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B Y T
TRNG I HC DC H NI
NG TH KHNH
NGHIN CU TNG HP V TH
TC DNG SINH HC MT S DN
CHT 2-ARYLBENZIMIDAZOL
KHA LUN TT NGHIP DC S
H NI 2013
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B Y T
TRNG I HC DC H NI
NG TH KHNH
NGHIN CU TNG HP V TH
TC DNG SINH HC MT S DN
CHT 2-ARYLBENZIMIDAZOL
KHA LUN TT NGHIP DC S
Ngi hng dn:
1. PGS. TS. Nguyn nh Luyn
2. DS. Phm Th Hin
Ni thc hin:
B mn Cng nghip Dc
Trng i hc Dc H Ni
H NI 2013
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LI CM N
Sau mt thi gian lm vic khn trng c s gip tn tnh ca cc
thy c gio, gia nh cng bn b, ti hon thnh Kha lun tt nghip Nghin
cu tng hp v th tc dng sinh hc mt s dn cht 2-arylbenzimidazol.
Vi tt c s knh trng, trc tin ti xin by t lng bit n su sc n
thy gio PGS. TS Nguyn nh Luyn trc tip hng dn, ch bo tn tnh v
to mi iu kin gip ti nghin cu thc hin kha lun ny.
Ti cng xin gi li cm n chn thnh ti thy gio DS. Nguyn Vn
Giang, DS. Phm Th Hin v CN. Phan Tin Thnh ca T mn Tng hp Ha
dc - B mn Cng nghip dc hng dn, gip v to mi iu kin tt
nht cho ti trong sut thi gian thc hin kha lun va qua.
Ti cng xin c gi li cm n n cc thy, c gio thuc B mn Cng
nghip Dc, cng nh cc thy c trong Trng i hc Dc H Ni dy bo
ti tn tnh trong sut nm nm hc v to iu kin thun li cho ti hon thnh
kha lun tt nghip ny.
Cui cng, ti xin gi li bit n su sc n gia nh, bn b nhng ngi
ng vin, gip ti sut thi gian i hc v trong sut qu trnh thc hin
ti Kha lun tt nghip.
H Ni, ngy 21 thng 05 nm 2013
Sinh vin
ng Th Khnh
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MC LC
T VN ............................................................................................................. 1
CHNG I: TNG QUAN ....................................................................................... 2
1.1. TNG QUAN V TC DNG SINH HC CA MT S DN CHT 2-
ARYL BENZIMIDAZOL ............................................................................... 2
1.1.1. Tc dng khng nm, khng khun ............................................................ 2
1.1.2. Tc dng chng ung th ............................................................................. 3
1.1.3. Tc dng chng tng huyt p .................................................................... 8
1.1.4. Tc dng chng virus ................................................................................. 8
1.2. CC PHNG PHP TNG HP MT S DN CHT 2-ARYL
BENZIMIDAZOL ........................................................................................... 9
1.2.1. ng vng to nhn 2-aryl-1H-benzimidazol t o-phenylendiamin hoc
dn cht ca o-phenylendiamin .................................................................. 9
1.2.1.1. Phn ng vi acid ................................................................................. 9
1.2.1.2. Phn ng vi aldehyd ......................................................................... 10
1.2.2. To cc dn cht trn nhn 2-aryl-1H-benzimidazol ............................... 12
1.2.2.1. Phn ng bromo ha ........................................................................... 12
1.2.2.2. Phn ng alkyl ha ............................................................................. 13
1.2.3. Phn ng th vo v tr N-1 trn nhn 2-aryl-1H-benzimidazol ............... 15
CHNG II: NGUYN LIU, THIT B, NI DUNG V PHNG PHP
NGHIN CU .......................................................................................................... 16
2.1. NGUYN LIU V THIT B .................................................................... 16
2.1.1. Ha cht v dung mi .............................................................................. 16
2.1.2. Thit b, dng c ...................................................................................... 17
2.2. NI DUNG NGHIN CU ......................................................................... 18
2.3. PHNG PHP NGHIN CU .................................................................. 19
2.3.1. Tng hp ha hc v kim tra tinh khit ............................................ 19
2.3.2. Xc nh cu trc ..................................................................................... 19
2.3.3. Th tc dng sinh hc .............................................................................. 20
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CHNG III: THC NGHIM, KT QU V BN LUN .............................. 21
3.1. HA HC ...................................................................................................... 21
3.1.1. Tng hp ha hc ..................................................................................... 21
3.1.1.1. Tng hp 4-(1H-benzimidazol-2-yl)-2-methoxyphenol (I) ............... 21
3.1.1.2. Tng hp 4-(1H-benzimidazol-2-yl)-2-bromo-6-methoxyphenol (II)
............................................................................................................ 22
3.1.1.3. Tng hp 2-(4-benzyloxy)-3-bromo-5-methoxyphenyl)-1H-
benzimidazol (III) ............................................................................... 24
3.1.1.4. Tng hp ethyl 2-[2-(4-benzyloxy-3-bromo-5-methoxyphenyl)-1H-
benzimidazol-1-yl] acetat (IV) ........................................................... 25
3.1.2. Kim tra tinh khit .............................................................................. 26
3.1.3. Xc nh cu trc ..................................................................................... 28
3.1.3.1. Kt qu ph hng ngoi (IR) .............................................................. 28
3.1.3.2. Kt qu ph khi lng (MS) ............................................................. 28
3.1.3.3. Kt qu ph cng hng t ht nhn proton (1H-NMR) .................... 29
3.2. TH TC DNG SINH HC ...................................................................... 31
3.2.1. Th hot tnh khng khun ...................................................................... 31
3.2.1.1. Ging vi sinh vt kim nh ............................................................... 31
3.2.1.2. Mi trng th nghim ...................................................................... 32
3.2.1.3. Nguyn tc .......................................................................................... 32
3.2.1.4. Tin hnh ............................................................................................ 32
3.2.1.5. Kt qu th nghim ............................................................................ 33
3.2.2. Th hot tnh khng nm ......................................................................... 34
3.2.2.1. Ging vi sinh vt kim nh ............................................................... 34
3.2.2.2. Mi trng th nghim ...................................................................... 34
3.2.2.3. Nguyn tc .......................................................................................... 35
3.2.2.4. Tin hnh ............................................................................................ 35
3.2.2.5. Kt qu th nghim ............................................................................ 36
3.2.3. Th hot tnh gy c t bo .................................................................... 37
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3.2.3.1. Nguyn tc .......................................................................................... 37
3.2.3.2. Cch tin hnh .................................................................................... 37
3.2.3.3. Kt qu ................................................................................................ 38
3.3. BN LUN ................................................................................................... 39
3.3.1. V tng hp ha hc ................................................................................ 39
3.3.1.1. Phn ng ng vng to nhn benzimidazol v phn ng th trn
nhn thm ........................................................................................... 39
3.3.1.2. Phn ng th v tr N-1 trn nhn benzimidazol ............................. 40
3.3.2. V tc dng sinh hc ................................................................................ 40
3.3.2.1. V tc dng khng khun ................................................................... 40
3.3.2.2. V tc dng khng nm ...................................................................... 41
3.3.2.3. V tc dng gy c t bo ................................................................. 41
KT LUN V XUT ...................................................................................... 42
TI LIU THAM KHO .............................................................................................
PH LC ......................................................................................................................
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CH GII CH VIT TT
1H-NMR
Ph cng hng t ht nhn proton (1H Nuclear Magnetic
Resonance proton)
ADN Acid 2-deoxynucleic
ATCC American Type Culture Collection
BrdU 5-bromo-2'-deoxyuridin
BVDV Virus gy tiu chy b (Bovine Viral Diarhoea)
CVB Coxsackie B virus
DMF N,N-dimethylformamid
DMSO Dimethyl sulfoxid
EC50 Nng 50% tc dng ti a (Effective Concentration of 50%)
EtOH Ethanol
HIV Virus suy gim mim dch ngi (Human immunodeficiency
virus)
HSV-1 Herpes Simplex Virus
IC50 Nng c ch ti thiu 50% s pht trin ca t bo (Inhibitory
concentration of 50%)
IR Ph hng ngoi (Infrared Spectroscopy)
LM Nng di micromol (Low Micromol)
MIC Nng c ch ti thiu (Minimum Inhibitory Concentration)
MMC Mitomycin C
MRSA T cu vng khng methicillin (Methicillin Resistant
Staphylococus aureus)
MS Ph khi lng (Mass Spectroscopy)
MTT Thuc nhum 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromid
MW Vi sng (Micro Wave)
NTCC National Type Culture Collection
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Rf H s lu gi (Retention factor)
RSKK Refik Saydam National Type Culture Collection
SKLM Sc k lp mng
SNU T bo ung th d dy ngi
THF Tetrahyrdofuran
to
Nhit
tonc Nhit nng chy
VSV Vesicular stomatitis virus
VV Vaccinia virus
YFV Virus gy st vng da (Yellow Fever Virus)
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DANH MC CC BNG
STT Tn bng Trang
1 Bng 1.1: Gi tr IC50 ca cc dn cht benzimidazol-4,7-dion trn 3
dng t bo ung th bng kho nghim MTT 5
2 Bng 1.2: Kt qu tng hp mt s dn cht 2-arylbenzimidazol 11
3 Bng 2.1: Danh mc cc dung mi, ha cht 16
4 Bng 2.2: Danh mc cc thit b 17
5 Bng 2.3: Danh mc cc dng c 17
6 Bng 3.1: Kt qu tng hp ha hc 26
7 Bng 3.2: Gi tr Rf v nhit nng chy (to
nc) ca cc cht 27
8 Bng 3.3: Kt qu ph hng ngoi ca cc cht I-IV 28
9 Bng 3.4: Kt qu ph khi lng ca cc cht I-V 28
10 Bng 3.5: Kt qu ph cng hng t ht nhn proton ca cc cht
I-V 29
11 Bng 3.6: Kt qu th tc dng khng khun 34
12 Bng 3.7: Kt qu th tc dng khng nm 36
13 Bng 3.8: Kt qu th hot tnh gy c t bo ca 2 cht III, IV 39
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DANH MC CC HNH V, TH
STT Tn hnh v, th Trang
1 Hnh 1.1: Mt s dn cht th v tr 2, 5 ca benzimidazol v
benzoxazol 2
2 Hnh 1.2: Cng thc cu to mt s dn cht 4-methoxyphenyl-
1H-benzimidazol 4
3 Hnh 1.3: Cng thc cu to ca mt s dn cht 2-aryl-1H-
benzimidazol 7
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DANH MC CC S
STT Tn s Trang
1 S 1.1: o-phenylendiamin ngng t vi acid carboxylic thm 9
2 S 1.2: 4-cloro-o-phenylenediamin ngng t vi acid 2-amino
benzoic
10
3 S 1.3: Tng hp 2-alkyl v 2-aryl-benzimidazol theo phng
php vi sng
10
4 S 1.4: o-phenylendiamin ng vng vi aldehyd 10
5 S 1.5: o-phenylendiamin ngng t vi aldehyd 11
6 S 1.6: Tng hp dn cht 4-(benzimidazol-2-yl)phenol 12
7 S 1.7: o-phenylendiamin ng vng vi aldehyd thm bng cng
ngh vi sng
12
8 S 1.8: Phn ng th N-1 ca dn cht 2-aryl-1H-benzimidazol 15
9 S 3.1: Cc giai on tng hp cc dn cht 2-phenyl
benzimidazol
21
10 S 3.2: Tng hp 4-(1H-benzimidazol-2-yl)-3-methoxyphenol (I) 22
11 S 3.3: Tng hp bromo vanillin 22
12 S 3.4: Tng hp 4-(1H-benzimidazol-2-yl)-2-bromo-6-
methoxyphenol (II)
23
13 S 3.5: Tng hp 4-benzyloxy-3-bromo-5-methoxybenzaldehyd 24
14 S 3.6: Tng hp 2-(4-benzyloxy)-3-bromo-5-methoxyphenyl)-
1H-benzimidazol (III).
25
15 S 3.7: Tng hp ethyl 2-[2-(4-benzyloxy-3-bromo-5-
methoxyphenyl)-1H-benzimidazol-1-yl] acetat (IV)
25
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1
T VN
Ngy nay, tnh trng lm dng thuc din ra ph bin trn th gii c bit l
cc nc ang hoc km pht trin, m Vit Nam nm trong s . in hnh l
vic s dng thuc khng sinh khng hp l to ra rt nhiu chng vi khun
khng v a khng thuc, khin cho vic iu tr tr nn ngy cng kh khn. Mt
khc, nhim mi trng, ch n ung, sinh hot khng hp l lm xut
hin thm nhiu bnh him ngho nh bnh ung th, bnh tim mch, bnh tiu
ng, bnh ni tit. Thc t ny i hi con ngi cn tm ra nhiu loi thuc mi
c tc dng iu tr hiu qu.
Nhiu cng trnh nghin cu gn y cho thy cc dn cht 2-
arylbenzimidazol l nhm hp cht d vng c tc dng sinh hc rt a dng nh:
khng nm, khng khun, khng t bo ung th, khng vim, h huyt pVic
nghin cu tng hp v nh gi tc dng sinh hc ca chng ha hn rt nhiu
trin vng.
Chnh v vy, chng ti thc hin ti: Nghin cu tng hp v th tc
dng sinh hc ca mt s dn cht 2-arylbenzimidazol vi cc mc tiu sau:
1. Tng hp mt s dn cht 2-arylbenzimidazol vi cc nhm th khc nhau
trn nhn thm v v tr N-1.
2. Th tc dng gy c t bo v khng nm, khng khun ca mt s dn
cht 2-arylbenzimidazol tng hp c.
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2
CHNG I: TNG QUAN
1.1. TNG QUAN V TC DNG SINH HC CA MT S DN CHT
2-ARYL BENZIMIDAZOL
1.1.1. Tc dng khng nm, khng khun
Nm 1997, Sener v cng s tng hp mt s dn cht th v tr 2, 5 ca
benzimidazol v benzoxazol, ng thi th tc dng c ch vi sinh vt in vitro theo
k thut pha long 2 ln lin tip. Tc dng khng khun c th vi 3 dng vi
khun Gram(+) l: Staphylococcus aureus ATCC 6538, Streptococcus faecalis
ATCC 10541, Bacillus subtilis ATCC 6033 v 3 dng vi khun Gram() l:
Escherichia coli ATCC 10536, Klepsiella pneumoniae NTCC 52211, Pseudomonas
aeruginosa RSKK 355. Kt qu cho thy cc cht tng hp c c tc dng khng
khun yu trn S. aureus, S. feacalis vi MIC nm trong khong 25-50 g/ml.
Ring 5-nitro-2-cyclohexylmethylbenzimidazol (X=NH, Y=CH2, R=NO2) c tc
dng tt hn trn S. aureus vi MIC = 12,5 g/ml.
X= O, NH; R= H, Cl, NO2, CH3; Y= CH2 hoc khng c.
Hnh 1.1: Mt s dn cht th v tr 2, 5 ca benzimidazol v benzoxazol
i vi tc dng khng nm, hu ht cc cht tng hp c u c tc
dng tt trn Candida albicans RSKK 628 vi MIC trong khong 12,5-50,0 g/ml
[21].
Nm 2005, G. Hakan cng cng s tin hnh tng hp v th tc dng
khng khun in vitro trn cc chng vi khun S. aureus v S. aureus khng
methicilin (MRSA), E. coli, E. faecalis, khng nm C. albicans ca 22 dn cht 1,2
ca 1H-benzimidazol-N-alkyl-5-carboxamidin. Kt qu cho thy hp cht 1-(2,4-
diclorobenzyl)-N-(2-diethylaminoethyl)-1H-benzimidazol-5-carboxamidin v hp
cht 3,4-diclorophenyl v tr C-2 (1) c hot tnh khng khun kh mnh, vi MIC
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3
= 3,12 mg/ml i vi S. aureus v MRSA, c tc dng tt trn E. coli, E. faecalis,
C. albican [13].
1
Nm 2006, Ayhan Kilchigil v cng s th tc dng chng nm Candida
albicans, Candida glabrata v Candida krusei ca mt s dn cht benzimidazol.
Kt qu th tc dng in vitro hp cht 2-(p-fluorophenyl)-5-nitro-1-
propylbenzimidazol (2) (MIC=12,5 g/ml) c hot tnh i vi C. albicans tng t
nh fluconazol. Tuy nhin cha c hp cht no tng hp c c tc dng tt vi
c ba loi nm nh cc cht i chiu (Fluconazol, Metronidazol) [14].
2
1.1.2. Tc dng chng ung th
ADN topoisomerase l enzym xc tc cho qu trnh tho xon ca ADN siu
xon c Wong pht hin nm 1971. ADN topoisomerase c vai tr quan trng
trong hu ht cc giai on ca chu trnh t bo, n gn vi ADN to thnh phc
hp thun nghch enzym-ADN. Da vo c ch xc tc c hai loi ADN
topoisomerase l ADN topoisomerase I v II. n nm 1984, Tewey v cng s
nghin cu v cng nhn vai tr ca ADN topoisomerase nh l mc tiu phn t
ca cc thuc chng ung th. Camptothecin l thuc u tin gy c vi
topoisomerase I (c chit tch t Camptotheca acuminata) [23]. Sau nm
1993, Chen v cng s nghin cu v tm ra nhm cht mi cha vng
benzimidazol c tc dng gy c vi ADN topoisomerase I, l Hoechst 33258
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4
v Hoechst 33342 thuc dn cht bis-benzimidazol. Bng cch gn vo ADN,
nhng cht ny n nh phc hp thun nghch enzym-ADN, gy mt hot tnh
enzym v cui cng gy cht t bo [7,9,27,28].
Nm 1996, Kim Jung Sun v cng s tng hp mt dy cc dn cht 4-
methoxyphenyl-1H-benzimidazol v th tc dng c ch topoisomerase I. Cht 5-
nitro-2-(4-methoxyphenyl)-1H-benzimidazol th hin tc dng c ch ln nht vi
topoisomerase I. Tri ngc vi cc dn cht bis-benzimidazol trn, nhng dn
cht trong nghin cu c rt t hoc khng c i lc gn vi ADN. Trong th
nghim gy c t bo, nhm cht ny khng c s khng cho vi dng t bo
khng topoisomerase I ca camptothecin. Tip tc, nghin cu mi lin quan cu
trc - tc dng ca 10 cht trong bng di y, cho thy tc dng gy c vi
topoisomerase I hiu qu hn khi nhm th v tr s 5 l cht nhn lin kt hydro
[15].
Hnh 1.2: Cng thc cu to mt s dn cht 4-methoxyphenyl-1H-benzimidazol
Nm 2000, Ahn Chan Mug v cng s nghin cu tc dng chng ung th
trn 3 dng t bo (dng t bo ung th bch cu chut P388, cc dng t bo ung
th biu m d dy ngi SNU-1 v SNU-16) ca mt dy cc dn cht 2-alkyl, 2-
X CN CHO CH2OH CH2NH2 CONH2 COOH H Br NO2 H
Y H H H H H H H H H NO2
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5
aryl, v 2-piperazinylbenzimidazol. Cc hp cht ny c kh nng gy c mnh
chng li tt c ba dng t bo th nghim [6].
Bng 1.1: Gi tr IC50 ca cc dn cht benzimidazol-4,7-dion trn 3 dng t
bo ung th bng kho nghim MTT
Cht IC50 (g/ml)
P388 SNU-1 SNU-2
6-aziridinyl-1,2,5-trimethylbenzimidazol-4,7(1H)-
dion
0,36 0,34 0,18
6-aziridinyl-2-ethy1-1,5-dimethylbenzimidazol-4,7
(1H)-dion
1,20 1,00 0,44
6-aziridinyl-l,5-dimethyl-3-propylbenzimidazol-4,7
(1H)-dion
1.36 1,05 0,37
6-aziridinyl-2-isopropyl-1,5-dimethylbenzimidazol-
4,7(1H)-dion
1,27 3,34 0,30
6-aziridinyl-2-butyl-l,5-dimethylbenzimidazol-
4,7(1H)-dion
2,05 0,95 0,34
6-aziridinyl-2-cyclohexyl-l,5-
dimethylbenzimidazol-
4,7(1H)-dion
1,46 1,43 0,46
2-(m-amino)phenyl-6-aziridinyl-l,5-
dimethylbenzimi
dazol-4,7(1H)-dion
0,21 0,32 0,40
2-(acetamidophenyl)-6-aziridinyl-1,5-
dimethylbenzimidazol-4,7(1H)-dion
0,38 0,26 0,30
6-aziridinyl-l,5-dimethyl-2-(4-methyl-l-piperazinyl)
benzimidazol-4,7(1H)-dion
0,21 0,30 0,09
MMC 0,43 0,40 0,86
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6
Nm 2008, Emine v cng s tip tc nghin cu hot tnh c ch ADN
topoisomerase I, II ca t bo nhn tht vi mt vi dn cht th v tr 2, 5 ca
benzoxazol v benzimidazol. Mi tm dn cht c th hot tnh tho xon ca
ADN topoisomerase I, II trong h thng t bo t do (pBR322 plasmid ADN).
Trong 2-(4-methoxyphenyl)-5-nitro-1H-benzimidazol (3) gy c ch c hai loi
ADN topoisomerase. ng thi vi IC50=248 M, cht ny c hiu lc gy c
ADN topoisomerase I tt hn cht i chiu l camptothecin [18].
3
Nm 2011, Romero Castro cng cng s tng hp v nh gi s b kh
nng chng ung th ca su dn cht 2-aryl-5-nitro-1H-benzimidazol. c tnh t
bo c nh gi trn by dng t bo ung th ngi bng cch s dng kho
nghim MTT. Hp cht 2-(4-cloro-3-nitrophenyl)-5-nitro-1H-benzimidazol (4) c
tc dng tt nht trn dng t bo A549 (dng t bo ung th phi). Hp cht ny
c kh nng gy cht t bo theo chu trnh trn dng t bo K562 (dng t ung th
bch cu), c ch hot ng ca poly-(ADP-ribose)-polymerase (PARP). Nhng kt
qu nghin cu trn cho thy hp cht 4 l mt tc nhn chng ung th mnh [19].
4
Nm 2012, Balram Soni v cng s tng hp v nh gi hot tnh gy c
t bo ca 11 dn xut 2-aryl-1H-benzimidazol chng li cc dng t bo ung th
ngi. Nghin cu c th trn hai dng t bo K-562 (bnh ung th bch cu
nguyn bo ty) v HL-60 (bnh bch cu promyelocytic). Trong s cc hp cht
th nghim vi dng t bo K-562, cc hp cht 1a, 7a v 10a cho thy hot ng
mnh v hp cht 5a tc dng tt c ch s pht trin trn dng t bo HL-60 [8].
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7
R R1
1a H H
2a H -2NO2
3a H -3NO2
4a H -2Cl
5a H -4Cl
6a -2NO2 -2NO2
7a -2NO2 -3NO2
8a -3NO2 -2NO2
9a -3NO2 -3NO2
10a -2Cl -2NO2
11 -2Cl -3NO2
Hnh 1.3: Cng thc cu to ca mt s dn cht 2-aryl-1H-benzimidazol
Nm 2012, Ju-Fang Liu v cng s thc hin nghin cu vi hp cht 1-
benzyl-2-phenyl-benzimidazol (BPB) (5) cho thy BPB c tc dng chng ung th
trn t bo sn ngi. BPB gy cht t bo theo chu trnh c nh gi bng kho
nghim MTT v phn tch dng cytometry. Trong nghin cu ny, ta thy BPB gy
cht t bo theo chu trnh trong dng t bo sn (JJ012 v SW 1353 ). Trong nghin
cu trn ng vt cho thy gim 40% khi lng khi u sau 21 ngy iu tr [16].
5
Nm 2013, Yurttas cng cng s nghin cu tng hp cc dn cht 1-(2-aryl-
2-oxoethyl)-2-[(morpholin-4-yl)thioxomethyl]benzimidazol v th tc dng chng
ung th ca chng. Tc dng chng ung th ca cc hp cht ny c nh gi
bng kho nghim MTT, phng php BrdU trn dng t bo A549 (dng t bo
ung th phi). Hu ht cc hp cht tng hp c tc dng chn lc ng k trn
dng t bo ny [26].
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8
1.1.3. Tc dng chng tng huyt p
Nm 2006, Samuel Estrada-Soto v cc cng s tin hnh nghin cu tc
dng ca ca cc dn cht v tr o-, p-ca nhn phenyl gn vo C2 trn nhn
benzimidazol vi cc nhm th (-H , -CH3 , -NO2 , -CF3) trn ng mch ch ca
chut. Cc dn cht 2-(5-nitro-1H-benzimidazol-2-yl)phenol (6) v 2-(4-
methoxyphenyl)-5-nitro-1H-benzimidazol (3) c tc dng gin mch, chng tng
huyt p [12].
6
1.1.4. Tc dng chng virus
Nm 2007, Vasquez cng cng s nghin cu tng hp cc dn xut ca 1-
benzoyl-2-(2-nitrophenyl)-1H-benzimidazol v th tc dng c ch s sao chp
ngc ca virus HIV-1. Trong , hp cht 1-(2,6-diclorobenzoyl)-2-(2-
nitrophenyl)-1H-benzimidazol (7) cho hot ng c ch chng li RT-HIV l tt
nht [24].
7
Nm 2010, Mechele Tonelli v cng s th tc dng chng virus ca 76 dn
cht 2-phenylbenzimidazol. Kt qu cho thy tc dng chng virus theo th t gim
dn CVB-2, BVDV, HSV-1 v YFV, trong khi khng c tc dng trn HIV-1,
VSV v RSV, VV ch nhy cm vi 1 s hp cht. Hp cht 5,6-dicloro-2-(4-
nitrophenyl)benzimidazol (8) c hot tnh cao v chn lc chng li VV (EC50 = 0,1
-
9
LM), kt qu cho thy c tc dng mnh hn 18 hoc 110 ln so vi cc thuc tham
chiu l acid mycophenolic v 6-azauridin (EC50 = 1,8 LM v 11 LM) v gim c
tc ng c hi ln cc t bo th [29].
8
1.2. CC PHNG PHP TNG HP MT S DN CHT 2-ARYL
BENZIMIDAZOL
1.2.1. ng vng to nhn 2-aryl-1H-benzimidazol t o-phenylendiamin hoc
dn cht ca o-phenylendiamin
1.2.1.1. Phn ng vi acid
Thng thng, o-phenylendiamin c ngng t vi cc acid mono
carboxylic, trong iu kin phn ng loi nc, s dng cc acid nh acid
polyphosphoric (PPA), acid hydrocloric, acid boric hoc acid p-toluensulfonic to
ra cc nhm th khc nhau v tr s 2 trn nhn benzimidazol. Tuy nhin vic s
dng tc nhn yu hn c bit l acid Lewis, mui v c, acid v c ci thin c
hiu sut v tinh khit ca sn phm [11].
S 1.1: o-phenylendiamin ngng t vi acid carboxylic thm
Trong mt s trng hp, phng php Phillips dng HCl 4N v un hi lu
cho php thc hin phn ng nhit thp hn v tng hiu sut phn ng. Vi
cc acid thm nh acid benzoic th hiu sut phn ng thng thp, tng hiu
sut cn thc hin phn ng trong ng kn vi nhit 180oC-185oC [25].
Theo phng php ca Sharma v cng s, tng hp nhn benzimidazol i
t 4-cloro-o-phenylendiamin c ngng t vi acid 2-aminobenzoic s dng acid
polyphosphoric (PPA) theo s sau [22]:
-
10
S 1.2: 4-cloro-o-phenylenediamin ngng t vi acid 2-amino benzoic
T nm 1995, nhiu dn cht benzimidazol c tng hp theo cng ngh
vi sng. Nm 2007, Dubey v Moorthy tng hp cc cht 2-alkyl v 2-
arylbenzimidazol theo phng php ny. Thi gian phn ng gim n pht, hiu
sut tng 10-50% so vi phng php trc y [11].
R= H, CH3, C6H5, 4-NH2C6H4, 4-ClC6H4
S 1.3: Tng hp 2-alkyl v 2-arylbenzimidazol theo phng php vi
sng
1.2.1.2. Phn ng vi aldehyd
Phn ng ca o-phenylendiamin vi aldehyd cn c cht oxy ha to nhn
benzimidazol. Nhng cht oxy ha c s dng nh: nitrobenzen, benzoquinon,
natri metabisulfit, thy ngn (I) oxyd, ch (IV) tetra acetat, iod, ng (II) acetat,
indium perfluorooctan sulfonat[11].
S 1.4: o-phenylendiamin ng vng vi aldehyd thm
Weidenhagen l ngi u tin thc hin phn ng gia diamin v aldehyd
s dng ng acetat l cht oxy ha trong dung mi nc hoc alcol. Theo phng
php ca Weidenhagen, nhiu dn cht benzimidazol th v tr s 2 c tng
hp [25].
Nm 2006, Nagawade v cng s nghin cu tng hp mt s dn cht
benzimidazol t o-phenylendiamin v dn cht vi aldehyd s dng cht xc tc
BF3.OEt2 cho hiu sut rt cao. Phng php ny, c tc dng rt tt vi cc
Cht oxy ha
-
11
aldehyd thm, khng no hoc bo [17]. Di y l mt s dn cht 2-
arylbenzimidazol c tng hp theo phng php ny:
Bng 1.2: Kt qu tng hp mt s dn cht 2-arylbenzimidazol
Diamin Aldehyd Sn phm
Hiu
sut
%
tonc (
oC)
90 288-290
89 292-293
84 310-311
90 198-200
86 220-221
Nm 2012, Chen v cng s nghin cu tng hp cc dn cht v tr C2
trn nhn benzimidazol s dng cht xc tc FeCl3/Al2O3. Kt qu cho thy phn
ng din ra iu kin nh nhng hn, thi gian phn ng ngn hn, hiu sut cao
hn, cht xc tc khng c v r tin [10].
S 1.5: o-phenylendiamin ngng t vi aldehyd
-
12
Nm 2009, Savall v cng s nghin cu tng hp dn cht 2-
arylbenzimidazol s dng cht xc tc l natri metabisulfit (Na2S2O5) cho hiu sut
kh cao trn 85% [20].
S 1.6: Tng hp dn cht 4-(benzimidazol-2-yl)phenol
Gn y, Navarrtete v cng s s dng cng ngh vi sng trong phn
ng ng vng ca o-phenylenediamin vi aldehyd trong tng hp dn cht 2-
(alkyloxyaryl)-1H-benzimidazol vi hiu sut cao. Phn ng c 3 thnh phn v
khng cn dung mi [11].
S 1.7: o-phenylendiamin ng vng vi aldehyd thm bng cng ngh vi sng
1.2.2. To cc dn cht trn nhn 2-aryl-1H-benzimidazol
1.2.2.1. Phn ng bromo ha
a. Phn ng bromo ha
Phn ng bromo ha l phn ng ha hc nhm a 1 hay nhiu nguyn t
brom vo hp cht hu c.
C th to dn xut brom theo 2 phng php chnh nh sau:
- Th hydro hay nhm nguyn t khc bng brom.
- Cng hp brom vo lin kt kp ca phn t hu c.
b. Tc nhn bromo ha
Brom lng
- Cht lng mu nu sm, d bay hi.
- Thng s dng bromo ha cc hp cht thm theo c ch i in t hoc
cng hp v lin kt bi ca hydrocarbon khng no.
-
13
Acid hydrobromic (HBr)
- ng dng cng hp v lin kt bi ca hydocarbon khng no theo c ch phn
ng cng hp, hoc th nhm OH alcol bng brom theo c ch i nhn.
Mui ca brom vi kim loi kim (NaBr)
- Thng dng th cc dn cht clo
Cc tc nhn khc
N-bromosuccinimid (NBS) l mt cht cung cp brom hay c
s dng. Cc phn ng bromo ha dng NBS l: cng brom vo
alken, bromo ha dn xut carbonyl v tr alpha (), bromo ha
hp cht thm [1,3].
1.2.2.2. Phn ng alkyl ha
a. Phn ng alkyl ha
Alkyl ha l qu trnh thay th mt hoc nhiu nguyn t hydro ca hp cht
hu c bng mt hoc nhiu nhm alkyl.
Nhm alkyl c th lin kt trc tip vi carbon, oxy, nit hoc lu hunh
tng ng ta c cc qu trnh C-alkyl ha, O-alkyl ha, N-alkyl ha hoc S-alkyl
ha,
b. Cc tc nhn alkyl ha
Cc alcol (R-OH)
Methanol v ethanol l nhng tc nhn alkyl ha quan trng. Hu nh trong
mi trng hp alkyl ha, phi s dng acid v c lm xc tc.
-
14
Alcol thng dng alkyl ha cc amin hoc alcol khc. Sn phm l cc
amin hoc ester.
Cc alkyl halogenid (R-X)
Cc alkyl halogenid mch carbon ngn (C=1-3) l cht lng c si thp.
Tc nhn hay dng l methyl halogen v ethyl halogen. Alkyl halogenid c s
dng alkyl ha cc amin.
Cc arakyl halogenid (Ar-(CH2)nX)
Benzyl clorid hoc benzyl bromid c s dng nhiu. N thng c
dng bo v nhm -OH ca ng hoc celluose trong ha hc cc
hydratcarbon.
Cc ester ca acid v c cha oxy
Gm cc dialkyl sulfat, alkyl nitrat, alkyl phosphat, dialkyl carbonat. Tc
nhn c s dng nhiu nht l dimethyl sulfat. Tc nhn ny c th alkyl ha
nhm OH phenol trong mi trng kim, nc, nc-alcol, thm ch c trong mi
trng acid. Cc alkyl nitrat rt t khi s dng v d gy n.
Cc ester ca acid sulfonic
Cc ester hu c ca acid sulfonic thm c ngha trong thc t. Vic s
dng n ging vi dialkyl-sulfat. Nhm tc nhn ny thng c dng alkyl
ha nhm OH phenol hoc NH amin. Mt s tc nhn in hnh l methyl-
benzensulfonat, benzyl-benzensulfonat hoc methyl, benzyl ester ca acid p-toluen
sulfonic.
Cc mui amoni bc 4
Trong phenyl-trimethyl-amoni clorid l tc nhn hay c s dng. Tc
nhn ny methyl ha chn lc nhm OH phenol trong mi trng kim.
Cc tc nhn alkyl ha khc
Cc epoxid: c s dng iu ch cc polyether-alcol, cht c tc dng
lm sc cng b mt.
Cc hp cht c kim: Hp cht c magnesi dng iu ch cc ether hn
tp [4].
-
15
1.2.3. Phn ng th vo v tr N-1 trn nhn 2-aryl-1H-benzimidazol
Cc dn cht 2-aryl-1H-benzimidazol thng c th vi tc nhn R-X (X
l halogen) to ra cc nhm th khc nhau v tr N-1 trn nhn 2-
arylbenzimidazol. Phn ng thng c thc hin trong cc dung mi nh: aceton,
DMF, THF Vi cc cht xc tc base: natri hydrid, natri hydroxid, kali carbonat
khan
S 1.8: Phn ng th N-1 ca dn cht 2-aryl-1H-benzimidazol
-
16
CHNG II: NGUYN LIU, THIT B, NI DUNG V PHNG
PHP NGHIN CU
2.1. NGUYN LIU V THIT B
Kha lun s dng mt s ha cht, dung mi v thit b ca phng th
nghim Tng hp Ha Dc B mn Cng Nghip Dc Trng i hc Dc
H Ni. Bao gm:
2.1.1. Ha cht v dung mi
Bng 2.1: Danh mc cc dung mi, ha cht
STT Dung mi, ha cht Ngun gc
1 Aceton Trung Quc
2 Acid acetic bng Merck
3 Acid hydrobromic Trung Quc
4 Benzyl clorid Trung Quc
5 Cloroform Merck
6 Dicloroethan Trung Quc
7 DMF Trung Quc
8 Ethanol Trung Quc
9 Ethyl acetat Trung Quc
10 Ethyl cloroacetat Trung Quc
11 Kali bromat Trung Quc
12 Kali carbonat khan Trung Quc
13 Kali iodid Trung Quc
14 Methanol Trung Quc
15 Methyl cloroacetat Trung Quc
16 Natri metabisulfit Trung Quc
17 Natri sulfat khan Trung Quc
18 n-hexan Trung Quc
19 o-phenylendiamin Merck
-
17
STT Dung mi, ha cht Ngun gc
20 Vanillin Vit Nam
2.1.2. Thit b, dng c
Bng 2.2: Danh mc cc thit b
STT Tn thit b, my mc Ngun gc
1 Cn k thut Sartorius BP2001S c
2 Cn phn tch Mettler Toledo AB204S Thy S
3 My ct quay Buchi B491 Thy S
4 My o nhit nng chy EZ-Melt M
5
My o ph cng hng t ht nhn proton 1H-NMR
Brucker AV-500MHz (Vin Hn lm Khoa hc v Cng
ngh Vit Nam)
M
6 My o ph hng ngoi Impact-410 (Vin Hn lm Khoa
hc v Cng ngh Vit Nam) M
7 My o ph khi lng 1100 Series LC/MSD Trap (Vin
Hn lm Khoa hc v Cng ngh Vit Nam) c
8
My o ph khi lng phn gii cao FT-ICR-MS-Varian
910MS (Vin Hn lm Khoa hc v Cng ngh Vit
Nam)
c
9 My khuy t gia nhit IKA c
10 T sy Memmert c
Bng 2.3: Danh mc cc dng c
STT Tn dng c Ngun gc
1 Bn mng Silicagel GF254 70-230 mesh c
2 Bnh cu 2 c 100ml c
3 Bnh cu y trn loi 50ml, 100ml c
Cc c m 100ml, 250ml, 500ml Trung Quc
-
18
STT Tn dng c Ngun gc
4 a thy tinh, pipet paster Trung Quc
5 Nhit k thy ngn Trung Quc
6 Phu lc thy tinh Trung Quc
7 Phu lc Buchner Trung Quc
8 Pipet chnh xc 1ml, 5ml, 10ml Trung Quc
9 Nhit k thy ngn Trung Quc
10 Phu lc thy tinh Trung Quc
11 Phu lc Buchner Trung Quc
12 Pipet chnh xc 1ml, 5ml, 10ml Trung Quc
2.2. NI DUNG NGHIN CU
- Tng hp ha dc: Tng hp 4 dn cht 2-phenylbenzimidazol, c cng thc
cu to nh sau:
Cht I
Cht II
Cht III Cht IV
- Kim tra tinh khit ca 4 cht I-IV bng SKLM v o nhit nng chy,
xc nh cng thc cu trc cc cht tng hp c bng ph hng ngoi IR, ph
khi lng MS, ph cng hng t ht nhn 1H-NMR.
-
19
- Th tc dng khng nm, khng khun vi cc cht I, II, III, IV.
- Th tc dng chng ung th vi cht III, IV.
2.3. PHNG PHP NGHIN CU
2.3.1. Tng hp ha hc v kim tra tinh khit
S dng phng php tng hp ha hc, da trn 4 phn ng:
- Phn ng ng vng to dn cht th 2-phenylbenzimidazol theo phng php
mc 1.2.1.
- Phn ng bromo ha to dn cht bromo theo phng php mc 1.2.2.
- Phn ng alkyl ha theo phng php mc 1.2.2
- Phn ng th vo v tr N-1 trn nhn 2-phenylbenzimidazol theo phng php
mc 1.2.3.
Dng sc k lp mng theo di tin trin ca phn ng vi 2 h dung mi
khai trin ethylacetat: n-hexan (7: 3) hoc cloroform: methanol (9: 1), hin mu
bng n t ngoi bc sng 254nm.
Kim tra tinh khit ca sn phm bng SKLM c tin hnh trn bn
nhm trng sn silicagel GF254 Merck 70-230 mesh, quan st di n t ngoi
bc sng 254nm, dung mi ha tan: ethanol 96o, h dung mi khai trin
ethylacetat: n-hexan (7: 3) hoc cloroform: methanol (9: 1). o nhit nng chy
bng my o nhit nng chy EZ-Melt.
2.3.2. Xc nh cu trc
Xc nh cu trc ca cc cht tng hp c da trn kt qu phn tch ph
hng ngoi (IR), ph khi lng (MS), ph cng hng t ht nhn proton (1H-
NMR).
Ti phng phn tch cu trc phn t Vin ha hc - Vin Hn lm Khoa hc
v Cng ngh Vit Nam, cc cht I-IV c ghi ph hng ngoi (IR) trn my
vi k thut vin nn KBr ghi trong vng 4000-400 cm-1.
Ti phng phn tch khi ph ca Vin Ha hc - Vin Hn lm Khoa hc v
Cng ngh Vit Nam cc cht I, IV c ghi ph khi lng trn my 1100 Series
-
20
LC/MSD Trap - Agilent, cht III c ghi ph khi lng phn gii cao FT-ICR-
MS-Varian 910MS. Ti phng Ha vt liu -Khoa Ha - i hc Khoa hc T
nhin - i hc quc gia H Ni ph MS ca cht II c ghi trn my LTQ
Orbitrap XL theo phng php EI.
Ti phng phn tch ph - Vin ha hc - Vin Hn lm Khoa hc v Cng
ngh Vit Nam, ph cng hng t ht nhn proton (1H-NMR) c ghi trn my
Brucker AV-500MHz, s dng dung mi l DMSO, ly mc l pic ca cht chun
ni tetramethylsilan (TMS).
2.3.3. Th tc dng sinh hc
Th tc dng khng nm, khng khun ti b mn Vi sinh v sinh hc -
Trng i hc Dc H Ni bng phng php khuch tn trn thch.
Th hot tnh gy c t bo ti Phng Sinh hc thc nghim - Vin ha hc
cc hp cht thin nhin - Vin Hn lm Khoa hc v Cng ngh Vit Nam theo
phng php ca Skehan cng cng s (1990) v Likiwitayawuid cng cng s
(1993) hin ang c p dng ti Vin nghin cu ung th Quc gia ca M
(NCI) v Trng i hc Dc, i hc tng hp Illinois, Chicago, M.
-
21
CHNG III: THC NGHIM, KT QU V BN LUN
3.1. HA HC
3.1.1. Tng hp ha hc
Sau khi tham kho cc ti liu v da vo iu kin phng th nghim, cc
ha cht cho php, cc dn cht 2-phenylbezimidazol c tng hp theo s sau:
S 3.1: Cc giai on tng hp cc dn cht 2-phenylbenzimidazol
3.1.1.1. Tng hp 4-(1H-benzimidazol-2-yl)-2-methoxyphenol (I)
S phn ng:
-
22
S 3.2: Tng hp 4-(1H-benzimidazol-2-yl)-2-methoxyphenol (I)
Tin hnh:
Thm 2,7 g (2,5 mmol) o-phenylendiamin v 3,8 g (2,5 mmol) vanillin vo
bnh cu cha 45 ml DMF, khuy cho n khi tan hon ton. Sau , cho t t 4,75
g (2,5 mmol) Na2S2O5 vo v un hn hp phn ng 900C trong 2 gi.
Sau khi kt thc phn ng, rt t t ton b hn hp vo cc c m cha
150ml nc lnh, khuy mnh trong 30 pht. Lc ly ta, sau em kt tinh li
trong ethanol 40o, sy kh 60-70oC.
Kt qu:
- Cm quan: tinh th mu trng xm.
- Rf = 0,51 (h dung mi khai trin ethylacetat:n-hexan = 7:3)
- tonc= 219oC 221oC.
- Khi lng: m = 5,41 g.
- Hiu sut phn ng t 90,2%.
3.1.1.2. Tng hp 4-(1H-benzimidazol-2-yl)-2-bromo-6-methoxyphenol (II)
a. Tng hp Bromovanillin
S phn ng:
S 3.3: Tng hp bromovanillin
Tin hnh:
Thm 1,0 g (6,6 mmol) vanillin v 0,4 g (2,4 mmol) kali bromat vo bnh cu
c cha 3ml acid acetic bng, khuy k trong 30 pht. Sau , nh t t 3ml (12,4
mmol) acid hydrobromic 40%, mu vng xut hin. Tip tc khuy hn hp phn
ng nhit phng trong 1 gi 30 pht.
-
23
Sau khi kt thc phn ng, chuyn hn hp phn ng sang cc c m cha
50ml nc xut hin kt ta, tip tc khuy trong 15 pht. Sau lc ta, ra ta
bng nc ct nhiu ln. Kt tinh li trong ethanol 60o. Sy ta 60-70oC.
Kt qu:
- Cm quan: cht bt mu trng.
- Rf = 0,79 (h dung mi khai trin ethylacetat:n-hexan = 7:3).
- tonc=162-165oC.
- Khi lng: m = 1,18 g.
- Hiu sut phn ng t 77,6%.
b. Tng hp 4-(1H-benzimidazol-2-yl)-2-bromo-6-methoxyphenol (II)
S phn ng:
S 3.4: Tng hp 4-(1H-benzimidazol-2-yl)-2-bromo-6-methoxyphenol
(II)
Tin hnh:
Thm 0,54 g (5 mmol) o-phenylendiamin v 1,18 g (5 mmol) bromovanillin
vo bnh cu 50 ml cha 10 ml DMF, khuy cho ti khi tan hon ton. Sau , thm
t t 0,95 g (5 mmol) natri metabisulfit vo hn hp phn ng. Duy tr phn ng
90oC trong 3 gi.
Sau khi kt thc phn ng, t t hn hp phn ng vo cc c m c cha
50ml nc , xut hin kt ta. Lc kt ta, kt tinh li trong ethanol 60o, sy kh
60-70oC.
Kt qu:
- Cm quan: cht bt mu vng xanh.
- Rf = 0,64 (h dung mi khai trin cloroform: methanol = 9:1).
- tonc = 164-166oC.
-
24
- Khi lng: m = 1,27 g.
- Hiu sut phn ng t 79,6%.
3.1.1.3. Tng hp 2-(4-benzyloxy)-3-bromo-5-methoxyphenyl)-1H-benzimidazol
(III)
a. Tng hp 4-benzyloxy-3-bromo-5-methoxybenzaldehyd
S phn ng:
S 3.5: Tng hp 4-benzyloxy-3-bromo-5-methoxybenzaldehyd
Tin hnh:
Cho 1,16 g (5 mmol) bromovanillin vo bnh cu 50 ml cha 10ml methanol,
khuy cho n khi tan hon ton. Tip tc cho 0,69 g (5 mmol) kali carbonat v
0,83 g (5 mmol) kali iodid vo hn hp, khuy trong 30 pht. Nh t t 0,6 ml (5
mmol) benzyl clorid vo hn hp phn ng. un hi lu 70oC trong 4 gi.
Sau khi kt thc phn ng t t hn hp phn ng vo cc c m cha
30ml nc lnh, khuy k thy xut hin cc git du khng tan. Dng 30 ml
diclomethan (CHCl2) chit (chit 3 ln mi ln 10 ml). Lm khan dch chit
diclomethan bng natri sulfat khan, ct loi dung mi thu c sn phm.
Kt qu:
- Cm quan: cht lng mu vng nu.
- Khi lng: m = 1,08 g.
- Hiu sut phn ng t 92,3%.
b. Tng hp 2-(4-benzyloxy)-3-bromo-5-methoxyphenyl)-1H-benzimidazol (III).
S phn ng:
-
25
S 3.6: Tng hp 2-(4-benzyloxy)-3-bromo-5-methoxyphenyl)-1H-
benzimidazol (III).
Tin hnh:
Thm 0,64 g (2 mmol) 4-benzyloxy-3-bromo-5-methoxybenzaldehyd vo
bnh cu 50 ml cha 10 ml DMF, cho 0,38 g (2 mmol) natri metabisulfit vo, khuy
k trong 30 pht. Sau , cho 0,22 g (2 mmol) o-phenylendiamin vo hn hp phn
ng. un phn ng 90oC trong 3 gi.
Sau khi kt thc phn ng, chuyn t t hn hp phn ng sang cc c m
cha 50ml nc lnh, xut hin kt ta, khuy mnh. Lc ta, kt tinh li trong
ethanol 70o, sy kh 60-70oC.
Kt qu:
- Cm quan: cht bt mu vng nht.
- Rf = 0,82 (h dung mi khai trin cloroform: methanol = 9: 1).
- tonc = 211-213oC.
- Khi lng: m = 0,68 g.
- Hiu sut phn ng t 83,1%.
3.1.1.4. Tng hp ethyl 2-[2-(4-benzyloxy-3-bromo-5-methoxyphenyl)-1H-
benzimidazol-1-yl] acetat (IV)
S phn ng:
S 3.7: Tng hp ethyl 2-[2-(4-benzyloxy-3-bromo-5-methoxyphenyl)-
1H-benzimidazol-1-yl] acetat (IV)
-
26
Tin hnh:
Thm 0,41 g (1 mmol) cht III vo bnh cu 50 ml c cha 10 ml DMF
khuy cho ti khi tan ht. Tip tc cho 0,14 g (1 mmol) kali carbonat vo bnh phn
ng, khuy trong 1 gi. Sau , nh t t 0,14 ml (1,3 mmol) ethyl cloroacetat
(d=1,1450 g/ml) vo hn hp phn ng. Khuy hn hp phn ng nhit phng
trong 24 gi.
Sau khi phn ng kt thc, lc hn hp phn ng loi mui v c.
dch lc vo cc c m cha 30 ml nc lnh, ri chit bng ethyl acetat (chit 3
ln, mi ln 20 ml). Lm khan dch chit ethyl acetat bng natri sulfat khan. Ct
quay chn khng n ht dung mi thu c sn phm.
Kt qu:
- Cm quan: cht rn mu nu .
- Rf = 0,87 (h dung mi khai trin cloroform: methanol = 9: 1).
- tonc = 78-80oC.
- Khi lng: m = 0,29 g.
- Hiu sut phn ng t 58,6%.
Nh vy, 4 cht I-IV c tng hp. Sau y l bng tm tt kt qu tng
hp ha hc:
Bng 3.1: Kt qu tng hp ha hc
STT Cht CTPT Cm quan Hiu sut
(%)
1 I C14H12N2O2 Tinh th mu trng xm 90,2
2 II C14H11BrN2O2 Cht bt mu vng xanh 79,6
3 III C21H17BrN2O2 Cht bt nu vng nht 83,1
4 IV C25H23BrN2O4 Cht rn mu nu 58,6
3.1.2. Kim tra tinh khit
Kim tra tinh khit ca 4 cht tng hp c bng SKLM v o nhit
nng chy nh sau:
-
27
Tin hnh chy SKLM bn cht I-IV vi 2 h dung mi khai trin:
ethylacetat : n-hexan (7: 3) v cloroform: methanol (9: 1).
Kt qu trn sc k i vi 2 h dung mi khai trin thy 1 vt r, gn,
khng c vt ph khi soi di nh sng t ngoi bc sng 254 nm.
Bn cht t I-IV sau khi tinh ch u dng rn v c o nhit nng
chy.
Gi tr Rf v nhit nng chy (tonc) ca cc cht c tm tt trong bng
sau:
Bng 3.2: Gi tr Rf v nhit nng chy (to
nc) ca cc cht
Cht H dung mi khai trin Rf Tonc (
oC)
I Ethylacetat: n-hexan = 7: 3 0,51 219-221
II Cloroform: methanol = 9: 1 0,64 164-166
III Cloroform: methanol = 9: 1 0,82 211-213
IV Cloroform: methanol = 9: 1 0,87 78-80
Nhn xt: Vi cc h dung mi khai trin, cc cht tng hp c cho vt
gn r trn bn sc k v nhit nng chy ca cc cht c khong dao ng hp
20C. Nh vy, c th kt lun s b cc cht tng hp c l tinh khit.
-
28
3.1.3. Xc nh cu trc
xc nh cu trc ha hc, cc cht I-IV c tin hnh o ph hng
ngoi (IR), ph khi lng (MS), ph cng hng t ht nhn proton (1H-NMR).
3.1.3.1. Kt qu ph hng ngoi (IR)
Thng qua vic phn tch ph IR ca cc cht tng hp, nhn bit c cc
di hp ph c trng ca dao ng ha tr (), dao ng bin dng () ca cc
nhm chc v cc lin kt in hnh c trong cu trc phn t ca chng nh C=C
thm, O-H, N-H, C=N, C-N, C=O ester[2,5] Ngoi ra vi cc nhm th khc
nhau, mi cht li c nhng pic c trng ring. S liu phn tch ph hng ngoi
ca 5 cht c trnh by trong bng sau:
Bng 3.3: Kt qu ph hng ngoi ca cc cht I-IV
Cht O-H C-O alcol N-H C=C C-O-C C=N C=O
I 3341,11 1217,47 3511,15 1504,25 1136,26 1656,52
II 3159,83 1463,61 1226,76 1628,60
III 3057,72 1464,20 1232,98 1630,57
IV 1459,22 1212,17 1562,63 1746,49
Nhn xt: Qua bng nhn thy s liu phn tch ph IR hon ton ph hp
vi cc ti liu tham kho [2,5] cng nh cu trc d kin ca 4 cht I-IV.
3.1.3.2. Kt qu ph khi lng (MS)
Tin hnh ghi ph MS ca cc cht I-IV kt qu thu c nh sau:
Bng 3.4: Kt qu ph khi lng ca cc cht I-IV
Cht Cng thc cu to CTPT Khi
lng m/z
I
C14H12N2O2 240 238,4
[M-H]-
-
29
Cht Cng thc cu to CTPT Khi
lng m/z
II
C14H11BrN2O2 318 319/321
[M+H]+
III
C21H17BrN2O2 408 409/411
[M+H]+
IV
C25H23BrN2O4 494 495/497
[M+H]+
Nhn xt: T bng trn cho thy cc cht c gi tr ph khi lng ph hp
vi khi lng phn t ca cc hp cht d kin [2,5]. Cc cht II, III, IV do c
nguyn t brom trong phn t nn ph khi lng xut hin pic ng v ca brom
ng vi t l t nhin (79Br: 81Br = 0,981)
3.1.3.3. Kt qu ph cng hng t ht nhn proton (1H-NMR)
khng nh chc chn cu trc ca cc cht tng hp c, chng ti tin
hnh ghi ph cng hng t ht nhn proton (1H-NMR) ca cc cht I-IV kt qu
c trnh by trong bng sau:
Bng 3.5: Kt qu ph cng hng t ht nhn proton ca cc cht I-IV
Cht Cng thc cu to 1H-NMR
I
1H-NMR (500MHz, DMSO,
ppm): 3,88 (3H, s, O-CH3),
6,9 (1H, d, J = 4,1 Hz, C5-H),
7,15 (2H, dd, C5-H + C6-H),
7,47 (1H, d, J = 3,5 Hz, C6-H),
-
30
Cht Cng thc cu to 1H-NMR
7,60 (2H, m, C4-H +C7-H),
7,74 (1H, d, C2-H),
9,53 (1H, s, O-H),
12,65 (1H, s, N-H)
II
1H-NMR (500MHz, DMSO,
ppm): 3,95 (3H, s, O-CH3),
7,18 (2H, dd, C5-H + C6-H),
7,56 (2H, s, C2-H + C6-H),
7,77 (1H, d, C4-H),
7,93 (1H, d, C7-H),
10,02 (1H, s, O-H)
III
1H-NMR (500MHz, DMSO,
ppm): 3,98 (3H, s, O-CH3),
5,10 (2H, s, O-CH2),
7,29 (2H, dd, C5-H + C6-H),
7,32 (1H, d, CHAr),
7,38 (2H, d, 2CHAr),
7,51 (2H, d, C4-H + C7-H),
7,66 (2H, dd, 2CHAr),
7,88 (1H, s, C6-H),
8,00 (1H, s, C2-H)
IV
1H-NMR (500MHz, DMSO,
ppm): 1,15 (3H, t, CH2-CH3),
3,92 (3H, s, O-CH3),
4,11-4,15 (2H, m, CH2-CH3),
5,08 (2H, s, N-CH2),
5,27 (2H, s, O-CH2),
7,28-7,32 (2H, m, CHAr),
-
31
Cht Cng thc cu to 1H-NMR
7,35-7,42 (4H, m, CHAr),
7,47 (1H, d, C2-H),
7,53 (2H, d, C5-H + C6-H),
7,61 (1H, d, C4-H),
7,70 (1H, d, C7-H)
Ghi ch: dch chuyn ha hc (ppm); s: singlet; d: double; t: triplet; m:
muntiplet.
Nhn xt: Qua vic phn tch ph , thy s c mt ca cc proton ph hp
vi cng thc cu to ca cc cht I-IV [2,5].
3.2. TH TC DNG SINH HC
Da vo mt s nghin cu v tc dng sinh hc ca mt s dn cht 2-
arylbenzimidazol, chng ti tin hnh th tc dng khng nm, khng khun ca
cc cht I, II, III, IV v th hot tnh gy c t bo vi cht III, IV.
3.2.1. Th hot tnh khng khun
3.2.1.1. Ging vi sinh vt kim nh
Th tc dng khng khun c thc hin i vi 5 vi khun Gram(+) v 5
vi khun Gram() sau:
Vi khun Gram(-) gm:
Escherichia coli ATCC 25992 (EC)
Proteus mirabilis BV 108 (Pro)
Shigella flexneri DT 112 (Shi)
Salmonella typhi DT 220 (Sal)
Pseudomonas aeruginosa VM 201 (Pseu)
Vi khun Gram(+) gm:
Staphylococus aureus ATCC 1128 (Sta)
Bacillus pumilus ATCC 10241 (Bp)
Bacillus subtilis ATCC 6633 (Bs)
-
32
Bacillus cereus ATCC 9946 (Bc)
Sarvina lutea ATCC 9341 (SL)
Khng sinh i chng: Streptomycin (Strep) 14 g/ml
Penicillin (Peni) 27 UI/ml
3.2.1.2. Mi trng th nghim
Thnh
. phn
MT
NaCl
(%)
Cao tht
(%)
Pepton
(%)
Thch
(%) Nc pH
Canh thang
nui cy VSV 0,5 0,3 0,5 0,0
Va
100 ml
7,0-7,4
Thch thng 0,5 0,3 0,5 1,6
3.2.1.3. Nguyn tc
Th tc dng khng khun tin hnh theo phng php khuch tn trn
thch. Mu th (c cha hot cht th) c t ln lp thch dinh dng cy vi
sinh vt kim nh, hot cht t mu th khuch tn vo mi trng thch s c ch
s pht trin ca vi sinh vt kim nh to thnh vng v khun.
Php th c thc hin ti B mn Vi sinh - Sinh hc, Trng i hc
Dc H Ni.
3.2.1.4. Tin hnh
Mu th c pha vo dung mi thch hp vi nng l 100 g/ml. Cc
khoanh giy lc v trng v c sy kh c tm 3 ln vi dung dch mu th
gm: mu cht I, II, III, IV, sau mi ln tm cc khoanh giy lc c cha mu th
u c sy nhit thp hn 60oC n kh ht dung mi.
Chun b mi trng v cy vi sinh vt kim nh: vi sinh vt kim nh
c cy vo mi trng canh thang, ri nui cy cho pht trin trong t m 37oC
trong thi gian 18 - 24 gi n nng 107 t bo/ml (kim tra bng pha long v
dy dch chun). Mi trng thch thng v trng (tit trng 118oC/30 pht) c
lm lnh v 45 - 500C v c cy ging vi sinh vt kim nh vo vi t l 2,5
-
33
ml/100 ml. Lc trn vi sinh vt kim nh phn tn u trong mi trng thch
thng, ri vo a petri v trng vi th tch 20 ml/a v cho thch ng li.
t khoanh giy lc: khoanh giy lc c tm cht th v x l nh trn
c t ln b mt mi trng thch thng cha vi sinh vt kim nh theo s
nh sn.
cc a petri c mu th c t nh trn trong t m to = 37oC trong
18 24 gi, ri sau ly ra c kt qu, o ng knh vng v khun nu c (s
dng thc kp Panmer chnh xc 0,02 mm).
nh gi kt qu: da trn ng knh vng v khun v c nh gi theo
cng thc:
2
1 1
( )
1
n n
i i
i i
D D D
D sn n
D : ng knh trung bnh vng v khun
iD : ng knh vng v khun th i
s: lch thc nghim chun c hiu chnh
n: s th nghim lm song song (n = 3).
3.2.1.5. Kt qu th nghim
Bng 3.6: Kt qu th tc dng khng khun
Mu ng knh vng v khun [mm] ( D , s)
Bc Bs Bp SL Sal EC Shi Pro Pseu Sta
I 9,31
0,69 - - - - - -
12,03
1,05 -
9,00
1,22
II - - 8,53
1,12 - - - - - -
8,47
0,83
III 8,24
1,20
9,45
0,72 - - -
12,37
0,82 -
10,06
0,47 - -
IV - - - - - 11,94 - - - -
-
34
Mu ng knh vng v khun [mm] ( D , s)
Bc Bs Bp SL Sal EC Shi Pro Pseu Sta
0,75
Peni 19,38
0,37
20,47
0,70 -
12,91
0,47 - - - - -
11,42
0,67
Strep 21,50
0,66
20,16
1,21 -
12,73
0,41
13,73
1,50
13,20
1,05
12,07
0,89
15,53
0,81
17,37
0,67
17,37
0,12
Kt lun:
- Mu I c tc dng c ch 3 chng vi khun th (Proteus mirabilis,
Staphylococus aureus, Bacillus cereus).
- Mu II c tc dng c ch 2 chng vi khun th (Staphylococus aureus,
Bacillus pumilus).
- Mu III c tc dng c ch 4 chng vi khun th (Escherichia coli, Proteus
mirabilis, Bacillus subtilis, Bacillus cereus).
- Mu IV c tc dng c ch 1 chng vi khun th (Escherichia coli).
3.2.2. Th hot tnh khng nm
3.2.2.1. Ging vi sinh vt kim nh
S dng 4 chng nm:
Candida albicans (Ca)
Aspergillus niger (As)
Saccharomyces cerevisiae (Sa)
Penicilium sp. (Pe)
Cht i chng: Intraconazol (Intra) 50 g/ml
3.2.2.2. Mi trng th nghim
Mi trng th nghim trong nghin cu c thnh phn nh sau:
-
35
Thnh .
. phn
MT
Glucose
(%)
Pepton
(%)
Thch
(%) Nc pH
Sabouraud nui cy 2,0 1,0 0,0 Va
100ml 7,0-7,4 Sabouraud th
nghim 2,0 1,0 1,6
3.2.2.3. Nguyn tc
Th tc dng khng nm tin hnh theo phng php khuch tn trn thch.
Mu th (c cha hot cht th) c t ln lp thch dinh dng cy vi sinh
vt kim nh, hot cht t mu th khuch tn vo mi trng thch s c ch s
pht trin ca vi sinh vt kim nh to thnh vng v nm.
Php th c thc hin ti B mn Vi sinh - Sinh hc, Trng i hc
Dc H Ni.
3.2.2.4. Tin hnh
Mu th c pha vo dung mi thch hp vi nng l 100 g/ml. Cc
khoanh giy lc v trng v c sy kh c tm 3 ln vi dung dch mu th
gm: mu cht I, II, III, IV, sau mi ln tm cc khoanh giy lc c cha mu th
u c sy nhit thp hn 60oC n kh ht dung mi.
Chun b mi trng v cy vi sinh vt kim nh: nm kim nh (C.
albicans) c cy vo mi trng Sabouraud lng, ri nui cy cho pht trin
trong t m 28 - 30oC trong thi gian 24 - 48 gi n nng 107 t bo/ml (kim
tra bng pha long v dy dch chun). i vi Aspergillus niger th s dng huyn
conidi trong dung dch Tween 80 0,2% v trng. Mi trng Sabouraud v trng
(tit trng 118oC/30 pht) c lm lnh v 45 - 50oC v c cy ging vi sinh vt
kim nh vo vi t l 2,5 ml/100 ml. Lc trn vi nm kim nh phn tn u
trong mi trng thch thng, ri vo a petri v trng vi th tch 20 ml/a
v cho thch ng li.
-
36
t khoanh giy lc: khoanh giy lc c tm cht th v x l nh trn
c t ln b mt mi trng thch thng cha VSV kim nh theo s nh
sn (4 th nghim song song).
cc a petri c mu th c t nh trn trong t m to = 28 - 30oC
trong 24 -48h, ri sau ly ra c kt qu, o ng knh vng v nm nu c (s
dng thc kp Panmer chnh xc 0,02 mm).
nh gi kt qu: da trn ng knh vng v nm v c nh gi theo
cng thc:
2
1 1
( )
1
n n
i i
i i
D D D
D sn n
D : ng knh trung bnh vng v nm
iD : ng knh vng v nm th i
s : lch thc nghim chun c hiu chnh
n : s th nghim lm song song (n = 3).
3.2.2.5. Kt qu th nghim
Tc dng i vi vi nm th hin bng sau:
Bng 3.7: Kt qu th tc dng khng nm
Mu ng knh vng v nm [mm] ( D , s)
Ca As Sa Pe
I - - 8,16
0,65 -
II - - 8,20
0,17 -
III - - 7,72
0,82 -
IV 8,78
0,72 -
8,30
0,83 -
-
37
Mu ng knh vng v nm [mm] ( D , s)
Ca As Sa Pe
Intra 16,33
2,01
15,26
0,41
9,73
0,43
12,39
0,75
Kt lun:
- Mu I, II, III c tc dng vi 1 chng nm (Saccharomyces cerevisiae).
- Mu IV c tc dng vi 2 chng nm (Saccharomyces cerevisiae, Candida
albicans) .
3.2.3. Th hot tnh gy c t bo
3.2.3.1. Nguyn tc
Th hot tnh gy c t bo theo phng php ca Skehan cng cng s
(1990) v Likiwitayawuid cng cng s (1993) hin ang c p dng ti Vin
nghin cu ung th Quc gia ca M (NCI) v Trng i hc Dc, i hc tng
hp Illinois, Chicago, M.
Php th c thc hin ti phng Sinh hc thc nghim (Vin ha hc cc
hp cht thin nhin Vin Hn lm Khoa hc v Cng ngh Vit Nam).
3.2.3.2. Cch tin hnh
Cc dng t bo ung th ngi c cung cp bi ATCC gm: Hep-G2
(Hepatocellular carcinoma t bo ung th gan), Lu (Lung cancer Ung th phi).
Cc dng t bo c nui cy ph hp: DMEM (Dulbeccos Modified
Eagle Medium) hoc MEME (Minimum Essential Medium with Eagles salt) c b
sung thm L-gluthamin, natri pyruvat, 10% huyt thanh b v cc thnh phn cn
thit khc iu kin tiu chun (5% CO2, 37oC, m 98%, v trng tuyt i).
Ty thuc vo c tnh tng dng t bo khc nhau, thi gian cy cng khc nhau.
T bo pht trin pha logarit s c s dng th c tnh.
Th c t bo: 200 M dung dch t bo pha logarit nng 3x104 t
bo/ml vo mi ging (a 96 ging) trong mi trng thch hp cho mi dng t
bo. Mu th c x l vi t bo cc nng pha long khc nhau sao cho t
-
38
n nng thch hp. 37oC, 5% CO2, 3 ngy. Ging iu khin gm 200 M
dung dch t bo nng 3x104 t bo/ml trong iu kin nh trn, thm 50 M
MTT (1 mg/ml pha trong mi trng nui cy khng huyt thanh), 37oC trong 4
gi, loi b mi trng, thm 100 M DMSO lc u, c kt qu bc sng
495-515nm trn my ELISA.
Cht chun i chng c kh nng dit t bo: Ellipithin, Vinblastin hoc
Taxol pha trong DMSO.
3.2.3.3. Kt qu
a. Cch tnh kt qu
Gi tr CS: l kh nng sng st ca t bo nng no ca cht th
tnh theo % so vi i chng. Da trn kt qu o c ca chng OD (ngy 0),
DMSO 10% v so snh vi gi tr OD khi trn mu tm gi tr CS (%) theo cng
thc:
( ) ( )
( ) ( )
Gi tr CS % sau khi tnh theo cng thc trn c a vo tnh ton excel
tm ra % trung bnh lch tiu chun ca php th c lp li 3 ln theo cng
thc ca Ducan nh sau: lch tiu chun
=( )
Cc mu c biu hin hot tnh (CS < 50%) s c chn ra th nghim
tip tm gi tr IC50.
b.aKt qu thc nghim
-
39
Bng 3.8: Kt qu th hot tnh gy c t bo ca 2 cht III, IV
STT K hiu mu Nng % t bo cn sng st
Kt lun Hep-G2 Lu
DMSO 100,000,0 100,00,0 m tnh
Chng(+)
Ellipithin 0,20,0 1,20,2
Dng
tnh
1 Cht III 5 g/ml 87,70,9 89,60,8 m tnh
2 Cht IV 20 g/ml 60,10,2 92,31,5 m tnh
Nhn xt: cc cht III, IV khng c hot tnh gy c t bo ng k trn c
2 dng t bo ung th Hep-G2 v Lu.
3.3. BN LUN
3.3.1. V tng hp ha hc
3.3.1.1. Phn ng ng vng to nhn benzimidazol v phn ng th trn nhn
thm
Phn ng ng vng ca o-phenylendiamin vi cc dn cht khc nhau ca
vanillin s dng cht xc tc Na2S2O5 din ra trong thi gian ngn vi hiu sut kh
cao.
Vic tng hp cc nhm th mi trn nhn phenyl nu i t cht I din ra rt
kh khn, sn phm thu c khng nh mong mun v nhim nhiu tp. Nu i t
vanillin, tin hnh phn ng bromo ha hoc alkyl ho trc sau ng vng vi
o-phenylendiamin cho sn phm nh mong mun vi hiu sut cao.
-
40
3.3.1.2. Phn ng th v tr N-1 trn nhn benzimidazol
Phn ng th v tr N-1 vi cc tc nhn ethyl cloroacetat c hiu sut phn
ng thp, c th l do cc tc nhn ny l tc nhn alkyl ha yu.
iu kin phn ng: phn ng cn thc hin trong mi trng khan nc, do
dung mi, ha cht, thit b cn c lm kh trc khi tin hnh phn ng.
iu ny c th gii thch nh sau: tc nhn alkyl ha y l ester, nu trong mi
trng c mt ca nc di s tc dng ca kim xc tc (K2CO3), chng s b
thy phn to thnh acid monocloroacetic, s lm gim hiu sut phn ng.
Cht xc tc: phn ng th v tr N-1 trn nhn benzimidazol s dng
K2CO3 lm xc tc va c vai tr hot ha, va c vai tr phn ng vi HCl sinh ra
to thnh KHCO3, do khng to ra nc trong qu trnh phn ng.
T l mol gia tc nhn th v dn cht benzimidazol: Khi tng hoc gim t
l ny u lm nh hng ti hiu sut phn ng. nh hng ny c th gii thch
nh sau: khi t l ny < 1, phn ng xy ra khng hon ton v lm gim hiu sut.
Mt khc nu t l ny > 1,5, c th xy ra qu trnh to mui amoni bc 4 ti nit
v tr s 3 trn nhn benzimidazol, hiu sut phn ng cng gim.
- Phn ng gia ethyl cloroacetat v cht III vi t l s mol 1,3 : 1 th sn
phm IV thu c l dn cht th vo v tr N-1.
Dung mi: s dng dung mi l DMF cho hiu sut cao hn dung mi
aceton. Tuy nhin, vic tinh ch ly sn phm t DMF tn nhiu thi gian hn
rt nhiu so vi aceton.
3.3.2. V tc dng sinh hc
3.3.2.1. V tc dng khng khun
C 4 cht I-IV u c tc dng khng khun, kt qu cho thy nhm dn cht
c tc dng trn VK Gram(-) tt hn Gram(+), c th nh sau:
- Cht I c tc dng c ch 3 VK th (1 chng Gram(-): Proteus mirabilis; 2
chng Gram(+): Staphylococus aureus, Bacillus cereus), tc dng c ch
trn VK Gram(-) tt hn VK Gram(+) (ng knh vng v khun trn VK
Gram(-) ln hn VK Gram(+)).
-
41
- Cht II c tc dng c ch 2 VK Gram(+): Staphylococus aureus, Bacillus
pumilus).
- Cht III c tc dng c ch 4 VK th (2 chng Gram(-): Escherichia coli,
Proteus mirabilis; 2 chng Gram(+): Bacillus subtilis, Bacillus cereus), tc
dng c ch trn VK Gram(-) tt hn VK Gram(+).
- Cht IV c tc dng c ch 1 VK th (chng Gram(-): Escherichia coli).
3.3.2.2. V tc dng khng nm
C 4 cht I-IV u c tc dng khng nm C. albicans, ring cht IV c tc
dng trn c Saccharomyces cerevisiae.
3.3.2.3. V tc dng gy c t bo
Cc cht III, IV khng c hot tnh gy c t bo ng k trn c 2 dng t
bo ung th Hep-G2 v Lu.
-
42
KT LUN V XUT
KT LUN
T nhng kt qu nghin cu trnh by trn y, chng ti rt ra mt s kt
lun sau:
1. tng hp c 4 dn cht ca 2-arylbenzimidazol vi cc nhm th khc
nhau trn nhn phenyl v v tr N-1. Cu trc cc cht trung gian v sn phm
c xc nh bng phng php phn tch cc ph IR, MS, 1H-NMR.
2. th c tc dng khng nm, khng khun ca cc cht I-IV v th c
tc dng khng t bo ung th ca cc cht III, IV trn 2 dng t bo ung th
Hep-G2 v Lu. Kt qu cho thy c 2 cht u khng c tc dng
XUT
Vi cc kt qu t c, hy vng nghin cu ny c th ng gp mt phn
vo cng trnh nghin cu rng hn v tng hp v th tc dng sinh hc ca dn
cht 2-arylbenzimidazol. tip tc pht trin kt qu, chng ti a ra mt s
xut sau:
1. Tip tc nghin cu phn ng tng hp dn cht 2-arylbenzimidazol vi cc
nhm th khc nhau trn nhn phenyl v v tr N-1.
2. Tip tc th nghim cc hot tnh sinh hc ca dn cht 2-arylbenzimidazol nh
tc dng khng t bo ung th, tc dng khng nm, khng khungp phn
vo vic tm ra nhm dn cht mi c tc dng sinh hc trin vng.
-
TI LIU THAM KHO
Ting Vit
1. Phan nh Chu (2005), Cc qu trnh c bn tng hp hu c, NXB khoa
hc & k thut, H Ni, tr. 30-44, 64-85.
2. Nguyn Hu nh, Trn Th (1999), ng dng mt s phng php ph
nghin cu cu trc phn t, Nh xut bn gio dc, H Ni, tr. 27-88, 199-
275, 296-333.
3. T Minh Kong v cng s (2006), K thut sn xut dc phm, Tp I,
NXB Y hc, H Ni, tr. 24-31, 42-48, 121-127.
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PH LC
Ph lc 1: Ph hng ngoi (IR) ca cht I
Ph lc 2: Ph khi lng (MS) ca cht I
Ph lc 3.1: Ph cng hng t ht nhn proton (1H-NMR) ca cht I
Ph lc 3.2: Ph gin cng hng t ht nhn proton (1H-NMR) ca cht I
Ph lc 4: Ph hng ngoi (IR) ca cht II
Ph lc 5.1: Ph khi lng (MS) ca cht II
Ph lc 5.2: Ph khi lng (MS) ca cht II
Ph lc 6.1: Ph cng hng t ht nhn proton (1H-NMR) ca cht II
Ph lc 6.2: Ph gin cng hng t ht nhn proton (1H-NMR) ca cht II
Ph lc 7: Ph hng ngoi (IR) ca cht III
Ph lc 8.1: Ph khi lng (MS) ca cht III
Ph lc 8.2: Ph khi lng (MS) ca cht III
Ph lc 9.1: Ph cng hng t ht nhn proton (1H-NMR) ca cht III
Ph lc 9.2: Ph gin cng hng t ht nhn proton (1H-NMR) ca cht III
Ph lc 9.3: Ph gin cng hng t ht nhn proton (1H-NMR) ca cht III
Ph lc 10: Ph hng ngoi (IR) ca cht IV
Ph lc 11.1: Ph khi lng (MS) ca cht IV
Ph lc 11.2: Ph khi lng (MS) ca cht IV
Ph lc 12.1: Ph cng hng t ht nhn proton (1H-NMR) ca cht IV
Ph lc 12.2: Ph gin cng hng t ht nhn proton (1H-NMR) ca cht IV
Ph lc13.1: Kt qu th tc dng gy c t bo ca cht III, IV
Ph lc13.2: Kt qu th tc dng gy c t bo ca cht III, IV
Ph lc13.3: Kt qu th tc dng gy c t bo ca cht III, IV
-
Ph lc 1: Ph hng ngoi (IR) ca cht I
-
Ph lc 2: Ph khi lng (MS) ca cht I
-
Ph lc 3.1: Ph cng hng t ht nhn proton (1H-NMR) ca cht I
-
Ph lc 3.2: Ph gin cng hng t ht nhn proton (1H-NMR) ca cht I
-
Ph lc 4: Ph hng ngoi (IR) ca cht II
-
Ph lc 5.1: Ph khi lng (MS) ca cht II
-
Ph lc 5.2: Ph khi lng (MS) ca cht II
-
Ph lc 6.1: Ph cng hng t ht nhn proton (1H-NMR) ca cht II
-
Ph lc 6.2: Ph gin cng hng t ht nhn proton (1H-NMR) ca cht II
-
Ph lc 7: Ph hng ngoi (IR) ca cht III
-
Ph lc 8.1: Ph khi lng (MS) ca cht III
-
Ph lc 8.2: Ph khi lng (MS) ca cht III
-
Ph lc 9.1: Ph cng hng t ht nhn proton (1H-NMR) ca cht III
-
Ph lc 9.2: Ph gin cng hng t ht nhn proton (1H-NMR) ca cht III
-
Ph lc 9.3: Ph gin cng hng t ht nhn proton (1H-NMR) ca cht III
-
Ph lc 10: Ph hng ngoi (IR) ca cht IV
-
Ph lc 11.1: Ph khi lng (MS) ca cht IV
-
Ph lc 11.2: Ph khi lng (MS) ca cht IV
-
Ph lc 12.1: Ph cng hng t ht nhn proton (1H-NMR) ca cht IV
-
Ph lc 12.3: Ph gin cng hng t ht nhn proton (1H-NMR) ca cht IV
-
Ph lc 13.1: Kt qu th tc dng gy c t bo ca cht III, IV
-
Ph lc 13.2: Kt qu th tc dng gy c t bo ca cht III, IV
-
Ph lc 13.3: Kt qu th tc dng gy c t bo ca cht III, IV