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B GIO DC V O TO

B Y T

TRNG I HC DC H NI

TRN TH BCH LAN

TNG HP V TH TC DNG SINH HC CA


MT S ACID HYDROXAMIC
MANG KHUNG 5-ARYL-1,3,4-THIADIAZOL 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

TRN TH BCH LAN

TNG HP V TH TC DNG SINH HC CA MT


S ACID HYDROXAMIC
MANG KHUNG 5-ARYL-1,3,4-THIADIAZOL 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:

TS. o Th Kim Oanh


PGS.TS. Nguyn Hi Nam

H NI 2014

LI CM N
Trong qu trnh thc hin lun vn, ti nhn c s hng dn tn tnh
v nhiu gip qu bu ca cc thy c, ng nghip, gia nh v bn b.
T tn y lng mnh, ti xin gi li cm n chn thnh v s bit n su
sc ti TS. o Th Kim Oanh v PGS.TS. Nguyn Hi Nam, nhng ngi thy
ch dn ti tn tnh v to mi iu kin tt nht cho ti hon thnh lun vn.
Ti xin chn thnh cm n cc anh ch em lm vic v thc hin ti ti
b mn Ha dc, c bit cc em Th Mai Dung v Lng Xun Huy,
ng h v gip ti rt nhit tnh trong sut qu trnh nghin cu.
Ti cng nhn c s phi hp v gip t cc cn b lm vic ti Khoa
Ha trng i hc Khoa hc t nhin i hc quc gia H Ni, Vin Ha
hc cc hp cht t nhin, Vin ha hc Vit Nam, Vin Khoa hc v Cng Ngh
Vit Nam, cng ton th cc thy c trong cc phng, ban, th vin. Ti xin chn
thnh cm n.
Cui cng, ti xin gi li cm n su sc ti gia nh v b bn nhng
ngi lun st cnh, ng vin v khch l ti trong cuc sng v hc tp.
H Ni, ngy 30 thng 08 nm 2014
Hc vin

Trn Th Bch Lan

MC LC
DANH MC CC K HIU, CH VIT TT
DANH MC CC BNG
DANH MC CC HNH V, S
T VN ......................................................................................................... 1
Chng 1. TNG QUAN ....................................................................................... 2
1.1. HISTON DEACETYLASE (HDAC) .......................................................... 2
1.1.1. Khi nim, phn loi ............................................................................. 2
1.1.2. Cu trc ca HDAC .............................................................................. 3
1.1.3. Mi lin quan gia ung th v s bt thng hot ng ca HDAC ... 3
1.2. CC CHT C CH HDAC ..................................................................... 4
1.2.1. Phn loi cc cht c ch HDAC (HDIs) ............................................. 4
1.2.2. C ch tc dng .................................................................................... 6
1.2.3. Cu trc ca cc cht c ch HDAC .................................................... 6
1.3. TNH HNH NGHIN CU TRN TH GII V CC ACID
HYDROXAMIC C CH HDAC ..................................................................... 7
1.4. TNH HNH NGHIN CU TRONG NC V CC CHT C CH
HDAC ............................................................................................................... 15
Chng 2. NGUYN VT LIU, TRANG THIT B, NI DUNG V
PHNG PHP NGHIN CU ......................................................................... 19
2.1. NGUYN VT LIU ............................................................................... 19
2.1.2. Ha cht chnh .................................................................................... 19
2.1.2. Cc ha cht v dung mi khc .......................................................... 19
2.2. THIT B ................................................................................................... 19
2.3. NI DUNG V PHNG PHP NGHIN CU .................................. 20
2.3.1. Ni dung nghin cu........................................................................... 20
2.3.2. Phng php nghin cu .................................................................... 21

Chng 3. THC NGHIM V KT QU ....................................................... 26


3.1. NGHIN CU DOCKING ....................................................................... 26
3.2. TNG HP HA HC ............................................................................ 27
3.2.1. Tng hp cht N1-[5-(furan-2-yl)-1,3,4-thiadiazol-2-yl]-N8hydroxyoctandiamid (Va) ............................................................................. 27
3.2.2. Tng hp cht N1-[5-(5-bromofuran-2-yl)-1,3,4-thiadiazol-2-yl]-N8hydroxyoctandiamid (Vb)............................................................................. 31
3.2.3. Tng hp cht N1-hydroxy-N8-[5-(5-methylfuran-2-yl)-1,3,4thiadiazol-2-yl]octandiamid (Vc) ................................................................. 33
3.2.4.... Tng hp cht N1-hydroxy-N8-[5-(thiophen-2-yl)-1,3,4-thiadiazol-2yl]octandiamid (Vd) ..................................................................................... 34
3.2.5. Tng hp cht N1-[5-(5-bromothiophen-2-yl)-1,3,4-thiadiazol-2-yl]N8-hydroxyoctandiamid (Ve) ....................................................................... 36
3.2.6. Tng hp cht N1-hydroxy-N8-[5-(5-methylthiophen-2-yl)-1,3,4thiadiazol-2-yl]octandiamid (Vf) .................................................................. 37
3.2.7. Tng hp cht N1-hydroxy-N8-[5-(pyridin-2-yl)-1,3,4-thiadiazol-2yl]octandiamid (Vg) ...................................................................................... 39
3.2.8. Tng hp cht N1-hydroxy-N8-[5-(pyridin-3-yl)-1,3,4-thiadiazol-2yl]octandiamid (Vh) ..................................................................................... 40
3.2.9. Tng hp cht N1-hydroxy-N8-[5-(pyridin-4-yl)-1,3,4-thiadiazol-2yl]octandiamid (Vi)....................................................................................... 42
3.2. KIM TRA TINH KHIT .................................................................. 43
3.3. KHNG NH CU TRC ..................................................................... 44
3.3.1. Ph hng ngoi (IR) ............................................................................ 44
3.3.2. Ph khi lng (MS) .......................................................................... 46
3.3.3. Ph cng hng t ht nhn (1H-NMR, 13C-NMR) ........................... 46
3.5. HOT TNH SINH HC .......................................................................... 50
3.5.1. Tc dng c ch HDAC ...................................................................... 50
3.5.2. Hot tnh khng t bo ung th in vitro .............................................. 51
Chng 4. BN LUN ........................................................................................ 52

4.1. TNG HP HA HC ............................................................................ 52


4.2. KHNG NH CU TRC ..................................................................... 53
4.2.1. Ph hng ngoi (IR) ............................................................................ 53
4.2.2. Ph khi (MS) ..................................................................................... 54
4.2.3. Ph cng hng t ht nhn (1H-NMR, 13C-NMR) ........................... 55
4.3. HOT TNH SINH HC .......................................................................... 58
KT LUN V KIN NGH............................................................................... 64
TI LIU THAM KHO

DANH MC CC K HIU, CH VIT TT


AsPC-1: T bo ung th ty ngi
13
1

C - NMR: Ph cng hng t ht nhn 13C

H - NMR: Ph cng hng t ht nhn 1H

dd: Dung dch


HAT: histon acetyltranferase
HDAC: Histon deacetylase
H460: T bo ung th phi ngi
HDI, HDIs: Histon deacetylase Inhibitor(s)
IR: Ph hng ngoi
MCF-7: T bo ung th v ngi
MS: Ph khi lng
MTT: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid)
NCI-H460: T bo ung th phi ngi
NST: Nhim sc th
PC-3: T bo ung th tin lit tuyn ngi
Rt: khuy u
SAHA: Acid suberoylanilid
SKLM: Sc k lp mng
SW620: T bo ung th i trng ngi
TSA: Trichostatin A

DANH MC CC BNG
Tn bng

STT
1

Bng 1.1:

Cc cht c ch HDAC ang th nghim trn lm


sng

Bng 1.2:

Tc dng khng cc t bo ung th in vitro ca


N25 (IC50SD [M])

Bng 1.3:

Hot tnh c ch HDAC cc tup v tc dng


khng t bo ung th in vitro ca cc dn cht
amido-lactam v tr 7 ca khung SAHA

Bng 1.4:

Cc acid hydroxamic mang khung 5-phenyl-1,3,4thiadiazol-2-yl v c tnh trn mt s dng t bo

Bng 3.1:

Kt qu docking ca cc cht Va-i vi HDAC8

Bng 3.2:

Gi tr Rf v T0nc ca cc cht Va-i

Bng 3.3:

S liu phn tch ph IR ca Va-i

Bng 3.4:

S liu phn tch ph khi lng ca cc cht

Bng 3.5:

S liu phn tch ph 1H-NMR

10

Bng 3.6:

S liu phn tch ph 13C-NMR

11

Bng 3.7:

Tc dng c ch HDAC ca cc dn cht tng hp

12

Bng 4.1:

Kt qu th hot tnh khng t bo ung th ngi


in vitro

13

Bng 4.2:

So snh tc dng khng t bo ung th ca Va-d,


Vf-h vi mt s dn cht acid hydroxamic mang
khung 5-phenyl-1,3,4-thiadiazol

DANH MC CC HNH V, S
HNH V
STT
1

Tn hnh
Hnh 1.1:

Vai tr sinh hc ca cc HDAC trong sinh l t


bo ung th

Hnh 1.2:

iu ha s pht trin v sng st ca t bo bi


cc cht c ch HDAC

Hnh 1.3:

Cng thc c in ca HDI

Hnh 1.4:

HDIs c cu trc acid hydroxamic

Hnh 1.5:

Cc acid phenylthiazol hydroxamic tng t


SAHA

Hnh 1.6:

Mt s acid phenylthiazol hydroxamic

Hnh 1.7:

Cc dn cht acid biphenyl-hydroxamic

Hnh 1.8:

Cc acid isoxazol-hydroxamic

Hnh 1.9:

Cc triazol-hydroxamic

10

Hnh 1.10:

Mt s dn cht saccarin ca acid hydroxamic mi

11

Hnh 1.11:

Cng thc mt s dn cht amido-lactam v tr 7


ca khung SAHA

12

Hnh 1.12:

Cc acid hydroxamic mang khung benzothiazol,


cu ni 6 carbon

13

Hnh 2.1:

Cc dn cht hydroxamic mang khung 5-aryl1,3,4-thiadiazol d kin tng hp

14

Hnh 3.1:

M hnh tng tc ca Va, Vg v SAHA vi


HDAC8

15

Hnh 4.1:

Ph hng ngoi ca cht Vb

16

Hnh 4.2:

Ph khi lng ca cht Vb

17

Hnh 4.3:

Ph 1H-NMR dn rng ca cht Vd

18

Hnh 4.4:

Ph 13C-NMR ca cht Vb

19

Hnh 4.5:

Kt qu phn tch Western blot ca cc cht Va-i

Tn s

STT
1

S 1.1:

Tng hp cc acid isoxazol-hydroxamic

S 1.2:

Tng hp acid triazol-hydroxamic (4)

S 2.1:

Tng hp cc dn cht hydroxamic mang khung


5-aryl-1,3,4-thiadiazol

S 3.1:

Quy trnh tng hp cht Va

S 3.2:

Tng hp cht IIa

S 3.3:

Tng hp cht IIIa

S 3.4:

Tng hp cht IVa

S 3.5:

Tng hp cht Va

S 3.6:

Quy trnh tng hp cht Vb

10

S 3.7:

Quy trnh tng hp cht Vc

11

S 3.8:

Quy trnh tng hp cht Vd

12

S 3.9:

Quy trnh tng hp cht Ve

13

S 3.10:

Quy trnh tng hp cht Vf

14

S 3.11:

Quy trnh tng hp cht Vg

15

S 3.12:

Quy trnh tng hp cht Vh

16

S 3.13:

Quy trnh tng hp cht Vi

17

S 4.1:

C ch phn ng ng vng tng hp 2-amino5-aryl-1,3,4-thiadiazol

18

S 4.2:

C ch hot ha ca CDI

T VN
Histon deacetylase (HDAC) l mt nhm enzym c nh hng ln qu trnh
sao chp v biu hin gen. Cc nghin cu cho thy s hot ng bt thng ca
HDAC l cn nguyn ca nhiu bnh ung th [7,11,15,24]. V th, vic tm ra cc
thuc c hot tnh c ch HDAC iu tr ung th ang l mt hng nghin cu
c rt nhiu nh khoa hc quan tm.
Mt trong cc nhm cht c ch HDAC c tp trung nghin cu hin nay
l cc dn cht acid hydroxamic. Nhm cht ny c hot tnh c ch mnh, cu trc
n gin d tng hp, trong acid suberoylanilid hydroxamic (SAHA) l cht u
tin c FDA ph duyt s dng trong iu tr u lympho da t bo T [31]. Tip
tc thnh cng ny, nhiu nhm nghin cu trn th gii vn ang n lc tm kim
cc dn cht mi ti u hn da trn phin mu ca SAHA v cc cht tm
c.
Ti Vit Nam, mt s nghin cu thit k, tng hp cc dn cht acid
hydroxamic hng c ch HDAC v ang c thc hin ti b mn Ha Dc
trng i hc Dc H Ni. Cc nghin cu ny tp trung vo hai hng: thay
i cu ni hoc nhm kha hot ng da trn khung ca SAHA. Trong lun n
tin s ca tc gi o Th Kim Oanh, dn cht mang khung benzothiazol vi vai
tr l nhm kha hot ng thay cho nhn phenyl ca SAHA v cu ni 6 carbon
cho hot tnh rt ng lu [3]. Ngoi ra, trong mt cng b gn y ca tc gi
Nguyn Hi Nam v cng s, cc dn cht acid hydroxamic mang khung 5-phenyl1,3,4-thiadiazol-2-yl vi cu ni 6 carbon cng cho hot tnh rt tt [21].
V vy, tip tc hng nghin cu ny, chng ti tin hnh ti Tng hp
v th tc dng sinh hc ca mt s acid hydroxamic mang khung 5-aryl-1,3,4thiadiazol hng c ch histon deacetylase vi hai mc tiu:
-

Tng hp c mt s dn cht acid hydroxamic mang khung 5-aryl-1,3,4thiadiazol.

Th tc dng c ch histon deacetylase v hot tnh khng mt s dng t


bo ung th in vitro ca cc cht tng hp c.

Chng 1. TNG QUAN

1.1. HISTON DEACETYLASE (HDAC)


1.1.1. Khi nim, phn loi
Histon l cc protein c bn cu to nn nhim sc th, trong bn cp
histon (H2A, H2B, H3 v H4) to nn li protein ca nucleosom. Cc cp histon li
c 2 phn quan trng: ui C nm bn trong li v u N nm bn ngoi
nucleosom. u amin ny mang nhiu in tch dng nn tng tc mnh vi
phn phosphat mang in m trn phn t ADN to nn cu trc nucleosom v cc
cu trc bc cao hn ca nhim sc th.
Vic tch in dng ca histon mnh hay yu thng qua qu trnh acetyl
ha u amin phn ui ca histon. dng deacetyl ha, histon tch in dng
ln, tng tc in tch vi ADN mnh lm ng xon nhim sc th gy c ch
qu trnh dch m v tng hp protein, c ch s biu hin gen. Ngc li, khi b
acetyl ha, in tch dng b trung ha, nhim sc th c tho xon, qu trnh
tng hp protein din ra v c tnh ca gen c biu hin. Trong t bo, qu trnh
acetyl ha ny c iu ha bi 2 enzym: histon acetyltranferase (HAT) v histon
deacetylase (HDAC) [1, 13, 27, 29].
Histon deacetylase (HDAC) l nhm gm 18 enzym xc tc cho s di
chuyn lm gim bt nhm acetyl trn phn ui lysin ca cc protein histon H2A,
H2B, H3 v H4 trong li nucleosom. Hin nay, 18 HDAC khc nhau ngi
c xc nh, chia lm 4 nhm [1, 5, 6, 27, 29].
-

Nhm I: HDAC 1, HDAC 2, HDAC 3, HDAC 8 : Nm nhn t bo, c


protein ch l cc histon.

Nhm IIa: HDAC 4, HDAC 5, HDAC 7, HDAC 9: Nm nhn hoc t bo


cht, c protein ch l cc protein histon v khng phi histon.

Nhm IIb: HDAC 6, HDAC 10 : Nm t bo cht, c ch l cc protein


khng phi histon.

Nhm III: Cc protein iu ho chui thng tin 2 (SIRT): SIRT 1 7, chng


c bo tng, ty th v nhn.

Nhm IV: HDAC 11 : Nm nhn t bo, c ch l cc histon.


Cc HDAC nhm I, II, IV c gi l cc HDAC kinh in ph thuc vo

Zn2+ v b c ch bi cc cht to phc chelat vi Zn2+ nh cc acid hydroxamic,


thiol... Trong khi cc HDAC nhm III li ph thuc vo NAD+ [6]. Thut ng
cc cht c ch HDAC thng c dng cho nhng cht c mc tiu phn t l
cc HDAC kinh in v hin ang c nghin cu trn lm sng [1, 6].
1.1.2. Cu trc ca HDAC
Bng phng php kt tinh to tinh th v chp tia X ngi ta xc nh
c cu trc 3D ca mt s HDAC v cc trung tm xc tc phn ng deacetyl
ca chng. V c bn cc HDAC c cu trc trung tm hot ng kh ging nhau,
chng u gm cc phn c bn sau:
+ Ion Zn2+ l coenzym ca HDAC nm trung tm xc tc ca chng. y l
thnh phn tham gia lin kt mnh nht vi phn ui histon bng lin kt phi tr.
Thng thng cc cht c ch HDAC lin kt cng mnh vi Zn2+ th tc dng c
ch HDAC v c tnh t bo cng mnh [12, 19, 28].
+ Knh enzym l ni cha ng c cht v tham gia lin kt Van der Walls vi c
cht. Knh ny c cu trc dng ti hnh ng hp, c cu to bi cc acid amin
thn du c bit l cc acid amin c nhn thm nh: Phe, Tyr, Pro, His. N c cu
trc kh linh ng c th thay i kch thc ph hp vi c cht v tham gia
phn ng deacetyl. Trn ming ti c mt vnh nh c to nn t 1 vi vng
xon protein (phn vnh s tng tc vi nhm nhn din b mt ca HDAC). i
vi cc hp cht acid hydroxamic chiu di ca knh ny l ti u vi khong 5-6
lin kt carbon [12, 28, 29].
1.1.3. Mi lin quan gia ung th v s bt thng hot ng ca HDAC
Nh trnh by trong phn khi nim, hot ng ca HDAC nh hng ti
s tch in trn phn u amin ca histon, t gy tc ng ln qu trnh phin
m. Cc sai lch ca qu trnh phin m l mt trong nhng nguyn nhn dn ti s
hnh thnh khi u [1]. Chng hn, mi tng quan gia hot ng ca HDAC v s

to thnh khi u c th hin r nht trong bnh ung th bch cu tin ty bo cp


tnh (APL). Nhng nghin cu trn phm vi rng ch ra rng s c ch phin m
khng thch hp ca HDAC l c ch ph bin to ra cc protein gy ung th
(oncoprotein). Ngoi ra trong cc t bo ung th ngi ta cn thy s hot ng
qu mc ca HDAC nh ung th bung trng (HDAC1-3), ung th d dy
(HDAC2); ung th phi (HDAC1, 3) [11, 24].
Cc nghin cu c ngha thng k ch ra rng cc HDAC lin quan n
nhiu giai on iu ha c bn ca qu trnh sinh hc trong t bo ung th nh
chu trnh t bo, s bit ha, s cht t bo theo chng trnh, k c s di chuyn,
s xm ln v s to mch. Vai tr chc nng ca cc HDAC trong qu trnh sinh
hc ca t bo ung th c tm tt hnh 1.1 [7, 11, 15, 24].

Hnh 1.1: Vai tr sinh hc ca cc HDAC trong sinh l t bo ung th

1.2. CC CHT C CH HDAC


1.2.1. Phn loi cc cht c ch HDAC (HDIs)
HDIs c chia thnh 5 nhm chnh da trn cu trc ha hc: cc acid
hydroxamic, cc peptid vng, cc acid bo, benzamid v cc dn cht ceton. [9]

Bng 1.1: Cc cht c ch HDAC ang th nghim trn lm sng


Cht

Cu trc

Cht

Cu trc

Cc acid hydroxamic
O

TSA
(Trichostatin A)

NHOH

Oxamflatin

NHOH

H
N

O
S

Acid

H
N
NHOH

suberoylanilid

NVP-

LAQ-824

hydroxamic
(SAHA)

CBHA
(Acid mcarboxycinnamic

Acid

bishydroxamid)

sulfonamid
hydroxamic

Scriptaid
Peptid vng
O

Depsipeptid

HN

NH

S
S

(FK-228)

CH3
O

H
N

CH3
CH3

H3C

O
HN

CH3
O

Acipidin

CHAP

Benzamid
O
O

MS-275

N
H

H
N

NH2

CI-994

N
O

Cc acid bo
O

Acid valproic

Phenyl

ONa

OH

butyrat

Cc dn cht ceton
Trifluoromethyl

Alpha-

ceton

cetoamid

Mi nhm 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, trong khi cc peptid vng c cu trc qu phc tp v gy
ra s chy mu kh cha v FK-228 trong cu trc c mt phn lin kt vi kim
loi c cha lu hunh khng mong mun [4, 14].
1.2.2. C ch tc dng
Cc cht c ch HDAC 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 , yu t then cht quyt nh hot tnh chng ung th ca HDIs l thc y
s bit ha, c ch chu trnh t bo v thc y s cht t bo.
Ngoi ra, hot ha p ng min dch v c ch to mch cng l vai tr rt
quan trng ca HDIs, gin tip c ch s pht trin in vivo ca khi u (hnh 1.2) [2,
13].

Hnh 1.2: iu ha s pht trin v sng st ca t bo bi cc cht c ch HDAC

1.2.3. Cu trc ca cc cht c ch HDAC


Cc HDIs chia thnh nhiu nhm khc nhau nhng c mt s c im
chung v mt cu trc gm 3 phn chnh [2, 8, 20]:

- Nhm gn vi ion Zn2+ (Zinc binding group - ZBG) (A): tng tc vi ion Zn2+
ti trung tm hot ng ca cc HDAC nh acid hydroxamic, cc thiol, nhm oaminoanilin ca benzamid, mercaptoceton..., quyt nh tnh c hiu v hiu lc
ca HDIs.
- Vng cu ni s nc (B): thng l nhng hydrocacbon thn du c th to cc
lin kt Van der Waals vi knh enzym.
- Nhm kha hot ng (capping group) hay vng nhn din b mt (surface
recognition group) (C): l cc vng thm hoc peptid vng, thng nm trn b
mt enzym.

Hnh 1.3: Cng thc c in ca HDI


Cu trc tinh th kt tinh ca cc HDAC lin kt vi mt s cht c ch
HDAC cho thy phn A, B v mt phn ca C nm trong ti enzym, lm lp y
khong trng trong lng knh enzym. Phn cn li ca nhm kha hot ng C
tng tc vi phn vnh trn b mt ming ti enzym. Nhm nhn din b mt C
c th lin kt vi phn cu ni thng qua mt s lin kt peptid lm tng kh nng
phn cc v gp phn ci thin dc ng hc cho cc cht c ch HDAC. Vic
nghin cu thit k cu trc cc cht mi u da trn cu trc c in ny.
1.3. TNH HNH NGHIN CU TRN TH GII V CC ACID
HYDROXAMIC C CH HDAC
Acid hydroxamic l nhm cht c ch HDAC c nghin cu rng ri
nht, vi nng c ch nm trong khong micromol n nanomol.
Trichostatin A ( (R)-TSA) l cht u tin c phn lp t Streptomyces
hygroscopicus c chng minh c tc dng c ch mnh, c hiu vi HDAC
(Ki=3,4nM), c tc dng rt tt trong vic lm gim s bit ha t bo trong bnh
bch cu Friend, v ng vai tr nh cht ngn cn s di cn trong ung th i
trng. Tuy nhin vic sn xut ra TSA rt tn km vi hiu sut thp (tri qua 20

bc vi hiu sut 2%) nn vic nghin cu ra cc cht c ch HDAC thay th


TSA ang c nhiu nh nghin cu quan tm. Hin nay, TSA ch yu c dng
lm cht i chiu trong nghin cu tm ra HDIs mi [23].
Mt cht c ch HDAC c nghin cu tng hp thnh cng l acid
suberoylanilid hydroxamic (SAHA). Cht ny c FDA ph duyt s dng
trong iu tr u lympho da t bo T. SAHA lm tng s acetyl ha ca cc histon
H2B, H3 v H4, ng thi thay i s biu hin ca mt s gen dn n s cht t
bo theo chng trnh [31].
Bn cnh , nhiu dn cht acid hydroxamic c ch HDAC ang c
nghin cu rng ri v c chia thnh nhiu phn nhm nh (hnh 1.4).

Hnh 1.4: HDIs c cu trc acid hydroxamic


c im chung ca cc cht ny l nhm hydroxamic d to phc vi Zn 2+
ca HDAC nn c ch khng chn lc c HDAC nhm I, II v b chuyn ha
nhanh [20, 30].
Chnh v vy cc nh khoa hc trn th gii vn ang n lc tm kim cc
dn cht mi da trn phin mu TSA, SAHA v cc cht tm c. Da trn
c im cu trc ca cc cht c ch HDAC dn cht hydroxamic (hnh 1.3), cc

nghin cu c th tin hnh thay i mt trong 2 phn cu trc: nhm kha hot
ng (C), cu ni (B).
Sau y l tng kt mt s nghin cu trn th gii v thay i nhm kha
hot ng ca cc acid hydroxamic.
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 trong SAHA (1, hnh 1.5) [10]. Mt
dn cht oxazol l WR301849 cng c tng hp [17].

Hnh 1.5: Cc acid phenylthiazol hydroxamic tng t SAHA


Kt qu nh gi tc dng trn HDAC cho thy dn cht WR301801 (1a)
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 (1b) (nhm th amino v tr ortho) cng c tc
dng mnh tng ng SAHA [30].
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 1.6) [16]. 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 1a-5 (hnh 10). 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 1a, 1b, 1a-2 cho thy cc dn cht ny
u c IC50 nh hn 3 M [16].

Hnh 1.6: Mt s acid phenylthiazol hydroxamic


Trong qu trnh nghin cu cc dn cht acid phenylthiazol-hydroxamic,
nhm nghin cu ca Alan P. Kozikowski cng ng thi tin hnh thit k v
tng hp mt dy cc dn cht acid biphenyl-hydroxamic tng t SAHA (hnh
1.7) [16]. Kt qu cc dn cht biphenyl c ch HDAC mnh hn SAHA trn 6 loi
HDAC (HDAC1, 2, 3, 8, 10, 6). Khi gn thm nhm -NH2 vo v tr ortho trn
vng phenyl th 2 hot tnh gim song khi nhm -NH2 ny c acyl ha bng
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. Mt s acid
biphenyl-hydroxamic cng cho c tnh trn 5 dng t bo ung th ty th nghim
nhng tc dng khng tt bng cc acid phenylthiazol hydroxamic.

Hnh 1.7: Cc dn cht acid biphenyl-hydroxamic


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 WR301849 (hnh 1.8) [17].
Dn cht isoxazol ny c tc dng c ch cc HDAC1, 3 v 6 rt mnh vi IC50
thp n 0,002 nM. Tip tc mch nghin cu ny, mt s dn cht trong vng
isoxazol c a vo v tr ngay st cnh nhm acid hydroxamic c thit k
v tng hp (s 1.1) [25].

10

S 1.1: Tng hp cc acid isoxazol-hydroxamic


* Tc nhn v iu kin: (a) acid 7-heptynoic, POCl3, pyridin, -13oCrt, 30 pht; (b) ethyl
clorooxamidoacetat, triethylamin, THF, rt, 16 h; (c) NH2OH.HCl, KOH, MeOH, rt, 15 pht.

Kt qu th hot tnh c ch HDAC cho thy mt s dn cht trong cc acid


isoxazol-hydroxamic tng hp c ch c 5 tup HDAC1, 2, 3, 6 v 10 vi IC50
trung bnh khong 150 nM [25]. Tc dng ny km hn nhiu so vi cc acid
phenylthiazol-hydroxamic (1) v acid biphenyl-hydroxamic (2). C th nhn xt,
khi c mt vng isoxazol cnh nhm hydroxamic, nhm kha hot ng l quinolin
hoc biphenyl khng ti u cho hot tnh, trong khi hai dn cht vi vng 5phenylthiazol (3a, 3b) c tc dng c ch HDAC kh mnh, gn tng ng
SAHA (hnh 1.8). y cng l hai dn cht th hin c tnh t bo mnh nht vi
IC50 thp n 1 M. iu ngc nhin l mc d 3b c ch HDAC mnh hn 3a
song 3a li c c tnh t bo mnh hn 3b [25]. C th s c mt ca nhm 3amino 3b lm gim tnh thm qua mng t bo ca cht ny, dn n c tnh
t bo thp hn so vi 3a. C th cc dn cht acyl ha ca 3b s ci thin c tc
dng trn HDAC v t bo, tng t nh vi cc dn cht acid phenylthiazolhydroxamic (1).

Hnh 1.8: Cc acid isoxazol-hydroxamic


Cng nhm mc tiu tm ra cu ni v nhm kha hot ng thch hp, cc
nh khoa hc ti Vin Parker H. Petit thuc Vin Cng ngh Georgia thit k v
tng hp dy cc dn cht hydroxamic cha vng triazol (4, s 1.2) [17].

11

S 1.2: Tng hp acid triazol-hydroxamic (4). Tc nhn v iu kin: (a) CuI,


Hunig base, THF, rt; (b) dd NH2OH, KCN, THF/MeOH (1/1), rt.
Kt qu sng lc hot tnh c ch HDAC cho thy cu ni gia phn
hydroxamic v triazol 5, 6C l ti u. Tuy nhin, khng thy c quy lut r rng
khi so snh hai dn cht c cng nhm kha hot ng Ar v khc nhau v di
cu ni. Vi nhm Ar cng knh, dn cht 5C thng c tc dng c ch HDAC
mnh hn trong khi nhng cht c Ar nh, dn cht 6C thng biu th hot tnh
trn HDAC mnh hn. Khi so snh vi SAHA, rt nhiu dn cht triazol chng t
c i lc mnh hn vi HDAC. In vitro, 4 dn cht 4t, 4v, 4y, 4w gy c i vi
dng t bo ung th tin lit tuyn ca ngi DU145 vi IC50 thp nht n 2,25
M, tng ng vi SAHA (hnh 1.9) [8].

Hnh 1.9: Cc triazol-hydroxamic


c nhiu nghin cu v cc dn cht acid hydroxamic c ch HDAC (cc
dn cht N-hydroxybenzamid, 1,3,4-thiadiazol v tetrahydroisoquinolin), gn y,
nhm cc nh khoa hc ca Trung Quc v M tip tc cng b kt qu tng hp
v th tc dng sinh hc ca mt dy cc dn cht saccarin (1,2dihydrobenzo[d]isothiazol-3-1-1,1-dioxid) ca acid hydroxamic mi (hnh 1.10)
[18].

12

Hnh 1.10: Mt s dn cht saccarin ca acid hydroxamic mi


Vic kho st di cu ni n=1-5 cng cho thy cc dn cht c cu ni 5C
cho hot tnh c ch HDAC mnh nht. c bit cc cht 5e, 5m, 5p c ch HDAC
tng t hoc tt hn SAHA (vi IC50 ln lt l 0,152; 0,113; 0,096M so vi
SAHA l 0,135M). Cc nh gi sinh hc su hn cho thy 5m c hot tnh khng
cc t bo ung th MDA-MB-231 (t bo ung th v); PC-3 (t bo ung th tin
lit tuyn) v KG1 (t bo ung th bch cu nguyn bo ty) mnh nht vi IC50 =
4,34; 9,28 v 1,66 M, tng ng SAHA [18]. Kt qu ny gi rng cc dn
cht hydroxamat mang khung saccarin c th l cc cht dn ng pht trin
cc hot cht khng ung th mi.
Mt nhm cc nh khoa hc Trung Quc khc cng va ng k bng sng
ch (s CN 103159646) cho mt dn cht mi ca SAHA l N1-(2,5dimethoxyphenyl)-N(8)-hydroxyoctandiamid (N25) [32]. Hot tnh khng ung th
ca N25 mnh hn SAHA trn cc dng t bo ung th phi H460, A549, H1299
v ung th thn kinh m U251 (bng 1.2).

13

Bng 1.2: Tc dng khng cc t bo ung th in vitro ca N25 (IC50SD [M])

Mt hng thay i nhm kha hot ng ca SAHA c thc hin bi


cc nh khoa hc Italia (Maurizio Taddei v cng s, cui nm 2013) bng cch
gn cc lactam-carboxyamid vo v tr s 7 ca khung suberoylanilid thu c
kt qu rt ng lu . Vic gn lactam vo nhm kha hot ng ny tng
cng ng k tc dng in vitro. Nhiu dn cht c to ra c kh nng c ch
HDAC cc tup vi IC50 mc nanomol, thp hn SAHA hng trm ln, in hnh
l 3 cht 6, 7, 8 (hnh 1.11, bng 1.3) [26].

Hnh 1.11: Cng thc mt s dn cht amido-lactam v tr 7 ca khung SAHA


Nghin cu tc dng khng t bo ung th (H460) cng cho thy cc dn
cht amido-lactam v tr 7 ca khung SAHA cho hot tnh rt mnh. Cc dn cht
th v tr meta-, para- v -lactam anilid v tr 7 u c c tnh cao vi IC50

14

di micromol, c bit cc cht 6, 7 v 8 c IC50 = 0.5 M, thp hn SAHA hn


60 ln (bng 1.3) [26].
Bng 1.3: Hot tnh c ch HDAC cc tup v tc dng khng t bo ung th in
vitro ca cc dn cht amido-lactam v tr 7 ca khung SAHA.

Nhn chung, cc dn cht SAHA cha (S)-7-amino carboxylactam c ch


cc tup HDAC vi gi tr IC50 mc nano, cho thy s c mt ca vng amid trn
vng kha hot ng c nh hng c bit ln i lc vi enzym. S bin i trn
nhm kha hot ng ca SAHA ny mang li cc cht c hot tnh cc mnh
trn cc HDAC nhm I v II, c chng thc thm bng kh nng khng ung th
y ha hn trn dng t bo th nghim. Cht 6 c th coi 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 [26].
1.4. TNH HNH NGHIN CU TRONG NC V CC CHT C
CH HDAC
Ti Vit Nam, ln u tin cc nghin cu thit k, tng hp cc cht c ch
HDAC v th hot tnh sng lc tm ra cht c hng tc dng khng t bo ung
th v ang c nhm nghin cu ti b mn Ha Dc, trng i hc Dc
H Ni thc hin.
Lun n tin s ca tc gi o Th Kim Oanh tin hnh thay th nhn
phenyl ca SAHA bng khung benzothiazol vi vai tr l nhm kha hot ng
(hnh 1.12). Kt qu kho st di cu ni t 2 6 carbon cho thy vi cu ni
6C cho hot tnh ti u [3].

15

Hnh 1.12: Cc acid hydroxamic mang khung benzothiazol, cu ni 6 carbon


Kt qu th c tnh t bo in vitro cho thy cht 9b, 9g c hot tnh mnh
tng ng SAHA. Thm ch trn 3 dng t bo SW620, AsPC-1 v NCI-H460,
cht 9g c tc dng mnh gn gp i so vi SAHA, th hin gi tr IC50 = 0,32;
0,34; 0,39 g/ml (tng ng vi tng dng t bo) so vi ca SAHA l IC50 = 0,50;
0,69; 0,68 g/ml. Th nghim hot tnh khng t bo ung th tin lit tuyn PC-3
in vivo vi m hnh ghp d chng chut tri lng cho thy cht 9g c ch
49,00% s pht trin ca khi u tng ng SAHA (48,30%) (liu 30 mg/kg).
Gi nguyn cu ni 6 carbon c kho st trong lun n trn, nhm
nghin cu ti b mn Ha Dc trng i hc Dc H Ni tip tc hng
nghin cu thay i nhm kha hot ng. Dy dn cht acid hydroxamic mang
khung 5-phenyl-1,3,4-thiadiazol-2-yl c thit k, tng hp v kt qu cng
cho hot tnh rt ng ch (bng 1.4) [21].

16

Bng 1.4: Cc acid hydroxamic mang khung 5-phenyl-1,3,4-thiadiazol-2-yl v c


tnh trn mt s dng t bo

Cht

10a

c tnh trn cc dng t bo (IC50,* M)


SW620

MCF-7

PC3

AsPC-1

NCI-H460

0,70

1,80

0,88

2,71

1,07

10b

2-Cl

0,34

1,23

1,42

0,63

0,11

10c

3-Cl

0,45

1,23

1,76

1,10

1,94

10d

4-Cl

1,62

0,73

0,84

1,34

1,50

10e

4-F

3,58

8,05

2,92

1,88

4,79

10f

4-Br

0,72

1,27

0,83

0,08

1,42

10g

4-CH3

11,52

20,78

14,90

6,38

13,16

10h

4-OCH3

1,46

2,46

1,63

0,80

1,97

10i

4-N(CH3)2

6,61

6,67

7,35

7,16

8,18

10j

2-NO2

19,23

31,18

14,69

33,47

16,69

10k

4-NO2

26,71

76,25

25,57

76,25

76,25

10l

2,6-Cl2

16,21

16,29

15,18

14,95

14,38

10m

3,4-CH2OCH2-

1,89

2,15

2,54

3,04

3,34

10n

2,3,4-(OCH3)3

2,29

4,15

1,77

2,11

3,21

10o

3,4,5-(OCH3)3

4,31

3,50

1,89

6,02

10,20

3,70

6,82

4,31

3,66

2,77

SAHA

*Nng ca cht gy ra s gim 50% s lng t bo, s liu c ly trung


bnh ba kt qu vi lch di 10%.
SAHA: acid suberoylanilid hydroxamic.

Nghin cu ny tng hp c nhiu dn cht cht mang khung 5-phenyl1,3,4 thiadiazol c hot tnh khng t bo ung th mnh. Cc cht 10a-c c IC50
thp hn SAHA 3-20 ln trn cc dng t bo th nghim, cn 10d-f, 10m-o cng
cho hot tnh thp hn hoc tng ng SAHA.
Cc nghin cu trn cho thy, vic gi cu ni 6C v thay nhn phenyl ca
SAHA bng cc dn cht vng benzothiazol, 5-phenyl-1,3,4-thiadiazol u to ra
c cc cht c tc dng khng ung th hiu qu vi hot lc mnh hn. V vy,

17

ti ny s tip tc hng nghin cu trn, thay khung 5-phenyl-1,3,4-thiadiazol


bng khung 5-aryl-1,3,4-thidiazol v vn gi nguyn cu ni 6 carbon.

18

Chng 2. NGUYN VT LIU, TRANG THIT B,


NI DUNG V PHNG PHP NGHIN CU
2.1. NGUYN VT LIU
Cc ha cht, dung mi s dng trong qu trnh thc nghim l loi dnh
cho tng hp c nhp t cng ty Merck hoc Sigma-Aldrich. Cc ha cht ny
c s dng trc tip khng qua tinh ch thm. Bao gm:
2.1.2. Ha cht chnh
Cc aldehyd:
Furan-2-carbaldehyd
Pyridin-2-carbaldehyd
Pyridin-3-carbaldehyd
Pyridin-4-carbaldehyd
Thiophen-2-carbaldehyd
5-Bromothiophen-2-carbaldehyd
5-Bromofuran-2-carbaldehyd

FeCl3.12H2O
Acid suberic monomethyl ester
Carbonyldiimidazol (CDI)
Hydroxylamin hydroclorid
Thiosemicarbazid
2.1.2. Cc ha cht v dung mi khc
DMF

Aceton

MeOH

Acid acetic bng

n-Hexan

HCl

EtOH

NaOH

Dicloromethan

Nc ct

2.2. THIT B
-

Bnh cu y trn dung tch 50 ml c nt mi, my khuy t gia nhit, sinh hn


hi lu, my ct quay chn khng, t lnh, t sy, pipet, phu thy tinh, giy

19

lc, cn phn tch, cn k thut, bnh chy sc k lp mng (SKLM), ch th


mu vn nng.
-

SKLM tin hnh trn bn mng silicagel 60 F254 trng sn.

Nhit nng chy (tonc) c xc nh bng my o nhit nng chy nhit


in (Electrothermal digital).

Ph hng ngoi (IR) c ghi trn my Perkin Elmer USA ti b mn Ha


vt liu Khoa ha Trng i hc Khoa hc t nhin i hc quc gia
H Ni.

Ph khi lng (MS) c ghi bng my khi ph LC-MSD-Trap-SL (ca


Vin ha hc Vit Nam) v Agilent 6310 Ion Trap (ca Vin ha hc cc hp
cht thin nhin).

Ph cng hng t ht nhn (1H - NMR) v carbon (13C - NMR) c ghi bng
my Bruker AV-500 ti Trung tm khoa hc v cng ngh Vit Nam.

2.3. NI DUNG V PHNG PHP NGHIN CU


2.3.1. Ni dung nghin cu
-

Kho st v tng hp 9 cht vi cng thc nh sau:

Hnh 2.1: Cc dn cht hydroxamic mang khung 5-aryl-1,3,4-thiadiazol d kin tng hp


STT
1

Cht
Va

Vb

Tn
N1-[5-(furan-2-yl)-1,3,4-thiadiazol-2-yl]-N8hydroxyoctandiamid.
N1-[5-(5-bromofuran-2-yl)-1,3,4-thiadiazol-2yl]-N8-hydroxyoctandiamid.

20

Vc

Vd

Ve

Vf

Vg
Vh
Vi

N1-hydroxy-N8-[5-(5-methylfuran-2-yl)-1,3,4thiadiazol-2-yl]octandiamid.
N1-hydroxy-N8-[5-(thiophen-2-yl)-1,3,4thiadiazol-2-yl]octandiamid.
N1-[5-(5-bromothiophen-2-yl)-1,3,4-thiadiazol2-yl]-N8-hydroxyoctandiamid.
N1-hydroxy-N8-[5-(5-methylthiophen-2-yl)1,3,4-thiadiazol-2-yl]octandiamid.
N1-hydroxy-N8-[5-(pyridin-2-yl)-1,3,4thiadiazol-2-yl]octandiamid.
N1-hydroxy-N8-[5-(pyridin-3-yl)-1,3,4thiadiazol-2-yl]octandiamid.
N1-hydroxy-N8-[5-(pyridin-4-yl)-1,3,4thiadiazol-2-yl]octandiamid.

Khng nh cu trc ca cc cht tng hp c

Th tc dng c ch HDAC v hot tnh khng mt s dng t bo ung th


(in vitro) ca cc cht tng hp c

2.3.2. Phng php nghin cu


2.3.2.1. Nghin cu Docking
Sau khi thit k dy dn cht, nghin cu s b tng tc ca cc cht vi
HDAC, chng ti tin hnh docking cho cc cht tng hp c vi HDAC8
[12]. Cu trc HDAC8 c ly t ngn hng d liu protein (Protein Data Bank
(PDB)) (PDB ID: 1T69). Cu trc ca HDAC8 c im tng ng cao vi
HDAC4 vi im DALI Z (Z-score im nh gi ging) = 40,4 v im r.m.s.d
(root-meansquare deviation, lch) = 2,1, th t acid amin ging nhau (46%) v
l HDAC ca ng vt c v u tin c cu trc khng gian c nghin cu k
nht. Chng ti thc hin kim sot th nghim lp ghp ca cc cht vo HDAC8
bng chng trnh AutoDock Vina [22]. Tip theo chng ti d on nng lng

21

ca s tng tc lin kt ca cc cht tng hp c vi HDAC8 t s lp ghp


trn.
Nghin cu docking c thc hin ti Phng Nghin cu cu trc i hc
Quc gia Seoul, Hn Quc.
Kt qu c minh ha bng hnh nh docking v s liu d on tng tc.
2.3.2.2. Tng hp ha hc
Tng hp 9 cht mc tiu theo s sau:

S 2.1: Tng hp cc dn cht hydroxamic mang khung 5-aryl-1,3,4-thiadiazol


a) Thiosemicarbazid, ethanol, un hi lu; (b) FeCl3.12H2O; c) Acid suberic monomethyl
ester, CDI, DMF; d) NH2OH.HCl, MeOH, NaOH.

Dng TLC theo di qu trnh tin trin ca phn ng.


2.3.2.3. Kim tra tinh khit
Nhit nng chy: o bng my o im chy nhit in (Electrothermal
digital).
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 60 F254 trng sn,
hot ha 110oC trong 30 pht. H dung mi ty thuc vo c im ca
tng cht. Mu th c ha tan trong dung mi thch hp. Chm khong
2l.
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.

22

2.3.2.4. Xc nh cu trc
S dng cc phng php ph IR, MS, NMR (1H v

13

C) xc nh cu

trc ca cc cht tng hp.


2.3.2.5. Th hot tnh sinh hc
* Th hot tnh c ch HDAC:
Phn tch da trn Western blot, c tin hnh ti khoa Dc, H
Chungbuk, Hn Quc. 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 cc t
bo ung th. 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.
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 37 C 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 dimethylsulfoxid (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 g/ml v 10 g/ml.
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 (t bo khng vi mu th). Sau , cc t bo c x l v ra
bng dung dch mui c b sung m phosphat. T bo c ly gii trong dung
dch 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 SDSPAGE 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

23

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 c tnh t bo in vitro c thc hin ti Khoa Dc, Trng i hc
Chungbuk, Hn Quc theo phng php MTT v gi tr IC50 c tnh theo phng
php GraphPad Prism.
Dng t bo th nghim: SW620 (t bo ung th i trng ngi), MCF-7
(t bo ung th v ngi), AsPC-1 (t bo ung th ty ngi), PC-3 (t bo ung th
tin lit tuyn ngi).
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 RPMI b sung 10% FBS (huyt thanh bo thai b). c tnh t
bo ca cc cht c th bng phng php MTT theo cc bc sau:
- Chun b: Cc t bo pha logarit c trypsin ha v phn tn vo hn dch n
t bo trong mi trng RPMI b sung 10% FBS v iu chnh n nng
khong 1,5.104 n 3,5. 104 t bo, sau chia u vo cc ging ca a 96 ging,
mi ging 200 l. Cc a sau c 37oC trong iu kin 5% CO2. Sau 24
gi , cc mu th c chun b trong 20 l mi trng RPMI b sung 10% FBS
t dung dch gc 10 mg/mL trong dimethyl sulfoxid (DMSO) ri thm 2 l mu
th vo cc ging nhiu nng khc nhau, cc a ny sau c thm 48
gi. Tt c cc mu c chun b sao cho nng cui cng ca DMSO khng
qu 0,1%.
- Tin hnh th: Sau khi 48 gi, thm vo mi ging 20 l thuc nhum MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid) vi nng MTT l 5
mg/mL trong mui m phosphat - PBS. Cc a c thm 3 gi 37oC trong
iu kin 5% CO2. Tip theo, mi ging c cho 100l dung dch DMSO, 5
pht cho MTT formazan c ha tan. hp th c c bc sng 510 nm.
- Tnh kt qu: Gi tr IC50 l nng ca mu th m hp th gim i
50% so vi nhm chng (trng m tnh l ging ch thm mi trng nui cy): kt

24

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


nhau khng qu 5%. Gi tr IC50 c tnh da trn phn mm GraphPad Prism.

25

Chng 3. THC NGHIM V KT QU


3.1. NGHIN CU DOCKING
tm hiu s b v kh nng tng tc ca cc cht vi mc tiu phn t
l cc HDAC, chng ti nghin cu v d on nng lng lin kt cc cht trong
thit k vi trung tm hot ng ca HDAC8. Kt qu nng lng lin kt d on
c trnh by trong bng 3.1.
Bng 3.1: Kt qu docking ca cc cht Va-i vi HDAC8

Nng lng tng tc


(kcal/mol)

STT

Cht

Va

-6.9

Vb

-7.1

Vc

-6.9

Vd

-6.8

Ve

-6.6

Vf

-6.8

Vg

-6.9

Vh

-6.4

Vi

-6.4

SAHA

-4.4

Nhn xt: C 9 cht thit k u c nng lng lin kt vi trung tm hot


ng ca HDAC8 (-7,1 n -6,4 kcal/mol) thp hn SAHA (-4,4 kcal/mol) chng
t cc cht ny c i lc vi HDAC8 mnh hn SAHA. iu ny ph hp vi

26

hng thit k tm kim cc cht c tc dng c ch HDAC nn chng ti tip tc


tin hnh tng hp v th tc dng sinh hc ca dy cht.
Di y l hnh nh minh ha bng m hnh tng tc ca cc cht vi
HDAC8 ca cht Va, Vg (hnh 3.1).

Hnh 3.1: M hnh tng tc ca Va, Vg v SAHA vi HDAC8


Ch thch: SAHA (mu vng); Va (mu xanh dng), Vg(mu vng nu); Ion Zn2+ (mu
xm)

3.2. TNG HP HA HC
3.2.1. Tng

hp

cht

N1-[5-(furan-2-yl)-1,3,4-thiadiazol-2-yl]-N8-

hydroxyoctandiamid (Va)
Cht Va c tng hp theo s 3.1.

S 3.1: S phn ng tng hp cht Va

3.2.1.1.

Tng hp cht IIa

Tng hp 2-(furan-2-ylmethyliden)hydrazincarbothioamid (IIa) t furan-2carbaldehyd (Ia) c thc hin bng phn ng ngng t vi xc tc l acid hu
c, tin hnh theo s 3.2.

27

S 3.2: Tng hp cht IIa


Tin hnh phn ng:
Cho 0,18ml (2mmol) furan-2-carbaldehyd (Ia) v 0,2180g (2,4mmol)
thiosemicarbazid vo bnh cu 50 mL. Thm 20 ml dung mi EtOH tuyt i v 2
git acid acetic bng lm xc tc. un hi lu v khuy 300 vng/pht trong 4h.
Thu sn phm:
Ct thu hi dung mi bng my ct quay nhit 400C sau thm 15
mL nc ct ta sn phm. Lc v ra ta 3 ln bng 15 mL nc ct. Sy kh
ta 600C (trong khong 24h).
Kt qu:
Cm quan: Bt tinh th mu trng .
Hiu sut: 90,5% (0,3059g).
T0nc: 165-1670C.
Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,66.
Ton b sn phm IIa c dng thc hin bc tip theo ca qu trnh
tng hp cht Va.
3.2.1.2.

Tng hp cht IIIa

Thc hin vic tng hp cht 5-(furan-2-yl)-1,3,4-thiadiazol-2-amin (IIIa)


t IIa bng phn ng ng vng ni phn t. Qu trnh tng hp c thc hin
theo s 3.3 [33].

S 3.3: Tng hp cht IIIa


Tin hnh phn ng:
Cn 1,14g (3 mmol) mui st FeCl3.12H2O, ha tan bng 3 mL dung mi
EtOH trong bnh cu 50 mL, gia nhit n hi lu. ng thi cn 0,1690g (1

28

mmol) cht IIa, ha tan bng 20 mL dung mi EtOH tuyt i trong cc c m,


un nng khong 70C.
dung dch cht IIa t cc c m vo bnh phn ng, tip tc un hi lu
v khuy 300 vng/pht. Kt thc phn ng sau 3 gi.
Thu sn phm:
Ct thu hi bt dung mi bng my ct quay nhit khong 400C trong 2
pht sau pha long hn hp phn ng bng nc ct. Kim ha hn hp bng
khong 10 mL dung dch NaOH 30% (kim tra bng giy qu).
Chit 3 ln bng cloroform (mi ln 5-10 mL), thu lp di. Gp dch chit
ca 3 ln chit li vi nc mui bo ha. Ct thu hi dung mi ca dch chit bng
my ct quay nhit 400C n khi ht dung mi, thu sn phm IIIa. Sy sn
phm 600C trong 4h.
Kt qu:
Cm quan: Bt kt tinh mu vng nht.
Hiu sut: 72,1% (0,1204g).
T0nc: 196-1970C.
Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,54.
3.2.1.3.

Tng hp cht IVa

Cht methyl 8-{[5-(furan-2-yl)-1,3,4-thiadiazol-2-yl]amino}-8-oxooctanoat


(IVa) c tng hp t cht IIIa bng phn ng acyl ha ca cht IIIa vi acid
suberic monomethyl ester. Quy trnh tng hp c thc hin nh sau:

S 3.4: Tng hp cht IVa


Tin hnh phn ng:
Cho 0,1620g (1 mmol) 1,1-carbonyldiimidazol (CDI) v 1mL (1 mmol)
acid suberic monomethyl ester vo bnh cu 50mL. Thm 3mL dung mi DMF.
Hot ha trong 5 pht nhit phng. Sau khi hn hp trong bnh phn ng

29

hot ha thi gian, cho 0,1670g (1 mmol) cht IIIa vo bnh phn ng, gia nhit
ln 600C, khuy 300 vng/pht trong vng 24h.
Thu sn phm:
Lm lnh bnh phn ng bng nc . t t 15 mL dung dch HCl
long, lnh vo bnh phn ng, va va lc. yn 15 pht.
Lc v ra ta 3 ln bng 15 mL dung dch acid HCl long, lnh. Sy ta
600C trong 24h.
Kt qu:
Cm quan: Bt kt tinh mu trng hi hng.
Hiu sut: 57,0% (0,1921g).
T0nc: 228-2300C.
Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,72.
3.2.1.4.

Tng hp cht Va

Cht N1-[5-(furan-2-yl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid (Va)


c tng hp bng phn ng gia cht IVa vi hydroxylamin hydroclorid. Tng
hp cht Va c thc hin nh sau:

S 3.5: Tng hp cht Va


Tin hnh phn ng:
Cho 0,1690g (0,5 mmol) cht IVa v 0,33g (5 mmol) hydroxylamin
hydroclorid vo bnh cu 50 mL, thm 20 mL dung mi MeOH, dng siu m
to hn dch ng nht. t bnh phn ng trong hn hp nc v mui n, duy
tr nhit di 00C, khuy 300 vng/pht.
Ha tan 0,40g (10mmol) NaOH trong 5 ml nc ct trong ng nghim v
lm lnh xung di 00C trong hn hp nc mui. dung dch NaOH vo
bnh phn ng, gi nhit di 00C, phn ng kt thc sau 1gi. Theo di tin

30

trnh phn ng v xc nh thi im kt thc bng cch dng dung dch FeCl3 v
TLC, c th lm nh sau:
-

Acid ha khong 0,1 mL hn hp phn ng bng dung dch HCl long trn

khay s, sau nh 1 git dung dch FeCl3 5%, xut hin mu vang chng t
c acid hydroxamic c to ra.
-

Acid ha khong 0,1 mL hn hp phn ng, kim tra TLC vi pha ng

DCM : MeOH (9:1). Kt thc phn ng khi ester ban u ht.


Thu sn phm:
Acid ha hn hp phn ng bng dung dch HCl long, lnh iu chnh pH
v khong 4-5 (kim tra bng giy qu vn nng). qua m sau lc v ra
ta 3 ln bng 15 mL dung dch HCl long. Sy ta 600C trong 24h.
Kt qu:
Cm quan: Bt kt tinh mu trng.
Hiu sut: 51,2% (0,0865g).
Kim tra bng TLC v xc nh cu trc (kt qu c th c trnh by trong
bng 3.1 v phn 3.3).
T0nc: 198-2010C.
3.2.2. Tng

hp

cht

N1-[5-(5-bromofuran-2-yl)-1,3,4-thiadiazol-2-yl]-N8-

hydroxyoctandiamid (Vb)
Cht Vb c tng hp theo s 3.6.

S 3.6: S phn ng tng hp cht Vb

31

3.2.2.1.

Tng hp cht IIb

Tng hp cht IIb t 0,3500g (2 mmol) cht 5-bromofuran-2-carbaldehyd


(Ib) c thc hin tng t nh tng hp cht IIa. Kt qu nh sau:

Cm quan: Sn phm thu c l bt kt tinh mu trng.

Hiu sut: 85,0% (0,4216g).

T0nc: 171-1740C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,65.

3.2.2.2.

Tng hp cht IIIb

Tng hp cht 5-(5-bromofuran-2-yl)-1,3,4-thiadiazol-2-amin (IIIb) t


0,2480g (1 mmol) cht IIb c tin hnh tng t nh tng hp cht IIIa.
Kt qu nh sau:

Cm quan: Bt kt tinh mu vng nht.

Hiu sut: 70,3% (0,1729g).

T0nc: 192-1940C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,53.

3.2.2.3.

Tng hp cht IVb

Tng

hp

cht

methyl

8-{[5-(5-bromofuran-2-yl)-1,3,4-thiadiazol-2-

yl]amino}-8-oxooctanoat (IVb) t 0,2460g (1 mmol) cht IIIb c tin hnh


tng t nh tng hp cht IVa. Kt qu ca qu trnh nh sau:

Cm quan: Bt kt tinh mu trng ng.

Hiu sut: 62,9% (0,2617g).

T0nc: 210-2120C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,77.

3.2.2.4.

Tng hp cht Vb

Tng hp cht ch N1-[5-(5-bromofuran-2-yl)-1,3,4-thiadiazol-2-yl]-N8hydroxyoctandiamid (Vb) t 0,2080g (0,5 mmol) cht IVb c tin hnh tng
t nh tng hp cht Va. Kt qu ca qu trnh nh sau:

Cm quan: Bt kt tinh mu trng.

32

Hiu sut phn ng: 52,0% (0,1084g).

T0nc: 200-2030C.

Kim tra bng TLC v xc nh cng thc (kt qu c th c trnh by


trong bng 3.1 v phn 3.3).

3.2.3. Tng hp cht N1-hydroxy-N8-[5-(5-methylfuran-2-yl)-1,3,4-thiadiazol2-yl]octandiamid (Vc)


Cht Vc c tng hp theo s 3.7.

S 3.7: S phn ng tng hp cht Vc

3.2.3.1.

Tng hp cht IIc

Tng hp cht IIc t 0,2220g (2 mmol) cht 5-methylfuran-2-carbaldehyd


(Ic) c thc hin tng t nh tng hp cht IIa. Kt qu nh sau:

Cm quan: Sn phm thu c l bt kt tinh mu trng.

Hiu sut: 86,6% (0,3170g).

T0nc: 169-1710C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,65.

3.2.3.2.

Tng hp cht IIIc

Tng hp cht 5-(5-methylfuran-2-yl)-1,3,4-thiadiazol-2-amin (IIIc) t


0,1830g (1 mmol) cht IIc c tin hnh tng t nh tng hp cht IIIa.
Kt qu nh sau:

Cm quan: Bt kt tinh mu vng nht.

Hiu sut: 71,0% (0,1285g).

T0nc: 186-1890C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,53.

33

3.2.3.3.

Tng hp cht IVc

Tng

hp

cht

methyl

8-{[5-(5-methylfuran-2-yl)-1,3,4-thiadiazol-2-

yl]amino}-8-oxooctanoat (IVc) t 0,1810g (1 mmol) cht IIIc c tin hnh


tng t nh tng hp cht IVa. Kt qu ca qu trnh nh sau:

Cm quan: Bt kt tinh mu trng ng.

Hiu sut: 58,8% (0,2064g).

T0nc: 199-2020C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,75.

3.2.3.4.

Tng hp cht Vc

Tng hp cht ch N1-hydroxy-N8-[5-(5-methylfuran-2-yl)-1,3,4-thiadiazol2-yl]octandiamid (Vc) t 0,1800g (0,5 mmol) cht IVc c tin hnh tng t
nh tng hp cht Va. Kt qu ca qu trnh nh sau:

Cm quan: Bt kt tinh mu trng.

Hiu sut phn ng: 54,9% (0,0966g).

T0nc: 178-1810C.

Kim tra bng TLC v xc nh cng thc (kt qu c th c trnh by


trong bng 3.1 v phn 3.3).

3.2.4. Tng hp cht N1-hydroxy-N8-[5-(thiophen-2-yl)-1,3,4-thiadiazol-2yl]octandiamid (Vd)


Cht Vd c tng hp theo s 3.8.

S 3.8: S phn ng tng hp cht Vd

34

3.2.4.1.

Tng hp cht IId

Tng hp cht IId t 0,19mL (2mmol) cht thiophen-2-carbaldehyd (Id)


c thc hin tng t nh tng hp cht IIa. Kt qu nh sau:

Cm quan: Bt kt tinh mu trng.

Hiu sut: 86% (0,3182g).

T0nc: 170-1720C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,63.

3.2.4.2.

Tng hp cht IIId

Tng hp cht 5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amin (IIId) t 0,185g (1


mmol) cht IId c tin hnh tng t nh tng hp cht IIIa. Kt qu nh sau:

Cm quan: Bt kt tinh mu vng nu.

Hiu sut: 70,0% (0,1281g).

T0nc: 182-1840C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,53.

3.2.4.3.

Tng hp cht IVd

Tng hp cht methyl 8-oxo-8-{[5-(thiophen-2-yl)-1,3,4-thiadiazol-2yl]amino}octanoat (IVd) c tin hnh tng t nh tng hp cht IVa. Kt qu
ca qu trnh nh sau:

Cm quan: Bt kt tinh mu trng .

Hiu sut: 60,3% (0,2120g).

T0nc: 196-1980C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,77.

3.2.4.4.

Tng hp cht Vd

Tng hp cht ch N1-hydroxy-N8-[5-(thiophen-2-yl)-1,3,4-thiadiazol-2yl]octanediamid (Vd) t 0,1775g ( 0,5 mmol) cht IVd c


tin hnh tng t nh tng hp cht Va. Kt qu nh sau:

Cm quan: Bt kt tinh mu trng.

Hiu sut phn ng: 55,2% (0,0977g).

T0nc: 177-1780C.

35

Kim tra bng TLC v xc nh cu trc (kt qu c th c trnh by trong


bng 3.1 v phn 3.3).

3.2.5. Tng hp cht N1-[5-(5-bromothiophen-2-yl)-1,3,4-thiadiazol-2-yl]-N8hydroxyoctandiamid (Ve)


Cht Ve c tng hp theo s 3.9.

S 3.9: S phn ng tng hp cht Ve


3.2.5.1.

Tng hp cht IIe

Tng hp cht

IIe t 0,24ml (2 mmol) cht 5-bromothiophen-2-

carbaldehyd (Ie) c thc hin tng t nh tng hp cht IIa. Kt qu nh sau:

Cm quan: Sn phm thu c l bt kt tinh mu trng.

Hiu sut: 90,1% (0,4757g).

T0nc: 175-1770C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,60.

3.2.5.2.

Tng hp cht IIIe

Tng hp cht 5-(5-bromothiophen-2-yl)-1,3,4-thiadiazol-2-amin (IIIe) t


0,2640g (1 mmol) cht IIe c tin hnh tng t nh tng hp cht IIIa.
Kt qu nh sau:

Cm quan: Bt kt tinh mu vng nht.

Hiu sut: 68,5% (0,1795g).

T0nc: 187-1900C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,52.

36

3.2.5.3.

Tng hp cht IVe

Tng hp cht methyl 8-{[5-(5-bromothiophen-2-yl)-1,3,4-thiadiazol-2yl]amino}-8-oxooctanoat (IVe) t 0,2620g (1mmol) cht IIIe c tin hnh tng
t nh tng hp cht IVa. Kt qu ca qu trnh nh sau:

Cm quan: Bt kt tinh mu trng ng.

Hiu sut: 55% (0,2376g).

T0nc: 203-2050C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,76.

3.2.5.4.

Tng hp cht Ve

Tng hp cht ch N1-[5-(5-bromothiophen-2-yl)-1,3,4-thiadiazol-2-yl]-N8hydroxyoctandiamid (Ve) t 0,2240g (0,5 mmol) cht IVe c tin hnh tng t
nh tng hp cht Va. Kt qu ca qu trnh nh sau:

Cm quan: Bt kt tinh mu trng.

Hiu sut phn ng: 51,8% (0,1121g).

T0nc: 181-1830C.

Kim tra bng TLC v xc nh cng thc (kt qu c th c trnh by


trong bng 3.1 v phn 3.3).

3.2.6. Tng

hp

cht

N1-hydroxy-N8-[5-(5-methylthiophen-2-yl)-1,3,4-

thiadiazol-2-yl]octandiamid (Vf)
Cht Vf c tng hp theo s 3.10.

S 3.10: S phn ng tng hp cht Vf

37

3.2.6.1.

Tng hp cht IIf

Tng hp cht

IIf t 0,22ml (2 mmol) cht 5-methylthiophen-2-

carbaldehyd (If) c thc hin tng t nh tng hp cht IIa. Kt qu nh sau:

Cm quan: Sn phm thu c l bt kt tinh mu trng.

Hiu sut: 89,2% (0,3550g).

T0nc: 173-1760C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,61.

3.2.6.2.

Tng hp cht IIIf

Tng hp cht 5-(5-mehtylthiophen-2-yl)-1,3,4-thiadiazol-2-amin (IIIf) t


0,1990g (1 mmol) cht IIf c tin hnh tng t nh tng hp cht IIIa.
Kt qu nh sau:

Cm quan: Bt kt tinh mu vng nht.

Hiu sut: 71,3% (0,1405g).

T0nc: 183-1840C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,50.

3.2.6.3.

Tng hp cht IVf

Tng hp cht methyl 8-{[5-(5-methylthiophen-2-yl)-1,3,4-thiadiazol-2yl]amino}-8-oxooctanoat (IVf) t 0,1970g (1mmol) cht IIIf c tin hnh tng
t nh tng hp cht IVa. Kt qu ca qu trnh nh sau:

Cm quan: Bt kt tinh mu trng ng.

Hiu sut: 56,7% (0,2081g).

T0nc: 200-2030C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,78.

3.2.6.4.

Tng hp cht Vf

Tng

hp

cht

ch

N1-hydroxy-N8-[5-(5-methylthiophen-2-yl)-1,3,4-

thiadiazol-2-yl]octandiamid (Vf) t 0,1915g (0,5 mmol) cht IVf c tin hnh


tng t nh tng hp cht Va. Kt qu ca qu trnh nh sau:

Cm quan: Bt kt tinh mu trng.

Hiu sut phn ng: 51,0% (0,0938g).

38

T0nc: 179-1800C.

Kim tra bng TLC v xc nh cng thc (kt qu c th c trnh by


trong bng 3.1 v phn 3.3).

3.2.7. Tng

hp

cht

N1-hydroxy-N8-[5-(pyridin-2-yl)-1,3,4-thiadiazol-2-

yl]octandiamid (Vg)
Cht Vg c tng hp theo s 3.11.

3.2.7.1.

S 3.11: S phn ng tng hp cht Vg


Tng hp cht IIg

Tng hp cht IIg t 0,19mL (2mmol) cht pyridin-2-carbaldehyd (Ig) c


thc hin tng t nh tng hp cht IIa. Kt qu nh sau:

Cm quan: Bt kt tinh mu trng.

Hiu sut: 91,5% (0,3294g).

T0nc: 183-1850C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,62.

3.2.7.2.

Tng hp cht IIIg

Tng hp cht 5-(pyridin-2-yl)-1,3,4-thiadiazol-2-amin (IIIg) t 0,1800g


(1mmol) cht IIg c tin hnh tng t nh tng hp cht IIIa. Kt qu nh sau:

Cm quan: Bt kt tinh mu vng nu.

Hiu sut: 69,0% (0,1228g).

T0nc: 193-1950C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,48.

39

3.2.7.3.

Tng hp cht IVg

Tng

hp

cht

methyl

8-oxo-8-{[5-(pyridin-2-yl)-1,3,4-thiadiazol-2-

yl]amino}octanoat (IVg) c tin hnh tng t nh tng hp cht IVa. Kt qu


ca qu trnh nh sau:

Cm quan: Bt kt tinh mu trng .

Hiu sut: 59,1% (0,2057g).

T0nc: 221-2230C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,75.

3.2.7.4.

Tng hp cht Vg

Tng hp cht ch N1-hydroxy-N8-[5-(pyridin-2-yl)-1,3,4-thiadiazol-2yl]octandiamid (Vg) t 0,1772g ( 0,5 mmol) cht IVg c tin hnh tng t nh
tng hp cht Va. Kt qu nh sau:

Cm quan: Bt kt tinh mu trng.

Hiu sut phn ng: 54,3% (0,0948g).

T0nc: 209-2110C.

Kim tra bng TLC v xc nh cu trc (kt qu c th c trnh by trong


bng 3.1 v phn 3.3).

3.2.8. Tng

hp

cht

N1-hydroxy-N8-[5-(pyridin-3-yl)-1,3,4-thiadiazol-2-

yl]octandiamid (Vh)
Cht Vh c tng hp theo s 3.12.

S 3.12: S phn ng tng hp cht Vh

40

3.2.8.1.

Tng hp cht IIh

Tng hp cht IIh t 0,19mL (2mmol) cht pyridin-3-carbaldehyd (Ih)


c thc hin tng t nh tng hp cht IIa. Kt qu nh sau:

Cm quan: Bt kt tinh mu trng.

Hiu sut: 88,4% (0,3182g).

T0nc: 185-1880C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,51.

3.2.8.2.

Tng hp cht IIIh

Tng hp cht 5-(pyridin-3-yl)-1,3,4-thiadiazol-2-amin (IIIh) t 0,1800g


(1mmol) cht IIh c tin hnh tng t nh tng hp cht IIIa. Kt qu nh sau:

Cm quan: Bt kt tinh mu vng nu.

Hiu sut: 68,5% (0,1219g).

T0nc: 191-1940C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,55.

3.2.8.3.

Tng hp cht IVh

Tng

hp

cht

methyl

8-oxo-8-{[5-(pyridin-3-yl)-1,3,4-thiadiazol-2-

yl]amino}octanoat (IVh) c tin hnh tng t nh tng hp cht IVa. Kt qu


ca qu trnh nh sau:

Cm quan: Bt kt tinh mu trng .

Hiu sut: 57,4% (0,1998g).

T0nc: 218-2200C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,72.

3.2.8.4.

Tng hp cht Vh

Tng hp cht ch N1-hydroxy-N8-[5-(pyridin-3-yl)-1,3,4-thiadiazol-2yl]octandiamid (Vh) t 0,1772g ( 0,5 mmol) cht IVh c tin hnh tng t nh
tng hp cht Va. Kt qu nh sau:

Cm quan: Bt kt tinh mu trng.

Hiu sut phn ng: 50,9% (0,0888g).

T0nc: 207-2100C.

41

Kim tra bng TLC v xc nh cu trc (kt qu c th c trnh by trong


bng 3.1 v phn 3.3).

3.2.9. Tng

hp

cht

N1-hydroxy-N8-[5-(pyridin-4-yl)-1,3,4-thiadiazol-2-

yl]octandiamid (Vi)
Cht Vi c tng hp theo s 3.13.

3.2.9.1.

S 3.13: S phn ng tng hp cht Vi


Tng hp cht IIi

Tng hp cht IIi t 0,19mL (2mmol) cht pyridin-4-carbaldehyd (Ii) c


thc hin tng t nh tng hp cht IIa. Kt qu nh sau:

Cm quan: Bt kt tinh mu trng.

Hiu sut: 87,8% (0,3161g).

T0nc: 186-1880C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,53.

3.2.9.2.

Tng hp cht IIIi

Tng hp cht 5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amin (IIIi) t 0,1800g


(1mmol) cht IIi c tin hnh tng t nh tng hp cht IIIa. Kt qu nh sau:

Cm quan: Bt kt tinh mu vng nu.

Hiu sut: 70,8% (0,1260g).

T0nc: 194-1970C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,54.

42

3.2.9.3.

Tng hp cht IVi

Tng

hp

cht

methyl

8-oxo-8-{[5-(pyridin-4-yl)-1,3,4-thiadiazol-2-

yl]amino}octanoat (IVi) c tin hnh tng t nh tng hp cht IVa. Kt qu


ca qu trnh nh sau:

Cm quan: Bt kt tinh mu trng .

Hiu sut: 57,0% (0,1984g).

T0nc: 222-2250C.

Kim tra bng TLC vi pha ng DCM : MeOH (9:1) cho Rf = 0,76.

3.2.9.4.

Tng hp cht Vi

Tng hp cht ch N1-hydroxy-N8-[5-(pyridin-4-yl)-1,3,4-thiadiazol-2yl]octandiamid (Vi) t 0,1772g ( 0,5 mmol) cht IVi c tin hnh tng t nh
tng hp cht Va. Kt qu nh sau:

Cm quan: Bt kt tinh mu trng.

Hiu sut phn ng: 52,7% (0,0920g).

T0nc: 206-2080C.

Kim tra bng TLC v xc nh cu trc (kt qu c th c trnh by trong


bng 3.1 v phn 3.3).

3.2. KIM TRA TINH KHIT


tinh khit cc cht Va-i c kim tra bng TLC v o nhit nng
chy (Tonc) ca chng.

TLC c tin hnh trn bn mng silicagel 60 F254 trng sn, quan st di

n t ngoi bc sng 254 nm. Ha tan trong dung mi DMF v sau kho st
vi cc h dung mi trin khai. Kt qu thu c trn cc sc k u thy mt
vt r, gn khi soi di nh sng n t ngoi.

o nhit nng chy: 9 cht sau khi c tinh ch bng cch kt tinh li

u c dng tinh th rn c o nhit nng chy bng my o nhit nng


chy nhit in (Electrothermal digital).
Nhit nng chy v gi tr Rf c tm tt trong bng 3.2.

43

Bng 3.2: Gi tr Rf v T0nc ca cc cht Va-i

Hqui trnh

T0nc
(0C)

Rf
H DCM : MeOH
(9:1)

STT

Cht

Va

19,0%

198-201

0,47

Vb

19,5%

200-203

0,45

Vc

19,8%

178-181

0,50

Vd

20,0%

177-178

0,52

Ve

17,6%

181-183

0,55

Vf

18,4%

179-180

0,51

Vg

20,3%

210-211

0,46

Vh

17,7%

207-210

0,51

Vi

18,7%

206-208

0,45

3.3. KHNG NH CU TRC


khng nh chc chn cu trc ca cc cht tng hp c, chng ti
tin hnh o 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)
Ph hng ngoi ca cc cht c ghi trn my Perkin Elmer vi k thut
lm vin nn KBr ghi trong vng 4000-600 cm-1. Cc ph c ph lc.

44

Bng 3.3: S liu phn tch ph IR ca Va-i

STT

Cht

Va

Vb

Vc

O-H
3350

3416

S sng ng vi dao ng ha tr (cm-1)


C-H
C-H
N-H
C=O
(-CH2;(benzen)

3152

C=C

CH3)

2923
2865
2853

1637
1673

1567
1504
1465
1561
1499
1464

3245
3158

3025

2931
2858

1690
1638

3158

3032

2925
2856

1718
1694

1567
1552

1674
1637

1563
1461
1436

Vd

3361

3154

2931
2854

Ve

3411

3143

2933
2730

1692
1631

1565
1450

Vf

Vg

3163

3035

2914
2853

1692
1642

1612
1573

3231

3161

3022

2930
2855

1693
1667

1557
1435

Vh

3251

3155

3008

2911
2852

1695
1638

1576
1554

Vi

3265

3139

3009

2852

1701
1646

1602
1547
1441

Nhn xt: S liu phn tch ph IR hon ton ph hp vi cc ti liu tham


kho cng nh cu trc d kin ca cc cht Va-i c th nh sau:
Cc cht ch Va-i u cho d liu ph ph hp vi cc nhm chc d kin trong
cng thc: Vng 1612-1435 cm-1 c trng cho dao ng C=C ca vng thm,
vng 1718-1637 cm-1 c trng cho dao ng C=O ca nhm carbonyl, vng 30353008 cm-1 c trng cho dao ng C-H trong vng benzen, vng 3361-3139 cm-1
c trng cho N-H v 3400 cm-1 cho O-H.

45

3.3.2. Ph khi lng (MS)


Bn cnh phng php o ph IR, phng php o ph khi lng ch
khng phn mnh ESI gip khng nh thm cu trc ca cc cht tng hp c.
Kt qu phn tch ph MS trnh by trong bng 3.4.
Bng 3.4: S liu phn tch ph khi lng ca cc cht

Cht

m/z

Va

338

337 [M-H]-

Vb

417

416 [M-H]-

Vc

352

351 [M-H]-

Vd

354

353 [M-H]-

Ve

433

432 [M-H]-

Vf

368

367 [M-H]-

Vg

349

348 [M-H]-

Vh

349

348 [M-H]-

Vi

349

348 [M-H]-

Nhn xt: S liu ph MS bng 3.4 cho thy rng cc cht kho st u c
pic phn t c s khi ng bng s khi d kin.
3.3.3. Ph cng hng t ht nhn (1H-NMR, 13C-NMR)
khng nh chc chn hn cu trc, chng ti tin hnh ghi ph 1H-NMR
v 13C-NMR ca 9 cht tng hp c. Kt qu ph 1H-NMR c trnh by trong
bng 3.5 v trong ph lc.

46

Bng 3.5: S liu phn tch ph 1H-NMR

Cht

(ppm) (500 MHz, DMSO-d6)

10,35 (1H, s, NH); 7,93 (1H, s, H5); 7,18 (1H, d, J = 3,0 Hz,
H3); 6,72 (1H, s, H4); 2,47-2,48 (2H, t, J = 7,5Hz, 2H-7);
Va

1,92-1,95 (2H, t, J = 7,5Hz, 2H-2); 1,58-1,61 (2H, t, J = 6,5 Hz,


2H-6); 1,47-1,49 (2H, t, J = 6,5Hz, 2H-3); 1,26-1,28 (4H, m,
2H-4, 2H-5).
12,70 (1H, s, NH); 10,33 (1H, s, NH); 7,23 (1H, d, J = 3,5 Hz,

Vb

H3); 6,83 (1H, d, J = 3,5 Hz, H4); 2,47-2,49 (2H, m, 2H-7);


1,92-1,95 (2H, m, 2H-2); 1,57-1,61 (2H, m, 2H-6); 1,45-1,51
(2H, m, 2H-3); 1,26-1,28 (4H, m, 2H-4, 2H-5).
12,64 (1H, s, NH); 10,36 (1H, s, NH); 7,04 (1H, d, J = 3,0Hz,
H3); 6,32 (1H, d, J = 3,0 Hz, H4); 2,47-2,48 (2H, t, J = 7,0Hz,

Vc

2H-7); 2,36 (3H, s, CH3); 2,18 (1H, t, J = 7,5Hz, CH2); 1,93


(1H, t, J = 6,0Hz, CH2); 1,58-1,60 (2H, m, 2H-6); 1,46-1,48 (2H,
m, 2H-3); 1,20-1,34 (4H, m, 2H-4, 2H-5).
12,65 (1H, s, NH); 10,34 (1H, s, NH); 8,67 (1H, s, OH); 7,75
(1H, d, J = 4,5 Hz, H5); 7,69 (1H, d, J = 2,5 Hz, H3); 7,19 (H,

Vd

s, H4); 2,47-2,48 (2H, t, J = 7,0Hz, 2H-7); 1,92-1,95 (2H, t, J =


7,0Hz, 2H-2); 1,58-1,60 (2H, t, J = 6,0Hz, 2H-6); 1,47-1,49 (2H,
t, J = 6,5Hz, 2H-3); 1,26 (4H, m, 2H-4, 2H-5).
12,68 (H, s, NH); 10,33 (1H, s, NH); 7,53 (1H, s, H3); 7,30 (H,

Ve

d, J = 2,5Hz, H4); 2,46-2,48 (2H, m, 2H-7); 1,90-1,93 (2H, t, J


= 6,5Hz, 2H-2); 1,57 (2H, m, 2H-6); 1,46 (2H, m, 2H-3); 1,24
(4H, m, 2H-4, 2H-5).
12,57 (1H, s, NH); 10,34 (1H, s, NH); 7,45-7,46 (1H, d, J = 3,5
Hz, H3); 6,87-6,88 (1H, d, J = 2,5 Hz, H4); 2,45-2,48 (5H, m,

Vf

CH3, 2H-7); 1,92-1,95 (2H, t, J = 7,5Hz, 2H-2); 1,57-1,60 (2H,


m, 2H-6); 1,46-1,49 (2H, m, 2H-3); 1,26-1,27 (4H, m, 2H-4,
2H-5).

47

12,73 (1H, s, NH); 10,44 (1H, s, NH); 9.12 (1H, d, J = 1,5Hz,


H6); 8,69 -8,70 (2H, OH, H5); 8,32 (1H, d, J = 8,0Hz, H3);
7,57 (1H, dd, J = 5,0Hz, J = 8,0Hz, H4); 2,50-2,52 (2H, m, 2H-

Vg

7); 1,92-1,95 (2H, t, J = 7,0Hz, 2H-2); 1,58-1,62 (2H, m, 2H-3);


1,45-1,51 (2H, m, 2H-6); 1,25-1,27 (4H, m, 2H-4, 2H-5).
12,62 (1H, s, NH); 10,35 (1H, s, NH); 8,67 (2H, OH, H6); 8,19
(1H, d, J = 7,5Hz, H4); 7,98 (1H, t, J = 7,0Hz, H5); 7,51 (1H,
s, H2); 2,50-2,48 (2H, m, 2H-7); 1,92-1,95 (2H, t, J = 7,0Hz,

Vh

2H-2); 1,59-1,62 (2H, t, J = 6,5Hz, 2H-3); 1,47-1,50 (2H, t, J =


6,5Hz, 2H-6); 1,20-1,27 (4H, m, 2H-4, 2H-5).
12,85 (1H, s, NH); 10,42 (1H, s, NH); 8,71 (3H, OH, H2, H6);
7,90 (2H, H3, H5); 2,50 (2H, 2H-7); 1,94 (2H, 2H-2); 1,60

Vi

(2H, 2H-3); 1,48 (2H, 2H-6); 1,26 (4H, 2H-4, 2H-5).

Nhn xt: S c mt ca cc proton ph hp vi cng thc ca 9 cht Va-i.


Trn ph c cc pic c trng ca cc proton trong c phn khung 5-aryl-1,3,4thiadiazol v proton ca cc nhm chc, nhm th.
Ph 13C-NMR c trnh by trong bng 3.6 v trong ph lc.
Bng 3.6: S liu phn tch ph 13C-NMR

Cht

Cng thc

(ppm) (125 MHz, DMSO-d6)


171,66 (C5); 169,10 (C8); 157,66 (C1); 152,38 (C2);

Va

145,17-145,31 (C5-C2); 112,52 (C3); 110,75 (C3);


34,80 (C7); 32,19 (C2); 28,21-28,25 (C3-C6); 24,40-24,93
(C4-C5).

Vb

171,71 (C5); 169,13 (C8); 157,82 (C1); 151,33 (C2);


147,07 (C2); 124,45 (C5); 114,59 (C3); 113,57 (C4);
34,81 (C7); 32,21 (C2); 28,25-28,21 (C3-C6); 24,41-24,94
(C4-C5).

48

174,47 (C5); 171,64 (C8); 157,15 (C1); 154,60 (C2);


152,54 (C5); 143,57 (C2);

Vc

108,85-112,03 (C3-C4);

34,81 (C7); 33,63 (C2); 32,21 (C3); 28,19 (C6); 24,31-24,95


(C4-C5); 13,38 (CH3).
171,61 (C5); 169,10 (C8); 157,79 (C1); 156,10 (C2);
132,32 (C2); 129,00-129,11 (C5-C3); 128,39 (C4);

Vd

34,80 (C7); 32,21 (C2); 28,20-28,28 (C3-C6); 24,45-24,96


(C4-C5).
171,68 (C5); 169,08 (C8); 158,08 (C1); 155,25 (C2);
134,04 (C2); 131,71-129,61 (C3-C4); 114,73 (C5);

Ve

34,79 (C7); 32,20 (C2); 28,27-28,22 (C3-C6); 24,95-24,43


(C4-C5).
171,58 (C5); 169,18 (C8); 157,40 (C1); 156,2 (C2),
142,97(C2), 129,91(C5), 129,10(C3), 126,83(C4), 34,81

Vf

(C7); 32,23 (C2); 28,28 (C3); 28,23 (C6); 24,47-24,97 (C4C5); 15,09 (CH3).
171,71 (C5); 169,12 (C8); 163,48 (C1); 160,03 (C2);

Vg

119,81-149,92 (C2-6); 34,89 (C7); 32,21 (C2); 28,2728,24 (C3-C6); 24,95-24,41 (C4-C5).
172,19 (C5); 169,56 (C8); 159,39 (C1); 151,64 (C2);
147,88 (C2-C6); 134,85(C3); 127,00-124,80 (C4-C5);

Vh

34,89 (C7); 32,21 (C2); 28,27-28,24 (C3-C6); 24,95-24,41


(C4-C5).
171,86 (C5); 169,10 (C8); 159,52 (C1); 150,70 (C2-C2C6); 137,20 (C4); 120,79 (C3-C5); 34,83 (C7); 32,19

Vi

(C2); 28,27-28,21 (C3-C6); 24,42-24,95 (C4-C5).

Nhn xt: S c mt ca cc tn hiu cng hng ht nhn carbon ph hp


vi cng thc cu to ca 9 cht Va-i. Trn ph , cc pic c trng ca cc
carbon trong c phn khung 5-aryl-1,3,4-thiadiazol v carbon ca cc nhm chc,
nhm th.

49

3.5. HOT TNH SINH HC


3.5.1. Tc dng c ch HDAC
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 cc t bo ung th. 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. Th nghim
c tin hnh ti khoa Dc, H Chungbuk, Hn Quc. Kt qu c ch HDAC
ca cc cht c trnh by trong bng 3.7.

Bng 3.7: Tc dng c ch HDAC ca cc dn cht tng hp

Tc dng
c ch
HDAC
(1M)

Cht

Va

Vf

Vb

Vg

Vc

Vh

Vd

Vi

Ve

SAHA*

Cht

(+): C tc dng c ch; (-): Khng c ch; (*): Chun dng tnh

50

Tc dng
c ch
HDAC
(1M)

3.5.2. Hot tnh khng t bo ung th in vitro


Th c tnh t bo in vitro c thc hin ti Khoa Dc, Trng i hc
Chungbuk, Hn Quc theo phng php MTT.
Dng t bo th nghim: SW620 (t bo ung th i trng ngi), MCF-7
(t bo ung th v ngi), PC-3 (t bo ung th tin lit tuyn ngi), AsPC-1 (t
bo ung th ty ngi).
Kt qu gi tr IC50 ca cc cht trn 4 dng t bo c trnh by trong bng
4.1 phn 4.3.

51

Chng 4. BN LUN
4.1. TNG HP HA HC
Cc acid hydroxamic mang khung 5-aryl-1,3,4-thiadiazol c tng hp
theo quy trnh 4 bc.
bc u tin, cc thiosemicarbazon IIa-i c to nn bng cch ngng
t cc benzaldehyd Ia-i vi thiosemicarbazid. Phn ng xy ra d dng v t hiu
sut cao.
Bc th 2, phn ng to vng ni phn t ca cc cht IIa-i vi tc nhn
FeCl3 to thnh cc dn xut 2-amino-5-aryl-1,3,4-thiadiazol (IIIa-i) [33]. Trong
phn ng ny, tc nhn oxi ha (v d cc acid Lewis nh FeCl3, NH4Fe(SO4)2,...)
ng vai tr quan trng.

S 4.1: C ch phn ng ng vng tng hp 2-amino-5-aryl-1,3,4-thiadiazol


Bc th 3, ghp ni IIIa-i vi acid suberic monomethyl ester s dng CDI
nh mt tc nhn hot ha to ra IVa-i.

52

S 4.2: C ch hot ha ca CDI


Thc hin bc ny dng c phi kh hon ton, cc ha cht v dung mi
phi khan nc do CDI d phn hy dn n khng cn kh nng hot ha acid
monomethyl suberic nu tip xc vi nc.
Bc cui cng, phn ng gia ester IVa-i vi hydroxylamin trong mi
trng kim cho ra sn phm cui cng Va-i. trnh phn hy ester thnh acid
carboxylic bi dung dch NaOH m c, cn lm lnh hn dch IVa-i v NaOH
trc khi cho vo bnh phn ng. Phn ng cng cn thc hin nhit thp v
thi gian ngn trnh phn hy nhm amid. Lng NaOH phi d chuyn ht
NH2OH.HCl thnh NH2OH tan v m bo acid hydroxamic to thnh tan ht trong
dung dch phn ng.
4.2. KHNG NH CU TRC
4.2.1. Ph hng ngoi (IR)
D liu v ph hng ngoi ca 9 cht tng hp c trnh by trong mc
3.3.1 v ph trong phn ph lc. Chng ti tp trung phn tch ph vng nhm
chc xc nh sn phm to ra vi cc nhm chc c trng.
- Trn ph ca mt s cht (Vc, Ve) c th quan st thy vn hp th ca
dao ng ha tr O-H acid mnh v t (3400 cm-1).
- Trong cu trc c 2 lin kt NH-CO- nn ph ca c 9 cht u xut
hin 1-2 vn hp th ca dao ng ha tr N-H amid cng nh vn hp th ca dao

53

ng ha tr C=O amid. Cc dao ng N-H c s sng t 3361-3139 cm-1, cc dao


ng C=O c s sng t 1718-1638 cm-1.
- Phn cu ni alkyl ca cc cht tng hp c cng cho thy s c mt
trn ph vi dao ng ha tr CH2 t 2730-2933 cm-1.
- Trn ph ca cc cht cng quan st thy dao ng ha tr ca cc lin
kt C=C vng thm t 1602-1435 v C-H vng thm t 3025-3008 cm-1.
Vic quan st c cc vn hp th c trng ca cc nhm chc OH, NH, C=O v cu ni alkyl cho thy phn ng to acid hydroxamic xy ra.
Chng ti tip tc phn tch cc d liu ph khi lng v ph cng hng t ht
nhn khng nh chc chn cu trc ca cc cht tng hp c.
Di y l hnh nh minh ha ph hng ngoi ca cht Vb.

Hnh 4.1: Ph hng ngoi ca cht Vb


4.2.2. Ph khi (MS)
Cc cht tng hp c c o ph khi lng ch khng phn mnh
ESI (-). Trn ph ca mi cht u xut hin mt pic ion cng mnh, c s
khi bng [M-H]- ng vi tng cht (d liu mc 3.3.1 v ph lc).
Di y l hnh nh minh ha ph khi lng ca cht Vb.

54

Hnh 4.2: Ph khi lng ca cht Vb


4.2.3. Ph cng hng t ht nhn (1H-NMR, 13C-NMR)
khng nh chc chn cu trc, ngoi ph hng ngoi v ph khi lng,
9 cht tng hp c o ph cng hng t ht nhn 1H, 13C. D liu ph c
trnh by trong mc 3.3.1 v ph phn ph lc. Kt qu ph ca c 9 cht
u th hin s c mt ca cc d vng v cu ni alkyl vi y tn hiu proton
v carbon.
* V ph cng hng t ht nhn 1H (1H-NMR)

- Cc cht u c cu trc cha nhm hydroxamic v lin kt amid nn quan


st thy s c mt ca proton NH ( = 10,33-12,85 ppm), -OH acid ( = 8,67-8,71
ppm). H ca NHCO- v tr 7 cng hng trng yu nht vi dch chuyn
ha hc trong khong 12,57-12,85 ppm, do hiu ng ht in t ca vng
thiadiazol lm ht nhn b gim s chn ti ch. H ca NH trong nhm chc
hydroxamic NHOH linh ng hn trong mi trng DMSO nn cng hng
trng mnh hn ( = 10,33-10,42 ppm). Cc proton ny linh ng v d b trao

55

i hoc h bin nn mt s cht khng cho tn hiu ca proton trong NH, -OH
trn ph (Va, Vb, Vc, Ve, Vf).
- Mt nhm cc proton quan trng trong cu trc ca dy cht l cc proton
mch nhnh. C 9 cht u th hin s proton mch nhnh vi dch chuyn
trong khong 1,20-2,52 ppm. Cc cht Vc, Vf c nhm CH3 gn trn d vng nn
cho tn hiu 3H dng singlet v tr 2,36-2,48 ppm.
- Trn ph ca dy cht cng quan st c y cc proton trong d
vng gn vi vng thiadiazol. dn cht d vng furan (Va-c), cc proton thm
dao ng trong khong 6,32-7,93 ppm; dn cht d vng thiophen (Vd-f), khong
ny l 6,87-7,75 ppm v i vi d vng pyridin (Vg-i) l 7,51-9,12 ppm.
Di y l hnh nh minh ha ph 1H-NMR ca cht Vd.

Hnh 4.3: Ph 1H-NMR ca cht Vd

56

* V ph cng hng t ht nhn 13C (13C-NMR)


Ph 13C-NMR c u im l ton b carbon trong phn t cc cht u cho
tn hiu c tch r rng dng mi tn n do c o dng ph 13C kh hon
ton tng tc spin-spin.
- Hai carbon ca khung thiadiazol (C2, C5) c dch chuyn ha hc trong
khong 171,58-172,19 ppm v 151,33-160,03 ppm.
- Hai carbon trong nhm carbonyl (C=O) (C1, C8) c dch chuyn trong
khong 157,15-169,65 ppm.
- Cc carbon trong d vng cng c quan st r: D vng furan c 4C dao
ng trong khong 108,85-154,60 ppm, d vng thiophen c 4C dao ng t
114,73-142,97 ppm, d vng pyridin c 5C dao ng t 120,79-150,70 ppm.
- Phn cu ni alkyl cho tn hiu cng hng ca carbon trong khong
24,31-34,89 ppm.
- Vc, Vf c thm nhm th CH3 gn trn d vng c dao ng v tr 13,3815,09 ppm.
Di y l hnh nh minh ha ph 13C-NMR ca cht Vb.

Hnh 4.4: Ph 13C-NMR ca cht Vb

T nhng kt qu bin lun cc loi ph , chng ti khng nh tng


hp c 9 cht c cu trc ng nh d kin v sn phm tinh khit.

57

4.3. HOT TNH SINH HC


h tr trong qu trnh thit k cu trc v tm hiu kh nng gn vo
trung tm hot ng trn enzym HDAC ca cc cht, chng ti tin hnh nghin
cu docking 9 cht Va-i. So snh m hnh tng tc vi HDAC8 ca cc cht Va-i
v SAHA c th thy phn cu ni gia nhm kha hot ng v nhm gn vi
Zn2+ ca 9 cht cng nh ca SAHA c cu trc tng i ging nhau. Nh vy
cu ni vi di 6C ca cc cht tng hp c c nhiu kh nng a c
nhm -NHOH tin gn ion Zn2+ ging nh SAHA. Bn cnh , nng lng lin
kt ca cc cht Va-i vi trung tm hot ng ca HDAC8 c d on t -7,1
n -6,4 kcal/mol, ngha l u thp hn SAHA (-4,4 kcal/mol), chng t cc cht
c i lc vi HDAC8 mnh hn SAHA. C th s thay i nhm nhn din b mt
lm tng lc lin kt ca cc cht vi phn vnh ca enzym HDAC8. Tm li,
kt qu docking cho thy cc cht Va-i ph hp vi hng thit k cu trc nhm
tm kim nhng cht c kh nng c ch HDAC. Trn c s chng ti tip
tc tin hnh tng hp ha hc v th tc dng sinh hc.
Bc u nghin cu hot tnh sinh hc, cc acid hydroxamic tng hp c
(Va-i) c nh gi kh nng c ch HDAC nng 1M. Kt qu hnh 4.5
cho thy Va, Vb, Vd, Vg c ch enzym r rt tng t cht chun dng tnh
SAHA. Vh, Vi th hin tc dng mc yu v rt yu, cn Vc, Ve, Vf hu nh
khng c ch enzym HDAC. Kt qu thu c gi rng: 1) Cc d vng (gn vo
vng thiadiazol) u cho kh nng c ch HDAC mnh, 2) Vic gn thm cc
nhm th vo d vng khng thch hp c ch enzym (gn 5-Br, 5-CH3 vo
thiophen hoc 5-CH3 vo furan lm mt hn hot tnh), 3) Tc dng ca dn cht
pyridin ph thuc vo v tr ca N (N ca pyridin cng xa v tr gn, kh nng c
ch HDAC cng gim).

58

Hnh 4.5: Kt qu phn tch Western blot ca cc cht Va-I (nng th 1 M)

c th gy ra c tnh vi t bo ung th, cc cht phi thm c qua


mng sinh hc tip cn vi HDAC. V th, chng ti tip tc tin hnh th c tnh
t bo in vitro ca 9 cht tng hp. Kt qu cho bng 4.1.
Bng 4.1: Kt qu th hot tnh khng t bo ung th ngi in vitro

Cht

KLPT
SW620

IC50 (M)*
MCF-7
PC-3

AsPC-1

Va

338

0,29

0,60

0,42

0,34

Vb

417

0,25

0,31

0,46

0,43

Vc

352

0,65

0,39

0,60

0,88

Vd

354

0,29

0,35

0,45

0,28

Ve

433

>20

>20

>20

>20

Vf

368

0,46

0,39

0,43

0,86

Vg

349

0,57

0,52

0,47

0,32

Vh

349

0,60

0,37

0,91

1,05

Vi

349

3,57

3,01

6,03

4,32

264

1,91

6,34

4,28

3,60

SAHA

*Nng ca cht gy ra s gim 50% s lng t bo, s liu c ly trung bnh ba kt


qu vi lch di 10%.
SAHA: acid suberoylanilid hydroxamic.

Kt qu th trn 4 dng t bo ung th (SW620 (t bo ung th i trng


ngi), MCF-7 (t bo ung th v ngi), PC-3 (t bo ung th tin lit tuyn

59

ngi), AsPC-1 (t bo ung th ty ngi)) cho thy tt c cc cht tng hp c

(tr Ve) u c c tnh mnh trn c 4 dng t bo th nghim (IC50 t 0,25-6,03


M), c bit:
- Trn dng t bo ung th i trng (SW620), Va, Vb, Vd th hin tc dng
mnh hn SAHA 6-7 ln (vi gi tr IC50 ln lt l 0,29; 0,25 v 0,29 M, so vi
SAHA l 1,91M).
- C Vb-d,Vf, Vh v Vi u c gi tr IC50 trn MCF-7 t 0,31-0,39M,
mnh hn SAHA (6,34M) 16-20 ln.
- Kh nng c ch t bo ung th tin lit tuyn (PC-3) ca Va, Vb, Vd, Vf,
v Vg mnh hn SAHA 9-10 ln (vi IC50 t 0,42-0,47M, so vi SAHA 4,28M).
- Tc dng khng t bo ung th ty (AsPC-1) ca Va, Vd, Vg (vi IC50 t
0,28-0,34M) cng mnh hn SAHA 10-12 ln (IC50=3,60M).
Kt qu ny kh tng ng vi kt qu th tc dng c ch enzym HDAC:
Ve khng c ch enzym HDAC c tc dng gy c t bo km nht vi
IC50>20M trn c 4 dng t bo, Vi cho IC50 cao hn hn cc cht cn li 5-15
ln. Tuy nhin, vi cc cht Vc, Vf, d tc dng c ch HDAC khng quan st
c nng 1M trn m hnh th nghim Western Blot nhng cho vn cho tc
dng khng t bo ung th tng i mnh. y l mt kt qu th v cn c
nghin cu thm.
Nh vy, cc acid hydroxamic mang khung 5-aryl-1,3,4-thiadiazol tng hp
c (Va-d, Vf-i) u cho hot tnh khng t bo ung th in vitro mnh. Vic gn
nhm th c hiu ng y in t (5-CH3) vo d vng lm gim nh hot tnh ca
dn cht: chng hn vi dn cht vng furan, Vc (gn thm CH3) lm gim tc
dng trn trn SW620, AsPC-1 hn 2 ln so vi Va (khng c nhm th), th hin
gi tr IC50 = 0,65; 0,88M (tng ng trn tng dng t bo) so vi Va l IC50 =
0,29; 0,34M; kt qu tng t vi dn cht 5-CH3 trn d vng thiophen. Gn
nguyn t halogen vi hiu ng ht in t (5-Br) gy ra cc tc ng khc nhau
ty thuc vo d vng. i vi d vng furan, nhm th ht in t (Br) khng nh
hng nhiu thm ch lm tng nh hot tnh: IC50 trn SW620, MCF-7, PC3,

60

AsPC-1 ca Vb gn Br so vi Va ln lt l 0.25 v 0,29M; 0,31 v 0,60M;


0,46 v 0,42M; 034 v 0,43M. Trong khi, gn 5-Br vo d vng thiophen (Ve)
li lm gim hn hot tnh (IC50>20M) so vi Vd (khng c Br).
C th d on rng cc nhm th trn vng khin cho cu trc phn t ca
cc cht tr nn cng knh hn v thay i s phn b in t trong vng to hiu
ng khng gian lm cn tr, hoc gim lc lin kt ca nhm kha hot ng (vng
thm) vi knh enzym ca HDAC. Nguyn t halogen (Br) khi gn vo d vng
furan v thiophen c th lm gim hoc mt hon ton kh nng lin kt hydro ca
d t trong vng vi phn vnh (c th l mt yu t quan trng vi hot tnh) gy
tng hoc mt hot tnh ca dn cht. Ty loi d vng la chn nhm th to ra
s phn b in t v cu trc khng gian ph hp.
D vng pyridin nhn chung c hot tnh yu hn d vng furan v thiophen.
Kh nng tc dng ca d vng pyridin ph thuc vo v tr gn vng: N cng xa
mch nhnh tc dng cng gim (Vg c N v tr ortho c IC50 trn PC3, AsPC-1
nh hn Vh (c m-N) 2-3 ln, v nh hn 5-7 ln IC50 ca Vi (c p-N) trn c 4
dng t bo th nghim).
So snh hot tnh ca mt s acid hydroxamic mang khung 5-aryl-1,3,4thiadiazol tng hp c (Va-i) vi mt s acid hydroxamic mang khung 5phenyl-1,3,4-thiadiazol cng b trn tp ch Journal of Enzyme Inhibition and
Medicinal Chemistry ca tc gi Nguyn Hi Nam v nhm nghin cu ti b mn
Ha Dc trng i hc Dc H Ni (bng 4.1) [21] cho thy nhn chung cc
dn cht d vng cho hot tnh tt hn vng phenyl. Chng hn, dn cht ca d
vng pyridin: Vh tc dng mnh hn dn cht phenyl (10a) gn 5 ln trn MCF-7
(IC50(Vh) = 0,37M, IC50(10a) = 1,80M), Vg mnh hn 10a hn 8 ln trn AsPC1 (vi IC50 ln lt l 0,32M v 2,71M). C th s gia tng cc nguyn t d
vng (O, S) trong nhm nhn din b mt ca 9 dn cht 5-aryl-1,3,4-thiadiazol
mi tng hp c lm tng tng tc Van der Waals hoc hydro ca cc cht vi
cc acid amin trn ming ti enzym HDAC, t tng cng hot tnh.

61

Bng 4.2: So snh tc dng khng t bo ung th ca Va-d, Vf-h vi mt s dn cht acid
hydroxamic mang khung 5-phenyl-1,3,4-thiadiazol

MCF-7

IC50 (M)
PC-3

AsPC-1

Va

0,60

0,42

0,34

Vb

0.132

0.191

0.179

Vc

0.139

0.211

0.311

Vd

0,35

0,45

0,28

Vf

0.144

0.159

0.316

Vg

0,52

0,47

0,32

Vh

0,37

0,91

1,05

Cht

10a

1,80

0,88

2,71

10b

2-Cl

1,23

1,42

0,63

10c

3-Cl

1,23

1,76

1,10

10d

4-Cl

0,73

0,84

1,34

10e

4-F

8,05

2,92

1,88

10f

4-Br

1,27

0,83

0,08

10g

4-CH3

20,78

14,90

6,38

10m

3,4-CH2OCH2-

2,15

2,54

3,04

6,34

4,28

3,60

SAHA

Nhn chung, ngoi tr Ve, 8 cht tng hp c u bc l tc dng


khng ung th rt tt. Ngoi nhng qui lut chung c rt ra hoc d on, kh
nng tc dng ca tng cht cn ty thuc vo tng dng ung th khc nhau (chng
hn, Va tc dng trn SW620, PC3 mnh hn Vd, Vg, nhng yu hn trn MCF-7,
AsPC-1 ). iu ny c th ph thuc vo cu trc ca tng loi t bo ung th,

62

loi v s lng HDAC cng nh mc nh hng ca HDAC trn tng loi


bnh l khc nhau.

63

KT LUN V KIN NGH


1. KT LUN
T nhng kt qu nghin cu trn c th rt ra mt s kt lun nh sau:
Tng hp v khng nh cu trc
tng hp c 9 dn cht acid hydroxamic mi:

N1-[5-(furan-2-yl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid (Va).

N1-[5-(5-bromofuran-2-yl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid
(Vb).

N1-hydroxy-N8-[5-(5-methylfuran-2-yl)-1,3,4-thiadiazol-2-yl]octandiamid
(Vc).

N1-hydroxy-N8-[5-(thiophen-2-yl)-1,3,4-thiadiazol-2-yl]octandiamid (Vd).
N1-[5-(5-bromothiophen-2-yl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid
(Ve).

N1-hydroxy-N8-[5-(5-methylthiophen-2-yl)-1,3,4-thiadiazol-2-yl]octandiamid
(Vf).

N1-hydroxy-N8-[5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl]octandiamid (Vg).
N1-hydroxy-N8-[5-(pyridin-3-yl)-1,3,4-thiadiazol-2-yl]octandiamid (Vh).
N1-hydroxy-N8-[5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl]octandiamid (Vi).
khng nh cu trc cc cht tng hp c bng phn tch cc d liu
ph IR, MS, 1H-NMR, 13C-NMR.
Th hot tnh sinh hc
- Th tc dng c ch HDAC bng phn tch Western blot cho thy cc cht
Va, Vb, Vd, Vg, Vh, Vi c hot tnh nng 1M.
- Kt qu th hot tnh khng t bo ung th in vitro theo phng php MTT
cho thy 8 cht Va-d, Vf-i c tc dng mnh trn c 4 dng t bo th nghim vi
IC50 = 0,25-6,03 M, trong Va, Vd c hot tnh tt nht vi IC50 = 0,280,60M.

64

2. KIN NGH
Kt qu nghin cu cho thy cc dn cht acid hydroxamic mang khung 5aryl-1,3,4-thiadiazol l cc cht khng ung th rt trin vng. Vi kt qu ny,
chng ti xin kin ngh:
- Tin hnh th c tnh ca cc dn cht Va-i trn cc t bo ung th khc.
Qua nh gi cc kt qu thu c lm c s cho vic th tc dng in vivo.
- Nghin cu tng hp thm cc dn cht mang khung 5-aryl-1,3,4thiadiazol khc hoc thay th khung 5-aryl-1,3,4-thiadiazol bng cc nhm ng
cu in t v vng thm khc, v th tc dng sinh hc ca chng.

65

TI LIU THAM KHO


TING VIT
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bn Y hc, H Ni.
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cu pht trin thuc iu tr ung th hin nay, tr. 103-186, Nh xut bn Y
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4. Nguyn nh Triu (2001), Cc phng php phn tch vt l v ha l, Nh
xut bn Khoa hc v k thut, H Ni.
TING ANH
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hydroxamic

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PH LC
Ph lc 1-1 n 1-9: Ph IR ca cc cht Va-i
Ph lc 2-1 n 2-9: Ph MS ca cc cht Va-i
Ph lc 3-1 n 3-9: Ph 1H-NMR ca cc cht Va-i
Ph lc 4-1 n 4-9: Ph 13C-NMR ca cc cht Va-i

Ph lc 1-1: Ph hng ngoi ca cht Va

Ph lc 1-2: Ph hng ngoi ca cht Vb

Ph lc 1-3: Ph hng ngoi ca cht Vc

Ph lc 1-4: Ph hng ngoi ca cht Vd

Ph lc 1-5: Ph hng ngoi ca cht Ve

Ph lc 1-6: Ph hng ngoi ca cht Vf

Ph lc 1-7: Ph hng ngoi ca cht Vg

Ph lc 1-8: Ph hng ngoi ca cht Vh

Ph lc 1-9: Ph hng ngoi ca cht Vi

Ph lc 2-1: Ph khi lng ca cht Va

Ph lc 2-2: Ph khi lng ca cht Vb

Ph lc 2-3: Ph khi lng ca cht Vc

Ph lc 2-4: Ph khi lng ca cht Vd

Ph lc 2-5: Ph khi lng ca cht Ve

Ph lc 2-6: Ph khi lng ca cht Vf

Ph lc 2-7: Ph khi lng ca cht Vg

Ph lc 2-8: Ph khi lng ca cht Vh

Ph lc 2-9: Ph khi lng ca cht Vi

Ph lc 3-1: Ph cng hng t ht nhn 1H-NMR ca cht Va

Ph lc 3-2: Ph cng hng t ht nhn 1H-NMR ca cht Vb

Ph lc 3-3: Ph cng hng t ht nhn 1H-NMR ca cht Vc

Ph lc 3-4: Ph cng hng t ht nhn 1H-NMR ca cht Vd

Ph lc 3-5: Ph cng hng t ht nhn 1H-NMR ca cht Ve

Ph lc 3-6: Ph cng hng t ht nhn 1H-NMR ca cht Vf

Ph lc 3-7: Ph cng hng t ht nhn 1H-NMR ca cht Vg

Ph lc 3-8: Ph cng hng t ht nhn 1H-NMR ca cht Vh

Ph lc 3-9: Ph cng hng t ht nhn 1H-NMR ca cht Vi

Ph lc 4-1: Ph cng hng t ht nhn 13C-NMR ca cht Va

Ph lc 4-2: Ph cng hng t ht nhn 13C-NMR ca cht Vb

Ph lc 4-3: Ph cng hng t ht nhn 13C-NMR ca cht Vc

Ph lc 4-4: Ph cng hng t ht nhn 13C-NMR ca cht Vd

Ph lc 4-5: Ph cng hng t ht nhn 13C-NMR ca cht Ve

Ph lc 4-6: Ph cng hng t ht nhn 13C-NMR ca cht Vf

Ph lc 4-7: Ph cng hng t ht nhn 13C-NMR ca cht Vg

Ph lc 4-8: Ph cng hng t ht nhn 13C-NMR ca cht Vh

Ph lc 4-9: Ph cng hng t ht nhn 13C-NMR ca cht Vi