B GIO DC V O TO

B Y T

TRNG I HC DC H NI

----- ------

L TH THO

TNG HP V TH TC DNG SINH HC

CA MT S ACID HYDROXAMIC MANG
KHUNG 3-METHOXIM-ISATIN HNG C
CH HISTON DEACETYLASE

LUN VN THC S DC HC

H NI 2014

B GIO DC V O TO

B Y T

TRNG I HC DC H NI

----- ------

L TH THO

TNG HP V TH TC DNG SINH HC

CA MT S ACID HYDROXAMIC MANG
KHUNG 3-METHOXIM-ISATIN HNG C
CH HISTON DEACETYLASE
LUN VN THC S DC HC

CHUYN NGNH CNG NGH

DC PHM V BO CH THUC
M S: 60720402
Ngi hng dn khoa hc:
1. TS. o Th Kim Oanh
2. PGS.TS. Nguyn Hi Nam

H NI 2014

LI CM N
Trong qu trnh thc hin ti Tng hp v th tc dng sinh hc ca mt
s acid hydroxamic mang khung 3-methoxim-isatin hng c ch histon
deacetylase, ti nhn c rt nhiu s gip ca cc thy c gio, bn b v
ng nghip.
Trc tin, ti xin c gi li cm n chn thnh v su sc nht ti TS.
o Th Kim Oanh, PGS. TS. Nguyn Hi Nam nhng ngi thy tn tm
hng dn, ch bo v to mi iu kin thun li cho ti trong sut qu trnh
nghin cu, thc hin ti ti B mn Ha dc Trng i hc Dc H Ni.
Vi lng knh trng v bit n, ti xin gi li cm n ti TS. Hunh Kim
Thoa ngi Thy to iu kin cho ti c hi c nng cao kin thc v c
thi gian thc hin c hi .
Ti cng xin gi li cm n ti cc thy c gio, cc anh ch k thut vin,
cc bn sinh vin nhm nghin cu Ha dc - B mn Ha dc Trng i
hc Dc H Ni, cc anh ch Khoa ha hc Trng i hc Khoa hc T nhin
i hc Quc gia H Ni, Vin ha hc Vin Khoa hc v Cng ngh Vit
Nam, Vin Ha hp cht thin nhin Vin Khoa hc v Cng ngh Vit Nam,
Trng i hc Quc gia Chungbuk (Cheongji, Hn Quc) nhit tnh gip ti
trong thi gian ti thc hin ti ny.
Cui cng, xin gi li tri n su sc n chng, gia nh, bn b v ng
nghip ng vin, h tr ti rt nhiu trong qu trnh hc tp, lm vic v hon
thnh lun vn.
H Ni, ngy 29 thng 8 nm 2014

L Th Tho

MC LC
DANH MC CC CH VIT TT

Trang

DANH MC HNH V
DANH MC BNG
DANH MC S
T VN

Chng 1. TNG QUAN

1.1. HISTON DEACETYLASE (HDAC)

1.1.1. Khi nim HDAC

1.1.2. Phn loi HDAC

1.1.3. Mi lin quan gia ung th v s hot ng bt thng ca HDAC

1.2. CC CHT C CH HDAC (HDACIs)

1.2.1. Phn loi cc cht c ch HDAC

1.2.2. C ch tc dng ca cc cht c ch HDAC

1.2.3. Cu trc ca cc cht c ch HDAC

1.3. TNH HNH NGHIN CU TNG HP CC ACID HYDROXAMIC

10

HNG C CH HDAC HIN NAY

1.3.1. Cc nghin cu tng hp cc acid hydroxamic trn th gii

10

1.3.1.1. Thay i cu ni

11

1.3.1.2. Thay i nhm nhn din b mt

16

1.3.2. Cc nghin cu trong nc

21

1.4. CC PHNG PHP TNG HP ACID HYDROXAMIC

24

1.4.1. Tng hp acid hydroxamic t ester

24

1.4.2. Tng hp acid hydroxamic t acid carboxylic

25

Chng 2. NGUYN LIU, THIT B, NI DUNG V PHNG PHP

26

NGHIN CU
2.1. NGUYN LIU

26

2.2. THIT B V DNG C

26

2.3. NI DUNG V PHNG PHP NGHIN CU

27

2.3.1. Ni dung nghin cu

27

2.3.2. Phng php nghin cu

28

2.3.2.1. Tng hp ha hc

28

2.3.2.2. Phng php kim tra tinh khit

28

2.3.2.3. Phng php phn tch cu trc

28

2.3.2.4. Phng php th hot tnh sinh hc

29

2.3.2.5. Docking

31

2.3.2.6. nh gi mc ging thuc ca cc cht tng hp c

31

Chng 3. THC NGHIM, KT QU

33

3.1. TNG HP HA HC

33

3.1.1. Tng hp 3-(methoxyimino)-2-oxoindolin v dn cht (IIa-g)

33

3.1.1.1. Tng hp 3-(methoxyimino)-2-oxoindolin (IIa)

34

3.1.1.2. Tng hp 5- fluoro-3-(methoxyimino)-2-oxoindolin (IIb)

34

3.1.1.3. Tng hp 5-cloro-3-(methoxyimino)-2-oxoindolin (IIc)

34

3.1.1.4. Tng hp 5-bromoro-3-(methoxyimino)-2-oxoindolin (IId)

34

3.1.1.5. Tng hp 3-(methoxyimino)-5-nitro-2-oxoindolin (IIe)

35

3.1.1.6. Tng hp 3-(methoxyimino)-5-methyl-2-oxoindolin (IIf)

35

3.1.1.7. Tng hp 7-cloro-3-(methoxyimino)-2-oxoindolin (IIg)

35

3.1.2. Tng hp ethyl-7-(3-(methoxyimino)-2-oxoindolin-1-yl)heptanoat v dn

35

cht (IIIa g)
3.1.2.1. Tng hp ethyl-7-(3-(methoxyimino)2-oxoindolin-1-yl)heptanoat (IIIa)

36

3.1.2.2.

ethyl-7-(5-fluoro-3-(methoxyimino)-2-oxoindolin-1-

36

3.1.2.3. Tng hp ethyl-7-(5-cloro-3-(methoxyimino)-2-oxoindolin-1-yl)heptanoat

36

Tng

hp

yl)heptanoat (IIIb)

(IIIc)
3.1.2.4.

Tng

hp

ethyl-7-(5-bromo-3-(methoxyimino)-2-oxoindolin-1-

37

3.1.2.5. Tng hp ethyl-7-(3-(methoxyimino)-5-nitro-2-oxoindolin-1-yl)heptanoat

37

yl)heptanoat (IIId)

(IIIe)

3.1.2.6.

Tng

hp

ethyl-7-(3-(methoxyimino)-5-methyl-2-oxoindolin-1-

37

3.1.2.7. Tng hp ethyl-7-(7-cloro-3-(methoxyimino)-2-oxoindolin-1-yl)heptanoat

37

yl)heptanoat (IIIf)

(IIIg)
3.1.3. Tng hp N-hydroxy-7-(3-methoxyimino-2-oxoindolin-1-yl)heptanamid v

38

dn cht (IVa-g)
3.1.3.2.

Tng

hp

N-hydroxy-7-(5-fluoro-(3-methoxyimino)-2-oxoindolin-1-

39

N-hydroxy-7-(5-cloro-(3-methoxyimino)-2-oxoindolin-1-

39

N-hydroxy-7-(5-bromo-(3-methoxyimino)-2-oxoindolin-1-

39

N-hydroxy-7-(3-(methoxyimino)-5-nitro-2-oxoindolin-1-

39

3.1.3.6. Tng hp N-hydroxy-7-(3-(methoxyimino)-5-methyl-2-oxoindolin-1-

40

yl)heptanamid (IVb)
3.1.3.3.

Tng

hp

yl)heptanamid (IVc)
3.1.3.4.

Tng

hp

yl)heptanamid (IVd)
3.1.3.5.

Tng

hp

yl)heptanamid (IVe)

yl)heptanamid (IVf)
3.1.3.7.

Tng

hp

N-hydroxy-7-(7-cloro-(3-methoxyimino)-2-oxoindolin-1-

40

yl)heptanamid (IVg)
3.2. KIM TRA TINH KHIT

41

3.3. XC NH CU TRC

42

3.3.1. Ph hng ngoi (IR)

42

3.3.2. Ph khi lng (MS)

43

3.3.3. Ph cng hng t ht nhn

43

3.3.3.1. Ph cng hng t ht nhn 1H-NMR

44

3.3.3.2. Ph cng hng t ht nhn 13C-NMR

45

3.4. KT QU TH HOT TNH SINH HC

47

3.4.1. Th tc dng c ch histon deacetylase

47

3.4.2. Kt qu th hot tnh khng t bo ung th in vitro

47

3.5. KT QU DOCKING

47

3.6. NH GI MC GING THUC CA CC CHT TNG HP

49

Chng 4. BN LUN

50

4.1. V HA HC

50

4.1.1. Tng hp 3-(methoxyimino)-2-oxoindolin v cc dn cht (IIa-g)

50

4.1.2. Phn ng tng hp dy ester trung gian (IIIa-g)

50

4.1.3. Phn ng tng hp dy cht acid hydroxamic (IVa-g)

51

4.2. V KHNG NH CU TRC

52

4.2.1. Ph hng ngoi

52

4.2.2. Ph khi lng

54

4.2.3. Ph cng hng t ht nhn

55

4.2.3.1. Ph 1H NMR

56

4.2.3.2. Ph 13C NMR

61

4.3. V HOT TNH SINH HC

64

4.3.1. V tc dng c ch HDAC

64

4.3.2. V tc dng khng t bo ung th in vitro

65

KT LUN V XUT

72

1. KT LUN

72

1.1. V tng hp ha hc v khng nh cu trc

72

1.2. V hot tnh sinh hc

72

2. XUT

72

TI LIU THAM KHO

PH LC PH

DANH MC CC CH VIT TT
AsPC-1

T bo ung th ty

13

Ph cng hng t ht nhn 13C

C-NMR

DCM

Dicloromethan

DMF

Dimethylformamid

DMSO

Dimethyl sulfoxid

FDA

Cc qun l dc phm v thc phm M

HAT

Histon acetyl transferase

HDAC

Enzym histon deacetylase

HDACIs

Cc cht c ch HDAC

Ph cng hng t ht nhn 1H

H-NMR

IC50

Nng c ch 50% s pht trin ca t bo

IR

Phng php ph t ngoi

LD50

Liu gy cht cho 50% s c th nghin cu

MCF-7

T bo ung th v

MeOH

Methanol

MS

Ph khi lng

NCI-H460

T bo ung th phi

NMR

Phng php ph cng hng t ht nhn

PC-3

T bo ung th tin lit tuyn

SAHA

Acid suberoylanilid hydroxamic

SW620

T bo ung th rut kt

THF

Tetrahydrofuran

TLC

Phng php sc k lp mng

TSA

Trichostatin A

DANH MC HNH V
Tn hnh

Trang

Hnh 1.1: S cu to nucleosom

Hnh 1.2: Cu to trung tm hot ng ca HDAC nhm I, II, IV

Hnh 1.3: Vai tr ca HDAC trong sinh l t bo ung th

H h 1.4: HDACIs c cu trc hydroxamat

10

Hnh 1.5: Cc dn cht th 2 ca SAHA

12

Hnh 1.6: Cc dn cht -alkoxy ca SAHA

12

Hnh 1.7: Cc dn cht 7-aminosuberoylamid hydroxamic acid

13

Hnh 1.8: SAR ca cc dn cht acid hydroxamic hng c ch HDAC

14

Hnh 1.9: Cc dn cht amid ngc ca SAHA

14

Hnh 1.10: Cc dn cht c gn thm O, S vo cu ni ca SAHA

16

Hnh 1.11: Cc dn cht phenyl-hydroxamic tng t SAHA

16

Hnh 1.12: Cc aryl-hydroxamic tng t SAHA

17

Hnh 1.13: Cc dn cht acid biphenyl-hydroxamic

17

Hnh 1.14: Cc acid phenylthiazol hydroxamic tng t SAHA

18

Hnh 1.15: Mt s acid phenylthiazol hydroxamic

19

Hnh 1.16: Dn cht acid phenylisoxazol-hydroxamic WR3018049

19

Hnh 1.17: Dn cht 1,3,4-thiadiazol hydroxamic acid

19

Hnh 1.18: Cc dn cht 5-phenyl-1,3,4-thiadiazol hydroxamic

20

Hnh 1.19: Cc dn cht 5-phenyl-1,3,4-oxadiazol hydroxamic

20

Hnh 1.20: Cu trc N1-(2,5-dimethoxyphenyl)-N(8)-hydroxyoctandiamid

21

Hnh 1.21: Cu trc cc hydroxamic tng t SAHA vi nhm kha hot ng

22

benzothiazol
Hnh 1.22: Cu trc cc hydroxamic tng t SAHA vi nhm kha hot ng

22

5-phenyl-1,3,4-thiadiazol
Hnh 1.23: Cu trc ca cc dn cht 3-oxim-isatin

24

Hnh 3.1: Kt qu phn tch Western blot ca cc cht IVa g

47

Hnh 3.2: Kt qu docking ca cht IVa v SAHA vi HDAC8

48

Hnh 3.3: Kt qu docking ca cht IVa v SAHA vi HDAC2

48

Hnh 4.1: Hin tng h bin ca nhm chc hydroxamic

53

Hnh 4.2: Ph hng ngoi ca cht IVa

54

Hnh 4.3: Ph khi lng ca cht IVa

55

Hnh 4.4: Ph 1H NMR dn rng ca IVa

57

Hnh 4.5: Ph 1H NMR dn rng ca IVg

58

Hnh 4.6: Ph cng hng t 1H NMR ca 19e

59

Hnh 4.7: Ph cng hng t 1H NMR ca IVe

59

Hnh 4.8: a, Ph 13C NMR ca cht IVf.

63

b, Ph 13C NMR dn rng ca cht IVf

Hnh 4.9: Kt qu phn tch Western blot ca 2 dy cht 19a-g v IVa-g

64

Hnh 4.10: Biu so snh tc dng khng t bo ung th in vitro ca cc dn

67

cht IVa-g

DANH MC BNG
Tn bng

Trang

Bng 1.1: Phn loi cc cht c ch HDAC

Bng 1.2: Kh nng c ch ca mt s HDACIs trn HDAC nhm I, II, IV

Bng 1.3: Hot tnh c ch HDAC v kt qu th hot tnh khng t bo ung th

13

in vitro ca cc dn cht 7-aminosuberoylamid hydroxamic acid

Bng 1.4: Tc dng khng cc t bo ung th in vitro ca N25

21

Bng 1.5: Kt qu th hot tnh khng t bo ung th in vitro v tc dng c ch

23

enzym HDAC ca cc cht 18a-d

Bng 3.1: Kt qu tng hp 3-(methoxyimino)-2-oxoindolin v dn cht

35

Bng 3.2: Kt

7-(3-(methoxyimino)-2-oxoindolin-1-

38

Bng 3.3: Kt qu tng hp N-hydroxy-7-(3-methoxyimino-2-oxoindolin-1-

40

qu

tng

hp

ethyl

yl)heptanoatv dn cht

yl)heptanamidv dn cht
Bng 3.4: Gi tr Rf v Tnc ca cc cht IVa-g

41

Bng 3.5: Kt qu phn tch ph IR ca cc cht IVa-g

42

Bng 3.6: Kt qu phn tch ph khi lng ca cc cht IVa-g

43

Bng 3.7: Kt qu phn tch ph 1H-NMR ca cc cht IVa-g

44

Bng 3.8: Kt qu phn tch ph 13C-NMR ca cc cht IVa-g

46

Bng 3.9: nh gi mc ging thuc ca cc cht IVa-g theo quy tc

49

Lipinsky
Bng 4.1: Bng so snh ph cng hng t 1H NMR ca 2 cht 19e v IVe

60

Bng 4.2: Kt qu th hot tnh khng t bo ung th in vitro ca cc cht IVa-g

66

Bng 4.3: So snh tc dng khng t bo ung th ca 2 dy IVa-g v 19a-g

69

Bng 4.4: So snh gi tr logP ca cc cht 19a-g v IVa-g

70

DANH MC S
T s

Trang

S 1.1: Tng hp cc dn cht -alkoxy ca

24

S 1.2: Tng hp acid biaryl hydroxamic

25

S 1.3: Tng hp cc acid phenylthiazol hydroxamic

25

S 3.1: S phn ng tng hp cc dn cht mang khung 3-methoximisatin

S 3.2: S phn ng tng hp cc cht IIa - g

33

S 3.3: S phn ng tng hp cc cht IIIa - g

36

S 3.4: S phn ng tng hp cc cht IVa-g

38

S 4.1: C ch phn ng th i nhn alkyl to IIIa-g

50

S 4.2: Phn ng th i nhn acyl

51

S 4.3: C ch phn ng tng hp acid hydroxamic IVa-g t ester

51

33

T VN
Ung th l bnh kh c th iu tr khi mc d c nhng tin b vt
bc trong y hc trong hai thp k qua. Theo c tnh v thng k ca t chc Y t
Th gii (WHO), hng nm c khong 9 10 triu ngi mc ung th mi v mt
na trong s cht v cn bnh ny. Ch tnh ring nm 2012 c 8,2 triu ngi
cht v ung th, y tr thnh cn bnh gy t vong nhiu nht trn Th gii [51].
gim thiu t l t vong trong ung th, vic phng v iu tr ung th
ng vai tr v cng quan trng. Vi nhng tin b trong y hc, di truyn hc v
sinh hc phn t th k 21, c bit khi tm ra bn gen ngi, iu tr ung th
c nhng bc tin mi trong phu thut, x tr, iu tr ha cht v gn y nht l
iu tr ung th hng ch. Phng php iu tr ny c hiu qu hn v t gy c
hn so vi cc phng php iu tr ung th c trc . Mc tiu phn t trong
iu tr ung th hng ch bao gm cc enzym c hiu, protein hoc th th khc
nhau c lin quan n s pht trin t bo ung th, v d nh: proteasome,
telomerase, histon deacetylase, hoc protein kinase,... [12]. Nghin cu thuc iu
tr ung th hng ch hin nay ch yu giai on tin lm sng, mt s ang
trong giai on lm sng v s t c FDA ph duyt a vo iu tr. Trong s
, histon deacetylase (HDAC) l mc tiu phn t em li nhiu kt qu kh quan
khi nghin cu, thit k v th nghim lm sng cc thuc tc dng hng c ch
enzym ny [5, 8, 33, 50]. Acid suberoylanilid hydroxamic (Vorinostat, Zolinza)
l cht c ch HDAC u tin c FDA cp php trong iu tr u lympho t bo
T di da [37]. Bn cnh , mt s cht c ch HDAC khc cng c nghin
cu v ang a vo th nghim lm sng nh NVP-LAQ824, MS-275,
cyclodepsipeptid FK-228,... ng ngc nhin, cc cht c ch HDAC th nghim
c hiu qu iu tr cao v c tnh thp i vi t bo lnh tnh [33], v th hin
nay cht c ch HDAC tr thnh mc tiu hp dn v mang y tnh kh quan trong
cng cuc nghin cu tm ra cht chng ung th ca cc cng ty dc phm v cc
t chc chnh ph.

Ti Trng i hc Dc H Ni, chng ti tin hnh thit k cng thc

v tng hp cc cht c ch HDAC. Trong cc cht tng hp, nghin cu v
hot tnh sinh hc ca mt s dn cht acid hydroxamic hng c ch histon
deacetylase cho thy c hot tnh tt trn t bo ung th trong th nghim in
vitro, c bit l mt s dy cht mang khung benzothiazol [1, 40, 41], 3-oximisatin [4]. Vi mong mun em li nhiu ng vin lm sng tm ra thuc iu
tr ung th mi, theo hng nghin cu ny, chng ti tin hnh thc hin ti:
Tng hp v th tc dng sinh hc ca mt s acid hydroxamic mang khung
3-methoxim-isatin hng c ch histon deacetylase vi mc tiu:
1. Tng hp N-hydroxy-7-(3-(methoxyimino)-2-oxoindolin-1-yl)heptanamid v
mt s dn cht.
2. Th tc dng c ch histon deacetylase v hot tnh khng t bo ung th in
vitro trn mt s dng t bo ung th ca cc cht tng hp c.

Ch g 1. TNG QUAN
1.2.

HISTON DEACETYLASE (HDAC)

1.2.1. Khi nim HDAC

Histon mt phn ca cu trc nhim sc th, l cc protein c bn giu acid
amin nh lysine, arginin, c chia thnh 5 nhm chnh (H1, H2A, H2B, H3, H4)
[26]. Cc cp histon li (H2A, H2B v H3, H4) c 2 phn quan trng: ui C nm
bn trong li v u N nm bn ngoi nucleosom. Phn u N tn ca histon, c
bit H3, H4 l ni din ra rt nhiu qu trnh bin i khc nhau trong phin m nh
acetyl ha/deacetyl ha lysin, methyl ha lysin v arginin, phosphoryl ha serin v
ubiquinin, sumoyl ha lysin [52].

Hnh 1.1. S cu to nucleosom [52]

Histon c th tn ti mt trong hai dng i lp nhau l acetyl ha hoc
deacetyl ha. Cc enzym ng vai tr trong s chuyn i ny l histon acetyl
transferase (HAT) v histon deacetylase (HDAC) [13, 50]. Histon acetyl
transeferase (HAT) xc tc chuyn nhm acetyl t acetyl coenzym A n lin kt
vi nhm -amino ca lysine phn u N ca histon, s chuyn i ny xy ra
nhiu hn trn histon H3 v H4. S acetyl ha histon lm tho xon nhim sc th
bng cch trung ha in tch dng ca phn u N ca histon, do vy lm gim i

lc ca histon vi phn in tch m trn ADN [45]. Ngc li, histon deacetylase
(HDAC) xc tc vic loi b nhm acetyl ca lysin phn u N ca histon, dn
n nhim sc th b ng xon v c ch qu trnh phin m [18].
1.1.2. Phn loi HDAC
HDAC c bo tn trong qu trnh tin ha v biu hin trong t chc ca
cc sinh vt t n bo nguyn thy cho ti loi ngi. Hin nay, 18 HDAC c
tm thy v chia thnh 4 nhm: I, II, III v IV [13, 35].
HDAC nhm I, II, IV l nhng enzym ph thuc Zn2+ v b c ch bi cc
cht to phc chelat vi Zn2+ [50], khc vi cc HDAC nhm III c c ch hot
ng ph thuc cofactor NAD+. Cc HDAC u c trung tm hot ng gm 2
phn chnh: ion Zn2+ l coenzym ca cc HDAC v knh enzym dng ti hnh ng.
Cu trc ti rt linh ng, c th bin i ph hp vi chiu di ca cc c cht
khc nhau. Trn ming ti c 1 vnh nh c to nn t 1 vi vng xon protein,
phn vnh ny s tng tc vi nhm nhn din b mt ca HDAC [25, 49].

Hnh 1.2. Cu to trung tm hot ng ca HDAC nhm I, II, IV [25]

(Ion Zn2+ biu th l hnh trn mu tm)
HDAC khng ch iu ha cc protein histon m rt nhiu protein khng
histon cng b nh hng bi hot tnh ca cc HDAC. Thut ng cc cht c ch
HDAC ch cc cht c kh nng c ch HDAC kinh in nhm I, II v IV [9]
mc tiu phn t m cc nghin cu iu tr ung th hng ch ang tin hng
n.

1.1.3. Mi lin quan gia u g th v s hot ng bt thng ca HDAC

Hot ng ca HDAC nh hng ti qu trnh acetyl ha histon. Do , s
mt cn bng trong hot ng ca enzym ny c th dn ti nhng thay i trong
cu trc ca NST v s ri lon iu ha phin m cc gen tham gia vo iu khin
chu trnh t bo, phn ha v/hoc gy cht t bo, do dn n ung th [13].
S gia tng bt thng ca HDAC1 v/hoc HDAC2 v/hoc HDAC6 c
quan st trong mt s bnh ung th tng c nh ung th tin lit tuyn, ung th d
dy, trc trng, ung th v v ung th no cng nh cc bnh l c tnh v mu
(bnh bch cu ty bo cp, bch cu t bo B, bnh u lympho t bo T ngoi vi,
bnh u lympho t bo B) v bnh u lympho da t bo T. Vic tm ra cc c cht ca
HDAC l cc protein nh p53, GATA1, GATA2, E2F, Rb, Bc16, Gli1, lin quan
n xu hng gy ung th v tin trin ca bnh ung th khng nh vai tr ca
HDAC trong ung th [16], chng c 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 trnh k c s xm ln, s di chuyn v s to mch [20,
28, 48, 50].

Hnh 1.3. Vai tr ca HDAC trong sinh l t bo ung th [50]

Nh vy c ch qu trnh phin m c iu ha bi s gia tng HDAC v
c th kim sot ung th bng cch c ch hot ng ca HDAC, chnh iu ny
m ra tng lai mi ha hn hn cho iu tr ung th hng ch.

1.2. CC CHT C CH HDAC (HDACIs)

1.2.1. Phn loi cc cht c ch HDAC
Trichostatin (TSA) l dn cht hydroxamat t nhin u tin c tc dng c
ch trc tip HDAC c Yoshida v cng s pht hin ra nm 1990 vi tc dng
chng nm. Sau , da vo nhng hiu bit v mi lin quan gia HDAC v ung
th ng thi xc nh c cu trc 3D ca cc HDAC, mt s dn cht c ch
HDAC c nghin cu v th nghim lm sng ng dng trong iu tr ung
th [36]. Nm 2006 v 2009, SAHA (Vorinostat, Merck) v Depsipeptid
(Istodax, Celgene) c FDA cp php dng trong iu tr u lympho t bo T
di da.
Cho ti nay, nhiu HDACIs c tm thy c ngun gc t nhin hoc tng
hp vi cu trc a dng. Da vo nhng cu trc ha hc ny, HDACIs c chia
thnh 5 nhm: cc acid hydroxamic, cc peptid vng, cc acid bo, benzamid v cc
dn cht ceton (bng 1.1).
Mi nhm HDACIs c nhng hn ch ring.Cc acid hydroxamic l nhng
cht b chuyn ha nhanh v c ch khng chn lc cc HDAC. Cc benzamid v
cc acid bo c hiu lc hn ch. Dn cht ceton d b kh ha trong huyt tng.
Trong khi cc peptid vng c cu trc phc tp, kh to thnh v mt ha hc, gy
ra s chy mu kh cha v FK-228 trong cu trc c mt phn lin kt vi ion
Zn2+ c cha lu hunh khng mong mun [24].
Khi nghin cu v hot tnh ca HDACIs trn cc HDAC kinh in, cc nh
khoa hc nhn thy hot tnh sinh hc ca cc HDACIs ph thuc vo kh nng
lin kt vi ti enzym v kh nng to phc vi ion Zn2+ phn y knh ca cc
cht ny. Do HDAC c bo v trong ti enzym, hu ht cc HDACIs u khng
th c ch chn lc ring mt HDAC no, chng c th c ch tt c cc HDAC
hoc c ch ng thi nhiu thnh vin HDAC khc nhau (bng 1.2).

Cht

Bng 1.1.Phn loi cc cht c ch HDAC [18]

Cu trc
Cht
Cu trc
Cc acid hydroxamic
O

TSA
(Trichostatin A)

O
NHOH

Oxamflatin

NHOH

H
N

O
S

Acid
suberoylanilid
hydroxamic
(SAHA)

OH

O
H
N
NHOH

O
H
N

NVP-LAQ824

NHOH
N

HN

CBHA (Acid mcarboxycinnamic

bishydroxamid)

Acid
sulfonamid
hydroxamic

Scriptaid
Peptid vng
O

Depsipeptid
(FK-228)

HN
O

CH3
O

H
N

NH

S
S

CH3
CH3

H3C

O
HN

CH3
O

Acipidin
CHAP
Benzamid
O

MS-275

N
H

H
N

NH2

CI-994

Cc acid bo
O

Acid valproic

Phenyl
butyrat

OH

Cc dn cht ceton
Trifluoromethyl
ceton

Alphacetoamid

ONa
O

Bng 1.2. Kh nng c ch ca mt s HDACIs trn HDAC nhm I, II, IV [50]

Nhm I

HDAC10

HDAC11

nd

nd

nd

NVP-LAQ824

nd

nd

nd

Panbinostat

nd

nd

nd

Belinostat

nd

nd

nd

nd

Cht c ch
khng chn lc

Depsipeptid

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

Apicidin
Valproic acid

nd

Trapoxin

nd

nd

SB-429201

nd

Bispyridinum diene

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

SHI-1:2
R306465

nd

SB-379278A

nd

nd

PCI-34051
Cpd2

nd

nd

APHA derivaties

nd

nd

nd

Tubacin

nd

nd

nd

NCT-10a/14a
Ghi ch:

nd

nd

MGCD0103

Mercaptoacetamide

HDAC6

Vorinostat (SAHA)

nd

HDAC9

nd

MS-275

HDAC7

nd

nd

HDAC5

HDAC4

HDAC8

HDAC3

HDAC2

HDAC1

nd

PCI-24781

Cht c ch nhm I

Nhm IV

TSA

HDACIs

Cht c ch
nhm II

Nhm II
B

Nhm II A

nd
nd nd
c ch mnh
c ch yu

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd
nd

nd

nd

nd

nd

nd

nd
nd

nd
nd
nd
Khng c ch
nd: khng c d liu cng b

Trong s cc HDACIs, nhiu cht ang c nghin cu v th nghim lm

sng pha I, II hoc III trn cc i tng bnh nhn ung th mu, ung th th rn
hoc ung th cc c quan khc trong c th nh: Phenyl butyrate, SAHA, LAQ824,
Acid valproic, PXD101, ITF-2357, Depsipeptid, MS275, CI-994, Pyroxamid,
[36]. Nhng d liu bc u ca cc th nghim lm sng cho thy, HDACIs c
hiu qu tt trong iu tr ung th v c tnh thp i vi cc t bo lnh tnh. Cc

u im trong lm sng ny khin HDACIs tr thnh mc tiu phn t y ha hn

cho tng lai iu tr ung th v l hng nghin cu kh quan cho cc nh khoa
hc.
1.2.2. C ch tc dng ca cc cht c ch HDAC
C nhiu bng chng cho rng HDACIs c tc dng chng ung th do tc
ng ln nhiu giai on quan trng ca chu trnh t bo, lm mt s iu ha trong
t bo c tnh. Trong , cc c ch chnh quyt nh hot tnh chng ung th ca
HDACIs l thc y s bit ha, c ch chu trnh t bo v gin tip gy ra s cht
t bo theo chng trnh. Ngoi ra, HDACIs cn c ch s pht trin ca khi u
bng cch ngn cn cung cp oxy v cht dinh dng, t c ch qu trnh to
mch ca khi u, lm gim kh nng hnh thnh v sng st ca khi u; ng thi
lm tng s nhn dng v hot ha ca t bo min dch, ngn cn r rt s pht
trin khi u ban u v s di cn [5, 26].
1.2.3. Cu trc ca cc cht c ch HDAC
Cc cht c ch HDAC c chia thnh nhiu nhm khc nhau, nhng cu
trc u c cc phn chnh sau [2, 16, 38]:

CAP (C)

Cu ni (B)

Nhm gn
Zn2+ (A, ZBG)

Trong :
-

Nhm kho hot ng (capping group) hay vng nhn din b mt (C):

thng l vng thm hoc peptid vng, nm trn b mt ca enzym.

-

Cu ni s nc (B): thng l cc hydrocarbon thn du, nm trong lng

mch enzym, quyt nh s ph hp v cu trc ca cc cht vi chiu di knh

enzym.
-

Nhm gn vi Zn2+ trn v tr tc dng ca cc enzym HDAC (A) nh acid

hydroxamic, cc thiol, benzamid, mercaptoceton... quyt nh tnh c hiu v hiu

lc tc dng ca cc HDACIs.
Cu trc tinh th kt tinh ca cc HDAC cho thy phn B, C v mt phn
ca A nm trong ti enzym, lm y khong trng trong lng knh enzym. Phn
9

cn li ca A tng tc vi phn vnh trn b mt ming ti enzym. Vic nghin

cu thit k cng thc cho cc HDACIs mi u da trn cu trc c in ny.
1.3. TNH HNH NGHIN CU TNG HP CC ACID HYDROXAMIC
HNG C CH HDAC HIN NAY
1.3.1. Cc nghin cu tng hp cc acid hydroxamic trn th gii
Nm 1986, TSA c Morioka v cng s chng minh tc dng rt tt
trong vic lm gim s bit ha t bo trong bnh bch cu Friend v c ch chu
trnh t bo bnh thng c pha G1 v G2 [53]. Bn cnh , TSA c tc dng c
ch mnh, c hiu vi HDAC (Ki = 3,4nM) [46, 48] v ng vai tr nh cht ngn
cn s di cn trong ung th i trng. Tuy nhin, hin nay TSA ch yu dng lm
cht i chiu trong nghin cu tm ra cc cht c ch HDAC mi [46] do vic sn
xut TSA rt tn km m li khng hiu qu (tri qua 20 bc v hiu sut 2%).
Nm 2006, sau khi tri qua cc pha trong tin trnh th nghim lm sng, SAHA
(Vorinostat, Zolinza) c FDA ph duyt s dng trong iu tr u lympho da
t bo T, c chng minh nhy cm vi cc dng t bo Hut78, HH, M [53].
Ngoi TSA, SAHA, nhiu dn cht acid hydroxamic c ch HDAC khc
ang c nghin cu v chia thnh nhiu phn nhm nh: acid hydroxamic mch
thng, dn cht cinnamic, dn cht phenyl... (hnh 1.4).

H h 1.4. HDACIs c cu trc hydroxamic

10

Cho ti nay, dn cht acid hydroxamic tr thnh nhm cht c ch HDAC

c nghin cu rng ri nht, vi nng c ch nm trong khong micromol n
nanomol. Ging nh cc HDACIs khc, cu trc ca cc dn cht acid hydroxamic
cng gm 3 phn chnh: Nhm kha hot ng (C), cu ni s nc (B) v nhm
gn Zn2+(Zinc Binding Group, ZBG) l acid hydroxamic. Nhiu nghin cu thay
i nhm gn Zn2+ ny bng nhiu nhm chc khc vi mong mun tm ra nhiu
cht c ch HDAC c hiu lc. Tuy nhin,khi thay nhm chc hydroxamic bng
mt s nhm chc nh sulfonamid, thiocarbonat, dithiocarbonat v trithiocarbonat,
hot tnh c ch HDAC ca cc cht tng hp c u gim so vi SAHA, thm
ch c cht cn khng c hot tnh [17, 24, 34]. Nh vy c th thy, cc acid
hydroxamic l nhm c cu trc n gin, hot tnh c ch HDAC mnh do rt d
to phc vi ion Zn2+. Tuy nhin, cng v nhm chc acid hydroxamic d to phc
vi ion Zn2+ ca HDAC nn cc dn cht ny c ch khng chn lc c HDAC
nhm I, II. Thm vo , chng b chuyn ha nhanh nn na i in vivo ngn [21,
38]. Chnh v vy, rt nhiu nhm nghin cu trn th gii ang n lc tm kim cc
dn cht mi da trn phin mu ca TSA, SAHA v cc dn cht acid hydroxamic
tm c trc nhm tm ra s lin quan v cu trc tc dng ca cc dn cht
ny, phc v cho vic nghin cu thit k v tng hp cc ng vin lm sng mi
cho iu tr ung th. Mt s nghin cu trn th gii tin hnh thay i cu trc
cu ni v nhm nhn din b mt ca SAHA v thu c cc kt qu di y.
1.3.1.1. Thay i cu ni
Nhm hiu r hn v vai tr ca cu ni trong cu trc hot ng ca cc
dn xut acid hydroxamic tng t SAHA, nhm nghin cu do Anton V.
Bieliauskas (M) ng u thit k v tng hp cc hydroxamic khc nhau bng
cch gn thm cc nhm th vo nguyn t C lin k vi nhm chc acid
hydroxamic ca SAHA (hnh 1.5). Kt qu khi th hot tnh khng t bo ung th
in vitro cho thy cc dn cht thu c c hot tnh gim 800 1000 ln (IC50 = 3,2
- 226 M) so vi SAHA (IC50 = 0,09 M) trong cng th nghim. iu ny cho
thy vng khng gian tham gia to phc vi nhm chc acid hydroxamic trong

11

trung tm hot ng ca cc HDAC c kch thc rt hn ch v cc nhm th v

tr gn vi nhm chc ny s lm gim hot tnh ca cc HDACIs [14].

Hnh 1.5. Cc dn cht th 2 ca SAHA

Phn tch cu to trung tm hot ng dng ti ca HDAC, nhm nghin cu
thuc trung tm nghin cu Sigma-Tau (Pomezia, ) c bit ch phn ming ti
vi cc vng acid amin c th tham gia nhiu tng tc quan trng vi nhm nhn
din b mt hoc cc nhm ln cn. Da trn c s ny, nhm nghin cu thit
k v tng hp dy dn cht -alkoxy ca SAHA (hnh 1.6) [23]. Kt qu sng lc
hot tnh c ch HDAC2 cho thy tt c cc dn cht -alkoxy (dng racemic) ca
SAHA u cho gi tr IC50 mc rt thp (0,05-0,5 M), nhiu cht tc dng tng
ng SAHA. Kt qu ny cho thy cc nhm th -alkoxy c hiu qu tng
cng tc dng sinh hc cho cc dn cht ny, c th do chng to thm cc
tng tc vi cc aminoacid thuc vng mp ti ti trung tm hot ng ca enzym,
ng thi cc dn cht -alkoxy c cu ni vi chui alkyl c 6C cho hot tnh tt
hn so vi cu ni c chui alkyl 5C. ng ch l cu hnh ca nhm chc alkoxy khng nh hng n hot tnh sinh hc.

Hnh 1.6. Cc dn cht -alkoxy ca SAHA [23]

Mt hng thay i cu ni ca SAHA c thc hin bi cc Maurizio
Taddei v cng s (Italia) bng cch gn cc lactam-carboxyamid vo C7 trong
chui alkyl ca SAHA (hnh 1.7) thu c kt qu rt ng lu . Vic gn
lactam vo cu ni ny tng cng ng k tc dng khng t bo ung th in
vitro. Nhiu dn cht c to ra c kh nng c ch HDAC nhm I, II B v IV vi

12

IC50 mc nanomol, thp hn SAHA hng trm ln, in hnh l 2 dn cht 3a, 3b
(bng 1.3) [39].

Hnh 1.7. Cc dn cht 7-aminosuberoylamid hydroxamic acid

Kt qu trn cho thy s c mt ca vng amid trn C7 ca cu ni alkyl gn
vi nhm kha hot ng c nh hng c bit ln i lc vi enzym. C th s
xut hin ca vng amid lm tng thm tng tc vi cc acid amin li vo
trung tm hot ng ca HDAC em li hot tnh c ch HDAC mnh cho cc dn
cht ny. iu ny c minh chng khi tip tc nghin cu tc dng khng t bo
ung th in vitro trn dng t bo H460, cc dn cht 7-aminosuberoylamid
hydroxamic acid cho hot tnh rt mnh, c bit cc cht 3a, 3b c IC50 = 0,5 M,
mnh hn SAHA 6 ln (bng 1.3).
Bng 1.3. Hot tnh c ch HDAC v kt qu th hot tnh khng t bo ung th in
vitro ca cc dn cht 7-aminosuberoylamid hydroxamic acid [39].
Cht

HDAC (IC50, nM)

IC50, M

HDAC1 HDAC2 HDAC3 HDAC8 HDAC6 HDAC10 HDAC11

H460

SAHA

258

921

350

243

29

456

362

3,4

3a

32,9

10,6

55,5

0,5

3b

27

11

52

30,3

15,5

0,5

T kt qu trn c th nhn thy 3a (ST8078AA1) l mt cht dn ng

hp dn, vi cc bin i n gin nhng to c c mt cht khng ung th
mi hng c ch HDAC y trin vng.
Da trn cu trc ca SAHA, Andrianov Victor v cng s thit k hng
lot cc dn cht hydroxamic c cu trc amid hng c ch HDAC nhm kho st
13

lin quan v mt cu trc tc dng ca cc dn xut ny. Nhm nghin cu thay

i cu trc nhm nhn din b mt ca cc HDACIs ny bng mt lot cc aryl rt
a dng, ng thi thay i chiu di ca cu ni s nc theo s nhm CH2 v
thu c kt qu v s lin quan cu trc tc dng ca cc dn cht ny nh sau:
Lin kt amid quan
trng nhng khng phi
l then cht

Acid hydroxamic cn
thit gn kt Zn2+
v hot tnh

di mch ti
u l 5 6 carbon

Cc hp cht thm tt
hn, c bit l cc lin
kt khng no

Hnh 1.8. SAR ca cc dn cht acid hydroxamic hng c ch HDAC [11]

Nhm nghin cu thuc cng ty TopoTarget (Anh) thit k v tng hp
gn 40 dn cht amid ngc ca SAHA (hnh 1.9) [10]. Nhiu cht trong s ny c
hot tnh c ch HDAC tng ng, thm ch mnh hn SAHA.

Hnh 1.9. Cc dn cht amid ngc ca SAHA

Trong cc dn cht ngc ca SAHA, 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. Lin quan cu trc - tc dng rt ra c t cc dy cht ny cho thy
nhng c im tng t SAHA, l: i) nhm acid hydroxamic l cn thit cho
hot tnh c ch HDAC mnh; ii) cu ni 5-6 C l ti u; iii) lin kt amid c vai tr
trong tng tc vi trung tm hot ng song c th thay i; iv) phn Ar l nhn
thm cho hot tnh mnh hn, nu c cu ni gia nhn thm vi nhm amid th
cu ni khng no tt hn [10].
14

Ngoi vic tin hnh gn thm cc nhm th, nhiu nh khoa hc cng tin
hnh gn thm cc nguyn t nguyn t khc C nh O, S,vo chui alkyl trong
cu ni ca SAHA (hnh 1.10) v tin hnh kho st hot tnh ca cc dy dn cht
ny. Hu ht hot tnh c ch HDAC ca cc dn cht ny u gim so vi SAHA
trong cng th nghim, c th nh cc nghin cu ca nhm Soon-Ai Kim v Dong
Hoon Kim (Hn Quc).
Nhm nghin cu ca Soon-Ai Kim nghin cu thit k v tng hp cc
dn cht oxo-SAHA (5) vi nguyn t O c thm vo phn cu ni alkyl ca
SAHA (hnh 1.10). Kt qu th hot tnh c ch enzym cho thy tt c cc cht tng
hp u c tc dng c ch HDAC vi IC50 = 0,64 37,69 M, thp hn so vi
SAHA trong cng th nghim [29].
Nhm nghin cu ca Dong Hoon Kim thay th nhm amid trong cu ni
ca SAHA bng nguyn t S, thu c N-hydroxy-7-(2-naphthylthio) heptanomide
(HNHA) (hnh 1.10) c hot tnh c ch HDAC in vitro mnh vi IC50 = 0,1 M,
nhng vn ch bng mt na hot tnh so vi SAHA (IC50 = 0,05M) [27]. Ngoi
ra, khi thay th S trong HNHA bng O hoc N vi chiu di ca chui alkyl l 5C
hoc 7C hot tnh c ch HDAC ca cc cht u gim [27].
Charles Marson v cc cng s (Anh) tin hnh tng hp cc dn cht
aryloxyalkanoic acid hydroxamic (7) (hnh 1.10) vi mong mun c th lm tng
kh nng lin kt vi ming ti ca knh enzym nh lin kt hydro ca O nm gia
vng thm v chui alkyl vi cc acid amin phn vnh ming ti, qua lm tng
hiu lc ca cc HDACIs hng c ch HDAC. Kt qu thu c nhiu dn cht c
hot tnh in vitro mnh, thm ch mnh hn c Trichostatin A v SAHA trong cng
th nghim vi IC50 = 0,9 - 70 nM, hot tnh mnh nht l 7a (IC50 = 0,9nM) [38].

15

Hnh 1.10. Cc dn cht c gn thm O, S vo cu ni ca SAHA

1.3.1.2. Thay i nhm nhn din b mt
Phn nhn din b mt ti trung tm hot ng ca HDAC chp nhn mt h
vng kh a dng, t indol, benzofuran, quinolin, benzodioxol Nhm Nit trong
cu trc ca phn nhn thm R c th to lin kt Hydro vi cc nhm aminoacid
trong cu trc ti, tng tng tc vi mp ti ti trung tm hot ng ca HDAC, do
tng hot tnh ca cc cht c ch HDAC [23]. V vy, cc nghin cu kho st
dn cht c ch HDAC bng cch thay i nhm kho hot tnh (thay th v tr
phenyl bng cc nhn thm nh benzothiazol, phenylthiazol, isoxazol...) trong
SAHA c tnh kh quan cao. Mt s nghin cu trn th gii v thay i nhm kha
hot ng ca dy cht ny thu c nhng kt qu bc u.
Cc acid phenyl-hydroxamic tng t SAHA
Mt lot cc dn cht thay i nhm kha hot ng ca SAHA bng cch
thay i v tr ca cc nhm th khc nhau trn khung phenyl ca SAHA c
Chanaz Salmi Smail v cc cng s (Php) nghin cu thit k tng hp (hnh
1.11). nh gi hot tnh khng ung th in vitro trn cc dng t bo ung th
ngi ca cc dn cht ny, c nhiu cht c hot tnh tng ng vi SAHA,
thm ch mnh hn SAHA ti 10 ln nh 8a [47].

Hnh 1.11. Cc dn cht phenyl-hydroxamic tng t SAHA

T kt qu nghin cu, nhm nghin cu nhn thy bn cht nhm th ht (NO2, -CN, -F) hay y in t (-NH2, -NMe2, -OH) trn khung phenyl khng nh
16

hng ti hot tnh ca cc cht, tuy nhin nhm th v tr ortho lm gim hot
tnh ca cc cht ny. iu ny chng t s cn tr v cu trc khng gian v bn
cht s nc ca cc nhm th l yu t ch yu nh hng ti hot tnh khng
HDAC ca cc dn cht 8. Vi cn c ny, tng kch thc vng s nc, nhm
nghin cu thay i nhm phenyl ca SAHA bng cc h vng thm khc nhau
v thu c dn cht 9a (hnh 1.12) c hot tnh khng HDAC in vitro mnh hn so
vi SAHA (EC50 = 1,2 M) [47].

Hnh 1.12. Cc aryl-hydroxamic tng t SAHA

Hai cht 8a v 9a c nh gi c tnh trn cc t bo ung th ca bnh
nhn ung th bch cu v cc t bo mu ngoi vi ca ngi tnh nguyn khe
mnh so snh vi SAHA trong cng th nghim. Kt qu cho thy 9a th hin
kh nng iu tr tt hn so vi 8a v SAHA. V th, cng vi SAHA, cc cht ny
c tip tc nh gi hot tnh khng HDAC in vivo trn dng t bo ung th bch
cu chut khi cho dng thuc bng ng ung. y l mt trong nhng ng vin
kh quan tip tc nghin cu su hn trong iu tr ung th, c bit l ung th
bch cu [47].
Cc acid biphenyl-hydroxamic tng t SAHA
Nhm nghin cu ca Alan P. Kozikowski tin hnh thit k v tng hp
mt dy cc dn cht acid biphenyl-hydroxamic tng t SAHA (hnh 1.13).

Hnh 1.13. Cc dn cht acid biphenyl-hydroxamic

Kt qu cc dn cht biphenyl c ch HDAC mnh hn SAHA trn 6 loi
HDAC (HDAC1, 2, 3, 8, 6, 10) [30]. Khi gn thm nhm -NH2 vo v tr ortho trn
vng phenyl th 2 hot tnh gim, xong khi nhm -NH2 ny c acyl ha bng

17

nhng nhm aminoacyl cng knh, tc dng c ch HDAC li c tng cng.

iu ny chng t phn nhn din b mt ca trung tm hot ng ca HDAC c
th chp nhn nhiu nhm c kch thc ln. Kt qu ny cng gi cc tng tc
c to lp thm t nhng nhm aminoacyl ny vi cc acid amin ti vnh ca ti
hot ng lm tng i lc i vi HDAC ca cc dn cht.
Cc acid phenylthiazol-hydroxamic tng t SAHA
Cc nh khoa hc thuc Vin nghin cu qun i Walter Reed (M)
thit k v tng hp hng trm dn cht acid hydroxamic mang hp phn
phenylthiazol thay th vo v tr ca phenyl ca SAHA (hnh 1.14), trong c
nhiu cht c kh nng c ch HDAC tng ng hoc mnh hn SAHA [19].
Kt qu nh gi tc dng trn HDAC cho thy dn cht WR301801 (11a)
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 WR301801 l WR301826 (11b) (nhm th amino v tr ortho) cng c tc
dng mnh tng ng SAHA [19].

Hnh 1.14.Cc acid phenylthiazol hydroxamic tng t SAHA

S dng hai cht WR301801 v WR301826 lm nhng cht dn ng mi,
nhm nghin cu ca Alan P. Kozikowski thuc i hc Illinois (Chicago, M) tip
tc thit k dy acid phenylthiazol-hydroxamic dn cht ha da trn nhm amino
ca vng phenyl (hnh 1.15) [30]. Kt qu cho thy, khi nhm amin th trn vng
phenyl c acyl ha bng nhng nhm cng knh, tc dng c ch nhiu tp
HDAC tng. Tc dng mnh nht thu c vi dn cht 11d (hnh 1.15). IC50 ca
dn cht ny vi HDAC2, HDAC3 thp di mc 0,2 nM. Kt qu th c tnh trn
5 dng t bo ung th ty cng b vi 11a, 11b, 11c cho thy cc dn cht ny u
c IC50 nh hn 3 M [30].
18

Hnh 1.15. Mt s acid phenylthiazol hydroxamic

Cc acid isoxazol-hydroxamic tng t SAHA
Trong qu trnh nghin cu cc dn cht acid phenylthiazol-hydroxamic,
nhm nghin cu ca Alan P. Kozikowski thuc i hc Illinois (Chicago, M)
tng hp dn cht acid phenylisoxazol-hydroxamic WR3018049 (hnh 1.16). Dn
cht isoxazol ny c tc dng c ch cc HDAC1, 3 v 6 rt mnh vi IC50 thp n
0,002 nM [31].

Hnh 1.16. Dn cht acid phenylisoxazol-hydroxamic WR3018049

Cc dn cht 1,3,4-thiadiazol hydroxamic acid
Nhm nghin cu ca Peng Guan (Trung Quc) tin hnh tng hp cc
dn cht acid hydroxamic vi nhm kha hot ng l 1,3,4-thiadiazol (hnh 1.17).
Kt qu nh gi tc dng trn HDAC cho thy nhiu cht c kh nng c ch
mnh tng ng vi SAHA, thm ch mnh hn SAHA nh 12a vi IC50 = 0,089
M [22].

Hnh 1.17. Dn cht 1,3,4-thiadiazol hydroxamic

Gi nguyn nhm kha hot ng 5-phenyl-1,3,4-thiazol, nhm nghin cu
ca Harish Rajak (n ) thit k tng hp dy dn cht acid hydroxamic vi
cu ni 2-amino pyrimidin, thu c 8 dn cht c nhm th khc nhau v tr
2,3,4 trn nhm phenyl ca nhm kha hot ng (hnh 1.18). Tt c cc dn cht
tng hp u c hot tnh mnh hn hoc tng ng vi SAHA, c th nh 13a,
19

13b u c hot tnh c ch HDAC vi IC50 = 0,007 M; 13, 13c vi IC50 = 0,008
M [44].

Hnh 1.18. Cc dn cht 5-phenyl-1,3,4-thiadiazol hydroxamic

Kt qu docking ca cc dn cht 1,3,4-thiazol cho thy 2 nguyn t N trn
nhm kha hot ng ny to lin kt hydro vi cc acid amin phn vnh ca
ming ti enzym. iu ny chng t nhm kha hot ng c khung 1,3,4-thiazol
c kh nng tng tc rt mnh vi trung tm hot ng ca HDAC, lm tng hot
tnh ca cc cht.
Cc dn cht 1,3,4-oxadiazol hydroxamic acid
Trong qu trnh tng hp cc dn cht 1,3,4-thiazol, nhm nghin cu ca
Harish Rajak cng tin hnh tng hp cc dn cht 5-phenyl-1,3,4-oxadiazol tng
t (hnh 1.19). Kt qu nh gi hot tnh c ch HDAC cho thy cc dn cht ny
cho thy dy dn cht ny c kh nng c ch mnh t 0,006 0,017 M, trong
mnh nht l cht 14 v 14a vi IC50 = 0,006 M [44].

Hnh 1.19. Cc dn cht 5-phenyl-1,3,4-oxadiazol hydroxamic

Tng t nh nhm kha hot ng 5-phenyl-1,3,4-thiadiazol, nhm nghin
cu nhn thy nhm kha hot ng 5-phenyl-1,3,4-oxadiazol tng tc mnh vi
cc acid amin li vo trung tm hot ng ca knh enzym theo tng tc Van der
Waals, lm tng hot tnh c ch HDAC.

20

Gn y nht, Zhang Song cng cc cng s (Trung Quc) ng k bng

sng ch (CN 103159646) cho mt dn cht mi ca SAHA l N1-(2,5dimethoxyphenyl)-N(8)-hydroxyoctandiamid (N25) (hnh 1.20) [54].

Hnh 1.20. Cu trc N1-(2,5-dimethoxyphenyl)-N(8)-hydroxyoctandiamid

Khi kim tra hot tnh khng t bo ung th in vitro, N25 cho thy hot tnh
mnh hn SAHA trn cc dng t bo ung th phi H460, A549, H1299 v ung th
thn kinh m U251 (bng 1.4). Thm vo , khi th nghim trn chut, N25 thm
qua c hng ro mu no vi c tnh thp (LD50 = 240,84 mg/kg). iu ny m
ra hi vng mi cho iu tr ung th, c bit l ung th thn kinh m.

Cht
N25

Bng1.4. Tc dng khng cc t bo ung th in vitro ca N25 [54]

IC50SD (M)
U251

U87

T98G

H460

6,280,89 11,330,51 15,441,01 2,440,31

SAHA 6,321,27

6,410,08

10,042,00 5,160,97

A549

H1299

3,460,84 2,930,64
7,201,29 6,871,13

1.3.2. Cc nghin cu tro g c

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. Vit Nam, vn cha c nh khoa hc no quan tm n thit k, tng
hp cc cht c ch HDAC ngoi cc cng b m nhm nghin cu ti B mn Ha
dc Trng i hc Dc ang tin hnh. Nhiu kt qu c cng b trong
cc tp ch trong nc v quc t, cng nh trong cc kha lun thc s, Dc s i
hc.

21

Khi kho st hot tnh c ch HDAC ca cc cht c cu trc tng t

SAHA vi nhm kha hot ng khc l benzothiazol, tc gi o Th Kim Oanh
ch ra rng khi thay i nhm nhn din b mt l vng benzothiazol th di
cu ni c 6 carbon l ti u cho hot tnh. iu ny cng hon ton ph hp vi
mt s nghin cu trc y v di cu ni ca cc hydroxamat tng t SAHA.
Quan trng hn, kt qu c ch HDAC2 cn cho thy s c mt ca vng
benzothiazol cho tc dng tt hn. C 8 cht 16a-h c ch HDAC2 vi IC50 = 0,013
0,262 g/ml, cao hn c SAHA (IC50 = 0,530 g/ml) [6].

Hnh 1.21. Cu trc cc hydroxamic tng t SAHAvi nhm kha hot ng

benzothiazol [6]
Nghin cu thit k dy cc hydroxamic tng t SAHA vi nhm th l 5phenyl-1,3,4-thiadiazol (17) ca nhm nghin cu ti B mn cng thu c mt
s cht c trin vng trong pht trin thuc mi (hnh 1.22) [1, 2, 6, 7].

Hnh 1.22. Cu trc cc hydroxamic tng t SAHAvi nhm kha hot ng 5phenyl-1,3,4-thiadiazol
Khi th hot tnh khng t bo in vitro ca cc dn cht 5-phenyl-1,3,4thiadiazol (17), cht 17b th hin c tnh mnh trn c 5 dng t bo, c bit l
cht c hot tnh mnh nht trn 3 dng t bo SW620 (IC50 = 0,34 M), AsPC-1
(IC50 = 0,63 M), NCI-H460 (IC50 = 0,11 M) so vi cc dn cht cn li v c
hiu lc mnh gp 11, 6, 25 ln so vi SAHA trn cc dng t bo trn trong cng

22

th nghim. Ngoi ra, 17a c hot tnh mnh trn dng t PC-3 vi IC50 = 0,88
M, mnh gp 5 ln so vi SAHA; 17c c hot tnh mnh trn dng t bo MCF-7
vi IC50 = 0,73M, mnh gp 9 ln so vi SAHA. iu ny cho thy cc cht c
cu trc 1,3,4 thiadiazol gia vng phenyl v nhm amid cho kt qu c tnh
tt hn SAHA.
Kt qu ny to iu kin cho nhm nghin cu tip tc pht trin hng
nghin cu trong thit k cng thc mi bng vic gi nguyn cu trc acid
hydroxamic vi d vng 5 cnh 1,3,4-thiadiazol gia vng thm v nhm amid,
v thay vng phenyl bng d vng cha O hoc S, cc cht thu c cho nhng kt
qu kh quan v c tnh trn t bo v tc dng c ch HDAC, c bit l cht
18c c kh nng khng t bo ung th mnh gp 15 ln SAHA (bng 1.5) [1].
Bng 1.5. Kt qu th hot tnh khng t bo ung th in vitro v tc dng c ch
enzym HDAC ca cc cht 18a-d
IC50 (M)1

18a

Tc dng c
ch HDAC
+

18b

0,28

18c

0,25

18d

0,65

3,7

Cht

SAHA

0,29

Ch thch: Nng c ch 50% s pht trin ca t bo.

1

Gn y nht, khi kho st cc hydroxamic tng t SAHA vi nhm kha

hot ng l 3-oxim-isatin, cu ni 6C (hnh 1.23), nhm nghin cu B mn
thu c mt lot cc dn cht c hot tnh khng t bo ung th in vitro mnh hn
SAHA trn c 5 dng t bo SW620, MCF-7, PC-3, AsPC-1, NCI-H460. Cht 19a
c hot tnh mnh trn dng t bo AsPC-1 vi IC50 = 0,08 M, gp 46 ln so vi
SAHA trong cng t nghim [4].
Nh vy, nhm kha hot ng 3-oxim-isatin lm tng hot tnh ca cc
cht c ch HDAC tng t SAHA nh tng tng tc vi cc acid amin li vo
trung tm hot ng ca knh enzym.

23

Hnh 1.23. Cu trc ca cc dn cht 3-oxim-isatin [4]

Tip tc hng nghin cu ny, chng ti tin hnh kho st hot tnh ca
dy dn cht tng t SAHA vi cu ni 6 carbon nhng nhm kha hot ng l
3-methoxim-isatin nhm khai thc s khc bit phn ming ca knh enzym khi
thay i nhm nhn din b mt gia cc HDAC. Cn c vo cc kt qu nghin
cu , c th thit k cng thc lm tng hot tnh v tng kh nng c ch chn
lc, nhm a ra cc ng vin th tin lm sng v lm sng cho iu tr ung th.
Cho ti hin nay, cha c cng b no s dng 3-methoxim-isatin lm nhm kha
hot ng thay th cho phenyl trong SAHA, v vy nghin cu m chng ti tin
hnh trong lun vn l ph hp vi xu hng nghin cu trn th gii.
1.4. CC PHNG PHP TNG HP ACID HYDROXAMIC
1.4.1. Tng hp acid hydroxamic t ester
y l phng php c nhiu nhm nghin cu ng dng tng hp cc
acid hydroxamic. Hanessian v cng s [23], Andrianov v cng s [11], Marson
v cng s [38] u tng hp cc acid hydroxamic t dn cht methyl ester bng
cch cho phn ng vi dung dch hydroxylamin trong hn hp NaOH, MeOH
iu kin 0oC n nhit phng vi hiu sut kh cao (s 1.1).

S 1.1. Tng hp cc dn cht -alkoxy ca SAHA [23]

Ty thuc vo bn cht ca cht tham gia phn ng, iu kin phn ng v
dung mi c th thay i cho ph hp. Trong nghin cu tng hp cc acid
hydroxamic c cu ni cha vng triazol, Chen v cng s [15] tin hnh tng hp
acid hydroxamic t dn cht methyl ester vi dung dch hydroxylamin trong hn
hp KCN:THF (1:1) nhit phng, hiu sut phn ng cng rt cao (>80%).
24

1.4.2. Tng hp acid hydroxamic t acid carboxylic

Trong mt s trng hp, phn ng trc tip gia dn cht methyl ester v
hydroxylamin khng th xy ra. Khi , nhiu nhm nghin cu la chn phng
php s dng tc nhn hot ha acid carboxylic ng thi s dng hydroxylamin c
nhm bo v -OH tng kh nng phn ng vo nhm -NH2.
tng hp cc dn cht acid 5-pyridin-2-yl-thiophen-2-hydroxamic, Price
v cng s s dng tc nhn hot ha HATU to ester hot ha ngay trong
phn ng, ng thi s dng hydroxylamin tetrahydropyran. Sau , nhm bo v OH c tch bi acid p-TSA hoc HCl 4M trong dioxan. Phn ng tri qua 2 bc
do vy hiu sut tng ch t c khong 40 50%.

S 1.2. Tng hp acid biaryl hydroxamic [43]

Song song, nhiu tc nhn hot ha khc cng c cc nhm nghin cu s
dng trong tng hp acid hydroxamic. Isobutyl cloroformat c Kozikowski v
cng s [31] dng trong phn ng tng hp cc acid phenylthiazol hydroxamic.
Phn ng tin hnh 0oC, to anhydrid hn tp ngay trong phn ng. y l tc
nhn acyl ha mnh nn hydroxylamin phn ng trc tip, khng cn s dng loi
c nhm bo v -OH. Tuy nhin, hiu sut phn ng khng cao, khong 26-30% (s
1.3).

S 1.3. Tng hp cc acid phenylthiazol hydroxamic [31]

25

Ch g 2. NGUYN LIU, THIT B, NI DUNG

V PHNG PHP NGHIN CU
2.1. NGUYN LIU
Cc ha cht, dung mi s dng trong qu trnh tng hp c nhp t
Mecrk, Sigma Aldrich v Trung Quc. Bao gm:
TT

Nguyn liu

Xut x

TT

Nguyn liu

Xut x

Isatin

Aldrich

10

Pyridin

Merck

5-Fluoroisatin

Aldrich

11

Ethanol

Trung quc

5-Cloroisatin

Aldrich

12

K2CO3

Trung quc

5-Bromoisatin

Aldrich

13

KI

Trung quc

5-Nitroisatin

Aldrich

14

Dimethylformamid

Merck

5-Methylisatin

Aldrich

15

Acid hydrocloric

Trung quc

7-Cloroisatin

Aldrich

16

Natri hydroxid

Trung quc

Methoxylamin HCl

Merck

17

Methanol

Trung quc

Ethyl 7-Bromoheptanoat

Merck

18

Tetrahydrofuran

Merck

2.2. THIT B V DNG C

-

Cc dng c thy tinh: bnh cu y trn dung tch 50 ml, bnh chit, sinh

hn hi lu, ng nghim, ng ong, pipet, cc c m cc loi.

-

Bnh sc k Camag, n t ngoi Camag bc sng 254 nm v 365 nm, bn

mng silicagel F254 (Merck).

-

Cn phn tch, cn k thut Shimazu. T lnh, t sy Memmert.

My ct quay Buchi R-210, my siu m, my khuy t gia nhit IKA-RTC,

my o nhit nng chy nhit in Gallenkamp Melting Point Apparatus.

-

My o ph hng ngoi: GX-Perkin Elmer-USA, khoa Ha hc - Trng

i hc Khoa hc t nhin, i hc Quc gia H Ni.

-

My o ph khi: Agilent 6310 Ion Trap LC/MS (Agilent Technologies),

Vin Ha hp cht thin nhin - Vin Khoa hc v Cng ngh Vit Nam.
-

My o ph cng hng t ht nhn Bruker AC-500 MHz, Vin Ha hc -

Vin Khoa hc v Cng ngh Vit Nam.

26

My c kt qu Western blot: molecular Imaging ChemiDocTM XRS+ (vi

phn mm ImageTM) kt ni my qut HP 4850.

2.3. NI DUNG V PHNG PHP NGHIN CU
2.3.1. Ni dung nghin cu
Tng hp 7 dn cht mang khung 3-methoxim-isatin:

N-hydroxy-7-(3-(methoxyimino)-2-oxoindolin-1-yl)heptanamid

(IVa)

7-(5-fluoro-3-(methoxyimino)-2-oxoindolin)-N-hydroxyheptanamid

(IVb)

7-(5-cloro-3-(methoxyimino)-2-oxoindolin)-N-hydroxyheptanamid

(IVc)

7-(5-bromo-3-(methoxyimino)-2-oxoindolin)-N-hydroxyheptanamid

(IVd)

N-hydroxy-7-(3-(methoxyimino)-5-nitro-2-oxoindolin)heptanamid

(IVe)

N-hydroxy-7-(3-(methoxyimino)- 5-methyl-2-oxoindolin)heptanamid

(IVf)

7-(7-cloro-3-(methoxyimino)-2-oxoindolin)-N-hydroxyheptanamid

(IVg)

Khng nh cu trc ca cc cht tng hp c da trn phn tch d liu

ph khi (MS), ph hng ngoi (IR) v ph cng hng t ht nhn (1H-NMR, 13CNMR).
Th tc dng c ch HDAC ca cc dn cht tng hp c.
Th hot tnh khng t bo ung th in vitro ca mt s dn cht c tc dng
c ch HDAC mnh.
nh gi mi lin quan cu trc tc dng v mc ging thuc ca cc
cht tng hp c.
2.3.2. Ph g php ghi cu
2.3.2.1. Tng hp ha hc
Tng hp 7 dn cht IVa-g bng phng php ha hc. S phn ng tng
hp c trnh by trong mc 3.1.
2.3.2.2. Phng php kim tra tinh khit

27

Sc k lp mng: Dng theo di qu trnh phn ng, xc nh thi im

kt thc phn ng v kim tra tinh khit ca sn phm sau khi tinh ch.
Sc k lp mng c tin hnh trn bn mng silicagel Merck 60 (240
400 mesh) trng sn, hot ha 110oC trong 30 pht. H dung mi DCM:MeOH
(9:1). Mu th c ha tan trong dung mi thch hp. bn mng trong bnh sc
k bo ha dung mi nhit phng, cho dung mi chy khong 8 cm. Quan
st kt qu di n t ngoi bc sng 254 nm.
Nhit nng chy: o bng my o nhit nng chy nhit in
Gallenkamp Melting Point Apparatus kim tra tinh khit ca cc dn cht.
2.3.2.3. Phng php phn tch cu trc
phn tch, khng nh cu trc ca cc cht tng hp, lun vn s dng
cc phng php ph hng ngoi (IR), ph khi lng (MS) v ph cng hng t
ht nhn (1H-NMR, 13C-NMR).
Ph hng ngoi: Ph hng ngoi c tin hnh ghi trn my GX-Perkin
Elmer-USA vi k thut vin nn KBr trong vng 4000 - 600 cm-1. Ghi ph
phn gii 4 cm-1. Cc mu rn c phn tn tn trong KBr sy kh vi t l
1:200 ri p di dng film mng di p lc cao c ht chn khng loi b hi
m.
Ph khi lng: Ph khi lng c tin hnh ghi trn my Agilent 6310
Ion Trap LC/MS (Agilent Technologies), Vin ha hp cht thin nhin Vin
Khoa hc v Cng ngh Vit Nam.
Ph cng hng t ht nhn:
Ph NMR c o trn my cng hng t ht nhn Bruker AC-500 MHz,
dung mi DMSO-d6. Ph 1H-NMR o tn s 500 MHz, ph 13C-NMR o tn
s 125 MHz. dch chuyn ha hc (, ppm) c tnh theo cht chun ni
tetramethylsilan (TMS), nhit ghi ph khong 300oK.
2.3.2.4. Phng php th hot tnh sinh hc
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
28

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 (fetal bovine serum)), gi l mi
trng nui cy hon chnh (complete medium). Tt c t bo c 37C 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 dimethyl sulfoxide (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 1 M.
b. Phn lp histon v Western Blot
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. T bo c dung gii
trong dung dch dung gii m [20 mM Tris-HCl (pH 7,5); 150 mM NaCl; 1 mM
Na2EDTA; 1 mM EGTA; 1% triton; 2,5 mM Na pyrophosphat; 1 mM glycerophosphat; 1 mM 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.
Th hot tnh khng t bo ung th in vitro
Th hot tnh khng t bo ung th ngi in vitro c thc hin ti Khoa
Dc, Trng i hc Quc gia Chungbuk, Cheongju, Hn Quc theo phng
php MTT [12, 15] v gi tr IC50 c tnh trn phn mm GraphPad Prism.
D ng t bo th nghim:
-

SW620: t bo ung th i trng

MCF-7: t bo ung th v
29

AsPC-1: t bo ung th ty

PC-3: t bo ung th tin lit tuyn

NCI-H460: t bo ung th phi

Cc dng 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 mi trng DMEM (Dulbeccos modified Eagle medium vi dng t bo
MCF-7) hoc RPMI (vi dng 4 t bo cn li) 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:
Bc 1: Chun b
Cc t bo pha logarit c trypsin ha v phn tn vo hn dch n t
bo trong mi trng DMEM hoc RPMI b sung 10% FBS v iu chnh n
nng khong 1,5.104 n 3,5.104 t bo, sau chia u vo cc ging ca a 96
ging, mi ging 200l. Cc a sau c 37oCtrong iu kin 5% CO2. Sau
24 gi , cc mu th c chun b trong 20l mi trng DMEM/RPMI b sung
10% FBS t dung dch gc 10 mg/ml trong dimethyl sulfoxid (DMSO) ri thm 2
l mu th vocc 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
l 0,1%.
Bc 2: Tin hnh th
Sau khi 48 gi, thm vo mi ging 20 l thuc nhum MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) nng 5 mg/ml trong
PBS. Cc a c thm 3 gi 37oCtrong iu kin 5% CO2. Tip theo, mi
ging c cho 100 l dung dch MTT trong DMSO, 5 pht cho thuc nhum
MTT c ha tan. hp th c c bc sng 510nm.
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

30

cng l gi tr trung bnh ca 4 ln o c lp vi gi tr hp th khc nhau

khng qu 5%.
2.3.2.5. Docking
kim tra s b tnh tng tc ca cc cht tng hp c vi HDAC, cht
IVa c tin hnh docking. Qu trnh docking c thc hin ti phng nghin
cu cu trc i hc quc gia Seul, Hn Quc, s dng mng li cu trc ca
HDAC8, HDAC2 tng tc vi SAHA.
Chng ti la chn HDAC2 v HDAC8 l hai enzym thuc HDAC nhm I
phn nhm HDAC m cc dn cht acid hydroxamic c tc dng c ch mnh nht
(bng 1.2) tin hnh docking nhm d on kh nng c ch ca IVa i vi cc
enzym ny cn c vo s ph hp v hnh dng, kch thc v nng lng tng
tc gia IVa v cc enzym. Docking c thc hin bng chng trnh AutoDock
Vina, sau d on nng lng ca tng tc lin kt t s gn kt trn khi
SAHA ri khi h tng tc. Kt qu s c minh ha bng hnh nh m hnh v
s liu d on nng lng lin kt (mc 3.5).
2.3.2.6. nh gi mc ging thuc ca cc cht tng hp c
Tnh gi tr logP ca cc cht tng hp c bng phn mm EPTsuite cung
cp bi US Environmental Protection Agencys 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 EPTsuite tnh cc gi tr logP.
nh gi mc ging thuc ca cc cht da trn quy tc Lipinsky [32]:
-

Khi lng phn t ca cht khng ln hn 500 g/mol

S trung tm nhn lin kt Hydro (N,O) khng ln hn 10

S trung tm cho lin kt Hydro (NH,OH) khng ln hn 5

Gi tr logP ca cht khng ln hn 5

S lin kt linh ng khng ln hn 15

31

Ch g 3. THC NGHIM, KT QU
3.1. TNG HP HA HC
T c im chung ca cc dn cht acid hydroxamic hng c ch HDAC
v kt qu ca cc nghin cu gn y, chng ti thit k dy dn cht vi cu trc
tng t SAHA vi cc c im v cu trc nh sau:
-

Gi nguyn phn cu ni carbon v nhm chc acid hydroxamic

Thay khung phenyl ca SAHA bng khung 3-methoxim-isatin

7 cht trong lun vn c tng hp theo s :

S 3.1. S phn ng tng hp cc dn cht mang khung 3-methoxim-isatin

Quy trnh tng hp gm 3 giai on:
-

Giai on 1: Thc hin phn ng cng hp i nhn vo nhm carbonyl, to

3-(methoxyimino)-2-oxoindolin v cc dn cht.
-

Giai on 2: Thc hin phn ng alkyl ha, tng hp ethyl 7-(3-

(methoxyimino)-2-oxoindolin-1-yl)heptanoat t 3-methoxim-isatin v cc dn cht

mang nhm th ti v tr s 5 v s 7 vi ethyl 7-bromoheptanoat trong dung mi
DMF.
-

Giai on 3: Thc hin phn ng th i nhn acyl to acid hydroxamic, tng

hp N-hydroxy-7-(3-(methoxyimino)-2-oxoindolin-1-yl)heptanamid v cc dn
cht t cc cht trung gian giai on 2 vi hydroxylamin hydroclorid.
3.1.1. Tng hp 3-(methoxyimino)-2-oxoindolin v dn cht (IIa-g)
Quy trnh tng hp 3-(methoxyimino)-2-oxoindolinv dn chtc thc
hin theo s sau:

32

S 3.2. S phn ng tng hp cc cht IIa - g

3.1.1.1. Tng hp 3-(methoxyimino)-2-oxoindolin (IIa)
* Tin hnh phn ng:
Cn 0,147 g isatin (1 mmol) cho vo bnh cu, ha tan trong hn hp dung
mi 5 ml ethanol v 2,5 ml methanol. Tip tc cho 0,501 g methoxylamin
hydroclorid (6 mmol) trong 5ml pyridin khan. un hi lu phn ng trong 3h.
Kim tra phn ng bng SKLM vi pha ng DCM:CH3OH (9:1).
* X l hn hp phn ng:
Hn hp phn ng c ngui n nhit phng, sau thm t t 30
ml acid HCl 5M lnh thy xut hin kt ta. Lc ly ta, ra ta bng nc ct cho
ti khi dch lc trung tnh, sy chn khng 40C trong 24h. Sn phm trung gian
IIa thu c c dng lm nguyn liu tng hp IIIa.
* Kt qu:

- Rf = 0,63. Cht rn thu c c mu vng.

- Khi lng: 0,138 mg
- Hiu sut: 93,9%.

3.1.1.2. Tng hp 5- fluoro-3-(methoxyimino)-2-oxoindolin (IIb)

Cht IIb c tng hp t 0,165 g 5-fluoroisatin (1 mmol) vi quy trnh tng
hp v cch x l phn ng tng t nh tng hp cht IIa.
Kt qu thu c c th hin trong bng 3.1
3.1.1.3. Tng hp 5-cloro-3-(methoxyimino)-2-oxoindolin (IIc)
Cht IIc c tng hp t 0,181 g 5-cloroisatin (1 mmol) vi quy trnh tng
hp v cch x l phn ng tng t nh tng hp cht IIa.
Kt qu thu c c th hin trong bng 3.1
3.1.1.4. Tng hp 5-bromo-3-(methoxyimino)-2-oxoindolin (IId)
Cht IId c tng hp t 0,226 g 5-bromoisatin (1 mmol) vi quy trnh
tng hp v cch x l phn ng tng t nh tng hp cht IIa.

33

Kt qu thu c c th hin trong bng 3.1

3.1.1.5. Tng hp 3-(methoxyimino)-5-nitro-2-oxoindolin (IIe)
Cht IIe c tng hp t 0,192 g 5-nitroisatin (1 mmol) vi quy trnh tng
hp v cch x l phn ng tng t nh tng hp cht IIa.
Kt qu thu c c th hin trong bng 3.1
3.1.1.6. Tng hp 3-(methoxyimino)-5-methyl-2-oxoindolin (IIf)
Cht IIf c tng hp t 0,163 g 5-methylisatin (1 mmol) vi quy trnh
tng hp v cch x l phn ng tng t nh tng hp cht IIa.
Kt qu thu c c th hin trong bng 3.1
3.1.1.7. Tng hp 7-cloro-3-(methoxyimino)-2-oxoindolin (IIg)
Cht IIg c tng hp t 0,181 g 7-cloroisatin (1 mmol) vi quy trnh tng
hp v cch x l phn ng tng t nh tng hp cht IIa.
Kt qu thu c c th hin trong bng 3.1
Bng 3.1. Kt qu tng hp 3-(methoxyimino)-2-oxoindolin v dn cht

TT Cht

KLPT

Mu

Dng
tn ti

Rf

Khi lng Hiu

(g)
sut (%)

IIa

-H

176

Vng

Cht rn

0,63

0,138

94

IIb

5-F

194

Vng

Cht rn

0,58

0,149

90

IIc

5-Cl

210,5

Vng

Cht rn

0,60

0,165

91

IId

5-Br

255

Vng da cam Cht rn

0,53

0,210

93

IIe

5-NO2

221

Vng

Cht rn

0,52

0,169

88

IIf

5-CH3

190

Vng

Cht rn

0,50

0,150

92

IIg

7-Cl

210,5

Vng

Cht rn

0,57

0,170

94

3.1.2. Tng hp ethyl-7-(3-(methoxyimino)-2-oxoindolin-1-yl)heptanoat v dn

cht (IIIa g)
Quy trnh tng hp ethyl-7-(3-(methoxyimino)-5-2-oxoindolin-1-yl)heptanoat
v dn cht c thc hin theo s sau:
34

S 3.3. S phn ng tng hp cc cht IIIa - g

3.1.2.1. Tng hp ethyl-7-(3-(methoxyimino)2-oxoindolin-1-yl)heptanoat (IIIa)
* Tin hnh phn ng:
Cho 0,176 g (1 mmol) cht IIa vo bnh cu dung tch 50 ml. Thm 3 ml
DMF vo bnh cu, siu m cho hn hp tan hon ton (7 pht), sau lm lnh
xung 5C. Thm 0,166 g K2CO3 khan (1,2 mmol). Khuy u hn hp -5C
trong 1h (s dng my khuy t). Sau khuy thm 45 pht nhit phng cho
hn hp ng nht. Thm 0,5 ml CH3OH v 0,008 g KI (0,05 mmol) vo hn hp,
tip tc khuy trong vng 15 pht. Nh t t 0,237 g ethyl 7-bromoheptanoat (1
mmol) ( c ha tan trong 1 ml DMF) vo hn hp phn ng. Nng nhit
phn ng ln 60C, khuy u hn hp phn ng trong 24h. Kim tra phn ng
bng SKLM vi pha ng DCM:CH3OH (9:1).
* X l hn hp phn ng:
Sau khi phn ng kt thc, lm lnh bnh phn ng. Acid ha hn hp phn
ng bng acid HCl 10%. Chit sn phm bng DCM (50 ml 2 ln). Thu lp dch
chit, ct quay thu hi DCM di p sut gim thu c dn cht IIIa.
Kt qu:

- Rf = 0,69. Ester thu c c dng du, mu nu vng.

- Khi lng: 0,137 mg.
- Hiu sut 77,7%

3.1.2.2.

Tng

hp

ethyl-7-(5-fluoro-3-(methoxyimino)-2-oxoindolin-1-

yl)heptanoat (IIIb)
Cht IIIb c tng hp t 0,194 g (1 mmol) 5-fluoro-3-(methoxyimino)-2oxoindolin (IIb) vi quy trnh tng hp v x l phn ng tng t khi tng hp
cht IIIa.
Kt qu ca qu trnh tng hp c th hin trong bng 3.2.

35

3.1.2.3. Tng hp ethyl-7-(5-cloro-3-(methoxyimino)-2-oxoindolin-1-yl)heptanoat

(IIIc)
Cht IIIc c tng hp t 0,211 g (1 mmol) 5-cloro-3-(methoxyimino)-2oxoindolin (IIc) vi quy trnh tng hp v x l phn ng tng t khi tng hp
cht IIIa.
Kt qu ca qu trnh tng hp c th hin trong bng 3.2.
3.1.2.4.

Tng

hp

ethyl-7-(5-bromo-3-(methoxyimino)-2-oxoindolin-1-

yl)heptanoat (IIId)
Cht IIId c tng hp t 0,255 g (1 mmol) 5-bromo-3-(methoxyimino)-2oxoindolin (IId) vi quy trnh tng hp v x l phn ng tng t khi tng hp
cht IIIa.
Kt qu ca qu trnh tng hp c th hin trong bng 3.2.
3.1.2.5. Tng hp ethyl-7-(3-(methoxyimino)-5-nitro-2-oxoindolin-1-yl)heptanoat
(IIIe)
Cht IIIe c tng hp t 0,221 g (1 mmol) 3-(methoxyimino)-5-nitro-2oxoindolin (IIe) vi quy trnh tng hp v x l phn ng tng t khi tng hp
cht IIIa.
Kt qu ca qu trnh tng hp c th hin trong bng 3.2.
3.1.2.6.

Tng

hp

ethyl-7-(3-(methoxyimino)-5-methyl-2-oxoindolin-1-

yl)heptanoat (IIIf)
Cht IIIf c tng hp t 0,190 g (1 mmol) 3-(methoxyimino)-5-methyl-2oxoindolin (IIf) vi quy trnh tng hp v x l phn ng tng t khi tng hp
cht IIIa.
Kt qu ca qu trnh tng hp c th hin trong bng 3.2.
3.1.2.7. Tng hp ethyl-7-(7-cloro-3-(methoxyimino)-2-oxoindolin-1-yl)heptanoat
(IIIg)
Cht IIIg c tng hp t 0,211 g (1 mmol) 7-cloro-3-(methoxyimino)-2oxoindolin (IIg) vi quy trnh tng hp v x l phn ng tng t khi tng hp
cht IIIa.
Kt qu ca qu trnh tng hp c th hin trong bng 3.2.
36

Bng 3.2. Kt qu tng hp ethyl 7-(3-(methoxyimino)-2-oxoindolin-1yl)heptanoatv dn cht

TT Cht

KLPT

Mu

Dng tn
ti

Rf

Khi lng Hiu sut

(g)
(%)

332

Nu vng Dng du

0,69

0,137

78

350

Nu vng Dng du

0,60

0,165

85

366,7

Nu vng Dng du

0,62

0,186

88

411

Nu vng Dng du

0,63

0,204

80

5-NO2

377

Vng nht Dng du

0,65

0,141

64

IIIf

5-CH3

346

Nu vng Dng du

0,55

0,160

84

IIIg

7-Cl

366,7

Nu vng Dng du

0,61

0,158

75

IIIa

-H

IIIb 5-F

IIIc

IIId 5-Br

IIIe

6
7

5-Cl

3.1.3. Tng hp N-hydroxy-7-(3-methoxyimino-2-oxoindolin-1-yl)heptanamid

v dn cht (IVa-g)
Quy

trnh

tng

hp

N-hydroxy-7-(3-methoxyimino-2-oxoindolin-1-yl)

heptanamid v dn cht c thc hin theo s sau:

S 3.4. S phn ng tng hp cc cht IVa-g

3.1.3.1. Tng hp N-hydroxy-7-(3-methoxyimino-5-2-oxoindolin-1-yl)heptanamid
(IVa)
* Tin hnh phn ng:
Cn 0,4 g (10 mmol) NaOH, ha tan trong 2 ml nc ct, lnh -5C.
Cho 0,332 g (1 mmol) ester IIIa vo bnh cu dung tch 50 ml. Thm vo
hn hp dung mi CH3OH/THF (3 ml/3ml). Lm lnh bnh phn ng xung -5C,
37

thm 0,695 g (10 mmol) hydroxylammonium clorid. Thm t t dung dch NaOH
c lm lnh vo hn hp phn ng. Duy tr nhit phn ng -5C (s dng
mui), khuy u hn hp cho n khi ester phn ng ht. Kim tra phn ng
bng SKLM vi pha ng DCM:CH3OH (9:1).
* X l hn hp phn ng:
Sau khi phn ng kt thc, trung ha phn ng bng dung dch HCl 15% ti
pH 7 to kt ta. Lc ly ta, ra sch bng nc ct. Sy kh ta 60C. Tinh
ch bng cch kt tinh li trong ethanol.
Kt qu:

- Rf = 0,71. Cht rn thu c c mu vng

- Khi lng: 0,249 mg
- Hiu sut: 75%

3.1.3.2.

Tng

hp

N-hydroxy-7-(5-fluoro-(3-methoxyimino)-2-oxoindolin-1-

yl)heptanamid (IVb)
Cht IVb c tng hp t 0,350 g (1 mmol) ethyl 7-(5-fluoro-3(methoxyimino)-2-oxoindolin-1-yl)heptanoat (IIIb) vi quy trnh v cch x l
phn ng tng t khi tng hp IVa.
Kt qu ca qu trnh tng hp c th hin trong bng 3.3.
3.1.3.3.

Tng

hp

N-hydroxy-7-(5-cloro-(3-methoxyimino)-2-oxoindolin-1-

yl)heptanamid (IVc)
Cht IVc c tng hp t 0,367 g (1 mmol) ethyl-7-(5-cloro-3(methoxyimino)-2-oxoindolin-1-yl)heptanoat (IIIc) vi quy trnh v cch x l
phn ng tng t khi tng hp IVa.
Kt qu ca qu trnh tng hp c th hin trong bng 3.3.
3.1.3.4.

Tng

hp

N-hydroxy-7-(5-bromo-(3-methoxyimino)-2-oxoindolin-1-

yl)heptanamid (IVd)
Cht IVd c tng hp t 0,411 g (1 mmol) ethyl-7-(5-bromo-3(methoxyimino)-2-oxoindolin-1-yl)heptanoat (IIId) vi quy trnh v cch x l
phn ng tng t khi tng hp IVa.
Kt qu ca qu trnh tng hp c th hin trong bng 3.3.

38

3.1.3.5.

Tng

hp

N-hydroxy-7-(3-(methoxyimino)-5-nitro-2-oxoindolin-1-

yl)heptanamid (IVe)
Cht IVe c tng hp t 0,377 g (1 mmol) ethyl-7-(3-(methoxyimino)-5nitro-2-oxoindolin-1-yl)heptanoat (IIIe) vi quy trnh v cch x l phn ng tng
t khi tng hp IVa.
Kt qu ca qu trnh tng hp c th hin trong bng 3.3.
3.1.3.6.

Tng

hp

N-hydroxy-7-(3-(methoxyimino)-5-methyl-2-oxoindolin-1-

yl)heptanamid (IVf)
Cht IVf c tng hp t 0,346 g (1 mmol) ethyl 7-(3-(methoxyimino)-5methyl-2-oxoindolin-1-yl)heptanoat (IIIf) vi quy trnh v cch x l phn ng
tng t khi tng hp IVa.
Kt qu ca qu trnh tng hp c th hin trong bng 3.3.
3.1.3.7.

Tng

hp

N-hydroxy-7-(7-cloro-(3-methoxyimino)-2-oxoindolin-1-

yl)heptanamid (IVg)
Cht IVg c tng hp t 0,367 g (1 mmol) ethyl 7-(7-cloro-3(methoxyimino)-2-oxoindolin-1-yl)heptanoat (IIIg) vi quy trnh v cch x l
phn ng tng t khi tng hp IVa.
Kt qu ca qu trnh tng hp c th hin trong bng 3.3.

39

Bng 3.3. Kt qu tng hp N-hydroxy-7-(3-methoxyimino-2-oxoindolin-1yl)heptanamid v dn cht

TT Cht

KLPT

Mu

Dng tn ti

Khi lng
(g)

Hiu sut (%)

1 IVa

-H

319

Vng

Cht rn

0,249

75

2 IVb

5-F

337

Vng nht

Cht rn

0,252

72

3 IVc

5-Cl

353

Vng nht

Cht rn

0,286

78

4 IVd

5-Br

397

Vng m

Cht rn

0,300

73

5 IVe

5-NO2

364

Vng m

Cht rn

0,264

70

5-CH3

333

Vng nht

Cht rn

0,266

77

7-Cl

353

Vng nht

Cht rn

0,275

75

IVf

7 IVg

3.2. KIM TRA TINH KHIT

Sau khi tng hp, cc dn cht IVa-g c kim tra tinh khit bng sc
k lp mng v o nhit nng chy (Tnc) ca chng.
Sc k lp mng (SKLM):
-

SKLM c tin hnh trn bn nhm trng sn silicagel Merck 60 (240

400 mesh). Cc cht IVa-g c ha tan trong dung mi DMF, tin hnh chy
SKLM vi h dung mi DCM:CH3OH (9:1)
-

Sau khi chy sc k, quan st bn mng di n t ngoi c bc sng

254nm thy cc cht em th u cho mt vt gn.

o nhit nng chy (Tnc):
Bn cnh vic kim tra tinh khit bng SKLM, chng ti tin hnh o
nhit nng chy ca cc cht tng hp c. Kt qu o nhit nng chy cho
thy: cc cht u c im chy r rng, khong chnh lch hp.
Gi tr Rf v Tnc ca cc cht IVa-g c th hin trong bng 3.4.

40

Bng 3.4. Gi tr Rf v Tnc ca cc cht IVa-g

Pha ng

Rf

Tnc (C)

-H

DCM/CH3OH = 9/1

0,71

191 - 193

IVb

5-F

DCM/CH3OH = 9/1

0,70

200 - 202

IVc

5-Cl

DCM/CH3OH = 9/1

0,70

198 - 199

IVd

5-Br

DCM/CH3OH = 9/1

0,76

205 - 207

IVe

5-NO2

DCM/CH3OH = 9/1

0,67

203 - 205

IVf

5-CH3

DCM/CH3OH = 9/1

0,75

193 - 195

IVg

7-Cl

DCM/CH3OH = 9/1

0,72

189 - 191

TT

Cht

IVa

Nhn xt: Thng qua sc k khi chy SKLM v nhit nng chy ca
cc cht, c th s b khng nh cc cht ny l tinh khit, iu kin o ph
v th tc dng sinh hc.
3.3. KHNG NH CU TRC
phn tch, khng nh cu trc ca cc cht tng hp c, 7 cht IVa-g
c tin hnh ghi v phn tch d liu ph hng ngoi (IR), ph khi lng (MS)
v ph cng hng t ht nhn (1H-NMR, 13C-NMR).
3.3.1. Ph hng ngoi (IR)
Ti phng th nghim khoa Ha Trng i hc Khoa hc T nhin, cc
cht IVa-g c ghi ph hng ngoi (IR) trn my Perkin Elmer vi k thut lm
vin nn KBr, ghi trong vng 4000- 600 cm-1.
Kt qu phn tch ph c th hin trong bng 3.5.

41

S sng ng vi cc dao ng (cm-1)

Bng 3.5. Kt qu phn tch ph IR ca cc cht IVa-g

Cht

IVa

IVb

IVc

IVd

IVe

IVf

IVg

5-F

5-Cl

5-Br

5-NO2

5-CH3

7-Cl

OH

3399

3331

3431

NH

3249

3234

3259

3222

3210

3217

3232

C-H, aren

3038
2933

3045

3047

3059

2931

2935

2935

2934

2926

2937

2861

2859

2856

2856

2856

2858

1607

1602

1609

1604

1611

1616

1605

1539

1529

1469

1466

1522

1594

1545

1464

1477

1442

1436

1474

1482

1465

1704

1702

1621

1622

1714

1722

1729

1706

1728

C=N, oxim

1647

1655

1645

1647

1638

Nhm th

1343

C-H, mch nhnh

(-CH2)

C=C, aren

C=O

2862
2824

1522

Ch thch: l dao ng ha tr

Nhn xt: Rt kh khng nh cu trc ca cc dn cht qua ph hng

ngoi, tuy nhin trn ph ca cc dn cht IVa-g thy xut hin mt s peak
rt c trng ca dao ng ha tr trong mt s nhm chc: -OH (acid), C=O, oxim,
C-H (-CH2), aryl.
3.3.2. Ph khi lng (MS)
S dng phng php o ph khi lng vi k thut ion ha ti p sut kh
quyn API (Atmospheric Pressure Ionization API), hnh thnh ion theo kiu ion
ha tia in (electrospray ionization ESI).
42

Bng 3.6. Kt qu phn tch ph khi lng ca cc cht IVa-g

TT

Cht

Nhm th

KLPT (M)

m/z (ESI-MS)

IVa

-H

319

318 [M H]-

IVb

5-F

337

335 [M 2H]-

IVc

5-Cl

353

352 [M H]-

IVd

5-Br

399

398 [M H]-

IVe

5-NO2

364

363 [M H]-

IVf

5-CH3

333

332 [M H]-

IVg

7-Cl

353

352 [M H]-

Nhn xt: Kt qu phn tch ph khi bng 3.6 cho thy cc cht kho st
u c gi tr pic ion phn t ng bng s khi ca phn t d kin vi cng
mnh.
3.3.3. Ph c g hng t ht nhn
Ph NMR c o trn my cng hng t ht nhn Bruker AC-500 MHz
ti phng phn tch ph - Vin ha hc Vin Khoa hc v Cng ngh Vit Nam
vi dung mi DMSO-d6 . Ph 1H-NMR o tn s 500 MHz, ph 13C-NMR o
tn s 125 MHz. dch chuyn ha hc (, ppm) c tnh theo cht chun ni
tetramethylsilan (TMS), nhit ghi ph khong 300oK.
Di y l kt qu phn tch ph .
3.3.3.1. Ph cng hng t ht nhn 1H-NMR
Kt qu phn tch ph cng hng t ht nhn 1H-NMR c th hin trong
bng 3.7.
Nhn xt: Kt qu phn tch ph 1H-NMR ca cc cht IVa-g cho thy s
lng proton ca nhn thm, mch nhnh v cc nhm th l ph hp vi cng thc
cu to d kin ca cc cht.

43

Bng 3.7. Kt qu phn tch ph 1H-NMR ca cc cht IVa-g

TT Cht

(ppm) (500 MHz, DMSO-d6)

10,33 (1H, s, NH); 8,66 (1H, s, OH); 7,87 (1H, d, JH4-H5 =

7,5 Hz, H-4); 7,46 (1H, t, JH5-H6 JH5-H4 = 7,5 Hz, H-5);
7,12 (1H, d, JH7-H6 = 8,0 Hz, H-7); 7,07 (1H, t, JH6-H7
1

IVa

-H

JH6-H5 = 7,5 Hz, H-6); 4,20 (3H, s, - OCH3); 3,66 (2H, t, J=

7,0 Hz, 2H-7); 1,92 (2H, t, J= 7,0 Hz, 2H-2); 1,56 (2H, m,
2H-6); 1,45 1,47 (2H, m, 2H-3); 1,26 (4H, m, 2H-4 & 2H5).
10,33 (1H, s, NH); 8,66 (1H, s, OH); 7,65 (1H, dd, JH4-H6 =
8,0Hz, JH4-H7 = 2,5 Hz, H-4); 7,33 (1H, td,JH6-F= JH6-H7=
9,25 Hz, JH6-H4 = 2,5 Hz, H-6); 7,14 (1H, dd, JH7-H6 = 8,5

IVb

5-F

Hz, JH7-H4 = 4,0 Hz, H-7); 4,21 (3H, s, - OCH3); 3,66 (2H,
t, J= 7,0 Hz, 2H-7); 1,91 (2H, t, J= 7,0 Hz, 2H-2); 1,53 - 1,56
(2H, m, 2H-3); 1,45 1,46 (2H, m, 2H-6); 1,25 - 1,26 (4H,
m, 2H-4 & 2H-5).
10,45 (1H, s, NH); 7,81 (1H, s, H-4); 7,50 (1H, d, JH6-H7 =
8,0 Hz, H-6); 7,16 (1H, d, JH7-H6 = 8,0 Hz, H-7); 4,22 (3H,

IVc

5-Cl

s, - OCH3); 3,65 (2H, t, J= 6,5 Hz, 2H-7); 1,93 (2H, t, J= 7,0

Hz, 2H-2); 1,53 - 1,55 (2H, m, 2H-3); 1,44 1,46 (2H, m,
2H-6); 1,25 - 1,27 (4H, m, 2H-4 & 2H-5).
7,94 (1H, s, H-4); 7,62 (1H, d, JH6-H7 = 7,5 Hz, H-6); 7,11

IVd

5-Br

(1H, d, JH7-H6 = 8,0 Hz, H-7); 4,22 (3H, s, - OCH3); 3,65

(2H, t, J= 6,5 Hz, 2H-7); 1,91 (2H, t, J= 7,0 Hz, 2H-2); 1,53

44

(2H, m, 2H-3); 1,44 (2H, m, 2H-6); 1,25 (4H, m, 2H-4 & 2H5).
10,33 (1H, s, NH); 8,66 (1H, s, OH); 8,49 (1H, d, JH4-H6 =
1,5 Hz, H-4); 8,35 (1H, dd, JH6-H7 = 9,0 Hz, JH6-H4 = 2,5
5

IVe

5-NO2

Hz, H-6); 7,35 (1H, d, JH7-H6 = 9,0 Hz, H-7); 4,29 (3H, s, OCH3); 3,73 (2H, t, J= 7,0 Hz, 2H-7); 1,92 (2H, t, J= 7,0 Hz,
2H-2); 1,54 - 1,58 (2H, m, 2H-3); 1,43 1,49 (2H, m, 2H-6);
1,26 - 1,27 (4H, m, 2H-4 & 2H-5).
10,34 (1H, s, NH); 7,70 (1H, s, H-4); 7,26 (1H, d, JH6-H7 =
8,0 Hz, H-6); 7,00 (1H, d, JH7-H6 = 8,0 Hz, H-7); 4,19 (3H,

IVf

5-CH3

s, - OCH3); 3,64 (2H, t, J= 7,0 Hz, 2H-7); 2,27 (3H, s, CH3);

1,91 (2H, t, J= 7,5 Hz, 2H-2); 1,53 - 1,55 (2H, m, 2H-3);
1,43 1,46 (2H, m, 2H-6); 1,24 - 1,25 (4H, m, 2H-4 & 2H5).
10,36 (1H, s, NH); 8,66 (1H, s, OH); 7,90 (1H, d, JH6-H5=
7,5 Hz, H-6); 7,47 (1H, d, JH4-H5= 8,0 Hz, H-4); 7,09 (1H,

IVg

7-Cl

t, JH5-H6 JH5-H4 = 8,0 Hz, H-5); 4,22 (3H, s, - OCH3);

3,95 (2H, t, J= 7,0 Hz, 2H-7); 1,93 (2H, t, J= 7,5 Hz, 2H-2);
1,56 1,60 (2H, m, 2H-3); 1,44 1,50 (2H, m, 2H-6); 1,26
1,28 (4H, m, 2H-4 & 2H-5).

Ghi ch: s: singlet (vch n); d: doublet (hai vch); t: triplet (ba vch); m: multiplet (a
vch).

3.3.3.2. Ph cng hng t ht nhn 13C-NMR

Nhn xt: Qua kt qu phn tch ph 13C-NMR bng 3.8 v ph phn
ph lc, c th nhn thy s lng C ca cc dn cht IVa-g xut hin y ,
tng ng vi cc gi tr ca dch chuyn ha hc .
Nh vy, d liu cc ph thc nghim IR, MS,1H-NMR, 13C-NMR cho php
khng nh cu trc cc cht tng hp IVa-g ng nh d kin v l sn phm tinh
khit. T , cc acid hydroxamic ny s c tin hnh th hot tnh sinh hc.

45

Bng 3.8. Kt qu phn tch ph 13C-NMR ca cc cht IVa-g

Cht

IVa

IVb

IVc

IVd

-H

5-F

5-Cl

5-Br 5-NO2 5-CH3

169,06

169,10

169,04 168,69 169,05 169,02 169,01

C2 162,15

162,03

161,77 161,66 162,43 162,11 162,57

C3 143,68

158,83

142,44 142,80 148,87 143,38 141,92

C=O

C1

(ppm) (125 MHz, DMSO-d6)

143,28
C

C4

(aryl)

C9

132,96
127,34
122,61
114,87
109,40

C(nhm C10
th)

C
(mch
nhnh)

156,93*

IVe

IVf

IVg
7-Cl

142,41 142,32 142,34 141,44 139,21

142,96

132,30 135,13 141,79 133,13 134,76

140,02
119,04-119,23
115,33-115,41
114,29-114,50
110,46-114,52

126,54 129,22 129,06 131,65 126,36

126,38 116,41 121,84 127,81 124,10
115,97 114,00 114,74 114,90 117,84
111,00 111,47 109,72 109,15 114,49

64,40

64,67

64,71 64,72 65,20

64,34 64,79

-CH3

20,40

C7

40,87

C2

32,14

32,15

32,09 28,15 32,11

32,12 32,10

C4

28,14

28,16

28,11 26,68 28,13

28,13 29,06

C6

26,77

26,71

26,67 26,18 26,77

26,76 28,13

C5

25,87

25,86

25,80 25,84 25,80

25,85 25,70

C3

24,95

24,95

24,91 25,03 24,91

24,93 24,95

Ghi ch: l dch chuyn ha hc, * dch chuyn ha hc ca C5-F.

3.4. KT QU TH HOT TNH SINH HC

3.4.1. Th tc dng c ch histon deacetylase
46

Sau khi khng nh cu trc, chng ti tin hnh nh gi kh nng c ch

histon deacetylase trn histon H3, H4 trong t bo ung th i trng SW620 nng
10 g/ml ca cc dn cht IVa-g theo phng php phn tch Western blot. Kt
qu thu c: IVa-c, IVg c ch mnh HDAC nng ny; cn li IVd-f c ch
khng ng k (hnh 3.1). Th nghim c thc hin ti khoa Dc, Trng i
hc Quc gia Chungbuk, Cheongju, Hn Quc.

Hnh 3.1. Kt qu phn tch Western blot ca cc cht IVa g

Ch thch: VH: mu trng, GAPDH: enzym chuyn ha glucosid

3.4.2. Kt qu th hot tnh khng t bo u g th in vitro

Th hot tnh khng t bo ung th ngi in vitro trn 5 dng t bo th
nghim: SW620: t bo ung th i trng, MCF-7: t bo ung th v, AsPC-1: t
bo ung th ty, PC-3: t bo ung th tin lit tuyn, NCI-H460: t bo ung th
phi theo phng php MTT. Th nghim c thc hin ti Khoa Dc, Trng
i hc Quc gia Chungbuk, Cheongju, Hn Quc. Gi tr IC50 c tnh trn phn
mm GraphPad Prism.
Kt qu th hot tnh khng t bo ung th in vitro ca cc cht IVa g c
trnh by trong bng 4.2 (mc 4.3.2).
3.5. KT QU DOCKING
kim tra s b tnh tng tc ca cc cht tng hp c vi HDAC
(docking), chng ti tin hnh docking cht IVa vi HDAC 8 v HDAC2. Qu
trnh docking c thc hin ti phng nghin cu cu trc i hc quc gia Seoul,
Hn Quc.
47

Kt qu docking ca cht IVa v SAHA vi HDAC8 (hnh 3.2) cho thy

IVa gn vi HDAC8 ti trung tm hot ng ca enzym tng t SAHA, nhm
acid hydroxamic lin kt vi Zn2+. Tuy nhin, nng lng lin kt IVa HDAC8
nh hn nng lng lin kt SAHA HDAC8, do i lc ca IVa vi HDAC8
mnh hn so vi SAHA.

Hnh 3.2. Kt qu docking ca cht IVa v SAHA vi HDAC8

Ch thch: SAHA (mu vng), cht IVa (mu tm) v HDAC8 (mu xanh l cy). Ion Zn2+
l hnh cu mu xm m.

Tng t, khi tin hnh docking cht IVa v SAHA vi HDAC2 thu c kt
qu nh sau:

Hnh 3.3. Kt qu docking ca cht IVa v SAHA vi HDAC2

Ch thch: SAHA (mu vng), cht IVa(mu xm bc) v HDAC8 (mu xanh l cy) . Ion
Zn2+ l hnh cu mu xm m.

Chng ti nhn thy: IVa gn vi HDAC2 ti trung tm hot ng ca enzym

tng t SAHA, nhm acid hydroxamic lin kt vi Zn2+. Chui alkyl (6C) gn
48

mnh vo vng thn du ca knh enzym. Nng lng lin kt IVa HDAC2 nh
hn nng lng lin kt SAHA HDAC2, do i lc ca IVa vi HDAC2 mnh
hn so vi SAHA.
3.6. NH GI MC GING THUC CA CC CHT TNG HP
Tnh gi tr logP ca cc cht tng hp c bng phn mm EPTsuite cung
cp bi US Environmental Protection Agencys Office of Pollution Prevention and
Toxics and Syracuse Research Corporation (SRC). Tt c cc cht IVa-g u tha
mn 5 yu cu ca quy tc Lipinsky v ging thuc.
Bng 3.9. nh gi mc ging thuc ca cc cht IVa-g theo quy tc Lipinsky

TT

Cht

LogP

S NH, OH

S N, O

Kt lun

IVa

-H

319.36

2,67

IVb

5-F

337.35

2,87

IVc

5-Cl

353.80

3,31

IVd

5-Br

398.25

3,56

IVe

5-NO2

364.35

2,89

IVf

5-CH3

333.38

3,22

IVg

7-Cl

353.80

3,31

49

Ch g 4. BN LUN
4.1. V HA HC
4.1.1. Tng hp 3-(methoxyimino)-2-oxoindolin v cc dn cht (IIa-g)
-

Phn ng xy ra theo c ch cng hp i nhn AN vo nhm carbonyl, sau

c s loi nc.
-

iu kin ca phn ng ty thuc vo hot tnh ca nhm carbonyl, ph

thuc vo c tnh base ca tc nhn i nhn.

-

Hiu sut phn ng cao 88 94% (bng 3.1), d thc hin.

4.1.2. Phn ng tng hp dy ester trung gian (IIIa-g)

Isatin v cc dn cht 5 (7) isatin phn ng vi ethyl-7 bromoheptanoat theo
c ch th i nhn alkyl (SN2) vi s c mt ca K2CO3 v xc tc KI.
C ch phn ng c trnh by theo s sau:

Trong :

S 4.1. C ch phn ng th i nhn alkyl to IIIa-g

- Phn ng xy ra kh d dng, hiu sut tng i cao, t 70 80% (bng
3.2). 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.
- K2CO3 c s dng hot ha isatin to tc nhn i nhn
- KI xc tc nhm lm tng tc phn ng do to RI c kh nng tham gia
phn ng th i nhn nhanh hn RBr.
- Phn ng th i nhn alkyl xy ra trong h dung mi phn cc DMF:MeOH.
H dung mi ny lm tng tan ca cc cht tham gia phn ng, gip phn ng
xy ra nhanh ng thi trnh nguy c thy phn nhm chc amid v ester.
50

- 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.
4.1.3. Phn ng tng hp dy cht acid hydroxamic (IVa-g)
- Phn ng c s dng tng hp acid hydroxamic trong lun vn l phn
ng th i nhn acyl vi tc nhn th i nhn l nhm amin, nn cn gi l phn ng
N-acyl ha (s 4.2).

S 4.2. Phn ng th i nhn acyl

Trong phn ng trn, tc nhn i nhn l amin. Phn ng tng hp cc acid
hydroxamic t ester v hydroxylamin mi trng trung tnh khng xy ra, n ch
c thc hin pH > 10. Phng php tng hp ch yu l dng xc tc base v
ester trong alcol [12].
Qua tham kho mt s ti liu nh trn v ph hp vi kh nng ha tan
trong dung mi ca cc cht cng nh ha cht sn c ti phng th nghim, phn
ng tng hp acid hydroxamic IVa-g t ester v hydroxylamin c tin hnh vi
xc tc NaOH, dung mi MeOH/THF -5oC trong thi gian t 30 pht n 1gi.
Phn ng din ra nhanh vi hiu sut thu c t 70 78% (bng 3.3). S 4.3
gii thch c ch ca phn ng.

S 4.3. C ch phn ng tng hp acid hydroxamic IVa-g t ester

- Lng NH2OH.HCl cho vo bnh phn ng phi d cn bng dch chuyn
theo chiu thun.
51

- Duy tr nhit thp (-5 C) lm cho phn ng xy ra nhanh v hon ton,

ng thi trnh c s thy phn ester thnh cc acid carboxylic khi c mt
NaOH.
- NaOH c lm lnh v cho t t vo bnh phn ng trnh qu nhit khi
pha long dung mi MeOH bng nc. Lng NaOH s dng cn d m bo
iu kin phn ng v chuyn NH2OH. HCl thnh dng NH2OH.
- Khi acid ha bng acid HCl x l hn hp phn ng, cn lu trnh to
pH vt qu pH ta. Khi pH vt qu pH ta, cc acid hydroxamic to thnh s
chuyn sang dng du, ng thi rt kh ta li.
4.2. V KHNG NH CU TRC
7 cht trong lun n sau khi tng hp xong u c nhn dng cu trc bng
cc phng php ph hng ngoi (IR), phi khi lng (MS) v ph cng hng t
ht nhn 1H v

13

C. D liu cc loi ph c trnh by trong mc 3.2 v ph

trong phn ph lc. Sau y l mt s bn lun v vic nhn dng cu trc thng
qua cc d liu ph.
4.2.1. Ph hng ngoi
Ph hng ngoi bn cht l ph dao ng ca cc nhm chc trong phn t,
cho thng tin v nh hp th ca cc kiu dao ng trong cc nhm chc. Ph
ca cc cht u bao gm hai vng: vng nhm chc c bc sng t 4000 - 1500
cm-1 v vng vn tay c bc sng t 1500 625 cm-1. Cc cht IVa-g trong lun
vn u l nhng cht c cu trc phc tp, v vy ph hng ngoi ca chng cha
rt nhiu di hp th khc nhau, c bit l cc di hp th trong vng vn tay, rt
kh bin gii cho tng nhm chc. V th, trong lun vn chng ti tp trung ch
yu vo vng nhm chc vng cho cc thng tin v nhm chc trong phn t vi
di hp th kh c trng xc nh cc nhm chc c trng ca cc cht tng
hp.
- Tt c 7 cht IVa-g u l acid hydroxamic nn u c vn hp th ca dao
ng ha tr O-H acid mnh t 3400-2500 cm-1[3]. Cc cht IVa-c c cc vn hp
th t ca dao ng ha tr O-H t 3431 3331 cm-1. Cc cht IVd-g khng ghi
nhn nh hp th m to thnh di hp th hnh qu i di 3200 cm-1.
52

- Cc cht tng hp IVa-d, IVf u xut hin vn hp th ca C=N trong hp

cht oxim. Ring IVe, IVg khng ghi nhn nh nhng to thnh vn hp th t
trong vng 1690 1630 cm-1 ca hp cht ny.
- Cc cht IVa-g trong cu trc u c lin kt NH-CO-, nn ph hng ngoi
ca tt c cc cht ny u xut hin vn hp th ca dao ng ha tr N-H trong
amid bc 2 t 3320 3150 cm-1 [3].
- Ngoi lin kt -NH-CO- trong nhm chc acid hydroxamic, cc cht IVa-g
cn c lin kt C=O ca khung 3-methoxim-isatin nn trong ph cc cht IVa,
IVb xut hin 2 vn hp th ca dao ng ha tr C=O. Tuy nhin lin kt -NH-COtrong nhm chc hydroxamic c th xy ra hin tng h bin (hnh 4.1), nn cc
cht IVc-g ch xut hin 1 vn hp th C=O trong khung isatin nm trong di 1725
1680 cm-1.

Hnh 4.1. Hin tng h bin ca nhm chc hydroxamic

- Ph ca c 7 cht u cho thy dao ng ha tr ca lin C=C trong aryl
vi di hp th t 1600 1450 cm-1. Ngoi ra, cc cht IVa, IVb, IVf, IVg cn
xut hin dao ng ha tr ca lin kt C-H (sp2) trong vng 3100 3000 cm-1 ca
aryl. Ph ca cc cht IVc-e khng thy xut hin vn hp th ca lin kt ny
do b vn hp th ca OH (acid hydroxamic) che lp.
- Phn cu ni alkyl ca cc cht tng hp c cng cho thy s c mt trn
ph vi dao ng ha tr ca -CH2 (sp3) nm trong di t 2970 2850 cm-1 [3].
- i vi cc nhm th trn aryl, chng ti nhn thy s xut hin ca vn hp
thu ca nhm -NO2 ti 1522 cm-1, nm trong di 1370 1300 cm-1 l vng hp thu
mnh ca dao ng ha tr NO2 trong hp cht thm. Cc nhm th halogen -F, -Cl,
-Br c dao ng ha tr nm trong vng vn tay, rt kh xc nh bng ph hng
ngoi [3].
Nh vy, ph IR cho thy mt s nhm chc v lin kt trong cu to cc
cht tng hp . Tuy nhin, s hp thu hng ngoi ca cc nhm chc c th thay

53

i trong mt min rng do s tng tc phc tp ca cc dao ng bn trong phn

t, v th gii on ph hng ngoi cn s ngoi suy t ph ca cc hp cht n
gin hn. iu ny l gii ti sao khng th c s x l chnh xc cc dao ng ca
mt phn t phc tp, ging nh cc cht m chng ti tng hp. V th,
khng nh chc chn cu trc ca cc cht cn nghin cu cc s liu ca ph khi
lng v ph cng hng t ht nhn.
Sau y l hnh nh minh ha ph hng ngoi ca cht IVa.
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
Ten may: GX-PerkinElmer-USA Resolution: 4cm-1 Date: 11/9/2012

Nguoi do: Phan Thi Tuyet Mai

Ten mau: 6A

100.0
95
90
85
80
75
70

811
1314

65

503

1193
1094

60

1539

1204

55
3038

%T

939

593
495
558

783

1126

1444

692
1049

616

1115

2824

50

864

1349

45

2862

745

1081

2933

40

1377

35

3399

30

1004

1464

1029

3249

25

1621

20

1647

15

1704
1607

10
5
0.0
4000.0

3600

3200

2800

2400

2000

1800

1600

1400

1200

1000

800

600

400.0

cm-1

Hnh 4.2. Ph hng ngoi ca cht IVa

4.2.2. Ph khi lng
Ph khi lng ng vai tr quan trng trong khng nh cu trc cc cht
tng hp v thng l ph c la chn u tin kim chng xem sn phm
phn ng c kh nng s l cht d kin hay khng.
Trn ph ca 7 cht u xut hin pic ion c s khi bng [M-H]- (ch
ESI (-)). Pic ion phn t ca cc hp cht khng phi l vch ring l m l mt
cm pic do cc nguyn t cha trong hp cht u tn ti cc ng v nh
54

13

C l

ng v ca 12C, 2H l ng v ca 1H, 33S,

15

N l ng v ca 14N, 37Cl l ng v

ca 35C, 18O l ng v ca 16O... Bi vy, bn cnh vch chnh ng vi pic ion phn
t M+ cn c cc vch [M-1]-, [M]-, [M+1]-...
Phn tch ph ca cht IVa lm v d. Cht IVa c CTPT d kin l
C16H21N3O4 tng ng vi s khi 319. Ph ca IVa (hnh 4.3) cho thy cm pic
ion phn t gm cc ion ng v c s khi l M-1, M, M+1 (hnh 4.3) v pic [M1]- cng ln nht (100%) c s khi 318. Nh vy, s b cho thy cht IVa c
s khi ng nh s khi d kin.

Hnh 4.3. Ph khi lng ca cht IVa

4.2.3. Ph c g hng t ht nhn
Cng vi d liu ph hng ngoi v ph khi, 7 cht tng hp cn c o
ph 1H-NMR v 13C-NMR khng nh chnh xc cu trc ca sn phm. D liu
ph cng hng t ht nhn ca cc cht c trnh by trong mc 3.2 v ph
trong phn ph lc. Sau y l mt s bn lun da trn d liu ph cng hng t
chng minh cu trc cc cht tng hp.

55

4.2.3.1. Ph 1H NMR

a. Khung 3-methoxim-isatin c cc proton c xc nh r rng trn ph

Proton H-4 c dch chuyn ha hc trong khong 7,49 7,94 ppm, vn
ph c dng singlet (s) hoc doublet (d).
Cht IVa, IVg khng c nhm th ti v tr s 5, do H-4 tng tc vi H5 (ortho) vi hng s tng tc Jortho= 7,5 8,0 Hz, vn ph dng doublet (d) (hnh
4.4).
Cc cht IVb-f u l c cc nhm th ti v tr 5, tnh cht ht y in t
ca nhm th c nh hng n chuyn dch ha hc cng nh dng vn ph ca
H-4. Cc nhm th ht in t mnh -NO2 hoc cc nguyn t c m din ln F, -Cl, -Br lm gim s chn ti ch nn tng dch chuyn ha hc. m in
ca cc nhm ny cng ln, th dch chuyn ha hc cng ln, proton c xu
hng chuyn dch sang pha t trng yu. Tuy nhin, khi cc nhm th ny bn
cnh proton, chng c th to dng in v to mt t trng xung quanh H-4, to
ra s chn bt ng hng. V th, thay v dch chuyn v pha trng yu khi
m in ca nguyn t bn cnh tng ln, cc cht tng hp ln lt dch chuyn
theo hng ngc li theo chiu t IVd, IVc, IVb, IVe v pha t trng mnh.
Ngoi ra, cc nguyn t - Cl, -Br to ra s cn tr khng gian gia H-4 v H-6,
nn vn ph c dng singlet (s), trong khi cc nhm th -NO2, -F c dng doublet
(d).
Cht IVf c nhm th -CH3 do tnh cht y in t yu nn dch chuyn
ha hc gn nh khng thay i 7,73 7,74 ppm, vn ph dng singlet (s).
Proton H-5 ca cc cht IVa, IVg c dch chuyn l 7,46 v 7,09 ppm,
vn ph c dng triplet do tng tc vi H-4 v H-6. Hng s tng tc H-4 v
H-6 (Jortho) ln, nm trong khong 7,5 8,0 Hz nn vn ph c dng triplet (hnh
4.4).
56

 Proton H-6c dch chuyn ha hc t 7,07 8,35 ppm (thay i ty

thuc vo nh hng ca nhm th 5-R hoc 7-R) v tng tc vi proton l H-7
(vi dn cht c nhm th 5-R) hoc H-5 (vi n cht c nhm th 7-R) th vn
ph c dng doublet (d) (IVc, IVd, IVf, IVg), mt s cht quan st thy c tng
tc vi H-4 nn vn ph c dng doublet of doublets (dd) (IVe), vn ph dng
triplet of doublets (td) do c thm tng tc vi F (IVb). Ring cht IVa khng c
nhm th trn khung 3-methoxim-isatin nn proton H-6 c th tng tc vi H-5
v H-4 (Jortho Jpara). Hng s tng tc ortho ln, nm trong khong 7,5 9,25 Hz
(IVa-g), cn tng tc meta c J thp, khong 2,5 Hz (IVb, IVe).
Proton H-7 ca cc cht IVa-f c dch chuyn ha hc t 7,00 7,35
ppm v tng tc vi proton l H-6 th vn ph c dng doublet (d), cht IVb quan
st thy c tng tc vi H-5 nn vn ph c dng doublet of doublets (dd).
Sau y l ph 1H NMR ca IVa.

Hnh 4.4. Ph 1H NMR dn rng ca IVa

Trn l thuyt, cc H trn vng thm u tng tc vi nhau to ra mt h
thng multiplet phc tp. Thc t, ph trong nhng trng hp trn cho thy
hng s tng tc gia cc proton l rt nh v c th b qua (Jmeta, Jpara).
57

b. Cc proton mch nhnh

Mt nhm cc proton quan trng khc trong cu trc ca cc dy cht trn l
cc proton mch nhnh. C 7 cht u c s proton mch nhnh vi dch
chuyn ha hc ca cc nhm -CH2- trong khong 1,24 3,95 ppm. Do hiu ng
ht in t mnh ca nhm chc amid v vng 3-oxim-isatin lm ht nhn b gim
s chn ti ch, dch chuyn ca H-7 l ln nht, sau n H-2 (hnh 4.5).

Hnh 4.5. Ph 1H NMR dn rng ca IVg

Chng ti tin hnh so snh ph 1H-NMR ph ca tng cp cht tng
ng vi nhau trong dy cht IVa-g tng hp c vi dy cht 19a-g trong lun
vn ca Thc s Nguyn Th M [4]. C th, khi so snh ph 1H-NMR ca 19e
(hnh 4.6) v IVe (hnh 4.7) thu c kt qu trong bng 4.1.

58

Hnh 4.6. Ph cng hng t 1H NMR ca 19e

Hnh 4.7. Ph cng hng t 1H NMR ca IVe

59

Bng 4.1. Bng so snh ph cng hng t 1H NMR ca 2 cht 19e v IVe

V tr cc proton

dch chuyn ha
hc
(ppm)
19e
IVe

Nhn xt
- dch chuyn ha hc ca

H-4

8,67

8,49

H-6

8,34

8,35

H-7

7,35

7,35

H-7

3,75

3,73

NH, -OH) ca 2 cht l tng

H-2

1,92

1,92

ng nhau.

H-3

1,58-1,59

1,54-1,58

H-6

1,46-1,47

1,43-1,49

H-4, H-5

1,27-1,29

1,26-1,27 - Pic OH (3-oxim) trn ph

-OH

14,13

-OCH3

4,29

Acid

-NH

10,34

10,33

hydroxamic

-OH

8,67

8,66

Khung isatin

H-6, H-7, cc proton trong

mch nhnh v proton trong

Mch nhnh
-CH2-

Oxim

nhm chc acid hydroxamic (-

- dch chuyn ha hc ca
proton H-4 2 cht khc
nhau.
cht 19e khng thy xut
hin trn ph cht IVe.
- Pic -OCH3 (3-oxim) ca
IVe khng xut hin trn ph
ca 19e.

So snh ph ca cc cp cht cn li trong 2 dy chng ti vn thu c kt

qu tng t. iu ny chng t nhm 3-oxo cc ester trung gian ca 19a-g
phn ng vi hydroxylamin, ng thi s thay i dch chuyn ha hc ca H-4
nh trn ch ra s hin din ca nhm oxim v tr 3 ca cc dn cht 19a-g.
60

iu ny cng c ngha, phn ng to oxim ca I xy ra (s 3.1, mc 3.1) .

Ngoi ra, ti v tr 3-oxim ca IVa-g khng thy xut hin pic dng singlet ti
13,20 14,32 ppm ca OH nh trong ph cc cht 19a-g, thay vo ph
ca cc cht IVa-g xut hin pic dng singlet ti 4,19 4,29 ppm ca OCH3.
Cu trc ca cc cht IVa-g c nhm chc hydroxamic v lin kt amid nn
cn quan st thy s c mt ca 2 singlet c dch chuyn ha hc 10,30 12,77
ppm v 8,62 8,78 ppm tng ng vi proton NH v -OH (acid hydroxamic).
Cc dn cht c nhm th cha proton trn khung 3-oxim-isatin nh -CH3, OCH3 cho tn hiu ca cc proton trong nhm th. C th nh cc cht IVa-g u
c nhm methoxy -OCH3 cho vn ph dng singlet ca 3H trng thp t 4,19
4,29 ppm. Ring cht IVf c nhm th -CH3 s quan st thy vn ph singlet ca
3H = 2,27 ppm.
4.2.3.2. Ph 13C NMR
Ph 13C-NMR cho thng tin trc tip v khung carbon ca phn t gip cho
vic xc nh cu trc d dng hn. S tng tc spin gia carbon carbon l rt
him (do ht nhn

13

C chim t l thp), cn tng tc spin C-H c kh ghp

spin hon ton nn tn hiu 13C-NMR l cc mi n, d phn tch.

Carbon trong nhm carbonyl (C=O ca acid hydroxamic) c dch chuyn

ha hc t 168,69 169,10 ppm.

-

8 carbon ca khung 3-methoxim-isatin c dch chuyn ha hc nm trong

khong 109,15 162,57 ppm. Trong , C2, C3 v C8 do gn vi d t O, N nn

cng hng trng thp hn ( cao hn) so vi cc carbon C4, C5, C6, C7 v
C9. Tuy nhin, ty thuc vo bn cht ca nhm th 5-R m dch chuyn ha
hc ca 8 carbon ca khung 3-oxim-isatin c th thay i tng hoc gim so vi cc
cht khng gn nhm th.

61

Phn cu ni alkyl cho tn hiu cng hng ca carbon trong khong 24,91

40,87 ppm. Trong , C7 v C2 gn vi nhm ht in t mnh nn dch chuyn v

pha trng yu, chng c dch chuyn ha hc ln hn so vi cc C cn li
trong cu ni.
- Ph cc cht cng u cho tn hiu cng hng ca cc proton c trn
nhm th -OCH3 ( = 64,34 65,20 ppm). Cht IVf c nhm -CH3 vi = 20,40
ppm. Ring cht IVb c gn nhm th 5-F nn cn xut hin tng tc C5-F.
Ngoi ra, do nh hng ca Flo, tn hiu ca C trong vng thm (C4, C6, C7, C9)
b tch i tn hiu thnh 4 cp t 110,46 119,23 ppm.
Sau y l minh ha ph 13C-NMR ca cht IVf.

62

Hnh 4.8. a, Ph 13C NMR ca cht IVf.

b, Ph 13C NMR dn rng ca cht IVf
63

4.3. V HOT TNH SINH HC

4.3.1. V tc dng c ch HDAC
Kt qu phn tch Western blot ca cc cht IVa-g cho thy IVa-c v IVg c
ch mnh HDAC nng 1 M. Hnh nh kt qu in di trn gel (hnh 3.1): vt
m ca IVa-c, IVg biu th s c mt ca acetyl-histon H3 v acetyl-histon H4,
chng t cc cht ny c ch c HDAC nn cc acetyl-histon H3 v acetylhiston H4 khng b deacyl ha. Trong khi , cc cht IVd, IVe c kt qu in di
trn gel l cc vt m, chng t 2 cht ny c kh nng c ch HDAC nhng hiu
lc c ch khng ng k. Vi cht IVf, khng thy vt ca acetyl-histon H3 v
acetyl-histon H4 trn hnh nh in di, cho thy 2 histon ny b deacetyl ha.
Nh vy nng 1 M, cht IVf khng th hin tc dng c ch HDAC.
Khi so snh kt qu phn tch Western blot ca cc cht IVa-g vi cc cht
19a-g c tng hp trong lun vn Thc s ca Nguyn Th M [4], chng ti
nhn thy:

Hnh 4.9. Kt qu phn tch Western blot ca 2 dy cht 19a-g v IVa-g

64

C 2 cht 19e v IVe (nhm th -NO2 v tr 5) u khng c tc dng c

ch HDAC nng 1 M. Nh vy c th kt lun rng s hin din ca nhm

th 5-NO2 trn khung 3-methoxim-isatin gy cn tr cho vic c ch HDAC.
-

Cc cht 19d, 19f c tc dng c ch HDAC nng 1 M, trong khi cc

cht IVd, IVf hu nh khng c tc dng nng ny. iu ny cho thy s hin
din ca nhm th cng knh nh -Br, -CH3 v tr s 5 trn khung 3-oxim-isatin
cng vi vic methyl ha ca nhm 3-oxim (IVd, IVf) khng thun li cho vic c
ch enzym.
4.3.2. V tc dng khng t bo u g th in vitro
Vi cc acid hydroxamic thit k, tc dng c ch HDAC l mt yu t
quan trng 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 enzym. V th, cc cht IVa-g tip tc c tin hnh th c tnh t bo in
vitro nhm tm ra nhng cht thc s c tc dng sinh hc tt.
Th hot tnh khng t bo ung th ngi in vitro trn 5 dng t bo th
nghim: SW620: t bo ung th i trng, MCF-7: t bo ung th v, AsPC-1: t
bo ung th ty, PC-3: t bo ung th tin lit tuyn, NCI-H460: t bo ung th
phi theo phng php MTT. Kt qu c trnh by trong bng 4.2.
Kt qu th c tnh t bo cho thy c 7 cht th nghim u c tc dng c
ch trn c 5 dng t bo, c bit l 2 dng t bo SW620 (0,26 2,32 M) v
MCF-7 (0,34 7,79 M).
Cc cht IVa-c v c bit l IVg th hin tc dng c ch HDAC mnh vi
biu hin histon-H3, histon-H4 khng b deacetyl ha trong phn tch Western blot
l nhng cht gy c mnh trn c 5 dng t bo th nghim. Cc cht IVd v IVf
l nhng cht hu nh khng gy ra tc dng c ch HDAC l nhng cht c c
tnh t bo thp nht trong dy IVa-g. Nhng ng ch , vi cht IVe chng ti
nhn thy c s khc bit v hot tnh sinh hc so vi cc dn cht cn li trong
dy. Mc d IVe khng gy tc dng c ch enzym HDAC nng 1 M th
nghim Western blot, nhng khi th nghim tc dng khng t bo ung th in vitro,
IVe cho thy kh nng c ch mnh trn 4 dng t bo (SW620, MCF-7, PC-3,
65

AsPC-1) vi nng 0,84 1,69 M. Cht ny ch c c tnh thp vi dng t bo

NCI-H460 vi nng 14,7 M. iu ny chng t IVe c th khng c ch
HDAC nhng c ch yu t khc trong chu trnh pht trin ca t bo.
Bng 4.2. Kt qu th hot tnh khng t bo ung th in vitro ca cc cht IVa - g

TT Cht

c tnh t bo (IC50)1 , M

KLPT
SW620

MCF-7

PC3

AsPC-1

NCI-H460

IVa

-H

319,36

0,73

1,71

1,67

1,22

0,75

IVb

5-F

337,35

1,11

2,42

1,15

0,97

0,97

IVc

5-Cl

353,80

0,49

1,56

2,33

0,49

0,76

IVd

5-Br

398,25

2,32

7,79

8,91

1,68

2,90

IVe

5-NO2

364,35

1,35

0,94

1,69

0,84

14,27

IVf

5-CH3

333,38

1,07

16,67

0,35

5,48

1,19

IVg

7-Cl

353,80

0,26

0,34

0,29

0,63

0,35

264,32

3,70

6,42

4,31

3,66

2,77

SAHA2

Ch thch:

Nng c ch 50% s pht trin ca t bo;

Acid suberoylanilid

hydroxamic

Khi so snh c tnh trn t bo ca 7dn cht IVa-g vi SAHA, chng ti

nhn thy i vi tng dng t bo, hot tnh khng t bo ung th in vitro ca cc
dn cht c nhm th trn khung 3-methoxim-isatin l khc nhau so vi SAHA.

66

IC50
(M)
18

16

14

IVa

12

IVb
IVc

10

IVd
IVe

IVf
IVg
6

SAHA

0
SW620

MCF-7

PC-3

AsPC-1

NCI-H460

Dng t bo

Hnh 4.10. Biu so snh tc dng khng t bo ung th in vitro ca cc dn cht

IVa-g
-

Trn dng t bo SW620: Tt c cc dn cht IVa-g u c hot tnh mnh

hn SAHA, trong IVg (nhm th 7-Cl) v IVc (nhm th 5-Cl) l hai dn cht
c hot tnh mnh nht vi IC50 ln lt l 0,26 M v 0,49 M, mnh hn SAHA
14 ln v 8 ln trong cng th nghim. IVd (nhm th 5-Br) l cht c hot tnh
khng t bo ung th thp nht trong dy vi IC50 = 2,32 M.
-

Trn dng t bo MCF-7: IVg (nhm th 7-Cl) l cht c hot tnh mnh

nht trong dy IVa-g (IC50 = 0,34 M) v mnh hn SAHA (IC50 = 6,42 M) 19

ln. Cc dn cht IVa-c, IVe u c hot tnh mnh hn SAHA, cn li IVf (nhm
th 5-CH3, IC50 = 16,67 M) v IVd (nhm th 5-Br, IC50 = 7,79 M) l hai cht c
hot tnh thp nht, u yu hn SAHA.
67

Trn dng t bo PC-3: IVg (nhm th 7-Cl) l cht c hot tnh mnh nht

(IC50 = 0,29 M), gp 15 ln so vi SAHA (IC50 = 4,31 M). Tt c cc cht cn li

u c hot tnh mnh hn SAHA, tr IVd (nhm th 5-Br, IC50 = 8,91 M).
-

Trn dng t bo AsPC-1: IVc (nhm th 5-Cl) v IIg (nhm th 7-Cl) l hai

dn cht c hot tnh mnh nht vi IC50 ln lt l 0,49 M v 0,63 M, cao gp 7

v 6 ln SAHA (IC50 = 3,66 M). IVf (nhm th 5-CH3, IC50 = 5,48 M) l cht c
hot tnh thp nht v l dn cht duy nht trong dy IVa-g c hot tnh thp hn
SAHA trn dng t bo ny.
-

Trn dng t bo NCI-H460: IVg (nhm th 7-Cl) l cht c hot tnh mnh

nht (IC50 = 0,35 M), gp 8 ln so vi SAHA (IC50 = 2,77 M). IVe (nhm th 5NO2, IC50 = 14,27 M) l cht c hot tnh thp nht trong dy.
Nh vy, trong dy IVa-g, tt c cc cht IVa-c, IVg u c hot tnh khng
t bo ung th in vitro mnh hn SAHA trn c 5 dng t bo th nghim, ch c
IVd v IVf l c hiu lc thp hn SAHA trn dng t bo MCF-7. iu cho
thy nhm th cng knh v tr s 5 trn vng isatin c nh hng bt li i vi
c tnh ca cc dn cht 3-methoxim-isatin (IVd, IVf).
T cc kt qu trn, c th thy nhm th 5-Cl, c bit 7-Cl trn vng isatin
ca dn cht 3-methoxim-isatin lm tng thm hot tnh ca cc dn cht ny. Cht
IVb c hot tnh km hn IVc 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.
Tin hnh so snh tc dng khng t bo ung th ca 2 dy IVa-g v 19a-g
c tng hp trong lun vn Thc s ca Nguyn Th M [4], chng ti thu c
kt qu trong bng 4.2.

68

Bng 4.3. So snh tc dng khng t bo ung th ca 2 dy IVa-g v 19a-g

TT

c tnh t bo (IC501, M)

Cht
SW620

MCF-7

PC3

AsPC-1

NCI-H460

IVa/19a 0,73

0,64

1,71

0,79 1,67

0,98

1,22

1,10

0,75

0,89

IVb/19b 1,11

0,11

2,42

1,55 1,15

2,73

0,97

1,72

0,97

1,50

IVc/19c 0,49

0,65

1,56

0,68 2,33

0,85

0,49

1,86

0,76

0,85

IVd/19d 2,32

0,29

7,79

1,77 8,91

2,21

1,68

0,08

2,90

0,97

IVe/19e 1,35

3,39

0,94

1,34 1,69 19,69 0,84

4,68

14,27

7,71

IVf/19f 1,07

0,99

1,19 0,35

0,62

5,48

2,44

1,19

2,09

IVg/19g 0,26

1,05

16,6
7
0,34

1,74 0,29

1,57

0,63

1,82

0,35

1,35

SAHA2
Ch thch:

3,70
1

6,42

4,31

3,66

Nng c ch 50% s pht trin ca t bo;

2,77
2

Acid suberoylanilid

hydroxamic

So vi tt c cc cht trong 2 dy trn, chng ti nhn thy cht IVg l cht c

hot tnh khng t bo ung th mnh nht trn c 5 dng t bo. Tuy nhin, nu xt
trn tng th, hot tnh khng t bo ung th ca cc cht dy IVa-g c xu hng
yu hn so vi cc cht dy 19a-g. iu ny c th d on c t:
Kt qu docking cc cht i din:
-

Cc cht dy 19a-g c nhm OH (3-oxim) c kh nng to lin kt hydro

ni phn t vi nhm carbonyl v tr 2, trong khi cc cht dy IVa-g
khng h c lin kt ny. l iu kin thun li hn cho cc cht 19a-g d
dng qua c mng sinh hc hn so vi cc cht IVa-g.
69

Nhm methyl (3-oxim) cng knh lm cho cc dn cht IVa-g b cn tr v

mt khng gian nn kh thm qua mng sinh hc hn so vi cc dn cht
19a-g.

Ch s logP ca cc cht trong dy 19a-g [4] v IVa-g cho thy cc cht

IVa-g u c logP thp hn so vi cht 19a-g tng ng trong dy (bng 4.4).
Bng 4.4. So snh gi tr logP ca cc cht 19a-g v IVa-g
TT

Cht

LogP

R
19

IV

5-H

2,69

2,67

5-F

2,90

2,87

5-Cl

3,34

3,31

5-Br

3,58

3,56

5-NO2

2,91

2,89

5-CH3

3,24

3,22

7-Cl

3,34

3,31

iu ny chng t, cc dn cht 19a-g c tnh thn du hn so vi cc dn

cht IVa-g, v th cc dn cht 19a-g c kh nng tng tc cht ch hn vi knh
enzym HDAC thn du.
Nhm OH (3-oxim) ngoi kh nng to lin kt hydro ni phn t, cn c
kh nng to lin kt hydro vi cc acid amin ming knh enzym, do kh nng
gn cht vi enzym ca dy cht 19a-g tt hn so vi dy cht IVa-g.
So snh c 2 dy cht trn vi SAHA, chng ti thy phn nhn din b mt
ca cc cht ny c thm 2 nguyn t N, s c mt ca chng c l lm tng kh
nng tng tcvi 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 chng.
Cc cht IVa-g trong lun vn tip tc duy tr phn cu ni 6C, nhm acid
hydroxamic gn Zn2+, nhng thay i nhm nhn din b mt. So vi SAHA, nhm
nhn din b mt phenyl c thay th bng nhm 3-methoxim-isatin. Mc tiu
70

ca nghin cu l thit k, tng hp c dy dn cht mi, ng thi xc nh kt

qu ca vic thay i nhm nhn din b mt, xa hn l xem xt nh hng ca
nhm th ti cc v tr khc nhau trn vng isatin. Kt qu cho thy hot tnh sinh
hc rt tt ca cc acid hydroxamic IVa-g. Ngoi ra, cc cht u tha mn c
yu cu ca quy tc Lipinsky v ging thuc.
Nh vy, cng vi cc kt qu nghin cu trc y [6, 10, 11] c th thy
cu ni 6C v nhm gn Zn2+ tng t SAHA l yu t quan trng quyt nh c
tnh t bo ca cc acid hydroxamic hng c ch histon deacetylase. Bn cnh ,
vic la chn nhm nhn din b mt l 3-methoxim-isatin trong nghin cu cho
hot tnh tt, mnh hn gp nhiu ln so vi SAHA, c bit l cc cht IVa-c, IVg.
iu ny m ra trin vng cho vic nghin cu cn lm sng v lm sng ca cc
dn cht sau ny.

71

KT LUN V XUT
1. KT LUN
1.1. V tng hp ha hc v kh g nh cu trc
tng hp c 7 dn cht ca acid hydroxamic cha thy cng b trong
bt c ti liu no trn th gii cng nh trong nc.

N-hydroxy-7-(3-(methoxyimino)-2-oxoindolin-1-yl)heptanamid

(IVa)

7-(5-fluoro-3-(methoxyimino)-2-oxoindolin)-N-hydroxyheptanamid

(IVb)

7-(5-cloro-3-(methoxyimino)-2-oxoindolin)-N-hydroxyheptanamid

(IVc)

7-(5-bromo-3-(methoxyimino)-2-oxoindolin)-N-hydroxyheptanamid

(IVd)

N-hydroxy-7-(3-(methoxyimino)-5-nitro-2-oxoindolin)heptanamid

(IVe)

N-hydroxy-7-(3-(methoxyimino)- 5-methyl-2-oxoindolin)heptanamid

(IVf)

7-(7-cloro-3-(methoxyimino)-2-oxoindolin)-N-hydroxyheptanamid

(IVg)

kim tra tinh khit v khng nh cu trc ca cc cht tng hp c

1.2. V hot tnh sinh hc
Kt qu th hot tnh sinh hc ca cc cht IVa-g cho thy IVa-c v IVg c
ch mnh HDAC; cht IVd, IVe c kh nng c ch HDAC nhng hiu lc c ch
khng ng k; IVf khng c tc dng c ch HDAC nng 1 M.
Tc dng khng t bo ung th in vitro: 7 cht u c tc dng khng 5 dng
t bo ung th, trong c 4 cht IVa-c, IVg tc dng mnh hn SAHA trn c 5
dng t bo. c bit, trn dng t bo SW620 cht IVg (nhm th v tr s 7 trn
khung isatin) vi IC50 = 0,26 M c tc dng mnh gp 14 ln SAHA (IC50 = 3,7
M), trn dng t bo MCF-7 vi IC50 = 0,34 M mnh gp 19 ln SAHA (IC50 =
6,42 M), trn dng PC-3 vi IC50 = 0,29 M mnh gp 15 ln SAHA (IC50 = 4,31
M).
2. XUT

72

Tip tc nghin cu v pht trin cc dn xut ca acid hydroxamic mang

khung 3-methoxim-isatin mang nhm th v tr s 7 trn khung isatin.

-

Thnh cng bc u cho thy cht 7-(7-cloro-3-(methoxyimino)-2-

oxoindolin)-N-hydroxyheptanamid (IVg) cn nghin cu tin lm sng vi hy vng

c th tr thnh ng c vin lm sng iu tr ung th.

73

TI LIU THAM KHO

Ting Vit
1. Th Mai Dung (2013), Tng hp v th hot tnh sinh hc ca mt s
acid hydroxamic mang khung 1,3.4 thiadiazol, Kha lun tt nghip dc
s, i Hc Dc H Ni.
2. V Phng ng (2012), Tng hp N1-hydroxy-N8-(5-phenyl-1,3,4thiadiazol-2-yl)octandiamid v mt s dn cht hng c ch histon
deacetylase, Kha lun tt nghip dc s, i Hc Dc H Ni.
3. 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.
4. Nguyn Th M (2013), Tng hp mt s acid hydroxamic mang khung 3oxim-isatin hng tc dng khng ung th, Lun vn thc s, i hc Dc
H Ni.
5. Nguyn Hi Nam (2012), Mt s mc tiu phn t v ng dng trong nghin
cu pht trin thuc iu tr ung th hin nay, NXB Y hc, trang 103-186.
6. o Th Kim Oanh (2013), Tng hp v th hot tnh sinh hc ca mt s
dn cht acid hydroxamic hng c ch enzym histon deacetylase, Lun n
tin s dc hc, i hc Dc H Ni.
7. V Th Thanh Tm (2013), Tng hp N1-hydroxy-N8-(5-phenyl-1,3,4thiadiazol-2-yl)octandiamid v mt s dn cht hng c ch histon
deacetylase, Kha lun tt nghip dc s, i Hc Dc H Ni.
Ting Anh
8. Ajamian F., et al. (2004), "Selective regulation of class I and class II histone
deacetylases expression by inhibitors of histone deacetylases in cultured
mouse neural cells", Neuroscience Letters, 365(1), p. 64-68.
9. Annemieke

J.M.,

et

al.

(2003),

"Histone

deacetylases

(HDACs):

characterization of the classical HDAC family", Biochem. J. , 370, p. 737

749.

10. Andrianov V., et al. (2008), Novel amide derivatives as inhibitors of histone
deacetylase: Design, synthesis and SAR, European Journal of Medicinal
Chemistry, 226, 1-19.
11. Andrianov V., et. al. (2009), Novel amide derivatives as inhibitors of
histone deacetylase: Design, synthensis and SAR, European Journal of
Medicinal Chemistry, 44, 1067-1085.
12. Bernard Miller (1980), Organic chemistry: The basic of life, The
Benjamin/Cummings Publishing Company, Inc, USA, 321-324, 355-364.
13. Betrand P. (2010), Inside HDAC with HDAC inhibitors, Eur. J. Med.
Chem, 45, 2095 2116.
14. Bieliauskas A.V., et. al (2007), Structural requirement of HDAC inhibitors:
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PH LC PH
Ph lc 1-1 n 1-4: Ph IR, MS, 1H-NMR, 13C-NMR ca cht IVa
Ph lc 2-1 n 2-4: Ph IR, MS, 1H-NMR, 13C-NMR ca cht IVb
Ph lc 3-1 n 3-4: Ph IR, MS, 1H-NMR, 13C-NMR ca cht IVc
Ph lc 4-1 n 4-4: Ph IR, MS, 1H-NMR, 13C-NMR ca cht IVd
Ph lc 5-1 n 5-4: Ph IR, MS, 1H-NMR, 13C-NMR ca cht IVe
Ph lc 6-1 n 6-4: Ph IR, MS, 1H-NMR, 13C-NMR ca cht IVf
Ph lc 7-1 n 7-4: Ph IR, MS, 1H-NMR, 13C-NMR ca cht IVg

Ph lc 1-1: Ph IR ca cht IVa

Ph lc 1-2: Ph MS ca cht IVa

Ph lc 1-3: Ph 1H-NMR ca cht IVa

Ph lc 1-4: Ph 13C-NMR ca cht IVa

Ph lc 2-1: Ph IR ca cht IVb

Ph lc 2-2: Ph MS ca cht IVb

Ph lc 2-3: Ph 1H-NMR ca cht IVb

Ph lc 2-4: Ph 13C-NMR ca cht IVb

Ph lc 3-1: Ph IR ca cht IVc

Ph lc 3-2: Ph MS ca cht IVc

Ph lc 3-3: Ph 1H-NMR ca cht IVc

Ph lc 3-4: Ph 13C-NMR ca cht IVc

Ph lc 4-1: Ph IR ca cht IVd

Ph lc 4-2: Ph MS ca cht IVd

Ph lc 4-3: Ph 1H-NMR ca cht IVd

Ph lc 4-4: Ph 13C-NMR ca cht IVd

Ph lc 5-1: Ph IR ca cht IVe

Ph lc 5-2: Ph MS ca cht IVe

Ph lc 5-3: Ph 1H-NMR ca cht IVe

Ph lc 5-4: Ph 13C-NMR ca cht IVe

Ph lc 6-1: Ph IR ca cht IVf

Ph lc 6-2: Ph MS ca cht IVf

Ph lc 6-3: Ph 1H-NMR ca cht IVf

Ph lc 6-4: Ph 13C-NMR ca cht IVf

Ph lc 7-1: Ph IR ca cht IVg

Ph lc 7-2: Ph MS ca cht IVg

Ph lc 7-3: Ph 1H-NMR ca cht IVg

Ph lc 7-4: Ph 13C-NMR ca cht IVc