Synthesis, characterization and antimicrobial studies of some new pyrazole incorporated imidazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.05.005

In the present study two series of novel imidazole derivatives containing substituted pyrazole moiety (3a-d and 5a-j) were synthesized. The first series were synthesized by the reaction of 3-aryl-1H-pyrazole-4-carbaldehyde thiosemicarbazones (2a-d) with D

Full text of DOI:10.1016/j.ejmech.2011.05.005

European Journal of Medicinal Chemistry 46 (2011) 3531e3536
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, characterization and antimicrobial studies of some new pyrazole
incorporated imidazole derivatives
,
,
c
d
e
A.M. Vijesh a b, Arun M. Isloor b , Sandeep Telkar , S.K. Peethambar , Sankappa Rai ,
*
Nishitha Isloor f
a SeQuent Scientific Ltd., No. 120 A & B, Industrial Area, Baikampady, New Mangalore 575 011, Karnataka, India
b Department of Chemistry, National Institute of Technology Karnataka, Surathkal, Mangalore 575 025, India
c Department of P.G. Studies and Research in Biotechnology and Bioinformatics, Jnanasahyadri, Kuvempu University, Shankaraghatta 577 451, Karnataka, India
d Department of Bio-Chemistry, Jnanasahyadri, Kuvempu University, Shankaraghatta 577 451, Karnataka, India
e Department of Chemistry, Manipal Institute of Technology, Manipal University, Manipal, India
f Biotechnology Division, Chemical Engineering Department, National Institute of Technology Karnataka, Surathkal, Mangalore 575 025, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study two series of novel imidazole derivatives containing substituted pyrazole moiety
(3aed and 5aej) were synthesized. The first series were synthesized by the reaction of 3-aryl-1H-pyr-
azole-4-carbaldehyde thiosemicarbazones (2aed) with DMAD and the second series by the reaction of
3-aryl-1H-pyrazole-4-carbaldehydes (1aee) with 1,2-diketones (4a,b) in the presence of ammonium
acetate. Structures of newly synthesized compounds were characterized by spectral studies. New
compounds were screened for antifungal and antibacterial activities. Among the synthesized
compounds, compound 3c was found to be potent antimicrobial agent. The acute oral toxicity study for
the compound 3c was carried out and the experimental studies revealed that compound 3c is safe up to
3000 mg/kg and no death of animals were recorded.
Received 4 February 2011
Received in revised form
15 April 2011
Accepted 4 May 2011
Available online 12 May 2011
Keywords:
Imidazoles
Pyrazoles
Antimicrobial studies
Toxicity study
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
agrochemical industry. Pyrazole derivatives have showed signifi-
cant biological activities, such as anti-microbial [6], analgesic [7],
Imidazole and its derivatives are an important class of hetero-
cycles. 2,4,5-Triaryl-1H-imidazole compounds have gained the
remarkable importance due to their widespread biological activi-
ties and their use in synthetic chemistry. Imidazole derivatives
possess a broad spectrum of pharmacological activities such as
anti-inflammatory [1], analgesic, anti-convulsant [2], antituber-
cular [3], antimicrobial [4], anticancer and anti-Parkinson [5]
activities. Imidazole and its derivatives are of great significance
due to their important roles in biological systems, particularly in
enzymes, as proton donors and/or acceptors, coordination system
ligands and the base of chargeetransfer processes. The imidazole
nucleus appears in a number of naturally occurring products like
the amino acids histidine and purines, which comprise many of the
most important bases in nucleic acids.
anti-inflammatory [8] and anticancer [9] activities. This gave
a great impetus to the search for potential pharmacologically active
drugs carrying pyrazole substituents.
Although we have newer less toxic antimicrobial agents that are
available for clinical use, their clinical efficacy in some invasive
fungal infections, is not optimal [10]. In recent years, the wide-
spread use of antimicrobial agents has resulted in the development
of resistance to these drugs by pathogenic microorganisms, causing
an increase in morbidity and mortality [11]. Thus, intense efforts in
antimicrobial drug discovery are still needed to develop more
promising and effective antifungal agents for use in the clinical
arena [10]. Selected azole drugs have supplied many effective
antifungal agents, which are currently in clinical use. Ketoconazole
and Omeprazole are well-known drugs contains imidazole ring
system.
The synthesis of pyrazoles remains of great interest due to the
wide applications of such heterocycles in the pharmaceutical and
Prompted by these observations and in continuation of our
research on biologically important heterocycles [12e15], we hereby
report the synthesis, characterization and antimicrobial studies of
some new substituted imidazoles carrying pyrazole moiety.
* Corresponding author. Tel.: þ91 824 2474000; fax: þ91 824 2474033.
E-mail address: isloor@yahoo.com (A.M. Isloor).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.005

3532
A.M. Vijesh et al. / European Journal of Medicinal Chemistry 46 (2011) 3531e3536
H
N
N
Ar
S
Ar
Ar
NH2
NH
NH2NHCSNH2
N
O
DMAD
Methanol
Reflux
1h
N
N
N
O
Ethanol
Reflux
8h
HN
N
S
N
H
N
H
O
O
H3C
1a-d
2a-d
3a-d
Scheme 1. Synthetic route for the compounds 3aed. Where Ar ¼ 2,4-dichlorophenyl, 4-SCH3eC6H4, 2,5-dichlorothiophene, 4-CH3eC6H4.
2. Results and discussion
2.1. Chemistry
of 3a showed molecular ion peak at m/z ¼ 423.9 (Mþ), which is in
agreement with the molecular formula C16H11Cl2N5O3S. For
second series, IR spectrum of compound 5a showed absorption at
3135 cmꢀ1 which is due to the NH stretching. Band at 1663 cmꢀ1 is
due to C]N. Formation of 2,4,5-trisubstituted imidazoles further
confirmed by the absence of CH stretching of aldehydic group
(2700 cmꢀ1) in the IR spectrum of the final compounds. The 1H
3-Substituted-1H-pyrazole-4-carbaldehydes
(1aee)
were
synthesized by the Vilsmayer Haack reaction of semicarbazones
[16]. The targeted imidazoles (3aed) were obtained in good yield
by refluxing substituted thiosemicarbazones (2aed) with dime-
thylacetylenedicarboxylate (DMAD) in methanol for 1 h [17]. The
starting material 2aed in turn were synthesized by refluxing
equimolar amount of 3-aryl-1-H-pyrazole-4-carbaldehyde with
thiosemicarbazide in the presence of anhydrous sodium acetate in
ethanol [17]. 2,4,5-Trisubstituted imidazoles (5aej) were obtained
in excellent yields by refluxing 3-substituted-1H-pyrazole-4-car-
baldehydes (1aee) with 1,2-diketones (4a,b) and ammonium
acetate in acetic acid for 6e7 h via Debus reaction [18]. The reaction
pathway has been summarized in Scheme 1 and Scheme 2. Newly
synthesized compounds (3aed and 5aej) were characterized by IR,
NMR, mass spectral and C, H, N elemental analyses.
NMR spectrum of 5a showed a singlet at
protons. Similarly multiplets at 7.32e7.99 are due to aromatic
protons. Pyrazole-5H appeared as a singlet at 8.10. Two singlets
at 12.40 and 13.30 are due to pyrazole-NH and imidazole-NH
d 2.53 is due to SCH3
d
d
d
d
respectively further confirmed the structure of the molecule.
The mass spectrum of 5a showed molecular ion peak at m/
z ¼ 409.2 (M þ 1), which is in agreement with the molecular
formula C25H20N4S. Similarly the spectral values for all the
compounds and C, H, N analyses are given in the experimental
part and the characterization is provided in Table 1.
2.2. Antimicrobial studies
Formation of methyl(2Z)-[3-({(E)-[3-(substituted)-1H-pyrazol-
4-yl]methylidene}amino)-5-oxo-2-thioxoimidazolidin-4-ylidene]
ethanoate (3aed) and 4-(4,5-aryl-1H-imidazol-2-yl)-3-[substitu-
ted]-1H-pyrazole (5aej) were confirmed by recording their IR, 1H
NMR, 13C NMR and mass spectra. For first series, IR spectrum of
compound 3a showed absorption at 3242 cmꢀ1 which is due to
the NH stretching. Bands at 1707, 1645 cmꢀ1 are due to C]O of
ester and cyclic amide respectively. Similarly, bands at 1607 cmꢀ1
and 1106 cmꢀ1 are due to C]N and C]S groups. The CeO
stretching frequency of ester appeared at 1238 cmꢀ1 and
1195 cmꢀ1 further confirms the structure. The 1H NMR spectrum
2.2.1. Antibacterial studies
The in vitro antibacterial activity of newly synthesized
compounds 3aed and 5aej were determined by well plate method
[19,20]. In this work, Escherichia coli, Staphylococcus aureus, Bacillus
subtilis, Salmonella typhimorium. Clostridium profingens and Pseu-
domonas aeruginosa were used to investigate the activity. The test
compounds were dissolved in dimethyl sulfoxide (DMSO) at
concentrations of 1 and 0.5 mg/mL.
The antibacterial screening revealed that some of the tested
compounds showed good inhibition against various tested micro-
bial strains. The result indicated that among the tested compounds,
3c showed excellent activity against P. aeruginosa at concentrations
of 1 and 0.5 mg/mL compared to standard drug streptomycin. 3c
Showed similar activity as that of standard, against C. profingens, at
1 and 0.5 mg/mL concentrations. The remaining compounds sho-
wed moderately good activity against all of the six tested bacterial
of 3a showed a singlet at
d
d
3.80 is due to OCH3 protons. A singlet at
6.58 is due to C]CH. Aromatic protons appeared as multiplet at
7.41e7.57. Pyrazole-5H appeared as a singlet at 7.96. Similarly
8.28 is due to eN¼CH protons. Two singlets
13.50 are due to pyrazole-NH and imidazole-NH
d
d
a singlet appeared at
at 12.64 and
d
d
d
respectively further confirms the structure. The mass spectrum
X
X
Ar
Ar
N
O
Ammonium acetate
O
N
+
N
H
Acetic acid
Reflux
6h
N
X
O
N
H
N
H
1a-e
5a-j
X
4a,b
Scheme 2. Synthetic route for the compounds 5aej. Where Ar ¼ 2,4-dichlorophenyl, 4-SCH3eC6H4, 2,5-dichlorothiophene, 4-CH3eC6H4, Biphenyl X ¼ H, Br.

A.M. Vijesh et al. / European Journal of Medicinal Chemistry 46 (2011) 3531e3536
3533
Table 1
Characterization data of the compounds 3aed and 5aej.
Compounds
Ar
X
Molecular Formula (Mol. wt.)
16H11Cl2N5O3S (424.2)
C14H9Cl2N5O3S2 (430.2)
C17H15N5O3S2 (401.4)
C17H15N5O3S (369.3)
C25H20N4S (408.5)
C24H16Cl2N4 (431.3)
C30H22N4 (438.5)
C25H20N4 (376.4)
C22H14Cl2N4S (437.3)
C25H18Br2N4S (566.3)
C24H14Br2Cl2N4 (589.1)
C30H20Br2N4 (596.3)
C25H18Br2N4 (534.2)
Yield (%)
M.p.(ꢁC)
3a
3b
3c
3d
5a
5b
5c
5d
5e
5f
2,4-Dichlorophenyl
2,5-Dichlorothiophene
4-SCH3eC6H4
4-CH3eC6H4
4-SCH3eC6H4
2,4-Dichlorophenyl
Biphenyl
4-CH3eC6H4
2,5-Dichlorothiophene
4-SCH3eC6H4
2,4-Dichlorophenyl
Biphenyl
e
C
81
86
84
80
73
72
75
68
70
77
73
75
72
78
282ꢀ284
280ꢀ282
230ꢀ232
286ꢀ288
214ꢀ216
210ꢀ212
140ꢀ142
180ꢀ182
188ꢀ190
190ꢀ192
168ꢀ170
158ꢀ160
194ꢀ196
140ꢀ142
e
e
e
H
H
H
H
H
Br
Br
Br
Br
Br
5g
5h
5i
4-CH3eC6H4
2,5-Dichlorothiophene
5j
C22H12Br2Cl2N4S (595.1)
Table 2
Antibacterial activity of the compounds 3aeb and 5aej.
Compound
name
Staphylococcus aureus
1000 500
6 ꢂ 0.02
Bacillus subtillis
1000 500
Escherichia coli
Clostridium profingens
Salmonella
typhimorium
Psedumonas
aureginosa
Concn
(mg/ml)
1000
500
1000
7 ꢂ 0.03
500
6 ꢂ 0.03
1000
500
1000
500
3a
3b
3c
3d
5a
5b
5c
5d
5e
5f
5 ꢂ 0.01
6 ꢂ 0.02
5 ꢂ 0.01 10 ꢂ 0.02
8 ꢂ 0.02
7 ꢂ 0.02
7 ꢂ 0.01
8 ꢂ 0.01
9 ꢂ 0.01
7 ꢂ 0.02
4 ꢂ 0.02
3 ꢂ 0.01
9 ꢂ 0.01
7 ꢂ 0.01
9 ꢂ 0.03
6 ꢂ 0.03
8 ꢂ 0.02
7 ꢂ 0.03
6 ꢂ 0.02
4 ꢂ 0.01
4 ꢂ 0.01
6 ꢂ 0.02
2 ꢂ 0.01
7 ꢂ 0.02
6 ꢂ 0.02
3 ꢂ 0.02
4 ꢂ 0.02
2 ꢂ 0.01
3 ꢂ 0.02
3 ꢂ 0.02
8 ꢂ 0.01
8 ꢂ 0.03 12 ꢂ 0.01 11 ꢂ 0.02 10 ꢂ 0.01
9 ꢂ 0.02 12 ꢂ 0.02
9 ꢂ 0.02 16 ꢂ 0.01 15 ꢂ 0.02 12 ꢂ 0.01 11 ꢂ 0.02 16 ꢂ 0.02 15 ꢂ 0.02
3 ꢂ 0.02
3 ꢂ 0.02
5 ꢂ 0.02
1 ꢂ 0.02
6 ꢂ 0.02
5 ꢂ 0.02
2 ꢂ 0.02
3 ꢂ 0.01
1 ꢂ 0.02
2 ꢂ 0.01
2 ꢂ 0.02
9 ꢂ 0.01
5 ꢂ 0.02
6 ꢂ 0.01
3 ꢂ 0.02
8 ꢂ 0.01
4 ꢂ 0.02
5 ꢂ 0.01
5 ꢂ 0.02
4 ꢂ 0.01
4 ꢂ 0.02
2 ꢂ 0.01
8 ꢂ 0.01
4 ꢂ 0.01
5 ꢂ 0.01
7 ꢂ 0.01
4 ꢂ 0.01
6 ꢂ 0.01
9 ꢂ 0.02
2 ꢂ 0.02
8 ꢂ 0.01
5 ꢂ 0.02
2 ꢂ 0.02
4 ꢂ 0.02
2 ꢂ 0.02
3 ꢂ 0.02
3 ꢂ 0.01
6 ꢂ 0.01
4 ꢂ 0.02
6 ꢂ 0.02
2 ꢂ 0.02
6 ꢂ 0.01
8 ꢂ 0.01
4 ꢂ 0.02
3 ꢂ 0.01
2 ꢂ 0.02
3 ꢂ 0.01
5 ꢂ 0.02
5 ꢂ 0.01
3 ꢂ 0.01
5 ꢂ 0.02
1 ꢂ 0.01
5 ꢂ 0.02
7 ꢂ 0.02
3 ꢂ 0.02
2 ꢂ 0.01
1 ꢂ 0.02
2 ꢂ 0.01
4 ꢂ 0.02
6 ꢂ 0.01
4 ꢂ 0.01
3 ꢂ 0.02
4 ꢂ 0.02
6 ꢂ 0.02
7 ꢂ 0.02
4 ꢂ 0.01
4 ꢂ 0.02
3 ꢂ 0.02
4 ꢂ 0.02
2 ꢂ 0.01
5 ꢂ 0.02
3 ꢂ 0.03
2 ꢂ 0.01
3 ꢂ 0.02
5 ꢂ 0.01
6 ꢂ 0.01
3 ꢂ 0.02
3 ꢂ 0.02
2 ꢂ 0.01
3 ꢂ 0.02
1 ꢂ 0.03
4 ꢂ 0.01
5 ꢂ 0.02
4 ꢂ 0.01
4 ꢂ 0.02
6 ꢂ 0.01
6 ꢂ 0.03
3 ꢂ 0.01
5 ꢂ 0.03
2 ꢂ 0.02
2 ꢂ 0.01
4 ꢂ 0.03
3 ꢂ 0.01
4 ꢂ 0.01
3 ꢂ 0.02
3 ꢂ 0.01
4 ꢂ 0.01
5 ꢂ 0.02
2 ꢂ 0.02
4 ꢂ 0.02
1 ꢂ 0.02
1 ꢂ 0.02
3 ꢂ 0.02
9 ꢂ 0.01
5 ꢂ 0.01 10 ꢂ 0.03
2 ꢂ 0.01
3 ꢂ 0.01
7 ꢂ 0.02 14 ꢂ 0.02
3 ꢂ 0.01
4 ꢂ 0.02
4 ꢂ 0.03
3 ꢂ 0.02
3 ꢂ 0.02
1 ꢂ 0.03
6 ꢂ 0.01
3 ꢂ 0.02
5 ꢂ 0.02
3 ꢂ 0.02
4 ꢂ 0.01
4 ꢂ 0.01
5g
5h
5i
5j
Streptomycin 15 ꢂ 0.02 10 ꢂ 0.01 21 ꢂ 0.02 11 ꢂ 0.02 16 ꢂ 0.02 10 ꢂ 0.01 17 ꢂ 0.02 16 ꢂ 0.01 18 ꢂ 0.02 17 ꢂ 0.01 13 ꢂ 0.02
(Std.)
strains compared to standard, streptomycin. Results of antibacterial
studies have been presented in Table 2.
antimicrobial activity was determined by well plate method [21,22]
at concentration of 1 and 0.5 mg/mL.
Among the tested compounds, the compound 3c has emerged as
active against T. rubrum compared with standard, fluconazole.
Whereas the other compounds showed less activity against all the
tested microorganisms compared to standard. It can be concluded
that none of the prepared compounds were superior to standard
against various tested microbial strains, but the antifungal activities
2.2.2. Antifungal studies
Newly synthesized compounds 3aed and 5aej were also
screened for their antifungal activity against Aspergillus flavus,
Aspergillus niger, Candida albicans, Microsporum gypseum, and
Trichophyton rubrum. The compounds were dissolved in DMSO and
Table 3
Antifungal activity of the compounds 3aeb and 5aej.
Compound name
Candida albicans
1000 500
7 ꢂ 0.03
Microsporum gypseum
1000 500
7 ꢂ 0.02 6 ꢂ 0.01
Aspergillus flavus
Aspergillus niger
1000 500
6 ꢂ 0.03
Trichophyton rubrum
Concn (
m
g/ml)
1000
500
1000
500
9 ꢂ 0.02
3a
3b
3c
3d
5a
5b
5c
5d
5e
5f
5g
5h
5i
6 ꢂ 0.01
5 ꢂ 0.01
4 ꢂ 0.02
3 ꢂ 0.01
9 ꢂ 0.01
11 ꢂ 0.01
8 ꢂ 0.01
9 ꢂ 0.02
9 ꢂ 0.01
11 ꢂ 0.01
8 ꢂ 0.01
2 ꢂ 0.01
3 ꢂ 0.02
5 ꢂ 0.01
21 ꢂ 0.01
12 ꢂ 0.01
10 ꢂ 0.02
7 ꢂ 0.01
6 ꢂ 0.02
5 ꢂ 0.01
8 ꢂ 0.02
9 ꢂ 0.01
10 ꢂ 0.02
10 ꢂ 0.01
13 ꢂ 0.02
5 ꢂ 0.02
4 ꢂ 0.01
3 ꢂ 0.01
4 ꢂ 0.01
22 ꢂ 0.01
11 ꢂ 0.01
9 ꢂ 0.01
6 ꢂ 0.03
5 ꢂ 0.01
4 ꢂ 0.02
7 ꢂ 0.03
7 ꢂ 0.02
9 ꢂ 0.01
9 ꢂ 0.01
12 ꢂ 0.01
4 ꢂ 0.02
3 ꢂ 0.01
2 ꢂ 0.02
3 ꢂ 0.03
21 ꢂ 0.02
5 ꢂ 0.03
5 ꢂ 0.01
9 ꢂ 0.03
9 ꢂ 0.01
9 ꢂ 0.01
7 ꢂ 0.01
9 ꢂ 0.02
9 ꢂ 0.02
9 ꢂ 0.01
5 ꢂ 0.03
4 ꢂ 0.01
2 ꢂ 0.01
2 ꢂ 0.01
3 ꢂ 0.02
18 ꢂ 0.02
10 ꢂ 0.01
13 ꢂ 0.02
16 ꢂ 0.02
10 ꢂ 0.02
12 ꢂ 0.01
10 ꢂ 0.02
9 ꢂ 0.03
12 ꢂ 0.01
10 ꢂ 0.01
8 ꢂ 0.03
10 ꢂ 0.01
4 ꢂ 0.02
3 ꢂ 0.01
6 ꢂ 0.01
24 ꢂ 0.01
6 ꢂ 0.02
5 ꢂ 0.03
4 ꢂ 0.01
10 ꢂ 0.01
12 ꢂ 0.02
9 ꢂ 0.01
10 ꢂ 0.03
10 ꢂ 0.02
12 ꢂ 0.02
9 ꢂ 0.01
3 ꢂ 0.02
4 ꢂ 0.01
6 ꢂ 0.01
22 ꢂ 0.03
6 ꢂ 0.01
5 ꢂ 0.01
4 ꢂ 0.02
6 ꢂ 0.01
4 ꢂ 0.02
5 ꢂ 0.01
3 ꢂ 0.02
5 ꢂ 0.03
4 ꢂ 0.02
4 ꢂ 0.01
2 ꢂ 0.01
2 ꢂ 0.01
3 ꢂ 0.03
15 ꢂ 0.03
5 ꢂ 0.01
4 ꢂ 0.01
3 ꢂ 0.01
5 ꢂ 0.01
3 ꢂ 0.01
4 ꢂ 0.02
2 ꢂ 0.01
4 ꢂ 0.02
3 ꢂ 0.01
3 ꢂ 0.03
1 ꢂ 0.02
1 ꢂ 0.01
2 ꢂ 0.02
14 ꢂ 0.02
6 ꢂ 0.02
10 ꢂ 0.02
10 ꢂ 0.02
10 ꢂ 0.02
9 ꢂ 0.01
10 ꢂ 0.01
10 ꢂ 0.01
10 ꢂ 0.02
6 ꢂ 0.02
5 ꢂ 0.03
3 ꢂ 0.01
3 ꢂ 0.02
4 ꢂ 0.01
20 ꢂ 0.01
12 ꢂ 0.01
15 ꢂ 0.01
9 ꢂ 0.01
11 ꢂ 0.02
8 ꢂ 0.02
8 ꢂ 0.02
11 ꢂ 0.02
9 ꢂ 0.02
7 ꢂ 0.02
9 ꢂ 0.01
3 ꢂ 0.02
2 ꢂ 0.01
5 ꢂ 0.01
22 ꢂ 0.01
5j
Flucanazole (Std.)

3534
A.M. Vijesh et al. / European Journal of Medicinal Chemistry 46 (2011) 3531e3536
of some of the compounds are comparable to those of standard.
Results of antifungal studies have been presented in Table 3.
4.2. General procedure for the synthesis of methyl(2Z)-[3-({(E)-[3-ar-
yl-1H-pyrazol-4-yl]methylidene}amino)-5-oxo-2-thioxoimidazolidin-
4-ylidene]ethanoate (3aed)
2.2.3. Acute toxicity and behavioral studies
The acute oral toxicity study for the test compound 3c was
carried out by following the OECD guidelines. Swiss albino female
mice weighing 25e30 g were used for the evaluation. Each group
consisting of 6 female mice (overnight fasted) was kept in the
colony cage at 25 ꢂ 2 ꢁC with 55% relative humidity and 12 h light/
An equimolar mixture of 3-aryl-1H-pyrazole-4-carbaldehyde
thiosemicarbazone 2aed (0.01 mol) and dimethylacetylenedi-
carboxylate (DMAD) (0.01 mol) in methanol (20 mL) were refluxed
for 1 h. After completion of the reaction, the reaction mixture was
allowed to cool to the room temperature. The solid thus separated
was collected by filtration and recrystallized using ethanol-DMF
mixture.
dark cycle was maintained.
A Different dose from 1000 to
3000 mg/kg was selected and administered orally as a single dose
as fine suspension prepared in double distilled water using Tween
80. The acute toxic symptoms and the behavioral changes produced
by the test compound were observed continuously for 4th h, 8th h,
12th h and 24th h onset of toxic symptoms and behavioral changes
were also recorded [23,24]. The experimental studies revealed that
the synthesized compound 3c is safe up to 3000 mg/kg and no
death of animals was recorded. Further, no significant behavioral
changes were observed in experimental animals.
4.2.1. Characterization of synthesized compounds
4.2.1.1. (Z)-Methyl 2-(3-((E)-(3-(2,4-dichlorophenyl)-1H-pyrazol-4-
yl)methyleneamino)-5-oxo-2-thioxoimidazolidin-4-ylidene)acetate
(3a). IR (KBr, nmax cmꢀ1): 3242 (NeH-str), 3068, 2951 (CeH-str),
1707 (C]O ester), 1645 (C]O cyclic amide), 1607 (C]N), 1106 (C]
S), 1238, 1195 (C-O ester); 1H NMR (400 MHz, DMSO-d6):
d 3.80 (s,
3H, OCH3), 6.58 (s, 1H, C]CH), 7.41e7.57 (m, 3H, AreH), 7.96 (s, 1H,
pyrazole-5H), 8.28 (s, 1H, N]CH), 12.64 (s, 1H, pyrazole-NH), 13.50
(s, 1H, imidazole-NH); MS: m/z ¼ 423.9 (Mþ), 425.9 (M þ 2), 427.9
(M þ 4); Anal. calcd. for C16H11Cl2N5O3S: C, 45.30; H, 2.61; N, 16.71;
Found: C, 45.23; H, 2.57; N, 16.67%.
3. Conclusion
Two series of novel substituted imidazole derivatives were
synthesized in reasonably good yields. They were characterized by
1H NMR, 13C NMR, mass spectrometry, IR studies and elemental
analyses. All the newly synthesized compounds were screened for
antimicrobial activity by well plate method. Among the screened
samples, compound 3c has showed excellent anti-microbial
activity at 1 and 0.5 mg/mL concentrations against tested micro-
bial strains as compared to the standard drug.
As regards the relationships between the structure of the
heterocyclic scaffold and the detected antibacterial properties, it
showed varied biological activity. Among the tested compounds,
compound 3c showed excellent activity against P. aeruginosa at
concentrations of 1 and 0.5 mg/mL compared to standard drug
streptomycin. 3c Showed similar activity as that of standard,
4.2.1.2. (Z)-Methyl 2-(3-((E)-(3-(2,5-dichlorothiophen-3-yl)-1H-pyr-
azol-4-yl)methyleneamino)-5-oxo-2-thioxoimidazolidin-4-ylidene)
acetate (3b). IR (KBr, nmax cmꢀ1): 3213 (NeH-str), 3051, 2953 (CeH-
str), 1713 (C]O ester), 1640 (C]O cyclic amide), 1602 (C]N), 1023
(C]S), 1247,1197 (CeO ester); 1H NMR (400 MHz, DMSO-d6):
d 3.79
(s, 3H, OCH3), 6.62 (s, 1H, C]CH), 7.23 (s, 1H, AreH), 8.35 (s, 1H,
pyrazole-5H), 8.38 (s, 1H, N]CH), 12.71 (s, 1H, pyrazole-NH), 13.60
(s, 1H, imidazole-NH); 13C NMR:
d 165.77, 165.65, 151.60, 142.92,
131.66, 128.72, 125.06, 114.89, 114.13, 52.38; MS: m/z ¼ 430.0 (Mþ),
432.0 (M þ 2). 434.0 (M þ 4); Anal. calcd. for C14H9Cl2N5O3S2: C,
39.08; H, 2.11; N, 16.28; Found: C, 39.03; H, 2.06; N, 16.26%.
4.2.1.3. (Z)-Methyl 2-(3-((E)-(3-(4-(methylthio)phenyl)-1H-pyrazol-
4-yl)methyleneamino)-5-oxo-2-thioxoimidazolidin-4-ylidene)acetate
(3c). IR (KBr, nmax cmꢀ1): 3121 (NeH-str), 3033, 2950 (CeH-str),
1711 (C]O ester), 1636 (C]O cyclic amide), 1598 (C]N), 1096 (C]
against C. profingens, at
1 and 0.5 mg/mL concentrations.
Compound 3c has thioanisyl moiety, which is accounted for the
enhanced antibacterial activity. However from the second series,
compounds showed moderate antimicrobial activity. Imidazole and
pyrazole nucleus which is present in both the series are responsible
for the biological activity. However the presence of other substit-
uents is responsible for the varied biological activity of the
compounds.
The acute oral toxicity study for the compound 3c was carried
out and the experimental studies revealed that compound 3c is safe
up to 3000 mg/kg and no death of animals were recorded.
S), 1240, 1188 (CeO ester); 1H NMR (400 MHz, DMSO-d6):
d 2.55 (s,
3H, SCH3), 3.79 (s, 3H, OCH3), 6.65 (s, 1H, C]CH), 7.40e7.77 (m, 4H,
AreH), 8.48 (s, 1H, pyrazole-5H), 8.64 (s, 1H, N]CH), 12.75 (s, 1H,
pyrazole-NH), 13.48 (s, 1H, imidazole-NH); MS: m/z ¼ 402.0
(M þ 1); Anal. calcd. for C17H15N5O3S2: C, 50.86; H, 3.77; N, 17.44;
Found: C, 50.79; H, 3.71; N, 17.41%.
4.2.1.4. (Z)-Methyl2-(5-oxo-2-thioxo-3-((E)-(3-p-tolyl-1H-pyrazol-
4-yl)methyleneamino) imidazolidin-4-ylidene)acetate (3d). IR (KBr,
nmax cmꢀ1): 3241 (NeH-str), 3029, 2950 (CeH-str), 1706 (C]O
ester), 1641 (C]O cyclic amide), 1611 (C]N), 1098 (C]S), 1240,
4. Experimental
4.1. Chemistry
1195 (CeO ester); 1H NMR (400 MHz, DMSO-d6):
d 2.38 (s, 3H, CH3),
3.80 (s, 3H, OCH3), 6.65 (s, 1H, C]CH), 7.35e7.65 (m, 4H, AreH),
7.96 (s, 1H, pyrazole-5H), 8.42 (s, 1H, N]CH), 12.72 (s, 1H, pyrazole-
NH), 13.41 (s, 1H, imidazole-NH); MS: m/z ¼ 370.1 (M þ 1); Anal.
calcd. for C17H15N5O3S: C, 55.27; H, 4.09; N, 18.96; Found: C, 55.21;
H, 4.05; N, 18.93%.
Melting points were determined by open capillary method and
were uncorrected. The IR spectra (in KBr pellets) were recorded on
a JASCO FT/IR-4100 spectrophotometer. 1H NMR and 13C NMR
spectra were recorded (DMSO-d6) on a Bruker (400 MHz) using
TMS as internal standard. Chemical shift values are given in d (ppm)
scales. The mass spectra were recorded on a JEOL JMS-D 300
spectrometer operating at 70 eV. Elemental analyses were per-
formed on a Flash EA 1112 series CHNS-O Analyzer. The completion
of the reaction was checked by thin layer chromatography (TLC) on
silica gel coated aluminium sheets (silica gel 60 F254) obtained
from Merck. Commercial grade solvents and reagents were used
without further purification.
4.3. General procedure for the synthesis of new derivatives of 2,4,5-
trisubstituted imidazoles (5aei)
A
mixture of 3-aryl-1H-pyrazole-4-carbaldehyde 1aed
(0.01 mol), 1,2-diketone 4a,b (0.01 mol) and ammonium acetate
(0.05 mol) in acetic acid (50 mL) were refluxed for 6e7 h at 120 ꢁC.
After completion of the reaction, the reaction mixture was allowed

A.M. Vijesh et al. / European Journal of Medicinal Chemistry 46 (2011) 3531e3536
3535
to cool and filtered to remove any precipitate. 300 mL of ice-water
was added to the filtrate and the precipitated product was collected
by filtration. The crude product was recrystallized using ethanol-
DMF mixture.
(s, 1H, pyrazole-NH), 13.28 (s, 1H, imidazole-NH); MS: m/z ¼ 589.0
(Mþ), 591.0 (M þ 2), 593.0 (M þ 4); Anal. calcd. for C24H14Br2Cl2N4: C,
48.93; H, 2.40; N, 9.51; Found: C, 48.88; H, 2.36; N, 9.49%.
4.3.1.8. 3-(biphenyl-4-yl)-4-(4,5-bis(4-bromophenyl)-1H-imidazol-
2-yl)-1H-pyrazole (5h). IR (KBr, nmax cmꢀ1): 3148 (NeH-str), 3020,
2923 (CeH-str), 1655 (C]N), 1599 (C]C); 1H NMR (400 MHz,
4.3.1. Characterization of synthesized compounds
4.3.1.1. 4-(4,5-Diphenyl-1H-imidazol-2-yl)-3-[4-(methylsulfanyl)phe-
nyl]-1H-pyrazole (5a). IR (KBr, nmax cmꢀ1): 3120 (NeH-str), 3058,
2920 (CeH-str),1663 (C]N),1602 (C]C); 1H NMR (400 MHz, DMSO-
DMSO-d6):
d 7.28e7.91 (m, 17H, AreH), 8.08 (s, 1H, pyrazole-5H),
12.36 (s, 1H, pyrazole-NH), 13.21 (s, 1H, imidazole-NH); MS: m/
z ¼ 596.9 (M þ 1), 597.9 (M þ 2); Anal. calcd. for C30H20Br2N4: C,
60.42; H, 3.38; N, 9.40; Found: C, 60.38; H, 3.33; N, 9.36%.
d6):
d 2.53 (s, 3H, SCH3), 7.32e7.99 (m, 14H, AreH), 8.10 (s, 1H,
pyrazole-5H), 12.40 (s, 1H, pyrazole-NH), 13.30 (s, 1H, imidazole-NH);
MS: m/z ¼ 409.2 (M þ 1); Anal. calcd. for C25H20N4S: C, 73.50; H, 4.93;
N, 13.71; Found: C, 73.41; H, 4.88; N, 13.69%.
4.3.1.9. 4-[4,5-bis(4-bromophenyl)-1H-imidazol-2-yl]-3-(4-methyl-
phenyl)-1H-pyrazole (5i). IR (KBr, nmax cmꢀ1): 3135 (NeH-str),
3040, 2916 (CeH-str), 1608 (C]N), 1552 (C]C); 1H NMR (400 MHz,
4.3.1.2. 3-(2,4-Dichlorophenyl)-4-(4,5-diphenyl-1H-imidazol-2-yl)-1H-
pyrazole (5b). IR (KBr, nmax cmꢀ1): 3135 (NeH-str), 3062, 2922 (CeH-
str), 1668 (C]N), 1598 (C]C); 1H NMR (400 MHz, DMSO-d6):
DMSO-d6):
pyrazole-5H), 12.42 (s, 1H, pyrazole-NH), 13.11 (s, 1H, imidazole-
NH); 13C NMR:
171.97, 141.29, 137.37, 131.48, 129.51, 128.62,
d 2.33 (s, 3H, CH3), 7.22e7.88 (m,12H, AreH), 8.05 (s,1H,
d
7.14e7.92 (m, 13H, AreH), 8.27 (s, 1H, pyrazole-5H), 12.22 (s, 1H,
d
pyrazole-NH), 13.24 (s, 1H, imidazole-NH); 13C NMR:
d
194.77, 184.39,
127.93, 120.34, 109.10, 21.02; MS: m/z ¼ 535.1 (M þ 1), 536.1
(M þ 2); Anal. calcd. for C25H18Br2N4: C, 56.20; H, 3.40; N, 10.49;
Found: C, 56.15; H, 3.34; N, 10.45%.
171.94, 140.16, 135.4, 134.5, 133.95, 133.27, 132.22, 131.38, 129.55,
129.46, 128.51, 128.05, 126.55, 120.97, 111.56; MS: m/z ¼ 431.2 (Mþ),
433.1 (M þ 2), 435.1 (M þ 4); Anal. calcd. for C24H16Cl2N4: C, 66.83; H,
3.74; N, 12.99; Found: C, 66.76; H, 3.68; N, 12.95%.
4.3.1.10. 4-[4,5-bis(4-bromophenyl)-1H-imidazol-2-yl]-3-(2,5-dichl-
orothiophen-3-yl)-1H-pyrazole (5j). IR (KBr, nmax cmꢀ1): 3123 (NeH-
str), 3010, 2922 (CeH-str), 1615 (C]N), 1575 (C]C); 1H NMR
4.3.1.3. 3-(Biphenyl-4-yl)-4-(4,5-diphenyl-1H-imidazol-2-yl)-1H-pyr-
azole (5c). IR (KBr, nmax cmꢀ1): 3129 (NeH-str), 3055, 2922 (CeH-
str), 1669 (C]N), 1599 (C]C); 1H NMR (400 MHz, DMSO-d6):
(400 MHz, DMSO-d6):
pyrazole-5H), 12.41 (s, 1H, pyrazole-NH), 13.36 (s, 1H, imidazole-
NH); 13C NMR:
206.46, 192.85, 140.50, 132.56, 131.73, 131.59,
d 7.37e7.85 (m, 12H, AreH), 8.27 (s, 1H,
d
7.34e8.14 (m, 19H, AreH), 8.17 (s, 1H, pyrazole-5H), 12.39 (s, 1H,
d
pyrazole-NH),13.19 (s,1H, imidazole-NH); 13C NMR:
d
140.52,139.63,
131.20,130.03,128.63,125.95,120.84,119.49,111.07; MS: m/z ¼ 594.9
(Mþ), 596.9 (M þ 2), 598.9 (M þ 4); Anal. calcd. for C22H12Br2Cl2N4S:
C, 44.40; H, 2.03; N, 9.41; Found: C, 44.35; H, 2.01; N, 9.38%.
139.46, 135.45, 129.52, 129.44, 128.89, 128.47, 127.45, 126.85, 126.54,
126.17, 109.74; MS: m/z ¼ 439.3 (M þ 1); Anal. calcd. for C30H22N4: C,
82.17; H, 5.06; N, 12.78; Found: C, 82.06; H, 5.01; N, 12.75%.
4.4. Antibacterial studies
4.3.1.4. 4-(4,5-Diphenyl-1H-imidazol-2-yl)-3-(4-methylphenyl)-1H-
pyrazole (5d). IR (KBr, nmax cmꢀ1): 3130 (NeH-str), 3054, 2919 (CeH-
The antibacterial activity of newly synthesized compounds 3aed
and 5aej were determined by well plate method in Mueller-Hinton
Agar. The in vitro antibacterial activity was carried out against 24 h
old cultures of bacterial strains. In this work, E. coli (ATTC-25922), S.
aureus, B. subtilis, S. typhimorium. profingens, and P. aeruginosa
(ATCC-27853) were used to investigate the activity. The test
compounds were dissolved in dimethyl sulfoxide (DMSO) at
concentration of 1 and 0.5 mg/mL. Twenty milliliters of sterilized
agar media was poured into each pre-sterilized Petri dish. Excess of
suspension was decanted and plates were dried by placing in an
str),1654(C]N),1604(C]C);1HNMR(400MHz,DMSO-d6):
d
2.07(s,
3H, CH3), 7.16e7.91 (m,14H, AreH), 7.92 (s,1H, pyrazole-5H),12.28 (s,
1H, pyrazole-NH), 13.10 (s, 1H, imidazole-NH); 13C NMR:
184.70,
d
140.62, 137.21, 129.53, 129.44, 128.54, 128.11, 127.91, 127.03, 109.39,
20.98; MS: m/z ¼ 377.2 (M þ 1); Anal. calcd. for C25H20N4: C, 79.76; H,
5.35; N, 14.88; Found: C, 79.66; H, 5.28; N, 14.55%.
4.3.1.5. 3-(2,5-Dichlorothiophen-3-yl)-4-(4,5-diphenyl-1H-imidazol-
2-yl)-1H-pyrazole (5e). IR (KBr, nmax cmꢀ1): 3135 (NeH-str), 3049,
2924 (CeH-str), 1676 (C]N), 1604 (C]C); 1H NMR (400 MHz,
incubator at 37 ꢁC for an hour. About 60
ml of 24 h old culture
DMSO-d6):
12.31 (s, 1H, pyrazole-NH), 13.34 (s, 1H, imidazole-NH); 13C NMR:
194.79,171.95,139.85,135.98,135.50,132.23,131.22,130.18,129.56,
d
7.16e7.64 (m, 11H, AreH), 8.24 (s, 1H, pyrazole-5H),
suspensionwere poured and neatly swabbed with the pre-sterilized
cotton swabs. Six millimeter diameter well were then punched
carefully using a sterile cork borer and 30 ml of test solutions of
d
129.47,128.62,128.15,127.61,126.57,123.25,111.34; MS: m/z ¼ 437.1
(Mþ), 439.1 (M þ 2), 441.1 (M þ 4); Anal. calcd. for C22H14Cl2N4S: C,
60.42; H, 3.23; N, 12.81; Found: C, 60.39; H, 3.17; N, 12.76%.
different concentrations were added into each labeled well. The
plates were incubated for 24 h at 37 ꢁC. The inhibition zone that
appeared after 24 h, around the well in each plate were measured as
zone of inhibition in mm. Experiments were triplicates and standard
deviation was calculated.
4.3.1.6. 4-[4,5-Bis(4-bromophenyl)-1H-imidazol-2-yl]-3-[4-(methyl-
sulfanyl)phenyl]-1H-pyrazole (5f). IR (KBr, vmax cmꢀ1): 3290 (NeH-
str), 3050, 2960 (CeH-str), 1658 (C]N), 1573 (C]C); 1H NMR
4.5. Antifungal studies
(400 MHz, DMSO-d6):
d 2.49 (s, 3H, SCH3), 7.30e7.95 (m, 12H,
AreH), 8.19 (s, 1H, pyrazole-5H), 12.45 (s, 1H, pyrazole-NH), 13.19 (s,
1H, imidazole-NH); MS: m/z ¼ 567.0 (M þ 1), 568.0 (M þ 2); Anal.
calcd. for C25H18Br2N4S: C, 53.02; H, 3.20; N, 9.89; Found: C, 52.95;
H, 3.16; N, 9.84%.
Antifungal studies of newly synthesized compounds 3aed and
5aej were carried out against A. flavus, A. niger, C. albicans, M. gyp-
seum, T. rubrum. Sabourands agar media was prepared by dissolving
peptone (10 g), D-glucose (40 g) and agar (20 g) in distilled water
(1000 mL) and adjusting the pH to 5.7. Normal saline was used to
make a suspension of spore of fungal strains for lawning. A loopful of
particular fungal strain was transferred to 3 mL saline to get
a suspension of corresponding species. Twenty milliliters of agar
media was poured into each petri dish. Excess of suspension was
4.3.1.7. 4-[4,5-bis(4-bromophenyl)-1H-imidazol-2-yl]-3-(2,4-dichlor-
ophenyl)-1H-pyrazole (5g). IR (KBr, nmax cmꢀ1): 3387 (NeH-str),
3068, 2961 (CeH-str), 1617 (C]N), 1485 (C]C); 1H NMR (400 MHz,
DMSO-d6): d7.25e7.68(m,11H, AreH),8.25(s,1H,pyrazole-5H),12.37

3536
A.M. Vijesh et al. / European Journal of Medicinal Chemistry 46 (2011) 3531e3536
decanted and plates were dried by placing in incubator at 37 ꢁC for
1 h. Using sterile cork borer punched carefully, wells were made on
these seeded agar plates different concentrations of the test
compounds in DMSO were added into each labeled well. A control
was also prepared for the plates in the same way using solvent
DMSO. The Petri dishes were prepared in triplicate and maintained
at 25 ꢁC for 72 h. Antifungal activity was determined by measuring
the diameter of inhibition zone. Activity of each compound was
compared with fluconazole as standard. Zones of inhibition were
determined for compounds 3ae3d and 5ae5j.
[4] I. Takeuchi, M. Sugiura, K. Yamamoto, T. Ito, Y. Hamada, Yakuga. Zasshi 105 (6)
(1985) 554e561.
[5] H. Miyachi, H. Kiyota, M. Segawa, Bioorg. Med. Chem. Lett. 8 (14) (1998)
1807e1812.
[6] A.M. Isloor, B. Kalluraya, P. Shetty, Eur. J. Med. Chem. 44 (2009) 3784e3787.
[7] A.M. Isloor, B. Kalluraya, M. Rao, J. Saudi Chem. Soc. 4 (3) (2000) 265e270.
[8] B. Kalluraya, A.M. Isloor, P.V. Frank, R.L. Jagadesha, S. Shenoy, Indian J. Het-
erocycl. Chem. (OcteDec 2001) 159e162.
[9] D. Sunil, A.M. Isloor, P. Shetty, Der Pharma Chemica 1 (2) (2009) 19e26.
[10] T.A. Vincent, J. Antimicrob. Chemother. 44 (1999) 151e162.
[11] M. Giulia, M. Luisa, F. Paola, S. Silvia, R. Angelo, M. Luisa, B. Francesco,
L. Roberta, M. Chiara, M. Valeria, L.C. Paolo, T. Elena, Bioorg. Med. Chem.
12 (20) (2004) 5465e5483.
[12] A.M. Isloor, B. Kalluraya, K.S. Pai, Eur. J. Med. Chem. 45 (2010) 825e830.
[13] B. Chandrakantha, P. Shetty, V. Nambiyar, N. Isloor, A.M. Isloor, Eur. J. Med.
Chem. 45 (2010) 1206e1210.
[14] U.S. Rai, A.M. Isloor, P. Shetty, A.M. Vijesh, N. Prabhu, S. Isloor,
M. Thiageeswaran, H.K. Fun, Eur. J. Med. Chem. 45 (2010) 2695e2699.
[15] A.M. Vijesh, A.M. Isloor, V. Prabhu, S. Ahmad, S. Malladi, Eur. J. Med. Chem.
45 (2010) 5460e5464.
[16] P.G. Baraldi, C. Barbara, S. Giampiero, R. Romeo, B. Giovanni, N.Z. Abdel,
J. Maria, D.L. Pineda, Synthesis 10 (19987) 1140e1142.
[17] M.M. Heravia, N. Namib, M.M. Heravic, N. Ramezanianc, J. Iran, Chem. Soc.
5 (2008) S21eS25.
[18] Z. Zang, R. He, P. Lei, Y. Gao, H. Chi, Z. Hu, J. Mater. Sci. Technol. 20 (1) (2004)
129e132.
Acknowledgments
AMI thank Department of Atomic Energy, Board for research in
Nuclear Sciences, Government of India for ‘Young Scientist’ award.
AMV thankful to Dr. Arulmoli, Vice President (R&D) and the
management, SEQUENT SCIENTIFIC LTD, New Mangalore, India for
their invaluable support and allocation of resources for this work.
The authors are also thankful to Head, NMR Research center, IISc,
Bangalore for providing spectral data.
[19] B.A. Arthington-Skaggs, M. Motley, D.W. Warnock, C.J. Morrison,
J. Clin.Microbiol 38 (2000) 2254e2260.
[20] L. Rocha, A. Marston, O. Potterat, M.A.C. Kaplan, H. Stoeckli-Evans,
K. Hostettmann, Phytochemistry 40 (5) (1995) 1447e1452.
[21] D.J. Mac. Lowry, M.J. Jaqua, S.T. Selepak, Appl. Microbiol. 20 (1970) 46e53.
[22] A. Portillo, R. Vila, B. Freixa, T. Adzet, S. Can-igueral, J. Ethnopharmacol 76 (1)
(2001) 93e98.
References
[1] F. Suzuki, T. Kuroda, T. Tamura, S. Sato, K. Ohmori, S. Ichikawa, J. Med. Chem
35 (15) (1992) 2863e2870.
[23] N. Pahari, D. Saha, V.K. Jain, B. Jain, D. . Mridha, Int. J. Pharma Sci. Res. 1 (9)
(2010) 399e408.
[24] J.T. Jaouhari, H.B. Lazrek, A. Seddik, M. Jana, J. Ethnopharmacol. 64 (1999)
211e217.
[2] M. Pinza, Z. Farina, A. Cerri, U. Pfeiffer, M.T. Riccaboni, S. Banfi, R. Biagetti,
O. Pozzi, M. Magnani, L. Dorigotti, J. Med. Chem 36 (26) (1993) 4214e4220.
[3] J. Pandey, V.K. Tiwari, S.S. Verma, V. Chaturvedi, S. Bhatnagar, S. Sinha,
A.N. Gaikwad, R.P. Tripathi, Eur. J. Med. Chem. 44 (2009) 3350e3355.