Tổng hợp và thử hoạt tính sinh học một số dẫn chất n hydroxypropenamid mang khung 3 methoxyimino 2 oxoindolin

B Y T
I HI



TRN TH THANH

TNG H HOC
MT S DN CHT
N-HYDROXYPROPENAMID MANG KHUNG
3-METHOXYIMINO-2-OXOINDOLIN


N TT NGHI






I - 2015

B Y T
I HI



TRN TH THANH


TNG H HOC
MT S DN CHT
N-HYDROXYPROPENAMID MANG KHUNG

3-METHOXYIMINO-2-OXOINDOLIN

N TT NGHI

ng dn:
1. GS.TS. Nguyn Hi Nam
2. 
c hin:
1. B c



I - 2015


Nh gi li ci
thn, nh em rt nhi
n.
c h c ti:
- GS.TS. Nguyn Hi Nam: i thy  n cho em cm h
u khoa hc tip ng dn, t, h tr

- TS.   : i th     em trong 

- DS Th Mai Dung ch bo, h tr, ng dn tn
 em thc hit.
-   a b 
di h- i hc Khoa hc t 
Ni, Vin Khoa h    Vi   c - i hc Quc gia
Chungbuk - uc  u ki em thc hi
lun ti B 
Cui li cc t
ghibc bic nghim B 
n lp M2K vui bu
trong sut thi gian qua.

i, 13  




Trn Th Thanh

MC LC
Trang
DANH M VIT TT
DANH MNG
DANH M
DANH M
T V
C1. TNG QUAN
1.1. HISTON DEACETYLASE (HDAC)
histon deacetylase (HDAC)
1.1.2.
T C CH HDAC
1.2.1t c ch HDAC
1.2.1
1.2.1
1.2.1
1.2.2t c ch  nghi


1.2.3.2. Li
1.3. MT S U TNG HC CH
 GI
n din b mt PXD101

t c ch cu b mdc, tng i
hi thit k u




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C U, THIT B, NI  

U
T LIU, THIT B
t
2.1.2. Thit b, dng c
2.2. NU
2.2.1. Tng hc
2.2.2. Th ng sinh hc ct tng hc
U
2.3.1. Tng h hc
2.3.2. Th ng sinh hc
 ging thuc ct tng hc
3. THC NGHIM, KT QU N
C
3.1.1. Tng hc
3.1.2. Ki tinh khit
nh c
3.2. TH HOHC
3.2.1. Th hoc
 ging thuc
N
3.3.1. Tng hc
3.3.2. Ho sinh hc
KT LUN NGH
U THAM KHO
PH LC




16

16
16
16
16
16
16
16
17
18
20
21
21
21
29
30
35
35
35
36
36
36
41



DANH M VIT TT
AML
: Bnh bch cu cy
AML-ETO


APL
ch cu
APAF1
: Yu t ho
CBHA
: Carboxy cinnamic acid bis-hydroxamic
CD
t t i ti
DCM
: Dicloromethan
DMF
: Dimethylformamid
MeOH
: Methanol
MHC
: Phc h 
ROS
: Gc oxy t do hong

: Receptor ca acid retinoic
SPB
: SulforhodamineB
SAHA
: Acid suberoylanilid hydroxamic
T
O
nc
: Nhi y
VEGF
: Yu t ch

WAF1
: Cht c ch kinase ph thuc cyclin









DANH MNG
STT
ng
Trang
1
Bng 1.1: t c ch  nghing
7
2
Bng 1.2: Kt qu th ng c ch 
  
11
3
Bng 1.3: mt s
IIa, IIb, IIc, IId
12
4
Bng 1.4:         c  
hydroxamic mang khung 3-methoxyimino-2-oxoindolin
14

5

t ester
29
6

f

o
nc
4a-d
30
7
Bng 3.3: 4a-d
31
8
Bng 3.4: t 4a-d
32
9
Bng 3.5:  ph
1
H-NMR ct 4a-d
33
10
Bng 3.6: 
13
C-NMR ct 4a-d
34
11

Bng 3.7:  ging thuc ct 4a-d theo quy
tc Lipinsky
35

12
Bng 3.8: t qu c ch4a-d
37
13

38
14
  4a,
4b, 4d IIIa, IIIb, IIId
40







STT

Trang
1
nucleosom
2
2

HAT

3
3
: 
8
4
H: S t ca cn thit acid hydroxamic
c ch  nghisng
9
5
: Cn cht acid indol - hydroxamic 
11
6
   c t   n cht N-hydroxy-3-(4-(5-
aryl/arylalkyl-1,3,4-oxadiazol-2-yl)phenyl)acrylamidN-hydroxy-3-
(4-(5-aryl/arylalkyl-1,3,4-oxadiazol-2-yl)methyl)benzamid
12
7

 Kt qu th ng c ch HDAC cn cht 4a-d

37


STT

Trang
1
.1:

15
2

21
3
2a
21
4
3a
22
5
4a
23
6
4b
25
7
4c
26
8
4d
28

1








        Acid suberoylanilid hydroxamic (Zolinza

,
2006), depsipeptid (Romidepsin

, 2009), belinostat (Beleodaq


, PXD
-

 [10].

        
, 
. Trong luthca c
   Th  t k  ng h     khung 3-
(methoxyimino)-2-oxoindolin cho kt qu kh  
mt, belinostat (PXD101)  ng minh hiu qu  n,
      n din b mt ca PXD101 vi khung 3-
methoxyimino-2-oxoindolin ng hth ho sinh
hc mt s dn cht N-hydroxypropenamid mang khung 3-methoxyimino-2-
 
1.  p N-hydroxy-3-[4-[{3-(methoxyimino)-2-oxoindolin-1-yl}methyl]
phenyl]
2. Th ng c ch HDAC  
cht tng hc.


2


C1. TNG QUAN
1.1. HISTON DEACETYLASE (HDAC)
  b gen c     him sc th (NST), 
-ADN.  cu t n cnucleosom.
Mt nucleosom m ma 4 cp histon (2 cp
ca H2A vp ca H3 vc qun quanh bi 146 cp nucleotid

[19,24,33] nh 1.1).

: Cca nucleosom
u amin ca histon mang nhinh vi
 ADN t 
c     a nhim sc th     u hin gen. Khi
n nhim sc th 
c ch ng hc ch s biu hic
l  u, nhim sc th  ng hp
protein diu hing. M n
 a histon ph thu     u amin  pha
n hot ng ca 2 en
acetyltransferase (HAT).
1.1.1.iston deacetylase (HDAC)
Histon deacetylase           
acetyl t -N-acetyl lysin amino acid ca histon. i lp vi histon
acetyltransferase (HAT)- enzym n chuy acetyl coenzym

histon
(147bp ADN)
Cu ni ADN

3


  -amino ca lysin  u N ca histon      
acetyl c h
 [33] (h).

:    HAT

1.1.2. Hi
    -NH
2
trong 

               




 ,26].


n.

 cho 

.

4


1.2.
1.2.1HDAC (HDACi)


 
 - [14].

 

 .
 


1.2.1




1,20,23].

in vitroin vivoa
-RAR
              
PLZF-RAR

--


 [22].
5



            -  

 -  - 


-




 (G2 checkpoint)





1.2.1.2. 


       c
enzym cystein [1,22,15].
             -
     -    
(TNF-related apotosis-inducing ligand). 
--
--
               
cytochrome C     
second mitochondria-derived activator     
6


 -



BCL- 


) [1,20].


CD95 (CD95L). 
butyrat


-


sin - 





                
SAHA.
1.2.1

      
               



7




in vitroin vivo.


c th nghi
t c ch c th nghi s dng trong
u tr  u t]:
- 
- 
- ch ng
- nostat, chidamide II
Mng hn ch nh
chuyc ch n l
u l m
nh  ng c ch   
acid c ch mc ch
m14 cht cc ch n nay
c th nghi  u tr  th 
bng 1.1).

Bng 1.1t c ch  nghi

Hp cht
Pha
Lo


AN-9
(tin thuc)
I, II

Tc, NSCLC

Acid valproic

I, II

Tc, 

Phenyl butyrate
II
Tc, AML/MDS
Acid
hydroxamic
Vorinostat
(SAHA)
I, II

T       

CTCL, u lympho Hodgkin
8


Practinostat
(SB-939)
I
T
Belinostat
(PXD101)
II

       


Panobinostat
(LBH-589)
III
T       
MDS, ALL
Givinostat
(ITF-2357)
II
      

Quisinostat
(JNJ-16241199)
I

Romidepsin
(4SC-201)
I, II
U lympho Hodgkin

benzamid
Chidamide
(CS055)
II
Tc, u lympho
Mocetinostat
(MGCD01030)
II

Tng 
Entinostat
(MS275)
II
T

Romidepsin
I, II
T     
Hodgkin
 ch cu tp; MDS: hi chng lon sn ty; CTCL: u
lympho da t ch cu cp.

1.2.3.


 1.3: Cn c cht c ch HDAC
C [23] .3):
- n din b mt (surface
recognition group) (A)ng nm  mt
enzym.
9


-  u n  c (B)      u mch
th  no ho tt Van der Waals vi

- n vi km (Zinc binding group - ZBG) (C)i
ion Zn
2+

t ng c
-aminoanilin ca benzamid, mercaptoceton
C kt tinh cu n
mt phn cng ny khong trng
i ci phn
 mt min din b m t vi phn
cu nt s  n
ci thing ht c ch HDAC. Viu thit k cu
t mu d  [17].

1.4: S t ca cn thit acid hydroxamic c ch
 nghi
: An din b mt; Bu ni; Ct vi Zn
2+

10


ng cc ch HDAC
Cm 3 ph
d22]:
- ng (A)c hiu lc c ch
u v t tng hc cho
thn s ng t.
- Cu nc (B)n kh c ch enzym, quynh s
p v ct vi chi
c   -   ch h ho     
c nh. Trong cu nt amid      
ch ch carbon t- 6C.
- t vi Zn

2+
(C):  thi 
ng c ch HDAC.
         


 ng tng h dn cht acid indol - hydroxamic:
u Nht Bt k thay th n din b
mt ca PXD 101, tng hp  dn cht acid indol - hydroxamic 
b 20.


11



. Cn cht acid indol - 
Kt qu th ng c ch H

Cht
1
IC
50

HDAC
2
KB
a
H460
b

PC3
c
HCT116
d
SAHA
0,48  0,02

0,82 0,11
1,74 0,31

Ia
0,51 0,03

3,01 0,25
4,01 0,22

Ib
0,23  0,11
0,28
1,21 0,24
3,21 0,54

Ic
0,50  0,09

2,79 0,54
3,36 0,58

Id
0,48  0,04


0,56 0,11
1,11 0,06

Ie
0,56  0,04

1,67 0,34
1,97 0,29

If
0,10  0,05
0,23
0,57 0,01
1,63 0,41

Ig
0,24  0,11

0,91 0,06
1,24 0,18

Ih
0,60  0,09

1,82 0,01
4,11 0,43

Ik
0,85  0,11


1,98 0,16


:
1
N c ch 50% s n ca t t qu c ly
n th nghim v sai l
2
HDAC c 
c t cung,
a

b
i,
c
n tin lit,
d
i tr.



n tin lit, ung .
c bit cht (E)-3-(3-((1H-pyrrolo[2,3-b]pyridin-1-yl)sulfonyl)phenyl)-N-hydroxy
acrylamid (If) vng 7-t qu ch
t n tin li i
 rt tt.  ra rng cht If ng c ch HDAC
vi IC
50
      ng chn l   p 5 ln cht

ACY-c th nghing. Cht If c th 
chu   t qu kh quan vi sinh kh d ng ung 33% trong khi
SAHA 8,33t 7-azaindolylsulfonylcinnamic hydroxamat
(If) y ti n thuc chi kh c ch chn l
12



PXD
 ng tng h-oxadiazol:

m N-hydroxy-3-(4-(5-aryl/arylalkyl-1,3,4-
oxadiazol-2-yl)phenyl)acrylamid  N-hydroxy-3-(4-(5-aryl/arylalkyl-1,3,4-
oxadiazol-2-yl)methyl)benzamid d PXD101, vi cu n
-oxadiazo  [30]c
t6.

 6:  c t   dn cht N-hydroxy-3-(4-(5-aryl/arylalkyl-
1,3,4-oxadiazol-2-yl)phenyl)acrylamid  -hydroxy-3-(4-(5-aryl/arylalkyl-1,3,4-
oxadiazol-2-yl)methyl)benzamid.

mt s dn cht IIa, IIb, IIc, IId

R
n
1
IC
50
+ 
HDAC1

HDAC4
HDAC6
IIa

0
0,8 +0,02
>20

IIb
1

>20

IIc

0

>20

IId
1



SAHA






:
1
N c ch 50% s trin ca t t qu c ly
n th nghim v sai l


C6c bit cht IIb, IId
vi vithgn vi cu nng tn
cht IIa, IIc. 

      N-hydroxy-4-((5-(naphtalen-1-ylmethyl)-1,3,4-
13


oxadiazol-2-yl)methyl)bezamid (IIb)     
 
methyl gu ni phenylmethyl ca PXD101.
1.3.2. t c ch u b ng
i hi thit k u.

g 3-methoxyimino-2-oxoindolin 
th Tho [4]. Kt qu c
ch   -n tin lit
y AsPC-i NCI-H460 c (bng 1.4) ht sc kh
quan, c 7 chu c ch t SW620 u rt tt vi khong
IC
50
thp ch t 0,26-1,35 M.

Bng 1.4 c

khung 3-methoxyimino-2-oxoindolin

TT
R
 
50
)
1

SW620
a
MCF-7
b
PC3
c
AsPC-1
d
NCI-H460
e
1
-H
0,73
1,71
1,67
1,22
0,75
2
5-F
1,11
2,42

1,15
0,97
0,97
3
5-Cl
0,49
1,56
2,33
0,49
0,76
4
5-Br
2,32
7,79
8,91
1,68
2,90
5
5-NO
2
1,35
0,94
1,69
0,84
14,27
6
5-CH
3
1,07
16,67

0,35
5,48
1,19
7
7-Cl
0,26
0,34
0,29
0,63
0,35
:
1
N c ch 50% s n ca t t qu c ly
n th nghim v sai l,
a

b

c
n tin lit,
d
y,
e
ui.

H chn n din b mt 3-methoxyimino-2-
oxoindolin nh t  phn mi
        c ch chn l   u cho thy
14

n din b mt  ng kh 
acid amin  ming thi ng tt
 ng t
 [4].
ng thit k
so vi PXD 101 ng tng hp dn cht hydroxamic 
khung 3-methoxyimino-2-oxoindolin n din b mt u c cht
c ch HDAC ttng thi c  
y, trong kn tt nghi
da tr cPXD101  ng 
khung 3-methoxyimino-2-oxoindolin vi m
 v mi
 . Nh c ch
c ch s n ca t 

1.4.1. T
Ta hay s dng KCN hophn ng acid hydroxamic t
n  [6, 7, 9, 16]:

Vu ki
kha ch ti
phn    :
15



: 
.

1.4.2. Tng hp amid vi 
 
 u hiu trong
vic tc s
dng nhng s  n
ng to amid cho hiu sut cao [1,7n ng t
RCOOH + R
1
NH
2
RCONHR
1












16


T LIU, THIT B, NI DUNG
U
T LIU, THIT B

t
 c nghi
tng hc nhp t c Sigma-c
s dng trc ti m:
 
- Isatin
- 5-Methylisatin
- 5-Methoxyisatin
- 5-Bromoisatin
 Methyl 3-[4-(bromomethyl)phenyl]prop-2-enoat
 Hydroxylamin hydroclorid
 Methoxyamin hydroclorid
       N,N-dimethylformamid (DMF), aceton,
c ct, FeCl
3
.
2.1.2. Thit b, dng c
- Dng c th
u thy sp mng (TLC).
- y t gia nhit, m-200.
-   thut Shimazu.
- T lnh, t s u nhit.
- Bn mng silicagel Merck 70   chy sp mng.
-  y nhin (Melting point Apparatus Smp3)  
nh nhi y.
-  nh ph IR.
- Mi ph Agilent 6310 Ion Trap  ghi ph MS
- Mng t Bruker AV- ghi ph
1
H-

13
C-NMR.
2.2. NU
2.2.1. Tng hc
* Tng hp 4 cht: