tổng hợp và thử hoạt tính sinh học của 3-(4-nitrophenyl) - hydantoin và một số...
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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
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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)
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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
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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
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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
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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
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4.1 KT LUN 40
4.2 KIN NGH 40
TI LIU THAM KHO
DANH MC PH LC
PH LC
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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.
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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
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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
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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
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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
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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
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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].
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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
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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,
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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%.
Nhit nng chy: 288-2890C.
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).
Ph IR (KBr), max (cm-1) : xem ph lc 3.1.
Ph 1H-NMR(500MHz, DMSO-d6), (ppm): xem ph lc 3.2.
-
21
Ph 13 C-NMR (125MHz, DMSO-d6), (ppm) : xem ph lc 3.3.
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%.
Nhit nng chy: 271-2720C.
Rf = 0,58 (TLC, silicagel 60 F254, h dung mi CHCl3 : MeOH (9:0,5)).
Ph LC-MS: [M-H]- (tnh ton: 343,72 [C16H10ClN3O4], tm thy 342,0)
(xem ph lc 4.4).
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.
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
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 0,60g cht kt tinh mu vng m.
Hiu sut 56,82%.
Nhit nng chy 264-2650C.
Rf = 0,65 (TLC, silicagel 60 F254, h dung mi CHCl3 : MeOH (9:0,5)).
Ph LC-MS: [M-H]- (tnh ton: 354,27 [C16H10N4O6], tm thy 353,0)
(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
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 0,79g kt tinh mu trng.
Hiu sut 74,81%.
Nhit nng chy 245-2460C.
Rf = 0,68 (TLC, silicagel 60 F254, h dung mi CHCl3 : MeOH (9:0,5)).
-
23
Ph LC-MS: [M-H]- (tnh ton: 354,27 [C16H10N4O6], tm thy 353,0)
(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%.
Nhit nng chy: 262-2630C.
Rf = 0,66 (TLC, silicagel 60 F254, h dung mi CHCl3 : MeOH (9:0,5)).
Ph LC-MS: [M-H]- (tnh ton: 327,27 [C16H10FN3O4], tm thy 326,0)
(xem ph lc 7.4).
Ph IR (KBr), max (cm-1) : xem ph lc 7.1.
Ph 1H-NMR(500MHz, DMSO-d6), (ppm): xem ph lc 7.2.
Ph 13 C-NMR (125MHz, DMSO-d6), (ppm) : xem ph lc 7.3.
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
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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.
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tr.393-394.
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nitrofurfural, Tp ch Dc hc,2003,1,18-20.
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ung th ca mt s dn cht 5-halogenoarylidenhydantoin, Lun vn
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dng, NXB Y hc.
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ha hc, NXB i hc Quc gia H Ni
Ting Anh
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18. Lamiri, M. and al. (2006), Microwave-Assisted Solvent-Free
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27. Thenmozhiyal, J.C. and al. (2004), Anticonvulsant Activity of
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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: sonhuco@yahoo.comResolution: 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
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
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
-
Ten may: GX-PerkinElmer-USA Resolution: 4cm-1
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
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
-
Ten may: GX-PerkinElmer-USA Resolution: 4cm-1
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
Nguoi do: Phan Thi Tuyet MaiTen mau: D3Date: 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
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
-
Ten may: GX-PerkinElmer-USA Resolution: 4cm-1
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
Nguoi do: Phan Thi Tuyet MaiTen mau: D4Date: 4/1/2013
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: sonhuco@yahoo.comResolution: 4cm-1
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
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
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