Design, Synthesis and anti
microbial, anti-inflammatory, Antitubercular
activities of some 2,4,5-trisubstituted imidazole
derivatives.
R.
S. Kalkotwar1* and R. B. Saudagar2
1Department of
Pharmaceutical Sciences, Bhagwant University,
Ajmer, Rajashthan, India
2Department of
Pharmaceutical Chemistry, R.G. Sapkal College
of Pharmacy, Nashik,
M.S. India.
*Corresponding Author E-mail: rsk_55@rediffmail.com.
ABSTRACT:
A
Novel Series of Some 2,4,5-trisubstituted imidazole derivatives
were prepared with the aim to get better antibacterial activity, antifungal
activity, antitubercular and anti-inflammatory
activity. The structures of synthesized compounds were supported by means of
IR, 1H NMR. Title compounds were evaluated for antibacterial
activity, antifungal activity, antitubercular and
anti-inflammatory activities. Among the synthesized compounds some compounds
found to possess all these activities. The QSAR study for anti-inflammatory
activity shows good results.
KEYWORDS: QSAR, Antibacterial, antifungal,
anti-inflammatory activity.
INTRODUCTION:
Imidazole derivatives are of wide interest because of their diverse
biological activity and clinical applications, they are remarkably effective
compounds both with respect to their inhibitory activity and their favorable
selectivity ratio. Imidazole are regarded as a
promising class of bioactive heterocyclic compounds that exhibit a range of
biological activities. This ring system is present in numerous antioxidant, antiparasitic, antihelmintics, antiproliferative, anti-HIV, anticonvulsant,
anti-inflammatory, antihypertensive, antineoplastic
and antitrichinellosis activities. Varied
bioactivities exhibited by Imidazole, efforts have
been made from time to time to generate libraries of these compounds and
screened them for potential biological activities. Also it is well documented
that imidazole nucleus is associated with a variety
of pharmacological actions. It displays pronounced anticonvulsant, antifungal
and antimycobacterial activities. Extensive
biochemical and pharmacological studies have confirmed that imidazole
molecules are effective against various strains of microorganisms.
Imidazole are regarded as a promising class of bioactive heterocyclic
compounds that exhibit a range of biological activities. Looking at the
importance of imidazole nucleus, it was thought that
it would be worthwhile to design and synthesize some new imidazole
derivatives and screen them for potential biological activities.
MATERIALS AND
METHODS:
Melting points were determined in
open capillary method and are uncorrected. The 1H-NMR spectra were
recorded on sophisticated multinuclear FT-NMR Spec-trometer
model Advance-II (Bruker) using dimethylsulfoxide-d6
as solvent and tetramethylsilane as internal
standard. IR spectra were recorded on Thermo Nicolet IR 200 spectrophotometer
using KBr disc method. Biological activity
(anti-inflammatory activity) values are reported as inhibitory activity on Carrageenan induced rat paw oedema
(% inhibition at 2 hr). Pharmacological screening values were by converted into
Log (% Inh) were used for multiple correlation
analysis with descriptors generated using TSAR 3.3 software.
QSAR METHODOLOGY
All molecules were drawn in Chem draw ultra 8.0 module in Chem
office 2004 software and imported into TSAR software. Charges were derived
using Charge 2-Derive charges option and optimized by using Cosmic-optimize 3 D
option in the structure menu of the project table. Substituent was defined and
descriptors were calculated for whole molecule as well as for the substituent.
Several equations were generated correlating both Log (% Inh)
with physicochemical parameters (descriptors) by multiple linear regression
analysis (MLR) method. Data was standardized by range and leave one out method
was used for cross validation. Models were excluded if correlation was
exceeding 0.9 for more rigorous analysis. Correlation matrix was generated to
find any Intercorrelation between the descriptors. Intercorrelation between the descriptors in the final
equation is less than 0.2. 1
ANTIMICROBIAL SCREENING
Antibacterial activity
The
newly synthesized compounds were screened for their antibacterial activity
against Escherichia coli (MTCC 443), Bacilus subtilis (ATCC12228) and Staphylococcus aureus (ATCC25923)
bacterial strains by disc diffusion method. In all the determinations tests
were performed in triplicate and the results were taken as a mean of three
determinations. Ciprofloxacin was used as a standard drug. 2
Anti fungal activity
The newly synthesized compounds were screened
for their antifungal activity against C. albicans and A. niger in DMSO by agar diffusion method. In all the
determinations tests were performed in triplicate and the results were taken as
a mean of three determinations. Amphotericin B was
used as a standard drug.
Anti-tubercular
activity
The antitubercular
screening was carried out by Middle brook 7H9 agar medium against H37Rv.
Strain. Middle brook 7H9 agar medium containing different derivatives, standard
drug as well as control, Middle brook 7H9 agar medium was inoculated with Mycobacterium tuberculosis of H37Rv
Strain. The inoculated bottles were incubated for 37°C
for 4 weeks. At the end of 4 weeks they were checked for growth.3
ANTI-INFLAMMATORY
ACTIVITY
Carrageenan Induced hind Paw Edema:
Anti-inflammatory
activity was carried out by Carrageenan Induced Rat
hind Paw method of winter et al. wistar rats (120-150 g) was used for the experiment. The
conventional laboratory diet was fed with adequate supply of drinking water.
The animals were randomly selected, marked to permit individual identification
and kept in polypropylene cages for one week prior to dosing to allow
acclimatization of them to laboratory conditions. The drugs were prepared as a
suspension by triturating with water and 0.5% sodium CMC. The standard group
received 50mg/kg body weight of Ibuprofen, test group received 200mg/kg body
weight of synthesized compounds and the control group received 1% w/v of CMC. 4
Procedure for
Scheme5
Synthesis of
2,4,5- trisubstituted imidazoles (B1-B12)
0.01 mole of benzyl or ethane dione was mixed with 0.01 mole of aromatic aldehyde along with ammonium acetate and PPA. And refluxed
for 3 hrs. Cool pour the reaction mixture on ice to offer solid mass, filtered
and recrystallized from hot ethanol to offer titled
compounds (B1-B12).
Scheme
Spectral data:
B1 IR (KBr)
cm-1: 3310.4(-CH=CH str.),3213.45
(-NH str.), 3010.23 (Ar-CH str.), 1682.11 (-C=O str.), 1525.32
(-C=N str), 1245.36 (-C-N str). 1H NMR: (δ ppm): 9.06 (CH,4-benzil )
8.0 (NH, sec. amide), 7.26 (CH -imidazole)
B2IR (KBr)
cm-1: 3310.43 (-CH=CH str.), 3010.23
(Ar-CH str.), 1682.11 (-C=O str.), 1525.32 (-C=N str), 1245.36 (-C-N str). 1H NMR: (δ ppm):
8.3 (CH, benzil 7.26 (CH, imidazole),
7.04
(CH,4-benzil).
B3 IR (KBr)
cm-1: 3310.43(-CH=CH str.), 3213.45 (-NH str.), 3010.23
(Ar-CH str.), 1682.11 (-C=O str.), 1525.32 (-C=N str), 1245.36 (-C-N str). 1H NMR: .8 (CL, 7.26 (CH, imidazole),
7.54 (CH, 1-Benzene
B4 IR (KBr)
cm-1: 3213.45 (-NH str.), 3010.23 (Ar-CH
str.), 1682.11 (-C=O str.), 1525.32 (-C=N str), 1245.36 (-C-N str). 1H NMR: . 8.3 (CH benzil triazole), 7.26
(CH, -imidazole),
B5 IR (KBr) cm-1: 3010.23
(Ar-CH str.), 1682.11 (-C=O str.), 1525.32 (-C=N str), 1245.36 (-C-N str). . 1H NMR: . 7.26 (CH, Benz-imidazole), 8.29 (methoxy)
B6 IR (KBr) cm-1: 3213.45 (-NH
str.), 3010.23 (Ar-CH str.), 1682.11 (-C=O str), 1525.32 (-C=N str), 1245.36
(-C-N str). 6.66 (CH, 1-Benzene 1H
NMR: 8.8 (Of OH 7.26 (CH imidazole)
B7 IR (KBr) cm-1: 3213.45
(-NH str.), 3010.23 (Ar-CH str.), 1682.11 (-C=O str.), 1525.32
(-C=N str), 1245.36 (-C-N str), 1016.11 (-C-O-C str.). 1H NMR: . .3 (CH)7.26 (CH, imidazole), 6.26 (CH, 1-Benzene), 8.0 (NH, sec. amide)
B8 IR (KBr)
cm-1: 3010.23 (Ar-CH str.), 1682.11 (-C=O
str.), 1525.32 (-C=N str), 1245.36 (-C-N str), 1016.11 (-C-O-C str.). . 1H NMR: . . 8.3 (CH, 7.26 (CH, imidazole),6.26 (CH, 1-Benzene), 4.0 (NH2, Aromatic C-NH )
B9 IR (KBr)
cm-1: 3213.45 (-NH str.), 3010.23
(Ar-CH str.), 1682.11 (-C=O str.), 1525.32 (-C=N str), 1245.36 (-C-N str),
1016.11 (-C-O-C str.). 1H NMR: . 8.3 (CH, 7.26 (CH, imidazole), 6.26 (CH, 1-Benzene), 4.0 OF Cl Aromatic C-NH),
B10 IR (KBr) cm-1:. 3310.23
CH=CH str.), 3010.23 (Ar-CH str.), 1689.78 (-C=O str), 1525.32 (-C=N
str), 1245.36 (-C-N str), 1016.38 cm-1(-C-O-C str), 3210.45 (-OH str.), , 3010.23
Ar-CH str.), 1689.78 (-C=O str), 1525.32 (-C=N str), 1245.36 (-C-N str),
1016.38 cm-1(-C-O-C str), 1H NMR: . 7.26 (CH, imidazole), 7.30 (Of OH)
B11 IR (KBr) cm-1:.
3310.23 (-CH=CH str.),3208.12 (-NH2 str. ), 3010.23
(Ar-CH str.), 1689.78 (-C=O str), 1525.32 (-C=N str), 1245.36 (-C-N str), 1016.38
cm-1(-C-O-C str 1H NMR: 7.26 (CH, imidazole), 8.29
(CH,4-pyridine), 6.30 (CH,
1-Benzene), 4.0 of Methoxy
B12 IR (KBr) cm-1 3310.23 (-CH=CH str.), 3208.12 (-NH2 str.
), 3210.45 (-OH str.), 3010.23 (Ar-CH str.), 1689.78 (-C=O str),
1525.32 (-C=N str), 1245.36 (-C-N str), 1016.38 cm-1(-C-O-C str), 1H NMR: . 77.26 (CH, imidazole),
8.29 (of Methoxy 6.30 (CH, 1-Benzil ) 4.0 Aromatic C-NH),
Table no1: Analytical and Physicochemical
data of the synthesized compounds (B1-B12)
|
Comp. |
Mol. Formula |
Mol. Wt. |
M.P. ° C |
Yield % |
Elemental
analyses Calcd. (found) |
||
|
C |
H |
N |
|||||
|
B1 |
C23H18N2 |
322 |
128-132 |
68 |
85.68 |
5.63 |
8.69 |
|
B2 |
C24H20N2 |
336 |
116-118 |
65 |
85.68 |
5.99 |
8.33 |
|
B3 |
C23H17ClN2 |
356 |
202-204 |
67 |
77.41 |
4.80 |
7.85 |
|
B4 |
C23H18N2O |
338 |
176-178 |
56 |
81.63 |
5.36 |
8.28 |
|
B5 |
C24H20N2O |
352 |
189-193 |
58 |
81.79 |
5.72 |
7.95 |
|
B6 |
C23H18N2O |
338 |
207-209 |
69 |
81.63 |
5.36 |
8.28 |
|
B7 |
C15H18N2 |
226 |
210-213 |
72 |
79.61 |
8.02 |
12.38 |
|
B8 |
C16H20N2 |
240 |
235-238 |
58 |
79.66 |
8.39 |
11.66 |
|
B9 |
C15H17ClN2 |
260 |
224-226 |
71 |
69.09 |
6.57 |
10.74 |
|
B10 |
C15H18N2O |
242 |
175-178 |
72 |
74.35 |
7.49 |
11.56 |
|
B11 |
C16H20N2O |
256 |
165-169 |
67 |
74.97 |
7.86 |
10.93 |
|
B12 |
C15H18N2O |
242 |
201-205 |
64 |
74.35 |
7.49 |
11.56 |
Table no:2 Antibacterial and antifungal activity of
synthesized compounds (Scheme-IV)
|
Compd. |
Zone of inhibition at 200µcg/mL (in mm.) |
||||
|
E. coli |
B. Subtilis |
S. aureus |
A. niger |
C. albicans |
|
|
B1 |
24 |
25 |
26 |
15 |
22 |
|
B2 |
20 |
23 |
25 |
16 |
21 |
|
B3 |
20 |
24 |
25 |
19 |
22 |
|
B4 |
25 |
26 |
23 |
20 |
21 |
|
B5 |
24 |
23 |
26 |
21 |
22 |
|
B6 |
20 |
22 |
24 |
18 |
23 |
|
B7 |
21 |
23 |
22 |
20 |
21 |
|
B8 |
22 |
24 |
25 |
20 |
22 |
|
B9 |
23 |
22 |
20 |
18 |
22 |
|
B10 |
24 |
26 |
23 |
19 |
21 |
|
B11 |
25 |
23 |
24 |
21 |
23 |
|
B12 |
26 |
22 |
24 |
20 |
22 |
|
Ciprofloxacin |
26 |
25 |
26 |
- |
- |
|
Amphotericin B |
- |
- |
- |
22 |
23 |
Table no. 3 : Antitubercular activity of the synthesized compounds
|
Compd. |
25 µcg/mL |
50 µcg/mL |
100 µcg/mL |
|
R |
S |
S |
|
|
B2 |
R |
R |
S |
|
B3 |
R |
R |
R |
|
B4 |
R |
S |
S |
|
B5 |
R |
R |
S |
|
B6 |
R |
R |
R |
|
B7 |
R |
S |
S |
|
B8 |
R |
R |
S |
|
B9 |
R |
R |
R |
|
B10 |
R |
S |
S |
|
B11 |
R |
R |
S |
|
B12 |
R |
R |
R |
|
Streptomycin |
S |
S |
S |
Table no.4:
Anti-inflammatory activity of Synthesized compounds (Scheme-IV)
|
Treatment |
Mean increase in
paw volume (ml)±SEM |
|||||||||
|
Time in minute |
||||||||||
|
0 |
% Inh. |
30 |
% Inh. |
60 |
% Inh. |
90 |
% Inh. |
120 |
% Inh. |
|
|
Carrageenan (Control) |
0.21±0.01 |
|
0.48±0.03 |
|
0.75±0.09 |
|
0.85±0.12 |
|
0.89±0.14 |
|
|
Zaltoprofen |
0.21±0.03 |
0 |
0.28±0.07 |
32.41 |
0.27±0.07 |
58.53 |
0.24±0.06 |
66.23 |
0.23±0.13 |
67.78 |
|
B1 |
0.21±0.01 |
0 |
0.29±0.03 |
30.33 |
0.28±0.01 |
57.25 |
0.27±0.01 |
62.88 |
0.24±0.01 |
66.66 |
|
B2 |
0.21±0.02 |
0 |
0.31±0.03 |
26.16 |
0.32±0.01 |
52.12 |
0.30±0.01 |
59.35 |
0.27±0.02 |
63.29 |
|
B3 |
0.21±0.01 |
0 |
.031±0.01 |
26.16 |
0.31±0.01 |
53.41 |
0.30±0.02 |
59.35 |
0.26±0.02 |
64.41 |
|
B4 |
0.21±0.02 |
0 |
0.38±0.01 |
11.58 |
0.45±0.02 |
35.46 |
0.49±0.02 |
38.00 |
0.39±0.01 |
49.80 |
|
B5 |
0.20±0.01 |
3.16 |
0.29±0.01 |
30.33 |
0.28±0.01 |
57.25 |
0.28±0.01 |
62.70 |
0.26±0.02 |
65.53 |
|
B6 |
0.21±0.02 |
0 |
0.30±0.01 |
28.25 |
0.31±0.02 |
53.41 |
0.30±0.01 |
60.35 |
0.27±0.03 |
64.41 |
|
B7 |
0.20±0.02 |
3.16 |
0.31±0.01 |
26.16 |
0.32±0.02 |
52.12 |
0.31±0.02 |
60.17 |
0.28±0.01 |
63.29 |
|
B8 |
0.21±0.02 |
0 |
0.29±0.02 |
30.33 |
0.27±0.03 |
58.53 |
0.27±0.02 |
62.88 |
0.26±0.02 |
65.53 |
|
B9 |
0.20±0.03 |
3.16 |
0.31±0.02 |
26.16 |
0.30±0.01 |
54.69 |
0.30±0.02 |
60.35 |
0.26±0.02 |
64.41 |
|
B10 |
0.21±0.01 |
0 |
0.31±0.02 |
26.16 |
0.31±0.02 |
53.41 |
0.29±0.01 |
61.52 |
0.28±0.01 |
63.29 |
|
B11 |
0.21±0.02 |
0 |
0.31±0.03 |
26.16 |
0.32±0.03 |
52.12 |
0.29±0.01 |
61.52 |
0.27±0.02 |
63.29 |
|
B12 |
0.20±0.03 |
3.16 |
0.28±0.04 |
32.41 |
0.27±0.01 |
58.53 |
0.28±0.02 |
61.70 |
0.25±0.03 |
65.53 |
Inh.=Inhibition
Table no. 5 -:
Structures and Log (% Inh) of Compounds
Sr. No. |
Comp. Name |
Structure |
% Inh |
Log (% Inh) |
|
1. |
B1 |
|
68.47 |
1.921245 |
|
2 |
B2 |
|
69.07 |
1.930762 |
|
3. |
B3 |
|
68.09 |
1.905281 |
|
4. |
B4 |
|
72.08 |
1.928124 |
|
5. |
B5 |
|
75.38 |
1.925689 |
|
6. |
B6 |
|
71.04 |
1.902387 |
|
7. |
B7 |
|
72.45 |
1.928213 |
|
8. |
B8 |
|
74.08 |
1.928945 |
|
9. |
B9 |
|
71.57 |
1.928076 |
|
10. |
B10 |
|
72.47 |
1.923487 |
|
11. |
B11 |
|
70.13 |
1.912367 |
|
12 |
B12 |
|
72.36 |
1.923489 |
RESULTS:
QSAR
Intercorrelation between the descriptors in the
final equations is less than 0.2. Best Equations correlating Log (% Inh) with descriptors for series (B1-B12)
generated are presented in Table no. 06
Table no. 6 : Equations
generated between Log (% Inh) and descriptors
|
Sr. No. |
Equation |
N |
S |
R |
r2 |
r2cv |
F |
|
series (A1-A9
and B1-B9) |
Y = - 0.198 * X3 - 1.478 * X2 –
13.018 |
12 |
0.357 |
0.889 |
0.598 |
0.459 |
13.736 |
Where
Y = Log (% Inh)
X1: ClogP -
X2 = VAMP HOMO (Whole Molecule)
X3 = Dipole Moment Z Component
(Whole Molecule)
X4 = Inertia Moment 2 Length
(Whole Molecule)
Significance of
the terms –
N= No. of Molecules
s = standard error --- less is better
r = correlation coefficient – higher is
better > 0.7,
r2cv = cross validated r2 -
higher is better > 0.5,
F Value = higher is better
Observed and predicted data and graphs are
presented in Table no. 06 and Graph
I for Series
Table no7 :
Observed and predicted log (% Inh) value data for 12
compounds
|
Comp. No. |
Observed Value |
Predicted Value |
Residual Value |
Residual
Variance |
|
B1 |
1.823865 |
1.92125 |
0.09738 |
0.004228 |
|
B2 |
1.801335 |
1.93076 |
0.129427 |
0.004383 |
|
B3 |
1.808953 |
1.90528 |
0.096328 |
0.004398 |
|
B4 |
1.697229 |
1.92812 |
0.230895 |
0.004629 |
|
B5 |
1.81644 |
1.92569 |
0.109249 |
0.003234 |
|
B6 |
1.808953 |
1.90239 |
0.093434 |
0.003264 |
|
B7 |
1.801335 |
1.92821 |
0.126878 |
0.00343 |
|
B8 |
1.81644 |
1.92895 |
0.112505 |
0.00331 |
|
B9 |
1.808953 |
1.92808 |
0.119123 |
0.003333 |
|
B10 |
1.801335 |
1.92349 |
0.122152 |
0.003255 |
|
B11 |
1.801335 |
1.91237 |
0.111032 |
0.003016 |
|
B12 |
1.81644 |
1.92349 |
0.107049 |
0.002865 |
Fig. No 1 : a) Correlation graph and b) Histogram of observed and
predicted log (% Inh) data for 12 compounds
DISCUSSION:
QSAR
Statistical evaluation of the equations is
in accepted range. The correlation coefficient is high with less standard
error. The residual value and residual variance for each series also is less
indicating good predictive power of models. From equation 1 it is observed that
two electronic parameters Dipole Moment Z Component (Whole Molecule) and VAMP
HOMO (Whole Molecule) contribute (-0.198 and – 1.478) negatively for the
activity so electron withdrawing groups may enhance the activity (% Inh).
The synthesized
derivatives were screened for anti bacterial activity using DMF as a solvent
against the organisms S.aureus, B. subtilis and
E.coli.,
and antifungal activity using C. albicans and A. niger by disc diffusion method on nutrient agar media.
The standard drug used was Levofloxacin and Amphotericin B for antibacterial and antifungal activity respectively.
Antibacterial activity:
The compounds B1,B2,B3,B5,B8,
has excellent Antibacterial activity
against S. aureus, the compounds B1,B11,B12
have shown Antibacterial activity against B. subtilis, while B4, B11,B12
shows Antibacterial activity against E.coli., when compared with standard
Ciprofloxacin
Antifungal
activity:
The compounds B5, B11 has
excellent antifungal activity against Aspergillus niger (NCIM 596), while the B1,B3,B5,B6,B8,B9,B11,B12
have shown Antifungal activity against Candida albicans (NCIM 3102) when
compared with standard Amphotericin B.
Antitubercular activity:
All the compounds were screened for antitubercular activity by Middle brook 7H9 agar medium as
described by Elmer WK et al. against H37Rv Strain. Compounds B1,B7,B10
has shown promising antitubercular activity.
Anti-Inflammatory
Activity:
All
the compounds were evaluated for Anti-inflammatory activity by Carrageenan Induced Rat hind Paw method. The synthesized
compounds showed anti-inflammatory activity and it was found comparable with
standard drug zaltoprofen.
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J. Pharm. Res. 3(4): Oct. - Dec.2013; Page 159-165