Synthesis, antimicrobial and antiviral evaluation of substituted imidazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2008.08.010

In the present study, we have synthesized 2-(substituted phenyl)-1H-imidazole (1-12) and (substituted phenyl)-[2-(substituted phenyl)-imidazol-1-yl]-methanone (13-26) analogues and screened them for their antimicrobial activity against Gram positive, Gram negative and fungal species. The results of antibacterial study indicated that compounds 15, 17 and 24 showed appreciable antibacterial activity and compound 26 emerged as the most potential antifungal agent. The results of SAR studies indicated that the presence of electron withdrawing groups is necessary for the antimicrobial activity of the synthesized compounds. The results of the present study indicated that compounds 15, 17 and 24 might be of interest for the identification of new antimicrobial molecules as their antibacterial activity is equivalent to the standard drug norfloxacin. Further, the antiviral screening of (substituted phenyl)-[2-(substituted phenyl)-imidazol-1-yl]-methanones (13-26) against a panel of viral strains indicated that compounds 16 and 19 can be selected as lead compounds for the development of novel antiviral agents.

Full text of DOI:10.1016/j.ejmech.2008.08.010

European Journal of Medicinal Chemistry 44 (2009) 2347–2353
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antimicrobial and antiviral evaluation of substituted imidazole
derivatives
,
a
a
Deepika Sharma a, Balasubramanian Narasimhan b , Pradeep Kumar , Vikramjeet Judge ,
*
Rakesh Narang a, Erik De Clercq c, Jan Balzarini c
a Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Hisar 125001, India
b Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak 124001, Haryana, India
c Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2008
Received in revised form 22 August 2008
Accepted 25 August 2008
Available online 11 September 2008
In the present study, we have synthesized 2-(substituted phenyl)-1H-imidazole (1–12) and (substituted
phenyl)-[2-(substituted phenyl)-imidazol-1-yl]-methanone (13–26) analogues and screened them for
their antimicrobial activity against Gram positive, Gram negative and fungal species. The results of
antibacterial study indicated that compounds 15, 17 and 24 showed appreciable antibacterial activity and
compound 26 emerged as the most potential antifungal agent. The results of SAR studies indicated that
the presence of electron withdrawing groups is necessary for the antimicrobial activity of the synthe-
sized compounds. The results of the present study indicated that compounds 15, 17 and 24 might be of
interest for the identification of new antimicrobial molecules as their antibacterial activity is equivalent
to the standard drug norfloxacin. Further, the antiviral screening of (substituted phenyl)-[2-(substituted
phenyl)-imidazol-1-yl]-methanones (13–26) against a panel of viral strains indicated that compounds 16
and 19 can be selected as lead compounds for the development of novel antiviral agents.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords:
Substituted imidazoles
Antibacterial activity
Antifungal activity
Antiviral activity
1. Introduction
actions, which are distinct from those of well known classes of
antimicrobial agents to which many clinically relevant pathogens
The incidence of invasive microbial infections caused by oppor-
tunistic pathogens, often characterized by high mortality rates, has
been increasing over the past two decades. Patients who become
severely immunocompromised because of underlying diseases such
as leukemia or recently acquired immunodeficiency syndrome or
patients who undergo cancer chemotherapy or organ trans-
plantation are particularly susceptible to opportunistic microbial
infection [1]. Almost all of the major classes of antibiotics have
encountered resistance in clinical applications [2]. The emergence of
are now resistant [5].
The herpes virus family contains eight known human viruses,
amongst them are herpes simplex virus-1 (HSV-1) and herpes
simplex virus-2 (HSV-2) that cause mucocutaneous infections
resulting in cold sores (HSV-1) and genital lesions (HSV-2),
respectively. Much research has been focused on HSV-1 and HSV-2
as these viruses have a high incidence rate and a high prevalence
[6]. Vesicular stomatitis virus (VSV), a member of the Rhabdoviridae
family, is an enveloped single-stranded RNA virus that causes an
economically important disease in cattle, horses and swine [7].
The alphaviruses are a genus of approximately 27 arthropod-
transmitted plus-strand RNA viruses found in the Togaviridae family
which is responsible for a wide range of diseases and many of them
are important human and animal pathogens. Several examples of
alphaviruses are Sindbis, Semliki Forest, and Venezuelan equine
encephalitis viruses. Infection can result in fever, rash, arthralgia or
arthritis, lassitude, headache, and myalgia. The prototypic alpha-
virus is Sindbis virus (SINV), which is transmitted to humans
through mosquito bites [8]. Respiratory syncytial virus (RSV),
a paramyxovirus, is an important cause of respiratory tract infection
in infants, young children and adults [9].
bacterial resistance to b-lactam antibiotics, macrolides, quinolones
and vancomycin is becoming a major worldwide health problem [3].
A matter of concern in the treatment of microbial infections is
the limited number of efficacious antimicrobial drugs. Many of the
currently available drugs are toxic, enable recurrence because they
are bacteriostatic/fungistatic and not bactericidal/fungicidal or lead
to the development of resistance due in part to the prolonged
periods of administration [4]. There is a real perceived need for the
discovery of new compounds that are endowed with antibacterial
and antifungal activities, possibly acting through mechanism of
Previous antiviral research on herpes simplex viruses has
primarily focused on the development of nucleoside analogues,
* Corresponding author. Tel.: þ91 1262 272535; fax: þ91 1262 274133.
E-mail address: naru2000us@yahoo.com (B. Narasimhan).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.08.010

2348
D. Sharma et al. / European Journal of Medicinal Chemistry 44 (2009) 2347–2353
such as acyclovir (Zovirax), valacyclovir, famciclovir, and penciclo-
vir. Recently, immunomodulators (imiquimod and resiquimod),
nonnucleoside viral polymerase inhibitors (4-hydroxyquinoline-3-
carboxamides), and viral helicase inhibitors (thiazolylphenyl and
thiazolylamide) have received considerable attention. Though
numerous strategies and considerable efforts have been spent in
search of the next generation antiviral therapy, it has proved
difficult to outperform acyclovir [6,10].
The incorporation of the imidazole nucleus is an important
synthetic strategy in drug discovery. The high therapeutic proper-
ties of the imidazole related drugs have encouraged the medicinal
chemists to synthesize a large number of novel chemotherapeutic
agents. Imidazole drugs have broadened scope in remedying
various dispositions in clinical medicines. Medicinal properties
Dahiya and Pathak [35] resulted in resinous products. Therefore,
coupling was carried out using sodium acetate along with stirring
in cold conditions for the initial 3 h followed by 48 h stirring
at room temperature which resulted in a solid product. For the
synthesis of (substituted phenyl)-[2-(substituted phenyl)-imida-
zol-1-yl]-methanones (13–26), the key intermediates (1–12) were
reacted with substituted benzoyl chloride which was prepared by
the reaction of substituted benzoic acid with thionyl chloride. The
physicochemical characteristics of the synthesized compounds are
presented in Table 1.
The structures of compounds 1–26 were assigned by IR and 1H
NMR spectroscopic data, which are consistent with the proposed
molecular structures. The appearance of medium out-of-plane
deformation bands (C–C bending) at 725–680 cmꢀ1 indicated the
presence of 1,3-disubstituted benzene ring in compounds 3 and 18.
Similarly, the appearance of the C–C out-of-plane band at 790–
810 cmꢀ1 indicated the presence of 1,4-disubstituted benzene ring
in compounds 2, 6, 13, 17, 18 and 25. The presence of 2-substituted
benzene in structures of compounds 17, 23, and 25 was confirmed
by strong out-of-plane deformation bands (C–H bending) around
740 cmꢀ1 which were visible from their IR spectra. Moreover,
the presence of NO2 group in compounds 4, 6, 13, 17 and 18
was indicated by the appearance of asymmetric and symmetric
include anticancer [11], b-lactamase inhibitors [12], 20-HETE syn-
thase inhibitors [13], carboxypeptidase inhibitors [14], hemeox-
ygenase inhibitors [15], antiaging agents [16], anticoagulants [17],
anti-inflammatory [18], antibacterial [19], antifungal [20], antiviral
[21], antitubercular [22], antidiabetic [23] and antimalarial [24], all
are unique characteristics known for imidazole derivatives.
In continuation with our research program concerning the
synthesis and antimicrobial evaluation of medicinally important
compounds [25–33], in the present study we have synthesized
2-(substituted phenyl)-1H-imidazoles (1–12). Further recent liter-
ature revealed that N1-substitution of imidazoles improves the
antibacterial activity [34]. This created interest among us to
synthesize (substituted phenyl)-[2-(substituted phenyl)-imidazol-
1-yl]-methanones (13–26) and screen them for their antimicrobial
and antiviral activity.
NO2 stretching bands at 1550–1510 cmꢀ1 and 1365–1335 cmꢀ1
,
respectively. The appearance of medium bands at 3696.2 cmꢀ1 in
the IR spectra of compound 17 indicated the presence of a free OH
in the carboxylic acid group of 2-substituted benzene. Further, the
appearance of strong C]O stretching bands at 1695–1670 cmꢀ1 in
the IR spectra of (substituted phenyl)-[2-(substituted phenyl)-
imidazol-1-yl]-methanones (13–26) demonstrated the presence of
tertiary amide linkage between substituted benzoic acid and
imidazole nucleus. Compounds 23 and 25 showed C–Br stretching
at 522.8 cmꢀ1 and 533.8 cmꢀ1, which confirms the presence of 2-
bromo substituted benzene ring. The presence of aliphatic CH
stretching at 2923.2 cmꢀ1 and CH stretching of aralkyl ether at
3054.5 cmꢀ1 in compound 25 confirms the existence of OCH3 group
stretching.
2. Chemistry
Synthesis of compounds 1–26 followed the general pathway
that is depicted in Scheme 1. Compounds 1–12 are the key inter-
mediates for the synthesis of compounds 13–26. The key inter-
mediates, 2-(substituted phenyl)-1H-imidazoles (1–12), were
prepared by the condensation of imidazoles with corresponding
substituted aryldiazonium chlorides which in turn were prepared
by the diazotization of substituted anilines. However, based on our
experience, the application of cupric chloride to the condensation
of aryldiazonium chloride with benzimidazole as suggested by
Compounds 13 and 17 showed multiplet signal at
d 7.69–
7.77 ppm corresponding to the protons of substituted benzene at C2
of imidazole. In contrast, compounds 18 and 23 showed the
multiplet signals at
d 6.98–7.42 ppm for the protons of substituted
H
N
R1
R1
-lC+N2
R2
R4
R1
R2
R3
H2N
R5
R2
R3
N
NaNO2/HCl
0-10°C
N
48 h
R3
R5
N
H
R5
1-12
R4
R4
COCl
COOH
SOCl2
24 h
X
X
R2
R1
N
N
R3
R5
R4
O
X
13-26
Scheme 1. Synthetic scheme for the synthesis of 2-(substituted phenyl)-1H-imidazoles and (substituted phenyl)-[2-(substituted phenyl)-imidazol-1-yl]-methanones.

D. Sharma et al. / European Journal of Medicinal Chemistry 44 (2009) 2347–2353
2349
Table 1
Physicochemical characteristics of synthesized 2-(substituted phenyl)-1H-imidazoles and (substituted phenyl)-[2-(substituted phenyl)-imidazol-1-yl]-methanones
R2
R1
N
N
R3
R1
R2
N
R5
R4
R3
O
N
H
R5
R4
X
1-12
13-26
Compound
R1
R2
R3
R4
R5
X
Mol. formula
Mol. wt
Mp (ꢂC)
Rf
%Yield
1
2
3
4
5
6
7
8
Cl
H
H
H
NO2
H
H
COOH
H
CH3
CH3
H
H
NO2
Cl
H
COOH
H
H
H
NO2
H
Cl
H
H
Cl
NO2
H
H
H
H
H
CH3
H
H
NO2
H
H
H
H
Cl
H
H
H
Cl
H
H
H
NO2
H
H
OCH3
H
H
Br
H
H
H
Cl
H
H
NO2
OCH3
H
H
H
Cl
OCH3
NO2
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
–
–
–
–
–
–
–
–
–
–
–
–
C9H7N2Cl
C9H7N2Cl
C9H7N3O2
C9H7N3O2
C9H7N2Cl
C9H8N2
C9H7N3O2
C10H8N2O2
C10H10N2O2
C11H12N2
C11H12N2
C9H7BrN2
C16H10N4O5
C16H10N4O5
C16H10N3O3Cl
C16H10N3O3Cl
C17H11N3O5
C16H10ClN3O3
C16H10N4O5
C17H13N3O4
C16H10BrN3O3
C16H10BrN3O3
C16H10BrClN2O
C16H10BrClN2O
C17H17BrN2O2
C16H10BrN3O3
178.66
178.66
189.23
189.23
178.66
144.17
189.23
188.24
190.25
172.23
172.23
123.07
338.28
338.28
327.73
327.73
337.29
327.73
338.28
323.31
372.18
372.18
361.63
361.63
357.21
372.18
100
>250
196–199
201–203
99–101
51–53
>250
>250
109–111
219–221
209–211
>250
139–141
149–151
221–223
219–221
194–196
214–216
209–211
229–231
119–121
114–116
149–151
139–141
139–141
109–111
0.80a
0.86a
0.41a
0.81a
0.80a
0.60a
0.20a
0.12a
0.29a
0.71b
0.53b
0.2b
28.67
59.13
12.45
53.13
44.31
34.65
47.07
41.79
57.89
43.70
74.32
53.61
35.73
41.09
56.07
65.72
19.56
76.54
59.80
78.65
60.98
58.65
64.23
38.65
75.86
49.78
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
2-Br
2-Br
2-Br
2-Br
2-Br
0.13a
0.14a
0.11a
0.33a
0.34a
0.11a
0.66a
0.10a
0.85a
0.73a
0.85a
0.82a
0.82a
0.78a
H
NO2
H
H
H
H
H
H
H
2-Br
a
Toluene:chloroform (7:3).
Benzene.
b
benzene at C2 of imidazole. This may be due to the interchange of
electron withdrawing groups in compounds 13 and 17 to electron
donating groups in compounds 18 and 23. Compounds 13,17 and 18
presented in Table 2. In general the compounds showed improved
antibacterial activity when compared to their antifungal activity.
The deduced patterns of antimicrobial activity of substituted
imidazoles are in the following order: antibacterial > antifungal.
All of the synthesized substituted imidazole derivatives showed
appreciable in vitro antimicrobial activity against the tested
microorganisms. Compounds 15, 17, 20, 21, 24 and 26 were found to
be highly active against S. aureus showing a bacterial inhibition at
2 ꢁ 10ꢀ3 mM/ml. The synthesized derivatives 15, 17, 20, 21 and 24
showed high antibacterial potential at 2 ꢁ 10ꢀ3 mM/ml against B.
subtilis. The antibacterial potential against E. coli was exhibited by
compounds 15, 17, 22 and 24 at concentrations ranging from
2 ꢁ 10ꢀ3 mM/ml to 18 ꢁ 10ꢀ3 mM/ml. Compounds 14, 16, 21 and 26
were found to be effective against A. niger having MIC of
4 ꢁ10ꢀ3 mM/ml. Compounds 22, 25 and 26 were found to possess
high antifungal potential against C. albicans when compared to
other derivatives.
showed signals at
NO2 acyl substituent at N1 of imidazole but in compounds 23 and
25 a downfield shift at 7.50–7.59 ppm due to the substitution of
d 8.07–8.39 ppm due to the presence of the p-
d
the electron withdrawing NO2 with bromine was observed. In
compounds 13 and 18 signal corresponding to C4 of imidazole was
observed at
of imidazole was seen at
d
7.33 ppm but in compounds 17 and 23 the signal of C4
7.15 ppm. This effect may probably be
d
due to the presence of the 3-substituted benzene in compounds 13
and 18 and 2-substituted benzene in compounds 17 and 23. The
appearance of signal at
d 3.90 ppm confirmed the presence of OCH3
group in compound 25.
3. Results and discussion
3.1. Antimicrobial activity
The most active antibacterial agents, 15, 17 and 24, with MIC
value of 2 ꢁ10ꢀ3 mM/ml have activities equivalent to that of the
standard drug norfloxacin (MIC ¼ 2 ꢁ 10ꢀ3 mM/ml). Further modifi-
cation of the above structures may provide compounds with better
antibacterial potential than the reference compound, norfloxacin.
Compound 21 emerged as the most effective antibacterial agent
against all of the tested microorganisms, except for E. coli and C.
albicans, basedonitsMICvaluesbut it failedto prove effective against
the tested strains on the basis of its MBC and MFC values (Table 3).
The synthesized compounds were screened for their in vitro
antimicrobial activities against two Gram-positive bacteria
–
Staphylococcus aureus, Bacillus subtilis; Gram-negative bacterium –
Escherichia coli and fungal strains – Aspergillus niger and Candida
albicans by the tube dilution method [36] using norfloxacin and
fluconazole as control drugs for antibacterial and antifungal activ-
ities, respectively. The results of the antimicrobial studies are

2350
D. Sharma et al. / European Journal of Medicinal Chemistry 44 (2009) 2347–2353
Table 2
Table 3
Antimicrobial activity of synthesized 2-(substituted phenyl)-1H-imidazoles and
MBC/MFC values of synthesized 2-(substituted phenyl)-1H-imidazoles and
(substituted phenyl)-[2-(substituted phenyl)-imidazol-1-yl]-methanones
(substituted phenyl)-[2-(substituted phenyl)-imidazol-1-yl]-methanones
Compound
Minimum inhibitory concentration (1 ꢁ 10ꢀ3
m
M/ml)
Compound
Minimum bactericidal/fungicidal concentration (1 ꢁ 10ꢀ3
mM/ml)
S. aureus
B. subtilis
E. coli
A. niger
C. albicans
S. aureus
B. subtilis
E. coli
A. niger
C. albicans
1
2
3
4
5
6
7
8
35
4
4
4
4
4
5
4
4
702
8
17
4
4
16
5
4
4
4
4
702
8
702
4
4
4
5
4
32
4
72
3
9
36
2
38
2
38
73
18
33
2
69
2
33
4
35
702
35
33
33
33
86
33
65
17
9
14
9
4
9
4
702
35
35
33
33
33
43
66
65
72
36
28
18
36
38
19
18
38
18
18
33
16
17
17
16
16
1
2
3
4
5
6
7
8
561
35
35
33
33
33
43
531
32
36
561
702
140
33
33
132
43
33
32
36
36
561
702
561
33
33
33
280
561
280
264
264
264
694
265
526
144
144
112
73
561
280
280
264
264
264
347
526
526
289
289
224
295
295
152
152
256
304
147
147
268
268
138
138
134
134
43
265
263
36
578
28
36
295
19
9
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
4
4
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
36
28
3
28
18
36
2
38
2
38
73
2
2
4
69
2
16
2
36
36
2
19
2
38
36
2
147
295
19
152
18
305
295
18
268
33
295
295
19
305
18
305
295
18
268
268
268
17
73
76
76
305
18
9
148
152
147
147
67
67
138
69
19
18
9
4
16
8
8
16
4
305
295
147
268
16
268
17
628
33
2
33
69
2
16
4
268
17
134
16
628
33
134
67
Std
2a
2a
2a
1b
1b
Std
19a
19a
19a
40b
40b
a
a
Norfloxacin.
Fluconazole.
Norfloxacin.
Fluconazole.
b
b
et al. [38] who stated that the p-substituted phenyl causes an
increase in activity than the unsubstituted phenyl ring in the
case of oxazoles.
In general, the MFC and MBC values of the synthesized imid-
azole derivatives were 3-fold higher than the MIC values which
indicated that the synthesized compounds were bacteriostatic and
fungistatic in action (a drug is considered to be bacteriostatic/
fungistatic when its MFC and MBC values are 3-fold higher than its
MIC value) [37].
(iv) The high antimicrobial activity of compounds 13–26 in
comparison to compounds 1–12 may be attributed to the
presence of carbonyl group in the former. This is supported
by the fact that removal of carbonyl group in bis-imidazole
derivatives resulted in loss of their antifungal activity [39].
(v) Compounds 15, 17 and 24 were found to be the most effective
antibacterial agents, whereas compound 26 emerged as the
most effective antifungal agent. This indicated that there are
different structural requirements for binding of drug to
bacterial or fungal targets, respectively [40].
3.2. Structure activity relationship (SAR) studies
From the results of the antimicrobial activity of the synthesized
substituted imidazole derivatives, the following structure activity
relationships can be derived:
(vi) In general, the most active compounds contain an electron
withdrawing substituent on phenyl ring at second position of
imidazole. The role of electron withdrawing group in
improving antimicrobial activities is supported by the studies
of Sharma et al. [41].
(vii) In contrast to point (vi) mentioned above, the presence of
electron donating methoxy group also showed significant
activity against S. aureus, B. subtilis and C. albicans. The role of
methoxy group in improving antifungal activity of imidazoles
is supported by the studies of Emami et al. [20].
(viii) As far as antifungal activity against A. niger is concerned,
the N1-substituted phenyl ring have both 4-NO2 and 2-Br
substituents as electron withdrawing groups (14, 16, 21
and 26) whereas in case of C. albicans the N1-substituted
phenyl ring has only a bromine substituent as an electron
withdrawing moiety. The results indicated that the presence
of the electron withdrawing NO2 substituent on the N1-
substituted phenyl ring may not be favourable for the
binding of compounds 22, 25 and 26 with the fungal target in
case of C. albicans. The above SAR results are summarized in
Fig. 1.
(i) N1-Substituted imidazole derivatives exhibited high antimi-
crobial activity in comparison to N1-non-substituted
imidazole derivatives. This was similar to the literature
reports [1,34] where little or no activity was found for
the non-N1-substituted derivatives of benzimidazoles and
imidazoles.
(ii) Compound 17, having COOH substitution i.e. 2-[1-(4-nitro-
benzoyl)-1H-imidazol-2-yl]-benzoic acid, showed profound
antibacterial activity without any significant activity against
tested fungal strains. The significant antibacterial activity
(2 ꢁ10ꢀ3 mM/ml) may be attributed to the presence of the
COOH group which is also present in the standard drug
norfloxacin. The lower antifungal activity is supported by the
work of Goker et al. [1] who reported that the presence of
trifluoromethyl, carboxylic, ester and amide groups does not
improve antifungal activity.
(iii) In general, it was observed that most of the compounds with
substituted phenyl rings showed better antimicrobial activity
than the ones with a non-substituted phenyl ring (compound
7). This was similar to the results observed by Tekiner-Gulbas

D. Sharma et al. / European Journal of Medicinal Chemistry 44 (2009) 2347–2353
2351
Table 5
Presence of COOH increased the
antibacterial activity
Presence of electron withdrawing groups
improved the antibacterial activity
Cytotoxicity and antiviral activity of (substituted phenyl)-[2-(substituted phenyl)-
imidazol-1-yl]-methanones in HeLa cell cultures
N
R
b
Compound
Minimum
EC50
(m
g/ml)
N
cytotoxic
Presence of H decreased the antibacterial
activity
Vesicular
stomatitis virus
Coxsackie
virus B4
Respiratory
syncytial virus
concentrationa
O
(mg/ml)
Necessary for enhanced antibacterial activity
13
14
15
16
17
18
19
>100
>100
>100
>100
>100
>100
100
>100
>100
>100
>100
>100
>100
>20
>100
>100
>100
45
>100
>100
>20
>100
>100
>100
>100
>100
>100
>20
The antifungal activity against C. albicans is
X
increased if X is 2-Br group
Fig. 1. SAR for antimicrobial activity of (substituted phenyl)-[2-(substituted phenyl)-
imidazol-1-yl]-methanones.
20
21
22
23
24
25
26
DS-5000
(S)-DHPA
Ribavirin
>100
>100
>100
>100
>100
>100
>100
>100
>250
>250
>100
>100
>100
>100
>100
>100
>100
>100
>250
29
>100
>100
>100
>100
>100
>100
>100
9
>100
>100
>100
>100
>100
>100
>100
0.8
3.3. Antiviral activity
The results of antiviral screening of (substituted phenyl)-[2-
(substituted phenyl)-imidazol-1-yl]-methanones (13–26) against
herpes simplex virus-1 (KOS) (HSV-1 KOS), herpes simplex virus-2
(G) (HSV-2G), vaccinia virus (VV), vesicular stomatitis virus (VSV),
herpes simplex virus-1 TKꢀ KOS ACVr (HSV-1 TKꢀ KOS ACVr) in HEL
cell cultures, vesicular stomatitis virus (VSV), Coxsackie virus B4
(CV-B4), respiratory syncytial virus (RSV) in HeLa cell cultures and
parainfluenza-3 virus (PI-3V), reovirus-1 (RV-1), Sindbis virus (SV),
Coxsackie virus B4 (CV-B4), Punta Toro virus (PTV) in Vero cell
cultures were determined using a CPE reduction assay [42].
Compound 19 emerged as the best antiviral agent against HSV-1
>250
146
>250
10
a
Required to cause
a microscopically detectable alteration of normal cell
morphology.
b
Required to reduce virus-induced cytopathogenicity by 50%.
vaccinia virus at a concentration of 2
tively, which was almost 50 times and 25 times less than that of
their cytotoxic concentration (>100 g/ml) which made them to be
mg/ml and 4 mg/ml, respec-
KOS (EC50 ¼ 59
m
g/ml, Table 4), HSV-2G (EC50 ¼ 50
m
g/ml, Table 4)
and HSV-1 TKꢀ KOS ACVr (EC50 ¼ 45
mg/ml, Table 4) among the
m
tested (substituted phenyl)-[2-(substituted phenyl)-imidazol-1-
yl]-methanones (13–26), but it was much less active than the
reference compounds brivudin, cidofovir and ganciclovir. Against
VSV none of the tested compounds were active in HEL cell culture.
Further, compounds 16 and 19 (having a p-substituted electron
withdrawing group at both 1 and 2 positions of imidazole) emerged
as the most promising antiviral agents against vaccinia virus tested
considered as compounds having a marked therapeutic index and
be selected as lead compounds for the synthesis of newer antiviral
agents. All other compounds tested in HEL cell culture were active
at a concentration which caused microscopically detectable alter-
ation of normal cell morphology.
In HeLa cell cultures (Table 5) none of the compounds proved
active against any of the tested strains of viruses (VSV, CV-B4, RSV)
except compound 19 which showed appreciable antiviral activity
against the all aforesaid viruses in HeLa cell culture at an
in HEL cell culture with an EC50 value of 2 and 4
(Table 4) and their anti-VV activity was equivalent to that of the
standard drugs brivudin (EC50 ¼ 5 g/ml) and cidofovir (EC50
g/ml). Compounds 16 and 19 inhibited the viral replication of
mg/ml, respectively
m
¼
EC50 > 20
concentration (100
m
g/ml, which was ꢃ 5 times lower than its cytotoxic
7
m
m
g/ml).
In Vero cell culture no activity was noted with any of the
compounds against parainfluenza-3 virus, reovirus-1, Sindbis virus,
Table 4
Cytotoxicity and antiviral activity of (substituted phenyl)-[2-(substituted phenyl)-
imidazol-1-yl]-methanones in HEL cell cultures
Table 6
Cytotoxicity and antiviral activity of (substituted phenyl)-[2-(substituted phenyl)-
imidazol-1-yl]-methanones in VERO cell cultures
b
Compound Minimum
cytotoxic
EC50
(
m
g/ml)
Herpes Vaccinia Vesicular Herpes
Herpes
concentrationa
Compound Minimum
cytotoxic
EC50
(
m
g/ml)
b
simplex simplex virus stomatitis simplex
virus-1
(KOS)
(mg/ml)
virus-2
(G)
virus
virus-1 TKꢀ
Parainfluenza- Reovirus- Sindbis Coxsackie Punta
3 virus
concentrationa
KOS ACVr
1
virus
virus B4 Toro
virus
(mg/ml)
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Brivudin
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>250
>100
>100
>100
>100
>100
>100
59
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
50
>100
>100
>100
>100
>100
>100
>100
29
>100
>100
>100
2
>100
45
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>250
146
>100
>100
>100
>100
>100
>100
45
>100
>100
>100
>100
>100
>100
>100
96
13
14
15
16
17
18
19
20
21
22
23
24
25
26
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>250
45
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>250
>250
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
59
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>250
>250
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
4
>100
>100
>100
>100
>100
>100
>100
5
0.04
>250
Ribavirin >250
Cidofovir >250
Ganciclovir >100
>250
2
>250
7
>250
2
DS-5000 >100
(S)-DHPA >250
Ribavirin >250
3
0.06
>250
>100
>250
>250
>250
146
0.06 >100
1
a
a
Required to cause
a
microscopically detectable alteration of normal cell
Required to cause a microscopically detectable alteration of normal cell
morphology.
morphology.
b
b
Required to reduce virus-induced cytopathogenicity by 50%.
Required to reduce virus-induced cytopathogenicity by 50%.

2352
D. Sharma et al. / European Journal of Medicinal Chemistry 44 (2009) 2347–2353
Coxsackie virus B4, Punta Toro virus at an EC50 value of 100
(Table 6). Of some importance, no cytotoxicity was noted with any
of the compounds in Vero cells.
m
g/ml
7.20–7.28 (m, 1H, CH of C4 of ArH), 7.34–7.37 (d, 1H, CH of C6 of
ArH), 7.50 (s,1H, CH of C2 of ArH); IR (KBr pellets) cmꢀ1: 3488.02 (NH
str, imidazole), 1507.27 (C–C str, Ar), 1660.60 (C–H str, Ar), 738.69
(C–Cl str).
4. Conclusion
Analytical data for compound 3: mp (ꢂC) – 197; yield – 12.45%; 1H
NMR (CDCl3):
d 6.55–6.57 (d, 2H, CH of C4 and C5 of imidazole),
During an effort to discover new 2-(substituted phenyl)-1H-
imidazoles and (substituted phenyl)-[2-(substituted phenyl)-
imidazol-1-yl]-methanone analogues with antimicrobial activity we
found that compounds 15, 17 and 24 showed appreciable antibac-
terial activity. Compounds 14, 16, 21 and 26 were found to be
significantly active against A. niger. Compounds 22, 25 and 26 were
found to have antifungal activity against C. albicans. The results of
SAR studies indicated that the presence of electron withdrawing
groups is necessary for the antimicrobial activity of the synthesized
compounds. Further, the results of the present study indicated that
compounds 15, 17, 24 and 26 might be of interest for the develop-
ment of new antimicrobial molecules. Especially, it could be noticed
that most of the compounds were more active as antibacterials than
as antifungals, which could be of some guidance to design new lead
antibacterial compounds. In fact, the most active antibacterial
compounds (15, 17 and 24) showed antibacterial activity equivalent
to that of the standard drug norfloxacin in both MIC and MBC
determinations. Further, the antiviral screening of (substituted
phenyl)-[2-(substituted phenyl)-imidazol-1-yl]-methanones (13–
26) against a panel of viral strains indicated that compounds 16 and
19 could be selected as lead compounds for the development of
novel antiviral agents active against pox viruses.
8.15–8.17 (d, 2H, CH of C3 and C5 of ArH), 7.54–7.56 (d, 2H, CH of C2
and C6 of ArH); IR (KBr pellets) cmꢀ1: 3481.27 (NH str, imidazole),
1519.80 (C–C str, Ar), 1653.52 (C–H str, Ar), 1342.25 (NO2 sym, str).
Analytical data for compound 6: mp (ꢂC) – >250; yield – 59.13%;
1H NMR (CDCl3):
d 7.21–7.26 (d, 2H, CH of C4 and C5 of imidazole),
7.34–7.36 (d, 2H, CH of C3 and C5 of ArH), 7.45–7.47 (d, 2H, CH of C2
and C6 of ArH); IR (KBr pellets) cmꢀ1: 3489.95 (NH str, imidazole),
1521.73 (C–C str, Ar), 1653.85 (C–H str, Ar), 749.29 (C–Cl str).
Analytical data for compound 13: mp (ꢂC) – 139–141; yield –
35.73%; 1H NMR (CDCl3):
d 7.69–7.77 (m, 3H, CH of C5 of imidazole
and CH of C5 and C6 of m-ArNO2), 7.33 (d, 1H, CH of C4 of imidazole),
8.19–8.39 (m, 6H, CH of ArNO2); IR (KBr pellets) cmꢀ1: 1604.4 (three
ring stretching bands, imidazole), 1540.8 (NO2 asym str, aromatic
NO2), 1349.6 (NO2 sym str, aromatic NO2), 715.8 (C–C out-of-plane
bending, 1,3-disubstituted benzene ring), 800.3 (CH out-of-plane
bending, 1,4-disubstituted benzene), 1694.0 (C]O str).
Analytical data for compound 17: mp (ꢂC) – 194–196; yield –
19.56%; 1H NMR (CDCl3):
d 7.69–7.71 (m, 4H, CH of ArCOOH), 7.15 (d,
1H, CH of C4 of imidazole), 8.07–8.38 (m, 4H, CH of ArNO2), 7.85 (d,
1H, CH of C5 of imidazole); IR (KBr pellets) cmꢀ1: 1604.3 (three ring
stretching bands, imidazole), 1540.6 (asym str, aromatic NO2),
1349.8 (sym str, aromatic NO2), 930.7 (OH out-of-plane bending,
COOH), 1290.0 (OH in plane bending, COOH), 3062.1 (CH str,
benzene), 799.6 (CH out-of-plane bending, 1,4-disubstituted
benzene), 3696.2 (OH str, COOH), 1693.2 (C]O str).
5. Experimental
Melting points were determined in open capillary tubes on
a Sonar melting point apparatus and are uncorrected. Reaction
progress was monitored by thin layer chromatography on silica gel
sheets (Merck silica gel-G) and the purity of the compounds was
ascertained by single spot on TLC sheet. 1H nuclear magnetic
resonance (1H NMR) spectra were recorded on a Bruker Avance II
400 NMR spectrometer using appropriate deuterated solvents and
are expressed in parts per million (d, ppm) downfield from tetra-
methylsilane (internal standard). Infrared (IR) spectra were recor-
ded on a Shimadzu FTIR spectrometer.
Analytical data for compound 18: mp (ꢂC) – 214–216; yield –
76.54%; 1H NMR (CDCl3):
d 8.21–8.26 (m, 4H, CH of ArNO2), 7.33 (d,
1H, CH of C4 of imidazole), 6.98–7.31 (m, 4H, CH of ArCl); IR (KBr
pellets) cmꢀ1: 1601.7 (three ring stretching bands, imidazole), 714.2
(C–C out-of-plane bending, 1,3-disubstituted benzene ring), 1536.2
(NO2 asym str, aromatic NO2), 1348.3 (NO2 sym str, aromatic NO2),
797.0 (CH out-of-plane bending, 1,4-disubstituted benzene), 1694.4
(C]O str).
Analytical data for compound 23: mp (ꢂC) – 149–151; yield –
64.23%; 1H NMR (CDCl3):
d 7.16–7.42 (m, 4H, CH of ArCl), 7.10 (d, 1H,
CH of C4 of imidazole), 7.50–7.70 (m, 4H, CH of ArBr), 7.87 (d, 1H, CH
of C5 of imidazole); IR (KBr pellets) cmꢀ1: 1586.4 (three ring
stretching bands, imidazole), 1683.8 (C]O str, Aryl COOH), 3010.30
(CH str, aromatic), 522.2 (C–Br str, aromatic), 684.0 (C–Cl str), 740.7
(CH out-of-plane bending, 1,2 disubstituted benzene ring), 791.1
(CH out-of-plane bending, 1,4-disubstituted benzene).
5.1. General procedure for the synthesis of 2-(substituted
phenyl)-1H-imidazoles (1–12)
Substituted anilines (0.13 mol) in hydrochloric acid/water
mixture (1:1) were diazotized using solution of sodium nitrite at 0–
10 ꢂC. To the diazotized mixture, imidazole (0.004 mol) was added
with vigorous shaking. A solution of sodium acetate (40 g in
100 ml) was added dropwise to the above mixture by maintaining
the temperature at 5–10 ꢂC. The above solution was stirred initially
for 3 h at cold condition followed by continuation of stirring at
room temperature for 48 h. The product obtained was filtered,
dried and recrystallized from alcohol.
Analytical data for compound 25: mp (ꢂC) – 144–146; yield –
75.86%; 1H NMR (CDCl3):
d
7.40–7.44 (m, 4H, CH of ArOCH3), 7.15 (d,
1H, CH of C4 of imidazole), 7.59–7.79 (m, 4H, CH of ArBr), 7.87 (d,1H,
CH of C5 of imidazole), 3.90 (s, 3H, OCH3); IR (KBr pellets) cmꢀ1
:
1587.6 (three ring stretching bands, imidazole), 2923.2 (CH str,
CH3), 3054.5 (CH str, ArOCH3), 533.8 (C–Br str, aromatic), 685.4 (C–
Cl str), 741.4 (CH out-of-plane bending, 1,2 disubstituted benzene
ring), 810.9 (CH out-of-plane bending, 1,4-disubstituted benzene
ring), 1684.1 (C]O str).
5.2. General procedure for the synthesis of (substituted phenyl)-[2-
(substituted phenyl)-imidazol-1-yl]-methanones (13–26)
5.3. Evaluation of antimicrobial activity
A
solution of 2-(substituted phenyl)-1H-imidazoles (1–12)
(0.002 mol) in diethyl ether (50 ml) was added to a solution of
corresponding substituted benzoyl chloride (0.002 mol) in diethyl
ether (50 ml). The above mixture was stirred for 24 h at room
temperature. The resultant product was isolated by evaporation of
ether and purified by recrystallization with methanol.
5.3.1. Determination of MIC
The antimicrobial activity was performed against Gram-positive
bacteria: S. aureus, B. subtilis, Gram-negative bacterium: E. coli and
fungal strains: C. albicans and A. niger. The standard and test
samples were dissolved in DMSO to give a concentration of 100 mg/
ml. The minimum inhibitory concentration (MIC) was determined
by tube dilution method. Dilutions of test and standard compounds
Analytical data for compound 2: mp (ꢂC) – 100; yield – 44.31%;
1H NMR (CDCl3):
d 7.00–7.03 (d, 2H, CH of C4 and C5 of imidazole),

D. Sharma et al. / European Journal of Medicinal Chemistry 44 (2009) 2347–2353
2353
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5.4.1. Antiviral assay
The antiviral activity of the (substituted phenyl)-[2-(substituted
phenyl)-imidazol-1-yl]-methanones (13–26) was determined
using a CPE reduction assay [42] against herpes simplex virus-1
(KOS), herpes simplex virus-2 (G), vaccinia virus, vesicular stoma-
titis virus, herpes simplex virus-1 TKꢀ KOS ACVr in HEL cell cultures,
vesicular stomatitis virus (VSV), Coxsackie virus B4, respiratory
syncytial virus in HeLa cell cultures and parainfluenza-3 virus,
reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus in
Vero cell cultures and the results were expressed as the 50%
effective concentration (EC50). Cells, grown to confluency in 96-
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The cytotoxicity of the compounds was evaluated in parallel
with their antiviral activity in uninfected cells, and is expressed as
minimum cytotoxic concentration to cause a microscopically
detectable alteration of normal cell morphology (HEL cells, HeLa
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We would like to thank Mrs. Leentje Persoons, Rega Institute for
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