Tổng hợp và thử tác dụng sinh học một số acid hydroxamic mang khung 3 - methoxin-...

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



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


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):


5-cloroisatin (0,182g; 1mmol).

- Kt qu: Cht rn mu vng


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):


5-bromoisatin (0,226g; 1mmol).

- Kt qu: Cht rn mu vng da cam


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


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):


cloro-3-(methoxyimino)-2-oxoindolin (0,211g; 1mmol)


Rf = 0,8 (DCM:MeOH = 9:1)

d. Tng hp Ethyl 7-(5-bromo-3-(methoxyimino)-2-oxoindolin-1-yl)

heptanoat (3d):


bromo-3-(methoxyimino)-2-oxoindolin (0,255g; 1mmol)


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


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):


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):


Ethyl 7-(5-cloro-3-methoxyimino-2-oxoindolin-1-yl) heptanoat

(0,367g; 1mmol).


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):


Ethyl 7-(5-bromo-3-methoxyimino-2-oxoindolin-1-yl) heptanoat

(0,411g; 1mmol).


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


ca nhm N-H trong nhm chc acid hydroxamic.


ca nhm C-H mch nhnh.


ca nhm C=O trong khung 3-methoxim-isatin.


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


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


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


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


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


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.

TI LIU THAM KHO

Phn 1: Ting Vit

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Nxb Y hc, trang 115-128.

2. Phm Khnh Phong Lan (2007), Cc phng php quang ph xc nh

cu trc hp cht hu c, tp 1, Gio trnh sau i hc, i hc Y

Dc TP.H Ch Minh, trang 21-45.

3. Phm Khnh Phong Lan (2007), Cc phng php quang ph xc nh

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hc, i hc Dc H Ni, trang 42-55.

5. o Th Kim Oanh (2013), Tng hp v th hot tnh sinh hc ca mt

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6. Trng Thanh Tng (2012), Tng hp mt s acid hydroxamic hng

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inhibitors: SAR studies, isoenzym selectivity, and pharmacological

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16. Drummond DC, Noble CO, Kirpotin DB, Guo Z, Scott GK, Benz CC

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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



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



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



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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