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BGIO DC V O TO BY T
TRNG I HC DC H NI
.................. *.
NGUYN THPHNG
NGHIN CU TNG HP V THHOT
TNH SINH HC MT SDN CHT 2-MERCAPTOBENZIMIDAZOL
LUN VN THC S DC HC
H NI 2013
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BGIO DC V O TO BY T
TRNG I HC DC H NI
.................. *.
NGUYN THPHNG
NGHIN CU TNG HP V THHOT
TNH SINH HC MT SDN CHT 2-MERCAPTOBENZIMIDAZOL
LUN VN THC S DC HC
H NI 2013
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- 2 -
BGIO DC V O TO BY T
TRNG I HC DC H NI
.................. *.
NGUYN THPHNG
NGHIN CU TNG HP V THHOTTNH SINH HC MT SDN CHT 2-MERCAPTOBENZIMIDAZOL
LUN VN THC S DC HC
CHUYN NGNH: CNG NGHDC PHM V BO CH
M S: 6072040
Ngi hng dn :PGS.TS. Nguyn nh Luyn
TS. Phan Thanh Bnh
H NI 2013
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DANH MC PHLC
Phlc 1: Phhng ngoi (IR) ca chtII
Ph
l
c 2:
Phhng ngoi (IR) ca chtIII
.Phlc 3: Phhng ngoi (IR) ca chtIII-a.
Phlc 4: Phhng ngoi (IR) ca chtIII-b.
Phlc 5: Phhng ngoi (IR) ca chtIII-c.
Phlc 6: Phhng ngoi (IR) ca chtIII-d.
Phlc 7: Phhng ngoi (IR) ca chtIV.
Phlc 8: Phhng ngoi (IR) ca chtV.
Phlc 9: Phhng ngoi (IR) ca chtV-a.
Phlc 10: Phhng ngoi (IR) ca chtV-b.
Phlc 11: Phhng ngoi (IR) ca chtV-c.
Phlc 12: Phkhi lng phn t(MS) ca chtII.
Phlc 13: Phkhi lng phn t(MS) ca chtIII.
Phlc 14: Phkhi lng phn t(MS) ca chtIII-a.
Phlc 15: Phkhi lng phn t(MS) ca chtIII-d.
Phlc 16: Phkhi lng phn t(MS) ca chtIV.
Phlc 17: Phkhi lng phn t(MS) ca chtV.
Phlc 18: Phkhi lng phn t(MS) ca chtV-a.
Phlc 19: Phkhi lng phn t(MS) ca chtV-b.
Phlc 20: Phkhi lng phn t(MS) ca chtV-c.
Phlc 21: Phcng hng tht nhn (1H-NMR) ca cht II.
Phlc 22: Phcng hng tht nhn (1H-NMR) ca cht III.
Phlc 23: Phcng hng tht nhn (1H-NMR) ca cht III-a.
Phlc 24: Phcng hng tht nhn (1H-NMR) ca cht III-b.
Phlc 25: Phcng hng tht nhn (1H-NMR) ca cht III-c.
Phlc 26: Phcng hng tht nhn (1H-NMR) ca cht III-d.
Phlc 27: Phcng hng tht nhn (1H-NMR) ca cht IV.
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Phlc 28: Phcng hng tht nhn (1H-NMR) ca cht V.
Phlc 29: Phcng hng tht nhn (1H-NMR) ca cht V-a.
Ph
l
c 30:
Phcng hng tht nhn (1
H-NMR) ca chtV-b
.Phlc 31: Phcng hng tht nhn (1H-NMR) ca cht V-c.
Phlc 32: Phiu trli kt quthc tnh tbo.
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LI CM N
hon thnh lun vn Nghin cu tng hp v thhot tnh sinh hc mt
sdn cht 2-mercaptobenzimidazol ti nhn c rt nhiu sgip
tthy c, gia nh v bn b. Nhn y ti xin by tlng bit n su sc
n PGS.TS. Nguyn nh Luyn- ngi thy tn tnh hng dn v gip
ti trong sut qu trnh thc hin lun vn.
Ti cng xin chn thnh cm n TS. Phan Thanh Bnh, DS Nguyn Vn
Giang, CN. Phan Tin Thnh, bmn Cng Nghip Dc- Trng i hc
Dc H Ni v DS Sar Vuthy, DS Koen Sorphin lun gip , ng
vin ti trong qu trnh thc nghim.
Trong qu trnh thc hin lun vn ti nhn c nhiu sgip ca cc
c nhn n v trong v ngoi trng, ti xin chn thnh cm n Ban Gim
hiu, cc thy c phng Sau i hc-Trng i hc Dc H Ni, bmn
Vi sinh-sinh hc-Trng i hc Dc H Ni, khoa Ha-Trng i hc
khoa hc tnhin, Ths. ng V Lng- Vin Ha Hc-Vin khoa hc v
cng nghVit Nam, TS. L Mai Hng-phng thc nghim sinh hc- Vin
ha hc cc hp cht thin nhin.
Cui cng ti xin chn thnh cm n gia nh v bn b nhng ngi lun
bn cnh quan tm, gip , ng vin ti ti hon thnh lun vn ny.
Ti xin chn thnh cm n.
H Ni, ngy 28 thng 8 nm 2013
Hc vin
Nguyn ThPhng.
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MC LC
DANH MC CC K HIU, CHVIT TT
DANH MC CC BNGDANH MC CC HNH V, HNH NH V S
Trang
T VN .................................................................................................. 1
CHNG 1: TNG QUAN........................................................................... 2
1.1.KHIQUTCHUNGVKHUNGBENZIMIDAZOL ...........................................2
1.1.1. Cu trc ha hc.................................................................................. 2
1.1.2. Tnh cht ha hc ................................................................................ 3
1.2.KHIQUTCHUNGV2-MERCAPTOBENZIMIDAZOLVDNCHT ....3
1.2.1. Cng thc cu to ca 2-mercaptobenzimidazol ................................ 3
1.2.2. Tnh cht l hc ca 2-mercaptobenzimidazol ................................... 3
1.3.TCDNGSINHHCCADNCHT2-MERCAPTOBENZIMIDAZOL ....3
1.3.1. Tc dng khng khun v khng nm................................................. 4
1.3.2. Tc dng gim cholesterol mu v chng x cng ng mch .......... 8
1.3.3. Tc dng chng ung th.................................................................... 10
1.3.4. Tc dng chng co git ..................................................................... 13
1.3.5. Tc dng chng bi tit acid dch v, c chbm proton H+/K+-
ATPase v chng vim lot ddy ................................................................. 15
1.3.6. Cc tc dng khc ca dn cht 2-mercaptobenzimidazol ............... 16
1.4.CCPHNGPHPTNGHPDNCHT2-MERCAPTOBENZIMIDAZOL ......................................................................................17
1.4.1. Phn ng ng vng to nhn 2-mercaptobenzimidazol to-
phenylendiamin hoc dn cht o-phenylendiamin .......................................... 17
1.4.2. Phn ng to dn cht 2-mercaptobenzimidazol .............................. 18
1.4.2.1. Phn ng alkyl ha .......................................................................... 19
1.4.2.2. Phn ng acyl ha ........................................................................... 19
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1.4.2.3. Phn ng ngng tto hp cht bis-2-mercaptobenzimidazol ...... 20
CHNG 2: I TNG V PHNG PHP NGHIN CU......... 21
2.1.NGUYNLIU,THITB .......................................................................................21
2.1.1. Ha cht............................................................................................. 21
2.1.2. Thit b, dng c ............................................................................... 22
2.2.NIDUNGNGHINCU ........................................................................................23
2.3.PHNGPHPNGHINCU ..............................................................................23
2.3.1.TNGHPHAHC.23
2.3.2. Xc nh cu trc .............................................................................. 24
2.3.3. Thtc dng sinh hc ....................................................................... 25
CHNG 3: THC NGHIM V KT QU......................................... 29
3.1.TNGHPHAHC ............................................................................................29
3.1.1. Tng hp 2-mercaptobenzimidazol .................................................... 31
3.1.2. Tng hp cc dn cht ca 2-mercaptobenzimidazol ......................... 31
3.1.2.1. Tng hp 2-benzylthio-1H-benzo[d]imidazol ................................. 31
3.1.2.2. Tng hp 1-benzyl-2-(benzylthio)-1H-benzo[d]imidazol ............... 323.1.2.3. Tng hp methyl 2-(2-benzylthio)-1-H-benzo[d]imidazol-1-yl)acetat
......................................................................................................................... 33
3.1.2.4. Tng hp ethyl 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetat
......................................................................................................................... 34
3.1.2.5. Tng hp 1,4-bis(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)butan
........................................................................................................................ .35
3.1.2.6. Tng hp ethyl 2-(1H-benzimidazol-2-ylthio)acetat.36
3.1.2.7. Tng hp 1,4-bis(2-benzimidazolthio)butan 36
3.1.2.8. Tng hp 1,4-bis(1-benzyl-1H-benzo[d]imidazol-2-ylthio)butan37
3.1.2.9. Tng hp 1,4-bis(1-methoxycarboylmethyl-1H-benzo[d]imidazol-
2-ylthio)butan..38
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3.1.2.10. Tng hp 1,4-bis(1-ethoxycarboylmethyl-1H-benzo[d]imidazol-2-
ylthio)butan..39
3.2. KIM TRA TINH KHIT CA CC DN CHT TNG HP C42.
3.3.XCNHCUTRCCACCCHTTNGHPC ..........................43
3.3.1. Kt quphn tch phhng ngoi (IR) .............................................. 43
3.3.2. Kt quphn tch phkhi lng (MS) ............................................ 45
3.3.3. Kt quphn tch phcng hng tht nhn (1H-NMR) ............... 46
3.4.THTCDNGSINHHC .................................................................................49
3.4.1. Thtc dng khng khun, khng nm ............................................. 49
3.4.2. Thhot tnh gy c tbo .............................................................. 52
CHNG4.BNLUN ................................................................................................55
4.1. Vtng hp ha hc ............................................................................... 55
4.2. Bn lun vxc nh cu trc.57
4.3. Vthtc dng sinh hc ........................................................................ 58
KT LUN V KIN NGH..................................................................... 60
KTLUN .......................................................................................................................60KINNGH ......................................................................................................................61
TI LIU THAM KHO
PHLC
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DANH MC CC K HIU, CHVIT TT
DMF : N,N-dimethylformamid.
DMSO : Dimethyl sulfoxidTHF : Tetrahydrofuran.
DMAc : Dimethylacetamid.
MeOH : Methanol.
EtOH : Ethanol.
SKLM : Sc k lp mng.
MS : Phkhi lng phn t(Mass spectrometry).
IR : Phhng ngoi (Infrared spectroscopy).1HNMR : Phcng hng tht nhn proton
(Proton Nuclear Magnetic Resonance Spectroscopy).
CTCT : Cng thc cu to.
to : Nhit .
tonc : Nhit nng chy.
Rf : Hslu gi(retention factor).
M : Micromol.
EtOCS2K : Kali ethyl xanthat.
AcOH : Acid acetic.
MCA : Methyl cloroacetat.
ECA : Ethyl cloroacetat.
VSV : Vi sinh vt.
IC50 : Nng c ch50% i tng th
(Inhibitory Concentration)
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DANH MCCC BNG
BNG Trang
Bng 1 Cc dn cht ca cng thc (1). 4Bng 2 Cc thng sca vng c ch. 5
Bng 3 Cc dn cht ca cng thc (2). 6
Bng 4 Cc dn cht ca cng thc (3). 7
Bng 5 Cc dn cht ca cng thc (4). 7
Bng 6 Cc dn cht ca cng thc (5). 9
Bng 7 Cc dn cht ca cng thc (22). 14Bng 8 Cc dn cht ca cng thc (23). 15
Bng 9 Cc dn cht ca cng thc (24). 16
Bng 2.1Cc ha cht, dung mi sdng trong qu trnh thc
nghim.21
Bng 2.2Cc my mc, dng csdng trong qu trnh thc
nghim.
22
Bng 2.3Cc mi trng trong thnghim khng khun, khng
nm26
Bng 3.1 Tm tt kt qutng hp ha hc. 40
Bng 3.2Gi trRfv nhit nng chy ca cc cht tng hp
c.42
Bng 3.3Kt quphn tch phhng ngoi ca 11 cht tng
hp c44
Bng 3.4Kt quphn tch phkhi lng ca 9 cht tng
hp c.45
Bng 3.5Kt quphn tch phcng hng tht nhn ca 11
cht tng hp c.47
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Bng 3.6 Kt quthtc dng khng khun. 50
Bng 3.7 Kt quthtc dng khng nm. 52
Bng
3.8-3.9
Kt quthc nghim (thtc dng gy c tbo)53-54
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DANH MC CC HNH V
HNH Trang
Hnh 1.1 Cu trc ha hc ca phn t1Hbenzimidazol. 2Hnh 1.2 Cu trc ha hc ca phn tvitamin B12. 2
Hnh 1.3 Cu trc ha hc ca phn t2-mercaptobenzimidazol. 3
Hnh 1.4CTCT chung ca dn cht 2-mercaptobenzimidazol theo
nghin cu ca Gigani Yaseen nm 2010.4
Hnh 1.5CTCT chung ca dn cht bis(2-benzimidazolthio) theo
nghin cu ca G. Srikanth nm 2010.
6
Hnh 1.6CTCT ca bis vtr N1 v N1 trong nghin cu ca G.
Srikanth nm 2010.6
Hnh 1.7CTCT chung ca dithiobis(1H-benzimidazol) trong
nghin cu ca G. Srikanth nm 2010.7
Hnh 1.8
CTCT chung ca dn cht bis(2-benzimidazolthio) theo
nghin cu ca Kozo Aoki v Kazuhiro Aikawa nm1999.
8
Hnh 1.9 Cng thc cu to ca Hoechst 33258 v Hoechst 33342. 11
Hnh
1.10
CTCT ca dn cht 2-(4-methoxyphenyl)-1H-
benzimidazol.12
Hnh
1.11
CTCT ca dn cht 2-cloromethyl-1,5,6-trimethyl-1H-
benzimidazol-4,7-dion.
13
Hnh
1.12
CTCT ca dn cht benzimidazol theo cc nh nghin
cu Trung Quc nm 2012.13
Hnh
1.13
CTCT chung ca dn cht 2-mercaptobenzimidazol theo
nghin cu ca K. Anandarajagopal v cng s.13
Hnh
1.14
CTCT chung ca dn cht benzimidazol theo nghin cu
ca Sung Yun Cho v cng snm 2001.15
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Hnh
1.15
CTCT chung ca dn cht benzimidazol theo nghin cu
ca B. Bhushan Lohray v cng s.16
Hnh1.16
Dng hbin ca cht 2-mercaptobenzimidazol. 45
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DANH MC CC S
S Trang
S
1.1
Phn ng to khung 2-mercaptobenzimidazol theo
Maw-Ling Wang v Biing-Lang Liu.17
S
1.2
Phn ng to khung 2-mercaptobenzimidazol ca Van
Allan v Deacon.18
S
1.3
Phn ng to khung 2-mercaptobenzimidazol to-
nitroamin.
18
S
1.4
Phn ng alkyl ha to dn cht 2-
mercaptobenzimidazol19
S
1.5Phn ng acyl ha to dn cht 2-mercaptobenzimidazol 19
S
1.6
Phn ng ngng tto hp cht bis-2-
mercaptobenzimidazol. 20
S
3.1
S tng qut tng hp cc dn cht 2-
mercaptobenzimidazol.30
S
3.2S tng hp cht II 31
S 3.3
S tng hp cht III. 32
S
3.4S tng hp cht III-a. 32
S
3.5S tng hp cht III-b 33
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S
3.6S tng hp cht III-c. 34
S 3.7
S tng hp cht III-d 35
S
3.8S tng hpcht IV 36
S
3.9S tng hp cht V 36
S 3.10
S tng hp cht V-a 37
S
3.11S tng hp cht V-b 38
S
3.12S tng hp cht V-c 39
S 4.1
C chphn ng ng vng 2-mercaptobenzimidazol. 55
S
4.2Vai tr ca KOH trong phn ng to cht II 55
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T VN Ngy nay, khi m hnh bnh tt ca con ngi ngy cng tr ln phc tp v
nhu cu chm sc sc khe ca ngi dn cng ngy mt nng cao, th vic
tm ra nhng loi thuc mi p ng vi nhu cu iu tr l ht sc cn thit.
tm c cc hot cht c hiu qu iu tr cao, t c v c th ng dng
trong iu tr, cc nh khoa hc thng da vo cu trc ca cc cht ang
c dng lm thuc hoc cc cht c tc dng sinh hc trin vng to ra
nhiu thuc mi. Vic to ra thuc mi l mt qu trnh lu di v c thc
hin theo cc con ng khc nhau, trong ng quan tm l con ng
tng hp ha dc. Ngy nay, nhng thuc c ngun gc tng hp ha dc
chim mt t l ln nht trong iu tr.
Vit Nam, hin nay nhm thuc khng khun, khng nm v chng ung
th ang chim t trng kh cao trong phng v iu tr bnh. Bn cnh
nhiu cng trnh nghin cu cho thy dn cht benzimidazol l nhm cht d
vng c tc dng sinh hc rt a dng nh: khng khun[34], khng nm[25,
26], chng vim [20, 22], gim au [7, 22], tr giun sn [15, 30], chng co
git,c ch virus, chng ung th[27] ..
gp phn lm phong ph thm cc nghin cu v tng hp v th tc
dng sinh hc ca cc dn cht benzimidazol, ng thi vi mong muna
dng ha nhm thuc ny Vit Nam, chng ti chn ti: Nghin cu
tng hp v th hot tnh sinhhc mt s dn cht 2-mercaptobenzimidazol
vi 2 mc tiu :1. Tng hp c mt s dn cht 2-mercaptobenzimidazol.
2. Th hot tnh khng khun, khng nm, chng ung thca mt s dn
cht tng hp c.
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CHNG 1: TNG QUAN
1.1. Khi qut chung vkhung benzimidazol
1.1.1. Cu trc ha hcBenzimidazol l mt hp cht hu c d vng thm, kt hp gia 2 nhn
benzen v imidazol. Trong lch s, benzimidazol c pht minh ra ln
u tin bi nh khoa hc Hoebrecher trong nm 1872 [17].
N
HN
2
34
71
5
6
Hnh 1.1.Cu trc ha hc ca phn t1Hbenzimidazol.
Trong tnhin c stn ti ca dn cht benzimidazol, trong ni bt
nht l N ribosyl dimethylbenzimidazol l mt thnh phn quan trng
trong cu trc ha hc ca phn tvitamin B12 [25, 31].
Hnh 1.2.Cu trc ha hc ca phn tvitamin B12.
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1.1.2. Tnh cht ha hc
Nhn chung, khung benzimidazol l mt base yu, c thha tan trong acid
v c long. Tnh base ca benzimidazol yu hn imidazol. Benzimidazolcng c y tnh cht ca mt acid, tan tt trong dung dch kim [17].
1.2. Khi qut chung v2-mercaptobenzimidazol v dn cht
1.2.1. Cng thc cu to ca 2-mercaptobenzimidazol
Tn khoa hc v cu trc:
N
HN
SH
1H-Benzoimidazol-2-thiol
1
234
5
6
7
Hnh 1.3.Cu trc ha hc ca phn t2-mercaptobenzimidazol.
Cng thc phn t : C7H6N2S
Khi lng phn t : 150,2 g/mol
1.2.2. Tnh cht l hc ca 2-mercaptobenzimidazol
- tan: Tan trong dung mi hu c nh: methanol, ethyl acetat, aceton, tan
rt tt trong ethanol; rt t tan trong nc.
- ha tan trong nc < 0,1g/100 mL 23,5C.
- Nhit nng chy: 300-305C.
- c im tinh th: Tinh thhnh phin mng, c mu vng nht hoc mu
trng.
1.3. Tc dng sinh hc ca dn cht 2-mercaptobenzimidazol
Trong nhiu thp k va qua, cc dn cht 2-mercaptobenzimidazol thu
ht c squan tm ca nhiu nh khoa hc do c tc dng sinh hc phong
ph nh: tc dng khng nm, khng khun, chng ung th, chng vim,
gim au, trgiun sn, chng co git, c chvirus, chng stng lipid mu,
chng x cng ng mch, chng vim lot, c chbm proton H+/K+-
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ATPase, khng acid dch v. Nhiu cht trong s c ng dng trong
lm sng [8, 25, 26].
1.3.1. Tc dng khng khun v khng nmNm 2010, Gigani Yaseen v cng s tin hnh nghin cu tng hp v
thtc dng khng khun ca mt sdn cht 2-mercaptobenzimidazol, cng
thc tng qut (1)nh sau:
Hnh 1.4. Cng thc cu to chung ca dn cht 2-mercaptobenzimidazol
theo nghin cu ca Gigani Yaseen nm 2010.
Bng 1: Cc dn cht ca cng thc (1)thhin bng sau:
K hiu ca cht Gc R
1aHN OH
1b HN NO2
1cHN
N
N
1dN
CO
CH3
1e HN NO2
O2N
1fHN C
N
O
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Tin hnh th tc dng khng khun ca nhng dn cht trn bng phng
php khuch tn trn thch. Cc thnghim viEscherichia coliATCC 3750
i din cho vi khun Gram (-) v Bacillus subtilisATCC 6633 l i dincho vi khun Gram (+), cc nng khc nhau 50g/ml, 100g/ml v
200g/ml (Sdng cht i chiu l Ciprofloxacin). Kt quc thhin
bng 2.
Bng 2. Cc thng sca vng c ch( mm).
Cht
B.subtilis 6633 E.coliACTT3750
50g/
ml
100g/
ml
200g/
ml
50g/ml 100g/
ml
200g/
ml
1a 4 9 14 - 8 9
1b 5 7 9 3 7 8
1c 5 11 16 8 9 14
1d 3 3 4 - 7 10
1e 4 10 13 5 9 161f - - 4 7 10 10
Ciprofloxacin 10 14 18 8 10 19
Kt qucho thy cht tt ccc cht 1a,1b,1c,1d,1ev 1fu c tc dng
c ch vi 2 vi khun trn. Trong cht 1f c tc dng rt km i viB.
subtilis, 1a v 1d khng c tc dng chng E. coli nng thp hn50g/ml, 1cv 1ethhin tc dng mnh nht [34].
Nm 2010, G. Srikanth v cng s nghin cu tng hp v thtc dng
sinh hc ca mt sdn cht bis(2-benzimidazolthio). Trong in hnh l
nhm cc dn cht sau:
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Hnh 1.5. Cng thc cu to chung (2) ca dn cht bis(2-
benzimidazolthio) theo nghin cu ca G. Srikanth nm 2010.
Bng 3.Cc dn cht ca cng thc (2) thhin bng sau:
TT K hiu cc cht n R = R
1 2a
2
H
2 2b H3CO-
3 2c F2HCO-
4 2d
3
H
5 2e H3CO-
6 2f F2HCO-
Hnh 1.6. Cng thc cu to (3) ca bis vtr N1 v N1 trong nghin cu
ca G. Srikanth nm 2010.
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Bng 4. Cc dn cht ca cng thc (3) thhin bng sau:
TTK hiu ca
cht
n R1= R1 R2= R2
1 3a
2
-COCH3
H
2 3b H3CO-
3 3c F2HCO-
4 3d
3
H
5 3e H3CO-
6 3f F2HCO-
Hnh 1.7. Cng thc cu to chung (4)ca dithiobis(1H-benzimidazol) trong
nghin cu ca G. Srikanth nm 2010.
Bng 5. Cc dn cht ca cng thc (4) c thhin bng sau:
TT K hiu ca cht R3= R3
1 6a H
2 6b CH3O-
3 6c F2HCO-
Tc dng khng nmBng phng php chn Candida albicansl nm i din thtc dng
v Ketoconazol l cht khng nm i chiu, kt qul tt ccc dn cht to
bis ca 2-mercaptobenzimidazol u c tc dng khng Candida albicans.
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Trong c cht 2b, 3b, 4b,5b,6b v6cc tc dng c chmnh Candida
albicansti nng 250 g/ml v 500 g/ml. Tuy nhin, tc dng c chny
vn cn km hn cht i chiu Ketoconazol [25, 26].Tc dng khng khun
S dng Staphylococus aureus l i din cho vi khun Gram (+) v
Escherichia coli l i din cho vi khun Gram (-) thnghim tc dng
khng khun ca cc dn cht trn vi cht i chiu l Ampicillin.
Ti nng 50g/ml th hot tnh khng E. coli v S. aureus ca cc dn
cht 2a, 3a,4a,5a v6al thp, nhng liu cao hn tc l nng 100
g/ml th cc dn cht ny li c tc dng khng khun trung bnh.
Cc dn cht 2b, 3b,4b,5b v6b u c tc dng trung bnh khng E. coli
v S. aureus ti nng 50g/ml, v tc dng ny rt tt khi nng
100g/ml.
Cn li, i vi cc dn cht 2c, 3c,4c,5c v6cc tc dng c chtrung
bnh trnE. coliv S. aureusnng 100g/ml [25, 26].
1.3.2. Tc dng gim cholesterol mu v chng x cng ng mch
Trong nm 1999, Kozo Aoki vKazuhiro Aikawa tin hnh nghin cu
tng hp v th tc dng chng tng lipid mu v x cng ng mch ca
mt sdn cht bis(2-benzimidazolthio) c cng thc cu to chung nh sau:
Hnh 1.8.Cng thc cu to chung (5)ca dn cht bis(2-benzimidazolthio)
theo nghin cu ca Kozo Aoki v Kazuhiro Aikawa nm 1999.
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Bng 6. Cc dn cht ca cng thc (5) c thhin bng sau:
K hiu
ca chtL1 R4 = R4
R5
= R5 R6 = R6
7a(CH2)4
H H H
7b -COC2H5 H H
8a
(CH2)5
H H H
8b -CH2C6H5 H H
8c -SO2CH3 H H
8d -COCH3 H H
8e -C3H7 H H
8f -COC2H5 H H
8g -COC2H5 H CH3O-
8h -COC2H5 -CN H
8i -CH3 -NO2 H
8j H H -CH3
8k -COC2H5 H -CH3
8l H -CH3 -CH3
8m -COC2H5 -CH3 -CH3
8n H H Cl
8o -COC2H5 H Cl
8p H Cl Cl
8q -COC2H5 Cl Cl
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8r -CH3 Cl Cl
8s -COC2H5 Cl -CF3
9a(CH2)6
H H H
9b -COC2H5 H H
10a(CH2)8
H H H
10b -COC2H5 H H
11a
O
H H H
11b -COC2H5 H H
11c -CH2C6H5 H H
11d -COC2H5 -CH3 H
11e -COC2H5 Cl Cl
12a -COC2H5 H H
13a -COC2H5 H H
Tc dng chng tng lipid mu v chng x cng ng mch c nh gi
thng qua t lhnh thnh Cholesterol Ester (CE%) trong mu ca chut v
th[8].
Kt qucho thy trong scc dn cht mang thth cc cht 7a,7b,8a-8s,
9a,9b,10a,10b,11a-11e,12a v13au c tc dng lm gim cholesterol
mu (thliu 5M).
1.3.3. Tc dng chng ung th
Nm 1971, Wong pht hin ra ADN topoisomerase I v II. y l enzym
xc tc trong qu trnh tho xon ca ADN siu xon v c vai tr quan trng
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trong cc giai on pht trin ca tbo ung th. Enzym ny gn vi ADN
to thnh phc hp thun nghch enzym-ADN. Nhiu nghin cu cho rng
bezimidazol gn vo phc hp thun nghch enzym-ADN, c chenzym, dnn c chshnh thnh cc acid nucleic v ngn chn vic sao chp ADN,
cui cng lm tbo cht.
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.
Hin nay, c rt nhiu cng trnh nghin cu vtc dng khng tbo ung
th ca dn cht benzimidazol c cng b. Hoechst 33258v Hoechst
33342 l cc dn cht benzimidazol u tin c tc dng trn ADN
topoisomerase I [5].
Hoechst 33342
(14)
Hoechst 33258
(15)
Hnh 1.9. Cng thc cu to ca Hoechst 33258 v Hoechst 33342.
Nm 1996, Jung Sun Kim v cc cng s tng hp mt lot dn cht 2-
(4-methoxyphenyl)-1H-benzimidazol v thhot ng khng tbo ung th
ca cc dn cht [19].
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N
N
H
OCH3O2N
X
NOCH3
O2N
(16)
NH
NOCH3
O2N
X = O, S(17)
(18)
Hnh 1.10. Cng thc cu to ca dn cht 2-(4-methoxyphenyl)-1H-
benzimidazol.
Kt qul cht 5-nitro-2-(4-methoxyphenyl)-1H-benzimidazol (16) thhintc dng c chtopoisomerase I ln nht. Shin din ca nhm nitro (nhm
thc khnng hot ng nh cht nhn lin kt hydro) vtr 5 trn khung
benzimidazol gy c chtopoisomerase I tt hn cc cc dn cht thvtr
4 hay 6.
n nm 2008, Armand Gellis v cng s tip tc cng b cht 2-
cloromethyl-1,5,6-trimethyl-1H-benzimidazol-4,7-dion (19) v cc dn cht
ca n c tc dng c chspht trin ca tbo ung th v, ung th rut kt
v ung th phi [9].
N
N
O
O
Cl
CH3
H3C
H3C (19)
N
N
O
O
SO2C6H5
CH3
H3C
H3C(20)
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Hnh 1.11. Cng thc cu to ca dn cht 2-cloromethyl-1,5,6-
trimethyl-1H-benzimidazol-4,7-dion.
Nm 2012 mt nhm cc nh nghin cu ca Trung Quc tin hnh
nghin cu v tc dng ung th ca mt snhm cht trong c dn cht
benzimidazol vi cng thc (hnh 1.12) :
R: N-methyl piperazine phenyl ; R: 2-methoxyphenyl.
Nghin cu chra cht ny c khnng c chspht trin ca tbo ung
th i trng HCT-116 [35].1.3.4. Tc dng chng co git
Nhng dn cht 2-mercaptobenzimidazol vi cng thc tng qut:
Hnh 1.13. Cng thc cu to chung (22) ca dn cht 2-
mercaptobenzimidazol theo nghin cu ca K. Anandarajagopal v cng s.
R7, R8 tng ng vi cc nhm thcho bi bng 7 di y.
Cc dn cht 22a-22k trong bng 7 c nghin cu v cho thy tc
dng chng co git khi tin hnh thnghim trn chut ( 20-30g) ti nhit
phng 25 3oC vi cht i chiu l Phenytoin (vi hiu qu86,84%) [6, 7].
Cc dn cht 22a-22k ny cng u cho tc dng chng vim khi tin hnh
th tc dng theo m hnh gy ph chn chut bng Carrageenin (biu th
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bng % c ch) vi cht chun Natri diclofenac (vi % c ch61,9%) [6].
Kt qunghin cu c thhin bng 7:
Bng 7.Cc dn cht ca cng thc (22)c tc dng chng co git, chngvim:
K hiu ca
chtN
R7
R8
% Hiu qu
chng co git %c ch
22a N
O
O 26,10 %
45,2%
22bN
75,96 % 37,5%
22cNH3C
O
38,49 % 47,6%
22dN
82,51 % 40,4%
22eHN
N 85,98 % 30,9%
22fO
N 80,24 % 26,2%
22gNH3C
O OH
42,76 % 52,4%
22h N
N
Ph
Ph
Ph
74,22 % 38,1%
22i N
O 82,02 % 33,3%
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22kN
O
O
68,81 % 32,9%
Phenytoin 86,84%
Natri diclofenac 61,9%
1.3.5. Tc dng chng bi tit acid dch v, c chbm proton H+/K+-
ATPase v chng vim lot ddy
Nm 2001, Sung Yun Cho v cng s nghin cu tng hp v th tc
dng chng bi tit acid dch v, c chbm proton H+/K+-ATPase v chng
vim lot ddy ca dn cht benzimidazol c cng thc cu to chung nh
sau:
Hnh 1.14. Cng thc cu to chung (23)ca dn cht benzimidazol theo
nghin cu ca Sung Yun Cho v cng s.
Bng 8.Mt sdn cht ca cng thc (23) c tc dng c chbm proton
H+/K+-ATPase v chng vim lot ddy:
X OA R
ShocSO
OC(CH3)2CH=CH
-
H
4-OCH3
5-OCH3
5-Cl
OC(CH3)2CH2CH2
-
H
4-OCH3
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5-OCH3
OCH=C(CH3)-H
4-OCH3
Cc dn cht trong bng 8 u c tc dng c chH+/K+-ATPase ddy.
Mt scht cng c tc dng chng bi tit dch vmn vddy chut. (S
dng Omeprazol l cht i chiu) [13, 25, 29].
Nm 2000, B. Bhushan Lohray v cng s tin hnh tng hp v thtc
dng chng vim lot v chng bi tit dch v d dy ca dn cht
benzimidazol c cng thc chung cu to nh sau:
Hnh 1.15. Cng thc cu to chung (24)ca dn cht benzimidazol theo
nghin cu ca B. Bhushan Lohrayv cng s.
Bng 9.Cc dn cht ca cng thc (24) c thhin bng sau:
R9 H F -OCH3 -OCH2CH3 -CH2SO2
R10 H -CH3
R11 H F -OCH2CF3 -OCH2(CH2)2 -OCH2CCl3
R12 H -CH3
Tt ccc dn cht trong bng 9u c tc dng chng lot, chng bi tit
dch v. Hn na, phn ln cc dn cht ny c tc dng chng bi tit dch
vmnh hn Omeprazol v c hiu lc cao trong vic chng vim lot d
dy, c bit l bo vnim mc ddy khng btn thng do ethanol gy
ra [21, 22, 29].
1.3.6. Cc tc dng khc ca dn cht 2-mercaptobenzimidazol
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Nhiu nghin cu cho thy cc dn cht 2-mercaptobenzimidazol cn c tc
dng khc nh: iu trbnh ri lon hthn kinh trung ng (trm cm) [4],
chng virus HIV [27], iu trgiun [15, 30], khng histamin [14, 23], chng dng [16, 24], chng oxy ha [10, 12], chng khun lao [18], dinh dng thn
kinh [11], chng vim [20, 22], gim au [7, 22]
1.4. Cc phng php tng hp dn cht 2-mercapto benzimidazol.
Hu ht cc phng php tng hp dn cht 2-mercaptobenzimidazol u i
tnguyn liu ban u l o-phenylendiamin v dn cht, trong sc mt ca
CS2, KOH, ethanol v nc to thnh khung 2-mercaptobenzimidazol. Sau
t dn cht trung gian ny, tin hnh tng hp cc dn cht 2-
mercaptobenzimidazol.
1.4.1. Phn ng ng vng to nhn 2-mercaptobenzimidazol t o-
phenylendiamin hoc dn cht ca o-phenylendiamin.
Theo nghin cu ca Maw-Ling Wang v Biing-Lang Liu (nm 1995) khung
2-mercaptobenzimidazol c tng hp bi nguyn liu l o-phenylendiamin
v carbon disulfid trong ethanol hoc methanol v nc, di xc tc ca cc
amin bc 3 (R3N) [32, 33]. Phn ng xy ra theo tng bc nh sau:
CS2 + R3N CS2R3N
NH2
NH2
+ CS2R3N
N
HN
SH + H2S + R3N
S 1.1. Phn ng to khung 2-mercaptobenzimidazol theo Maw-Ling
Wang v Biing-Lang Liu.
Van Allan v Deacon nghin cu tng hp 2-mercaptobenzimidazol t
nguyn liu l o-phenylendiamin v carbon disulfid trong dung mi ethanol
(hoc methanol) v nc di xc tc l KOH [25, 26, 32]. Phng trnh xy
ra theo tng bc nh sau:
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NH2
NH2
R + CS2EtOH , H2O
KOH
N
HN
SHR + H2S + H2O
R
NH2
NH2N
HN
SKREtOCS2K
EtOH, H2O,
N
HN
SKN
HN
SHR
R2. AcOH, H2O
CH3CH2OH + CS2+ KOH CH3CH2OCS2K + H2O
S 1.2 :Phn ng to khung 2-mercaptobenzimidazol ca Van Allan v
Deacon.
Ngoi ra cn c rt nhiu phng php tng hp dn cht 2-
mercaptobenzimidazol cng i t nguyn liu ban u l o-phenylendiamin
trong cng dung mi EtOH/nc. V dnh: o-phenylendiamin phn ng vi
Thiourea SC(NH2)2 dng xc tc l acid poly-phosphoric (PPA), hoc o-
phenylendiamin phn ng vi lu hunh (S8) dng xc tc l Bu4NOH [28].
Nhn 2-mercaptobenzimidazol cng c th to ra t o-nitroamin bng cch
alkyl ha nhm NH2trc v khha nhm NO2sau, sau l phn ng to
2-mercaptobenzimidazol [8]. S ca phn ng nh sau:NO2
NH2
NO2
NH
R N
N
SH
R
S 1.3:Phn ng to khung 2-mercaptobenzimidazol to-nitroamin
1.4.2. Phn ng to dn cht 2-mercaptobenzimidazol
S dng cc tc nhn khc nhau tng hp dn cht 2-
mercaptobenzimidazol da trn cc phn ng alkyl ha, acyl ha, phn ng
ngng tto hp cht bis 2-mercaptobenzimidazol.
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1.4.2.1. Phn ng alkyl ha:
Cc dn cht 2-mercaptobenzimidazol thng c th vi tc nhn R-X
(X l halogen) to ra cc nhm thkhc nhau vtr N-1, vtr S ( S-alkylha) trn nhn 2-mercaptobenzimidazol. Phn ng thng c thc hin
trong cc dung mi nh: aceton, DMF, THF Vi cc xc tc base: natri
hydrid, natri hydroxyd, kali carbonat khan [1, 8]
S 1.4.Phn ng alkyl ha to dn cht 2-mercaptobenzimidazol.
1.4.2.2. Phn ng acyl ha:
Phn ng N-acyl ha th vo v tr ca N ca dn cht 2-
mercaptobenzimidazol thng c to thnh khi cho cc dn cht trn tc
dng vi clorid acid hoc anhydrid acid v thc hin trong mi trng khan
ca dung mi nh sau: acetonitril, ethyl acetat, THF, DMF, DMAc,...vi sc
mt ca xc tc nh: pyridin, triethylamin, K2CO3khan,...Phn ng N-acyl ny
c ththc hin nhit t30-150oC, nhng nhit tt nht l t50-100oC
[1, 8]
S 1.5.Phn ng acyl ha to dn cht 2-mercaptobenzimidazol.
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1.4.2.3. Phn ng ngng tto hp cht bis-2-mercaptobenzimidazol
Thng qua cc ti liu tham kho, tng hp hp cht bis-2-
mercaptobenzimidazol v dn cht thng thng sdng tc nhn Br-(CH2)n-Br v phn ng c thc hin trong nhng dung mi nh: ethanol, aceton,
acetonitril, ethyl acetat, DMF, THF...vi s c mt ca xc tc base nh:
aOH, K2CO3, C2H5ONa, triethylamin... Phn ng ny cng c th xy ra
trong mi trng ethanol v nhit vi svng mt ca bt knhng xc tc
no trn. Nhit thc hin phn ng ny ty thuc vo bn cht ca
cht tham gia phn ng v dung mi, thng thng l t 0-150oC nhng tt
nht l t20-100oC. Cn nu phn ng thc hin trong mi trng ethanol vi
svng mt ca cht xc tc th nhit tt nht l t50-120oC [8, 25, 26].
S 1.6. Phn ng ngng tto cc dn cht bis 2-mercaptobenzimidazol.
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CHNG 2: I TNG V PHNG PHP NGHIN CU
2.1. Nguyn liu, thit b
Thc hin lun vnny chng ti sdng mt sha cht, dung mi vthit bca phng th nghim Tng hp ha dc- Bmn Cng nghip Dc,
bao gm:
2.1.1. Ha cht
Cc ha cht v dung mi s dng trong qu trnh thc nghim c
thng k trong bng 2.1 sau:
Bng 2.1.Cc ha cht, dung mi sdng trong qu trnh thc nghim.
TT Tn ha cht, dung mi Xut x
1 O-Phenylendiamin Merck, c
2 Acid acetic Merck, c
3 Dicloromethan Trung Quc
4 Aceton Trung Quc5 Acetonitril Trung Quc
6 Cloroform Trung Quc
7 Tetrahydrofuran Trung Quc
8 Methanol Trung Quc
9 Ethanol tuyt i Trung Quc10 Kali carbonat Trung Quc
11 N,N-dimethylformamid (DMF) Trung Quc
12 Acid sulfuric c Trung Quc
13 Acid bromhydric Trung Quc
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14 Natri hydroxyd Trung Quc
15 Natri sulfat Trung Quc
16 Kali hydroxyd Trung Quc
17 Carbon disulfid Trung Quc
18 Nc ct Vit Nam
2.1.2. Thit b, dng c
Cc my mc, dng csdng trong qu trnh thc nghim c thng
k trong bng 2.2 sau:Bng 2.2. Cc my mc, dng csdng trong qu trnh thc nghim.
T
TTn my mc, dng c Xut x
1 Sinh hn hi lu c
2Bnh cu 25ml, 50ml, 100ml, 250ml,
500ml
c
3 Bnh cu 2 c100ml c
4 Nhit kthy ngn Trung Quc
5 Blc ht chn khng Buchner Trung Quc
6 My khuy tgia nhit IKA c
7 My ct quay Buchi B480 Thy s
8 Cn kthut Sartorius BP 2001S Thy s
9 My o nhit nng chy EZ-Melt M
10 Tsy Memmert c
11 Bn mng silicagel GF254Merck c
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12 Pipet 0,5ml, 1ml, 5ml, 10ml c
13 ng ong 20ml, 25ml, 100ml Trung Quc
14 Bnh chit 50ml, 100ml, 250ml Trung Quc
15 Bnh sc k Trung Quc
16 a petri Trung Quc
17 Cc c m50ml, 100ml, 250ml Trung Quc
18 n tngoi (soi bn mng) c
19 Tlnh Hn Quc
20 Bp bc (Heating Mantles) Hn Quc
21 My o phhng ngoi GX-Perkin Elmer M
22My o phkhi lng Agilent 6310 Ion
Trap v Autospec PremierM
23 My o ph cng hng t ht nhnBruker AV-500
M
2.2. Ni dung nghin cu
1. Tng hp mt sdn cht 2-mercaptobenzimidazol.
- Sdng phn ng alkyl ha vi cc tc nhn benzyl clorid, ECA, MCA to
nhm thmi vtr S, N1 trn khung 2-mercaptobenzimidazol.
- Thc hin phn ng ngng tto hp cht bis-2-mercaptobenzimidazol.2. Kim tra tinh khit v xc nh cu trc ca cc dn cht tng hp
c.
3. Th hot tnh khng khun, khng nm, chng ung th ca mt s dn
cht 2-mercaptobenzimidazol tng hp c.
2.3. Phng php nghin cu
2.3.1. Tng hp ha hc
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S dng cc phng php thc nghim c bn trong ha hc hu c
tng hp cc cht dkin.
Dng SKLM theo di tin trin ca phn ng. Soi n tngoi bcsng 254nm nhn bit vt sc k.
Xc nh tinh khit ca cc sn phm tng hp c bng phngphp
SKLM v o nhit nng chy.
2.3.2. Xc nh cu trc
Xc nh cu trc ca cc cht tng hp c da trn kt quphn tch
ph hng ngoi (IR), Ph khi lng phn t (MS), Ph cng hng t htnhn (1HNMR).
Phhng ngoi (IR):
c ghi ti phng Phn tch hu c - Vin ha hc Vit Nam, trn my
Perkin Elmer vi k thut vin nn KBr trong vng 4000-400cm-1. Mu rn
c phn tn trong KBr sy kh vi tlkhong 1:200 ri p di dng
vin nn di p lc cao c ht chn khng loi bhi m.
Di hp th nm trong vng 1640-1590 cm-1c trng cho lin kt C=N;
vng 1512-1403 cm-1c trng cho lin kt C=C v vng 1371-1257 cm-1c
trng cho lin kt C-N.
Di hp thtrong vng 3179-3136 cm-1c trng cho lin kt N-H.
Di hp th trong vng 1750-1738 cm-1c trng cho lin kt C=O, vng
1220-1179 cm-1c trng cho lin kt C-O.
Phkhi lng phn t(MS):
c ghi theo chkhng phn mnh (ESI) trn my Autospec Premier
ti khoa Ha Trng i hc Khoa hc tnhin - H Ni v trn my Agilent
6310 Ion Trap ti phng Phn tch ha c - Vin Ha hc cc hp cht thin
nhin - Vin Hn lm khoa hc v cng nghVit Nam. Phcng hng t
ht nhn (1HNMR):
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c ghi trn my Bruker AV-500 ti phng Phn tch ph- Vin Ha hc
- Vin Hn lm khoa hc v cng nghVit Nam.
dch chuyn ha hc ca proton vng thm ca cc cht dao ng trongkhong 7,09-7,65 ppm
dch chuyn ha hc ca proton nhm CH2trong lin kt -CH2-S- dao
ng trong khong 3,33-3,62 ppm.
dch chuyn ha hc ca proton nhm CH2trong lin kt -CH2-N- dao
ng trong khong 4,04-5,35 ppm.
2.3.3. Thtc dng sinh hc
Thtc dng khng nm, khng khun:
Th tc dng khng khun v khng nm tin hnh theo phng php
khuch tn trn thch, thc hin ti bmn Vi sinh- Sinh hc ca Trng i
hc Dc H Ni
2.3.3.1. Nguyn tc
Thtc dng khng khun v khng nm c 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 tmu thkhuch tn vo
mi trng thch sc chspht trin ca vi sinh vt kim nh to thnh
vng v khun hoc vng v nm.
2.3.3.2. Cch tin hnh
a. Cc vi sinh vt sdng trong thnghim
Bmn Vi sinh - Sinh hc, Trng i hc Dc H Ni tin hnh thtc
dng khng khun vi 5 vi khun Gram (+) v 5 vi khun Gram (-), th tc
dng khng nm vi 4 vi nm:
+ Vi khun Gram (-) gm:
- EscherichiacoliATCC 25922 (EC)
- ProteusmirabilisBV 108 (Pro)
- ShighellaflexneriDT 112 (Shi)
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- SalmonellatyphiDT 220 (Sal)
- Pseudomonas aeruginosaVM 201 (Pseu)
+ Vi khun Gram (+) gm:- StaphylococcusaureusATCC 1128 (Sta)
- BacilluspumilusATCC10241 (Bp)
- Bacillussubtilis ATCC6633 (Bs)
- Bacillus cereusATCC9946 (Bc)
- SarcinaluteaATCC9341 (SL)
+ Vi nm kim nh gm:
- Candida albicans (Ca)- Aspergillus niger (As)- Saccaromyces ceravitiae (Sa)- Penicilium sp. (Pe)
Khng sinh i chiu: Streptomycin (Strep) 14g/ml v Penicillin (Peni) 27
UI/ml i vi thnghim khng khun v Itraconazol (Itra) 50g/ml i vi
thnghim khng nm.
b. Mi trng thnghim
Bng 2.3.Cc mi trng trong thnghim khng khun v khng nm.
Khng khun Khng nm
Canh thang
nui cy VK
Thch
thng
Sabouraud
lng nui cy
C. albicans
Th nghim
Sabouraud
- NaCl 0,5%
- Cao tht
0,3%
- Pepton
0,5%
- Nc va
- NaCl 0,5%
- Thch
1,6%
- Pepton
0,5%
- Nc va
- Pepton 1,0%
-Glucose 2,0%
-Nc va
100ml.
- Pepton
1,0%
- Glucose
2,0%
- Thch
1,6%
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100ml. 100ml. - Nc va
100ml.
+nh gi kt qu: Da trn ng knh vng v khun hoc 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 khun hoc vng v nm,
iD : ng knh vng v khun hoc vng v nm thi,
s : lch thc nghim chun c hiu chnh,
n : sth nghim lm song song (n=3).Thtc dng khng tbo ung th:
Theo phng php ca Skehan & cng s ( 1990 ) v Likhiwitayawuid &
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, thc hin ti Vin Ha hc cc hp cht thin nhin- phng Sinh hc thc
nghim.
Dng t bo th: Dng Hep-G2: Ung th gan v dng MCF7: ung th
biu m v.
Mi trng nui cy tbo:DMEM (Dulbeccos Modified Eagle Medium)
hoc MEME (Minimum Essential Medium with Eagles salt). C bsung L-glutamine, Sodium piruvat, NaHCO3, PSF(Penixillin- Streptomycin sulfate-
Fungizone); NAA (Non-Essential Amino Acids); 10% BCS (Bovine Calf
Serum).Tripsin-EDTA 0,05%; DMSO (Dimethyl Sulfoside); TCA(Trichloro
Acetic acid); Tris Base; PBS (Phosphate Buffered Saline); SRB (Sulfo
Rhodamine B); Acid Acetic.
Cc dng cdng 1 ln: Bnh nui cy t bo, phin vi lng 96 ging,
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pipet pasteur, cc u tup cho micropipet
Cht chun chng dng tnh: Dng cht chun c khnng dit tbo:
Ellipithine, Vinblastine hoc Taxol pha trong DMSO.c trn my ELISA bc sng 495-515nm
Tnh kt qu: Gi trCS: l khnng sng st ca tbo nng no
ca cht thtnh theo % so vi i chng. Da trn kt quo c ca chng
OD (ngy 0), DMSO 10% v so snh vi gi trOD khi trn mu tm gi
trCS(%) theo cng thc:
OD (mu)OD (ngy 0)
CS% = x 100
OD (DMSO)OD (ngy 0)
Gi trCS% sau khi tnh theo cng thc trn, c a vo tnh ton Excel
tm ra % trung bnh lch tiu chun ca php thc lp li 3 ln
theo cng thc ca Ducan nh sau: lch tiu chun
(xi- x )2 =
n-1
Cc mu c biu hin hot tnh (CS < 50%) s c chn ra th
nghim tip tm gi trIC50
Gi trIC50: dng gi trCS ca 10 thang nng , da vo chng trnh
Table curve theo thang gi trlogarit ca ng cong pht trin tbo v nng
cht th tnh gi tr IC50.(Mu tinh c IC50 10g l c hot tnh)
Cng thc: 1/y=a+blnX
Trong Y: nng cht th; X: Gi trCS (%)
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CHNG 3: THC NGHIM V KT QU3.1. Tng hp ha hc
Thng qua cc ti liu tham kho v da vo iu kin ca phng th nghim,
cc ha cht cho php, chng ti tin hnh tng hp mt sdn cht ca 2-
mercaptobenzimidazol i tngun nguyn liu ban u l o-phenylendiamin
theo s sau:
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3.1.1. Tng hp 2-mercaptobenzimidazol (cht II)
Phng php ca Van Allan v Deacon :
S 3.2:S tng hp 2-mercaptobenzimidazol [25].
Tin hnh: Cho vo bnh cu hn hp gm c: 7,03g (65 mmol) O-
phenylendiamin v 3,64g (65 mmol) KOH, 70ml ethanol, 12ml nc ct, sau
thm tt3,93ml (65 mmol) carbon disulfid (CS2) v thm vi vin bt
(lm hn hp phn ng si u). Tin hnh hi lu trong 3 gi. Theo di
tin trin phn ng bng sc k lp mng vi hdung mi l dicloromethan:
methanol (20: 1) v hin mu bng n tngoi bc sng 254nm.
X l hn hp phn ng: Sau khi phn ng kt thc, thm vo hn hp
phn ng 0,7g than hot, tip tc un trong vng 10 pht, sau lc nng v
ra 3 ln bng ethanol (bthan hot v bt). Dch thu c thm 70ml ncnng (60-70oC), acid ha n pH khong 5-6, tip tc khuy khong 1 gi. Lc
ly ta (ra bng nc ct) v sy kh di n hng ngoi, thu c sn
phm th. Tinh chbng cch kt tinh li trong EtOH 50%, thu c 9,15g sn
phm.
Kt qu: - Cm quan: sn phm l tinh thhnh phin mng, mu trng bc
- Nhit nng chy to
nc302-3060
C [25]- Hiu sut phn ng 93,89%, Rf(CH2Cl2: CH3OH=20:1)=0,32.
3.1.2. Tng hp cc dn cht ca 2-mercaptobenzimidazol.
.1.2.1. Tng hp 2-benzylthio-1H-benzo[d]imidazol (cht I I I)
S phn ng:
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S 3.3: Tng hp 2-benzylthio-1H-benzo[d]imidazol
Tin hnh: Cho hn hp gm: 3 g (0,02 mol) 2-mercaptobenzimidazol (II);
2,9 g K2CO3khan v 70 ml aceton vo trong bnh cu 2 cdung tch 100 ml,
kt hp khuy t. Thm tt2,3 ml (0,02 mol) benzylclorid vo hn hp trn,
khuy nhit phng trong 1 gi. Sau hi lu hn hp trn trong 7 gi.
Theo di tin trin phn ng bng sc k lp mng vi h dung mi
dicloromethan: methanol = 100:1, hin mu bng n t ngoi bc sng
254 nm.
Xl hn hp sau phn ng: Hn hp sau khi kt thc phn ng c lm
ngui n nhit phng ri lc loi bmui v c. Dch lc c thm 40ml
nc ct, khuy k, s xut hin ta mu trng. Sau lc ly ta, ra ta
bng nc ct (15ml x 3 ln), sy ta nhit 50-60C, thu c 3,60g sn
phm.
Kt qu: - Cm quan: sn phm l bt mu trng, xp.
- Nhit nng chy: tonc186-1870C.
- Hphn ng=75%. Rf (CH2Cl2:CH3OH=100:1) =0,67.
3.1.2.2.Tng hp 1-benzyl-2-(benzylthio)-1H-benzo[d]imidazol (cht I I I -a).
S phn ng:
S 3.4: Tng hp 1-benzyl-2-(benzylthio)-1H-benzo[d]imidazol
Tin hnh: Thm 0,5 g (2 mmol) 2-benzylthio-1H-benzo[d]imidazol (III) v
0,5 g K2CO3khan trong 10 ml DMF khan vo bnh cu 2 cdung tch 100 ml,
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kt hp khuy t. Thm tt0,14 ml (2 mmol) benzylclorid vo hn hp trn.
Khuy nhit phng trong 1 gi. Sau un hn hp trn nhit 110
C
o
trong 20 gi. Theo di tin trin phn ng bng sc k lp mng vi hdung mi dicloromethan: methanol = 100:1, hin mu bng n t ngoi
bc sng 254 nm.
Xl hn hp sau phn ng: Hn hp sau khi kt thc phn ng c lm
ngui n nhit phng ri lc loi bmui v c. Dch lc c thm 30
ml nc ct v khuy kkhong 4 gi. Xut hin ta mu trng, lc ly ta,
ra bng nc ct (10 ml x 3 ln). Sy ta 50-60C, thu c 0,62g sn
phm.
Kt qu: - Cm quan: sn phm l bt mu trng, xp.
- Nhit nng chy: tonc80-900C.
- Hphn ng=90,18%. Rf (CH2Cl2:CH3OH=100:1) =0,86.
3.1.2.3. Tng hp methyl 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetat( cht I I I -b).
S phn ng
S 3.5: Tng hp methyl 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetat
Tin hnh: Cho vo bnh cu dung tch 50 ml hn hp gm: 0,5 g (2 mmol)2-benzylthio-1H-benzo[d]imidazol (III), 0,5 g K2CO3 v 10 ml DMF khan,
khuy nhit phng. Thm t t 0,183 ml (2 mmol) methyl cloroacetat
(MCA) vo hn hp trn. Khuy tip hn hp trn nhit thng trong 1
gi. Sau un hn hp trn nhit 105oC trong 5 gi. Theo di tin trin
phn ng bng sc k lp mng vi h dung mi
dicloromethan:methanol=100:1, hin mu bng n tngoi bc sng 254
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nm.
X l hn hp sau phn ng: Hn hp sau phn ng c x l bng
cch lc loi b K2CO3, dch lc c thm 20 ml nc ct lnh kt hpkhuy t. Sau chit bng ethyl acetat 3 ln, mi ln 10 ml. Sau thm
nc ct kt hp khuy tmnh nhit phng cho ta mu trng. Lc ly
ta v ra bng nc ct (10 ml x 3 ln). Sy kh ta, thu c 0,35g sn
phm.
Kt qu: - Cm quan: sn phm l bt mu trng.
- Nhit nng chy: tonc58-590C.
- Hphn ng=53,93%. Rf (CH2Cl2:CH3OH=100:1) =0,77.
3.1.2.4. Tng hp ethyl 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetat(cht I I I - c).
S phn ng:
S 3.6: Tng hp ethyl 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-
yl)acetat
Tin hnh: Cho vo bnh cu dung tch 50 ml hn hp gm: 0,5 g (2 mmol)
2-benzylthio-1H-benzimidazol (III); 0,5 g K2CO3v 10 ml DMF khan khuy
nhit phng. Thm tt0,214 ml (2 mmol) ethyl cloroacetat( ECA) vo
hn hp trn. Khuy tip hn hp trn nhit thng trong 1 gi. Sau
un hn hp trn nhit 105oC trong 5 gi. Theo di tin trin phn ng
bng sc k lp mng vi hdung mi dicloromethan: methanol=100: 1, hin
mu bng n tngoi bc sng 254 nm.
Xl hn hp sau phn ng: Hn hp sau phn ng c xl bng cch
lc loi bK2CO3v dch lc c thm 20 ml nc lnh kt hp khuy t.
Sau chit bng ethyl acetat 3 ln, mi ln 10 ml. Dch chit c lm khan
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qua m bng 5g Na2SO4. Lc, ct loi ethyl acetat. Sau thm nc ct kt
hp khuy tmnh nhit phng cho ta mu trng. Lc ly ta v ra
bng nc ct (10 ml/ ln x 3 ln). Sy kh ta, thu c 0,33g sn phmKt qu: - Cm quan: sn phm l bt mu trng.
- Nhit nng chy: tonc62-630C.
- Hphn ng=50,61%. Rf (CH2Cl2:CH3OH=100:1) =0,76.
3.1.2.5. Tng hp 1,4-bis(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)butan(cht I I I -d)
S tng hp:
S 3.7: Tng hp 1,4-bis(2-(benzylthio)-1H-benzo[d]imidazol-1-
yl)butan.
Tin hnh: Cho vo bnh cu dung tch 50 ml hn hp gm: 0,3 g (1,32
mmol) 2-benzylthio-1H-benzimidazol (III); 0,3 g K2CO3; 15 ml C2H5OH tuyt
i; kt hp khuy t. Thm tt0,078 ml (0,66 mmol) 1,4-dibromobutan
vo hn hp. Tin hnh un hi lu trong 14h. Theo di tin trin phn ng
bng sc k lp mng vi hdung mi dicloromethan: methanol=20: 1, hin
mu bng n tngoi bc sng 254 nm.
Xl hn hp sau phn ng: Thm 50 ml nc ct vo hn hp sau phnng ha tan ht K2CO3, ng thi khuy tqua m xut hin ta vng.
Lc thu ta th. Ta ny c kt tinh li trong 3 ml MeOH. Lc thu tinh th
v ra li bng MeOH lnh. Sy thu c 0,27g sn phm.
Kt qu: - Cm quan: sn phm l tinh thmu trng, hi vng.
- Nhit nng chy: tonc166-1680C.
- Hphn ng=40,80%. Rf (CH2Cl2:CH3OH=20:1) =0,85.
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3.1.2.6. Tng hp ethyl 2-(1H-benzimidazol-2-ylthio)acetat (cht I V)
S tng hp:
S 3.8: Tng hp ethyl 2-(1H-benzimidazol-2-ylthio)acetat
Tin hnh: Cho vo bnh cu dung tch 50 ml hn hp gm : 0,3g (2
mmol) 2-mercaptobenzimidazol (II); 0,3g K2CO3; 15 ml aceton. Khuy t
nhit phng. Thm 0,22 ml ECA(2 mmol) vo hn hp trn, tip tc khuynhit phng 24h. Theo di tin trin phn ng bng sc k lp mng vi
h dung mi dicloromethan: methanol=20: 1, hin mu bng n t ngoi
bc sng 254 nm.
Xl hn hp sau phn ng: Thm 50 ml nc ct vo hn hp sau phn
ng ha tan ht K2CO3,kt hp khuy t5h nhit thng. Lc thu ta,
kt tinh li ta trong 3 ml EtOH 96%. qua m trong tlnh sau lc thu
tinh th. Sy thu c 0,29g sn phm.
Kt qu: - Cm quan: sn phm l tinh thhnh kim, mu trng.
- Nhit nng chy: tonc102-1030C.
- Hphn ng=61,44%. Rf (CH2Cl2:CH3OH=20:1) =0,56.
3.1.2.7. Tng hp 1,4-bis(2-benzimidazolthio)butan (cht V)
Phng trnh phn ng :
S 3.9:Tng hp 1,4-bis(2-benzimidazolthio)butan.
Tin hnh: Cho vo bnh cu 2 cdung tch 250 ml: 3,51g (23,4 mmol) 2-
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mercaptobenzimidazol (II) trong 70ml ethanol tuyt i. Sau nh t t
1,39ml (11,7 mmol) 1,4-dibromobutan vo dung dch trn. Tin hnh hi lu
trong 14 gi. Theo di tin trin phn ng bng sc k lp mng vi hdungmi l dicloromethan: methanol (20: 1) v hin mu bng n tngoi bc
sng 254nm.
Xl hn hp sau phn ng: Sau khi phn ng kt thc, bnh cu c
ngui vnhit phng, thm khong 50ml nc ct vo trong bnh cu v
khuy, kim ha bng cch va khuy va nhttdung dch NaOH 2N vo
n pH khong 12-13, sau tin hnh lc ly ta, ra ta bng ethanol lnh.
Sy kh di n hng ngoi v em cn th thu c 3,83g sn phm.
Kt qu: - Cm quan: sn phm l bt mu trng.
- Nhit nng chy: tonc274-2770C.
- Hphn ng=92,31%. Rf (CH2Cl2:CH3OH=20:1) =0,42.
3.1.2.8. Tng hp 1,4-bis(1-benzyl-1H-benzo[d]imidazol-2-ylthio)butan
(cht V-a).
Phng trnh phn ng:
S 3.10: S tng hp 1,4-bis(1-benzyl-1H-benzo[d]imidazol-2-
ylthio)butan.
Tin hnh: Cho vo bnh cu 2 c dung tch 250ml hn hp gm: 0,42g
(1,19 mmol) 1,4-bis(2-benzimidazolthio)butan (V) trong 13ml DMF khan, sau
thm 0,45g K2CO3 v tin hnh khuy hot ha nhit phng trong
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vng 1 gi, tip theo l nht t0,28ml (2,37 mmol) benzyl clorid vo hn
hp phn ng. Tin hnh khuy 100oC, sau 1 gichm sc k xem th thy
c phn ng chy, tip tc un 100
o
C trong 10 gina th kt thc phn ng.Theo di tin trin phn ng bng sc k lp mng vi h dung mi l
dicloromethan: methanol (20: 1) v hin mu bng n tngoi bc sng
254nm
X l hn hp phn ng: Sau khi phn ng kt thc, bnh cu c
ngui vnhit phng. Chun bmt cc c mkhc cha khong 200ml
nc ct v 1 con khuy, va khuy va tthn hp phn ng trong bnh
cu vo cc c m trn, ra bnh cu bng t nc ct. Tip tc khuy trong
khong 2 gi ( tch sn phm khi dung mi v ha tan ht K2CO3 trong
nc). Sau tin hnh lc ly ta (ra ta bng nc ct). Sy kh di n
hng ngoi v em cn th thu c 0,5g sn phm.
Kt qu: - Cm quan: sn phm l bt mu trng, hi vng.
- Nhit nng chy: tonc166-1680C.
- Hphn ng=78,92%. Rf (CH2Cl2:CH3OH=20:1) =0,64.
3.1.2.9. Tng hp 1,4-bis(1-methoxycarbonylmethyl-1H-benzo[d]imidazol-2-
ylthio)butan (cht V-b).
Phng trnh phn ng:
S 3.11: S tng hp 1,4-bis(1-methoxycarbonylmethyl-1H-
benzo[d]imidazol-2-ylthio)butan.
Tin hnh: Cho vo bnh cu dung tch 250ml hn hp gm: 0,41g
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(1,16mmol) 1,4-bis(2-benzimidazolthio) butan (V) trong 13ml DMF khan, sau
thm 0,48g K2CO3 v tin hnh khuy hot ha nhit phng trong
vng 1 gi, nh t t 0,21ml (2,32mmol) methyl cloroacetat vo. Tin hnhkhuy nhit phng trong 3 gi30 pht. Theo di tin trin phn ng bng
sc k lp mng vi hdung mi l dicloromethan : methanol (20 : 1) v hin
mu bng n tngoi bc sng 254nm.
X l hn hp phn ng: Sau khi phn ng kt thc, ly bnh cu cha
hn hp phn ng ra. Chun bmt cc c mkhc cha khong 200ml nc
ct v 1 con khuy, va khuy va tthn hp phn ng trong bnh cu
vo cc c m trn, ra bnh cu bng t nc ct. Tip tc khuy trong
khong 2 gi ( tch sn phm khi dung mi v ha tan ht K2CO3 trong
nc). Sau tin hnh lc ly ta (ra ta bng nc ct). Sy kh di n
hng ngoi v em cn th thu c 0,37g sn phm.
Kt qu: - Cm quan: sn phm l bt mu trng.
- Nhit nng chy: tonc130-1320C.
- Hphn ng=64,18%. Rf (CH2Cl2:CH3OH=20:1) =0,46.
3.1.2.10. Tng hp 1,4-bis(1-ethoxycarbonylmethyl-1H-benzo[d]imidazol-2-
ylthio)butan (cht V-c).
Phng trnh phn ng:
S 3.12: S tng hp 1,4-bis(1-ethoxycarbonylmethyl-1H-
benzo[d]imidazol-2-ylthio)butan.
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Tin hnh: Cho vo bnh cu dung tch 250 ml hn hp gm: 0,51g (1,44
mmol) 1,4-bis(2-benzimidazolthio)butan (V) trong 13ml DMF khan, sau
thm 0,55g K2CO3v tin hnh khuy hot ha nhit phng trong vng 1gi, nhtt0,31ml (2,88 mmol) ethyl cloroacetat vo. Tin hnh un nhit
90oC trong 2 gi. Theo di tin trin phn ng bng sc k lp mng vi h
dung mi l dicloromethan: methanol (20: 1) v hin mu bng n tngoi
bc sng 254nm.
Xl hn hp phn ng: Sau khi phn ng kt thc, ly bnh cu hn hp
phn ng ra. Chun bmt cc c mkhc cha khong 200ml nc ct v 1
con khuy, va khuy va tthn hp phn ng trong bnh cu vo cc
c mtrn, ra bnh cu bng t nc ct. Tip tc khuy trong khong 2 gi
( tch sn phm khi dung mi v ha tan ht K2CO3 trong nc). Sau
tin hnh lc ly ta (ra ta bng nc ct). Sy kh di n hng ngoi v
em cn th thu c 0,62g sn phm.
Kt qu: - Cm quan: sn phm l bt mu trng.
- Nhit nng chy: tonc101-1030C.
- Hphn ng=81,81%. Rf (CH2Cl2:CH3OH=20:1) =0,5
Bng 3.1.Tm tt kt qutng hp ha hc.
STT Cht CTCT Cm quantonc
(oC)
Hiu sut
(%)
1 II N
HN
SH
Tinh th hnh
phin mng,mu trng bc.
302-306 93,89
2 IIIBt mu trng,
xp186-187 75
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3 III-aBt mu trng,
xp89-90 90,18
4 III-b Bt mu trng 58-59 53,93
5 III-c Bt mu trng 62-63 50,61
6 III-dTinh th mu
trng, hi vng166-168 40,80
7 IVTinh th hnh
kim, mu trng102-103 61,44
8 VN
HN
S (CH2)4 S
N
NH
Bt mu trng 274-277 92,31
9 V-aN
N
S (CH2)4 S
N
N
Bt mu trng
hi vng.166-168 78,92
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10 V-bN
N
S (CH2)4 S
N
N
CH2COOCH3
H3COOCH2C
Bt mu trng 130-132 64,15
11 V-cN
N
S (CH2)4 S
N
N
CH2COOC2H5
C2H5OOCH2C
Bt mu trng 101-103 81,81
3.2. Kim tra tinh khit ca cc dn cht tng hp c.
Sau khi tng hp c, chng ti tin hnh kim tra tinh khit ca
cc cht tng hp c bng phng php sc k lp mng v o nhit
nng chy nh sau:
SKLM c tin hnh trn bn nhm trng sn silicagel Merck 70-230 mesh,
quan st di n tngoi bc sng 254 nm.
- Dung mi ha tan: Ethanol hay DMF.
- Hdung mi khai trin l: Dicloromethan : methanol (20 : 1).
Dicloromethan : methanol (100 : 1).
Kt quthu c trn sc k i vi hdung mi trin khai trn u thy
mt vt r, gn, khng c vt ph, Rfkhc bit khi soi di nh sng n t
ngoi c bc sng 254nm.
o nhit nng chy bng my EZ-Melt: 11 cht sau khi tinh chu c
dng cht rn, c sy kh v o nhit nng chy.
Gi trRfv nhit nng chy (tnc) ca cc cht c c tm tt trong
bng sau:
Bng 3.2.Gi trRfv nhit nng chy (tnc) ca cc cht tng hp
c.
Cht Hdung mi khai trin Rf tnc (C)
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II Dicloromethan : methanol (20:1) 0,32 302-306
III Dicloromethan : methanol (100:1) 0,67 186-187
III-a Dicloromethan : methanol (100:1) 0,86 89-90
III-b Dicloromethan : methanol (100:1) 0,77 58-59
III-c Dicloromethan : methanol (100:1) 0,76 62-63
III-d Dicloromethan : methanol (20:1) 0,85 166-168
IV Dicloromethan : methanol (20:1) 0,56 102-103
V Dicloromethan : methanol (20:1) 0,42 274-277
V-a Dicloromethan : methanol (20:1) 0,64 166-168
V-b Dicloromethan : methanol (20:1) 0,46 130-132
V-c Dicloromethan : methanol (20:1) 0,50 101-103
Nhn xt: Vi hdung mi trin khai, cc cht tng hp c c vt gn,
r trn bn sc k, nh vy, c thkt lun s bl cc cht tng hp c
kh tinh khit.
3.3. Xc nh cu trc ca cc cht tng hp c
xc nh cu trc ca cc cht tng hp c, chng ti tin hnhphn tch ph hng ngoi (IR), ph khi lng phn t (MS) v ph cng
hng tht nhn proton (1H-NMR).
3.3.1. Kt quphn tch phhng ngoi (IR)
Thc hin ti phng phn tch cu trc phn tVin Ha hc Vit Nam,
tng c11 cht trn c ghi phhng ngoi (IR) trn my o phhng
ngoi Perkin Elmer vi k thut vin nn KBr ghi trong vng 4000-400cm-1
.
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Thng qua vic phn tch phca cc cht tng hp, chng ti nhn bit c
cc di hp phc 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 tca chngnh C=C thm, C=S, N-H, C=O ester, ceton... Ngoi ra vi cc nhm thkhc
nhau, mi cht li c nhng pic c trng ring. T xc nh c b
khung ca phn t.Phca cc cht c trnh by cc phlc 1-11. Kt
quphn tch phhng ngoi (IR) c ghi trong bng 3.3. Cc sliu phn
tch ph hp vi cc ti liu tham kho [2, 3]
Bng 3.3. Kt quphn tch phhng ngoi (IR) ca 11 cht tng hp
c.
Chtmax ca cc cht (cm
-1)
N-H C-HC=O
C=N
C=C
C-N C-O C=S C-S
II 3179 1618 1512 1257 1180 603
III 3136 1640 1403 1349 541
III-a 1590 1491 1361 567III-b 1743 1605 1447 1371 1218 573
III-c 1750 1604 1444 1371 1206 553
III-d 3076 1603 1427 1281 541
IV 3142 2966 1738 1621 1505 1301 1179 597
V 3136 3050 1613 1504 1268 601
V-a 3026 1612 1458 1352 576
V-b 3074 1745 1634 1450 1364 1217V-c 3052 1741 1608 1453 1372 1220 575
Nhn xt: Da vo bng 3.3 trn chng ti thy sliu phn tch phIR ca
11 cht hon ton ph hp vi ti liu tham kho cng nh cu trc dkin
ca cc cht.
Lu : Ring phca cht II cn c di hp thc trng ca lin kt C=S
(1200-1050 cm-1) v cht IIc thhbin nh sau:
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N
HN
SH
HN
NH
S
Hnh 1.16:Dng hbin ca 2-mercaptobenzimidazol.
3.3.2. Kt quphn tch phkhi lng (MS)
Tng c 9 cht: II, III, III(a, d), IV, V, V(a, b, c) c ghi phtheo ch
khng phn mnh (ESI) trn my Autospec Premier ti khoa Ha - Trng
i hc Khoa hc tnhin - H Ni v trn my Agilent 6310 Ion Trap ti
phng Phn tch hu c - Vin Ha hc cc hp cht thin nhin - Vin Hn
lm khoa hc v cng nghVit Nam.Phca cc cht c trnh by
cc phlc 12-20. Kt quphn tch ph MS c ghi trong bng 3.4. Cc s
liu phn tch ph hp vi cc ti liu tham kho [2, 3]
Bng 3.4. Kt quphn tch phkhi lng (MS) ca 9 cht tng hp
c.
Cht Cng thc cu to
Khi lng (MS)
Gii thchDkin
Thct oc
IIN
HN
SH
150 149,08 [M-H]-
III 240 241,28 [M+H]+
III-a 330 331,18 [M+H]+
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III-d 534 535,13 [M+H]+
IV 236 237,14 [M+H]+
VN
HN
S (CH2)4 S
N
NH
354 355,25 [M+H]+
V-aN
N
S (CH2)4 S
N
N
534 1091 [2M+Na]+
V-bN
N
S (CH2)4 S
N
N
CH2COOCH3
H3COOCH2C
498 499,18 [M+H]+
V-cN
N
S (CH2)4 S
N
N
CH2COOC2H5
C2H5OOCH2C
526 525,2 [M-H]-
Nhn xt: Tkt quphn tch phkhi lng c thnhn thy cc cht
tng hp c u c cc pic phn t, cc pic phn tny c skhi ng
bng khi lng phn ttnh theo cng thc ca cht dkin.
3.3.3. Kt quphn tch phcng hng tht nhn (1H-NMR)
khng nh chc chn cu trc ca cc cht tng hp c, chng ti
o phcng hng tht nhn proton (1H-NMR) c11 cht: II, III, III(a,
b, c, d), IV, V, V(a, b, c)ti phng Phn tch ph- Vin ha hc- Vin khoa
hc v Cng nghVit Nam. Phca cc cht c trnh by cc phlc
21-31. Kt quphn tch ph1H-NMR c ghi trong bng 3.5. Cc s liu
phn tch ph hp vi cc ti liu tham kho [2, 3].
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Bng 3.5.Kt quphn tch phcng hng tht nhn (1H-NMR) ca 11
cht tng hp c.
Cht CTCT1
H-NMR (500MHz, DMSO-d6 ),(ppm)
IIN
HN
SH
1
2
345
6
7
7,09-7,14(4H, m, 4CHAr)
12,51(2H, s, SH+NH)
III
4,56(2H, s, CH2-);
7,12-7,44(9H, m, CHAr);
III-a
4,62(2H, s, -CH2-S-);
5,35(2H, s, -CH2-N);
7,11-7,62(13H, m, CHAr)
III-b
3,33(3H, s, -CH3);
4,47(2H, s, -CH2-S-);4,93(2H, s, -CH2-N);
7,24-7,65(9H, m, CHAr).
III-c
1,17(3H, t, CH3-);
4,13(2H, q, CH2);
4,58(2H, s, -CH2-S-);
5,05(2H, s, -CH2-N);
7,17-7,61(9H, m, CHAr).
III-d
1,16(2H, s, -CH2-CH2-N-)
4,04(2H, s, -CH2-N)
4,56(2H, s, -CH2-S-)
7,13-7,58(9H, m, CHAr).
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IV
1,17(3H, t, -CH3)
4,09-4,14(2H, q, -OCH2-)
4,20(2H, s, -CH2-S-)
7,10-7,49(4H, m, CHAr)12,59(1H, s, -NH-).
VN
HN
S
1
2
34
5
6
7
(CH2)4 S
N
NH
1,89 (4H, s, 2-CH2)
3,47 (4H, s, 2S-CH2)
7,37 (4H, dd, 2(C5-H+C6-H))
7,60 (4H, dd, 2(C4-H+C7-H))
V-aN
N
S (CH2)4
N
N
S
1
2
345
67
1,85 (4H, s, 2-CH2)
5,34 (4H, s, 2N-CH2)
7,15 (8H, m, 8CHAr)
7,22- 7,28 (6H, m, 6CHAr)
7,45 (2H, s, 2CHAr)
7,55 (2H, s, 2CHAr)
V-bN
N
S
N
N
S
1
2
34
5
6
7 CH2COOCH3
H3COOCH2C
(CH2)4
1,83 (4H, t, 2-CH2)
3,33 (4H, s, 2S-CH2)
3,68 (3H, s, CH3)
5,08 (4H, s, 2N-CH2)
7,15 - 7,19 (4H, m, 2(C5-H + C6-H))
7,47 - 7,48 (2H, m, 2C4-H)
7,55 - 7,56 (2H, m, 2C7-H)
V-cN
N
S (CH2)4
N
N
S
1
2
34
5
6
7 CH2COOC2H5
C2H5OOCH2C
1,18 (6H, t, 2-CH3)
1,84 (4H, s, 2-CH2)
3,33 - 3,75 (4H, d, 2S-CH2)
4,12 - 4,16 (4H, q, 2-COOCH2)
5,06 (4H, s, 2N-CH2)
7,17 (4H, dd, 2(C5-H + C6-H))
7,48 (2H, d, 2C4-H)
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7,56 (2H, d, 2C7-H)
Ghi ch: : chuyn dch ha hc c ly theo gi tr trung bnh
(ppm); s: Singlet; d: Doublet; dd: Doublet doublet; t: Triplet; q: Quartet, m:Multiplet.
Nhn xt: Ph cng hng t ht nhn proton cho php nhn bit dng
proton v slng proton tng dng ca cc cht. Tbng 3.5 trn chng ti
nhn thy s c mt ca cc proton ph hp vi cng thc cu to ca cc
cht.
3.4. Thtc dng sinh hc3.4.1. Thtc dng khng khun, khng nmtm kim cc cht c hot tnh sinh hc cao, c trin vng trong sdng
lm thuc, chng ti tin hnh thtc dng khng khun v khng nm ca
cc cht tng hp c. Php thc thc hin ti bmn Vi sinh-Sinh hc,
Trng i hc Dc H Ni.
Tin hnh
Mu thc pha vo dung mi thch hp (ethanol) vi nng 100g/ml.
Cc cht i chiu cng c pha vo dung mi thch hp gm: Penicillin 27
UI/ml, Streptomycin 14g/ml v Intraconazol 50g/ml.
+Cc khoanh giy lc v trng v c sy kh c tm 3 ln vi
dung dch mu thgm: mu cht III, III-a, III-b, III-c,III-d, IV, V, V-a,
V-b, V-c, sau mi ln tm cc khoanh giy lc c cha mu thu c
sy nhit thp hn 60oC n kh ht dung mi.
+Chun bmi trng v cy VSV kim nh:
Vi vi khun kim nhc cy vo mi trng canh thang, ri nui cy
cho pht trin trong tm 37oC trong thi gian 18-24 gin nng 107t
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 VSV kim nh vo vi tl2,5ml/ 100ml. Lc trn VSV kim nh
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phn tn u trong mi trng thch thng, ri vo a petri v trng vi
thtch 20ml/a v cho thch ng li.
Vi vi nm: Vi nm kim nh (C. albicans) c cy vo mi trngSabouraud lng, ri nui cy cho pht trin trong tm 28-300C trong 18-24h
n nng 107tbo/ml (kim tra bng pha long v dy dch chun). i
viAspergillus nigerth sdng huyn conidi trong dung dch Tween 80 nng
0,2% v trng. Mi trng Sabouraud c v trng (tit trng 1180C/30
pht) c lm lnh v45-500C v c cy ging VSV kim nh vo vi t
l2,5 ml/ 100 ml. Lc u VSV kim nh phn tn u trong mi trng
thch, ri vo cc apetri v trng vi thtch 20 ml/a v cho thch
ng li.
+t khoanh giy lc: Khoanh giy lc c tm cht thv x l
nh trn c t ln bmt mi trng thch thng cha VSV kim nh
theo s nh sn (vi vi nm th phi lm 4 thc nghim song song).
+trong 18 - 24h, cc a petri c mu thc t nh trn trong tm
toC = 37oC i vi vi khun v toC = 28-30oC i vi vi nm, ri sau ly
ra c kt qu, o ng knh vng v khun hoc vng v nm nu c (s
dng thc kp Panmer chnh xc 0,02 mm).
Kt quthnghim
Tc dng khng khun, khng nm thhin bng sau:
Bng 3.6.Kt quthtc dng khng khun.
VK
Mu
ng knh vng v khun [mm] ( D , s)
Bc. Bs. Bp. SL. Sal EC Shi Pro Pseu Sta.
III - - - - - - - - - -
III-a11,34
0,31-
15,30
0,57-
14,62
1,17
14,62
1,17-
14,36
1,40
15,34
1,09-
III-b8,60
0,57-
13,07
0,32- - - - - - -
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III-c - - - - - - - - - -
III-d - -12,10
0,86-
16,46
0,74
11.70
0,54- - -
14,76
0,58
IV - - - - - - - - - -
V - -12,36
1,25-
16,92
0,61
12,10
0,31
10,10
0,66- -
14,62
0,69
V-a -10,65
0,76- -
15,45
0,95
10,75
0,40
7,35
0,34- - -
V-b - - - - - - - -12,54
0,84-
V-c - - - - - - -12,31
0,84- -
Peni19,38
0,37
20,47
0,70-
12,91
0,47- - - - -
11,42
0,67
Strep21,50
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
Vi () l khng c tc dng khng khun.
Kt lun:
Cht III-ac tc dng khng khun trn 2 chng vi khun G(+) l Bc, Bp
v 4 chng vi khun G(-) l Sal, EC, Pro, Pseu.
Cht III-bc tc dng khng khun trn 2 chng vi khun G(+) l Bc v Bp.
Cht III-d c tc dng khng khun trn 2 chng vi khun G(+) l Bp, Sta
v 2 chng vi khun G(-) l EC, Sal.
Cht V c tc dng khng khun trn 2 chng vi khun G(+) l Bp, Sta v 3
chng vi khun G(-) l Sal, EC, Shi.
Cht V-ac tc dng khng khun trn 1 chng vi khun G(+) l Bs v 3
chng vi khun G(-) l Sal, EC, Shi.
Cht V-bchc tc dng khng khun trn chng vi khun G(-) Pseu cn
cht V-cli c tc dng trn chng G(-) Pro.
Cc cht III, III-c, IV khng c tc dng khng khun trn cc chng vi
khun th.
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Bng 3.7. Kt quthtc dng khng nm.
Vi nm
Mu th
ng knh vng v nm [mm] ( D , s)
Ca As Sa Pe
III - -
8,26
1,17 -
III-a - -9,07
0,59-
III-b - -8,47
1,41-
III-c
9,36
0,37 -
8,32
0,65 -
III-d7,35
0,55-
10,75
0,53-
IV7,73
0,64- - -
V7,08
1,02-
11,26
0,60-
V-a8,45
1,15
-8,62
0,41
-
V-b7,98
0,55-
9,20
0,96-
V-c8,94
0,23- - -
Itra16,33
2,01
15,26
0,41
9,73
0,43
12,39
0,75
Vi ()l khng c tc dng khng nm.
C7 cht III-c, III-d, IV, V, V-a, V-b, V-cu c tc dng khng Ca.Tt ccc cht u c tc dng khng Sa trcht IV, V-c.
Khng c mu no em thc tc dng trn As v Pe.
3.4.2. Thhot tnh gy c tbo:
Vi mong mun cc cht tng hp c c gi trthc tin hn na, chng
ti tin hnh thtc dng gy c tbo ca 11 cht tng hp c. Php
thc thc hin ti phng sinh hc thc nghim ca Vin Ha hc cc hp
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cht thin nhin.
Phng php phn tch: Theo phng php ca Skehan & CS (1990) v
Likhiwitayawuid & CS(1993) hin ang c p dng ti Vin nghin cuung th Quc gia ca M(NCI) v Trng i hc Dc, i hc Tng hp
Illinois, Chicago, M.
Dng tbo: Dng Hep-G2 (Hepatocellular carcinoma- Ung th gan),
Dng MCF7: Ung th biu m v.
Bng 3.8-3.9. Kt quthtc dng khng tbo ung th cho bi bng sau:
STTK hiu
mu
Nngmu(g/ml)
Dng tbo
Cell survival (%) Kt lun
Hep-G2 MCF7
DMSO100,00,
0100,00,0
Chng (+) 10 1,20,3 0,70,09 Dng tnh
M1 (V) 10 70,10,6 90,20,7 m tnh
M2 (II) 10 79,50,7 96,71,1 m tnh
M3 (V-a) 10 95,21,3 99,10,7 m tnh
M4 (V-b) 10 97,30,4 92,41,3 m tnh
M5 (V-c) 10 87,61,1 96,50,7 m tnh
M6 (III-d) 10 59,30,5 80,60,4 m tnh
M7 (III-a) 10 46,80,9 76,20,9Dng tnh vi
dng Hep-G2
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M8 (III) 10 99,20,7 98,70,5 m tnh
M9 (III-b) 10 94,41,2 87,50,7 m tnh
M10 (III-c) 10 98,71,5 99,50,9 m tnh
M11 (IV) 10 97,60,3 99,20,6 m tnh
Bng 3.9
STTK hiu
mu
Dng tbo
Gi trIC50(g/ml) Kt lun
Hep-G2 MCF7
Chng (+) 0,275 0,237 Dng tnh
1 M7 (III-a) 9,471 >10 Dng tnh vi dng
Hep-G2
Vi cc mu M1n M11tng ng vi cc cht V, II,V-a, V-b, V-c, III-d,
III-a, III, III-b, III-c, IV.
Kt lun: nh vy cht III-a dng tnh vi dng Hep-G2 hay cht nyc
tc dng khng li tbo ung th gan.
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CHNG 4. BN LUN
4.1. Vtng hp ha hc
Qua vic tng hp 11 cht: II, III, III(a, b,c, d), IV,V, V(a, b, c) chng tirt ra mt snhn xt nh sau:
a. Phn ng ng vng to nhn 2-mercaptobenzimidazol
Phn ng ngng t ca o-phenylendiamin vi carbon disulfid din ra d
dng vi hiu sut kh cao. C chca phn ng nh sau:
NH2
NH2
CS S
NH2
NH2
CS2
N
NH2
C S
S
H H
HN
NH2
C
SH
S
- H2S
NH
HN
S
N
HN
SH
S 4.1: C chphn ng ng vng 2-mercaptobenzimidazol.
Trong phn ng ny, vai tr ca xc tc KOH c thc gii thch nh
sau: u tin l hot ha vi dung mi ethanol to thnh kali ethyl xanthat
(CH3CH2OCS2K), sau phn ng xy ra gia ion kali ethyl xanthat ny vi
1 nguyn tnit ca o-phenylendiamin nhs sau:
R
NH2
NH2
EtOCS2K
EtOH, H2O,R
HN
C
NH2
S
SK
- H2S
N
HN
SKR
N
HN
SHR2. AcOH, H2O
CH3CH2OH + CS2 + KOHCH3CH2OCS2K +
H2O
S 4.2: Vai tr ca KOH trong phn ng.
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Mt syu tc thnh hng n hiu sut phn ng ng vng:
Tlmol ca CS2v KOH sdng:
Thng qua nghin cu phn ng ny, phn ng xy ra tt nht khi sdngt lmol gia 3 cht l o-phenylendiamin : CS2 : KOH (1 : 1 : 1). Khi tng
hoc gim tlCS2hay KOH u lm gim hiu sut phn ng.
Nhit phn ng:
Nhit phn ng c nh hng n hiu sut v thi gian phn ng. Nu
nhit thp, phn ng din ra chm v khng hon ton. Nu nhit qu
cao sto ra nhiu tp do phn ng oxy ha amin, v vy slm gim hiu sut
phn ng.
Lng than hot sdng, nhit v thi gian ty mu:
Thng thng trong qu trnh ty mu lng than hot sdng tt nht l 5-
10% so vi lng sn phm, nhit tt nht l 60oC v thi gian l 10 pht.
Vic gim hoc tng lng than hot, nhit v thi gian ty mu u lm
gim hiu sut phn ng.
b. Phn ng to dn cht 2-mercaptobenzimidazol.
Tlmol:
T lmol gia cc cht tham gia phn ng l rt quan trng, v n quyt
nh n vic ti thnh sn phm mong mun hiu sut phn ng, qu trnh
tinh ch.
to thnh cc hp cht II, IV t lmol 2-mercaptobenzimidazol v tc
nhn l 1:1. Tlny nhhn hay ln hn 1 th hn hp sau phn ng hoc l
tha nhiu nguyn liu hay to ra nhiu sn phm phu gy kh khn cho
vic tinh chthu sn phm.
Phn ng to cht III-dv V, V(a, b, c)rt dto sn phm phl dn cht
mono. Chnh v vy tlmol gia nguyn liu v tc nhn l rt quan trng.
Nhit :
Nhit cng l yu tnh hng ng kn hiu sut phn ng. Nhit
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qu cao hoc qu thp u lm gim hiu sut phn ng. Khi nhit phn
ng thp, phn ng xy ra chm, khng hon ton nn hiu sut thp. Khi
nhit qu cao c thlm tng qu trnh to mui amoni bc 4 trn N3, do lm gim hiu sut phn ng.
Thi gian ca phn ng:
Cn phi theo di phn ng trong sut thi gian din ra phn ng, nu thi
gian khng slm gim hiu sut phn ng hay phn ng ko di c thto
iu kin thun li hnh thnh cc sn phm ph.
Lu : phn ng alkyl ha ny phi tin hnh trong iu kin khan nc.
u trong hn hp phn ng c nc th tc nhn alkyl ha dbthy phn,
do slm gim hiu sut phn ng.
4.2. Vxc nh cu trc:
Da trn kt quphn tch phhng ngoi (IR), phkhi lng (MS), ph
cng hng tproton (1H-NMR) ca cc cht tng hp c cho php chng
ti kt lun cc cht m chng ti tng hp c tinh khit v c cu trc ng
nh dkin.
V ph hng ngoi (IR): xut hin cc di hp th c trng cho cc
nhm chc v cc lin kt in hnh ca cc cht tng hp c nh linkt
-H; C=O; C-O-C;C=S.
C11 cht u c di hp thnm trong vng 1640-1590 cm-1c trng
cho lin kt C=N; vng 1512-1403 cm-1c trng cho lin kt C=C v vng
1371-1257 cm-1c trng cho lin kt C-N.
Cht II, III, IV, V xut hin di hp th trong vng 3179-3136 cm-1c
trng cho lin kt N-H.
Cht III-b, III-c, IV, V-b, V-c xut hin di hp thtrong vng 1750-1738
cm-1c trng cho lin kt C=O, vng 1220-1179 cm-1c trng cho lin kt
C-O.
Vphkhi lng (MS):cho thy cc cht tng hp c u c cc pic
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phn t, cc pic phn tny c skhi ng bng khi lng phn ttnh theo
cng thc ca cht dkin.
V ph cng hng t proton (1
H-NMR): cho thy s c mt ca ccproton l ph hp vi cng thc cu to ca cc cht.
dch chuyn ha hc ca proton vng thm ca cc cht dao ng trong
khong 7,09-7,65 ppm
dch chuyn ha hc ca proton nhm CH2trong lin kt -CH2-S- dao
ng trong khong 3,33-3,62 ppm.
dch chuyn ha hc ca proton nhm CH2trong lin kt -CH2-N- dao
ng trong khong 4,04-5,35 ppm.
4.3. Vthtc dng sinh hc:
Vtc dng khng khun:
C 4 cht III-a, III-d, V, V-a va c tc dng trn chng vi khun G(+) va
c tc dng trn chng vi khun G(-) th. Trong cc chng vi khun ny
nhy cm vi cht III-a nht.
Cht III-bchc tc dng trn chng vi khun G(+) cn V-b, V-c li chc
tc dng trn chng vi khun G(-).
Cc cht III, III-c, IV khng c tc dng khng khun trn cc chng vi
khun th.
Vtc dng khng nm:
C 5 cht III-c, III-d, V, V(a, b) c tc dng trn 2 chng vi nm l
Candida albicans, Saccaromyces ceravitiae.
C 5 cht III, III(a, b) c tc dng trn chng vi nm Saccaromyces
ceravitiae,IV,V-cc tc dng chng vi nm hoc Candida albicans.
Khng c cht no em thc tc dng trnAspergillus nigervPenicilium
p.
Vhot tnh gy c tbo:
Trong s11 mu em thhot tnh gy c tbo- thtrn 2 dng tbo l
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tbo ung th gan v tbo ung th v th chc cht III-ac tc dng khng
li tbo ung th gan.
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KT LUN V KIN NGH
Kt lun:
Tnhng kt qunghin cu trnh by trn y, chng ti rt ra mt sktlun nh sau:
tng hp c 11 cht bao gm cht 2-mercaptobenzimidazol (II)v cc
cht dn:
ChtIII: 2-benzylthio-1H-benzo[d]imidazol.
Cht III-a: 1-benzyl-2-(benzylthio)-1H-benzo[d]imidazol.
Cht III-b: Methyl 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetat.
Cht III-c: Ethyl 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetat.
Cht III-d: 1,4-bis(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)butan.
Cht IV: Ethyl 2-(1H-benzimidazol-2-ylthio)acetat.
Cht V: 1,4-bis(2-benzimidazolthio)butan.
Cht V-a:1,4-bis(1-benzyl-1H-benzo[d]imidazol-2-ylthio)butan.
Cht V-b: 1,4-bis(1-methoxycarbonylmethyl-1H-benzo[d]imidazol)butan
Cht V-c: 1,4-bis(1-ethoxycarbonylmethyl-1H-benzo[d]imidazol-
2ylthio)butan
thtc dng khng nm, khng khun v chng ung thca cc dn cht
2-mercaptobenzimidazol tng hp c, trong
C 4 cht III-a, III-d, V, V-a va c tc dng trn chng vi khun G(+) va
c tc dng trn chng vi khun G(-) th.
Cht III-bchc tc dng trn chng vi khun G(+) trong khi V-b, V-c li
chc tc dng trn chng vi khun G(-).
C 5 cht III-c, III-d, V, V(a, b) c tc dng trn 2 chng vi nm l
Candida albicans, Saccaromyces ceravitiae.
C 5 cht III, III(a, b), IV,V-c c tc dng ch trn 1 chng vi nm hoc
Candida albicans hay Saccaromyces ceravitiae
Trong s11 mu em thhot tnh gy c tbo- thtrn 2 dng tbo l
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tbo ung th gan v tbo ung th v th chc cht III-ac tc dng khng
li tbo ung th gan.
Kin ngh:Vi kt qut c, chng ti hy vng nghin cu ny c thng gp
mt phn vo vic lm phong ph hn vtng hp v thtc dng sinh hc
ca dn cht 2-mercaptobenzimidazol Vit Nam. Do thi gian v iu kin
thtc dng sinh hc c hn ch, v vy tip tc pht trin kt quca
ti ny, chng ti a ra mt sxut sau:
1. Tip tc tng hp cc dn cht 2-mercaptobenzimidazol vi cc nhmth khc nhau vtr C5, C6 v N1, S trn khung 2-mercaptobenzimidazol.
2. Tip tc thnghim cc hot tnh sinh hc trin vng ca dn cht 2-
mercaptobenzimidazol nh tc dng khng tbo ung thtrn cc dng tbo
ung th khc ngoi dng tbo ung th gan v ung th v, tc dng khng
khun, khng nm trn cc chng vi khun, vi nm khc, tc dng h
cholesterol mu
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PHLC
Phlc 1: Phhng ngoi (IR) ca chtII
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Phlc 2: Phhng ngoi (IR) ca chtIII.
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Phlc 3: Phhng ngoi (IR) ca chtIII-a.
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Phlc 4: Phhng ngoi (IR) ca chtIII-b.
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Phlc 5: Phhng ngoi (IR) ca chtIII-c.
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Phlc 6: Phhng ngoi (IR) ca chtIII-d.
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Phlc 7: Phhng ngoi (IR) ca chtIV.
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Phlc 8: Phhng ngoi (IR) ca chtV.
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Phlc 9: Phhng ngoi (IR) ca chtV-a.
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Phlc 10: Phhng ngoi (IR) ca chtV-b.
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Phlc 11: Phhng ngoi (IR) ca chtV-c.
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Phlc 12: Phkhi lng phn t(MS) ca chtII.
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Phlc 13: Phkhi lng phn t(MS) ca chtIII.
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Phlc 14: Phkhi lng phn t(MS) ca chtIII-a.
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Phlc 15: Phkhi lng phn t(MS) ca chtIII-d.
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Phlc 16: Phkhi lng phn t(MS) ca chtIV.
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Phlc 17: Phkhi lng phn t(MS) ca chtV.
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Phlc 18: Phkhi lng phn t(MS) ca chtV-a.
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Phlc 19: Phkhi lng phn t(MS) ca chtV-b.
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Phlc 20: Phkhi lng phn t(MS) ca chtV-c.
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Phlc 21: Phcng hng tht nhn (1H-NMR) ca cht II.
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Phlc 22: Phcng hng tht nhn (1H-NMR) ca cht III.
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Phlc 23: Phcng hng tht nhn (1H-NMR) ca cht III-a
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Phlc 24: Phcng hng tht nhn (1H-NMR) ca cht III-b.
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Phlc 25: Phcng hng tht nhn (1H-NMR) ca cht III-c.
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Phlc 26: Phcng hng tht nhn (1H-NMR) ca cht III-d.
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Phlc 27: Phcng hng