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B Y T
TRNG I HC DC H NI
TH MAI DUNG
TNG HP V TH HOT TNH
SINH HC CA 1 S ACID
HYDROXAMIC MANG KHUNG
1,3,4-THIADIAZOL
KHA LUN TT NGHIP DC S
H NI - 2013
B Y T
TRNG I HC DC H NI
TH MAI DUNG
TNG HP V TH HOT TNH
SINH HC CA 1 S ACID
HYDROXAMIC MANG KHUNG
1,3,4-THIADIAZOL
KHA LUN TT NGHIP DC S
Ngi hng dn :
1.PGS.TS. Nguyn Hi Nam
2.DS. Nguyn Xun Phong
Ni thc hin :
1. B mn Ha Dc
H NI - 2013
LI CM N
Sau mt thi gian thc hin ti vi nhiu n lc v c gng, thi im
hon thnh kha lun l lc ti xin php c by t lng bit n chn thnh vi
nhng ngi dy d, hng dn v gip ti trong sut thi gian qua.
Trc ht vi lng knh trng v bit n su sc nht ti xin by t li cm
n chn thnh n PGS.TS. Nguyn Hi Nam, DS. Nguyn Xun Phong - B mn
Ha Dc - trng i hc Dc H Ni, nhng ngi thy trc tip hng dn
v tn tnh ch bo ti trong thi gian thc hin kha lun ny.
Ti cng xin gi li cm n n cc thy gio, c gio v cc anh ch k
thut vin ca B mn Ha Dc - trng i hc Dc H Ni, Khoa Ha - i
hc Khoa hc t nhin H Ni, Vin Khoa hc v Cng ngh Vit Nam, Khoa
Dc - i hc Quc gia Chungbuk - Hn Quc lun gip to iu kin
thun li cho ti thc hin kha lun tt nghip.
Cui cng ti xin c gi li cm n su sc n b m, gia nh v bn b
lun ng vin khch l ti trong sut qu trnh hc tp v nghin cu.
H Ni, ngy 21 thng 5 nm 2013
Sinh vin
Th Mai Dung
MC LC
Trang
DANH MC CC K HIU, CC CH VIT TT
DANH MC CC BNG
DANH MC CC HNH V
DANH MC CC S
T VN ...................................................................................................... 1
Chng 1. TNG QUAN .................................................................................... 2
1.1. HISTON DEACETYLASE (HDAC) .............................................................. 2
1.1.1. Khi nim v histon deacetylase .................................................................. 2
1.1.2. Phn loi cc HDAC .................................................................................... 3
1.1.3. Mi lin quan gia ung th v hot ng bt thng ca HDAC ................. 4
1.2. CC CHT C CH HDAC......................................................................... 6
1.2.1. Phn loi cc cht c ch HDAC ................................................................. 6
1.2.2. Cu trc ca cc cht c ch HDAC ............................................................ 8
1.3. MT S HNG NGHIN CU TNG HP CC ACID HYDROXAMIC
C CH HDAC TRN TH GII ....................................................................... 9
1.3.1. Lin quan cu trc tc dng ca cc acid hydroxamic c ch HDAC ........... 9
1.3.2. Mt s hng thit k nghin cu v tng hp trn th gii ......................... 10
1.4. PHNG PHP TO LIN KT AMID V TNG HP ACID
HYDROXAMIC ................................................................................................... 13
1.4.1. Tng hp amid vi tc nhn acyl ha l acid carboxylic .............................. 13
1.4.2. Tng hp acid hydroxamic t ester .............................................................. 14
Chng 2. NGUYN LIU, THIT B, NI DUNG V PHNG PHP
NGHIN CU ..................................................................................................... 15
2.1. NGUYN VT LIU, THIT B .................................................................. 15
2.1.1. Ha cht ...................................................................................................... 15
2.1.2. Thit b, dng c .......................................................................................... 15
2.2. NI DUNG NGHIN CU ........................................................................... 16
2.2.1. Tng hp ha hc ........................................................................................ 16
2.2.2. Th tc dng sinh hc ca cc cht tng hp c ...................................... 16
2.3. PHNG PHP NGHIN CU ................................................................... 16
2.3.1. Tng hp ha hc ........................................................................................ 16
2.3.2. Th tc dng sinh hc .................................................................................. 17
2.3.3. nh gi mc ging thuc ca cc cht tng hp c ........................... 19
Chng 3. THC NGHIM, KT QU V BN LUN ............................... 20
3.1. HA HC ...................................................................................................... 20
3.1.1. Nghin cu Docking....................................................................................... 20
3.1.2. Tng hp ha hc ........................................................................................ 21
3.1.3. Kim tra tinh khit .................................................................................. 34
3.1.4. Xc nh cu trc ......................................................................................... 34
3.2. TH HOT TNH SINH HC ...................................................................... 40
3.2.1. Th hot tnh sinh hc ................................................................................. 40
3.2.2. nh gi mc ging thuc ...................................................................... 40
3.3. BN LUN ................................................................................................... 41
3.3.1. Tng hp ha hc ........................................................................................ 41
3.3.2. Tc dng sinh hc ........................................................................................ 41
KT LUN V KIN NGH ............................................................................. 44
TI LIU THAM KHO
PH LC
DANH MC CC K HIU, CC CH VIT TT ALL : Bnh ung th nguyn bo lympho cp tnh
AML : Bnh ung th bch cu dng ty cp tnh
APL
CDI
CLL
CTCL
:
:
:
:
Bnh ung th bch cu ty bo cp tnh
Carbonyldiimidazol
Bnh lympho mn tnh (Chronic lymphocytic leukemia)
T bo lymphoT di da (Cutaneous T cell lymphoma) 13C-NMR : Cng hng t ht nhn 13C
DCM : Dicloromethan
DMF : Dimethyl formamid
DMSO : Dimethyl sulfoxid
EtOH : Ethanol
HAT : Histon acetyltranferase
HDAC : Histon deacetylase 1H-NMR : Cng hng t ht nhn 1H
IC50 : Nng c ch hot t bo xung mt na
IR
MTT
:
:
Phng php ph t ngoi
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid
MeOH : Methanol
MS
NSCLC
:
:
Ph khi lng
Ung th phi t bo khng nh (Non-smali lung cell)
NST : Nhim sc th
SAHA : Acid suberoylanilid hydroxamic
T0nc : Nhit nng chy
TLC : Phng php sc k lp mng
TMS : Tetramethylsilan
TSA : Trichostatin A
DANH MC CC BNG
STT Tn bng Trang
1 Bng 1.1: Cc cht c ch HDAC ang th nghim trn lm
sng 8
2 Bng 1.2: Tc dng c ch HDAC ca cc acid isoxazol-
hydroxamic 13
3 Bng 3.1: Kt qu docking ca cc cht 5a-f vi HDAC8 20
4 Bng 3.2: Ch s l ha v hiu sut tng hp cc acid
hydroxamic t ester 33
5 Bng 3.3: Gi tr Rf v nhit nng chy ca cc cht 5a-f 34
6 Bng 3.4: Kt qu phn tch ph MS ca cc cht 4a-f 35
7 Bng 3.5: Kt qu phn tch ph IR ca cc cht 5a-f 36
8 Bng 3.6: Kt qu phn tch ph MS ca cc cht 5a-f 37
9 Bng 3.7: Kt qu phn tch ph 1H-NMR ca cc cht 5a-f 37
10 Bng 3.8: Kt qu phn tch ph 13C-NMR ca cc cht 5a-f 39
11 Bng 3.8: nh gi mc ging thuc ca cc cht 5a-f theo
quy tc Lipinsky 40
12 Bng 3.10: Kt qu th tc dng c ch HDAC v hot tnh
khng t bo ung th 42
DANH MC CC HNH V
STT Tn hnh Trang
1 Hnh 1.1: Cu trc ca histon trong nucleosom 2
2 Hnh 1.2: M t c im ca cc loi HDAC 4
3 Hnh 1.3: Vai tr sinh hc ca cc HDAC trong sinh l t bo
ung th 6
4 Hnh 1.4: Mt s cht c ch HDAC ang th nghim trn lm
sng 7
5 Hnh 1.5: Cu trc ca SAHA v trung tm hot ng ca
HDAC 9
6 Hnh 1.6: Cu trc ca TSA, oxamflatin v cc dn cht N-
hydroxy-2-propenamid 10
7 Hnh 1.7: Cc dn cht amid ngc ca SAHA 11
8 Hnh 1.8: Cc dn cht -alkoxy ca SAHA 11
9 Hnh 1.9: Cc acid phenylthiazol-hydroxamic tng t SAHA 11
10 Hnh 1.10: Cc dn cht aicd biphenyl-hydroxamic 12
11 Hnh 1.11: Cng thc cu to ca WR301849 12
12 Hnh 3.1: M hnh tng tc ca 6 dn cht 5a-f vi HDAC8 21
DANH MC CC S
STT Tn s Trang
1 S 1.1: C ch xc tc ca CDI 14
2 S 3.1: Quy trnh tng hp chung 21
3 S 3.2: Quy trnh tng hp cht 2a 22
4 S 3.3: Quy trnh tng hp cht 3a 23
5 S 3.4: Quy trnh tng hp cht 4a 24
6 S 3.5: Quy trnh tng hp cht 5a 25
7 S 3.6: Quy trnh tng hp cht 5b 26
8 S 3.7: Quy trnh tng hp cht 3b 27
1
T VN
Acid suberoylanilid hydroxamic (Zolinza, 2006) l cht c ch histon
deacetylase u tin c cc qun l thc phm v dc phm M (US-FDA) ph
duyt trong iu tr u lympho da t bo T. Sau , nm 2009 depsipeptid
(Romidepsin) mt cht c ch HDAC khc cng c cp php trong iu tr ung
th ti M. Nh vy c th thy, HDAC l mt mc tiu phn t quan trng trong
iu tr ung th v cc cht c ch HDAC l cc tc nhn chng ung th y trin
vng.
Nhm nghin cu ti b mn Ha Dc - i hc Dc H Ni cng thit
k, tng hp v cng b nhiu dy cht vi nh hng c ch HDAC c hot tnh
khng t bo ung th tt [40,41]. Cc nghin cu gn y ti b mn v cc acid
hydroxamic c tc dng c ch HDAC cho thy c th thay th nhm nhn din b
mt ca SAHA l nhn phenyl bng vng thm khc nh benzothiazol cho hot tnh
in vitro rt kh quan [3]. Tip theo , trong lun vn thc s ca Th nh Tuyt
[4] khung 5-phenyl-1,3,4-thiadiazol c s dng lm nhm nhn din b mt
thay cho benzothiazol. Tuy nhin nghin cu trn mi ch tng hp c 5 dn
cht, cha nh gi kh nng tng tc ca nhm nhn din b mt mi vi
HDAC, cng nh cha tm c cht c hot tnh khng t bo ung th in vitro tt
tin hnh cc th nghim su hn. Trn c s thay i cc nhm th vng
thm s lm thay i tng tc ca cht vi mc tiu phn t, chng ti thit k cc
nhm th mi trn khung 5-phenyl-1,3,4-thiadiazol v tin hnh ti Tng hp
v th hot tnh sinh hc ca mt s acid hydroxamic mang khung 1,3,4-
thiadiazol vi hai mc tiu:
1. Tng hp N1-hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)octandiamid v 5
dn cht.
2. Th c tnh t bo v tc dng c ch HDAC ca cc cht tng hp c.
2
Chng 1. TNG QUAN
1.1. HISTON DEACETYLASE (HDAC)
Tt c b gen ca ngi c gi trong nhim sc th (NST), mt phc hp
i phn t protein-ADN. Nhim sc th c cu trc ng, c t chc cao, bao
gm: ADN, cc protein histon v protein khng phi histon. n v cu trc c bn
ca NST l nucleosom. Mt nucleosom in hnh bao gm mt octamer hnh a
ca 4 cp histon (2 cp ca H2A vi H2B v 2 cp ca H3 vi H4) c qun
quanh bi 146 cp nucleotid [1,48] (hnh 1.1):
Hnh 1.1: Cu trc ca histon trong nucleosom
Khi u amin ca histon tch in dng s tng tc vi phn phosphat mang
in m trn phn t ADN lm ng xon NST gy c ch dch m v tng hp
protein, lm c ch s bin hin gen. Ngc li khi tng tc in tch ny yu
NST tho xon , qu trnh tng hp protein din ra, c tnh ca gen c biu hin
thng qua cc tnh trng. Vic tch in dng ca histon mnh hay yu thng qua
qu trnh acetyl ha u amin phn ui ca histon lin quan n hot ng ca 2
emzym histon deacetylase (HDAC) v histon acetyltransferase (HAT).
1.1.1. Khi nim v histon deacetylase
3
Histon deacetylase l mt nhm cc enzym xc tc qu trnh loi b nhm
acetyl t -N acetyl lysine amino acid ca histon. HDAC c tc dng i lp vi
histon acetyltransferase (HAT) - enzym xc tc chuyn nhm acetyl t acetyl
coenzym A n -amino ca lysin u N ca histon [49].
Histons ngng t cc nhim sc th
ngn cn phin m
Acetyl-histons ko gin cc nhim sc th
kch thch phin m
1.1.2. Phn loi cc HDAC
C 18 HDAC ngi c chia thnh 4 nhm da trn s tng ng cu
trc ca chng ln lt vi Rpd3, Hdal v Sir2 trong nm men [22,43] (hnh 1.2):
Nhm I: HDAC1, HDAC2, HDAC3, HDAC8.
Nhm II: HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, HDAC10.
Nhm III: Cc protein iu ha chui thng tin bao gm:
- Cht ng ng ca Sir2 trong nm men Saccharomyces cerevisiae.
- Sirtuin trong ng vt c v (SIRT1, SIRT2, SIRT3, SIRT4,
SIRT5, SIRT6, SIRT7).
Nhm IV: HDAC11.
Cc HDAC nhm I, II, IV c coi l cc HDAC kinh in gm 11 thnh
vin, trong khi cc thnh vin nhm III c gi l cc sirtuin [22]. Cc HDAC
kinh in v sirtuin c c ch xc tc khc nhau. Cc HDAC kinh in l nhng
enzym ph thuc Zn2+, chng c cha 1 ti xc tc vi 1 ion Zn2+ y ti. Do
nhng enzym ny c th b c ch bi cc hp cht to phc vi Zn2+ nh cc acid
hydroxamic, cc thiol Ngc li cc sirtuin khng b c ch bi cc hp cht to
chelat vi Zn2+ v c ch hot ng ca chng ph thuc vo NAD+ nh 1 cofactor
thit yu [43]. Thut ng cc cht c ch HDAC thng c s dng cho nhng
hp cht nhm mc tiu vo cc HDAC kinh in v nhng hp cht ny ang
c nh gi da trn cc th nghim lm sng cc giai on khc nhau.
HATs
HDACs
4
Ghi ch: Vng xc tc Vng tn hiu nhn din nhn t bo
Hnh 1.2: M t c im ca cc loi HDAC
1.1.3. Mi lin quan gia ung th v hot ng bt thng ca HDAC
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 NST, kt qu l c ch
qu trnh phin m do ngn cn cc nhn t sao m tin n ch ca chng trn
ADN. Ngc li, HAT acetyl ha histon lm trung ha cc dng trn lysin v gip
tho xon cu trc ca nucleosom, do gip cho cc yu t sao m d dng tip
cn ADN. Nh vy s acetyl ha v deacetyl ha NST 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
[18,27,30,35,39].
5
HDAC 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 gen) 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 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 [18,21,27]. V 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 [7]. 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, nn 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 [14]. 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 [23,45,47] 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) [37]. 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
6
HDAC trong ung th [12,36]. 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.
Cc nghin cu c tnh thng k cn ch ra rng cc HDAC lin quan n
nhiu giai on iu ha c bn ca qu trnh sinh hc trong t bo ung th nh chu
trnh t bo, s bit ha, s cht t bo theo chng trnh k c s xm ln, s di
chuyn v s to mch. Vai tr chc nng ca cc HDAC trong qu trnh sinh hc
ca t bo ung th c tm tt trong hnh 1.3 [9,20,28,44].
Nh vy c phin m c iu ha bi s ra tng HDAC v c th kim sot
ung th bng cch c ch hot ng ca HDAC. 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.
Hnh 1.3: Vai tr sinh hc ca cc HDAC trong sinh l t bo ung th
1.2. CC CHT C CH HDAC
1.2.1. Phn loi cc cht c ch HDAC
7
T nhng pht hin u tin v tc dng c ch HDAC ca natri butyrat, cho
ti nay cc nh nghin cu tm ra rt nhiu hp cht mi c tc dng c ch
HDAC. Trong trichostatin A (TSA), mt sn phm ln men ca Streptomyces, l
cht c tc dng c ch mnh nht tuy nhin vic sn xut n li khng kh thi [38].
Hin nay, c 2 cht c tc dng c ch HDAC c FDA cp php lu hnh trn
th trng iu tr u lympho t bo T di da l SAHA (vorinostat, Zolinza) v
Romidepsin (Istodax). Ngoi ra, cn khong 15 cht c ch HDAC khc cng
ang c th nghim tc dng iu tr ung th trn lm sng cc giai on khc
nhau (hnh 1.4) v c chia lm 4 nhm da theo cu trc (bng 1.1).
Hnh 1.4: Mt s cht c ch HDAC ang c th nghim lm sng
Mi nhm dn cht ny u c nhng hn ch ring nh: cc acid hydroxamic
b chuyn ha nhanh, c ch khng chn lc trn cc loi enzym HDAC; cc
benzamid v cc acid bo c hiu lc km; cc peptid vng kh to thnh v mt
ha hc. Cc nhm khc nhau c tc dng c ch cc loi HDAC khc nhau: cc
acid hydroxamic c ch mnh HDAC nhm I v II, cc acid carboxylic v peptid
vng c ch mnh HDAC nhm I.
8
Bng 1.1: Cc cht c ch HDAC ang th nghim trn lm sng Nhm Hp cht Pha Loi ung th
Acid carboxylic
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 JNJ-16241199
I, II II I II, III II I I I
Tng c, ung th mu Ung th mu Tng c, ung th mu Tng c, AML, MDS, ALL U lympho Hodgkin Tng c, ung th mu
Cc benzamid
SNDX-275 CI-994 MGCD-0103
I, II I, II II
Tng c, u lympho, AML Tng c, NSCLC, t bo thn,ty Tng c, ung th bch cu, MDS
Peptid vng Depsipeptid (FK228)
I, II Tng c, CLL, AML, u a ty xng, NHL t bo T ngoi vi, RAI khng thyroid, ung th i trng tin trin
Ghi ch: 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.
1.2.2. Cu trc ca cc cht c ch HDAC
Cu trc ca cc cht c ch HDAC rt a dng nhng nhn chung u gm 3
phn chnh:
- Nhm kha hot ng (capping group) hay vng nhn din b mt (surface
recognition group): l cc vng thm hoc peptid vng, thng nm trn b mt
enzym.
- Vng cu ni s nc: thng l nhng hydrocacbon thn du mch thng
hay vng, c th no hoc khng no to cc lin kt Van der Waals vi knh
enzym.
9
- Nhm kt thc gn vi km (Zinc binding group - ZBG): tng tc vi ion
Zn2+ ti trung tm hot ng ca cc HDAC nh acid hydroxamic, cc thiol, nhm
o-aminoanilin ca benzamid, mercaptoceton...
Cu trc tinh th kt tinh ca cc HDAC cho thy nhm kt thc, cu ni v
mt phn ca nhm kha hot ng nm trong ti enzym lm lp y khong trng
trong lng knh enzym. Phn cn li ca nhm kha hot ng tng tc vi phn
vnh trn b mt ming ti enzym. Nhm nhn din b mt c th lin kt vi phn
cu ni thng qua mt s lin kt peptid lm tng kh nng phn cc v gp phn
ci thin dc ng hc cho cc cht c ch HDAC. Vic nghin cu thit k cu
trc cc cht mi u da trn cu trc c in ny.
1.3. MT S HNG NGHIN CU TNG HP CC ACID
HYDROXAMIC C CH HDAC TRN TH GII
1.3.1. Lin quan cu trc tc dng ca cc acid hydroxamic c ch HDAC
Cc cht c ch HDAC da trn cu trc amid-alkyl-acid hydroxamic c
bit n nhiu, v d nh SAHA (hnh 1.5).
Hnh 1.5: Cu trc ca SAHA v trung tm hot ng ca HDAC
Cu trc ca nhm cht ny cng gm 3 phn chnh c lin quan n tc dng
nh sau [38]:
- Nhm kha hot ng: lin quan n tnh c hiu v hiu lc c ch enzym
HDAC, thng l cc aryl, cc nghin cu v cc cht tng hp c cho thy kch
thc vng nhn thm ln s cho tc dng tt hn vng nh.
10
- Cu ni s nc: lin quan n kh nng c ch enzym, quyt nh s ph
hp v cu trc ca cc cht vi chiu di ca knh enzym. Phn ny thng c cu
trc amid - alkyl, l cc hydrocarbon mch h hoc cc vng thm c kch thc
nh. Trong cu ni lin kt amid l quan trng nhng khng phi l then cht,
di mch carbon ti u l 5 - 6C.
- Nhm lin kt vi Zn2+: acid hydroxamic, l phn khng th thiu c tc
dng c ch HDAC.
1.3.2. Mt s hng thit k nghin cu v tng hp trn th gii
1.3.2.1. Thay i cu ni
- Cc N-hydroxy-2-propenamid, cc acid hydroxamic tng t TSA
Nhm cc N-hydroxy-2-propenamid (hnh 1.6) c thit k v tng hp da
trn c s nghin cu cu trc ca TSA v oxamflatin (hnh 1.6). Cc cht ny c
tc dng c ch HDAC yu hn TSA song tng t oxamflatin [29].
Hnh 1.6: Cu trc ca TSA, oxamflatin v cc dn cht N-hydroxy-2-propenamid
- Cc amid ngc ca SAHA
Nhm nghin cu thuc cng ty Topo Target (Anh) thit k v tng hp
gn 40 dn cht amid ngc ca SAHA (hnh 1.7) [6]. Nhiu cht trong s ny c
hot tnh c ch HDAC tng ng thm ch mnh hn SAHA. Trong c 2
cht c th nghim m hnh ung th in vivo trn chut cho kt qu kh quan,
nh hng cho cc nghin cu tip theo.
11
Hnh 1.7: Cc dn cht amid ngc ca SAHA
- Cc dn cht -alkoxy ca SAHA
Nhm nghin cu thuc trung tm nghin cu Sigma-Tau (Pomezia, )
thit k v tng hp dy dn cht -alkoxy ca SAHA (hnh 1.8) [24]:
Hnh 1.8: Cc dn cht -alkoxy ca SAHA
Kt qu sng lc hot tnh c ch HDAC2 cho thy tt c cc dn xut -
alkoxy ca SAHA u cho gi tr IC50 mc rt thp (0,05 - 0,5 M). c tnh ca
cc cht vi t bo ung th th hin mnh hn so vi SAHA trn c 3 dng t bo
NB4 (ung th bch cu tin ty bo cp), H460 (ung th phi ngi), HCT-116
(ung th i trng).
1.3.2.2. Thay i nhm kha hot ng
- Cc acid phenylthiazol-hydroxamic tng t SAHA
Vin nghin cu Walter Reed (M) thit k v tng hp hng trm dn cht
acid hydroxamic mang hp phn phenylthiazol thay th vo v tr phenyl ca SAHA
(hnh 1.9). Trong c nhiu cht c tc dng c ch HDAC tng ng hoc
mnh hn SAHA [19].
Hnh 1.9: Cc acid phenylthiazol - hydroxamic tng t SAHA
- Cc acid biphenyl-hydroxamic tng t SAHA
Nhm nghin cu ca Alan P. Kozikowski thit k v tng hp mt dy
cc dn cht acid biphenyl-hydroxamic tng t SAHA (hnh 1.10) [31]. Kt qu
12
cc dn cht ny c ch HDAC mnh hn SAHA trn 6 loi HDAC (HDAC1, 2, 3,
6, 8, 10). Mt s acid biphenyl-hydroxamic cng c c tnh trn 5 dng t bo ung
th ty th nghim nhng tc dng khng tt bng cc acid phenylthiazol-
hydroxamic.
Hnh 1.10: Cc dn cht acid biphenyl-hydroxamic
- Cc acid isoxazol-hydroxamic tng t SAHA
Trong qu trnh nghin cu cc dn cht ca 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 1.11) [32].
Dn cht isoxazol ny c tc dng c ch HDAC1, 3 v 6 rt mnh vi IC50 thp
n nng nanomol (0,002 nM).
Hnh 1.11: Cng thc cu to ca WR301849
Tip tc mch nghin cu ny, mt s dn cht trong vng isoxazol c
a vo v tr ngay st nhm acid hydroxamic c thit k v tng hp. Kt
qu th hot tnh c ch HDAC cho thy 4 dn cht I-IV c ch c 5 loi HDAC1,
2, 3, 6 v 10 vi IC50 trung bnh khong 150 nM, thp nht l 25 nM (bng 1.2).
13
Bng 1.2: Tc dng c ch HDAC ca cc acid isoxazol-hydroxamic
Cht Ar IC50 (nM)/HDAC
HDAC1 HDAC2 HDAC3 HDAC6 HDAC10
I
303 430 30 68 254
II
139 164 25 82 250
III
328 469 102 51 471
IV
885 3750 7410 885 #
SAHA 96 282 17 14 72 Ghi ch: # : Khng th hot tnh
Nhn chung, cc nghin cu u cho thy acid hydroxamic l nhm cht c
kh nng c ch HDAC tt, mt s cht c tc dng c tnh vi t bo ung th
nng rt thp. Tng hp cc acid hydroxamic hng c ch HDAC ang l mt
hng nghin cu mi v c nhiu trin vng trong vic tm kim cc hp cht c
tc dng khng t bo ung th chn lc, c hiu qu iu tr cao v t gy tc dng
khng mong mun.
1.4. PHNG PHP TO LIN KT AMID V TNG HP ACID
HYDROXAMIC
Cc cht tng hp trong ti ny u qua 4 bc phn ng. Trong , c 2
phn ng quan trng l phn ng to lin kt amid v phn ng tng hp acid
hydroxamic. Qua tham kho ti liu chng ti la chn cc phng php tng
hp ph hp vi iu kin phng th nghim nh sau:
1.4.1. Tng hp amid vi tc nhn acyl ha l acid carboxylic
Acid carboxylic l tc nhn acyl ha yu, acyl ha amin cn c cht xc
tc. Cc tc nhn hot ha acid carboxylic l imidazolium t ra kh hu hiu trong
vic to thnh lin kt amid, trong carbonyldiimidazol (CDI) l cht hay c s
14
dng nht trong nhm. Victor Andrianov v cng s dng CDI xc tc phn
ng to amid cho hiu sut cao [6], phng trnh phn ng tng qut nh sau:
RCOOH + R1NH2 RCONHR1
C ch xc tc ca CDI c biu din trong s 1.1:
S 1.1: C ch xc tc ca CDI
1.4.2. Tng hp acid hydroxamic t ester
C nhiu cch khc nhau tng hp acid hydroxamic t ester nhng hay
dng nht l s dng KCN hoc NaOH lm xc tc. Cc phn ng c phng trnh
nh sau [5,13,16,24]:
Vi iu kin ha cht, dung mi ti phng th nghim v tham kho ti liu,
chng ti la chn xc tc NaOH v dung mi MeOH tin hnh phn ng ny.
15
Chng 2. NGUYN LIU, THIT B, NI DUNG V
PHNG PHP NGHIN CU
2.1. NGUYN VT LIU, THIT B
2.1.1. Ha cht
Cc ha cht, dung mi dng trong qu trnh thc nghim l loi dng trong
tng hp c nhp t cng ty Merck hoc Sigma-Aldrich. Cc ha cht ny c
s dng trc tip khng qua tinh ch thm. Bao gm:
Cc benzaldehyd:
- Benzaldehyd
- 2,6-Diclorobenzaldehyd
- 4-Bromobenzaldehyd
- 4-Flourobenzaldehyd
- 4-Methylbenzaldehyd
- 4-Dimethylaminobenzaldehyd
Thiosemicarbazid
FeCl3.6H2O
Acid monomethyl suberic
Carbonyldiimidazol (CDI)
Hydroxylamin hydroclorid
Cc ha cht v dung mi khc: DMF, aceton, acid acetic bng,
dicloromethan, ethanol, methanol, NaOH, HCl, nc ct.
2.1.2. Thit b, dng c
- Dng c thy tinh: bnh cu y trn dung tch 50 ml c nt mi, sinh hn
hi lu, pipet, bnh chit, phu thy tinh, bnh chy sc k lp mng (TLC).
- My khuy t gia nhit.
- My ct quay chn khng Buchi R-210.
- Cn phn tch, cn k thut Shimazu.
- T lnh, t sy, my siu m.
- Bn mng silicagel Merck 70-230 mesh chy sc k lp mng.
- My o nhit nng chy nhit in (Electrothermal digital) xc nh
nhit nng chy.
- My Perkin Elmer xc nh ph IR.
- My khi ph HP 5989B-MS ghi ph MS.
- My cng hng t Bruker AV-500 ghi ph 1H-NMR, 13C-NMR.
16
2.2. NI DUNG NGHIN CU
2.2.1. Tng hp ha hc
* Nghin cu Docking vi 6 acid hydroxamic d kin tng hp.
* Tng hp 6 acid hydroxamic:
- N1-hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)octandiamid (5a).
- N1-{5-[4-(dimethylamino)phenyl]-1,3,4-thiadiazol-2-yl}-N8-
hydroxyoctandiamid (5b).
- N1-[5-(4-flourophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid (5c).
- N1-[5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid (5d).
- N1-hydroxy-N8-[5-(4-methylphenyl)-1,3,4-thiadiazol-2-yl]octandiamid (5e).
- N1-[5-(2,6-diclorophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid
(5f).
2.2.2. Th tc dng sinh hc ca cc cht tng hp c
- Th tc dng c ch HDAC.
- Th c tnh trn t bo ung th i trng (SW620).
- S b nh gi tnh ging thuc ca cc cht tng hp c.
2.3. PHNG PHP NGHIN CU
2.3.1. Tng hp ha hc
2.3.1.1. Nghin cu Docking
- Vic nghin cu s b tnh tng tc ca cc cht vi HDAC (docking) c
thc hin ti Phng Nghin cu cu trc i hc Quc gia Seul, Hn Quc.
- tm hiu s b tng tc ca cc cht vi HDAC, chng ti tin hnh
docking cho cc cht tng hp c da trn c s mng li ca HDAC8 tng
tc vi SAHA [26]. Cu trc ca HDAC8 c im tng ng cao vi HDAC4 vi
im DALI Z (Z-score im nh gi ging) = 40,4 v im r.m.s.d (root-mean-
square deviation, lch) = 2,1, th t acid amin ging nhau (46%) v l HDAC
ca ng vt c v u tin c cu trc khng gian c nghin cu k nht. Chng
ti thc hin kim sot th nghim lp ghp ca cc cht vo HDAC8 bng chng
17
trnh AutoDock Vina [42]. Tip theo chng ti d on nng lng ca s tng
tc lin kt ca cc cht tng hp c vi HDAC8 t s lp ghp trn.
Kt qu c minh ha bng hnh nh m hnh v s liu d on tng tc.
2.3.1.2. Tng hp
- Da trn nhng nguyn tc v phng php c bn ca ha hc hu c
tng hp cc dn cht theo thit k.
- Theo di qu trnh phn ng bng sc k lp mng (TLC).
2.3.1.3. Kim tra tinh khit
- Kim tra tinh khit ca sn phm bng chy sc k lp mng v o nhit
nng chy.
2.3.1.4. Xc nh cu trc cc cht tng hp c
Cc cht tng hp c c xc nh cu trc bng cc ph: ph hng ngoi,
ph khi, ph cng hng t ht nhn 1H-NMR v 13C-NMR.
- Ph hng ngoi (IR): c ghi ti khoa Ha, trng i hc Khoa hc t
nhin H Ni trn my Perkin Elmer vi k thut vin nn KBr trong vng 4000-
600 cm-1. Cc mu rn c phn tn trong KBr sy kh vi t l khong 1: 200
ri p di dng film mng di p lc cao c ht chn khng loi b hi m.
- Ph khi lng (MS): c o bng my khi ph HP 5989B-MS ca Vin
Ha hc Vit Nam, ch o LC-MS, dung mi methanol.
- Ph cng hng t ht nhn proton 1H-NMR v carbon 13C-NMR c ghi
trn my Bruker AV-500 ti Trung tm Khoa hc v Cng ngh Vit Nam, dung
mi DMSO-d6. chuyn dch ha hc () c biu th bng n v phn triu
(parts per million - ppm), ly mc l pic ca cht chun ni tetramethylsilan (TMS).
2.3.2. Th tc dng sinh hc
2.3.2.1. Th tc dng c ch HDAC
Tc dng c ch HDAC ca cc dn cht tng hp c nh gi gin tip
thng qua xc nh mc acetyl ha histon H3, H4 trong t bo ung th i trng
(SW620). Phn tch da trn Western blot c th khng nh c tc dng ca cc
18
mu th lm tng hoc gim s acetyl ha histon H3, H4 khi so snh vi mu trng.
Chi tit cch tin hnh da trn phng php c cng b trc y [4].
2.3.2.2. Th c tnh t bo
* Nguyn tc th
Th hot tnh khng t bo ung th (th c tnh t bo) in vitro c thc
hin ti Khoa Dc, trng i hc Quc gia Chungbuk, Cheongju, Hn Quc theo
phng php MTT v gi tr IC50 c tnh trn phn mm GraphPad Prism [8,34].
Dng t bo th nghim: SW620 (t bo ung th i trng).
Cc t bo ung th c ly t Ngn hng t bo ung th ca Vin nghin cu
Sinh hc v Cng ngh sinh hc Hn Quc (KRIBB) v c nui cy trong mi
trng RPMI b sung 10% FBS (huyt thanh bo thai b). c tnh t bo ca cc
cht c th bng phng php MTT theo cc bc sau:
Chun b: Cc t bo pha logarit c trypsin ha v phn tn vo hn dch
n t bo trong mi trng RPMI b sung 10% FBS v iu chnh n nng
khong 1,5.104 n 3,5.104 t bo, sau chia u vo cc ging ca a 96 ging,
mi ging 200 l. Cc a sau c 37oC trong iu kin 5% CO2. Sau 24
gi , cc mu th c chun b trong 20 l mi trng RPMI b sung 10% FBS
t dung dch gc 10 mg/mL trong dimethyl sulfoxid (DMSO) ri thm 2 l mu th
vo cc ging nhiu nng khc nhau, cc a ny sau c thm 48 gi.
Tt c cc mu c chun b sao cho nng cui cng ca DMSO khng qu
0,1%.
Tin hnh th: Sau khi 48 gi, thm vo mi ging 20 l thuc nhum MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid) vi nng MTT l
5 mg/mL trong mui m phosphat - PBS. Cc a c thm 3 gi 37oC trong
iu kin 5% CO2. Tip theo, mi ging c cho 100 l dung dch DMSO, 5
pht cho MTT formazan c ha tan. hp th c c bc sng 510 nm.
* Tnh kt qu
Gi tr IC50 l nng ca mu th m hp th gim i 50% so vi
nhm chng (trng m tnh l ging ch thm mi trng nui cy): kt qu cui
19
cng l gi tr trung bnh ca 4 ln o c lp vi gi tr hp th khc nhau
khng qu 5%. Gi tr IC50 c tnh da trn phn mm GraphPad Prism. Trong
phng php th ny, IC50 20 g/mL c coi l c hot tnh.
2.3.3. nh gi mc ging thuc ca cc cht tng hp c
Tnh gi tr logP ca cc cht tng hp bng phn mn EPTsuite cung cp bi
US Environmental Protection Agency's Office of Pollution Prevention and Toxics
and Syracuse Research Corporation (SRC). Quy trnh bao gm v cu trc 2D, to
Smile notation bng phn mm ChemSketch 4.0 ca ACD labs sau a Smile
notation vo phn mm EPIsuite tnh cc gi tr logP.
nh gi mc ging thuc ca cc cht da trn quy tc Lipinsky [33]:
+ Khi lng phn t ca cht khng ln hn 500 g/mol.
+ S trung tm nhn lin kt hydro (N, O) khng ln hn 10.
+ S trung tm cho lin kt hydro (NH, OH) khng ln hn 5.
+ Gi tr logP ca cht khng ln hn 5.
+ S lin kt linh ng khng ln hn 15.
20
Chng 3: THC NGHIM, KT QU V BN LUN
3.1. HA HC
3.1.1. Nghin cu Docking
tm hiu s b v kh nng tng tc ca cc cht vi mc tiu phn t l
cc HDAC, chng ti nghin cu v d on nng lng lin kt cc cht trong
thit k vi trung tm hot ng ca HDAC8. Kt qu c minh ha bng m hnh
tng tc ca cc cht vi HDAC8 (hnh 3.1), nng lng lin kt d on c
trnh by trong bng 3.1
Bng 3.1: Kt qu docking ca cc cht 5a-f vi HDAC8
Cht 5a 5b 5c 5d 5e 5f
SAHA R H 4-N(CH3)2 4-F 4-Br 4-CH3 2,6-Cl2
Nng lng tng tc (kcal/mol)
-6,7 -7,1 -6,9 -6,6 -7,4 -6,8 -4,4
21
Hnh 3.1: M hnh tng tc ca 6 dn cht 5a-f vi HDAC8
3.1.2. Tng hp ha hc
Qu trnh tng hp 6 cht trong kha lun c thc hin theo s chung
nh sau (s 3.1):
S 3.1. Quy trnh tng hp chung
Tc nhn v iu kin: i) Thiosemicarbazid, ethanol, acid acetic bng, hi lu, 4 h; ii) FeCl3.6H2O, ethanol, hi lu, 3 h; iii) Acid monomethyl suberic, carbonyldiimidazol, DMF, 60oC, 24 h; iv) Hydroxylamin hydroclorid, NaOH, methanol, -10oC, 1 h.
22
3.1.2.1. Tng hp N1-hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)octandiamid
(5a)
a. Tng hp cht 2a
Cht 2-benzylidenhydrazincarbothioamid (2a) c tng hp t benzaldehyd
(1a) qua phn ng ngng t vi thiosemicarbazid c xc tc l acid hu c theo s
3.2:
S 3.2: Quy trnh tng hp cht 2a
Tin hnh phn ng:
+ Ly 0,2 ml (2 mmol) benzaldehyd cho vo bnh cu y trn dung tch 50
ml, thm 20 ml ethanol khan.
+ Thm 0,218 g (2,4 mmol) thiosemicarbazid v 3-4 git acid acetic bng vo
bnh phn ng.
+ un hi lu kt hp khuy t 300 vng/pht, tin hnh phn ng trong 4 h.
X l phn ng:
+ Ct loi dung mi bng my ct quay chn khng trong 5 pht nhit
40oC.
+ Thm 20 ml nc ct ta sn phm.
+ Lc, ra ta 3 ln bng 15 ml nc ct lnh.
+ Sy kh ta 60oC v thu c 0,32 g sn phm.
Kt qu
+ Cm quan: bt kt tinh mu trng ng.
+ Hiu sut: 90%.
+ Tonc: 164-165oC.
+ Rf = 0,63 (DCM/MeOH = 9/1).
b. Tng hp cht 3a
23
Tng hp cht 5-phenyl-1,3,4-thiadiazol-2-amin (3a) t 2a bng phn ng
ng vng ni phn t theo s 3.3 nh sau:
S 3.3: Quy trnh tng hp cht 3a
Tin hnh phn ng:
+ Ha tan 1,22 g (4,5 mmol) FeCl3.6H2O vo 3 ml ethanol trong bnh cu y
trn dung tch 50 ml.
+ Ha tan 0,268 g (1,5 mmol) cht 2a vo 20 ml ethanol trong cc c m, rt
vo bnh phn ng.
+ un hi lu kt hp khuy t 300 vng/pht, tin hnh phn ng trong 3 h.
X l phn ng:
+ Ct loi dung mi bng my ct quay trong 5 pht nhit 40oC.
+ Thm 20 ml nc ct lnh vo hn hp phn ng.
+ Kim ha hn hp bng dung dch NaOH 30% n pH = 8-9.
+ Chit thu sn phm bng DCM (chit 3 ln mi ln 20 ml DCM), gp cc
dch chit v lc vi nc mui bo ha, thu v lm khan dch chit bng Na2SO4
khan, ct quay chn khng 40oC n khi ht dung mi v sn phm kt tinh
dng rn.
+ Sy sn phm 60oC trong 4 h, thu c 0,19 g.
Kt qu
+ Cm quan: bt kt tinh mu vng nht.
+ Hiu sut: 72%.
+ Tonc: 192-194oC.
+ Rf = 0,54 (DCM/MeOH = 9/1).
c. Tng hp cht 4a
24
Cht methyl 8-oxo-8-(5-phenyl-1,3,4-thiadiazol-2-ylamino)octanoat (4a) c
tng hp bng phn ng acyl ha hp cht 3a vi acid monomethyl suberic theo s
3.4 nh sau:
S 3.4: Quy trnh tng hp cht 4a
Tin hnh phn ng:
+ Ha tan 0,162 g (1 mmol) CDI v 0,17 ml (1 mmol) acid monomethyl
suberic vo 1 ml DMF trong bnh phn ng, hot ha nhit phng trong 1 h.
+ Tip tc cho 0,177 g (1 mmol) cht 3a vo bnh phn ng.
+ Duy tr nhit phn ng 60oC kt hp khuy t 300 vng/pht, tin hnh
phn ng trong 24 h.
X l phn ng:
+ Lm lnh bnh phn ng, thm t t 20 ml dung dch HCl 5% lnh vo bnh,
va thm va lc u. lnh 30 pht ta sn phm.
+ Lc ly ta, ra ta bng nc ct n khi dch lc trung tnh.
+ Sy kh ta 60oC, thu c 0,22 g sn phm.
Kt qu
+ Cm quan: bt kt tinh mu trng hi hng.
+ Hiu sut: 65%.
+ Tonc: 236-238oC.
+ Rf = 0,81 (DCM/MeOH = 9/1).
d. Tng hp cht 5a
Cht N1-hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)octandiamid (5a) c
tng hp bng phn ng to acid hydroxamic gia cht 4a vi hydroxylamin
hydroclorid theo s 3.5 nh sau:
25
S 3.5: Quy trnh tng hp cht 5a
Tin hnh phn ng:
+ Cho 0,174 g (0,5 mmol) cht 4a v 0,35 g (5 mmol) hydroxylamin
hydroclorid vo bnh cu dung tch 50 ml, thm 5 ml methanol, em siu m 5 pht
to hn dch ng nht.
+ Ha tan 0,8 g (20 mmol) NaOH vo 3 ml nc ct trong ng nghim.
+ ng thi lm lnh bnh phn ng v dung dch NaOH xung di 0oC
bng hn hp nc mui. Thm t t dung dch NaOH vo bnh phn ng. Duy
tr nhit phn ng -10oC kt hp khuy t 300 vng/ pht, phn ng kt thc
sau 1 h. Theo di tin trnh phn ng v xc nh thi im kt thc bng cch dng
dung dch FeCl3 5% v TLC, c th lm nh sau:
- Acid ha khong 0,1 ml hn hp phn ng bng dung dch HCl long trn
khay s, sau nh 1 git dung dch FeCl3 5%, nu xut hin mu vang chng
t c acid hydroxamic c to ra.
- Acid ha khong 0,1 ml hn hp phn ng trong vial, thm methanol
ha tan ta, kim tra bng TLC vi pha ng DCM/MeOH/CH3COOH = 90/10/1.
Kt thc phn ng khi kt qu cho thy phn ng ht ester ban u.
X l phn ng:
+ Acid ha hn hp phn ng bng dung dch HCl 5% lnh n pH = 6-7,
lnh 15 pht ta sn phm.
+ Lc ly ta, ra ta bng nc ct lnh.
+ Sy kh ta 60oC, thu c 0,09 g sn phm.
Kt qu
+ Cm quan: bt kt tinh mu trng.
+ Hiu sut: 52%.
26
+ Nhit nng chy v Rf c xc nh sau khi kt tinh li trong hn hp
dung mi methanol - nc, kt qu c th c trnh by trong bng 3.2.
+ Xc nh cu trc bng ph IR, MS, 1H-NMR, 13C-NMR kt qu c th
c trnh by trong phn 3.1.4.
3.1.2.2. Tng hp N1-{5-[4-(dimethylamino)pheny]-1,3,4-thiadiazol-2-yl}-N8-
hydroxyoctandiamid (5b)
Cht 5b c tng hp theo cc phn ng trnh by trong s 3.6:
S 3.6: Quy trnh tng hp 5b
Tc nhn v iu kin: i) Thiosemicarbazid, ethanol, acid acetic bng, hi lu, 4 h; ii) FeCl3.6H2O, HCl 5%, ethanol, hi lu, 3 h; iii) Acid monomethyl suberic, carbonyldiimidazol, DMF, 60oC, 24 h; iv) Hydroxylamin hydroclorid, NaOH, methanol, -10oC, 1 h. a. Tng hp cht 2b
tng hp cht 5b cn tng hp cht 2-[4-(dimethylamino)benzyliden]
hydrazincarbothioamid (2b). Qu trnh tng hp cht 2b t 0,298 g (2 mmol) cht
4-dimethylaminobenzaldehyd (1b) c thc hin tng t nh tng hp cht 2a.
Kt qu nh sau:
+ Cm quan: bt kt tinh mu trng hi vng.
+ Hiu sut: 88% (0,391 g).
+ Tonc: 169-170oC.
+ Rf = 0,65 (DCM/MeOH = 9/1).
b. Tng hp cht 3b
Tng hp cht 5-[4-(dimethylamino)phenyl]-1,3,4-thiadiazol-2-amin (3b) t
2b bng phn ng ng vng ni phn t theo s 3.7 nh sau:
27
S 3.7: Quy trnh tng hp 3b
Tin hnh phn ng:
+ Ha tan 1,22 g (4,5 mmol) FeCl3.6H2O vo 5 ml ethanol trong bnh cu
dung tch 50 ml.
+ Ha tan 0,333 g (1,5 mmol) cht 2b vo 20 ml HCl 5% trong cc c m, rt
vo bnh phn.
+ un hi lu kt hp khuy t 300 vng/pht, tin hnh phn ng trong 3 h.
X l phn ng:
+ Ct loi dung mi bng my ct quay trong 1 pht nhit 40oC.
+ Thm 10 ml nc ct lnh vo hn hp phn ng.
+ Kim ha hn hp bng dung dch NaOH 30% n pH = 8-9.
+ Chit thu sn phm bng DCM, chit 3 ln mi ln 20 ml DCM, gp cc
dch chit v lc vi nc mui bo ha, thu v lm khan dch chit bng Na2SO4
khan, ct quay chn khng 40oC n khi ht dung mi v sn phm kt tinh
dng rn.
+ Sy sn phm 60oC trong 4 h, thu c 0,23 g.
Kt qu
+ Cm quan: bt kt tinh mu vng nht.
+ Hiu sut: 70%.
+ Tonc: 190-191oC.
+ Rf = 0,50 (DCM/MeOH = 9/1).
c. Tng hp cht 4b
Tng hp cht methyl 8-{5-[4-(dimethylamino)phenyl]-1,3,4-thiadiazol-2-
ylamino}-8-oxooctanoat (4b) t 0,220 g (1 mmol) cht 3b c tin hnh tng t
nh tng hp cht 4a. Kt qu ca qu trnh nh sau:
+ Cm quan: bt kt tinh mu trng hi hng.
28
+ Hiu sut: 60% (0,23 g).
+ Tonc: 206-208oC.
+ Rf = 0,83 (DCM/MeOH = 9/1).
d. Tng hp cht 5b
Tng hp cht N1-{5-[4-(dimethylamino)phenyl]-1,3,4-thiadiazol-2-yl}-N8-
hydroxyoctandiamid (5b) t 0,195 g (0,5 mmol) cht 4b c tin hnh tng t
nh tng hp cht 5a. Kt qu nh sau:
+ Cm quan: bt kt tinh mu vng nu nht.
+ Hiu sut: 62% (0,12 g).
+ Nhit nng chy v Rf c xc nh sau khi kt tinh li trong hn hp
dung mi methanol - nc, kt qu c th c trnh by trong bng 3.2.
+ Xc nh cu trc bng ph IR, MS, 1H-NMR, 13C-NMR kt qu c th
c trnh by trong phn 3.1.4.
3.1.2.3. Tng hp N1-[5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxy
octandiamid (5c)
a. Tng hp cht 2c
tng hp cht 5c cn tng hp cht 2-(4-fluorobenzyliden)
hydrazincarbothioamid (2c). Qu trnh tng hp cht 2c t 0,21 ml (2 mmol) cht
4-fluorobenzaldehyd (1c) c thc hin tng t nh tng hp cht 2a. Kt qu
nh sau:
+ Cm quan: bt kt tinh mu trng.
+ Hiu sut: 90% (0,354 g).
+ Tonc: 166-167oC.
+ Rf = 0,60 (DCM/ MeOH = 9/1).
b. Tng hp cht 3c
Tng hp cht 5-(4-fluorophenyl)-1,3,4-thiadiazol-2-amin (3c) t 0,289 g (1,5
mmol) cht 2c bng phn ng ng vng ni phn t tng t nh tng hp cht
3a. Kt qu nh sau:
+ Cm quan: bt kt tinh mu xanh en.
29
+ Hiu sut: 71% (0,21 g).
+ Tonc: 185-186oC.
+ Rf = 0,55 (DCM/ MeOH = 9/1).
c. Tng hp cht 4c
Tng hp cht methyl 8-[5-(4-fluorophenyl)-1,3,4-thiadiazol-2-ylamino]-8-
oxooctanoat (4c) t 0,195 g (1 mmol) cht 3c c tin hnh tng t nh tng hp
cht 4a. Kt qu ca qu trnh nh sau:
+ Cm quan: bt kt tinh mu trng.
+ Hiu sut: 62% (0,23 g).
+ Tonc: 196-198oC.
+ Rf = 0,75 (DCM/MeOH = 9/1).
d. Tng hp cht 5c
Tng hp cht N1-[5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl]-N8-
hydroxyoctandiamid (5c) t 0,182 g (0,5 mmol) cht 4c c tin hnh tng t
nh tng hp cht 5a. Kt qu nh sau:
+ Cm quan: bt kt tinh mu trng.
+ Hiu sut: 60% (0,11 g).
+ Nhit nng chy v Rf c xc nh sau khi kt tinh li trong hn hp
dung mi methanol - nc, kt qu c th c trnh by trong bng 3.2.
+ Xc nh cu trc bng ph IR, MS, 1H-NMR, 13C-NMR kt qu c th
c trnh by trong phn 3.1.4.
3.1.2.4. Tng hp N1-[5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxy
octandiamid (5d)
a. Tng hp cht 2d
tng hp cht 5d cn tng hp cht 2-(4-
bromobenzyliden)hydrazincarbothioamid (2d). Qu trnh tng hp cht 2d t 0,370
g ( 2 mmol) cht 4-bromobenzaldehyd (1d) c thc hin tng t nh tng hp
cht 2a. Kt qu nh sau:
+ Cm quan: bt kt tinh mu trng.
30
+ Hiu sut: 85% (0,44 g).
+ Tonc: 171-172oC.
+ Rf = 0,67 (DCM/MeOH = 9/1).
b. Tng hp cht 3d
Tng hp cht 5-(4-bromophenyl)-1,3,4-thiadiazol-2-amin (3d) t 0,387 g
(1,5 mmol) cht 2d bng phn ng ng vng ni phn t tng t nh tng hp
cht 3a. Kt qu nh sau:
+ Cm quan: bt kt tinh mu vng nht.
+ Hiu sut: 75% (0,28 g).
+ Tonc: 192-194oC.
+ Rf = 0,50 (DCM/MeOH = 9/1).
c. Tng hp cht 4d
Tng hp cht methyl 8-(5-(4-bromophenyl)-1,3,4-thiadiazol-2-ylamino)-8-
oxooctanoat (4d) t 0,254 g (1 mmol) cht 3d c tin hnh tng t nh tng
hp cht 4a. Kt qu nh sau:
+ Cm quan: bt kt tinh mu trng hng.
+ Hiu sut: 65% (0,28 g).
+ Tonc: 216-218oC.
+ Rf = 0,80 (DCM/MeOH = 9/1).
d. Tng hp cht 5d
Tng hp cht N1-[5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl]-N8-
hydroxyoctandiamid (5d) t 0,214 g (0,5 mmol) cht 4d c tin hnh tng t
nh tng hp cht 5a. Kt qu nh sau:
+ Cm quan: bt kt tinh mu trng.
+ Hiu sut: 60% (0,12 g).
+ Nhit nng chy v Rf c xc nh sau khi kt tinh li trong hn hp
dung mi methanol - nc, kt qu c th c trnh by trong bng 3.2.
+ Xc nh cu trc bng ph IR, MS, 1H-NMR, 13C-NMR, kt qu c th
c trnh by trong phn 3.1.4.
31
3.1.2.5. Tng hp N1-hydroxy-N8-[5-(4-methylphenyl)-1,3,4-thiadiazol-2-yl]
octandiamid (5e)
a. Tng hp cht 2e
tng hp cht 5e cn tng hp cht 2-(4-methylbenzyliden)
hydrazincarbothioamid (2e). Qu trnh tng hp cht 2e t 0,24 ml (2 mmol) cht
4-methylbenzaldehyd (1e) c thc hin tng t nh tng hp cht 2a. Kt qu
nh sau:
+ Cm quan: bt kt tinh mu trng.
+ Hiu sut: 87% (0,335 g).
+ Tonc: 170-172oC.
+ Rf = 0,63 (DCM/MeOH = 9/1).
b. Tng hp cht 3e
Tng hp cht 5-(4-methylphenyl)-1,3,4-thiadiazol-2-amin (3e) t 0,289 g
(1,5 mmol) cht 2e bng phn ng ng vng ni phn t tng t nh tng hp
cht 3a. Kt qu nh sau:
+ Cm quan: bt kt tinh mu trng.
+ Hiu sut: 75% (0,21 g).
+ Tonc: 182-184oC.
+ Rf = 0,50 (DCM/MeOH = 9/1).
c. Tng hp cht 4e
Tng hp cht methyl 8-[5-(4-methylphenyl)-1,3,4-thiadiazol-2-ylamino]-8-
oxooctanoat (4e) t 0,191 g (1 mmol) cht 3e c tin hnh tng t nh tng hp
cht 4a. Kt qu nh sau:
+ Cm quan: bt kt tinh mu trng.
+ Hiu sut: 60% (0,22 g).
+ Tonc: 196-198oC.
+ Rf = 0,77 (DCM/MeOH = 9/1).
d. Tng hp cht 5e
32
Tng hp cht N1-hydroxy-N8-[5-(4-methylphenyl)-1,3,4-thiadiazol-2-
yl]octandiamid (5e) t 0,180 g (0,5 mmol) cht 4e c tin hnh tng t nh
tng hp cht 5a. Kt qu nh sau:
+ Cm quan: bt kt tinh mu vng nht.
+ Hiu sut: 65% (0,11 g).
+ Nhit nng chy v Rf c xc nh sau khi kt tinh li trong hn hp
dung mi methanol - nc, kt qu c th c trnh by trong bng 3.2.
+ Xc nh cu trc bng ph IR, MS, 1H-NMR, 13C-NMR kt qu c th
c trnh by trong phn 3.1.3.
3.1.2.6. Tng hp N1-[5-(2,6-diclorophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxy
octandiamid (5f)
a. Tng hp cht 2f
tng hp cht 5f cn tng hp cht 2-(2,6-diclorobenzyliden)
hydrazincarbothioamid (2f). Qu trnh tng hp cht 2f t 0,35 g (2 mmol) cht
2,6-diclorobenzaldehyd (1f) c thc hin tng t nh tng hp cht 2a. Kt qu
nh sau:
+ Cm quan: bt kt tinh mu trng.
+ Hiu sut: 85% (0,42 g).
+ Tonc: 170-172oC.
+ Rf = 0,66 (DCM/MeOH = 9/1).
b. Tng hp cht 3f
Tng hp cht 5-(2,6-diclorolphenyl)-1,3,4-thiadiazol-2-amin (3f) t 0,372 g
(1,5 mmol) cht 2f bng phn ng ng vng ni phn t tng t nh tng hp
cht 3a. Kt qu nh sau:
+ Cm quan: bt kt tinh mu trng.
+ Hiu sut: 70% (0,26 g).
+ Tonc: 192-194oC.
+ Rf = 0,50 (DCM/MeOH = 9/1).
c. Tng hp cht 4f
33
Tng hp cht methyl 8-[5-(2,6-diclorophenyl)-1,3,4-thiadiazol-2-ylamino]-8-
oxooctanoat (4f) t 0,246 g (1 mmol) cht 3f c tin hnh tng t nh tng hp
cht 4a. Kt qu nh sau:
+ Cm quan: bt kt tinh mu trng.
+ Hiu sut: 62% (0,26 g).
+ Tonc: 212-213oC.
+ Rf = 0,73 (DCM/MeOH = 9/1).
d. Tng hp cht 5f
Tng hp cht N1-[5-(2,6-diclorophenyl)-1,3,4-thiadiazol-2-yl]-N8-
hydroxyoctandiamid (5f) t 0,209 g (0,5 mmol) cht 4f c tin hnh tng t
nh tng hp cht 5a. Kt qu nh sau:
+ Cm quan: bt kt tinh mu vng nht.
+ Hiu sut: 61% (0,13 g).
+ Nhit nng chy v Rf c xc nh sau khi kt tinh li trong hn hp
dung mi methanol - nc, kt qu c th c trnh by trong bng 3.2.
+ Xc nh cu trc bng ph IR, MS, 1H-NMR, 13C-NMR kt qu c th
c trnh by trong phn 3.1.4.
* Sau khi tng hp c 6 cht 5a-f, chng ti tin hnh kt tinh li cc cht
bng hn hp dung mi methanol nc. Di y l bng tng hp cc ch s l
ha v hiu sut tng hp ca cc cht 5a-f:
Bng 3.2: Ch s l ha v hiu sut tng hp cc acid hydroxamic t ester
Cht R CTPT KLPT Mu H(%)
5a H C16H20N4O3S 348 Trng 52 5b 4-N(CH3)2 C18H25N5O3S 391 Vng nu nht 62 5c 4-F C16H19FN403S 366 Trng 60 5d 4-Br C16H19BrN403S 427 Trng 60 5e 4-CH3 C17H22N4O3S 362 Vng nht 65 5f 2,6-Cl2 C16H18Cl2N4O3S 417 Vng nht 61
34
3.1.3. Kim tra tinh khit
* Sc k lp mng (TLC)
Sc k lp mng c tin hnh trn bn nhm trng sn silicagel Merck 70-
230 mesh, vi 3 h dung mi khc nhau l DCM/MeOH (9/1), DCM/MeOH (5/1),
DCM/MeOH/AcOH (90/10/1), quan st di n t ngoi bc sng 254 nm. Kt
qu cho thy cc cht u c vt gn, r, khng c vt ph. Ch s Rf ca cc dn
cht 5a-f khi s dng 1 h dung mi c th c trnh by trong bng 3.2.
* o nhit nng chy
Cc cht sau khi c kt tinh li u c dng tinh th rn, c th tin hnh o
c nhit nng chy bng my o nhit nng chy nhit in. Kt qu trnh
by trong bng 3.2 cho thy cc cht 5a-f u c nhit nng chy xc nh
(khong dao ng nhit hp t 1-2oC).
Nh vy t kt qu chy sc k lp mng v o nhit nng chy ca 6 dn
cht 5a-f, chng ti khng nh cc cht tng hp c l tinh khit v c th s
dng o ph IR, MS, 1H-NMR, 13C-NMR.
Bng 3.3: Gi tr Rf v nhit nng chy (tonc) ca cc cht 5a-f
Cht R H dung mi Rf T
onc (
oC) 5a H DCM:MeOH:AcOH=90:10:1 0,32 202-204
5b 4-N(CH3)2 DCM:MeOH:AcOH=90:10:1 0,40 197-198
5c 4-F DCM:MeOH:AcOH=90:10:1 0,35 223-225
5d 4-Br DCM:MeOH:AcOH=90:10:1 0,37 216-217
5e 4-CH3 DCM:MeOH:AcOH=90:10:1 0,40 194-196
5f 2,6-Cl2 DCM:MeOH:AcOH=90:10:1 0,32 206-207
3.1.4. Xc nh cu trc
khng nh cu trc ca cc hp cht tng hp c, chng ti tin hnh
ghi ph khi lng (MS) ca dy cht 4a-f; ph khi lng (MS), ph hng ngoi
(IR) v ph cng hng t ht nhn proton (1H-NMR) v carbon (13C-NMR) ca
35
dy cht 5a-f. Kt qu ghi ph ca cc cht c th c trnh by trong phn phn
tch ph v ph lc.
3.1.4.1. Kt qu phn tch ph khi lng ca dy cht 4a-f
Bng 3.4: Kt qu phn tch ph MS ca cc cht 4a-f
Cht R KLPT m/z (ESI-MS)
4a H 347 347 [M]-
4b 4-N(CH3)2 390 388 [M-2H]-
4c 4-F 365 363 [M-2H]-
4d 4-Br 426 425 [M-H]-
4e 4-CH3 361 359 [M-2H]-
4f 2,6-Cl2 416 415 [M-H]-
Nhn xt: Qua kt qu phn tch ph bng 3.3 v ph ca 6 cht 4a-f
(ph lc 7-12), chng ti thy trn tt c cc ph u c pc phn t c s khi
ng bng s khi d kin ca cht tng ng vi cng mnh. Ph t c pc
phn mnh, cc pc phn mnh c cng nh. V vy c th khng nh ph
l ph hp vi cng thc ca 6 cht d kin 4a-f [25].
3.1.4.2. Kt qu phn tch ph ca dy cht 5a-f
a. Ph hng ngoi (IR)
Kt qu phn tch ph hng ngoi c trnh by trong bng 3.4:
36
Bng 3.5: Kt qu phn tch ph IR ca cc cht 5a-f
Cht R S sng (cm-1) ng vi cc dao ng
N-H CH
(benzen) CH
(CH2) C=O
C=C (benzen)
5a H 3251 3050 2927 2865
1629 1558
5b 4-N(CH3)2 3227 2926 2853
1652 1609
1570 1539
5c 4-F 3239 3054 2939 2853
1687 1628
1597 1557
5d 4-Br 3163 3049 2923 2850
1691 1616
1571 1557
5e 4-CH3 3162 3023 2929 2863
1702 1615
1561
5f 2,6-Cl2 3155 2927 2856
1701 1629
1534
Ghi ch: l dao ng ha tr.
Nhn xt: Ph IR ca cc cht 5a- f khng c nhiu tn hiu khng nh
chc chn cu trc ca cc cht. Nhng da trn ph (ph lc 1-6) v tham kho
mt s ti liu [10,11,15], cng c th nhn dng s b s hin din ca 1 s nhm
chc nh NH, CO... thng qua cc gi tr ca di hp thu, cng pic, hnh dng
pic. C th, cc cht u c nh hp thu ca nhm NH trong khong 3140-3250
cm-1, c nh hp thu ca C=O amid t 1600-1700 cm-1. Bn cnh , cc di hp
thu c trng cho vng benzen cng c ghi nhn nh di ko cng Caren-H xut
hin gia 3020-3060 cm-1 v di ko cng ca C=C trong vng thm hp thu trong
vng 1500-1600 cm-1. Trn ph cn c cc nh hp thu trong khong 2850-
2950 cm-1 c trng cho dao ng ko cng C-H ca cc nhm methylen. Nh vy
c th khng nh d liu ph IR ph hp vi cng thc cu to d kin ca 6 cht
5a-f.
b. Ph khi lng (MS)
37
Bng 3.6: Kt qu phn tch ph MS ca cc cht 5a-f
Cht R KLPT m/z (ESI-MS)
5a H 348 347 [M-H]-
5b 4-N(CH3)2 391 389 [M-2H]-, 374 [M-OH]-
5c 4-F 366 364 [M-2H]-
5d 4-Br 427 426 [M-H]-
5e 4-CH3 362 360 [M-2H]-, 345 [M-OH]-
5f 2,6-Cl2 417 416 [M-H]-
Nhn xt: Qua kt qu phn tch ph bng 3.5 v ph (ph lc 13-18)
chng ti thy cc ph u c pc phn t c s khi ng bng s khi d kin vi
cng mnh nht, ph t pc phn mnh, cc pc phn mnh u c cng
nh. V vy c th khng nh ph l ph hp vi cng thc phn t d kin ca
6 cht 5a-f [25].
c. Ph cng hng t ht nhn 1H (1H-NMR)
Bng 3.7: Kt qu phn tch ph 1H-NMR ca cc cht 5a-f
Cht R S liu phn tch 1H-NMR
5a H
1H-NMR (500 MHz, DMSO-d6, ppm): 1,26 (4H, m,
CH2); 1,47-1,49 (2H, m, CH2); 1,59-1,61 (2H, m, CH2);
1,92-1,95 (2H, m, CH2); 2,47-2,50 (2H, m, CH2); 7,51-7,52 (3H, m, H-2,H-4,H-6); 7,92-7,93 (2H, m, H-3, H-5);
8,65 (1H, s, OH); 10,36 (1H, s, NH); 12,61 (1H, s, NH).
5b 4-N(CH3)2
1H-NMR (500 MHz, DMSO-d6, ppm): 1,27 (4H, m,
CH2); 1,47-1,50 (2H, m, CH2); 1,58-1,61 (2H, m, CH2);
1,95 (2H, t, CH2); 2,46-2,50 (2H, m, CH2); 2,98 (6H, s, -CH3); 6,79 (2H, d, H-3, H-5); 7,71 (2H, d, H-2, H-6);
10,37 (1H, s, NH); 12,41 (1H, s, NH).
38
5c 4-F
1H-NMR (500 MHz, DMSO-d6, ppm): 1,25-1,26 (4H, m, CH2); 1,46-1,50 (2H, m, CH2); 1,56-1,60 (2H, m, CH2);
1,93-1,96 (2H, m, CH2); 2,47-2,50 (2H, m, CH2); 7,34
(2H, t, H-3, H-5); 7,95-7,98 (2H, m, H-2, H-6); 10,42(1H,
s, NH); 12,63 (1H, s, NH).
5d 4-Br
1H-NMR (500 MHz, DMSO-d6, ppm): 1,27-1,29 (4H, m,
CH2); 1,45-1,49 (2H, m, CH2); 1,59-1,61 (2H, m, CH2);
1,94 (1H, t, CH2); 2,19 (1H, t, CH2); 2,48-2,50 (2H, m,
CH2); 7,71 (2H, d, H-3, H-5); 7,86-7,88 (2H, m, H-2, H-6); 10,37(1H, s, NH); 12,68 (1H, s, NH).
5e 4-CH3
1H-NMR (500 MHz, DMSO-d6, ppm): 1,27-1,29 (4H, m,
CH2); 1,47-1,50 (2H, m, CH2); 1,58-1,61 (2H, m, CH2);
1,94 (1H, t, CH2a); 2,19 (1H, t, CH2b); 2,35 (3H, s, -CH3); 2,47-2,50 (2H, m, CH2); 7,32 (2H, d, H-3, H-5); 7,80 (2H,
d, H-2, H-6); 10,36 (1H, s, NH); 12,59 (1H, s, NH).
5f 2,6-Cl2
1H-NMR (500 MHz, DMSO-d6, ppm): 1,22-1,29 (4H, m,
CH2); 1,47-1,49 (2H, m, CH2); 1,59-1,61 (2H, m, CH2);
1,95 (2H, t, CH2); 2,50-2,54 (2H, m, CH2); 7,61 (1H, t, H-4); 7,67 (2H, d, H-3, H-5); 8,75 (1H, s ,OH); 10,46 (1H,
s, NH). Ghi ch: : chuyn dch ha hc (ppm); s: singlet, vch n; d: doublet, vch i; t: triplet, vch ba; m: multiplet, a vch.
Nhn xt: T ph 1H-NMR ca cc cht tng hp c c th thy s lng
proton, dch chuyn, bi tn hiu l ph hp vi cng thc cu to d kin
ca c 6 cht 5a-f [2]. C th, cc cht u c cc proton ca vng benzen nm
trong vng dch chuyn t 6,8-7,9 ppm v 12 proton ca phn cu ni nm trong
vng dch chuyn t 1,2-2,5 ppm. Hai proton ca 2 nhm -NH-CO- u xut hin
r, di dng 2 vch n khong 10,3-12,7 ppm. Tn hiu ca proton nhm OH
xut hin dng vch n nhng b gin rng khong 8,6-8,8 ppm trn ph
ca cht 5a v 5f, nhng ph ca cht 5b-e khng c tn hiu ny. C th do
proton ca nhm OH linh ng d b trao i hay h bin nn cho tn hiu rt yu
hoc khng cho tn hiu trn ph . Ngoi ra, hai dn cht 5b v 5e c nhm th
cha proton trn vng benzen tng ng l -N(CH3)2 v -CH3 u cho tn hiu ca
cc proton trong nhm th rt r rng.
39
d. Ph cng hng t ht nhn13C (13C-NMR)
Bng 3.8: Kt qu phn tch ph 13C-NMR ca cc cht 5a-f
Cht R S liu phn tch 13C-NMR
5a H
13C-NMR (125 MHz, DMSO-d6, ppm): 171,65 (C5);
169,22 (C1); 161,74 (C8); 158,37 (C2); 130,59 (C1); 130,26 (C4); 129,40 (C2-C6); 126,92 (C3-C5); 34,87 (C2);
30,25 (C7); 28,30 (C4); 28,25 (C5); 24,99 (C3); 24,50
(C6).
5b 4-N(CH3)2
13C-NMR (125 MHz, DMSO-d6, ppm): 171,26 (C5); 169,09 (C1); 162,25 (C8); 156,64 (C2); 151,52 (C4);
127,89 (C2-C6); 117,52 (C1); 112,06 (C3-C5); 34,78 (C2);
32,16 (C7); 28,23 (C4); 28,18 (C5); 24.91 (C3); 24,48
(C6).
5c 4-F
13C-NMR (125 MHz, DMSO-d6, ppm): 171,62 (C5);
169,21 (C1); 162,30 (C4); 160,60 (C8); 158,39 (C2);
129,22 (C2); 129,15 (C6); 126,88 (C1); 116,47 (C3); 116,30
(C5); 34,83 (C2); 32,22 (C7); 28,28 (C4); 28,23 (C5); 24,97 (C3); 24,47 (C6).
5d 4-Br
13C-NMR (125 MHz, DMSO-d6, ppm): 174,37 (C5);
171,63 (C1); 160,58 (C8); 158,56 (C2); 132,26 (C3-C5);
129,34 (C1); 128,71 (C2-C6); 123,76 (C4); 34,80 (C2);
33,59 (C7); 32,17 (C4); 28,18 (C5); 24,91 (C3); 24,42 (C6).
5e 4-CH3
13C-NMR (125 MHz, DMSO-d6, ppm): 174,40 (C5);
171,53 (C1); 161,71 (C8); 157,98 (C2); 140,39 (C4);
129,85 (C3-C5); 127,53 (C1); 126,78 (C2-C6); 34.80 (C2); 33,61 (C7); 28,18 (C4-C5); 24.94 (C6); 24,43 (C3);
20,93 (C, CH3).
5f 2,6-Cl2
13C-NMR (125 MHz, DMSO-d6, ppm): 172,04 (C5);
169,07 (C1); 160,46 (C8); 155,32 (C2); 135,05 (C2-C6); 132,86 (C1); 128,66 (C3-C5); 128,34 (C4); 34,82 (C2);
32,16 (C7); 28,24 (C4-C5); 24,92 (C3); 24,42 (C4). Ghi ch: : chuyn dch ha hc (ppm)
40
Nhn xt: Qua kt qu phn tch ph bng 3.7 v ph (ph lc 25-30)
chng ti nhn thy: s lng carbon, dch chuyn ha hc ca cc carbon l
ph hp vi cng thc cu to d kin [2]. Cc cht u c 2 pc ca 2 nhm C=O,
2 pc ca vng thiadiazol, cc pc ca nhn benzen v cc pc ca 6 nhm CH2.
* Nh vy, kt hp kt qu phn tch cc ph IR, MS, 1H-NMR v 13C-NMR
chng ti c th khng nh 6 cht 5a-f tng hp c c cng thc cu to ng
nh d kin. Trn c s , cc acid hydroxamic ny tip tc c tin hnh th
hot tnh sinh hc.
3.2. TH HOT TNH SINH HC
3.2.1. Th hot tnh sinh hc
3.2.1.1. Tc dng c ch HDAC
Cc acid hydroxamic 5a-f c th nghim tc dng c ch HDAC ti khoa
Dc, i hc Chungbuk (Cheongju, Hn Quc). Kt qu c trnh by bng
3.10 trong phn bn lun.
3.2.1.2. Hot tnh khng t bo ung th in vitro
Cc cht 5b-f c nh gi hot tnh khng t bo ung th ngi trn dng
t bo ung th i trng (SW620). Kt qu hot tnh ca cht 5a trch dn t lun
vn thc s ca Th nh Tuyt [4]. Kt qu gi tr IC50 c th ca cc cht c
trnh by bng 3.10 trong phn bn lun.
3.2.2. nh gi mc ging thuc
Bng 3.9: nh gi mc ging thuc ca cc cht 5a-f theo quy tc Lipinsky
Cht R LogP KLPT S NH,
OH S N, O
S lin kt linh ng
5a H 1,35 348 3 7 3 5b 4-N(CH3)2 1,53 391 3 8 3 5c 4-F 1,55 366 3 7 3 5d 4-Br 2,24 427 3 7 3 5e 4-CH3 1,90 362 3 7 3 5f 2,6-Cl2 2,64 417 3 7 3
41
Nhn xt :Tt c cc cht u tha mn c 5 yu cu ca quy tc Lipinsky
v ging thuc. Cng vi kt qu hot tnh sinh hc in vitro tt, cc cht c
nhiu trin vng cho vic nghin cu tc dng in vivo.
3.3. BN LUN
3.3.1. Tng hp ha hc
Cc cht 5a-f v cc cht trung gian m chng ti tng hp nhn chung l cc
cht c cu to phc tp, c mch C di, c cc nhm chc nhy cm vi cc yu t
ca mi trng phn ng. V vy khi thc hin phn ng chng ti cn m bo
nghim ngt v iu kin phn ng. C th:
- Vi phn ng tng hp cc ester 4a-f, cn m bo dng c phi kh hon
ton, cc ha cht v dung mi phi khan nc. V CDI d b phn hy v khng
cn kh nng hot ha acid monomethyl suberic nu tip xc vi nc.
- Vi phn ng tng hp acid hydroxamic 5a-f: trnh phn hy ester thnh
acid carboxylic bi dung dch NaOH m c, cn lm lnh hn dch cht 4a-f v
dung dch NaOH trc khi cho vo bnh phn ng. Phn ng cng cn tin hnh
nhit thp vi thi gian phn ng ngn trnh phn hy nhm amid. Lng
NaOH phi dng d chuyn ht NH2OH.HCl thnh NH2OH tan v ng thi
m bo cc acid hydroxamic to thnh tan ht trong dung dch phn ng.
3.3.2. Tc dng sinh hc
h tr trong qu trnh thit k cu trc v tm hiu kh nng gn vo trung
tm hot ng trn enzym HDAC ca cc cht, chng ti tin hnh nghin cu
docking vi 6 cht 5a-f. So snh m hnh tng tc vi HDAC8 ca cc cht 5a-f
v SAHA c th thy phn cu ni gia nhm kha hot ng v nhm gn vi
Zn2+ ca 6 cht cng nh ca SAHA c cu trc tng i ging nhau. Nh vy cu
ni vi di 6C ca cc cht tng hp c c nhiu kh nng a c nhm -
NHOH tin gn ion Zn2+ ging nh SAHA. Bn cnh , nng lng lin kt ca
cc cht 5a-f vi trung tm hot ng ca HDAC8 c d on t -7,4 n -6,6
kcal/mol, ngha l u ln hn so vi SAHA (-4,4 kcal/mol), chng t cc cht c
i lc vi HDAC8 mnh hn SAHA. C th gii thch do trong nhm nhn din b
42
mt ca c 6 cht ny u c nguyn t S v N lm tng tng tc Van der Waals
ca cc cht vi cc acid amin trn ming ti ca enzym HDAC8. Tm li, kt qu
docking cho thy cc cht 5a-f ph hp vi hng thit k cu trc nhm tm kim
nhng cht c kh nng c ch HDAC. Trn c s chng ti tip tc tin
hnh tng hp ha hc v th tc dng sinh hc.
Bng 3.10: Kt qu th tc dng c ch HDAC v hot tnh khng t bo ung th
Cht R Tc dng c ch HDAC1
c tnh t bo (IC50) 2
(g/ml) (M) 5a H +3 4,690 13,477 5b 4-N(CH3)2 + 1,070 2,737 5c 4-F + 0,394 1,077 5d 4-Br + 0,857 2,007 5e 4-CH3 + 1,878 5,188 5f 2,6-Cl2 + 3,296 7,904
SAHA + 0,976 3,697 Ghi ch: 1Hot tnh ca enzym b c ch hon ton khi th ti 10g/mL bng phng php Western Blot; 2Nng c ch 50% s pht trin ca t bo ung th SW620; 3Hin tng deacetyl
ha khng xy ra (hot tnh ca enzyme b c ch hon ton).
Kt qu th tc dng trn enzym HDAC cho thy c 6 cht 5a-f u lm cho
hot tnh enzym b c ch hon ton khi th ti nng 10 g/ml bng phng
php Western Blot. T kt qu ny c th kt lun c 6 cht tng hp c u c
tc dng c ch HDAC.
Tc dng c ch HDAC quyt nh tc dng khng t bo ung th, tuy nhin
gy ra c tnh vi t bo cc cht phi c kh nng thm qua mng sinh hc vo
trong t bo tip cn vi HDAC. Do chng ti tip tc tin hnh th c tnh t
bo in vitro nhm tm ra nhng cht thc s c tc dng sinh hc tt. Th nghim
tin hnh trn dng t bo ung th i trng SW620 cho thy IC50 ca cc u nh
hn 20 g/ml, tc l c 6 cht u c hot tnh khng t bo ung th SW620. Do
cc cht tc dng theo c ch phn t nn so snh hot tnh gia cc cht chng
43
ti quy i gi tr IC50 t g/ml sang M. Khi gi tr IC50 ca 5a-f l t 1,007
M n 13,477 M, so vi SAHA (IC50= 3,697 M) chng ti c 3 cht 5b, 5c, 5d
c tc dng tt hn, cc cht cn li nng c ch 50% s pht trin ca t bo
ung th u tng i thp. Kt qu th c tnh t bo cng cho thy s thay i
nhm th trn khung 5-phenyl-1,3,4-thiadiazol mang li hot tnh khng t bo ung
th khc nhau r rt. C th, cht 5a khng mang nhm th trn nhn phenyl c
hot tnh thp nht (IC50= 13,477 M), nhng khi c nhm th d l nhm ht hay
y in t th hot tnh cng tng ln ng k. So snh hot tnh ca cc cht vi
5a c th thy, cht 5b c thm 1 nhm y in t ( -N(CH3)2) trn nhn phenyl
th IC50 gim khong 5 ln, cht 5e cng mang nhm y in t -CH3 trn nhn
phenyl c IC50 gim 2,5 ln. Vi cht 5c, 5d c nhm th ht in t (-F, -Br) gi tr
IC50 cn thp hn rt nhiu v thp hn c SAHA. So snh vi cht c nhm th 4-
Cl cng b trc y trong lun vn thc s ca Th nh Tuyt th 5c cng c
c tnh trn dng t bo SW620 cao hn (IC50 = 1,007 M vi 1,63 M). Tuy
nhin, cht 5f c 2 nhm th trn nhn phenyl tc dng c tnh t bo li gim so
vi cc cht c 1 nhm th, mc d vn cao hn cht khng c nhm th. C th do
2 nhm th ca cht 5f lm mt phng ca vng benzen xoay i nhiu so vi vng
thiadiazol v cu trc ny khng ph hp vi trung tm hot ng ca HDAC. Tm
li, s c mt ca 1 nhm th trn nhn phenyl l cn thit i vi cc dn cht N1-
hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)octandiamid c hot tnh sinh hc tt
hn SAHA.
Vi nhng kt qu thu c c th khng nh cu ni gia khung 5-phenyl-
1,3,4-thiadiazol v nhm NHOH c di 6C l ph hp em hot tnh c ch
HDAC cng nh c tnh trn t bo ung th v khung 5-phenyl-1,3,4-thiadiazol c
nhiu trin vng thay th nhn phenyl ca SAHA nhm tm ra cc cc cht c ch
HDAC mi.
Thm vo , cc cht ch 5a-f tng hp c khng ch c hot tnh sinh
hc cao m cn tha mn c 5 yu cu ca quy tc Lipinsky, iu ny m ra trin
vng cho vic nghin cu cn lm sng v lm sng ca cc cht sau ny.
44
KT LUN V KIN NGH
I. KT LUN
T cc kt qu nghin cu trnh by c th rt ra mt s kt lun sau:
1.1. V tng hp v khng nh cu trc
* tng c 6 cht nh d kin, trong 5 cht 5b-f cha tng c cng
b trong bt k ti liu no:
- N1-hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)octandiamid (5a).
- N1-{5-[4-(dimethylamino)phenyl]-1,3,4-thiadiazol-2-yl}-N8-
hydroxyoctandiamid (5b).
- N1-[5-(4-flourophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid (5c).
- N1-[5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid (5d).
- N1-hydroxy-N8-[5-(4-methylphenyl)-1,3,4-thiadiazol-2-yl]octandiamid (5e).
- N1-[5-(2,6-diclorophenyl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctandiamid
(5f).
* khng nh cu trc cc cht tng hp c nh phn tch cc d liu
ph IR, MS, 1H-NMR, 13C-NMR.
1.2. V hot tnh sinh hc
* th tc dng c ch HDAC v hot tnh khng t bo ung th ca cc
cht tng hp c, kt qu cho thy:
- V tc dng c ch HDAC: c 6 cht u c tc dng c ch HDAC.
- V tc dng khng t bo ung th: c 6 cht u c hot tnh c ch mnh
vi dng t bo ung th i trng SW620. Trong cht 5c (IC50 = 1,007 M) c
tc dng mnh nht, mnh gp 3 ln so vi SAHA (IC50 = 3,697 M).
II. KIN NGH
- Tin hnh th hot tnh khng t bo ung th in vitro ca cc cht tng hp
c trn cc dng t bo ung th khc, tm ra cht c tc dng mnh lm c s cho
vic th tc dng in vivo.
- Nghin cu tng hp dy cht thay th khung 5-phenyl-1,3,4-thiadiazol bng
cc nhm ng cu in t v cc vng thm khc.
TI LIU THAM KHO
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3. Trng Thanh Tng, (2012), Tng hp mt s acid hydroxamic hng c ch
histon deacetylase, Kha lun Dc s i hc, i hc Dc H Ni.
4. Th nh Tuyt, (2012), Tng hp mt s acid hydroxamic mang khung 5-
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thc s Dc hc, i hc Dc H Ni.
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PH LC
Ph lc 1 : Ph hng ngoi IR ca 5a
Ph lc 2 : Ph hng ngoi IR ca 5b
Ph lc 3 : Ph hng ngoi IR ca 5c
Ph lc 4 : Ph hng ngoi IR ca 5d
Ph lc 5 : Ph hng ngoi IR ca 5e
Ph lc 6 : Ph hng ngoi IR ca 5f
Ph lc 7 : Ph khi MS ca 4a
Ph lc 8 : Ph khi MS ca 4b
Ph lc 9 : Ph khi MS ca 4c
Ph lc 10 : Ph khi MS ca 4d
Ph lc 11 : Ph khi MS ca 4e
Ph lc 12 : Ph khi MS ca 4f
Ph lc 13 : Ph khi MS ca 5a
Ph lc 14 : Ph khi MS ca 5b
Ph lc 15 : Ph khi MS ca 5c
Ph lc 16 : Ph khi MS ca 5d
Ph lc 17 : Ph khi MS ca 5e
Ph lc 18 : Ph khi MS ca 5f
Ph lc 19: Ph 1H-NMR ca 5a
Ph lc 20: Ph 1H-NMR ca 5b
Ph lc 21: Ph 1H-NMR ca 5c
Ph lc 22: Ph 1H-NMR ca 5d
Ph lc 23: Ph 1H-NMR ca 5e
Ph lc 24: Ph 1H-NMR ca 5f
Ph lc 25: Ph 13C-NMR ca 5a
Ph lc 26: Ph 13C-NMR ca 5b
Ph lc 27: Ph 13C-NMR ca 5c
Ph lc 28: Ph 13C-NMR ca 5d
Ph lc 29: Ph 13C-NMR ca 5e
Ph lc 30: Ph 13C-NMR ca 5f
Ph lc 1: Ph hng ngoi IR ca 5a
Ten may: GX-PerkinElmer-USA Resolution: 4cm-1
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
Nguoi do: Phan Thi Tuyet MaiTen mau: T1Date: 3/5/2012
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.0
0.0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100.0
cm-1
%T
3251
3050
2927
2865
1629
1558
1384
1339
1311
1179
1094
974
841
759
689
668
Ph lc 2: Ph hng ngoi IR ca 5b
Ten may: GX-PerkinElmer-USA Resolution: 4cm-1
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
Nguoi do: Phan Thi Tuyet MaiTen mau: 3IDate: 11/26/2012
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.0
0.0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80.0
cm-1
%T
3227
2926
2853 1652
16091570
1539
1459
1439
1424
1371
1344
1305
1234
1189
1121
1095
1059
1040
974
946
890
870
851
833
809
758
741
723
704
686
663N N
SHN
O
O
NH
OH
N
Ph lc 3: Ph hng ngoi IR ca 5c
Ten may: GX-PerkinElmer-USA Resolution: 4cm-1
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
Nguoi do: Phan Thi Tuyet MaiTen mau: 3EDate: 11/26/2012
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0
0.0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100.0
cm-1
%T
3239
3054
2939
2853 1687
1628
1597
1557
1515
1465
14451416
1383
1342
1312
1301
1272
1232
1178
1159
1110
1098 991
974
841
728
660
606
584
517
Ph lc 4: Ph hng ngoi IR ca 5d
Ten may: GX-PerkinElmer-USA Resolution: 4cm-1
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
Nguoi do: Phan Thi Tuyet Maiten mau: 3FDate: 11/26/2012
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0
0.0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100.0
cm-1
%T
3163
3049
2923
2850
1691
1616
1571
1557
1496
1443
1409
1394
1340
1317
1302
1252
1230
1175
1116
1103
1069
1041
1011
985
964
829
792
727
705
637
619
603
500
Ph lc 5: Ph hng ngoi IR ca 5e
Ten may: GX-PerkinElmer-USA Resolution: 4cm-1
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
Nguoi do: Phan Thi Tuyet MaiTen mau: 3GDate: 12/7/2012
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.0
0.0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100.0
cm-1
%T
3162
3023
2929
2863
1702
1615
1561
1463
1442
1410
1385
1335
1306
1270
1242
1177
1101
985
966
883
819
792 709
658
630
606
Ph lc 6: Ph hng ngoi IR ca 5f
Ten may: GX-PerkinElmer-USA Resolution: 4cm-1
BO MON HOA VAT LIEU-KHOA HOA-TRUONG DHKHTN
Nguoi do: Phan Thi Tuyet MaiTen mau: 12lDate: 10/15/2012
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0
0.0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90.0
cm-1
%T 3155
2927
2856 1701
1629
1534
1431
1383
1302
1242
1192
1166
1130
1091 982
787
734
494
409
Ph lc 7: Ph khi MS ca 4a Ph lc 8: Ph khi MS ca 4b
S
NN
NH
O
OCH3
O
Ph lc 9: Ph khi MS ca 4c Ph lc 10: Ph khi MS ca 4d
Ph lc 11: Ph khi MS ca 4e Ph lc 12: Ph khi MS ca 4f
Ph lc 13: Ph khi MS ca 5a Ph lc 14: Ph khi MS ca 5b
Ph lc 15: Ph khi MS ca 5c Ph lc 16: Ph khi MS ca 5d
Ph lc 17: Ph khi MS ca 5e Ph lc 18: Ph khi MS ca 5f
Ph lc 19: Ph 1H-NMR ca 5a
Ph lc 19: Ph 1H-NMR ca 5a (m rng)
Ph lc 20: Ph 1H-NMR ca 5b
Ph lc 20: Ph 1H-NMR ca 5b (m rng)
Ph lc 21: Ph 1H-NMR ca 5c
Ph lc 21: Ph 1H-NMR ca 5c (m rng)
Ph lc 22: Ph 1H-NMR ca 5d
Ph lc 22: Ph 1H-NMR ca 5d (m rng)
Ph lc 23: Ph 1H-NMR ca 5e
Ph lc 23: Ph 1H-NMR ca 5e (m rng)
Ph lc 24: Ph 1H-NMR ca 5f
Ph lc 24: Ph 1H-NMR ca 5f (m rng)
Ph lc 25: Ph 13C-NMR ca 5a
Ph lc 26: Ph 13C-NMR ca 5b
Ph lc 27: Ph 13C-NMR ca 5c
Ph lc 28: Ph 13C-NMR ca 5d
Ph lc 29: Ph 13C-NMR ca 5e
Ph lc 30: Ph 13C-NMR ca 5f