tổng hợp và thử hoạt tính sinh học của một số dẫn chất ức chế hdac-...
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Tổng Hợp Và Thử Hoạt Tính Sinh Học Của Một số dẫn chất ức chế HDAC- 3-hydroxyTRANSCRIPT
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B GIO DC V O TO B Y T
TRNG I HC DC H NI
O TH KIM OANH
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
H NI 2013
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B GIO DC V O TO B Y T
TRNG I HC DC H NI
O TH KIM OANH
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
CHUYN NGNH HA DC
M S: 62.72.04.03
Ngi hng dn khoa hc: PGS.TS. Nguyn Hi Nam
GS.TS. Sang-Bae Han
H NI 2013
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i
LI CAM OAN
Ti xin cam oan y l cng trnh nghin cu ca ring ti.
Cc s liu, kt qu c trnh by trong lun n l trung thc, khch quan v
cha tng c ai cng b trong bt k cng trnh no khc.
Tc gi lun n
o Th Kim Oanh
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ii
LI CM N
Trong qu trnh nghin cu v hon thnh lun n, ti nhn c s gip
qu bu ca cc thy c gio, cc nh khoa hc thuc nhiu lnh vc cng ng
nghip, gia nh v bn b.
u tin, ti xin c gi li cm n chn thnh v s bit n su sc ti
PGS.TS. Nguyn Hi Nam, GS.TS. Sang-Bae Han, nhng ngi thy tn tnh
hng dn v to mi iu kin gip ti trong sut qu trnh nghin cu c Vit
Nam v Hn Quc.
Ti xin chn thnh cm n cc ng nghip ti b mn Ha dc ng h,
ng vin ti trong qu trnh nghin cu.
Trong thi gian thc hin lun n, ti nhn c s phi hp, gip ca
cc c nhn, n v trong v ngoi trng. Ti xin chn thnh cm n cc anh ch
Phng th nghim trung tm Trng i hc Dc H Ni, Khoa ha hc Trng
i hc Khoa hc t nhin i hc Quc gia H Ni, Phng cng hng t - Vin
ha hc Vin Khoa hc v Cng ngh Vit Nam, Phng khi ph - Vin ha hc cc
hp cht thin nhin - Vin Khoa hc v Cng ngh Vit Nam, cc bn nghin cu
sinh ca b mn Dc l, Khoa Dc, Trng i hc Quc gia Chungbuk
(Cheongju, Hn Quc).
Ti xin chn thnh cm n ng y, Ban gim hiu, Phng o to sau i hc,
cc b mn v phng ban chc nng Trng i hc Dc H Ni to iu kin
thun li cho ti trong thi gian hc tp v hon thnh lun n ny.
Cui cng, xin gi li cm n su sc ti chng v hai con trai, ngi thn, bn
b lun l nhng ngi ng vin, l ng lc gip ti phn u hon thnh lun
n.
Mt ln na, xin chn thnh cm n tt c nhng s gip qu bu m mi
ngi dnh cho ti.
o Th Kim Oanh
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iii
MC LC
Trang
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 2
1.1.1. Histon acetyltransferase 4
1.1.2. Histon deacetylase 4
1.1.2.1. Phn loi 5
1.1.2.2. Cu trc ca HDAC v c ch phn ng deacetyl ha 7
1.1.3. Mi lin quan gia ung th v s bt thng hot ng ca HAT
hoc HDAC
10
1.2. Cc cht c ch HDAC 11
1.2.1. Phn loi 11
1.2.2. C ch tc dng ca cc cht c ch HDAC 14
1.2.3. Cu trc ca cc cht c ch HDAC 17
1.3. Tnh hnh nghin cu trn th gii v cc cht c ch HDAC 18
1.3.1. Cc peptid vng 19
1.3.2. Dn cht benzamid 20
1.3.3. Cc acid bo mch ngn 21
1.3.4. Cc dn cht ceton 22
1.3.5. Cc hydroxamat v dn cht 22
1.3.5.1. Thay i cu ni 24
1.3.5.2. Thay i nhm kha hot ng 29
1.3.5.3. Thay i nhm chc hydroxamic 34
1.4. Cc phng php to lin kt amid v tng hp acid hydroxamic 39
1.4.1. Cc phng php to lin kt amid 39
1.4.1.1. Acyl halid 40
1.4.1.2. Acyl azid 41
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iv
1.4.1.3. Acylimidazol 41
1.4.1.4. Anhydrid 42
1.4.1.5. Ester 43
1.4.2. Cc phng php tng hp acid hydroxamic 45
1.4.2.1. Tng hp acid hydroxamic t ester 45
1.4.2.2. Tng hp acid hydroxamic t acid carboxylic 45
CHNG 2. NGUYN LIU, THIT B, NI DUNG V PHNG
PHP NGHIN CU
47
2.1. Nguyn liu 47
2.2. Thit b 48
2.3. Ni dung v phng php nghin cu 49
2.3.1. Ni dung nghin cu 49
2.3.2. Phng php nghin cu 49
2.3.2.1. Phng php tng hp 49
2.3.2.2. Phng php kim tra tinh khit 51
2.3.2.3 Phng php phn tch cu trc 52
2.3.2.4. Phng php th hot tnh sinh hc 53
2.3.2.5. Docking 56
CHNG 3. KT QU NGHIN CU 57
3.1. Tng hp ha hc v phn tch d liu ph 57
3.1.1. Cc dn cht N1-(benzo[d]thiazol-2-yl)-N4-hydroxysuccinamid v
N1-(benzo[d]thiazol-2-yl)-N5-hydroxyglutaramid
57
3.1.1.1. Kt qu tng hp 57
3.1.1.2. Kt qu phn tch ph ca cc dn cht 3a-f v 5a-f 63
3.1.2. Cc dn cht N1-(benzo[d]thiazol-2-yl)-N6-hydroxyadipamid v
N1-(benzo[d]thiazol-2-yl)-N8-hydroxyoctandiamid
66
3.1.2.1. Kt qu tng hp 66
3.1.2.2. Kt qu phn tch ph ca cc dn cht 7a-f v 9a-h 73
3.1.3. Tng hp cht N1-(thiazol-2-yl)-N8-hydroxyoctandiamid (23) 76
3.1.4. Cc dn cht N1-(benzo[d]thiazol-2-yl)-N4-(3-(hydroxyamino)-3-
oxopropyl)succinamid v N1-(benzo[d]thiazol-2-yl)-N5-(2-(hydroxy
amino)-2-oxoethyl)glutaramid
77
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v
3.1.4.1. Kt qu tng hp 77
3.1.4.2. Kt qu phn tch ph ca cc dn cht 11a-d v 13a-f 82
3.1.5. Cc dn cht N1-(3-(hydroxyamino)-3-oxopropyl)-N4-
phenylsuccinamid v N1-(2-(hydroxyamino)2-oxoethyl)-N5-phenyl
glutaramid
85
3.1.5.1. Kt qu tng hp 85
3.1.5.2. Kt qu phn tch ph ca cc dn cht 17a-c, f, h v 20a-h 93
3.1.6. Tng hp N1-(4-clorophenyl)-N6-(3-(hydroxyamino)-3-oxopropyl)
adipamid (26)
97
3.2. Hot tnh sinh hc 102
3.2.1. Tc dng c ch HDAC 102
3.2.2. Hot tnh khng t bo ung th in vitro 105
3.2.3. Hot tinh khng t bo ung th in vivo 107
CHNG 4. BN LUN 110
4.1. Tng hp ha hc 110
4.1.1. Tc nhn acyl ha l anhydrid acid 110
4.1.2. Tc nhn acyl ha l acid carboxylic 111
4.1.3. Tc nhn acyl ha l ester 117
4.2. Khng nh cu trc 118
4.2.1. Ph hng ngoi 118
4.2.2. Ph khi lng 120
4.2.2.1. Phn tch cm pic ion phn t 121
4.2.2.2. C ch ph mnh ca phn t theo cu trc d kin 122
4.2.3. Ph cng hng t ht nhn 126
4.2.3.1. Ph cng hng t ht nhn ca cc acid hydroxamic mang
khung benzothiazol
126
4.2.3.2. Ph cng hng t ht nhn ca cc acid hydroxamic mang
vng phenyl
134
4.2.3.3. Ph cng hng t ht nhn ca acid hydroxamic mang vng
thiazol
137
4.3. Hot tnh sinh hc 138
4.3.1. Cc acid hydroxamic mang khung benzothiazol 138
4.3.2. Cc acid hydroxamic mch alkyl c lin kt amid 143
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4.3.3. Docking 147
4.3.4. Hot tnh khng t bo ung th in vivo 148
KT LUN V KIN NGH 151
DANH MC CC CNG TRNH CNG B
TI LIU THAM KHO
PH LC
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vii
DANH MC CC K HIU, CH VIT TT
(ppm) dch chuyn ha hc (phn triu) 13C-NMR Ph cng hng t ht nhn carbon 13 (13C-Nuclear Magnetic
Resonance) 1H-NMR Ph cng hng t ht nhn proton (1H-Nuclear Magnetic Resonance)
AcOH Acid acetic
ADN Acid desoxyribonucleic
AsPC-1 Dng t bo ung th ty ngi
BCL2 B-cell lymphoma 2
CBFb Core-binding factor subunit beta
CBP Cyclic-AMP response element-binding protein
CDI Carbonyl diimidazol
CTPT Cng thc phn t
DCC Dicyclohexyl carbodiimid
DCM Dicloromethan
DMEM Dulbeccos modified Eagle medium
DMF Dimethylformamid
DMSO-d6 Dimethylsulfoxid deutri ha
ESI Ion ha phun bi in t (Electron Spray Ionization)
FBS Huyt thanh bo thai b (Fetal bovine serum)
FDA Cc qun l Thc phm v Dc phm m
HAT Histon acetyltransferase
HATU N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridin-1ylmethylen]-N-
methylmethanaminium hexafluorophosphat
HDAC Histon deacetylase
HDIs Cc cht c ch HDAC
HDLP Histone deacetylase-like protein
HMBC Ph tng tc a lin kt d nhn
HOBt 1-hydroxybenzotriazol
HSQC Ph tng tc d nhn qua mt lin kt
IC50 Nng c ch 50%
IR Ph hng ngoi (Infrared Spectrometry)
i Dch chuyn in tch
J Hng s tng tc (Hz)
MCF-7 T bo ung th v ngi
MeOH Methanol
MOZ Monocytic leukemia zinc-finger protein
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MS Ph khi lng (Mass Spectrometry)
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid
NCI-H460 T bo ung th phi ngi
PBS m phosphat (Phosphat buffered saline)
PC-3 T bo ung th tin lit tuyn ngi
PCl3 Phosphor triclorid
PCl5 Phosphor pentaclorid
POCl3 Phosphor oxyclorid
PVDF Mng polyvinyliden difluorid
rH Dch chuyn hydro
RAR Receptor acid retinoic
ROS Reactive oxygen species
RPMI Mi trng nui cy t bo
SAHA Acid suberoylanilid hydroxamic
SDS-PAGE Gel SDS-PAGE (Sodium dodecyl sulfat polyacrylamid gel
electrophoresis)
Sin 3 Protein c ch phin m
SMMHC T bo c (Smooth muscle myosin heavy chain)
SW620 T bo ung th i trng ngi
TSA Trichostatin A
toC Nhit nng chy
WAF1 Cht c ch kinase ph thuc cyclin
ZBG Nhm gn ion Zn2+
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DANH MC CC BNG
TT Tn bng Trang
1 Bng 1.1 Phn loi cc cht c ch HDAC 13
2 Bng 1.2 Cc cht c ch HDAC v ang c th lm sng 19
3 Bng 1.3 Tc dng c ch HDAC2 v c tnh t bo ca dn cht
-alkoxy (AH10)
28
4 Bng 3.1 Kt qu phn tch ph khi ca cc cht 3a-f, 5a-f 64
5 Bng 3.2 Kt qu phn tch ph 1H-NMR ca cc cht 3a-f, 5a-f 64
6 Bng 3.3 Kt qu phn tch ph 13C-NMR ca cc cht 5a-f 66
7 Bng 3.4 Kt qu phn tch ph khi ca cc cht 7a-f, 9a-h 73
8 Bng 3.5 Kt qu phn tch ph 1H-NMR ca cc cht 7a-f, 9a-h 73
9 Bng 3.6 Kt qu phn tch ph 13C-NMR ca cc cht 7a-f, 9a-h 75
10 Bng 3.7 Kt qu phn tch ph khi ca cc cht 11a-d, 13a-f 82
11 Bng 3.8 Kt qu phn tch ph 1H-NMR ca cc cht 11a-d, 13a-f 83
12 Bng 3.9 Kt qu phn tch ph 13C-NMR ca cc cht 11a-d, 13a-f 85
13 Bng 3.10 Kt qu phn tch ph khi ca cc cht 17a-c, f, h, 20a-h 93
14 Bng 3.11 Kt qu phn tch ph 1H-NMR ca cc cht 17a-c,f,h,
20a-h
93
15 Bng 3.12 Kt qu phn tch ph 13C-NMR ca cc cht 17a-c,f,h,
20a-h
96
16 Bng 3.13 Tm tt kt qu tng hp cc dn cht trong lun n 98
17 Bng 3.14 Tc dng c ch HDAC ca cc dn cht tng hp 102
18 Bng 3.15 Kt qu nh lng tc dng c ch HDAC2 ca cc cht
9a-h, 23
104
19 Bng 3.16 Kt qu th hot tnh khng t bo ung th ngi in vitro 105
20 Bng 3.17 S thay i kch thc v khi lng ca khi u trn chut
nhm th, nhm trng i chiu v SAHA
107
21 Bng 3.18 Phn trm thay i cn nng ca chut trong qu trnh th
nghim
108
22 Bng 4.1 Cng cm pic ion phn t ca cht 7a 122
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x
TT Tn bng Trang
23 Bng 4.2 D liu cc ph cng hng t ht nhn ca cht 9g 132
24 Bng 4.3 D liu ph 1H-NMR v 13C-NMR ca cht 23 137
25 Bng 4.4 Tc dng c ch HDAC v c tnh t bo in vitro ca cc
cht 7a-f
140
26 Bng 4.5 Tc dng c ch HDAC v c tnh t bo in vitro ca cc
cht 9a-h
141
27 Bng 4.6 Nng lng lin kt vi trung tm hot ng ca HDAC 147
28 Bng 4.7 Kt qu c ch s pht trin khi u in vivo ca cht 9g
cc liu khc nhau vi dng t bo ung th PC-3
149
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xi
DANH MC CC HNH V, S
HNH V
TT Tn hnh Trang
1 Hnh 1.1 S cu to nucleosom 3
2 Hnh 1.2 HAT v HDAC iu ha qu trnh phin m 3
3 Hnh 1.3 Phn loi HDAC ngi 7
4 Hnh 1.4 Cu trc HDAC8 8
5 Hnh 1.5 Cu trc v tr hot ng ca HDLP khi lin kt vi phn
acetyl-lysin ca histon (tri) v Trichostatin A (phi)
9
6 Hnh 1.6 C ch phn ng deacetyl ha theo Finnin 9
7 Hnh 1.7 iu ha s pht trin v sng st ca t bo bi cc cht
c ch HDAC
14
8 Hnh 1.8 Cc cht c ch HDAC thc y s cht t bo 16
9 Hnh 1.9 Cng thc c in ca HDI v v tr ca HDI trong ti
enzym HDAC
18
10 Hnh 1.10 HDIs c cu trc peptid vng 20
11 Hnh 1.11 HDIs l cc benzamid 21
12 Hnh 1.12 HDIs l cc acid bo mch ngn 21
13 Hnh 1.13 HDIs l cc dn cht ceton 22
14 Hnh 1.14 HDIs c cu trc hydroxamat 23
15 Hnh 1.15 Cc dn cht N-hydroxy-2-propenamid 24
16 Hnh 1.16 a) Cc acid biphenyl-4-yl-acrylohydroxamic (AH2); b)
Cc dn cht vi cu ni c hai lin kt i (AH3) v
dng kh ha ca AH3 (AH4)
25
17 Hnh 1.17 Mt s dn cht c lin quan ca AH2b 25
18 Hnh 1.18 Cc dn cht amid ngc ca SAHA 26
19 Hnh 1.19 Cc aryloxyalkanoic N-hydroxyamid (AH9) 26
20 Hnh 1.20 Cu trc ca amamistatin A, B 27
21 Hnh 1.21 Cc dn cht -alkoxy ca SAHA 27
22 Hnh 1.22 Cu trc ca dn cht p-methoxybenzyl ether (AH10) 28
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TT Tn hnh Trang
23 Hnh 1.23 Mt s dn cht -alkyl ca SAHA 29
24 Hnh 1.24 Cc acid phenylthiazol hydroxamic tng t SAHA 29
25 Hnh 1.25 Mt s acid phenylthiazol hydroxamic 30
26 Hnh 1.26 Cc dn cht acid biphenyl-hydroxamic 30
27 Hnh 1.27 Cc acid isoxazol-hydroxamic 31
28 Hnh 1.28 ADS100380 32
29 Hnh 1.29 Cc nh hng ti u ha cu trc ca ADS102550 32
30 Hnh 1.30 Cc arylthiophen hydroxamat 33
31 Hnh 1.31 Cc acid pyridin-thiophen-hydroxamic 33
32 Hnh 1.32 Cc dn cht biphenyl sulfamid 34
33 Hnh 1.33 Mt s dn cht sulfamid khc 34
34 Hnh 1.34 Cc dn cht sulfamid khng mang cu ni amid 35
35 Hnh 1.35 Mt s dn cht trithiocarbonat 35
36 Hnh 1.36 Mt s trithiocarbonat khc v cht tng t 36
37 Hnh 1.37 Cc dn cht thiol 36
38 Hnh 1.38 T disulfid n KD5170 37
39 Hnh 1.39 S thy phn v to chelat vi Zn2+ ca KD5170 37
40 Hnh 1.40 a) Tng hp amid thng qua to ester hot ha; b) Mt s
alcol hay dng
44
41 Hnh 3.1 Cng thc cu to ca 23 77
42 Hnh 3.2 Cng thc cu to ca 26 98
43 Hnh 3.3 Hnh nh khi u ca nhm th, nhm trng i chiu v
SAHA
108
44 Hnh 4.1 Phn ng th i nhn acyl 110
45 Hnh 4.2 Mt s hydroxylamin c gn nhm bo v 115
46 Hnh 4.3 Cng thc cu to chung ca 51 cht tng hp c 118
47 Hnh 4.4 Ph hng ngoi ca cht 5e 120
48 Hnh 4.5 Ph khi lng ca cht 7a 121
49 Hnh 4.6 Ph khi lng ca cht 20f 124
50 Hnh 4.7 Ph 1H-NMR dn rng ca cht 5a 127
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xiii
TT Tn hnh Trang
51 Hnh 4.8 Ph 1H-NMR dn rng ca cht 9f 128
52 Hnh 4.9 a) Ph 1H-NMR; b) Ph 13C-NMR ca cht 9g 130
53 Hnh 4.10 Ph tng tc n lng t d nhn HSQC ca cht 9g 130
54 Hnh 4.11 Ph tng tc a lin kt d nhn - HMBC ca cht 9g 132
55 Hnh 4.12 Ph 1H-NMR dn rng ca cht 20f 135
56 Hnh 4.13 nh hng ca t trng flo ln carbon v hng s tng
tc (J)
136
57 Hnh 4.14 Ph 13C-NMR dn rng ca cht 20b 136
58 Hnh 4.15 Cu trc ca TSA v SAHA 138
59 Hnh 4.16 Cng thc cu to ca cc acid hydroxamic 3a-f 139
60 Hnh 4.17 Cc acid hydroxamic 5a-f, 7a-f 139
61 Hnh 4.18 Kt qu phn tch Western blot ca cc cht 7a-f 139
62 Hnh 4.19 Cu trc cc acid hydroxamic 9a-h 140
63 Hnh 4.20 Kt qu phn tch Western blot ca cc cht 9a-f 141
64 Hnh 4.21 Tng hp cc aryltriazolylhydroxamat 27a-d 142
65 Hnh 4.22 Cu trc ca cc acid hydroxamic 11a-d v 13a-f 143
66 Hnh 4.23 Cu trc ca cc acid hydroxamic 17a-c, f, h v 20a-h 144
67 Hnh 4.24 Kt qu phn tch Western blot ca mt s cht i din
dy 13 v 20
144
68 Hnh 4.25 Cu trc khng gian ca SAHA, 17a v 20a 145
69 Hnh 4.26 Cu trc cc acid -lactam-hydroxamic 146
70 Hnh 4.27 Cu trc chung ca cc dn cht homo-oxa-SAHA 146
71 Hnh 4.28 Docking ca cht 9g (mu cam), 9h (mu tm hng) v
SAHA (mu xanh l) vi HDAC8
148
72 Hnh 4.29 S thay i kch thc khi u trung bnh ca nhm th so
vi SAHA
149
73 Hnh 4.30 S thay i cn nng ca chut trong qu trnh th nghim 150
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xiv
S
TT Tn s Trang
1 S 1.1 Phn ng to lin kt amid trc tip 39
2 S 1.2 Phn ng to lin kt amid thng qua acid hot ha 40
3 S 1.3 a) Phn ng to acyl clorid; b) Tng hp amid 40
4 S 1.4 Vai tr xc tc ca pyridin 41
5 S 1.5 Tng hp amid thng qua to acyl azid 41
6 S 1.6 Tng hp amid s dng tc nhn hot ha CDI 42
7 S 1.7 Tng hp amid s dng tc nhn hot ha DCC 43
8 S 1.8 Tng hp amid s dng tc nhn hot ha ethyl
cloroformat
43
9 S 1.9 Tng hp amid s dng tc nhn BOP 44
10 S 1.10 Tng hp mt s dn cht amid ngc ca SAHA 45
11 S 1.11 Tng hp acid biaryl hydroxamic 46
12 S 1.12 Tng hp cc acid phenylthiazol hydroxamic 46
13 S 3.1 Tng hp cc dn cht 3a-f, 5a-f 57
14 S 3.2 Tng hp N1-(6-nitrobenzo[d]thiazol-2-yl)-N5-
hydroxyglutaramid (5f)
63
15 S 3.3 Tng hp cc dn cht 7a-f v 9a-h 66
16 S 3.4 Tng hp N1-(thiazol-2-yl)-N8-hydroxyoctandiamid (23) 76
17 S 3.5 Tng hp cc dn cht 11a-d v 13a-f 77
18 S 3.6 Tng hp cc dn cht 17a-c, f, h v 20a-h 86
19 S 3.7 Tng hp N1-(4-clorophenyl)-N6-(3-(hydroxyamino)-3-
oxopropyl)adipamid (26)
97
20 S 4.1 Tng hp cc cht trung gian 2a-f, 4a-f, 15a-c, 15f, 15h,
18a-h
110
21 S 4.2 Tng hp cc acid hydroxamic 3a-f v 5a-f 111
22 S 4.3 Tng hp acid hydroxamic 3a-f bng tc nhn hot ha
DCC
112
23 S 4.4 Vai tr ca HOBt trong qu trnh to ester hot ha 113
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xv
TT Tn s Trang
24 S 4.5 Tng hp 3a-f s dng tc nhn hot ha isobutyl
cloroformat
113
25 S 4.6 Tng hp 3a-f s dng tc nhn hot ha CDI 114
26 S 4.7 Tng hp acid hydroxamic 5f 115
27 S 4.8 Tng hp cc ester trung gian ca cc dy cht 7, 9, 11,
13, 17, 20 v cht 22
116
28 S 4.9 Tng hp ester trung gian 25 116
29 S 4.10 Tng hp cc aryltriazolylhydroxamat 117
30 S 4.11 Tng hp mt s dn cht -alkoxy ca SAHA 117
31 S 4.12 C ch phn ng tng hp cc acid hydroxamic dy 7, 9,
11, 13, 17 v 20, cht 23, 26 t ester
117
32 S 4.13 S ph mnh ca cht 7a 123
33 S 4.14 S ph mnh ca cht 20f 125
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1
T VN
Nhng tin b trong cc ngnh khoa hc c bn nh di truyn hc, sinh hc
phn t, sinh hc t bo v c bit l s ra i ca bn gen ngi gip cho cc
nh khoa hc c nhng hiu bit su sc v khi u cp phn t cng nh cc qu
trnh quyt nh s pht trin ca khi u. Do , hng lot cc protein ng vai tr
quan trng trong ung th ng thi cng l ch m cc thuc iu tr ung th hng
ti c pht hin nh cc protein kinase ph thuc cyclin, protein gy ung th
Bcl-2, p53, cc farnesyltransferase, histon deacetylase (HDAC), telomerase,
STAT[27]. Nh vy, vic nghin cu v pht trin thuc iu tr ung th theo
phng php mi hin nay, phng php thit k cng thc da trn ch tc dng
phn t, ngy cng t c nhiu thnh tu ng k.
Mt trong nhng ch tc dng phn t ang c ch hin nay l cc histon
deacetylase (HDAC). Nghin cu v cc HDAC xc nh hot ng bt thng ca
HDAC c lin quan n nhiu bnh ung th. V vy, cc cht c ch HDAC ang tr
thnh cc tc nhn chng ung th y trin vng. Acid suberoylanilid hydroxamic
(Zolinza, 2006) v depsipeptid (Romidepsin, 2009) l hai cht c ch HDAC
c Cc qun l thc phm v dc phm M (US-FDA) ph duyt trong iu tr u
lympho da t bo T [21]. Bn cnh , mt s cht c ch HDAC khc cng ang
c nghin cu v tri qua cc pha th lm sng nh NVL-LAQ824, MS-275, CI-
994, PXD-101...[21,33,65]. Cc cht c ch HDAC c chia thnh 5 nhm da theo
cu trc ha hc, trong cc dn cht acid hydroxamic c cc nh khoa hc trn
th gii tp trung nghin cu nhiu nht do cu trc n gin d tng hp, hot tnh c
ch HDAC mnh.
Hi nhp vi xu hng nghin cu ca th gii v tm kim cht c ch HDAC
c hot tnh khng t bo ung th tt, lun n Tng hp v th hot tnh sinh hc
ca mt s dn cht acid hydroxamic hng c ch enzym histon deacetylase
c thc hin vi 2 mc tiu:
1. Thit k v tng hp c khong 40 - 50 dn cht acid hydroxamic mi
hng c ch HDAC.
2. Th tc dng c ch HDAC v tc dng khng t bo ung th ca cc cht
tng hp c.
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Chng 1. TNG QUAN
1.1. HISTON DEACETYLASE
Cc nghin cu v ung th xc nh cn nguyn ca bnh khng ch do c
ch di truyn hc m cn do c ch di truyn biu hin gen. C ch di truyn biu hin
gen lin quan n nhng thay i trong qu trnh biu hin gen m khng nh hng
n cu trc chui ADN. C ch di truyn biu hin gen gm cc bin i v ADN v
histon nh s methyl ha, acetyl ha [25,55,71,90]. Nhng bin i ny dn n cc
bt thng ca qu trnh biu hin gen th hin s pht trin, s bit ha v s cht
t bo theo chng trnh, kt qu l tng kh nng bin i ca t bo.
Cho n nay, cc nghin cu chng minh cu trc ca nhim sc th l yu
t quan trng trong iu ha qu trnh biu hin gen [11,22,63,71]. Cu trc ca nhim
sc th l mt phc hp cu to bi ADN, cc histon v cc protein khng phi histon
[22,40]. Histon l cc protein c bn giu acid amin nh lysin, arginin, c chia
thnh 5 nhm chnh (H1, H2A, H2B, H3, H4). Tng cp ca H2A, H2B v H3, H4
cng nhau to nn li protein octomer hnh a. Li protein ny c qun quanh bi
146 cp ADN to nn nucleosom (hnh 1.1) [11,22,63,71,77]. Cc nucleosom ni vi
nhau nh phn amino tn ca cc histon. Bn cp histon li c 2 phn quan trng:
phn ui C nm bn trong li ca nucleosom v phn u N vi acid amin kt thc l
lysin nm bn ngoi nucleosom [63]. Cu trc ny chu nh hng chnh bi s bin
i phn u N ca histon. Phn u N 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
[22,40,71].
C ch ca phn ln cc bin i trn u cha sng t. So vi s methyl ha
v phosphoryl ha, dng nh s acetyl ha phn li histon l qu trnh bin i
c nghin cu v hiu bit tng tn hn. 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 acetyltransferase (HAT) v histon deacetylase (HDAC)
[11,22,40,63,71]. Khi xy ra s acetyl ha histon, nhim sc th s c tho xon v
hot ha qu trnh phin m, trong khi deacetyl ha phn u N ca histon s lm
gim qu trnh phin m thng qua s ng xon nhim sc th. Ni chung, khi tng
acetyl histon dn n thc y qu trnh phin m v ngc li khi s acetyl ha histon
gim lm ngn cn qu trnh phin m (hnh 1.2) [63,71].
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Hnh 1.1. S cu to nucleosom [71]
Hnh 1.2. HAT v HDAC iu ha qu trnh phin m [71]
* Ch thch: HAT: histon acetyltransferase; HDAC: histon deacetylase; Ac: acetyl.
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1.1.1. Histon acetyltransferase (HAT)
Histon acetyltransferase (HAT) xc tc chuyn nhm acetyl t acetyl coenzym
A n lin kt vi nhm -amino ca lysin phn u N ca histon. S chuyn i
ny xy ra nhiu hn trn histon H3, H4. V tr acetyl ha quan trng l Lys9 v Lys14
trn histon H3; Lys5, Lys8, Lys12 v Lys16 trn histon H4 [71,77]. 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 tch in m trn ADN [70]. Chnh
v vy, vic tng acetyl ha histon lm ni lng cu trc nhim sc th v hot ha
gen.
Cc histon acetyltransferase c chia thnh 5 nhm bao gm khong 20
isoenzym [15,22,40,70]. C s ca vic phn nhm ny l da trn s tng t nhau
ca cu trc chui c bn v thng c c cht c hiu. Nhm HAT u tin l cc
GNAT (Gcn5-related N-acetyltransferase), bao gm cc protein lin quan n s bt
u phin m, nh l Gcn5 v PCAF (p300/cyclic-AMP-response-element binding
protein-associated factor). Nhm th hai l cc MYST, c t tn theo protein gn
Zn trong bnh bch cu n nhn to (MOZ-monocytic leukemia zinc-finger protein),
YBF2/SAS3, SAS2 v HIV-1 TAT-interactive protein 60 (TIP60). Nhm th ba l 2
enzym lin quan cht ch n protein p300 v CBP (cyclic-AMP response element-
binding protein), chng hot ng nh cht ng hot ha mt s phc hp ca nhn
t sao chp m. Hai nhm HAT khc l cc cht ng hot ha receptor nhn nh
phc hp TFIID (general transcription factor IID) (cu trc di phn t l TAFII250)
v ACTR (amplified in breast cancer), SRC1 (avian sarcoma viral oncogene
homologue 1) [20]. Khng ch acetyl ha ui histon, cc HAT cn c th acetyl ha
cc cht ng hot ha, cht ng c ch sao chp m nh E2F, p53, GATA1 [40,76].
Nh vy, cc HAT ng vai tr quan trng trong hot ha cng nh c ch gen. Cc
HAT khng gn kt trc tip vi ADN m to thnh phc hp vi cc HAT khc cng
nh vi cc nhn t sao chp m.
1.1.2. Histon deacetylase (HDAC)
Histon deacetylase c tc dng ngc vi HAT, n 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 [22,40,43,63,71,81]. HDAC c bo tn trong qu trnh tin
ha v biu hin trong cc t chc ca cc sinh vt t n bo nguyn thy cho n
loi ngi.
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Tng t nh HAT, cc HDAC khng gn kt trc tip vi chui ADN m to
phc hp vi cc cht ng c ch phin m khc. Cc HDAC khc nhau to cc phc
hp khc nhau. Hot tnh ca HDAC c iu ha bi s bin i sau phin m. Cc
HDAC khng ch deacetyl ha histon, m cn deacetyl ha cc protein khng histon
nh p53, Ku70, pRB v E2F-1 [16,59,81].
1.1.2.1. Phn loi
HDAC1 ngi l histon deacetylase u tin c xc nh bng cch s
dng cht c ch HDAC trapoxin vo nm 1996 [80]. Cho n nay, 18 HDAC khc
nhau ngi c xc nh v chia thnh 4 nhm (hnh 1.3) [11,22,33,34,40,56,71,
89]. Cc HDAC khc nhau tnh tng ng chui, c cht c hiu v cofactor ph
thuc [33,34,89].
* Nhm I (HDAC1, 2, 3, 8) (hnh 1.3): tng ng vi Rdp3 t bo nm men
S.cerevisiae, c chc nng c ch phin m, ch hot ng khi nm trong phc hp
protein [2,16]. HDAC nhm I c nm men, ng vt c v v thc vt. ng vt
c v, cc HDAC ny c chia thnh 2 phn nhm l HDAC1/2 v HDAC3.
Cu trc ca HDAC1 v 2 kh tng ng (82%). Vng xc tc nm u N
to nn phn chnh ca protein. Khi c to ra bng k thut ti t hp, HDAC1 v 2
khng c hot tnh chng t cc cofactor l cn thit cho hot ng ca HDAC.
HDAC1, 2 hot ng khi nm trong phc hp nh phc hp SIN3, NuRD/NRD/Mi2
v CoREST. Phosphoryl ha serin trn HDAC1, 2 iu ha hot ng ca chng v
to nn phc hp vi cc cofactor khc [29,71].
HDAC3 c tm thy trong phc hp vi N-CoR (nuclear receptor copressor)
v phc hp vi SMRT (silencing mediator for retinoic acid and thyroid hormon
receptors). HDAC3 c cng cu trc vng ging nh cc HDAC nhm I. Khong 68%
vng cc acid amin ca HDAC3 ng nht vi HDAC1, 2. Phn ui C khng o
ngc ca HDAC3 cn thit cho c hot tnh deacetyl ha v c ch phin m [71].
HDAC8 khng c xp vo phn nhm no v n ch yu c ng vt c
xng sng. HDAC8 c 37% tng ng vng cc acid amin vi HDAC3. Hot ng
ca HDAC8 gim khi phosphoryl ha bi protein kinase A, trong khi HDAC1, 2 li
hot ng bi s phosphoryl ha [30]. HDAC8 l HDAC u tin ng vt c v
xc nh c cu trc 3 chiu [74].
* Nhm II gm HDAC4, 5, 7, 9 (nhm IIa) v HDAC6,10 (nhm IIb) tng ng vi
HDA1 trong t bo nm men (hnh 1.3). HDAC nhm II c kch thc phn t ln
(855-1122 aa) v khc bit so vi HDAC nhm I do c u N tham gia vo qu trnh
c ch phin m. Nhm ny cha tn hiu nh v ngoi nhn NES nn c th di
chuyn t nhn ra bo tng v ngc li.
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Hn 70% vng cc acid amin ca HDAC4, 5 tng ng vi nhau, cn
HDAC7 ch tng ng khong 57 - 58% vi HDAC4, 5. C 3 HDAC ny u c
vng xc tc nm ui C ca protein. Trong khi , HDAC9 li c vng xc tc
u N, ging nh cc HDAC nhm I. Cc HDAC nhm IIa c u N tng tc c
hiu vi yu t phin m MEF2 (myogenic transcription factor 2). Yu t ny ng vai
tr quan trng trong s bit ha c. Do vy, cc HDAC ny khi kt hp vi MEF2 gy
c ch chc nng phin m ca MEF2, h qu l t bo c khng bit ha c. Cc
HDAC ny c th b sung cho nhau kim sot s iu ha bit ha ca qu trnh
biu hin gen trong cc giai on khc nhau ca s bit ha trong t bo c [71].
HDAC6 v 10 c vng acid amin tng ng khong 37%. im khc bit ca
HDAC6 v 10 so vi cc HDAC khc l chng c 2 vng xc tc, tuy nhin HDAC10
c 1 vng xc tc b bt hot. Mt c im ring ch c HDAC6 l s c mt ca
HUB (HDAC6-, USP3- v Brap2-related zinc finger motif) ui C. c im ny l
du hiu cho s ubiquitin ha v cho thy HDAC ny thin v s thoi ha. Nghin
cu in vitro v in vivo xc nh HDAC6 lin quan cht ch n cc bnh thoi ha
m thn kinh nh bnh Parkinson, bnh Hutington. HDAC6 ch yu c bo tng,
tuy nhin n cn c tm thy nhn trong phc hp vi HDAC11. HDAC10 c mt
vng xc tc u N v vng xc tc b bt hot ui C. HDAC 10 c kh nng
tng tc vi HDAC1, 2, 3, 4, 5, 7, nhng khng tng tc vi HDAC6 v vn th
hin hot tnh deacetyl ha khi khng to phc. HDAC6, 10 khng li trapoxin v
butyrat natri tt hn cc HDAC nhm I, IIa. [21,45,71].
* Nhm III: (silent information regulator genes - Sirtuins), gm SIRT1-7 tng t
Sir2 t bo nm men. HDAC nhm III ny khng lin quan n cc nhm khc,
chng c trong nhn (SIRT1,6,7), bo tng (SIRT2) hoc ty th (SIRT3,4,5).
HDAC nhm III t c nghin cu ngi, nhng c xc nh c c ch hot
ng ph thuc vo cofactor NAD+, khc vi HDAC nhm I, II, IV (HDAC kinh in)
ph thuc Zn2+ v b c ch bi cc cht to phc chelat vi Zn2+ [45,71,89].
* Nhm IV: HDAC 11 (ch c ngi). Nghin cu h thng loi thy rng HDAC11
lin quan gn hn vi HDAC3, 8, nn c th gi nh HDAC11 lin quan mt thit vi
HDAC nhm I hn l nhm II. HDAC 11 c vng xc tc u N v c th b c ch
bi trapoxin (dn cht ca TSA). HDAC11 cha c tm thy trong cc phc hp
HDAC bit c th xc nh chc nng sinh hc [45,71,89].
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Hnh 1.3. Phn loi HDAC ngi [21]
* Ch thch: Hnh ch nht mu xanh dng l vng xc tc c bo tn ca HDAC; N:
nhn; C: bo tng; Mit: ty th; Ac: acetyl ha; P: phosphoryl ha; S: sumoyl ha; Ub:
ubiquitin ha. N.D: cha xc nh.
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 nhm I, II v IV [45,89].
1.1.2.2. Cu trc ca HDAC v c ch phn ng deacetyl ha
Vic xc nh cu trc ca HDAC rt cn thit xc nh c ch tc dng ca
HDAC, ng thi da vo cu trc HDAC thit k cng thc cho cc cht c ch
HDAC. Phng php kt tinh to tinh th v chp tia X c p dng tm ra cu
trc tinh th ca cc HDAC khc nhau, c bit l trung tm hot ng ca n.
HDAC8 l HDAC u tin ng vt c v xc nh c cu trc 3 chiu [74].
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Hnh 1.4. Cu trc HDAC8 ( ion Zn2+ biu th l hnh trn mu xanh l) [86]
Cc HDAC u c trung tm hot ng gm 2 phn chnh: ion Zn2+, knh
enzym dng ti hnh ng. Bao quanh ion Zn2+ l 2 cp acid amin Histidin-Aspartic
(HDLP l His131-Asp166 v His132-Asp173, HDAC 8 l His142-Asp176 v His143-
Asp183), mt phn t Tyrosin ng vai tr cho proton (HDLP l Tyr297, HDAC8 l
Tyr306) v 2 acid aspartic (HDLP l Asp258 v Asp168, HDAC8 l Asp178 v
Asp267), mt phn t Histidin (HDLP l His170, HDAC8 l His180) [11].
- Ion Zn2+ l coenzym ca HDAC, nm y knh enzym. Trong phn t HDAC,
ion Zn2+ c th to 5 lin kt phi tr: 4 lin kt vi nguyn t oxy, nit ca cc acid
amin, 1 lin kt phi tr vi nguyn t oxy ca nhm acetyl ca phn t acetyl-lysin
phn u N ca histon, t xc tc tch loi nhm acetyl. Khi c mt cc cht c ch
HDAC nh cc acid hydroxamic, ion Zn2+ to 2 lin kt phi tr vi 2 nguyn t oxy
ca nhm hydroxamic (hnh 1.5) [11].
- Knh enzym c dng ti hnh ng hp, to nhiu lin kt Van der Waals vi c
cht (lysin tn ca histon/phn cu ni ca cc cht c ch HDAC). Ti c cu to
t cc acid amin thn lipid: Phenylalanin, Tyrosin, Prolin, Histidin. y ti cn c 1
vi phn t nc lm nhim v vn chuyn nhm acetyl trong phn ng deactyl ha v
tham gia to lin kt hydro khi khng c mt nhm -OH ca Tyrosin. Cu trc ti rt
linh ng, c th bin i ph hp vi chiu di ca cc c cht khc nhau. B rng
ca ti c gii hn bi 2 vng thm c tm thy trn cng mt v tr nhiu
HDAC khc nhau. Trn ming ti c 1 vnh nh c to nn t 1 vi vng xon
protein (phn vnh s tng tc vi nhm nhn din b mt ca HDAC) [11].
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Hnh 1.5. Cu trc v tr hot ng ca HDLP khi lin kt vi phn acetyl-lysin ca
histon (tri) v Trichostatin A (phi) [13]
Finnin l ngi u tin nghin cu chi tit tng tc gia cht c ch HDAC
v enzym HDAC nm 1999 [27]. Nghin cu s dng HDLP (histone deacetylase like
protein), mt cht ng ng phn lp t vi khun Aquiflex aeolicus. HDLP c vng
acid amin tng ng 30% vi HDAC1 [11].
Hnh 1.6. C ch phn ng deacetyl ha theo Finnin [11,27]
u tin, nguyn t oxy ca nhm carbonyl ca phn N-acetyl lysin trn u N
ca histon lin kt phi tr vi Zn2+, lm nhm carbonyl phn cc v pha oxy v
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carbon tr nn i in t hn. Mt phn t nc s to lin kt phi tr vi Zn2+. Nh
h thng chuyn tip Asp173 - His132 v Asp166 His131, nguyn t oxy ca nc
tr nn i nhn hn. Sau khi c hot ha, oxy ca nc tn cng vo nguyn t C
ca nhm carbonyl to ra oxyanion t din trung gian (oxy ca nhm carbonyl), c
n nh bi Tyr297 v Zn2+. Cui cng lin kt C-N b b gy, 1 proton c chuyn
t His132 cho nguyn t nit to thnh acetat v lysin [11].
1.1.3. Mi lin quan gia ung th v s bt thng hot ng ca HAT hoc
HDAC
Rt nhiu bnh c nguyn nhn do s bin i bt thng di truyn biu hin
gen (epigenetic) cng nh t bin gen, c bit l bnh ung th.
HAT acetyl ha histon lm trung ha cc dng trn lysin v gip tho xon
cu trc ca nucleosom, do gip cho cc nhn t sao m d dng tip cn ADN.
Ngc li, cc HDAC xc tc loi b nhm acetyl t histon v protein khng phi
histon, lm tng s tch in dng trn u N ca histon v ng xon nhim sc th,
kt qu l c ch qu trnh phin m do ngn cn cc nhn t sao m tin n ch ca
chng trn ADN. Nh vy, s acetyl ha v deacetyl ha nhim sc th ng vai tr
quan trng trong iu ha qu trnh biu hin gen. Vic mt cn bng hot ng gia
HAT v HDAC c th dn n nhng bt thng biu hin gen v do dn n ung
th [21,40,45,56,63].
Hot ng ca HAT lin quan n s di chuyn, xm ln, s biu th qu mc
hoc s t bin trong rt nhiu loi ung th k c ung th mu v ung th biu m.
Hai HAT b bin i r nht trong mt s bnh ung th do t bin hoc di chuyn l
p300 v CBP. S t bin v ct on ca p300 c chng minh trong mt s
bnh nh u m thn kinh (glioblastommas), ung th carcinoma gan (hepatocellular
carcinomas), bnh bch cu. Hi chng Rubinstein Taybi, c xu hng pht trin
thnh ung th, lin quan n s t bin ca CBP [31,70]. Ngoi ra, CBP c tm
thy dng phi hp vi MOZ trong bnh bch cu ty bo cp (AML).
Tng t, cc HDAC cng c xc nh b ri lon iu ha trong ung th. S
huy ng bt thng ca cc HDAC vo chui iu ha ca gen ch c th xy ra
thng qua tng tc bin i ca chng vi vic tng cng vai tr ca cc protein gn
kt ADN gy ung th. V d nh receptor RAR gn kt gen PML (promyelocytic
leukemia gene) hoc PLZF (promyelocytic leukemia zinc finger) trong bnh bch cu
tin ty bo cp (APL). Trong iu kin sinh l, RAR l nhn t sao m hot ha
acid retinoic gip gii phng phc hp ng c ch cha HDAC v lm gia tng cc
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cht ng hot ha sao m (bao gm c HAT). iu ny dn n s acetyl ha histon
v c ch gen y mnh s bit ha t bo. Trong bnh bch cu tin ty bo cp
(APL), protein gn kt RAR/PML hoc RAR/PLZF gi HDAC v phi hp vi
phc hp ng c ch. Do tng methyl transferase histon v ADN, dn n ngn
cn s phin m [21,26,40]. Do vy, t bo ung th khng tri qua giai on bit ha
v pht trin qu mc. S gia tng bt thng ca HDAC cn c quan st khi gen
t bin gy ung th Bcl6 c biu th qu mc trong bnh u lympho t bo B [9].
Tng t, protein gn kt AML1-ETO tm thy trong bnh bch cu ty bo cp
(AML) c chc nng nh cht c ch sao m ch yu thng qua ETO, do vy c vai
tr nh v tr gn kt cho phc hp ng c ch N-Cor/Sin3/HDAC1. Vic chuyn i
AML1 t cht hot ha phin m thnh cht c ch c iu khin bi protein gn
kt CBFb-SMMHC thng qua s gia tng phc hp ng c ch mSin3A/HDAC.
Cui cng, biu th qu mc nhn t sao m SCL/Tal1 trong bnh u lympho t bo T
cp c nguyn nhn l do gia tng bt thng HDAC1 nm trong phc hp ng c
ch, dn n ngn cn gen ch iu ha E47/HEB [18]. Biu th qu mc HDAC1
v/hoc HDAC2 v/hoc HDAC6 cn gp 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
[35,75,88] cng nh trong 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) [62]. Hn na, vic tm ra cc c cht ca HDAC l cc protein
nh p53, E2F, Rb, Bcl6, Gli1 lin quan n xu hng gy ung th v tin trin bnh
ung th khng nh vai tr ca HDAC trong ung th [16,59,61]. Tm li, cc bin
i sau phin m ca HDAC c th lm thay i tng tc ca chng vi phc hp
ng c ch m cc phc hp ny lin quan n qu trnh phin m ca cc gen gy
ung th.
Nh vy, c ch 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. y chnh l mt trong mc tiu
phn t hp dn cho chin lc iu tr ung th. Cc cht c ch HDAC v ang
c nhiu nh khoa hc trn th gii nghin cu nhm tm ra cht c tc dng c ch
chn lc tng loi HDAC ng dng trong iu tr ung th.
1.2. CC CHT C CH HDAC (HDIs)
1.2.1. Phn loi
Nm 1990, Yoshida v cng s pht hin ra dn cht hydroxamat t nhin
u tin c tc dng c ch trc tip HDAC l Trichostatin A (TSA), vn l cht c
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tc dng chng nm [91]. Sau , da trn 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 cht
c ch HDAC c nghin cu v th nghim trn lm sng ng dng trong
iu tr ung th. Cho n nay, nhiu cht c ch HDAC (HDIs) c cng b.
Chng c th c ngun gc thin nhin hay tng hp ha hc. FDA ph duyt 2
cht c ch HDAC c s dng trong iu tr u lympho da t bo T l vorinostat
(2006), v depsipeptid (2009). y chnh l ngun ng lc thc y cc nh nghin
cu tip tc tm ra cc cht c ch HDAC mi.
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 (bng 1.1).
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Bng 1.1. Phn loi cc cht c ch HDAC [22]
Cht Cu trc Cht Cu trc
Cc acid hydroxamic
TSA (Trichostatin A)
NHOH
O
N
O
Oxamflatin
O
NHOHHN
S
O
O
Acid
suberoylanilid
hydroxamic (SAHA)
HN
NHOH
O
O
NVP-LAQ-
824
HN
NHOH
O
O
N
OH
HN
CBHA (Acid M-carboxycinnamic
bishydroxamid)
HN
NHOH
O
O
HOHN
O
Acid
sulfonamid
hydroxamic Scriptaid
Peptid vng
Depsipeptid
(FK-228)
HN
O CH3
HN
O
H3C
CH3
O
NH
O
CH3
CH3
OHN
O
S
S
Acipidin
CHAP
Benzamid
MS-275 N
O
O
NH H
N
O
NH2
CI-994
Cc acid bo
Acid valproic
O
OH
Phenyl
butyrat O
ONa
Cc dn cht ceton
Trifluoromethyl
ceton
Alpha-
cetoamid
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Mi nhm c nhng hn ch ring: cc acid hydroxamic 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 b kh ha trong huyt tng; trong khi cc peptid vng c cu
trc qu phc tp [41].
1.2.2. C ch tc dng ca cc cht c ch HDAC
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 s to mch cng l vai tr rt quan trng ca HDIs, gin tip c
ch s pht trin in vivo ca khi u (hnh 1.7) [3,40].
Hnh 1.7. iu ha s pht trin v sng st ca t bo bi cc cht c ch HDAC
[40]
* Cc cht c ch HDAC thc y s bit ha
Khi s dng n c, cc cht c ch HDAC thc y bit ha v ngng tng
sinh t bo cc bnh bch cu v cc khi u tng c. c ch chu trnh t bo l vic
cn thit trong s bit ha t bo. Phn tch cc kiu chu trnh t bo ca t bo ung
th c tip xc vi cc cht c ch HDAC cho thy cc t bo thng b ngng
li pha G1, nhng i khi li tch t trong t bo n pha G2 ca chu trnh t bo
(hnh 1.7) [40,57].
V d trong bnh bch cu tin ty bo cp (APL), cc cht c ch HDAC c
th hip ng tc dng vi acid retinoic thc dy s bit ha in vitro v in vivo ca
-
15
protein gy ung th PLZF-RAR. S bit ha t bo ty ph thuc vo RAR
(receptor acid retinoic) hot ha qu trnh phin m v cc protein gy ung th PLZF
RAR cn tr chng trnh bit ha bnh thng. Qu trnh tng t c th xy ra
trong bnh bch cu ty bo cp (AML). Cc cht c ch HDAC c ch chc nng ca
cc protein gn kt AML1-ETO v TEL-AML1, khi phc s nhy cm ca cc t bo
c tnh vi acid retinoic. Ngoi ra, cc cht c ch HDAC c th gy bit ha nhiu
loi t bo, nhng qu trnh ny vn cha c bit r [40].
Hu ht cc cht c ch HDAC c kh nng hot ha qu trnh phin m gy ra
bi cht c ch kinase ph thuc cyclin (cyclin-dependent kinase inhibitor), l WAF1
(cn gi l CIP1, p21; c m ha bi v tr gen CDKN1A). WAF1 c th c ch
cyclin E-CDK2 v cyclin A-CDK2. S c mt ca CDKN1A cn thit cho cc cht c
ch HDAC thc y s ngng pha G1. S tip xc ca cc t bo thiu ht
CDKN1A vi cc cht c ch HDAC dn n tch t cc t bo c ADN a bi v lm
cho cc t bo ny nhy cm vi s cht t bo. S phi hp hot ng ca cc cyclin,
cc CDK (cyclin-dependent kinase), nhng cht c ch CDK v cc thnh vin RB
(retinoblastoma protein) rt cn thit cho s bit ha ca nhiu h thng [40].
Sau khi tip xc vi HDIs, thng thng cc t bo ung th dng li pha G1
sao chp ADN, sau i vo s cht t bo. Nhng mt s t bo ung th li tch t
n im kim sot G2 (G2 checkpoint), v tr m ti chu trnh t bo tm thi dng
li, ch n khi cc iu kin tr nn ph hp th chu trnh li tip tc. Nhng t bo
bnh thng tri qua im kim sot G2 (G2 checkpoint) v tip tc pht trin, trong
khi nhng t bo ung th ti to ADN thnh dng ADN a bi (4nADN) v chuyn
sang giai on gy cht t bo theo chng trnh. Tuy nhin, c ch phn t cht c
ch HDAC thc y G2 checkpoint vn cn cha sng t. C th trong mt s t bo,
HDIs thc y trc tip hoc gin tip gen hot ha im kim sot G2 (G2
checkpoint) [40].
* Cc cht c ch HDAC gin tip gy ra s cht t bo theo chng trnh
Cc t bo ung th khc nhau sau khi c tip xc in vitro vi cc cht c ch
HDAC c th dn n s cht t bo theo chng trnh. S cht t bo l mt chng
trnh cht sinh l ca t bo kim sot s lng cc t bo bnh thng trong sut
qu trnh pht trin v bnh tt. Hin nay xc nh c hai con ng gy cht t
bo v c hai con ng u c s tham gia ca cc enzym cystein protease (cc
caspase) (hnh 1.8) [33,40].
Con ng th nht c hot ha bi cc receptor gi l cc death-receptor
nh CD95, receptor TNF (tumour-necrosis factor) v receptor TRAIL (TNF-related
-
16
apoptosis-inducing ligand). Cc yu t ny sau khi gn vi cc phi t bt u hot
ha cc caspase gn mng t bo (caspase-8 v -10). Cc caspase ny sau s hot
ha cc caspase kch thch (caspase-3 v -7) v dn n s cht t bo.
Con ng th hai cn c s ph v mng ty th, gin tip gii phng
cytochrom c, cc protein h tr s cht t bo khc (nh SMAC/DIABLO (the second
mitochondria-derived activator of caspase), HTRA2 (high temperature requirement 2)
v yu t thc y s cht t bo (AIF)) v s hot ha caspase-9 thng qua yu t
hot ha protease gy cht t bo (APAF1). S ton vn ca mng ty th c kim
sot bi cc protein trong h protein BCL2 (bao gm cc protein h tr s cht t bo
(BAX, BAK) v cc protein khng li s cht t bo (BCL2, BCL-XL) [33,40].
Hnh 1.8. Cc cht c ch HDAC thc y s cht t bo [40]
* Ch thch: thnh phn c ch s cht t bo c mu vng, thnh phn y mnh s cht t
bo c mu tm.
Cc cht c ch HDAC c ghi nhn c kh nng hot ha c receptor gy
cht t bo v qu trnh cht t bo ni sinh (hnh 1.8). V d, apicidin v CBHA c
th kch thch biu hin ca CD95 v gc kt hp CD95 (CD95L). Tuy nhin, butyrat
lm cho t bo nhy cm vi s cht t bo gin tip bi CD95 m khng thc y s
phin m ca gc kt hp hay receptor. Depsipeptid c ch s biu hin CD95L sau
khi hot ha t bo T v ngn cn s cht t bo T sau . V vy, qu trnh death-
-
17
receptor c th quan trng cho s cht t bo gy ra bi cc cht c ch HDAC, ph
thuc vo loi t bo v nhm cht c ch HDAC c s dng.
Vic biu hin qu mc ca BCL2 (yu t ngn cn qu trnh cht t bo ni
sinh) trong khi qu trnh death-receptor cn nguyn vn cng vi kh nng c ch s
cht t bo gin tip bi cc cht c ch HDAC cho thy tm quan trng ca cc qu
trnh ni sinh i vi vai tr ca cc cht c ch HDAC. Nhng d kin ny c
chng minh bng vic pht hin ra s cht t bo do cc cht c ch HDAC trng
khp vi s tng cng cc protein BCL2 h tr s cht t bo (BAX, BAK) v s
gim cc protein h BCL2 khng li s cht t bo (BCL2, BCL-XL) [40]. Cc cht c
ch HDAC c th vn gy ra s cht t bo khng km theo hot ha caspase. SAHA
hot ha qu trnh cht t bo ni sinh bng cch thc y b gy lin kt v hot ha
cht ch vn s cht t bo tng tc vi BH3 (BID), dn n ph v mng ty th v
to thnh cc dng oxy hot ng (ROS). Ngc li, cc cht c ch ROS ngn cn s
cht t bo do SAHA [40].
* Cc tc dng khc lin quan n c ch khng ung th ca cc cht c ch
HDAC
Ngoi tc dng trc tip ln s pht trin v sng st ca t bo ung th, cc
cht c ch HDAC cn tc dng gin tip n s pht trin khi u. Cc cht c ch
HDAC c th hot ha phc hp ha hp m (MHC) nhm I v II lin quan n phin
m, nhng phn t ng kch thch CD40, CD80 v CD86, phn t gn vo khong
gian bo ICAM1, v cc interferon loi I v II, nhm lm tng s nhn dng v hot
ha ca cc t bo min dch (hnh 1.7) [40]. Hn na, s pht trin v sng st ca
ung th tng c pht trin nhanh i hi s hnh thnh mch trong khi u duy tr
cung cp lin tc oxy v cht dinh dng. Trichostatin A (TSA) c th c ch s biu
hin gim oxy m ca yu t pht trin mng trong mch mu (VEGF) v ngn cn s
to mch c in vitro v in vivo. V vy, tng cng p ng min dch v c ch s to
mch ca khi u c th ngn cn r rt s pht trin khi u ban u v s di cn [40].
1.2.3. Cu trc ca cc cht c ch HDAC
Cc HDIs c chia thnh nhiu nhm khc nhau nhng c mt s c im
chung v mt cu trc, bao gm 3 phn chnh [3,6,17,58]:
- Nhm gn vi ion Zn2+ (A - Zinc Binding Group, ZBG): nh acid hydroxamic,
thiol, nhm o-aminoanilin ca benzamid, -cetoester, ceton thn du... quyt nh tnh
c hiu v hiu lc ca HDIs.
- Vng cu ni s nc (B): thng l mch hydrocarbon thn du c th to nhiu
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18
lin kt Van der Waals vi knh enzym.
- Nhm kha hot ng (capping group) hay vng nhn din b mt (C): thng l
cc aryl hoc 1 s vng khc, nm trn b mt enzym.
Hnh 1.9. Cng thc c in ca HDI v v tr ca HDI trong ti enzym HDAC
Cu trc tinh th kt tinh ca cc HDAC lin kt vi 1 s cht c ch HDAC
cho thy phn A, B v 1 phn ca C nm trong ti enzym, lm y khong trng trong
lng knh enzym. Phn cn li ca 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 1 s lin
kt peptid, lm tng kh nng phn cc v gp phn ci thin dc ng hc cho HDIs.
Vic nghin cu thit k cng thc cho cc HDIs mi u da trn cu trc c in
ny.
1.3. TNH HNH NGHIN CU TRN TH GII V CC CHT C CH
HDAC
Vic pht hin tc dng 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 ca TSA m ra mt
hng nghin cu trong con ng tm thuc mi iu tr ung th [91]. T nhng nm
90 ca th k trc, rt nhiu nh khoa hc trn th gii tp trung nghin cu v
hng trm bi bo c cng b trong qu trnh tm kim cc cht c ch HDAC.
Cho n nay, bn cnh 2 cht l vorinostat (Zolinza) v depsipeptid (Romidepsin)
c FDA ph duyt s dng trong iu tr u lympho da t bo T, cn c khong
hn 10 cht ang c nghin cu pha lm sng I, II (bng 1.2). 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 ca chng ti ang tin hnh.
C
A B
ArNH
NH
OH
n
OO
B AC
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19
Bng 1.2. Cc cht c ch HDAC v ang c th lm sng [56]
Nhm Hp cht Pha lm
sng
Loi ung th
Peptid vng Depsipeptid
(FK228)
I, II Tng c, CLL, AML, CTCL, u
a ty xng, NHL t bo T
ngoi vi, RAI khng thyroid, ung
th i trng giai on mun
Cc benzamid MS-275
CI-994
MGCD-0103
I, II
I, II
II
Tng c, u lympho, AML, u
hc sc t c tnh di cn giai
on mun
Tng c, NSCLC, t bo thn,
tu
Tng c, ung th bch cu,
MDS
Acid bo mch
ngn
Butyrat
AN-9 (tin thuc)
Acid valproic
Phenyl butyrat
I, II
I, II
I, II
I
Ung th i trng
Tng c, NSCLC
Tng c, ung th mu, AML,
MDS, CTCL, u trung biu m
Tng c, AML/MDS
Acid
hydroxamic
SAHA
PXD101
NVP-LAQ824
LBH-589
ITF-2357
SB-939
CRA 024781
ph duyt
I, II
II
I
II, III
II
I
I
I
CTCL
Tng c, ung th mu
Ung th mu
Tng c, ung th mu
Tng c, AML, ALL, MDS
U lympho Hodgkin
Tng c, ung th mu
* Ch thch: NSCLC, carcinom t bo phi khng nh; AML, ung th bch cu ty bo cp; MDS, hi chng lon sn ty; CTCL, u lympho da t bo T; ALL, ung th bch cu cp; CLL, ung th bch cu mn.
1.3.1. Cc peptid vng
y l nhm cc cht c cu trc phc tp nht trong HDIs, tc dng c ch
HDAC mnh tng t nh cc acid hydroxamic gm: Depsipeptid (FK-228), apicidin,
trapoxin A... v cc CHAP (cyclic hydroxamic acid-containing peptide) (hnh 1.10).
a s cc cht trong nhm ny c tc dng c ch HDAC nng nanomol.
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20
Depsipeptid l polypeptid t nhin c phn lp t nm Chromobacterium violaceum
v l cht c ch HDAC th 2 c FDA ph duyt trong iu tr u lympho da t bo
T (thng 11/2009). Cu trc ca depsipeptid kh phc tp vi 4 lin kt peptid.
Depsipeptid l tin thuc, khi vo c th c kh ha to ra nhm sulfydryl ca
redFK c tc dng tt vi HDAC nhm I, c bit HDAC1, 2. Nhm -SH ca redFK
c th phn ng vi cystein tn trong ti enzym to lin kt cng ha tr disulfid. Tnh
n nh v k nc ca FK228 gip cho n d dng qua c mng t bo, do tc
dng hiu qu hn cc HDIs khc [45].
Cc CHAP l sn phm kt hp peptid vng v acid hydroxamic. Trong cu
trc ca chng, chui acid hydroxamic c gn vi vng peptid thay cho nhm
epoxyketon trong trapoxin hay cc hp cht t thin nhin khc. Mt s hp cht c
tng hp bng cch thay i s lng acid amin trong cu trc vng, s khng i
xng ca cc acid amin v chiu di chui hydroxamic. Trong s , CHAP31 c tc
dng tt nht (c ch HDAC1 vi IC50 = 3,32 nM), v bn hn TSA trong t bo (t1/2 di hn) [63].
Hnh 1.10. HDIs c cu trc peptid vng
1.3.2. Dn cht benzamid
Dn cht benzamid c tc dng c ch HDAC in vitro v in vivo c cng b
nm 1999 bi Suzuki v cng s thuc cng ty dc Mitsui [63]. Nhm ny bao gm
mt s cht MS-275, CI-994, MGCD-103 (hnh 1.11), chng c hot tnh km hn cc
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21
acid hydroxamic v cc peptid vng, c ch HDAC nng micromol [39].
Hnh 1.11. HDIs l cc benzamid
MS-275 v CI-994 ang c th nghim lm sng pha 2. MS-275 lm gim
s acetyl ha qu mc ca histon trong nhiu loi ung th, gim s biu th qu mc
ca p21WAF1/CIP1 v gelsolin. CI-994 l 4-acetylamino-N-(2-aminophenyl)benzamid,
c chng minh c tc dng trn t bo ung th ngi v chut. Nghin cu in
vitro cho thy CI-994 c tc dng thc y s cht t bo theo chng trnh trn cc t
bo lympho chut. u im ln ca CI-994 l thuc chng phn bo ng ung [32].
1.3.3. Cc acid bo mch ngn
Cc acid bo mch ngn nh acid valproic, natri butyrat, phenylbutyrat (hnh
1.12) c dng trong lm sng t rt lu (acid valproic l thuc iu tr ng kinh)
Gn y, tc dng c ch HDAC ca chng mi c nghin cu. Acid butyric l sn
phm t nhin c tng hp trong c th ngi do chuyn ha acid bo v vi khun
ln men cht x i trng. Nghin cu cc cht c ch HDAC nhm ny i hi s
cn trng do chng c th iu ha qu trnh biu hin gen theo cc cch v cng tinh
vi. Thm vo , nhc im ln ca cc cht ny l p ng hn ch trn lm sng do
thi gian bn thi ngn v nng t c tc dng cao ( mc nng milimol
so vi TSA l nanomol) [40,71].
Hnh 1.12. HDIs l cc acid bo mch ngn
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22
1.3.4. Cc dn cht ceton
Nhm ny bao gm cc trifluoromethyl ceton v -ketoamid vi hot tnh c
ch HDAC mc nng micromol (hnh 1.13) [28].
CF3
HN
O
O
HN
HN
O
O
O
Trifluoromethyl ceton HDAC IC50 = 6,7 M
-cetoamid HDAC IC50 = 340 nM
Hnh 1.13. HDIs l cc dn cht ceton
1.3.5. Cc hydroxamat v dn cht
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 (TSA) l cht u tin c phn lp t Streptomyces
hygroscopicus vi tc dng chng nm bi Tsuji v cng s nm 1976 (hnh 1.14).
Mi nm sau , Morioka v cng s cng Yoshida v cng s chng minh tc
dng rt tt ca TSA 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 [91]. Tuy nhin,
nghin cu su hn cho thy ch cu trc (R)-TSA c tc dng c ch s pht trin ca
chu trnh t bo, gim s bit ha v s bin i ca histon trn chut c t bo ung
th tuyn v FM3A. TSA c tc dng c ch mnh, c hiu vi HDAC (Ki = 3,4nM)
v th nghim in vivo trn chu trnh pht trin v bit ha t bo khng nh chc chn
tc dng c ch enzym ny [63]. Nghin cu gn y xc nh TSA ging nh ligand
ca receptor hot ha sn sinh peroxisome (troglitazon v BRL46593) v ca
receptor retinoid X (LG268), c th iu ha s biu hin ca gen Drg-1. Do , TSA
ng vai tr nh cht ngn cn s di cn trong ung th i trng. Vic sn xut TSA
rt tn km m li khng hiu qu (tri qua 20 bc v hiu sut 2%) nn nghin cu
tm ra cc cht c ch HDAC thay th TSA v ang c nhiu nh nghin cu
quan tm. Hin nay, TSA ch yu dng lm cht i chiu trong nghin cu tm ra cc
cht c ch HDAC mi [71].
Mt cht c ch HDAC tng hp l acid suberoylanilid hydroxamic (SAHA)
(hnh 1.14) c nghin cu. Bo co cho thy SAHA c ch s pht trin t bo,
dn n kt thc s bit ho v ngn nga hnh thnh khi u trn chut. 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. SAHA
-
23
c chng minh nhy cm vi cc dng t bo Hut78, HH, M v nhy cm vi t
bo lympho mu ngoi vi nguyn pht bnh nhn u lympho da t bo T [92]. SAHA
lm tng s acetyl ho ca cc histon H2B, H3 v H4, ng thi lm tng s biu th
ca p21 v Bax trong khi li lm gim s biu th ca STAT6 v lm gim mc
ca STAT6 phospho. Tt c cc nh hng dn n hot ho caspase 3, s phn
chia ca PARP v s cht t bo theo chng trnh. Hin nay, Zolinza ang tip tc
c th nghim lm sng dng phi hp vi cc ho tr liu khc nh tamoxifen,
bortezomib, temozolomid...
Bn cnh , nhiu cht c ch HDAC dn cht acid hydroxamic ang c
nghin cu rng ri v c chia thnh nhiu phn nhm nh: acid hydroxamic mch
thng, dn cht cinnamic (CBHA, LAQ 824, LBH 589, PXD101), dn cht
phenyl...(hnh 1.14). c im chung ca nhm ny l nhm chc acid hydroxamic d
to phc vi ion Zn2+ ca HDAC nn c ch khng chn lc c HDAC nhm I, II v
b chuyn ha nhanh [58,89].
Hnh 1.14. HDIs c cu trc hydroxamat
Nh trn cp n, cc acid hydroxamic c hiu lc c ch HDAC tt
song chng c nhc im l c ch khng chn lc v na i in vivo ngn. 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 cht tm c. Da trn c im cu
trc ca cc cht c ch HDAC (mc 1.2.3), cc nghin cu c th tin hnh thay i
-
24
mt trong 3 phn cu trc: nhm kha hot ng, cu ni hoc nhm chc
hydroxamic. Sau y l tng kt mt s nghin cu trn th gii v thay i cu trc
ca cc acid hydroxamic.
1.3.5.1. Thay i cu ni
* Cc N-hydroxy-2-propenamid cc acid hydroxamic tng t TSA
Nhm cc N-hydroxy-2-propenamid (AH1, hnh 1.15) c thit k v tng
hp da trn c s nghin cu cu trc ca TSA, oxamflatin v MS-275. Nhn chung,
cc dn cht ny c tc dng c ch HDAC yu hn TSA song mnh hn MS-275 v
tng t oxamflatin. Cht c IC50 mnh nht l 172 nM [44].
Hnh 1.15. Cc dn cht N-hydroxy-2-propenamid
Trong mt nghin cu tng t, cc nh khoa hc ti Trung tm nghin cu
Sigma-tau, Pomezia () thit k v tng hp mt dy cc acid biphenyl-4-yl-
acrylohydroxamic (AH2) [19]. nh gi hot tnh ca cc cht tng hp c cho
thy nhiu cht trong dy c tc dng c ch HDAC2 tng ng vi SAHA. Khi
ko di cu ni thm mt lin kt i, hot tnh c ch HDAC2 gim ng k (AH3,
hnh 1.16). Tuy nhin, dn cht kh ha ca 7 li c ch HDAC2 kh mnh vi IC50 =
0,72 M (AH4, hnh 1.16). ng phn v tr ca AH2b (R l 4-hydroxyphenyl) vn
duy tr tc dng c ch HDAC2 (AH5, hnh 1.17). Tng t, dn cht AH6 vi mt
vng phenyl b kh ha cng cho tc dng c ch HDAC2 tng ng (hnh 1.17).
Tuy nhin, kh ha ng thi c lin kt i mch nhnh lm mt hon ton i lc
vi HDAC2 (AH7, hnh 1.17).
-
25
Hnh 1.16. a) Cc acid biphenyl-4-yl-acrylohydroxamic (AH2); b) Cc dn cht vi
cu ni c hai lin kt i (AH3) v dng kh ha ca AH3 (AH4)
Hnh 1.17. Mt s dn cht c lin quan ca AH2b
* Cc amid ngc ca SAHA
Nhm nghin cu thuc cng ty TopoTarget (Anh) thit k v tng hp gn
40 dn cht amid ngc (AH8) ca SAHA (hnh 1.18) [6]. Nhiu cht trong s ny c
hot tnh c ch HDAC tng ng, thm ch mnh hn SAHA. Trong , hai cht
c th nghim m hnh ung th in vivo trn chut v cho kt qu rt kh quan,
nh hng cho cc nghin cu tip theo. 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;
-
26
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 [6].
Hnh 1.18. Cc dn cht amid ngc ca SAHA
* Cc aryloxyalkanoic N-hydroxyamid: dn cht ca SAHA khng c nhm amid
Nhm amid ni gia mch alkyl v nhn phenyl ca SAHA c cho l cn
thit song c th thay th. Chnh v vy, y l v tr c nhiu nghin cu quan tm
kho st thay th bng nhng phn cu trc hoc nhm chc khc nhau. Mt trong
nhng nghin cu u tin c cng b l thay nhm amid bng oxy (-O-) to ra cc
aryloxyalkanoic N-hydroxyamid (AH9) (hnh 1.19) [58].
Hnh 1.19. Cc aryloxyalkanoic N-hydroxyamid (AH9)
Kt qu th hot tnh in vitro cho thy dn cht AH9a c mt nhm th n
gin nh N,N-dimethylamino (thch hp cho vng kha hot ng ca TSA) c th
mang li hiu lc c ch tt. Tuy nhin, m rng vng s nc ca nhm kha hot
ng c th lm tng hot tnh ln nhiu; iu ny c th ci thin bng vic dng
nhm 4-clorophenyl nh trong AH9b, hoc bng s hp nht vng nh trong AH9p
v AH9q. Cc vng phng ca AH9p v AH9q gp phn to nn hiu lc, c v nh
do s u tin chim gi phn s nc v cu trc phng tng ng ca vng kha hot
ng ca cc enzym [58].
Nh vy, cc aryloxyalkanoic hydroxyamid nu trn l mt vi cht c ch
HDAC c hiu qu nht c bit lin quan n tnh n gin v cu trc. Hiu lc
ca cc aryloxyalkanoic hydroxyamid cho thy nhm carbonyl (nh c mt trong
-
27
trichostatin A v SAHA) ln mt cu ni rn chc u khng cn thit thu c tc
dng c ch HDAC mc nng nanomol. Cc cht c cu trc aryloxy ny chng
t s n nh in vivo tt hn khi so snh vi cc cht c ch HDAC cu trc amid.
Thm vo , cht AH9q c ch s tng sinh ca HeLa (IC50 = 7 M) v dng t bo
MCF-7 (IC50 = 5 M) [58].
* Cc dn cht -alkoxy 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, ) [37] 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. Hai hp cht thin nhin amamistatin A v B vi
cu trc cha nhm N-formyl-N-hydroxylysin c bo co c tc dng c ch
HDAC ng k (hnh 1.20).
Hnh 1.20. Cu trc ca amamistatin A, B [37]
Da trn c s ny, nhm nghin cu thit k v tng hp dy dn cht -
alkoxy ca SAHA (hnh 1.21).
Hnh 1.21. Cc dn cht -alkoxy ca SAHA
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. c bit, c tnh i vi t bo ung th ca
cc dn cht -benzyloxy th hin mnh hn r rt so vi SAHA trn c 3 dng t bo
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28
NB4 (ung th bch cu tin ty bo cp), H460 (ung th phi ngi), HCT-116 (ung
th i trng). 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 ch l
cu hnh ca nhm chc -alkoxy khng nh hng n hot tnh sinh hc, th hin
qua tc dng ca dn cht p-methoxybenzyl ether AH10 (racemic) (hnh 1.22), AH10
(dng R) v AH10 (dng S). y l im ht sc c ngha i vi tng hp cc dn
cht ny v vn tch cc ng phn s khng cn t ra.
Hnh 1.22. Cu trc ca dn cht p-methoxybenzyl ether (AH10) [37]
Bng 1.3. Tc dng c ch HDAC2 v c tnh t bo ca dn cht -alkoxy (AH10)
Cht HDAC2 (IC50 M) IC50 (M)
0,05 NB4 H460 HCT-116
SAHA + 0,70 3,40 1,20
AH10 + 0,12 0,52 0,22
AH10 + 0,11 0,45 0,25
AH10 + 0,07 0,50 0,39
Tng phn vi cc dn cht -alkoxy trn, cc dn cht -alkyl ca SAHA
li hu nh khng c tc dng c ch HDAC (hnh 1.23) [12]. Nh vy, c th thy
vng lin kt vi Zn2+ ca HDAC l tng i nh v khng chp nhn cc nhm th
cng knh. Mi thay i v cu trc vng ln cn vi nhm acid hydroxamic cn
ch im ny.
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29
Hnh 1.23. Mt s dn cht -alkyl ca SAHA
1.3.5.2. Thay i nhm kha hot ng
* Cc acid phenylthiazol-hydroxamic tng t SAHA
Bn cnh cc nghin cu thay i phn cu ni, hng nghin cu kho st
thay i phn nhm kha hot ng (nhm nhn din b mt, SRG) cng c rt
nhiu nh khoa hc quan tm. 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 (AH11, hnh 1.24)
[23]. Mt dn cht oxazol l WR301849 cng c tng hp [52].
Hnh 1.24. Cc acid phenylthiazol hydroxamic tng t SAHA
Kt qu nh gi tc dng trn HDAC cho thy dn cht WR301801 (AH11a)
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 (AH11b) (nhm th amino v tr ortho) cng c tc dng
mnh tng ng SAHA [23].
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 1.25) [51]. 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 AH11a-5 (hnh 1.25). IC50 ca dn cht
ny vi HDAC2, HDAC3 thp di mc 0,2 nM. Kt qu th c tnh trn 5 dng t
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30
bo ung th ty cng b vi AH11a, AH11b, AH11a-2 cho thy cc dn cht ny u
c IC50 nh hn 3 M [51].
Hnh 1.25. Mt s acid phenylthiazol hydroxamic
* Cc acid biphenyl-hydroxamic tng t SAHA
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 1.26) [51]. 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 1.26. Cc dn cht acid biphenyl-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 WR301849 (hnh 1.24) [52]. 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
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31
vo v tr ngay st cnh nhm acid hydroxamic c thit k v tng hp (hnh
1.27) [79].
Hnh 1.27. Cc acid isoxazol-hydroxamic
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 tp HDAC1, 2, 3, 6 v 10 vi IC50 trung
bnh khong 150 nM [79]. Tc dng ny km hn nhiu so vi cc acid phenylthiazol-
hydroxamic AH11 v acid biphenyl-hydroxamic AH12. 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 5-phenylthiazol
(AH13a, AH13b) c tc dng c ch HDAC kh mnh, gn tng ng SAHA
(hnh 1.27). 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 AH13b c ch HDAC mnh hn AH13a song
AH13a li c c tnh t bo mnh hn AH13b [79]. C th s c mt ca nhm 3-
amino AH13b lm gim tnh thm t bo ca cht ny, dn n c tnh t bo
thp hn so vi AH13a. C th cc dn cht acyl ha ca AH13b s ci thin c tc
dng trn HDAC v t bo, tng t nh vi cc dn cht acid phenylthiazol-
hydroxamic AH11.
* Cc dn cht acid thiophen-2-hydroxamic
Mt trong nhng nghin cu ng ch cng b gn y l cc dn cht acid
thiophen-2-hydroxamic ca cng ty Argenta Ltd. (Anh) [67]. Bng phng php sng
lc o da trn cu trc tinh th ca HDLP (HDAC-like protein) (PDB: 1C3R), tin
hnh vi 644 acid hydroxamic sn c, nhm nghin cu la chn c 98 ng vin
tng hp v th tc dng c ch HDAC. Kt qu tm c cht dn ng vi
cu trc hon ton mi ADS100380 (hnh 1.28). ADS100380 c ch HDAC vi IC50 =
0,75 M v c tnh trn t bo MCF7 vi IC50 = 11,40 M. Docking ADS100380 vo
trung tm hot ng ca HDAC cho thy phn hydroxamic ca cht ny trng kht vi
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32
phn hydroxamic ca SAHA cn cu trc thiophen nm dc theo ng cu ni thn
du ca SAHA ti trung tm hot ng ca HDAC [67].
SN
N
F3C
H3CNHOH
O
Hnh 1.28. ADS100380
Nhm tm kim cc dn cht vi hot tnh mnh hn, nhm nghin cu tin
hnh thit k dy cc dn cht acid 5-(1H-pyrazol-3-yl)-thiophen-2-hydroxamic. Kt
qu thu c cht ADS102550 c hot tnh tt hn cht dn ng, c ch HDAC vi
IC50 = 0,029 M, c tnh trn MCF-7 vi IC50 = 0,30 M. ADS102550 c t1/2 = 2,5
gi (tim tnh mch), di gp ba ln ca SAHA (0,7 gi). Khi dng ng ung, c
SAHA v ADS102550 u c sinh kh dng km. Tuy nhin, khi tim vo mng
bng, ADS102550 c sinh kh dng cao. Tip tc nh gi tc dng chng ung th in
vivo s dng m hnh chut tri lng (nude mice) mang t bo ung th ngi
HCT116, kt qu cho thy ADS102550 c ch 33% s pht trin ca khi u. Tuy
nhin, ng tic l ADS102550 cn c ch cytochrom P450 kh mnh vi IC50 = 0,51
M [67].
Nhm tm kim cc dn cht mi ci thin c nhc im ca ADS102550,
nhm nghin cu ti Argenta tip tc thit k, thay i cu trc theo cc hng i) thay
i vng pyrazol bng cc vng khc nhau, sau ii) kho st cc nhm th v cu ni
trn vng mi (hnh 1.29).
Hnh 1.29. Cc nh hng ti u ha cu trc ca ADS102550
Theo hng th nht, nhm nghin cu tin hnh tng hp cc dn cht
trong pyrazol c thay bng cc nhm imidazol, thiophen, phenyl, 2-pyridyl, 3-
pyridyl, 4-pyridyl v pyrimidyl. Cc cht c sng lc hot tnh c ch HDAC v s
-
33
b nh gi c tnh t bo trn dng t bo ung th v MCF7. Kt qu sng lc dy
ny cho thy dn cht AH14c (mang vng 2-pyridin) v AH14f (mang vng
pyrimidin) c hot tnh mnh nht trn HDAC v t bo MCF7 (hnh 1.30) [67].
Hnh 1.30. Cc arylthiophen hydroxamat
T kt qu dy dn cht arylthiophen hydroxamat, nhm nghin cu la
chn AH14c lm nhng cht dn ng mi v kho st cc nhm th gn trn vng
2-pyridin. Hn 50 dn cht khc nhau c thit k, tng hp (hnh 1.31) [67].
Hnh 1.31. Cc acid pyridin-thiophen-hydroxamic
Tc dng sinh hc ca dy dn cht AH14ca AH14cm cho thy: i) Nhm th
trn vng pyridin v tr 5 tt hn v tr 6; ii) Khong cch gia nhm phenyl mch
nhnh v vng pyridin c th dao ng t 2-5 nguyn t m vn cho tc dng tt; iii)
C th chn thm O hoc N vo mch nhnh m vn gi c tc dng. T dy dn
cht ban u ny, nhm nghin cu ti Argenta Ltd. chn AH14cb (ADS102770)
nghin cu su hn. ADS102770 c tc dng mnh hn SAHA trn c HDAC v t
bo. ADS102770 cng mnh hn hai ln so vi ADS102550 trn HDAC v 3-6 ln
trn t bo (MCF7 v MDA-MB231). Tc dng c ch cytochrom P450 ca
ADS102770 gim 10 ln so vi ADS102550. Khi th nghim vi t bo gan ca chut
cng, ADS102770 bn hn ADS102550 v SAHA. Tuy nhin, n nh ca
ADS102770 li km hn so vi ADS102550 khi th vi microsom ca chut nht
(tim tnh mch, t1/2 = 0,17 gi so vi 2,5 gi ca ADS102550 v 0,8 gi ca SAHA).
B li, ADS102770 c sinh kh dng ng ung tng i cao hn so vi SAHA v
ADS102550 khi th nghim trn chut cng (AUC, 3,3 g h/mL so vi 0,4 g h/mL
ca SAHA v 0,1 g h/mL ca ADS102550) [67].
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34
1.3.5.3. Thay i nhm chc hydroxamic
* Cc dn cht sulfonamid
Gn y, cc nh khoa hc ti Trung tm nghin cu ca MethylGene (Canada)
cng b cc dn cht sulfamid trong nhm chc sulfonamid c kho st thay th
cho phn acid hydroxamic. Trong dy dn cht u tin vi nhm nhn din b mt l
biphenyl, hu nh khng dn cht no c kh nng c ch HDAC, IC50 trn HDAC1
v HDAC6 u > 10 M (hnh 1.32).
Hnh 1.32. Cc dn cht biphenyl sulfamid
Ch khi thay nhm biphenyl bng mt s nhm nhn din b mt khc nh
phenyl, quinolin, 5-phenylthiazol, tc dng c ch HDAC6 mc va phi (IC50,
0,39-1,50 M) mi c ghi nhn (AH16, hnh 1.33). ng ch nht l hai dn cht
trong phn cu ni 6-aminohexanoyl c thay bng cu ni lysyl (AH17, hnh
1.33), c tc dng c ch c HDAC1 v HDAC6 vi gi tr IC50 mc di 1 M
[87]. T kt qu ny, mt dy cc dn cht sulfamid khc vi cu ni lysyl v phn Ar
l nhm 5-phenylthiazol c thit k, tng hp (AH18, hnh 1.33) [54]. Nhn
chung, hu ht cc dn cht u c tc dng c ch c 2 loi HDAC1 v 6 vi IC50 < 1
M. C th nhm -amin ca phn lysin c tng tc vi cc aminoacid phn ming
ti ca trung tm hot ng ca HDAC.
Hnh 1.33. Mt s dn cht sulfamid khc [54]
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35
Mt kt qu ng ngc nhin khc l cc dn cht sulfamid ny, cu ni amid
c v khng ng vai tr quan trng i vi hot tnh c ch HDAC. Kt qu ny th
hin mt dy cc dn cht AH19 khng mang cu ni amid (hnh 1.34) nhng hot
tnh c ch HDAC1 v 6 vn rt tt (HDAC1, IC50 t 0,1-4,9 M; HDAC6, IC50 t
0,1-1,8 M) [54].
Hnh 1.34. Cc dn cht sulfamid khng mang cu ni amid
Cc kt qu trn cho thy nhm sulfamid hon ton c trin vng thay th cho
nhm acid hydroxamic trong tng tc vi Zn2+. Xt v mt chuyn ha, nhm
sulfamid c th bn hn in vivo so vi nhm chc acid hydroxamic.
* Cc dn cht thiocarbonat, dithiocarbonat v trithiocarbonat
Cng trong n lc tm kim cc cht c ch HDAC khng phi acid
hydroxamic, cng ty Nycomed GmbH (c) thit k, tng hp v nh gi hot
tnh ca hng lot dn cht thiocarbonat, dithiocarbonat v trithiocarbonat. Kt qu
dn cht trithiocarbonat c trin vng hn c khi cn nhc c hai kha cnh l tc dng
c ch HDAC v tc dng c ch s pht trin ca t bo (hnh 1.35) [20].
Hnh 1.35. Mt s dn cht trithiocarbonat
Nghin cu su hn v dy trithiocarbonat thu c mt s dn cht c ch
HDAC tim nng vi gi tr IC50 mc thp n di M (hnh 1.36) [20]. Cht
AH21a (R = 4-AcNH, Z = S) cho tc dng c ch HDAC (IC50 = 0,857 M) v c
tnh trn t bo Hela (IC50 = 1,46 M) tt nht trong dy AH21. Vi dy cht AH22,
cht AH22g (RRN l 4-methoxy benzylamin) c IC50 trn HDAC l 0,600 M, c
tnh trn t bo Hela l 1,45 M [20]. Nu so snh vi SAHA th nhn chung cc dn
cht trithiocarbonat mnh nht vn km SAHA khong 100 ln. Tuy nhin, xt v kha
cnh dc ng hc, cc trithiocarbonat c trin vng tt hn in vivo.
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36
Hnh 1.36. Mt s trithiocarbonat khc v cht tng t
* Cc dn cht thiol v mercaptoceton
Tip tc hng nghin cu tm kim cc cht c ch HDAC khng phi acid
hydroxamic, nhm nghin cu ti cng ty Daiichi Pure Chemicals ca Nht thit
k, tng hp (hnh 1.37) v cng b tc dng c ch HDAC ca mt dy cc thiol y
trin vng [78]. Bt u bng vic thay th nhm -CONHOH bng -SH, nhm nghin
cu thu c hp cht AH23a (Ar = Phenyl; X = NHCO; n = 4) vi tc dng c ch
HDAC IC50 = 0,21 M. Tip tc ti u ha cu trc ny, nhm nghin cu thu
c cc dn cht trong nhm nhn din b mt Ph c th thay th bng cc nhm
3-PhPh, 3-quinolyl, 2-naphthyl v 2-benzofuranyl to ra nhng cht c ch HDAC
mi vi hot lc tng t SAHA nh cht AH23i (Ar = 3-PhPh; X = NHCO; n = 4),
AH23s (Ar = 2-benzofuranyl; X= CONH; n = 4) [78].
Hnh 1.37. Cc dn cht thiol
Gn y, nhm nghin cu thuc cng ty Kalypsys (M) sng lc mt th
vin ha cht vi hn 600 000 cht. Kt qu tm c cht dn ng disulfid (hnh
1.38). Tuy nhin, thc cht disulfid ny khng phi l cht c hot tnh m trong iu
kin th nghim, cht disulfid nhanh chng b kh ha thnh dn cht -
mercaptoceton (-MRC) mi l dng hot ng. -MRC c ch HDAC vi IC50 =
0,135 M, tng ng vi nhiu dn cht acid hydroxamic [38]. N lc ti u ha
cu trc da trn -MRC dn ti ng vin lm sng KD5170 (hnh 1.38). KD5170
th hin hot tnh c ch HDAC vi IC50 = 0,045 M, mnh hn c SAHA. Ngoi ra,
KD5170 cn c ch s acetyl ha -tubulin, mt mc tiu phn t khc ca thuc iu
tr ung th. Trn 60 dng t bo th nghim chun ca Vin nghin cu ung th M
(NCI), KD5170 th hin c tnh vi EC50 = 0,1-7,7 M. Cc t bo ung th mu c
bit nhy cm hn vi KD5170 (EC50 0,5 M). Trn m hnh chut mang t bo ung
th i trng ngi (dng HCT-116), liu 42 mg/kg x ngy, KD5170 c ch s pht
trin ca khi u ti 56% so vi nhm trng [38].
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37
NH
O
S
S
O O
O
N
KD5170
O
SS
O
O
Br
BrDisulfid
SH
O
Br-MRC
Hnh 1.38. T disulfid n KD5170
Nghin cu chuyn ha in vivo cho thy KD5170 thc cht vn l tin thuc.
Khi vo trong c th KD5170 nhanh chng b thy phn thnh dn cht chuyn ha
dng -mercaptoceton. Chnh dng -mercaptoceton ny to chelat vi Zn2+ (hnh
1.39) [38].
NH
O
S
S
O O
O
N
KD5170
O
NH
O
SH
S
O O
O
N
NH
O
SH
S
O O
O
N
Zn
NH
O
SH
S
O O
O
N
Zn
hoc
Hnh 1.39. S thy phn v to chelat vi Zn2+ ca KD5170
Nh vy, nghin cu thay th nhm chc hydroxamic gn ion Zn+ bng cc
nhm khc nh thiol, sulfamid, -mercaptoceton ang c nhiu nh khoa hc
quan tm vi hy vng tm ra cht c ch chn lc tp HDAC khc nhau, vi mc tiu
chn lc trong iu tr ung th.
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38
Tm hiu v cc cht c ch HDAC c bit n v tnh hnh nghin cu
trn th gii trong nhng nm gn y v cc cht c ch HDAC, c th rt ra mt s
nhn xt sau:
- Cu trc ca cc cht c ch HDAC rt a dng, chng t cu trc trung tm
hot ng dng ti ca HDAC rt linh ng, c th bin i ph hp vi cu trc
rt khc nhau ca c cht.
- Cc peptid vng tc dng c ch HDAC mnh mc nng nanomol. Tuy
nhin cu trc ca chng rt phc tp nn kh khn cho qu trnh tng hp cng nh
khng th thu c hiu sut cao. Mt i din trong nhm ny l depsipeptid c
FDA ph duyt trong iu tr u lympho da t bo T nm 2009. Nhng tc dng ca
depsipeptid rt hn ch i vi cc loi ung th tng c.
- Cc dn cht benzamid c cu trc n gin hn nhiu so vi peptid vng.
Nhng hot tnh km cc acid hydroxamic v peptid vng. Mt s dn cht benzamid
c th lm sng pha II vi liu php n tr liu. Tuy nhin, kt qu th nghim
cho thy p ng lm sng rt hn ch.
- Cc acid bo mch ngn v ceton i in t c u im l cu trc n gin,
d tng hp. Tuy nhin, hot tnh c ch HDAC yu, p ng hn ch trn lm sng.
- Cc acid hydroxamic l nhm cht c ch HDAC c nghin cu rng ri
nht do hot tnh c ch HDAC mnh, cu trc n gin. Tuy nhin, do d to phc
vi ion Zn2+ nn cc acid hydroxamic c ch HDAC khng chn lc v thi gian bn
thi in vivo ngn. Mt cht trong nhm ny l SAHA thu c thnh cng ng k
nht trong iu tr, c FDA ph duyt trong iu tr u lympho da t bo T nm
2006. Nhng i vi ung th tng c, kt qu th nghim lm sng cho thy SAHA
rt t hoc khng c hiu qu khi s dng n tr liu. Nh vy, thay i cu trc
SAHA thu c cht c thi gian bn thi di hn, c hiu qu trn ung th tng
c l hng nghin cu rt ng ch .
Nh trnh by mc 1.3.5, cc nghin cu v cc acid hydroxamic hng c
ch HDAC hin nay tp trung thay i mt trong ba phn cu trc sau: thay i nhm
kha hot ng, thay i di v bn cht cu ni, thay i nhm chc acid
hydroxamic u vi mc ch thu c cht c hot tnh c ch HDAC tt hn ng
thi mong mun tm kim c ng vin cho th lm sng. V thay i phn cu ni,
kt qu tng hp cc nghin cu (mc 1.3.5.1) cho thy rt nhiu nghin cu c
tin hnh vi vic a thm d t nh O, gn thm vng hoc nhm th vo cu ni
tng tng tc ca cu ni vi knh enzym. Do , lun n cng thit k cc dn
cht acid hydroxamic c nhm kha hot ng l nhn phenyl c gn cc nhm th
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39
khc nhau, a thm lin kt amid vo cu ni vi hy vng tng tng tc vi knh
enzym. Thay i tip theo l nhm kha hot ng. Rt nhiu bi bo thay i nhn
phenyl ca SAHA bng cc hp phn khc c cng b, nhng cha c nhm
nghin cu no s dng khung benzothiazol. Da trn cc cng b v tc dng khng
ung th ca dn cht benzothiazol, hp phn benzothiazol c la chn thay cho
nhn phenyl ca SAHA. Mc ch l tng tng tc ca nhm kha hot ng vi
vng acid amin ming knh enzym. Nh tng hot tnh c ch HDAC v c ch
s pht trin t bo in vitro, in vivo.
Tm li, tng hp tnh hnh nghin cu trn th gii v cc cht c ch HDAC
cho thy hng nghin cu ca lun n ph hp vi xu th nghin cu ca th gii v
nh hng thit k cc dn cht acid hydroxamic u l cht mi, cha thy cng b
trong ti liu no.
1.4. CC PHNG PHP TO LIN KT AMID V TNG HP ACID
HYDROXAMIC
Cc cht tng hp trong lun n u c quy trnh tng hp tri qua 2 bc phn
ng tr ln. Phn ng chnh tng hp cc cht ny u l phn ng to lin kt
amid. Bc cui cng l phn ng to acid hydroxamic. Qua tham kho ti liu, c
nhiu phng php to lin kt amid cng nh tng hp acid hydroxamic. la
chn c phng php ph hp, sau y l tng kt mt s phng php to lin kt
amid v tng hp acid hydroxamic.
1.4.1. Cc phng php to lin kt amid
Lin kt amid c to thnh khi cho mt acid phn ng vi mt amin. Phn
ng trc tin l to dng mui bn, sau lin kt amid c to thnh khi thng
c nhit ng hc phn ng th hin trong s 1.1. Phn ng trc tip ny thng
din ra nhit cao 160 180oC [10,64]. Nhit ny thng khng thch hp cho
phn ng tng hp cc cht c mt mt s nhm chc khc v tng hp peptid. ng
thi, phn ng trc tip ny cn gp phi mt s nhc im nh hiu sut thp, s
racemic ha, kh tinh ch
S 1.1. Phn ng to lin kt amid trc tip
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40
V vy, hot ha nhm chc acid bng cch gn nhm d tch loi vo carbon
acyl ca acid trc khi nhm amin tn cng to lin kt amid l cn thit (s
1.2) [64].
S 1.2. Phn ng to lin kt amid thng qua acid hot ha
C nhiu cch hot ha acid carboxylic nh to acyl halid, acyl azid,
acylimidazol, anhydrid, ester bng cch s dng cc tc nhn hot ha khc nhau.
Cho n nay, nhiu loi tc nhn hot ha c bit n v c s dng thng
thng vi mc ch nng cao hiu sut, gim to thnh cc sn phm ph, tng tnh
chn lc cng nh d tinh ch sn phm. Sau y l mt s phng php to lin kt
amid thng qua acid hot ha.
1.4.1.1. Acyl halid
Cc acyl halid l acyl clorid, acyl fluorid v acyl bromid, trong hay s dng
nht l acyl clorid (cn c gi l acid clorid). y l mt trong nhng phng php
u tin hot ha acid v c rt nhiu acyl clorid khc nhau c bn trn th
thng. Cc tc nhn hay c s dng to acyl clorid l thionyl clorid (SOCl2),
oxalyl clorid (COCl)2, PCl3, POCl3, PCl5. Sau , lin kt amid c to thnh bng
cch cho acyl clorid phn ng vi amin (s 1.3). Trong phn ng ny, s c mt
ca mt base l cn thit phn ng vi HCl to thnh, trnh chuyn nhm amin
thnh mui HCl khng hot ng [42,64].
S 1.3. a) Phn ng to acyl clorid; b) Tng hp amid
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Cc base hay dng l amin bc 3 nh triethylamin (NEt3), iPr2Net, N-methyl
morpholin hay pyridin (s 1.4).
S 1.4. Vai tr xc tc ca pyridin
1.4.1.2. Acyl azid
Phng php tng hp amid theo con ng to acyl azid l phng php u
tin to lin kt peptid bi Curtius t nm 1902 [64]. Acyl azid c th c to
thnh t dn cht methyl ester, qua 2 bc phn ng. Nhm methoxy c thay th
bi hydrazin to acyl hydrazid, sau tc dng vi acid nitr thu c acyl azid
(s 1.5).
S 1.5. Tng hp amid thng qua to acyl azid
1.4.1.3. Acylimidazol
Cc tc nhn hot ha l imidazolium t ra kh hiu qu trong vic to lin kt
amid. Carbonyl diimidazol (CDI) l cht hay c s dng nht trong nhm ny. C
ch phn ng th hin trong s 1.6. Imidazol c to thnh ngay trong phn ng
nn thng khng cn thm mt base khc. Bn cnh , vi cc bc hot ha tng
t CDI, N,N-carbonylbis(3-methylimidazolium) triflat (CBMIT) cng rt hiu qu
trong vai tr to tc nhn hot ha tng hp peptid [42,64].
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S 1.6. Tng hp amid s dng tc nhn hot ha CDI
1.4.1.4. Anhydrid
Mt trong nhng phng php hay c dng hot ha acid carboxylic l
to anhydrid. Tc nhn acyl to thnh ny hot ng tng i mnh. Cc anhydrid
ny a s khng sn c, c iu ch ngay khi s dng. C rt nhiu tc nhn hot
ha to anhydrid nh dicyclohexyl carbodiimid (DCC), diisopropyl carbodiimid
(DIC), 1-ethyl-3-(3-dimethylamino)carbodiimid.HCl (EDC), ethyl cloroformat,
isobutyl cloroformat [36,42,64] Trong , hay c s dng nht l cc carbodimid
(s 1.7). Tuy nhin, nhc im ca phn ng s dng tc nhn hot ha
carbodiimid l ch mt na lng acid tham gia phn ng to lin kt amid. iu ny
s gy lng ph v tn km nu acid gi thnh cao.
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43
S 1.7. Tng hp amid s dng tc nhn hot ha DCC
S dng tc nhn hot ha ethyl cloroformat, isobutyl cloroformat to
anhydrid hn tp s khc phc c nhc im ca cc carbodiimid nu trn (s
1.8).
S 1.8. Tng hp amid s dng tc nhn hot ha ethyl cloroformat
1.4.1.5. Ester
Mt phng php hot ha acid carboxylic to tc nhn acyl ha hot ng
trung bnh l to ester. y cng l phng php to acid hot ha kh ph bin. Vic
la chn alcol to ester ty thuc vo mc ch ca ngi s dng. V d, trong
tng hp peptid, alcol hay c s dng nht l benzotriazol hydroxy (HOBt), p-
nitrophenol (PNP), pentafluorophenol (PFP) (hnh 1.40) [36,42,64]. Bn cnh , cn
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c mt s alcol khc c s dng vi cc u im ring nh N-hydroxysuccinimid
(HOSu) tan trong nc nn d dng loi b giai on tinh ch, hydroxy-7-
azabenzotriazol (HOAt) hiu qu hn HOBt trong mt s trng hp base amin yu.
Hnh 1.40. a) Tng hp amid thng qua to ester hot ha; b) Mt s alcol hay dng
Gn y, mt phng php thng dng trong tng hp peptid l to ester hot
ha trung gian ngay trong phn ng. Tc nhn hot ha l cc mui uronium,
phosphonium v immonium ca HOBt, HOAtTc nhn u tin c cng b v
a vo s dng l benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorophosphat (BOP) (s 1.9) [64]. Sau , mt lot cc mui uronium v
immonium cng c bit n vi vai tr tc nhn hot ha nh O-(1H-benzotriazol-
1-yl)-N,N,NN-tetramethyluronium hexafluorophosphat (HBTU), HATU, N-(1H-
benzotriazol-1-ylmethylen)-N-methylmethanaminium hexacloro antimonat (BOMI),
BDMP [36,64].
S 1.9. Tng hp amid s dng tc nhn BOP [64]
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1.4.2. Cc phng php tng hp acid hydroxamic
Acid hydroxamic l sn phm ca phn ng gia dn cht ca acid carboxylic
v hydroxylamin. Dn cht ca acid carboxylic c th l ester, acid clorid, anhydrid
Cho n nay, nhiu phng php tng hp acid hydroxamic s dng cc xc tc khc
nhau c cng b [7,68,69]. Sau y l tng kt mt s phng php hay dng
tng hp cc acid hydroxamic hng c ch HDAC.
1.4.2.1. 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 [37], Andrianov v cng s [6], Marson v
cng s [58], Belvedere v cng s [8] 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 1.10).
S 1.10. Tng hp mt s dn cht amid ngc ca SAHA [6]
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 [17] 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%).
1.4.2.2. 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 [67] 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%.
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S 1.11. Tng hp acid biaryl hydroxamic [67]
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 [51] 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 1.12).
S 1.12. Tng hp cc acid phenylthiazol hydroxamic [51]
Nh vy, c hai phng php ch yu tng hp acid hydroxamic vi cc tc
nhn hot ha khc nhau. Ty vo nguyn liu ban u v sn phm, cc nh ha hc
c th la chn phng php v cc tc nhn hot ha ph hp.
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Chng 2
NGUYN LIU, THIT B, NI DUNG
V PHNG PHP NGHIN CU
2.1. NGUYN LIU
Cc ha cht, dung mi dng trong thc nghim c trnh by trong bng di
y:
TT Nguyn liu Xut x TT Nguyn liu Xut x
1 2-Aminobenzothiazol Aldrich 21 Acid adipic monomethyl ester Aldrich
2 2-Amino-6-methylbenzothiazol Aldrich 22 Acid suberic monomethyl ester Aldrich
3 2-Amino-6-methoxybenzothiazol Aldrich 23 Anhydrid succinic Trung quc
4 2-Amino-6-ethoxybenzothiazol Aldrich 24 Anhydrid glutaric Merck
5 2-Amino-6-methylsulfonylbenzothiazol
Aldrich 25 N,N-dicyclohexylcarbodiimid Merck
6 2-Amino-6-nitrobenzothiazol Aldrich 26 Hydroxylamin HCl Merck
7 2-Amino-6-clorobenzothiazol Aldrich 27 Dimethylformamid Merck
8 2-Amino-6-trifluoromethylbenzothiazol
Aldrich 28 Triethylamin Merck
9 2-Aminothiazol Merck 29 Isobutylcloroformat Merck
10 Anilin Merck 30 Tetrahydrofuran Merck
11 2-Cloroanilin Merck 31 Aceton Trung quc
12 3-Cloroanilin Merck 32 Ethanol Trung quc
13 4-Cloroanilin Merck 33 n-Hexan Trung quc
14 4-Fluoroanilin Merck 34 Methanol Trung quc
15 4-Methoxyanilin Merck 35 Acid hydrocloric c Trung quc
16 4-Nitroanilin Merck 36 Natri hydroxid Trung quc
17 4-Cyanoanilin Merck 37 Pyridin Merck
18 -Alanin methyl ester.HCl Aldrich 38 O-tetrahydropyranyl hydroxylamin Aldrich
19 Glycin methyl ester.HCl Aldrich 39 Acid para-toluensulfonic Aldrich
20 Carbonyldiimidazol Merck 40 Liti hydroxyd Trung quc
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2.2. THIT B
- Cc dng c thy tinh: bnh cu y trn dung tch 50 - 100 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 366 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 khuy t gia nhit IKA-RTC, my o nhit nng
chy nhit in Electrothermal digital.
- My o ph hng ngoi:
- Perkin Elmer, phng th nghim trung tm - Trng i hc Dc H N