B Y T
TRNG I HC DC H NI
----- ------
HONG C MINH
TNG HP V TH TC DNG SINH
HC MT S ACID HYDROXAMIC
MANG KHUNG 3-METHOXIM-ISATIN
HNG C CH HISTON
DEACETYLASE
KHA LUN TT NGHIP DC S
H NI - 2014
B Y T
TRNG I HC DC H NI
----- ------
HONG C MINH
TNG HP V TH TC DNG SINH
HC MT S ACID HYDROXAMIC
MANG KHUNG 3-METHOXIM-ISATIN
HNG C CH HISTON
DEACETYLASE
KHA LUN TT NGHIP DC S
Ngi hng dn:
1. TS. o Th Kim Oanh
2. DS. L Th Tho
Ni thc hin:
B mn Ha Dc
H NI-2014
LI CM N
Li u tin em xin chn thnh by t lng cm n v knh trng su
sc n PGS.TS Nguyn Hi Nam, TS. o Th Kim Oanh, DS. L Th
Tho - B mn Ha Dc - Trng i hc Dc H Ni. Nhng thy c
tn tnh hng dn em trong sut qu trnh hon thnh kha lun tt
nghip ny.
Em cng xin gi li cm n n cc thy c gio v cc anh ch k
thut vin ca b mn Ha dc Trng i hc Dc H Ni, Khoa
Ha i hc KHTN H Ni, Trung tm Khoa hc v Cng ngh Vit
Nam, Vin Ha hc cc hp cht t nhin Vit Nam, Khoa Dc i
hc Quc gia Chungbuk lun gip , to iu kin thun li cho em
trong thi gian thc hin kha lun tt nghip ny.
Cui cng, em xin c gi li bit n su sc n b m, gia nh
v bn b lun lun bn khch l, ng vin, gip em trong hc
tp cng nh trong cuc sng.
H Ni, ngy 10 thng 5 nm 2014
Hong c Minh
MC LC
DANH MC CC K HIU, CC CH VIT TT
DANH MC CC HNH V
DANH MC CC BNG
DANH MC CC S
T VN 1
PHN I. TNG QUAN 3
1.1. HISTON DEACETYLASE 3
1.1.1. Khi nim v histon deacetylase (HDAC) 4
1.1.2. Phn loi HDAC 4
1.1.3. Cu to ca HDAC 5
1.1.4. HDAC v ung th 6
1.2. CC CHT C CH HDAC 7
1.2.1. Cu trc ca cc cht c ch HDAC 7
1.2.2. Lin quan cu trc v tc dng ca cc cht c ch HDAC 8
1.3. MT S NGHIN CU TNG HP CC ACID
HYDROXAMIC C CH HDAC TRN TH GII V VIT
NAM............... 9
1.3.1. Thay i cu ni... 11
1.3.2. Thay i nhm kha hot ng 12
PHN II. NGUYN LIU, THIT B, NI DUNG V PHNG
PHP NGHIN CU. 15
2.1. NGUYN VT LIU, THIT B 15
2.1.1. Ha cht 15
2.1.2. Thit b v dng c 16
2.2. NI DUNG NGHIN CU 16
2.3. PHNG PHP NGHIN CU 17
2.3.1. Tng hp ha hc 17
2.3.2. Th tc dng sinh hc 18
2.3.3. nh gi mc ging thuc ca cc cht tng hp c. 20
PHN III. THC NGHIM, KT QU V BN LUN 22
3.1. HA HC 22
3.1.1.Tng hp ha hc 22
3.1.1.1. Tng hp 3-methoxyimino-2 -oxoindolin v cc dn cht 22
3.1.1.2. Tng hp Ethyl 7-(3-methoxyimino-2-oxoindolin-1-yl)
heptanoat v cc dn cht 25
3.1.1.3. Tng hp N-hydroxy-7-(3-(methoxyimino)-2-oxoindolin-1-
yl) heptanamide v cc dn cht 28
3.1.2. Kim tra tinh khit 31
3.1.3. Xc nh cu trc 32
3.2. TH TC DNG SINH HC 37
3.3. NH GI MC GING THUC 37
3.4. BN LUN 38
3.4.1. Ha hc 38
3.4.2. Tc dng c ch histon deacetylase v hot tnh khng t bo
ung th in vitro... 39
3.4.3. nh gi mc ging thuc 42
PHN IV. KT LUN V KIN NGH 43
4.1. KT LUN 43
4.2. KIN NGH 43
TI LIU THAM KHO
PH LC
DANH MC CC K HIU, CC CH VIT TT
ALL : Bnh ung th nguyn bo lympho cp tnh
APL : Bnh ung th bch cu ty bo cp tnh
CTCL : (Cutaneous T cell lymphoma) T bo lympho T di da
CTCT : Cng thc cu to
CTPT : Cng thc phn t
DCM : Dicloromethan
DMF : N,N-Dimethylformamid
DMSO : Dimethylsulfoxid
EtOH : Ethanol
GAPDH : Glyceraldehyd 3-phosphat dehydrogenase
HAT : Histon acetyltranferase
HDAC : Histon deacetylase
IC50 : Nng c ch 50% s pht trin ca t bo
IR : Ph hng ngoi
MeOH : Methanol
MS : Ph khi lng
NMR : Ph cng hng t ht nhn
RA : Acid retionic
RAR : Receptor ca acid retionic
SAHA : Acid suberoylanilid hydroxamic
SAR : Lin quan gia cu trc v tc dng
SKLM : Sc k lp mng
SW620 : T bo ung th i trng
TLC : Phng php sc k lp mng
TSA : Trichostatin A
DANH MC CC HNH V
STT Tn hnh Trang
Hnh 1 S cu to ca nucleosome 3
Hnh 2 Vai tr cn bng ng ca HDAC v HAT 4
Hnh 3 Bng phn loi HDAC 5
Hnh 4 Cu to trung tm hot ng ca HDAC 6
Hnh 5 Vai tr sinh hc ca cc HDAC trong sinh l t bo
ung th
7
Hnh 6 Cu trc c bn ca cc cht c ch HDAC 8
Hnh 7 SAR ca cc cht c ch HDAC dn cht acid
hydroxamic
9
Hnh 8 Cng thc cu to ca TSA v SAHA 11
Hnh 9 Cc acid hydroxamic mang khung benzothiazol 12
Hnh 10 Cc dn cht amid ngc ca SAHA 12
Hnh 11 Cc acid phenylthiazol hydroxamic tng t SAHA 12
Hnh 12 Cu trc ca cc cht tng hp 17
Hnh 13 Tc dng c ch HDAC ca cc cht 4a-d 39
DANH MC CC BNG
STT Tn bng Trang
Bng 1 Cc cht c ch HDAC ang th nghim trn lm sng 9
Bng 2 Cc gi tr IC50 ca cc acid hydroxamic mang khung
benzothiazol
14
Bng 3 Hiu sut v cc ch s ha l ca 3-methoxyimino-2-
oxoindolin v cc dn cht
24
Bng 4 Hiu sut v cc ch s ha l ca Ethyl 7-(3-
methoxyimino-2-oxoindolin-1-yl) heptanoat v cc dn
cht
27
Bng 5 Hiu sut v cc ch s ha l ca N-hydroxy-7-(3-
(methoxyimino)-2-oxoindolin-1-yl) heptanamide v cc
dn cht
30
Bng 6 Gi tr Rf v nhit nng chy To
nc ca cc acid
hydroxamic 4a-d
31
Bng 7 Kt qu phn tch ph hng ngoi ca cc cht 4a-d 32
Bng 8 Kt qu phn tch ph khi lng ca cc cht 4a-d 33
Bng 9 Kt qu phn tch ph 1H-NMR ca cc cht 4a-d 34
Bng 10 Kt qu phn tch ph 13C-NMR ca cc cht 4a-d 35
Bng 11 nh gi mc ging thuc ca cc cht 4a-d theo
quy tc Lipinsky
37
Bng 12 Kt qu th c tnh t bo in vitro ca cc cht tng
hp c
40
Bng 13 Kt qu so snh tc dng c ch dng t bo SW620 42
DANH MC CC S
STT Tn s Trang
S 1 Quy trnh tng hp chung 22
S 2 Quy trnh tng hp cht 2a 22
S 3 Quy trnh tng hp cht 3a 25
S 4 Quy trnh tng hp cht 4a 28
1
T VN
Bnh ung th v sc khe cng ng l nhng vn ngy cng c
quan tm hu ht cc quc gia trn th gii. Theo c tnh v thng k ca
T chc y t th gii (WHO) th hng nm trn ton cu c khong 9-10 triu
ngi mc bnh ung th mi v mt na trong s cht v cn bnh ny
[10].
T chc Y t Th gii cng c tnh mi nm c khong trn 6 triu ngi
cht do ung th. T l cht do ung th chim ti 12% trong s cc nguyn
nhn gy t vong ngi [19].
cc nc pht trin, ung th l nguyn nhn gy t vong ng hng th
hai sau bnh tim mch. cc nc ang pht trin ung th ng hng th ba
sau bnh nhim trng/ k sinh trng v tim mch [19].
gim thiu t l t vong do ung th th vic nghin cu thuc chng
ung th c vai tr quan trng hng u [19].
Trong nhng nm gn y, cc cht c ch enzym HDAC ang tr thnh
cc tc nhn chng ung th y trin vng. Acid suberoylanilid hydroxamic
(Vorinostat, Zolinza) l cht c ch enzym HDAC u tin c FDA
cp php trong iu tr u lympho t bo T di da. Vorinostat l mt cht c
ch HDAC c cha nhm chc acid hydroxamic trong phn t. y l mt
trong s cc cht c nghin cu pht trin t TSA l mt cht c ch
HDAC t nhin cng c cha nhm chc acid hydroxamic trong phn t.
iu ny cho thy cc cht c ch HDAC c cha nhm chc acid
hydroxamic rt c trin vng trong iu tr ung th [10,19]. V vy, chng ti
tin hnh ti :
Tng hp v th tc dng sinh hc mt s acid hydroxamic mang
khung 3-methoxim-isatin hng c ch histon deacetylase vi 2 mc
tiu:
2
- Tng hp N-hydroxy-7-(3-(methoxyimino)-2-oxoindolin-1-yl)
heptanamid v 3 dn cht.
- Th tc dng c ch HDAC v hot tnh khng t bo ung th in
vitro ca cc cht tng hp c.
3
PHN I. TNG QUAN
1.1. HISTON DEACETYLASE (HDAC)
Hnh 1: S cu to ca nucleosom
Nucleosom l n v c bn cu to nn nhim sc th (NST). Mi
nucleosom bao gm 146 cp base ca ADN qun quanh li histon octamer
(hnh 1). u amin ca histon mang in tch dng nn tng tc mnh vi
u phosphat mang in m ca ADN to nn cu trc ca nucleosom v cu
trc bc cao ca NST quy nh qu trnh biu hin gen. Khi u amin ca
histon tch in dng cng ln tng tc ny cng mnh, NST ng xon
cng cht c ch qu trnh phin m. Ngc li th qu trnh phin m din ra
v gen c biu hin. Mc tch in dng ca histon ph thuc vo qu
trnh acetyl ha u amin ca histon. S acetyl ha lm trung ha bt in
tch dng u amin ca histon. Trong t bo c 2 enzym ng vai tr
chnh trong qu trnh acetyl ha l HDAC v HAT. Hai enzym ny c vai tr
tri ngc nhau. S cn bng trong hot ng ca chng m bo mc
tho xon ca nhim sc th din ra bnh thng [20,32,36].
4
1.1.1. Khi nim v histon deacetylase
Histon deacetylase l mt nhm cc enzym xc tc qu trnh loi b
nhm acetyl t -N acetyl lysin amino acid ca histon. HDAC c tc dng i
lp vi histon acetyltransferase (HAT) - enzym xc tc chuyn nhm acetyl
t acetyl coenzym A n -amino ca lysin u N ca histon (hnh 2) [39].
(DNMTs-DNA methyl transferase, HMT-histon methyltransferase, hnh trn: nhm
methyl, hnh tam gic: nhm acetyl, hnh vung: nhm methyl trn ui histon)
Hnh 2: Vai tr cn bng ng ca HDAC v HAT
1.1.2. Phn loi
C 18 HDAC ngi c chia thnh 4 nhm [11,13,14,27,32] (hnh 3):
- Nhm I: HDAC 1, HDAC 2, HDAC 3, HDAC 8.
- Nhm IIa: HDAC 4, HDAC 5, HDAC 7, HDAC 9. Nhm IIb: HDAC
6, HDAC 10.
- Nhm III: Cc protein iu ho chui thng tin 2 (SIRT): SIRT 1 7,
chng c bo tng, ty th v nhn.
- Nhm IV: HDAC 11
Cc HDAC nhm I, II, IV c gi l cc HDAC kinh in ph
thuc vo Zn2+ v b c ch bi cc cht to phc chelat vi Zn2+ nh cc
5
acid hydroxamic, thiol... Trong khi cc HDAC nhm III li ph thuc vo
NAD+ [13,28]. Thut ng cc cht c ch HDAC thng c dng cho
nhng cht c mc tiu phn t l cc HDAC kinh in v hin ang c
nghin cu trn lm sng [18,28].
Hnh 3: Bng phn loi HDAC
1.1.3. Cu to ca HDAC
V c bn cc HDAC c cu trc trung tm hot ng kh ging nhau,
chng u gm cc phn chnh (hnh 4):
Ion Zn2+ l coenzym nm trung tm hot ng. y l phn tham gia lin
kt mnh nht vi u amin ca histon thng qua lin kt phi tr [7].
Knh enzym l ni cha ng c cht v tham gia lin kt Van der Walls
vi c cht. Knh ny c cu trc dng ti. N c cu to bi cc acid
amin thn du, c bit l cc acid amin c cha vng thm nh: Phe, Tyr,
Pro, His. N c cu trc kh linh ng nn c th thay i kch thc
ph hp vi kch thc ca c cht tham gia phn ng deacetyl ha [33].
Vng xc tc
Vng nhn din
nhn t bo
6
Hnh 4: Cu to trung tm hot ng ca HDAC
1.1.4. HDAC v ung th
HAT xc tc cho phn ng acetyl ha nhm -NH2 trong phn t lysin
phn ui ca histon lm hot ha qu trnh phin m, trong khi , chc
nng ca HDAC l xc tc cho phn ng deacetyl ha lysin to ra nhm NH3+
phn ui ca histon. Nhm ny mang in tch dng s lin kt vi nhm
mang in m trn nhim sc th gy ng xon nhim sc th, lm gim s
tip xc ca cc yu t phin m vi nhim sc th gy c ch qu trnh phin
m. Cc sai lch ca qu trnh phin m l mt trong nhng nguyn nhn dn
ti s hnh thnh khi u [28].
Cc nghin cu c ngha thng k ch ra rng cc HDAC lin quan
n nhiu giai on iu ha c bn ca qu trnh sinh hc trong t bo ung
th nh chu trnh t bo, s bit ha, s cht t bo theo chng trnh, k c
s xm ln, s t tiu v s to mch. Vai tr chc nng ca cc HDAC trong
qu trnh sinh hc ca t bo ung th c tm tt hnh 5 [36].
7
Hnh 5: Vai tr sinh hc ca cc HDAC trong sinh l t bo ung th
1.2. CC CHT C CH HDAC
1.2.1. Cu trc ca cc cht c ch HDAC
Cu trc ca cc cht c ch HDAC rt a dng nhng nhn chung u
gm 3 phn chnh (hnh 6) [10,31]:
- Nhm kha hot ng (capping group) hay vng nhn din b mt
(surface recognition group): l cc vng thm hoc peptid vng,
thng nm trn b mt enzym.
- Vng cu ni s nc: thng l hydrocacbon thn du mch
thng hay vng, c th no hoc khng no to cc lin kt Van
der Waals vi knh enzym.
- Nhm kt thc gn vi km (Zinc binding group - ZBG): tng tc
vi ion Zn2+ ti trung tm hot ng ca cc HDAC nh acid
hydroxamic, cc thiol, nhm o-aminoanilin ca benzamid,
mercaptoceton...
Cu trc tinh th kt tinh ca cc HDAC cho thy nhm kt thc, cu
ni v mt phn ca nhm kha hot ng nm trong ti enzym lm lp y
8
khong trng trong lng knh enzym. Phn cn li ca nhm kha hot ng
tng tc vi phn vnh trn b mt ming ti enzym. Nhm nhn din b
mt c th lin kt vi phn cu ni thng qua mt s lin kt peptid lm tng
kh nng phn cc v gp phn ci thin dc ng hc cho cc cht c ch
HDAC. Vic nghin cu thit k cu trc cc cht mi u da trn cu trc
c in ny.
Hnh 6: Cu trc c bn ca cc cht c ch HDAC
1.2.2. Lin quan gia cu trc v tc dng ca cc cht c ch HDAC
Cc cht c ch HDAC da trn cu trc amid alkyl acid
hydroxamic c bit n nhiu, v d nh SAHA. Cu trc cng bao gm
3 phn chnh A-B-C:
- Phn A lin quan n hiu lc v tnh c hiu (thng l aryl).
Cha c nghin cu y v nhm ny nhng trn cc dn cht
tng hp c cho thy kch thc vng nhn thm ln s c tc
dng tt hn vng nh.
- Phn B l cu ni nh amid alkyl. Thng l hydrocarbon mch
h hoc cc vng thm c kch thc nh
- Phn C l nhm lin kt vi Zn2+ nh acid hydroxamic.
Sau y l tng kt lin quan cu trc tc dng ca mt dy cc dn
cht ca acid hydroxamic c nghin cu [8] (hnh 7):
9
Hnh 7: SAR ca cc cht c ch HDAC dn cht acid hydroxamic
1.3. MT S NGHIN CU TNG HP CC ACID
HYDROXAMIC C CH HDAC TRN TH GII V VIT NAM:
T nhng nm 90 ca th k trc, rt nhiu nh khoa hc trn th gii
tp trung nghin cu v hng trm bi bo c cng b trong qu trnh
tm kim cc cht c ch HDAC. Cho n nay, bn cnh 2 cht l vorinostat
(Zolinza) v depsipeptid (Romidepsin
) c FDA ph duyt s dng
trong iu tr u lympho da t bo T, cn c khong hn 10 cht ang c
nghin cu pha lm sng I, II (bng 1).
Bng 1: Cc cht c ch HDAC ang th nghim trn lm sng
Nhm Hp cht Pha Loi ung th
Acid bo
mch ngn Butyrat
AN-9 (tin thuc) Acid valproic
Phenyl butyrat
I, II
I, II
I, II
I
Ung th i trng Th rn, NSCLC
Th rn, ung th mu, AML, MDS, CTCL, u trung
biu m Th rn, AML/MDS
10
Acid
hydroxamic
SAHA
PXD 101
NVP-LAQ824
LBH-589
ITF-2357
SB-939
CRA-024781
JNJ-16241199
I, II
II
I
II, III
II
I
I
I
Th rn, ung th mu Ung th mu Th rn, ung th mu Th rn, AML, ALL, MDS U lympho Hodgkin
Th rn, ung th mu
Cc
benzamid
SNDX-275
CI-994
MGCD-0103
I, II
I, II
II
Th rn, u lympho, AML Th rn, NSCLC, t bo thn, ty Th rn, ung th bch cu, MDS
Peptid
vng
Depsipeptid
(FK-228)
I, II Th rn, CLL, AML, u a ty xng, NHL t bo T ngoi vi, RAI khng thyroid, ung th i trng tin trin
Ghi ch:NSCLC: carcinoma t bo phi khng nh; AML: ung th bch cu ty bo cp; MDS:hi chng lon sn ty; CTCL: u lympho da t bo T; ALL: ung th bch cu cp; CLL:ung th bch cu mn
Cc cht c ch HDAC ang c th nghim lm sng s dng
trong iu tr ung th c th c chia lm 4 nhm da trn cu trc [15]:
- Cc acid hydroxamic: TSA, SAHA.
- Cc peptid vng: depsipeptid, CHAPs.
- Cc acid bo mch ngn: butyrat, phenylbutyrat, valproic acid.
- Cc benzamid: N-acetyldinalin, MS-275.
Mi nhm nu trn u c nhng hn ch nht nh nh:
- Cc benzamid v acid bo c hiu lc km.
- Cc peptid vng kh tng hp v cu trc phc tp.
- Cc acid hydroxamic b chuyn ha nhanh v c ch khng chn lc
ln cc loi enzym HDAC.
11
Trong s cc nhm trn, nhm cc acid hydroxamic to chelat bn
vng vi Zn2+ nht v hu ht cc dn cht ca acid hydroxamic u c ch
HDAC nng nM (cc hp cht cn li a phn c ch HDAC vi IC50
mc M). Chnh v vy, cc acid hydroxamic ang c nghin cu rng ri
trn th gii. Nhiu nhm nghin cu ang n lc tm kim cc dn cht mi
da trn phin mu ca TSA , SAHA (hnh 8) v cc cht tm c.
TSA SAHA
Hnh 8: Cng thc cu to ca TSA v SAHA
Da trn c im cu trc ca cc cht c ch HDAC (hnh 6- trang
8), cc nghin cu c th tin hnh thay i mt trong cc phn cu trc l
nhm kha hot ng hoc cu ni. Sau y l tng kt mt s nghin cu
trn th gii v thay i cu trc ca cc acid hydroxamic.
1.3.1. Thay i cu ni:
TSA v SAHA l hai in hnh cho phn cu ni 5-6 carbon. Chng th
hin tc dng c ch mnh HDAC [20]. Hu ht cc nghin cu u ch ra
rng khong cch tt nht gia nhm acid hydroxamic v phn (A) l 5, 6, 7
nguyn t carbon, trong kh nng c ch ca dn cht mang cu ni 6C
ln hn so vi cu ni 5C hay 7C v tc dng tt hn rt nhiu so vi cu ni
3C hoc 4C [2,3,6].
Trong lun n Tin s Dc hc ca o Th Kim Oanh (Vit Nam)
[5] tng hp cc acid hydroxamic mang khung benzothiazol (hnh 9), vi
di cu ni thay i, kt qu l:
Cc cht cha vng benzothiazol vi mch alkyl 2-3 nhm methylen
khng c tc dng c ch HDAC. Nhng khi mch alkyl tng thnh 4 nhm
12
methylen, cht thu c bt u c tc dng trn HDAC nhng cn yu
(nng khong 10g/ml). Phn cu ni c 6 nhm methylen cho hot tnh
tt nht trn HDAC (c ch HDAC2 vi IC50=0,009-0,53 g/ml v hu ht
u c ch HDAC1 1g/ml) [5].
n = 2-6
Hnh 9: Cc acid hydroxamic mang khung benzothiazol
Trong cc cht c ch HDAC nguyn mu, nhm kha hot ng c
th lin kt vi phn cu ni thng qua cc nhm cho hoc nhn lin kt
hydro nh amid, ceton. i vi cc cht c ch HDAC c lin kt amid cu
ni, lin kt ny c vai tr quan trng. Vic o chiu lin kt amid cng lm
thay i tc dng ca cc cht c ch HDAC [8].
Nhm nghin cu thuc cng ty TopoTagret (Anh) thit k v tng
hp gn 40 dn cht amid ngc ca SAHA (hnh 10) [8]. Nhiu cht trong
s ny c hot tnh c ch HDAC tng ng, thm ch mnh hn SAHA.
Trong , hai cht c th nghim m hnh ung th in vivo trn chut v
cho kt qu rt kh quan, nh hng cho cc nghin cu tip theo.
Hnh 10: Cc dn cht amid ngc ca SAHA
1.3.2. Thay i nhm kha hot ng:
Bn cnh cc nghin cu thay i cu ni, hng nghin cu kho st
thay i nhm kha hot ng cng rt c nhiu nh khoa hc quan tm.
13
Cc nh khoa hc thuc Vin nghin cu qun i Walter Reed (M)
thit k v tng hp rt nhiu dn cht acid hydroxamic mang hp phn
phenylthiazol thay th vo v tr phenyl ca SAHA (hnh 11) [37].
A1 A2
Hnh 11: Cc acid phenylthiazol hydroxamic tng t SAHA
Kt qu nh gi tc dng trn HDAC cho thy dn cht A1 vi nhm
kha hot ng l 3-aminophenyl-5-thiazolyl c tc dng c ch mnh nht
vi IC50=10,4 nM, mnh hn c SAHA trn cng th nghim. ng phn v
tr ca A1 l A2 (nhm th amino v tr ortho) cng c tc dng mnh tng
ng vi SAHA [37].
Cn ti Vit Nam, trong nhng nm gn y cng c kh nhiu kt qu
nghin cu thay i nhm kha hot ng c cng b trong cc tp ch
trong nc v quc t.
Trong lun vn Thc s Dc hc ca Nguyn Th M (Vit Nam) [4],
thay th nhm phenyl ca SAHA bng mt lot dn cht mang khung 3-
oxim-isatin. Kt qu c ch dng t bo SW620 thu c (bng 2) ht sc
kh quan, c 6 cht u c ch SW620 mnh hn SAHA.
14
R IC50(g/ml)/SW620
-H 0,64
-F 0,11
-Cl 0,65
-Br 0,29
-NO2 3,39
-CH3 0,99
SAHA 3,70
Bng 2: Cc gi tr IC50 ca cc acid hydroxamic mang khung 3-oxim-isatin
Nhn chung, vic thay i nhm nhn din b mt nhm khai thc s
khc bit phn ming knh enzym gia cc HDAC, t c th thit k
cng thc lm tng hot tnh hoc tng kh nng c ch chn lc. Nhm nhn
din b mt ch yu l cc vng, c th l vng thm hoc peptid vng. Cc
nghin cu cho thy nhm nhn din b mt l vng thm th tc dng tt hn
vng no. Mt khc vng ln thng tt hn vng nh (khi thay nhm phenyl
ca SAHA bng vng indol th IC50 ca dn cht indol nh hn SAHA nhiu ln
[33] ). Khi vng thm c mt cc halogen cng c th lm tng hot tnh [9].
Tm li, cc nghin cu v acid hydroxamic hng c ch HDAC hin
nay tp trung thay i mt trong hai phn cu trc l thay i nhm kha hot
ng hoc cu ni, u vi mc ch thu c cht c hot tnh c ch HDAC
tt hn, ng thi mong mun tm c ng vin cho th lm sng.
i vi cu ni, hu ht cc nghin cu chng minh c kh nng
c ch HDAC ca cc dn cht acid hydroxamic mang cu ni 6C l ti u.
Chnh v vy, trong Kha lun tt nghip ny, chng ti tp trung vo
mc tiu thay i nhm kha hot ng, thay i nhn phenyl ca SAHA
bng b khung 3-methoxim-isatin vi mc ch l tng tng tc ca nhm
kha hot ng vi vng acid aminh ming knh enzym. Nh tng hot
tnh c ch HDAC v c ch s pht trin ca t bo in vitro v in vivo.
15
Phn II: NGUYN LIU, THIT B, NI DUNG V
PHNG PHP NGHIN CU
2.1. NGUYN VT LIU, THIT B
2.1.1. Ha cht
Cc ha cht, dung mi dng trong qu trnh thc nghim bao gm:
Nguyn liu Tn nguyn liu Xut x
Ha cht
chnh
Isatin Merck
5-floro isatin Merck
5-cloro isatin Merck
5-bromo isatin Merck
Hydroxylamin hydroclorid Sigma-aldrich
O-methylhydroxylamin hydroclorid Sigma-aldrich
Ethyl 7-bromoheptanoat Sigma-aldrich
Dung mi v
ha cht
khc
Dimethyl formamid Merk
Ethanol Vit Nam
TEA Vit Nam
Kali carbonat Vit Nam
Dicloromethan Vit Nam
Methanol Vit Nam
Natri hydroxyd Vit Nam
Kali iodid Vit Nam
Dd HCl 5% Vit Nam
Natri sulfat Vit Nam
Ethyl acetat Vit Nam
Acid acetic bng Vit Nam
Dd FeCl3 5% Vit Nam
16
2.1.2. Thit b v dng c
- Bnh cu y trn dung tch 50 ml c nt mi, bnh chit, bnh chy sc
k lp mng (TLC), ng nghim, cc c m, phu lc.
- My khuy t gia nhit, sinh hn hi lu, my ct quay chn khng,
my siu m, my o nhit nng chy nhit in (Electrothermal
digital).
- T lnh, t sy.
- Cn phn tch, cn k thut.
- Giy cn, giy ch th vn nng, giy lc.
- Bn mng silicagel Merck 70-230 mesh.
- Ph hng ngoi (IR) c ghi trn my Perkin Elmer ti phng th
nghim b mn Ha vt liu khoa Ha Trng i hc Khoa hc t
nhin i hc Quc gia H Ni.
- Ph khi (MS) c ghi bng my khi ph Agilent 6310 Ion Trap MS
v my khi ph HP 5989B MS ca Vin Ha hc cc hp cht t
nhin Vit Nam.
- Ph cng hng t ht nhn proton (1H-NMR) v carbon (13C-NMR)
c ghi bng my Bruker AV-500 ti Trung tm Khoa hc v cng
ngh Vit Nam.
2.2. NI DUNG NGHIN CU
2.2.1. Tng hp ha hc
* Tng hp 4 cht:
- N-hydroxy-7-(3-(methoxyimino)-2-oxoindolin-1-yl) heptanamid (4a)
- N-hydroxy-7-(5-floro-3-(methoxyimino)-2-oxoindolin-1-yl) heptanamid (4b)
- N-hydroxy-7-(5-cloro-3-(methoxyimino)-2-oxoindolin-1-yl) heptanamid (4c)
- N-hydroxy-7-(5-bromo-3-(methoxyimino)-2-oxoindolin-1-yl) heptanamid (4d)
* Kim tra tinh khit v xc nh cu trc ca cc cht tng hp c.
17
(Trong : 4a. R=-H; 4b.R=-F; 4c. R=-Cl; 4d. R=-Br)
Hnh 12: Cu trc ca cc cht tng hp
2.2.2. Th tc dng sinh hc ca cc cht tng hp c
- Th tc dng c ch HDAC bng phng php Western Blot.
- Th hot tnh khng t bo ung th in vitro trn dng t bo ung
th i trng (SW620).
2.2.3. S b nh gi tnh ging thuc ca cc cht tng hp c.
2.3. PHNG PHP NGHIN CU
2.3.1. Tng hp ha hc
2.3.1.1. Tng hp ha hc v kim tra tinh khit:
- Tng hp N-hydroxy-7-(3-(methoxyimino)-2-oxoindolin-1-yl)
heptanamid v 3 dn cht.
- Dng TLC theo di qu trnh tin trin ca phn ng v tinh
khit ca cc dn cht.
- o nhit nng chy kim tra tinh khit ca cc dn cht.
2.3.1.2. Khng nh cu trc:
Cc cht tng hp c xc nh cu trc bng cc loi ph: Ph hng
ngoi (IR), ph khi (MS), ph cng hng t proton (1H NMR), ph cng
hng t carbon (13C NMR)
* Ph hng ngoi (IR) c ghi trn my Perkin Elmer, s dng k
thut vin nn KBr, ghi vng 4000-600 cm-1 ti phng th nghim
b mn Ha vt liu Khoa Ha Trng i hc Khoa hc t
nhin H Ni. Cc mu rn c phn tn trong KBr sy kh
18
vi t l khong 1:200 ri p di dng film mng di p lc cao
c ht chn khng loi b hi m.
* Ph khi lng (MS) c ghi bng my khi ph Agilent 6310
Ion Trap MS v my khi ph HP 5989B MS ca Vin Ha hc
cc hp cht t nhin Vit Nam.
* Ph cng hng t ht nhn proton (1H-NMR) v carbon (13C-
NMR) c ghi bng my Bruker AV-500, dng DMSO-d6 lm
dung mi. dch chuyn ha hc () c biu th bng n v
phn triu (parts per million ppm), ly mc l pic ca cht chun
ni tetramethylsilan (TMS).
2.3.2. Th tc dng sinh hc
2.3.2.1. Th tc dng c ch histon deacetylase
Tc dng c ch HDAC ca cc dn cht tng hp c nh gi gin
tip thng qua xc nh mc acetyl ha histon H3, H4 trong t bo ung th
i trng SW620. Phn tch da trn Western Blot c th khng nh c tc
dng ca cc mu th lm tng hoc gim s acetyl ha histon H3, H4 khi so
snh vi mu trng.
a. Nguyn liu v nui cy t bo:
Cc t bo ung th i trng SW620 c nui cy trong mi trng
RPMI (b sung L-glutamin, 10% huyt thanh bo thai b FBS (fetal bovine
serum)), gi l mi trng nui cy hon chnh (complete medium). Tt c
cc t bo c 37oC vi 5% (w/v) CO2 v 95% (w/v) khng kh.
Trc khi s dng, cc mu th nghim dng bt c ha tan trong
DMSO to dung dch gc nng 10 mg/mL. Cc dung dch gc sau
c pha long bng mi trng nui cy hon chnh to cc dung dch
lm vic nng 1g/mL v 10g/mL.
b. Phn lp histon v Western Blot:
19
T bo SW620 (khong 1 x 106) c vi mu th trong 24 gi, song
song lm mu trng (mu t bo khng vi mu th). Sau , cc t bo
c x l v ra bng dung dch nc mui sinh l c m phosphat.
Tip theo, t bo c dung gii trong dung dch dung gii m (20
mM Tris-HCl (pH 7,5); 150mM NaCl; 1mM Na2EDTA; 1mM EGTA; 1%
triton; 2,5% Na pyrophosphat; 1 mM -glycerophosphat; 1mM Na3VO4; 1
g/mL leupeptin), trong 15 pht. Hn dch c ly tm v phn dch
c thu hi tin hnh in di trn gel.
Histon c in di qua gel SDS-PAGE 10% v chuyn vo mng
PVDF. Cc mng c qua m cng khng th 1 l khng acetyl histon
H3, khng acetyl histon H4 (milipore), tip theo vi khng th 2 l khng
th IgG th lin hp peroxidase ca nga trong 2 gi. Cc di c phn ng
dng tnh c pht hin nh s pht quang c tng cng. Cc th
nghim c lm lp li t nht 3 ln mt cch c lp
2.3.2.2. Th hot tnh khng t bo ung th in vitro
a. Nguyn tc th
Th hot tnh khng t bo ung th in vitro c thc hin ti khoa
Dc, trng i hc Chungbuk, Cheongju, Hn Quc theo phng php
MTT v gi tr IC50 c tnh trn phn mm GraphPad Prism.
Dng t bo th nghim SW620: t bo ung th i trng.
T bo ung th c ly t Ngn hng t bo ung th ca Vin nghin
cu sinh hc v cng ngh sinh hc Hn Quc (KRIBB) v c nui cy
trong RPMI b sung 10 FBS (huyt thanh bo thai b). c tnh t bo ca
cc cht c th bng phng php MTT theo cc bc sau:
b. Chun b
Cc t bo pha logarit c trypsin ha v phn tn vo hn dch
n t bo trong mi trng RPMI b sung 10 FBS v iu chnh n nng
20
khong 1,5.104 n 3,5.104 t bo, sau chia u vo cc ging ca a
96 ging, mi ging 200 l. Cc a sau c 370C trong iu kin 5%
CO2. Sau 24 gi , cc mu th c chun b trong 20 L mi trng
DMEM/RPMI b sung 10% FBS t dung dch gc 10 mg/mL trong DMSO
ri thm 2 L mu th vo cc ging nhiu nng khc nhau, cc a ny
sau c thm 48 gi. Tt c cc mu c chun b sao cho nng
cui cng ca DMSO khng qu 0,1 .
c. Tin hnh th
Sau khi 48 gi, thm vo mi ging 20 l thuc nhum MTT (nng
MTT 5 mg/mL trong mui m phosphat - PBS). Cc a c thm 3
gi 37oC trong iu kin 5% CO2. Tip theo, mi ging c cho 100 l
dung dch DMSO, 5 pht cho MTT formazan c ha tan. hp th
c c bc sng 510 nm.
d. Tnh kt qu
Gi tr IC50 l nng ca mu th m , hp th gim i 50
so vi nhm chng (trng m tnh l ging ch thm mi trng nui cy): kt
qu cui cng l gi tr trung bnh ca 4 ln o c lp vi gi tr hp th
khc nhau khng qu 5 . Gi tr IC50 c tnh da trn phn mm
GraphPad Prism. Trong phng php th ny, IC50 20 g/mL c coi l
c hot tnh.
2.3.3. nh gi mc ging thuc ca cc cht tng hp c
Tnh gi tr logP ca cc cht tng hp bng phn mn EPTsuite cung cp
bi US Environmental Protection Agency's Office of Pollution Prevention
and Toxics and Syracuse Research Corporation (SRC). Quy trnh bao gm v
cu trc 2D, to Smile notation bng phn mm ChemSketch 4.0 ca ACD
labs sau a Smile notation vo phn mm EPIsuite tnh cc gi tr
logP.
21
nh gi mc ging thuc ca cc cht da trn quy tc Lipinsky [34]:
- Khi lng mol phn t ca cht < 500 g/mol.
- S trung tm nhn lin kt hydro (N, O) phi < 10.
- S trung tm lin kt hydro (NH, OH) < 5.
- Cn bng h s thn du/nc: logP < 5.
22
PHN III. THC NGHIM, KT QU V BN LUN
3.1. HA HC
3.1.1. Tng hp ha hc
Quy trnh tng hp c tin hnh qua 3 giai on:
1a-d
i
2a-d
ii
3a-d
iii
4a-d
a.R=-H
b.R=-F
c. R=-Cl
d.R=-Br
S 1: Quy trnh tng hp chung
Tc nhn v iu kin: i) O-methylhydroxylamine hydroclorid, Triethylamin, ethanol, methanol, un hi lu 70oC/24h; ii)7-bromoheptanoat, KI, K2CO3, DMF, tin hnh 80oC/3h; iii)Methanol, NH2OH.HCl, NaOH, -5
oC/ 30 pht.
3.1.1.1.Tng hp 3-(methoxyimino)-2-oxoindolin v cc dn cht (2a-d):
a. Tng hp 3-(methoxyimino)-2-oxoindolin (2a)
Quy trnh tng hp 3-(methoxyimino)-2-oxoindolin (2a) c thc hin nh
s di y:
1a
NH2OCH3.HCl
TEA
2a
S 2: Quy trnh tng hp cht 2a
Tin hnh
- Cho 1mmol (0,147g) isatin vo bnh cu, ha tan trong hn hp
ng dung mi 5ml ethanol khan v 2,5ml methanol.
23
- Lm lnh bng nc 10 pht.
- Thm 1,2 mmol O-methylhydroxylamin hydroclorid
(NH2OCH3.HCl)
- Thm t t 1,2 mmol triethylamin (bnh lun t trong nc lnh).
- Sau tin hnh un hi lu trong 24h, 70oC, kt hp khuy t.
X l:
- Lm lnh bnh phn ng
- Thm t t 20 ml nc lnh s c ta 2a xut hin. Lm lnh bnh
30 pht.
- Lc ly ta, ra ta bng nc ct ti dch lc trung tnh.
Kt qu: Cht rn mu vng
Tan trong aceton, DCM, DMF
Khi lng: 0,165g. Hiu sut: 93,96 %
Rf = 0,58 (DCM:MeOH = 9:1)
b. Tng hp 5-floro-3-(methoxyimino)-2-oxoindolin (2b):
- Tin hnh: Nh quy trnh tng hp ca cht 2a vi cht ban u l
5-floroisatin (0,165g; 1mmol).
- Kt qu: Cht rn mu vng
Tan trong aceton, DCM, DMF
Khi lng: 0,175g. Hiu sut: 90,33 %
Rf = 0,53 (DCM:MeOH = 9:1)
c. Tng hp 5-cloro-3-(methoxyimino)-2-oxoindolin (2c):
- Tin hnh: Nh quy trnh tng hp ca cht 2a vi cht ban u l
5-cloroisatin (0,182g; 1mmol).
- Kt qu: Cht rn mu vng
Tan trong aceton, DCM, DMF
Khi lng: 0,191g. Hiu sut: 90,70 %
24
Rf = 0,55 (DCM:MeOH = 9:1)
d. Tng hp 5-bromo-3-(methoxyimino)-2-oxoindolin (2d):
- Tin hnh: Nh quy trnh tng hp ca cht 2a vi cht ban u l
5-bromoisatin (0,226g; 1mmol).
- Kt qu: Cht rn mu vng da cam
Tan trong aceton, DCM, DMF
Khi lng: 0,237g. Hiu sut: 93,03 %
Rf = 0,48 (DCM:MeOH = 9:1)
Bng 3: Hiu sut v cc ch s ha l ca 3-(methoxyimino)-2-oxoindolin v cc dn cht:
Cht CTCT CTPT KLPT Mu Hiu sut (%)
2a
C9H8N2O2 176 Vng 93,96
2b
C9H7N2O2F 194 Vng 90,33
2c
C9H7N2O2Cl 210,5 Vng 90,70
2d
C9H7N2O2Br 255 Vng da cam 93,03
25
3.1.1.2. Tng hp Ethyl 7-(3-(methoxyimino)-2-oxoindolin-1-yl)
heptanoat v cc dn cht (3a-d):
a. Tng hp Ethyl 7-(3-(methoxyimino)-2-oxoindolin-1-yl) heptanoat
(3a):
Quy trnh tng hp Ethyl 7-(3-(methoxyimino)-2-oxoindolin-1-yl)
heptanoat (3a) c tin hnh nh s di y:
2a
DMF, K2CO3, KI
3a
S 3: Quy trnh tng hp cht 3a
Tin hnh:
- Cho 1mmol (0,176g) cht 2a v 1ml DMF vo bnh cu dung tch
50ml. Kt hp siu m cho tan hon ton (siu m 7 pht).
- Thm 2-2,2 mmol K2CO3 khan. Khuy trong 1h30 pht cho n khi
hn hp ng nht.
- Cho 0,5 mmol KI vo bnh phn ng. Khuy thm 15 pht.
- Thm t t 1,2 mmol Ethyl 7-bromoheptanoate ( c ha tan ha
trong 1ml DMF) vo hn hp phn ng.
- Nng nhit ln 80oC.
- Khuy hn hp phn ng trong 3h.
- Kim tra phn ng bng TLC vi pha ng DCM : CH3OH (9:1).
X l:
- Sau khi phn ng kt thc, lm lnh bnh phn ng.
- Sau hn hp phn ng vo 20ml dd HCl 5% lnh.
- Chit sn phm bng 50 ml DCM (chia lm 3 ln chit), thu lp DCM.
26
- Sau khi lng, lm khan bng Na2SO4 khan.
- Ct loi DCM di p sut gim, thu c dn cht 3a.
Kt qu: Sn phm ester thu c c dng du mu gch
Rf = 0,89 (DCM:MeOH = 9:1)
b. Tng hp Ethyl 7-(5-floro-3-(methoxyimino)-2-oxoindolin-1-yl)
heptanoat (3b):
- Tin hnh: Nh quy trnh tng hp ca cht 3a vi cht ban u l 5-
floro-3-(methoxyimino)-2-oxoindolin (0,194g; 1mmol).
- Kt qu: Sn phm ester thu c c dng du mu gch
Rf = 0,77 (DCM:MeOH = 9:1)
c. Tng hp Ethyl 7-(5-cloro-3-(methoxyimino)-2-oxoindolin-1-yl)
heptanoat (3c):
- Tin hnh: Nh quy trnh tng hp ca cht 3a vi cht ban u l 5-
cloro-3-(methoxyimino)-2-oxoindolin (0,211g; 1mmol)
- Kt qu: Sn phm ester thu c c dng du mu gch
Rf = 0,8 (DCM:MeOH = 9:1)
d. Tng hp Ethyl 7-(5-bromo-3-(methoxyimino)-2-oxoindolin-1-yl)
heptanoat (3d):
- Tin hnh: Nh quy trnh tng hp ca cht 3a vi cht ban u l 5-
bromo-3-(methoxyimino)-2-oxoindolin (0,255g; 1mmol)
- Kt qu: Sn phm ester thu c c dng du mu gch
Rf = 0,83 (DCM:MeOH = 9:1)
27
Bng 4: Hiu sut v cc ch s ha l ca Ethyl 7-(3-(methoxyimino)-2-oxoindolin-1-yl) heptanoat v cc dn cht:
Cht R CTPT KLPT Mu
3a -H C18H24O4N2 332 gch
3b -F C18H23O4N2F 350 gch
3c -Cl C18H23O4N2Cl 366,5 gch
3d -Br C18H23O4N2Br 411 gch
28
3.1.1.3. Tng hp N-hydroxy-7-(3-(methoxyimino)-2-oxoindolin-1-yl)
heptanamid v cc dn cht (4a-d)
a. Tng hp N-hydroxy-7-(3-(methoxyimino)-2-oxoindolin-1-yl)
heptanamid (4a):
Quy trnh tng hp N-hydroxy-7-(3-(methoxyimino)-2-oxoindolin-1-yl)
heptanamid (4a) c tin hnh nh s di y:
NH2OH.HCl
CH3OH,NaOH
3a 4a
S 4: Quy trnh tng hp cht 4a
Tin hnh:
- Chun b dung dch NaOH bng cch ha tan 3,2g NaOH (~80 mmol)
trong 2 ml nc trong ng nghim, lm lnh.
- Ester 3a (~1mmol; 0,332g) ho vi 3ml CH3OH trong bnh cu dung
tch 50ml.
- Bnh cu t trong hn hp nc mui (-5oC), kt hp khuy t.
- Thm 10 mmol NH2OH.HCl vo bnh cu.
- Rt t t dung dch NaOH vo bnh phn ng, tin hnh trong 30 pht.
Ch lun duy tr nhit phn ng -5oC.
- Sau 30 pht, kim tra phn ng bng TLC vi pha ng DCM:CH3OH
(9:1), khi ht nguyn liu ester th dng phn ng.
X l:
- Acid ha hn hp phn ng bng HCl 5% lnh, iu chnh v pH 4-
5, lnh 15 pht ta sn phm.
- Lc v ra ta bng nc ct n khi dch lc trung tnh.
- Sy kh ta 60oC, thu c sn phm.
29
Kt qu: Cht rn mu vng
Hiu sut 56%
Rf = 0,63 (DCM:CH3OH =9:1)
b. Tng hp N-hydroxy-7-(5-floro-3-(methoxyimino)-2-oxoindolin-1-
yl) heptanamid (4b):
- Tin hnh: Nh quy trnh tng hp ca cht 4a vi cht ban u l
Ethyl 7-(5-floro-3-methoxyimino-2-oxoindolin-1-yl) heptanoat
(0,350g; 1mmol).
- Kt qu: Cht rn mu vng m
Hiu sut 50,52%
Rf = 0,48 (DCM:CH3OH =9:1)
c. Tng hp N-hydroxy-7-(5-cloro-3-(methoxyimino)-2-oxoindolin-1-
yl) heptanamid (4c):
- Tin hnh: Nh quy trnh tng hp ca cht 4a vi cht ban u l
Ethyl 7-(5-cloro-3-methoxyimino-2-oxoindolin-1-yl) heptanoat
(0,367g; 1mmol).
- Kt qu: Cht rn mu vng m
Hiu sut 49,90%
Rf = 0,51 (DCM:CH3OH =9:1)
d. Tng hp N-hydroxy-7-(5-bromo-3-(methoxyimino)-2-oxoindolin-
1-yl) heptanamid (4d):
- Tin hnh: Nh quy trnh tng hp ca cht 4a vi cht ban u l
Ethyl 7-(5-bromo-3-methoxyimino-2-oxoindolin-1-yl) heptanoat
(0,411g; 1mmol).
- Kt qu: Cht rn mu vng m
Hiu sut 55,20%
Rf = 0,52 (DCM:CH3OH =9:1)
30
Bng 5: Hiu sut v cc ch s ha l ca N-hydroxy-7-(3-(methoxyimino)-2-oxoindolin-1-yl) heptanamid v cc dn cht
.
Cht R KLPT CTPT Mu Hiu sut (%)
4a -H 319 C16H21O4N3 Vng 56,00
4b -F 337 C16H20O4N3F Vng m 50,52
4c -Cl 353,5 C16H20O4N3Cl Vng m 49,90
4d -Br 398 C16H20O4N3Br Vng m 55,20
31
3.1.2. Kim tra tinh khit:
Sau khi tng hp cc dn cht 4a-d chng ti kim tra tinh khit
cc cht bng sc k lp mng v o nhit nng chy (Tonc) ca chng.
* Sc k lp mng (SKLM)
SKLM c tin hnh trn bn nhm trng sn silicagel Merck 70-230
mesh, quan st di n t ngoi bc sng 254 nm. Cc cht u c vt
gn, r, khng c vt ph vi 2 h dung mi khc nhau..
* o nhit nng chy:
Bn cnh vic kim tra tinh khit bng SKLM, chng ti tin
hnh o nhit nng chy ca bn cht tng hp c. Kt qu o nhit
nng chy cho thy: cc cht u c im chy r rng, khong chnh lch
hp.
Nhit nng chy v gi tr Rf ca cc cht vi mt h dung mi khai
trin c tm tt trong bng 6.
Bng 6: Gi tr Rf v nhit nng chy To
nc ca cc acid hydroxamic 4a-d
Cht R Pha ng Rf Tonc(
oC)
4a -H DCM : MeOH=9:1 0,63 166-167oC
4b -F DCM : MeOH=9:1 0,48 198-199oC
4c -Cl DCM : MeOH=9:1 0,51 196-198oC
4d -Br DCM : MeOH=9:1 0,52 183-184oC
Nhn xt: Thng qua sc k khi chy SKLM v nhit nng chy
ca cc cht, chng ti c th khng nh cc cht ny l tinh khit, iu
kin o ph v th tc dng sinh hc.
32
3.1.3. Xc nh cu trc
khng nh cu trc ca cc cht tng hp c chng ti tin hnh
ghi ph hng ngoi (IR), ph khi lng (MS), ph cng hng t proton
(1H-NMR), ph cng hng t carbon (13C-NMR) ca cc cht 4a-d. Kt qu
ph ca cc cht c trnh by c th phn phn tch ph v ph lc.
3.1.3.1. Ph hng ngoi (IR)
Bng 7: Kt qu phn tch ph hng ngoi ca cc cht 4a-d
Cht R S sng (cm-1) ng vi cc dao ng
O-H N-H C-H C=O
4a -H 3399 3249 2933
2862
1704
1647
4b -F 3331 3234 2931
2861
1702
1655
4c -Cl 3431 3259 2935
2859
1714
1645
4d -Br - 3222 2935
2856
1722
1647
Nhn xt: Cn c vo kt qu phn tch ph bng 7 v ph (ph
lc 1-4), chng ti nhn thy c s xut hin:
- Di hp th khong 3431 3331 cm-1 c trng cho dao ng ha tr
ca nhm O-H trong nhm chc acid hydroxamic
33
- Di hp th khong 3259 3222 cm-1 c trng cho dao ng ha tr
ca nhm N-H trong nhm chc acid hydroxamic.
- Di hp th khong 2935 2856 cm-1 c trng cho dao ng ha tr
ca nhm C-H mch nhnh.
- Di hp th khong 1722 1702 cm-1 c trng cho dao ng ha tr
ca nhm C=O trong khung 3-methoxim-isatin.
- Di hp th khong 1655 1645 cm-1 c trng cho dao ng ha tr
ca nhm C=O trong nhm chc acid hydroxamic.
Trong c di hp th ca nhm OH t 3431-3331 cm-1 v di hp
th ca nhm NH t 3259-3222 cm-1 l c trng nht cho thy sn phm
c tng hp.
3.1.3.2. Ph khi lng (MS)
Bng 8: Kt qu phn tch ph khi lng ca cc cht 4a-d
Cht CTCT v M m/z
4a R = -H (M=319) 318,18[M-H]-
4b R = -F (M=337) 334,7[M-2H]-
4c R = -Cl (M=353,5) 352,2[M-H]-
4d R = -Br (M=398) 398,1[M]-
Nhn xt: Qua kt qu phn tch ph bng 8 v ph (ph lc 5-8)
chng ti u thy c pic phn t c s khi ng bng s khi d kin vi
cng mnh, ph c t pic phn mnh, cc pic phn mnh a s c
cng nh.
34
3.1.3.3. Ph cng hng t proton (1H-NMR)
Bng 9: Kt qu phn tch ph 1H-NMR ca cc cht 4a-d
Cht CTCT S liu phn tch
1H-NMR
4a R = -H
1H-NMR (500MHz, DMSO-d6, ppm): 10,33 (1H, s,
NH); 8,66 (1H, s, OH); 7,87 (1H, d, H4, J=7,5 Hz);
7,46 (1H, t, H5, J=7,5 Hz); 7,12 (1H, d, H7, J=8,0
Hz); 7,07 (1H, t, H6, J=7,5 Hz); 4,20 (3H, s, H10);
3,37 (2H, t, H2, J=7,0 Hz); 1,93-1,90 (2H, t, H7,
J=7,0 Hz); 1,56 (2H, m, H3); 1,47-1,45 (2H, m, H6);
1,26 (2H, m, H4,5).
4b R = -F
1H-NMR (500MHz, DMSO-d6, ppm): 10,33 (1H, s,
NH); 8,66 (1H, s, OH); 7,65 (1H, dd, H4, J=8,0; 2,5
Hz); 7,33 (1H, td, H6, J=9,25; 2,5 Hz); 7,13 (1H, dd,
H7, J=8,5; 4,0 Hz), 4,21 (3H, s, H10); 3,38 (2H, t,
H2, J=7,0 Hz); 1,93-1,90 (2H, t, H7, J=7,0 Hz); 1,56-
1,53 (2H, m, H3); 1,46-1,44 (2H, m, H6); 1,26-1,25
(2H, m, H4,5).
4c R = -Cl
1H-NMR (500MHz, DMSO-d6, ppm): 10,45 (1H, s,
NH); 7,81 (1H, s, H4); 7,50 (1H, d, H6, J=8,0 Hz);
7,16 (1H, d, H7, J=8,0 Hz); 4,22 (3H, s, H10); 3,37
(2H, t, H2, J=6,5 Hz); 1,93-1,91 (2H, t, H7, J=7,0
Hz); 1,53 (2H, m, H3); 1,44 (2H, m, H6); 1,25 (2H,
35
m, H4,5).
4d R = -Br
1H-NMR (500MHz, DMSO-d6, ppm): 7,94 (1H, s,
H4); 7,63 (1H, d, H6, J=7,5 Hz); 7,10 (1H, d, H7,
J=8,0 Hz); 4,22 (3H, s, H10); 2,88 (2H, t, H2, J=6,5
Hz); 2,73 (2H, t, H7); 1,93-1,90 (2H, t, H3); 1,53
(2H, m, H6); 1,44 (2H, m, H4,5).
Ghi ch: : chuyn dch ha hc (ppm); s: singlet; d: doublet; t: triplet; q: quartet, m: multiplet
Nhn xt: Da vo kt qu phn tch ph bng 9 v cn c vo ph
(ph lc 9-12) chng ti nhn thy:
- S lng proton, bi ca tn hiu l ph hp vi cng thc cu to
ca 4 cht d kin [2,3]. Cc cht u c cc proton ca khung 3-
methoxim-isatin nm trong vng dch chuyn t 7,06-7,94 v 12 proton
ca cu ni nm trong vng dch chuyn 1,25-3,38.
- Ngoi ra cn xut hin tn hiu proton ca nhm OH gi tr 8,66 v
nhm NH khong 10,45-10,33 ph hp vi cng thc cu to d
kin ca 4 cht 4a-d.
3.1.3.4. Ph cng hng t 13C (13C-NMR)
Bng 10: Kt qu phn tch ph 13C-NMR ca cc cht 4a-d
Cht CTCT S liu phn tch ph 13C-NMR
4a R = -H 13C-NMR (125MHz, DMSO-d6, ppm): 169,06 (C1);
36
162,15 (C2); 143,68 (C3) 143,28 (C8); 132,96 (C6);
127,34 (C4); 122,61 (C5); 114,87 (C9); 109,40 (C7);
64,40 (C10); 40,00-39,00 (C7); 32,14 (C2); 28,14
(C4); 26,77 (C6); 25,87 (C5); 24,95 (C3).
4b R = -F
13C-NMR (125MHz, DMSO-d6, ppm): 169,10 (C1);
162,03 (C2); 158,83-156,93 (C5); 142,96 (C3);
140,02 (C8); 119,23-119,04 (C6); 115,41-115,33
(C7); 114,50-114,29 (C4); 110,52-110,46 (C9);
64,67 (C10); 40,00-39,00 (C7); 32,15 (C2); 28,16
(C4); 26,71 (C6); 25,86 (C5); 24,95 (C3).
4c R = -Cl
13C-NMR (125MHz, DMSO-d6, ppm): 169,04 (C1);
161,77 (C2); 142,44 (C3); 142,41 (C8) 132,30 (C6);
126,54 (C5) 126,38 (C4); 115,97 (C7); 111,00 (C9);
64,71 (C10); 40,00-38,48 (C7); 32,09 (C2); 28,11
(C4); 26,67 (C6); 25,80 (C5);24,91 (C3).
4d R = -Br
13C-NMR (125MHz, DMSO-d6, ppm): 168,69 (C1);
161,66 (C2); 142,80 (C3); 142,32 (C8) 135,13 (C6);
129,22 (C4); 116,41 (C9); 114,00 (C5); 111,47 (C7);
64,72 (C10); 40,00-39,00 (C7); 32,21 (C2); 28,15
(C4); 26,68 (C6); 25,84 (C5);25,03 (C3).
Nhn xt: Qua kt qu phn tch ph bng 10 v ph cc cht
phn ph lc (ph lc 13-16) chng ti nhn thy: s lng carbon, dch
chuyn ha hc ca cc pic l ph hp vi cng thc cu to d kin [2,3].
Kt hp vi kt qu ph IR, MS, 1H-NMR c th khng nh cc cht chng
ti tng hp c c cng thc cu to ng nh d kin.
37
3.2. TH TC DNG SINH HC
3.2.1. Th tc dng c ch HDAC
Th tc dng c ch enzym HDAC 3,4 c thc hin ti Khoa Dc,
Trng i hc Quc gia Chungbuk, Cheongju, Hn Quc. Kt qu s c
trnh by phn bn lun.
3.2.2. Th hot tnh khng t bo ung th in vitro
Th hot tnh khng t bo ung th in vitro cng c thc hin ti
Khoa Dc, Trng i hc Quc gia Chungbuk theo phng php MTT v
gi tr IC50 c tnh trn phn mm GraphPad Prism. Chng ti s b
kho st hot tnh ca cc cht 4a, 4b, 4c, 4d trn dng t bo ung th
SW620. Kt qu gi tr IC50 c th ca cc cht s c trnh by trong phn
bn lun.
3.3. NH GI MC GING THUC
Bng 11: nh gi mc ging thuc ca cc cht 4a-d theo quy tc Lipinsky
Cht R LogP KLPT S
NH,OH S N,O
4a -H 2,67 319 2 7
4b -F 2,87 337 2 7
4c -Cl 3,31 353,5 2 7
4d -Br 3,56 398 2 7
V ging thuc ca cc cht trn theo quy tc Lipinsky, cc cht u
tha mn c cc yu cu ca quy tc.
38
3.4. BN LUN
3.4.1. Ha hc:
3.4.1.1. Tng hp 3-(methoxyimino)-2-oxoindolin v cc dn cht:
- y l phn ng cng hp i nhn vo nhm carbonyl, cho hiu sut
cao, d thc hin.
- iu kin ca phn ng ty thuc vo hot tnh ca nhm carbonyl,
ph thuc vo c tnh base ca tc nhn i nhn.
3.4.1.2. Phn ng tng hp dy ester trung gian
- y l phn ng alkyl ha.
- Phn ng xy ra kh d dng, hiu sut cao (t 90,33% ti 93,96%).
Kim tra bng SKLM cho thy sn phm ester thu c tng i tinh
khit, c th s dng cho giai on tng hp tip theo.
- Chng ti la chn s dng dung mi DMF trnh nguy c thy phn
nhm chc amid v ester.
- Cn kim sot thi gian phn ng v theo di kt qu phn ng bng
SKLM trnh to sn phm ph.
- Cc dung mi v dng c thc hin phn ng ny phi m bo yu
cu khan nc trnh thy phn chc ester to ra.
3.4.1.3. Phn ng tng hp dy cht acid hydroxamic
- L phn ng acyl ha vi tc nhn ester.
- Cn duy tr nhit thp (-5oC) trnh thy phn nguyn liu ester
thnh acid carboxylic do s c mt ca NaOH.
- Cn lu thi gian phn ng trnh lm phn hy nhm chc amid
(lu theo di bng SKLM v th phn ng mu vi FeCl3/HCl).
- Lng NaOH s dng cn d m bo tan ca ester trong dung
mi v chuyn NH2OH. HCl thnh dng NH2OH ng thi sn phm
to thnh (cc acid hydroxamic) tan ht trong dung dch phn ng.
39
- Dung dch NaOH phi c cho t t vo vo bnh cu trnh qu
nhit khi pha long dung mi MeOH bng H2O.
3.4.2. Tc dng c ch histon deacetylase v hot tnh hng t bo ung
th in vitro:
3.4.2.1. Tc dng c ch HDAC:
Sau khi tng hp v khng nh cu trc ca 4 cht tng hp c,
chng ti tin hnh th tc dng c ch HDAC bng phng php Western
Blot ti Khoa Dc, i hc Chungbuk. Kt qu th tc dng c ch HDAC
nng 10g/ml trong 24 gi c trnh by hnh 13.
Hnh 13: Tc dng c ch HDAC ca cc cht 4a-d
Trn hnh 13, cc di protein rt m tng ng vi acetyl-histon H3
v acetyl-histon H4. iu ny chng t hot tnh ca HDAC b kha bi 3
dn cht 4a, 4b, 4c.
3.4.2.2. Th hot tnh hng t bo ung th in vitro:
Tc dng c ch HDAC quyt nh tc dng khng t bo ung th, tuy
nhin gy ra c tnh vi t bo, cc cht phi c kh nng thm qua mng
sinh hc vo trong t bo tip cn vi HDAC. Do chng ti tip tc tin
40
hnh th c tnh t bo in vitro nhm tm ra nhng cht tht s c tc dng
sinh hc tt. Th nghim tin hnh trn dng t bo ung th i trng SW620.
Kt qu c trnh by bng 12:
Bng 12. Kt qu th c tnh t bo in vitro ca cc cht tng hp c
Cht R Khi lng
phn t IC50
1 (M)/SW620
4a -H 319,36 0,73
4b -F 337,35 1,11
4c -Cl 353,80 0,49
4d -Br 398,25 2,32
SAHA2 264,32 3,70
Ghi ch: 1IC50: Nng (M) lm c ch 50% S pht trin ca t bo, Cc kt qu c ly
trung bnh qua 3 ln th nghim vi sai lch khng qu 10%; 2SAHA: acid suberoylanilid
hydroxamic, cht i chiu.
.
K tha thnh qu ca cc cng trnh khoa hc trn th gii v tip tc
pht trin cc nghin cu trc y c thc hin ti b mn Ha Dc
trng i hc Dc H Ni [4,5,6], chng ti nhn thy trong cc dn cht
acid hydroxamic hng c ch enzym HDAC c nghin cu th cc cht c
phn cu ni 6C c kt qu hot tnh rt tt. V vy so vi SAHA, cc cht
chng ti tng hp c vn duy tr cu to phn cu ni (6C) v nhm to
chelat vi Zn2+ (dng acid hydroxamic) trong kho st ca mnh. Nhng c s
thay i v cu to ha hc ca nhm nhn din b mt.
Cc kt qu c tnh t bo ti bng 12 cho thy cc cht 4a-4d u th
hin tt hot tnh trn dng t bo ung th SW620.Trong cht 4c cho hot
41
tnh tt nht vi IC50 l 0,49M, mnh hn 7,5 ln so vi gi tr IC50 ca
SAHA (3,7M), gi tr IC50 ca cht 4b l 1,11M mnh hn 3 ln SAHA,
cht 4a (vi IC50 l 0,73M) mnh hn 5 ln, cn li cht 4d (IC50=2,32M)
gn nh tng ng vi SAHA.
gii thch kt qu thu c, chng ti so snh cu to dy cht
chng ti tng hp vi SAHA. Phn nhn din b mt ca 4 cht 4a-d c
thm 2 nguyn t N, s c mt ca chng c l lm tng kh nng tng
tc vi cc acid amin ming ti ca HDAC, ng thi mch carbon thn
du vi di ph hp (6C) gip a nhm chc acid hydroxamic tip xc
d dng vi Zn2+, to nn kh nng c ch HDAC mt cch hiu qu cho c 4
cht. Tuy nhin kh nng c ch t bo ung th cn ph thuc vo kh nng
thm qua mng t bo ca cc cht. Ba cht 4a, 4b v 4c c hot tnh c ch
t bo ung th tt. Cht 4c mang nhm th -Cl c tc dng c ch mnh nht.
Cht 4b c hot tnh km hn 4c do c nhm th ht in t rt mnh (-F)
lm gim mt in t trn khung 3-methoxim-isatin nn gim tng tc
Van der Waals ca chng vi cc acid amin thm dn n gim hot tnh.
Ngoi ra, cht 4d vi nhm th -Br c tc dng c ch t bo ung th yu
nht c th do cu trc cng knh ca -Br ngn cn cht qua mng t bo.
So vi dy cht trong lun vn thc s Dc hc ca Nguyn Th M
tng hp N-hydroxy-7-(3-(hydroxyimino)-2-oxoindolin-1-yl) heptanamid
v cc dn cht [4], chng ti thy tc dng c ch dng t bo SW620 ca
cc cht c cng nhm th R c xu hng yu hn. C th do 2 nguyn nhn
sau y:
- Cc cht 4a-d vi s c mt ca =NOCH3 lm cho cu trc tr ln
cng knh hn, v vy chng kh thm qua mng sinh hc hn.
42
- N-hydroxy-7-(3-(hydroxyimino)-2-oxoindolin-1-yl) heptanamid v cc
dn cht c cha =NOH c kh nng to lin kt hydro, nn kh nng gn
cht vi enzym tt hn so vi dy cht 4a-d.
Kt qu so snh c trnh by trong bng 13
Cht R IC50 (M)/ SW620
-H 0,73
-F 1,11
-Cl 0,49
-Br 2,32
[4]
-H 0,64
-F 0,11
-Cl 0,65
-Br 0,29
Bng 13: Kt qu so snh tc dng c ch dng t bo SW620
Tm li, chng ti tin rng vic la chn b khung ca cng thc c 2
phn ging vi SAHA l ph hp. kha lun ny chng ti thc hin
vic chn phn nhn din b mt l 3-(methoxyimino)-2-oxoindolin-1-yl
thay th cho nhm phenyl ca SAHA. Trong nghin cu ln ny cho cc
cht c hot tnh rt tt. Thm vo , vic a lin kt amid vo trong cu
trc vng lin hp vi vng benzen cng lm n gin ha cu trc, thun li
cho vic tng hp. Do y s l 1 hng i kh quan trong vic tm ra cc
cht c ch HDAC c hot tnh tt.
3.4.3. nh gi mc ging thuc:
V ging thuc ca cc cht trn theo quy tc Lipinsky, cc cht u
tha mn c cc yu cu ca quy tc. Cng vi hot tnh sinh hc tt, iu
ny m ra trin vng cho vic nghin cu cn lm sng v lm sng ca cc
dn cht sau ny.
43
PHN IV. KT LUN V KIN NGH
4.1. KT LUN
4.1.1. V tng hp ha hc v khng nh cu trc
tng hp c N-hydroxy-7-(3-(methoxyimino)-2-oxoindolin-1-yl)
heptanamide v 3 dn cht nh d kin, cc cht u cha c cng b
trong bt k ti liu no, bao gm:
- N-hydroxy-7-(3-(methoxyimino)-2-oxoindolin-1-yl) heptanamid (4a)
- N-hydroxy-7-(5-floro-3-(methoxyimino)-2-oxoindolin-1-yl) heptanamid (4b)
- N-hydroxy-7-(5-cloro-3-(methoxyimino)-2-oxoindolin-1-yl) heptanamid (4c)
- N-hydroxy-7-(5-bromo-3-(methoxyimino)-2-oxoindolin-1-yl) heptanamid (4d)
4.1.2. V hot tnh sinh hc:
th tc dng c ch HDAC v hot tnh khng t bo ung th ca
cc cht tng hp c. Kt qu cho thy :
a) V tc dng c ch HDAC: C 3 cht (4a-4c) c tc dng c ch
mnh HDAC nng 10g/ml.
b) V tc dng khng t bo ung th: c 4 cht (4a-4d) u c tc dng
c ch dng t bo ung th SW620. Trong cht 4c c tc dng mnh nht
(mnh hn 7,5 ln so vi SAHA), y l mt cht c trin vng trong tng
lai.
4.1.3. V ging thuc ca cc cht trn theo quy tc Lipinsky: Cc cht
u tha mn c cc yu cu ca quy tc Lipinsky.
4.2. KIN NGH
Vi kt qu kh quan v tc dng sinh hc ca cc cht tng hp c
chng ti a ra mt s kin ngh sau:
- Tin hnh th c tnh t bo in vitro ca cc cht tng hp c
trn cc dng t bo khc, tm ra cht c tc dng mnh lm c s
cho vic th tc dng in vivo.
44
- Tip tc nghin cu v pht trin cc dn xut ca acid hydroxamic
trn cng b khung vi cc nhm th -CH3, -OCH3 v NO2.
- Thay i vng phenyl ca SAHA bng mt s vng thm khc.
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PH LC
Ph lc 1: Ph hng ngoi IR ca cht 4a
Ph lc 2: Ph hng ngoi IR ca cht 4b
Ph lc 3: Ph hng ngoi IR ca cht 4c
Ph lc 4: Ph hng ngoi IR ca cht 4d
Ph lc 5: Ph khi lng MS [M-H]- ca cht 4a
Ph lc 6: Ph khi lng MS [M-2H]- ca cht 4b
Ph lc 7: Ph khi lng MS [M-H]- ca cht 4c
Ph lc 8: Ph khi lng MS [M]- ca cht 4d
Ph lc 9: Ph 1H-NMR ca cht 4a
Ph lc 10: Ph 1H-NMR ca cht 4b
Ph lc 11: Ph 1H-NMR ca cht 4c
Ph lc 12: Ph 1H-NMR ca cht 4d
Ph lc 13: Ph 13C-NMR ca cht 4a
Ph lc 14: Ph 13C-NMR ca cht 4b
Ph lc 15: Ph 13C-NMR ca cht 4c
Ph lc 16: Ph 13C-NMR ca cht 4d
Ph lc 1: Ph hng ngoi IR ca cht 4a
Ten may: GX-PerkinElmer-USA Resolution: 4cm-1
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
Nguoi do: Phan Thi Tuyet Mai Ten mau: 7ADate: 11/9/2012
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0
0.0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100.0
cm-1
%T
3399
3249
3038
2933
2862
2824
1704
1647
1621
1607
1539
1464
1444
1377
1349
1314
1204
1193
1126
1115
1094
1081
1049
1029
1004
939
864
811
783
745
692
616
593
558
503
495
Ph lc 2: Ph hng ngoi IR ca cht 4b
Ten may: GX-PerkinElmer-USA Resolution: 4cm-1
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
Nguoi do: Phan Thi Tuyet MaiTen mau: 7BDate: 11/26/2012
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0
0.0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
96.4
cm-1
%T
3331
3234
3045
2931
2861
1702
1655
1622
1602
1529
1477
1353
1277
1197
1142
1127
1082
1058
1045
1018
973
909
871
835
744
728
681
633
584
499
Ph lc 3: Ph hng ngoi IR ca cht 4c
Ten may: GX-PerkinElmer-USA Resolution: 4cm-1
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
Nguoi do: Phan Thi Tuyet MaiTen mau: 7CDate: 12/7/2012
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0
30.0
35
40
45
50
55
60
65
70
75
80
85
90
95
99.2
cm-1
%T
3431
3259
2935
2859
1714
1645
1609
1469
1442
1348
1270
1203
1159
1094
1072
1018
935
882
861
833
731
712
646
499
Ph lc 4: Ph hng ngoi IR ca cht 4d
Ten may: GX-PerkinElmer-USA Resolution: 4cm-1
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
Nguoi do: Phan Thi Tuyet MaiTen mau: 7DDate: 12/7/2012
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.0
0.0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100.1
cm-1
%T
3222
2935
2856
1722
1647
1604
1466
1436
1342
1269
1187
1157
1134
1113
1086
1061
1014
889
853
812
775
729
707
635
Ph lc 5: Ph khi lng MS [M-H]- ca cht 4a
Ph lc 6: Ph khi lng MS [2M+Na]+ ca cht 4b
Ph lc 7: Ph khi lng MS [M-H]- ca cht 4c
Ph lc 8: Ph khi lng MS [M]- ca cht 4d
Ph lc 9: Ph 1H-NMR ca cht 4a
Ph lc 10: Ph 1H-NMR ca cht 4b
Ph lc 11: Ph 1H-NMR ca cht 4c
Ph lc 12: Ph 1H-NMR ca cht 4d
Ph lc 13: Ph 13C-NMR ca cht 4a
Ph lc 14: Ph 13C-NMR ca cht 4b
Ph lc 15: Ph 13C-NMR ca cht 4c
Ph lc 16: Ph 13C-NMR ca cht 4d
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