1158
NH
20
1653
15
1235
1597 1514
1188
1375
10
1485
1729
5
0.0
4000.0
1093
1298
3600
3200
2800
2400
2000
1800
cm-1
1600
Ph lc 4.1: Ph IR ca cht II
1400
1200
1000
800
600.0
S
HN
F
NH
O
Ph
lc 4.2: Ph 1H-NMR ca cht II
S
HN
F
N
NH
O
Ph
Ph lc 4.3: Ph 13C-NMR ca cht II
S
HN
F
N
NH
O
Ph
Ph lc 4.4: Ph HSQC ca cht II
S
HN
F
NH
O
Ph
Ph lc 4.5: Ph HMBC ca cht II
S
HN
F
NH
O
Ph lc 4.6: Ph MS ca cht II

Ten mau:
Tran Anh-T4
100.0
95
90
85
3439
80
830
846
75
728
874
70
969
738
792
1034
65
2564
693
1156
1130
60
663
645
1278
635
1435
55
1050
1095
%T
50
2861
1470
757
45
1338
3128
40
1245
1193
35
Cl
HN
30
1666
NH
1380
25
1521
20
15
10
1729
5
0.0
4000.0
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
Ph lc 5.1: Ph IR ca cht III
1200
1000
800
600.0
S
Cl
HN
NH
O
Ph lc 5.2: Ph1H-NMR ca cht III
S
Cl
HN
NH
O
Ph lc 5.3: Ph13C-NMR ca cht III
S
Cl
H
HN
NH
O
Ph lc 5.4: Ph MS ca cht III

Ten may: GX-PerkinElmer-USA
Nguoi do: Nguyen Thi Son
Resolution: 4cm-1
Mail: sonhuco@yahoo.com
Date: 4/21/2014
DT: 0912140352
MAU T8
100.0
95
1001
90
85
80
1096
964
75
1255
1283
70
65
722
681
638
907
2826
782
60
55
1196
%T
50
3007
1372
45
3151
40
1648
1503
35
30
Cl
25
HN
20
15
1721
NH
O
10
5
0.0
4000.0
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
Ph lc 6.1: Ph IR ca cht IV
1200
1000
800
600.0
S
Cl
HN
NH
O
Ph lc 6.2: Ph 1H-NMR ca cht IV
S
Cl
H
HN
NH
O
Ph lc 6.3: Ph 13C-NMR ca cht IV
S
Cl
H
HN
NH
O
Ph lc 6.4: Ph MS ca cht IV

Ten mau: T2
100.5
95
90
635
85
80
948
623
886
75
70
717
727
1010
2532
839
696
65
1070
2825
60
757
969
872
1413
819
55
%T
1389
50
671
1269
1104
1281
45
1091
3220
40
1309
35
3323
30
HN
25
Cl
1190
NH
20
1647
1360
15
1502
1713
10
1482
5
0.0
4000.0
3600
3200
2800
2400
2000
1800
cm-1
1600
Ph lc 7.1: Ph IR ca cht V
1400
1200
1000
800
600.0
O
5'
Cl
6'
1'
4'
3'
2'
CH
H
3NH
1 2
4
5
N
H
Ph lc 7.2: Ph 1H-NMR ca hp cht V
O
5'
Cl
6'
1'
4'
3'
2'
CH
H
3NH
2
1
4
5
N
H
Ph lc 7.3: Ph 13C-NMR ca hp cht V
O
5'
Cl
6'
1'
4'
3'
2'
CH
3NH
1 2
4
5
N
H
Ph
Ph lc 7.6: Ph MS ca hp cht V
O
5'
Cl
6'
1'
4'
3'
2'
CH
3NH
1 2
4
5
N
H
Ph lc 7.4: Ph HSQC ca hp cht V
O
5'
Cl
6'
1'
4'
3'
CH
2'
Ph lc 7.5: Ph HMBC ca hp cht V
3NH
1 2
4
5
N
H

Ten mau:
T11
102.0
95
90
85
875
1121
80
968
1043
848
722
791
1027
75
2922
3440
70
1433
1279
741
645
1095
2853
663
1468
65
1193
1340
756
1247
60
55
3145
1664
%T 50
45
1380
40
1522
35
S
Br
30
HN
25
20
NH
O
1730
15
10
5
0.0
4000.0
3600
3200
2800
2400
2000
1800
cm-1
1600
Ph lc 8.1: Ph IR ca cht VI
1400
1200
1000
800
600.0
S
Br
HN
NH
O
Ph
Ph lc 8.2: Ph 1H-NMR ca cht VI
S
Br
HN
NH
O
Ph
Ph lc 8.3: Ph 13C-NMR ca cht VI
S
Br
HN
NH
O
Ph
Ph lc 8.4: Ph MS ca cht VI

Resolution: 4cm-1
DT: 0912140352
Date: 5/8/2014
T7
101.7
90
3415
80
2829
704
1560
859
1073
70
753
965
681
1096
637
1256
1281
60
895
781
3183
1471
%T
50
1195
40
Br
1370
S
HN
30
NH
1646
1723
1715
1499
20
10
0.0
4000.0
3600
3200
2800
2400
2000
1800
cm-1
1600
Ph lc 9.1: Ph IR ca cht VII
1400
1200
1000
800
600.0
S
Br
HN
NH
O
Ph lc 9.2: Ph 1H-NMR ca chtVII
S
Br
HN
NH
O
Ph lc 9.3: Ph13C-NMR ca chtVII
S
Br
HN
NH
O
Ph lc 9.4: Ph MS ca chtVII

Resolution: 4cm-1
DT: 0912140352
Date: 4/21/2014
T9
100.0
90
1575
1003
962
1272
80
751
892
1186
1169 1078
70
822
635
60
3379
%T
2935
1645
1343
50
40
3226
30
HN
Br
20
1477
NH
O
10
0.0
4000.0
1727
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
Ph lc 10.1: Ph IR ca cht VIII
1200
1000
800
600.0
S
HN
Br
N
NH
O
Ph lc 10.2: Ph 1H-NMR ca cht VIII
S
HN
Br
NH
O
Ph lc 10.3: Ph 13C-NMR ca cht VIII
S
HN
Br
NH
O
Ph lc 10.4: Ph MS ca cht VIII

Resolution: 4cm-1
Date: 4/21/2014
DT: 0912140352
T10
100.0
90
80
1198
1165
70
1098
1010 929
898
869
961
1600
679
814
761
632
60
%T
1647
50
1385
3344
1256
40
3148
1486
30
S
O2N
20
HN
10
1724
NH
O
0.0
4000.0
3600
3200
2800
2400
2000
1800
1600
1400
1200
cm-1
Ph lc 11.1: Ph IR ca cht IX
1000
800
600
400.0
S
O2N
HN
NH
O
Ph lc11.2: Ph 1H-NMR ca cht IX
S
O2N
HN
NH
O
Ph lc11.3: Ph 13C-NMR ca cht IX
S
O2N
HN
NH
O
Ph lc 11.4: Ph MS ca cht IX

Ten mau: T4
128.8
126
124
903
122
1110
120
826
745
845
622
1396
3007
874
118
1375
1593
116
759
968
1255
706
1085
670
114
1195
112
1655
3212
3309
110
1496
1177
108
%T 106
1482
104
S
102
HN
100
O2N
98
1319
NH
1756
96
1745
94
92
1522
90
88
85.0
4000.0
1336
3600
3200
2800
2400
2000
1800
cm-1
1600
Ph lc 12.1: Ph IR ca cht X
1400
1200
1000
800
600.0
S
HN
O2N
NH
O
Ph lc 12.2: Ph1H-NMR ca cht X
S
HN
O2N
NH
O
Ph
Ph lc 12.3: Ph13C-NMR ca cht X
S
HN
O2N
NH
O
Ph lc 12.4: Ph MS ca cht X

Resolution: 4cm-1
Date: 4/21/2014
MAU T12
DT: 0912140352
100.0
90
80
882
1255
70
811
2816
753
60
1657
1102
%T
50
3018
1174
40
1526
S
OH
3206
HN
30
629
677
NH
1730
1350
1484
20
10
0.0
4000.0
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
Ph lc 13.1: Ph IR ca cht XI
1200
1000
800
600.0
S
OH
HN
NH
O
Ph lc 13.2: Ph 1H-NMR ca cht XI
S
OH
HN
NH
O
Ph lc 13.3: Ph13C-NMR ca cht XI
S
OH
HN
NH
O
Ph lc 13.4: Ph MS ca cht XI

Ten mau:
Tran Anh-T5
100.0
95
90
731
85
627
80
864
686
938 879
997
75
849
983
70
806
826
65
967
60
55
%T
1314
50
1348
1648
45
1365
3170
40
1707
3322
35
HN
30
HO
25
1719
1263
1252
1586
1603
NH
1174
20
1499
15
10
5
0.0
4000.0
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
Ph lc 14.1: Ph IR ca cht XII
1200
1000
800
600.0
S
HN
HO
NH
O
Ph lc 14.2: Ph 1H-NMR ca cht XII
S
HN
HO
NH
O
Ph lc 14.3: Ph 13C-NMR ca cht XII
S
HN
HO
NH
O
Ph lc 14.6: Ph MS ca cht XII
S
HN
HO
NH
O
Ph lc 14.4: Ph HSQC ca cht XII
S
HN
HO
NH
O
Ph lc 14.5: Ph HMBC ca cht XII

Resolution: 4cm-1
Date: 5/8/2014
DT: 0912140352
T13
104.4
100
90
2885
884
622
1304
80
683
796
766
1015
70
957
1096
822
3038
60
3248
3142
1514
%T 50
1370
3434
1643
1596
40
1722
S
HN
30
H3C O
20
1172
1484
1258
NH
O
10
0.0
4000.0
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
Ph lc 15.1: Ph IR ca cht XIII

XIII
1200
1000
800
600.0
S
HN
H3C O
NH
O
Ph lc 15.2: Ph 1H-NMR ca cht XIII
S
HN
H3C O
NH
O
Ph lc 15.3: Ph 13C-NMR ca cht XIII
S
HN
H3C O
NH
O
Ph lc 15.4: Ph MS ca cht XIII
PH LC 16:
PHIU TR LI KT QU TH HOT TNH
GY C T BO (CYTOTOXICITY ASSAY)
Ch thch:
K hiu mu
ti
Phiu tr li kt qu
ti
K hiu mu
Phiu tr li kt qu
T1
VIII
T9
II
T6
IX
T10
III
IV
V
VI
VII
T3
T8
T2
T11
T7
X
XI
XII
XIII
T4
T12
T5
T13
NGHIN CU TC DNG C T BO IN VITRO CA CC MU DO

TS. TRN ANH I HC DC CUNG CP
n v thc hin: Khoa Dc l Sinh ha, Vin Dc liu
1. Nguyn vt liu th nghim:

Mu th: 22 mu, bao gm:
T1, T2, T3, T4, T5, T6, Y7, T8, T9, T10, T11
B, D1, D2, D3, D4, D5, D6, D7, D8, D9
T bo: gm 2 dng t bo:
-
Dng t bo A549 (t bo ung th phi)
Dng t bo HTC116 (t bo ung th i trng)
Ha cht:
-
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) 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.
2. Phng php:
2.1. Nguyn tc:
- p dng phng php MTT c bin son bi tc gi Mosmann (1983) nh
gi c tnh trn t bo [1]. 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,
bng.
Hp
cht
MTT
3-(4,5-Dimethyl-2-thiazolyl)-2,5-
diphenyltetrazolium bromid, 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 540 600 nm. 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.
2.2. 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.
- 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
[2]. Adriamycin 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.
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.
- Gi tr IC50 < 20m/ml c coi l c tc dng.
3. Kt qu
Bng 1: 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
HTC116
100
17,5
51,2
31,6
1
2
3
4
DMSO
Adriamycin
T1
T2
A549
100
29,2
53,0
53,1
T3
30,6
29,7
T4
62,4
28,7
7
8
T5
T6
74,6
39,9
53,6
34,0
T7
81,1
48,8
10
11
T8
T9
66,5
56,1
78,6
35,1
12
T10
79,7
35,5
13
T11
42,8
27,5
14
15
16
17
D1
D2
D3
D4
74,9
85,0
87,6
87,9
56,3
51,4
52,0
46,3
18
D5
68,3
44,3
19
20
21
22
D6
D7
D8
D9
75,1
71,4
56,2
57,7
50,5
53,9
55,7
40,0
23
79,8
52,5
Kt lun
Dng tnh
Dng tnh trn dng Hep-G2
Dng tnh trn 3 dng MCF7, Hep-G2, HTC116
Dng tnh trn c 4 dng
Dng tnh trn dng
HTC116
m tnh
Dng tnh trn 3 dng A549,
Hep-G2, HTC116
Dng tnh trn dng
HTC116
m tnh
Dng tnh trn 1 dng
HTC116
Dng tnh trn 1 dng
HTC116
Dng tnh trn 2 dng A549
v HTC116
m tnh
m tnh
m tnh
Dng tnh trn 1 dng
HTC116
Dng tnh trn 1 dng
HTC116
m tnh
m tnh
m tnh
Dng tnh trn 1 dng
HTC116
m tnh
Bng 2: Gi tr IC50 (g/ml) ca mu th

Mu th
Dng t bo (Gi tr IC50/g/ml)

A549
HTC116
Adriamycin
1,46
0,6
T1
T2
27,08
T3
24,4
T4
Khng tun theo quy lut nng
T6
43,22
T7
26,36
T9
41,26
T10
52,01
T11
78,4
D4
D5
10,99
D9
15,25
Ti liu tham kho:

1. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J Immunol Methods: 65, 55-63, 1983.
2. Beverly A. Teicher. Anticancer drug development guide: Preclinical screening, clinical
trials, and approval. Humana Press, Totowa, New Jersey, 1997.
H Ni, ngy 2 thng 5 nm 2013
Ngi vit bo co
TS. Phm Th Nguyt Hng
PH LC 17:
PHIU TR LI KT QU TH HOT TNH
KHNG VI SINH VT KIM NH (ANTIMICROBIAL ASSAY)
Ch thch:
K hiu mu
ti
Phiu tr li kt qu
ti
K hiu mu
Phiu tr li kt qu
T1
VIII
T9
II
T6
IX
T10
III
IV
V
VI
VII
T3
T8
T2
T11
T7
X
XI
XII
XIII
T4
T12
T5
T13

Tổng Hợp Và Thử Hoạt Tính Sinh Học Của Một Số Dẫn Chất 5-Aryliden-2-Thiohydantoin

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Tổng Hợp Và Thử Hoạt Tính Sinh Học Của Một Số Dẫn Chất 5-Aryliden-2-Thiohydantoin

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B GIO DC V O TO

TNG HP V TH HOT TNH

TNG HP V TH HOT TNH

TS. V Trn Anh

CHNG 1. TNG QUAN

1.1 Tc dng sinh hc ca cc dn cht 2-thiohydantoin

1.1.1 Tc dng c ch t bo ung th

1.1.2 Tc dng khng khun, khng nm

1.1.3 Cc tc dng khc

1.2. Cc phng php tng hp 2-thiohydantoin v dn cht

1.2.1 Phn ng tng hp 2-thiohydantoin

1.2.2 Phn ng tng hp dn cht 5-aryliden-2-thiohydantoin

CHNG 2. NGUYN VT LIU, TRANG THIT B V

PHNG PHP NGHIN CU

2.2 Thit b th nghim

2.3 Phng php nghin cu

2.3.1 Phng php tng hp 2-thiohydantoin, cc 5-aryliden-2-

2.3.3 Phng php th hot tnh sinh hc

2.3.3.1 Th hot tnh gy c t bo ung th

2.3.3.2 Th tc dng khng khun, khng nm

CHNG 3. THC NGHIM V KT QU NGHIN CU

3.1.1 Tng hp 2-thiohydantoin

3.1.2 Tng hp mt s dn cht 5-aryliden-2-thiohydantoin

3.2 Xc nh cu trc ca cc cht tng hp c

3.2.1 Ph hng ngoi

3.2.2 Ph cng hng t ht nhn

3.2.3 Ph khi lng

3.3 Th hot tnh sinh hc

3.3.1 Th hot tnh gy c t bo ung th

3.3.2 Th tc dng khng khun, khng nm

4.1.1 V phn ng tng hp 2-thiohydantoin

4.1.2 V phn ng ngng t to 5-aryliden-2-thiohydantoin

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

4.2.1 V ph hng ngoi (IR)

4.2.2 V ph cng hng t ht nhn1H-NMR

4.2.3 V xc nh ng phn hnh hc ca dn cht 5-aryliden-2-

4.3 V hot tnh sinh hc

4.3.2 V tc dng khng khun, khng nm

KT LUN V KIN NGH

TI LIU THAM KHO

DANH MC CC K HIU, CH VIT TT

: Ph cng hng t ht nhn carbon 13

: T bo ung th phi ngi

: Epidermal growth factor receptor

: Phng php phun m in t

: T bo ung th gan ngi

: T bo ung th i trng ngi

: Ph tng tc d ht nhn qua nhiu lin kt

: Ph tng tc d ht nhn qua mt lin kt

: T bo ung th biu m v ngi

: T bo ung th tuyn tin lit

S liu ph 1H-NMR ca A, BM, BT

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

: Phiu tr li kt qu th hot tnh gy c

: Phiu tr li kt qu th hot tnh khng vi

Tng hp mt s dn cht 5-aryliden-2-thiohydantoin.

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

CHNG 1.TNG QUAN

Da trn s tng t v cu trc ca 2-thiohydantoin, Blanc v Cussac

Nm 2010, mt s dn cht ca 2-thiohydantoin (2, 3, 4, 5, 6, 7, 8)

Nguyn nhn chnh ca ung th khng thuc l do nng qu cao ca

imidazolidin-4-on (10) c tc dng c ch bm tng thuc c yu t P-gp

Tc gi Spengler v Hadzlik [16] tin hnh tng hp mt s dn cht

Cc tc gi th tc dng c ch bm tng thuc ca cc dn cht

bm tng thuc t bo ung th th nghim, tc dng yu hn cht 4. Hp

c tc dng tt trn chng vi khun v tc dng trung bnh trn chng nm

Nm 2010, tc gi Abd E.l v cng s [6] tng hp mt s dn cht