Ultrasound-assisted one-pot four-component synthesis of novel 2-amino-3-cyanopyridine derivatives bearing 5-imidazopyrazole scaffold and their biological broadcast

Article abstract of DOI:10.1016/j.ejmech.2014.06.071

An alternative and environmentally caring way for the synthesis of novel 2-amino-3-cyanopyridine derivatives bearing 5-imidazopyrazole nucleus is reported by one-pot four-component cyclocondensation reaction of substituted 5-(1H-imidazol/4-methyl-1-yl)-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (3a-b), malononitrile (4), ammonium acetate (5) and aromatic (6a-f)/heterocyclic methyl ketones (7a-d) under ultrasonic irradiation. The newly synthesized compounds were evaluated for their in vitro antibacterial activity against a panel of pathogenic stains of bacteria and fungi, in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv stain and in vitro antioxidant activity by ferric-reducing antioxidant power method. Compounds 8e, 8h, 8l, 9c, 9g and 9h exhibited excellent antibacterial activity and compounds 3a, 8k, 9a and 9bshowed moderate antituberculosis activity as compared with the first line drugs. Majority of the compounds showed excellent antioxidant activity. This approaches claimed to be an environment friendly protocol as it afforded numerous advantages i.e. excellent yields, cleaner reaction profile and shorter reaction time.

Full text of DOI:10.1016/j.ejmech.2014.06.071

European Journal of Medicinal Chemistry 83 (2014) 655e664
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Ultrasound-assisted one-pot four-component synthesis of novel 2-
amino-3-cyanopyridine derivatives bearing 5-imidazopyrazole
scaffold and their biological broadcast
Piyush N. Kalaria*, Shailesh P. Satasia, Jemin R. Avalani, Dipak K. Raval
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar 388 120, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
An alternative and environmentally caring way for the synthesis of novel 2-amino-3-cyanopyridine
derivatives bearing 5-imidazopyrazole nucleus is reported by one-pot four-component cyclo-
condensation reaction of substituted 5-(1H-imidazol/4-methyl-1-yl)-3-methyl-1-phenyl-1H-pyrazole-4-
carbaldehyde (3aeb), malononitrile (4), ammonium acetate (5) and aromatic (6aef)/heterocyclic methyl
ketones (7aed) under ultrasonic irradiation. The newly synthesized compounds were evaluated for their
in vitro antibacterial activity against a panel of pathogenic stains of bacteria and fungi, in vitro antitu-
bercular activity against Mycobacterium tuberculosis H37Rv stain and in vitro antioxidant activity by
ferric-reducing antioxidant power method. Compounds 8e, 8h, 8l, 9c, 9g and 9h exhibited excellent
antibacterial activity and compounds 3a, 8k, 9a and 9bshowed moderate antituberculosis activity as
compared with the first line drugs. Majority of the compounds showed excellent antioxidant activity.
This approaches claimed to be an environment friendly protocol as it afforded numerous advantages i.e.
excellent yields, cleaner reaction profile and shorter reaction time.
Received 30 December 2013
Received in revised form
28 June 2014
Accepted 28 June 2014
Available online 30 June 2014
Keywords:
Ultrasound
Pyridine derivatives
5-Imidazopyrazole
Antimicrobial activity
Antituberculosis activity
FRAP assay
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
evergreen area characterized by energetic search with the intention
of conquering episode of abundant drug resistance. To establish
Mycobacterium tuberculosis is a deadly pathogen and contribu-
tory agent of tuberculosis (TB) which remains a chief reason of
death worldwide [1e3]. Almost one third of the world's population
is contaminated with TB bacilli and each year approximately 8
million people are added in the list suffering from active TB and 2
million die as a result [4]. HIV-positive patients are more likely to be
infected with TB. The co-infection with HIV is considered
accountable for these serious circumstances. The difficulty in
treating drug-resistant TB, such as multidrug- resistant TB (MDR-
TB) and extensively drug-resistant TB (XDR-TB) also contributes to
the increased morbidity and mortality. There has been remarkable
raise in the frequency of bacterial and fungal infections over the
past few decades. These organisms possess the ability to withstand
attack by currently available antimicrobial drugs. The uncontrolled
rise in drug resistant pathogens have threatened lives [5]. Innova-
tion of novel drug molecules for the action of complete mycoses is
one of the most imperative challenges in infectious disease
research. The investigation of new antimicrobial drugs is an
preeminent ways to build up efficient therapy, the urgency for the
need of novel, unique and persuasive antimicrobial and antituber-
cular agents attracts immediate attention.
Synthesis of the pyrazole ring system and its derivatives occupy
an important place in the realm of synthetic organic chemistry, due
to their pharmaceutical activities such as, anticancer [6], antibac-
terial [7], analgesic [8], antiviral [9] and anti-inflammatory [10]
activities. Imidazole derivatives are known to be allied with
diverse biological properties, such as antimicrobial [11], antitu-
bercular [12], anti-inflammatory [13], anti-Parkinson [14], anal-
gesic, anti-convulsant and anticancer activities [15]. In recent years,
pyridine derivatives represent an imperative class of compounds
which possesses high activity profile due to their therapeutic and
pharmacological properties [16e18]. Pyridine ring is an essential
part for the anticancer and anti-inflammatory agents [19,20]. The
biologically active compounds having carbonenitrogen bond
especially the natural and synthetic compounds with cyanopyr-
idine moiety are proved to display significant antifungal [21],
antibacterial [22] and anticancer [23] activities.2-Amino-3-
cyanopyridine motifs are known to have numerous biological ac-
tivities, such as anti-microbial [24], anti-inflammatory [25],
* Corresponding author.
E-mail address: piyush_kalaria@yahoo.com (P.N. Kalaria).
http://dx.doi.org/10.1016/j.ejmech.2014.06.071
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

656
P.N. Kalaria et al. / European Journal of Medicinal Chemistry 83 (2014) 655e664
cardiotonic [26], anti-parkinsonism properties [27] and potent in-
hibitor of HIV-1 integrase [28].
VilsmeiereHaack reaction of 3-methyl-1-phenyl-1H-pyrazol-
5(4H)-one [42]. The final aldehydes 5-(1H-imidazol-1-yl)-3-
methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 3aeb were pre-
pared by nucleophilic displacement of chloro group at C5 in 5-
Literature survey revealed that
a number of 2-amino-3-
cyanopyridine derivatives have been synthesized using a variety
of aldehydes [25,26,29e31]. 5-(1H-imidazol/4-methyl-1-yl)-3-
methyl-1-phenyl-1H-pyrazole-4-carbaldehydes has not been re-
ported so far for the purpose. Thus, with a view to obtain biologi-
cally more potent heterocyclic systems, our aim was focused on the
prologue of chemical diversity in the molecular framework to
synthesize pharmacologically interesting compounds of different
composition by green protocol.
chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde
1
with
secondary amine of imidazole 2aebin refluxing DMF using anhy-
drous potassium carbonate as a base. The targeted compounds
substituted 4-(5-(1H-imidazol/4-metyl-1-yl)-3-methyl-1-phenyl-
1H-pyrazol-4-yl)-2-aminonicotinonitrile 8ael and 9aeh were
prepared in moderate to good yield (64e84%) by the reaction of
substituted
5-(1H-imidazol/4-methyl-1-yl)-3-methyl-1-phenyl-
Ultrasonic-assisted organic synthesis (UAOS) offers a resource-
ful and too easy pathway for a large variety of scaffolds. The sig-
nificant features of the ultrasound approach are formation of pure
products in prominent yields, improved rate of reaction, easier
handling and also considered as a processing aid in terms of energy
conservation compared with conventional methods [32,33].
The single-pot synthesis proceeds over shorter reaction time
without the need for isolation of the intermediates [34]. Hence, as a
part of programme directed towards environmentally friendly
methodologies for the preparation of heterocyclic compound
[35e41], herein we report a simple, cost-effective, green and
expeditious method for the synthesis of novel 2-amino-3-
cyanopyridine derivatives bearing 5-imidazopyrazole moieties
under ultrasound irradiation and investigation on their antimi-
crobial, antituberculosis and antioxidant activities (Scheme 1).
1H-pyrazole-4-carbaldehyde 3aeb, malononitrile 4, ammonium
acetate 5 and aromatic/heterocyclic methyl ketone 6aef/7aed in
absolute alcohol by one-pot four-component cyclocondensation
reaction (Scheme 1 , Table 1). The formation of compounds 8ael
and 9aeh took place via imine formed from ketone and ammonium
acetate. Imine reacted with alkylidene malononitrile formed from
Knoevenagel condensation of aldehyde and malononitrile, fol-
lowed by cycloaddition, isomerization and aromatization to give
the targeted compounds 8ael and 9aeh. The mechanism of com-
pound 6c is illustrated in Scheme 2.
3. Pharmacology
3.1. Antimicrobial activity
The minimal inhibitory concentration (MIC) of all the synthe-
sized compounds 8ael and 9aeh was determined by broth
microdilution method according to National Committee for Clinical
Laboratory Standards (NCCLS) [43]. Antibacterial activity was
screened against three Gram positive (Streptococcus pneumoniae
MTCC 1936, Clostridium tetani MTCC 449 and Bacillus subtilis MTCC
441) and three Gram negative (Escherichia coli MTCC 443, Vibrio
cholerae MTCC 3906 and Salmonella typhi MTCC 98) bacteria by
2. Chemistry
The synthetic approach adopted to acquire the targeted 4-(5-
(1H-imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2-
aminonicotinonitrile derivatives 8ael and 9aeh is summarized in
Scheme 1. The starting material 5-chloro-3-methyl-1-phenyl-1H-
pyrazole-4-carbaldehyde
1
was prepared according to
Scheme 1. Synthesis of the substituted 4-(5-(1H-imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2-aminonicotinonitrile 8ael and 9aeh. (i) DMF, K2CO3, Reflux 2 h (ii) Ethanol,
ultrasound irradiation, room temperature Or Ethanol, Reflux 1.5e3 h.

P.N. Kalaria et al. / European Journal of Medicinal Chemistry 83 (2014) 655e664
657
Scheme 2. Plausible mechanistic pathway for the synthesis of compound 8c.
using ampicillin as the standard antibacterial drugs. Antifungal
activity was screened against two fungal species (Candida albicans
MTCC 227 and Aspergillus fumigats MTCC 3008) where griseofulvin
was used as the standard antifungal agents. All MTCC cultures were
collected from Institute of Microbial Technology, Chandigarh and
tested against above mentioned known drugs. The results of anti-
microbial screening are summarized in Table 2.
of 2212e2179 cmꢀ1could be attributed to the presence of eC^N
group. The CeH stretching in methyl group was observed around
3015e2951 cmꢀ1. The 1H NMR spectra of the targeted compounds
8ael and 9aeh showed the absence of the aldehydic proton,
moreover a sharp singlet due to methyl proton appeared around
d
2.47e2.55 ppm. The aromatic protons resonate as multiplet at
around d 6.93e8.72 ppm. The mass spectrum of all the compounds
showed molecular ion peak at Mþ corresponding to their molecular
weights, which confirmed the respective chemical structures. The
obtained elemental analysis values are in good agreement with
theoretical data.
3.2. Antituberculosis activity
In vitro antituberculosis activity of all newly synthesized com-
pounds 8ael and 9aeh was conducted at 250 mg/mL against M.
tuberculosis H37Rv stain by using LowensteineeJensen medium as
described by Rattan [44].The observed results are presented in
Table 3 in the form of %inhibition. Rifampicin and Isoniazid were
used as the standard antitubercular drugs.
4.2. Biological results
4.2.1. In vitro antimicrobial assay
The antibacterial screening of compounds 3a, 3b and their
pyridine derivatives 8ael and 9aeh (Table 2) indicated that
3.3. Antioxidant activity
compound 8h exhibited outstanding inhibitory effect i.e. 62.5
mL against B. subtilis as compared to ampicillin i.e. 250 g/mL and
norfloxacin i.e. 100 g/mL respectively. Compounds 3b, 8a, 8b, 8g,
8i, 8k, 9a, 9c and 9f showed excellent activity against gram positive
bacteria B. subtilis as that of the ampicillin i.e. 250 g/mL. Among
them, compounds 3b, 8b, 8k, and 9a showed comparable activity
i.e. 100 g/mL against B. subtilis as compared tonorfloxacin. While
compounds 3a, 8d, 8l and 9h were found to be equipotent against
B. subtilis as compared to ampicillin i.e. 250 g/mL. Against C. tetani,
compounds 3b, 8g, 8h, 8k, 9a, 9c, and 9f were found to be more
potent than ampicillin i.e. 250 g/mL. Among them, compound
8hwas found to be equipotent to ciprofloxacin i.e. 100 g/mL. The
compounds 3a, 8a, 8c, 8d, 8j and 8l showed comparable activity
against C. tetani as compared to ampicillin i.e. 250 g/mL. Against
S. pneumoniae, compounds 8e, 8j, 8l, 9a and 9ce9f were found to
have identical activity as compared to ampicillin i.e. 100 g/mL. In
case of inhibiting gram negative bacteria, the compounds 9g, 9h
and 9a, 9c showed superior activity i.e. 62.5 g/mL against S. typhi
and V. cholera relative to ampicillin i.e. 100 g/mL respectively and
compound 8e also illustrated marvellous activity i.e. 62.5 g/mL
mg/
m
In vitro antioxidant activity of the newly synthesized com-
pounds 8ael and 9aeh was carried out by using ascorbic acid as the
standard antioxidant and the results are summarized in Table 5. The
antioxidant potential of the compounds was estimated as their
power to reduce the TPTZ-Fe (III) complex to TPTZ-Fe (II) complex.
Ferric reducing antioxidant power (FRAP) was measured by
modified method of Benzie and Strain [45].The absorbance of
intensive blue colour [Fe (II)eTPTZ] complex was measured at
593 nm. The results are expressed as ascorbic equivalent (mmol/
100 g compound).
m
m
m
m
m
m
4. Result and discussion
m
4.1. Analytical results
m
The identity of all the synthesized compounds was confirmed by
FT-IR, 1H NMR, mass spectroscopy and elemental analysis. IR
spectra of the compounds showed absorption in the range of
3413e3312 cmꢀ1 due to symmetric and asymmetric eNH stretch-
ing of an amine. The CeH rocking in eCH3was observed around
1376e1368 cmꢀ1. The strong absorption band observed in the range
m
m
m
against E. coli as compared to ampicillin. Compounds 3a, 8c, 8g, 9g
and 9a showed the same inhibitory effects as that of the standard
ampicillin i.e. 100 mg/mL against E. coli and S. typhi respectively.

658
P.N. Kalaria et al. / European Journal of Medicinal Chemistry 83 (2014) 655e664
Table 1
Reaction parameters under sonication and by conventional method (8ael and 9aeh).
Entry
R
R1/Het.
Sonication methoda
Time (min)
Conventional methodb
Time (h)
Yieldc (%)
Yieldc (%)
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
9a
9b
9c
9d
9e
9f
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
H
H
Ph
Ph
Cl
Cl
Br
Br
60
45
75
60
45
60
30
30
45
30
30
30
75
60
90
75
90
90
45
45
84
85
93
92
81
80
82
84
81
83
91
88
82
83
79
78
81
85
91
93
2.0
1.5
2.5
2.5
2.0
2.5
2.5
2.5
2.0
2.0
1.5
1.5
3.0
2.5
3.0
3.0
3.0
3.0
1.5
1.5
78
79
84
81
71
69
72
76
73
72
79
75
71
68
64
67
71
73
77
77
F
F
OCH3
OCH3
2-furyl
2-furyl
2-thienyl
2-thienyl
3-coumaryl
3-coumaryl
CH3
H
CH3
H
9g
9h
4-(3',4'-Methylene dioxy)phenyl
4-(3',4'-Methylene dioxy)phenyl
CH3
a
Reaction condition: Reaction of aldehydes, malononitrile, aromatic/heterocyclic methyl ketones and ammonium acetate in ethanol at room temperature under ultrasound
irradiation.
b
c
Reaction condition: Reaction of aldehydes, malononitrile, aromatic/heterocyclic methyl ketones and ammonium acetate in ethanol under reflux condition.
Yields of isolated products.
Against V. cholera, compounds 8d, 8j, 9a and 9d showed compa-
rable activity i.e. 100 g/mL as compared to ampicillin.
activity i.e. 250
mg/mL when compared with griseofulvin i.e.
m
500 g/mL and compounds 8c, 8d, 8g, 8l, 9a and 9e showed same
m
The antifungal screening data (Table 2) revealed that against
potency as that of griseofulvin. None of the compounds were
C. albicans, compounds 3b, 9f and 9g were found to have significant
observed to be active against A. fumigatus.
4.2.2. In vitro antituberculosis assay
Table 2
The encouraging results from the antimicrobial activity
impelled us to set out for the preliminary screening of the syn-
thesized compounds for their in vitro antituberculosis activity
against M. tuberculosis H37Rv bacteria (Table 3). The antitubercu-
losis broadcast data revealed that all the tested compounds showed
moderate inhibitory effect. The conclusion of the antitubercular
result exposed that, compounds 3a, 8k, 9a and 9b found to have
In vitro antimicrobial activity (MICs,
mg/mL) of compounds (3ae3b, 8ae8l, 9ae9h).
Entry Gram positive bacteria
Gram negative bacteria
Fungi
S.P.
B.S.
C.T.
E.C.
S.T.
V.C.
C.A.
A.F.
MTCC
1936
MTCC
441
MTCC
449
MTCC
443
MTCC
98
MTCC
3906
MTCC MTCC
227 3008
>1000 >1000
3a
3b
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
9a
9b
9c
9d
9e
9f
9g
9h
A
500
500
250
500
500
125
100
200
200
250
200
100
125
100
100
250
100
100
100
100
250
500
100
n.t.
250
100
200
100
500
250
500
500
200
62.5
200
500
100
250
100
500
200
500
500
200
500
250
250
n.t.
250
150
250
500
250
250
500
500
200
100
500
250
200
250
150
500
200
500
500
200
500
500
250
n.t.
100
250
250
200
100
250
62.5
200
100
500
500
500
125
125
250
250
500
250
200
250
100
125
100
n.t.
125
200
200
250
125
200
200
250
200
500
500
250
200
250
100
200
500
250
500
250
62.5
62.5
100
n.t.
250
250
250
200
500
100
500
250
200
250
200
100
200
62.5
100
200
62.5
100
125
125
200
500
100
n.t.
moderate activity (i.e. 91%, 94%, 96% and 92% respectively at 250 mg/
250
1000
>1000 >1000
500
500
1000
mL) against M. tuberculosis H37Rv. While compounds 8b, 8h and 9g
are fairly active i.e. 84% against M. tuberculosis H37Rv. Remaining all
other compounds showed poor inhibition of M. tuberculosis growth.
The compounds 3a, 8k, 9a and 9b which showed higher inhibition
against M. tuberculosis H37Rv, they are further screened for their
500
500
500
500
500
>1000
1000
500
1000
1000
500
1000
1000
>1000 1000
>1000 1000
500
500
>1000 >1000
>1000 500
>1000 >1000
500
250
250
1000
n.t.a
500
MICs. Among them compound 9a MIC ¼ 25
mg/mL found to possess
highest potency against M. tuberculosis with 96% inhibition
Table 3
In vitro antituberculosis activity (% inhibition) of compounds (3ae3b, 8ae8l, 9ae9h)
against M. tuberculosis H37Rv (at concentration 250 mg/mL).
1000
>1000
Entry
% Inhibition
Entry
% Inhibition
3a
3b
8a
8b
8c
8d
8e
8f
91
65
25
84
45
23
16
22
37
84
15
62
8k
8l
94
56
96
92
41
23
61
45
84
78
98
99
1000
500
500
1000
n.t.
9a
9b
9c
9d
9e
9f
B
100
S.P.: Streptococcus pneumoniae, B.S.: Bacillus subtilis, C.T.: Clostridium tetani, E.C.:
Escherichia coli, S.T.: Salmonella typhi, V.C.: Vibrio cholerae, C.A.: Candida albicans,
A.F.: Aspergillus fumigatus, MTCC: Microbial Type Culture Collection. A: Ampicillin, B:
Griseofulvin.
8g
8h
8i
9g
9h
Rifampicin
Isoniazid
8j
a
n.t.: not tested.

P.N. Kalaria et al. / European Journal of Medicinal Chemistry 83 (2014) 655e664
659
Table 4
phenyl group (7d) elevated the antibacterial potency against
S. typhi (9g, 9h) whereas 7a, 7b and 7c groups caused remarkable
reduction in activity.
In vitro antituberculosis activity of title compounds exhibiting higher % inhibition
against M. tuberculosis H37Rv (MICs, mg/mL).
Entry
% Inhibition
MIC,
100
62.5
25
100
0.15
0.20
mg/mL
It is interesting to point out that the compounds 9a and 9b
carrying 2-furyl group showed reasonable antituberculosis potency
towards H37RV. Electron donating eOCH3 group inhibited the
antituberculosis potency. Electronegative groups also did not pro-
mote the antitubercular activity. In case of antioxidant activity, the
compounds with electron donating phenyl group possessed supe-
rior inhibition than the compounds with electron withdrawing and
other heterocyclic groups. The compounds which showed highest
potency in all activities are recognized in Fig. 2.
3a
8k
9a
9b
91
94
96
92
98
99
Rifampicin
Isoniazid
(Table 4). Compounds 8k and 3a, 9b MIC ¼ 62.5
mg/mL and
MIC ¼ 100
mg/mL exhibited better inhibition of 94% and 91%, 92%
respectively. From the above results, it can be accomplished that,
compound 9a may prove themselves as an innovative category of
antitubercular agents in future.
5. Conclusion
The aim of the present study was to design and synthesize novel
5-imidazopyrazole incorporated 2-amino-3-cyano pyridine de-
rivatives through a facile one-pot multicomponent reaction by
green method and to test for their in vitro antimicrobial, antitu-
berculosis and antioxidant activity. The spectral data supported the
structures of all newly synthesized compounds. This synthetic
strategy allows the assembly of relatively complicated nitrogen
containing heterocyclic system as well as the introduction of
diverse aromatic and heterocyclic substitutions into position-6 of
pyridine ring. Compounds 8e, 8h, 8l, 9c, 9g and 9h showed sig-
nificant antibacterial potency and compounds 3b, 9f and 9g dis-
played promising antifungal activity. Compounds 3a, 8k, 9a and 9b
displayed moderate inhibition against antitubercular activity as
compared to standard drugs. Majority of the compounds displayed
good antioxidant activity. The present library model can be further
explored to design the new class of antimicrobial, antitubercular
and antioxidant agents under ultrasound irradiation.
4.2.3. In vitro antioxidant assay
The ferric reducing antioxidant power (FRAP) of all the title
compounds were determined. The FRAP assay of more than half of
the compounds (8ae8f, 8i, 8k, 9a and 9de9f) was found to range
from 401.22 to 497.10 mmol/100 g. This indicated that the com-
pounds are excellent antioxidant. The compounds 8g and 8h
illustrated pitiable antioxidant power, while remaining all other
compounds (3a, 3b, 8j, 8l, 9b, 9c, 9g and 9h) exhibited moderate
antioxidant activity (Table 5).
4.2.4. Structureeactivity relationship (SAR)
The outcome of biological evaluation revealed that the activity
was significantly affected by replacement of eH with lipophilic
group eCH3 at R position in imidazole ring. The antibacterial po-
tency against E. coli was found to increase by more than three times
(8e and 8f). In the same way, it also raised antibacterial activity by
three times towards Bacillus. subtilis in 8g and 8h. Decrease in
antibacterial potency by one third folds against V. cholera was
observed by the replacement of eCH3 with eH (9c and 9d). In
contrast, replacement of lipophilic group eCH3 with eH improved
antifungal activity by four times against C. albicans (9g and 9h). The
same replacement was found to decrease antifungal activity by two
times against C. albicans (9e and 9f). The antituberculosis activity
was also affected by substituting eCH3 with eH. Presence of eCH3
group as R in compounds 8b and 8h exhibited increased antitu-
bercular activity, while compounds 8a and 8g having eH showed
lower antitubercular activity. In disparity, compound 9a containing
eH and 9b containing eCH3 illustrated good antitubercular activity.
In case of antioxidant activity, majority of the compounds showed
terrific activity. The effect of substituting eH by eCH3 was found to
increase antioxidant activity also by two times i.e. 9c and 9d
whereas the presence of eH in place of eCH3 decreased antioxidant
activity by two times i.e. 8k and 8l.
The activity was also considerably affected by various hetero-
cyclic and substituted aromatic motifs present at six positions in
pyridine ring (Fig. 1). Compounds 8e and 8h exhibited strong
inhibitory effects against E. coli and B. subtilis. This tremendous
inhibition could be attributed to the strong electron negativity of
eCl and eBr atoms. The activity is observed to decrease by two
times and four times against E. coli and B. subtillis respectively (8k
and 8l) on replacement of electronegative groups with electron
donating eOCH3 group. The presence of electron donating eOCH3
group raised the antibacterial potency against V. cholerae; while
electron negative groups were observed to be responsible for
depleting the activity. 2-thienyl group (7b) enhanced antibacterial
activity towards V. cholerae (9c), whereas other three heterocyclic
groups (7a, 7c, 7d) depleted the potency. 4-(3',4'-methylenedioxy)-
6. Experimental section
6.1. Chemistry
All the reagents were obtained commercially and used without
further purification. Ultrasonication was performed in D-Compact
ultrasonic cleaner with a frequency of 50 kHz and power of 250 W.
The reaction flask was suspended at the center of ultrasonic bath so
as surface of the reactants remained slightly lower than the level of
water in the bath. All reactions were monitored by thin-layer
chromatography (TLC, on aluminium plates coated with silica
gel60F254, 0.25 mm thickness, Merck) carried on fluorescent coated
plates and detection of the components was made by exposure to
iodine vapours or UV light. Melting points were taken in melting
point apparatus mThermoCal10 (Analab Scientific Pvt. Ltd, India) and
are uncorrected. 1H Nuclear Magnetic Resonance spectra were
recorded in DMSO-d6 on a Bruker Avance 400F (MHz) spectrometer
(Bruker Scientific Corporation Ltd., Switzerland) using residual
solvent signal as an internal standard at 400 MHz. Chemical shifts
are reported in parts per million (ppm). Splitting patterns were
designated as follows: s, singlet; d, doublet; dd, doublet of doublet
and m, multiplet. Mass spectra were recorded on Shimadzu LCMS
2010 spectrometer (Shimadzu, Tokyo, Japan) at Sardar Patel Uni-
versity (PURSE programme of DST), Vallabh Vidyanagar. The IR
spectra were recorded in KBr on a PerkineElmer Spectrum GX FT-IR
Spectrophotometer (PerkineElmer, USA) and only the character-
istic peaks are reported in cmꢀ1. Elemental analysis (% C, H, N) was
carried out on Perkin Elmer 2400 CHN elemental analyzer at So-
phisticated Instrumentation Centre for Applied Research & Training
(SICART), Vallabh Vidyanagar. All compounds are within 0.4% of
the theoretical compositions. Yields are not optimized. Ampicillin,

660
P.N. Kalaria et al. / European Journal of Medicinal Chemistry 83 (2014) 655e664
Table 5
ciprofloxacin, norfloxacin, chloramphenicol, griseofulvin, nystatin,
isoniazid and rifampicin were purchased from the local market.
In vitro antioxidant activity of the compounds (3ae3b, 8ae8l, 9ae9h).
Entry
OD (593 nm)
FRAP valuea
Entry
OD (593 nm)
FRAP valuea
3a
3b
8a
8b
8c
8d
8e
8f
1.221
1.072
2.147
2.020
2.499
2.493
2.465
2.035
0.340
0.355
2.037
1.974
242.88
213.24
427.08
401.81
497.10
495.90
490.33
404.80
67.63
8k
8l
9a
9b
9c
9d
9e
9f
9g
9h
A.A.
2.012
0.899
2.294
1.997
1.033
2.123
2.017
2.179
1.665
1.935
2.501
400.22
178.82
456.32
397.24
205.48
422.30
401.22
433.44
331.20
384.91
6.2. General procedure for the synthesis of substituted 5-(1H-
imidazol/4methyl-1-yl)-3-methyl-1-phenyl-1H-pyrazole-4-
carbaldehyde (3aeb)
5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde
1
(1.1 g, 5 mmol), imidazole 2a/4-methyl imidazole 2b(7.5 mmol) and
anhydrous potassium carbonate (0.6 g, 10 mmol) in dime-
thylformamide (5 mL) were charged in a 50 mL round bottom flask
equipped with mechanical stirrer and condenser. The reaction
mixture was refluxed for 2 h and the progress of the reaction was
monitored by TLC. After the completion of reaction (as evidenced
by TLC), the reaction mixture was cooled to room temperature and
then poured into ice cold water (50 mL) with continuous stirring
8g
8h
8i
70.61
405.20
392.66
8j
A.A. ¼ Ascorbic acid, Concentration of compounds used ¼ 200 mg/mL, Concentra-
tion of standard (A.A.) ¼ 176 mg/mL.
a
A.A. mm/100 g sample.
Fig. 1. Structureeactivity relationship for antimicrobial, antituberculosis and antioxidant activity of the synthesized compounds.
Fig. 2. Most active compounds of the series.

P.N. Kalaria et al. / European Journal of Medicinal Chemistry 83 (2014) 655e664
661
followed by neutralization with 1 N HCl until pH 7. The separated
1371 (eCH3 stretching); 1H NMR
d
ppm (DMSO-d6):
d
2.13 (s, 3H,
precipitates of substituted 5-(1H-imidazol/4-methyl-1-yl)-3-
methyl-1-phenyl-1H-pyrazole-4 carbaldehyde 3aeb were filtered,
thoroughly washed with water, dried, and recrystallized from hot
ethanol.
CH3), 2.56 (s, 3H, CH3), 7.26e8.23 (m, 15H, AreH þ NH2); 13C NMR
(100 MHz, DMSO-d6) d: 12.8 (CH3), 13.4 (CH3), 88.6 (CeCN), 109.1,
115.5, 116.2, 116.9, 123.3, 124.2, 125.4, 127.9, 128.5, 128.8, 129.5,
130.2, 131.1, 131.9, 140.4, 141.3, 141.9, 145.3, 155.2, 159.2, 160.9 (21C,
AreC); ESI-MS (m/z): 432.2 (Mþ); Anal. Calcd. (%) for C26H21N7: C,
72.37; H, 4.91; N, 22.72. Found: C, 72.19; H, 5.05; N, 22.79.
6.2.1. 5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazole-4-
carbaldehyde (3a)
Yield 79%; m.p. 204 ꢁC; 1H NMR (400 MHz, DMSO-d6):
d
2.59 (s,
6.4.3. 4-(5-(1H-imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-
yl)-6-([1,1'-biphenyl]-4-yl)-2-aminonicotinonitrile (8c)
3H, CH3), 7.07e7.46 (m, 7H, AreH), 7.94 (s, 1H, imidazole), 9.74 (s,
1H, CHO); ESI-MS (m/z): ¼ 252.0 (Mþ)
m.p. 231e232 ꢁC; IR (KBr, nmax, cmꢀ1): 3420 & 3318 (asym. &
sym. stretching of eNH2), 3006 (Ar CeH), 2201 (C^N stretching),
6.2.2. 3-Methyl-5-(4-methyl-1H-imidazol-1-yl)-1-phenyl-1H-
1375 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 2.30 (s, 3H,
pyrazole-4-carbaldehyde (3b)
CH3), 7.11e8.06 (m, 20H, AreH þ NH2); 13C NMR (100 MHz, DMSO-
Yield 82%; m.p. 215 ꢁC; 1H NMR (400 MHz, DMSO-d6):
d
2.05 (s,
d6) d: 12.6 (CH3), 89.7 (CeCN), 107.4, 115.2, 116.1, 122.6, 123.5, 123.9,
3H, CH3), 2.52 (s, 3H, CH3), 7.10e7.58 (m, 7H, AreH), 7.97 (s, 1H,
124.4, 125.1, 126.2, 126.8, 127.5, 127.9, 128.0, 128.2, 128.7, 128.9,
129.5, 132.4, 137.7, 138.6, 139.0, 140.6, 141.1, 141.7, 146.9, 155.5, 160.2,
162.5 (28C, AreC); ESI-MS (m/z): 494.4 (Mþ); Anal. Calcd. (%) for
imidazole), 9.59 (s, 1H, CHO); ESI-MS (m/z): ¼ 267.0 (Mþ)
6.3. General procedure for the synthesis of substituted 4-(5-(1H-
imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2-
C31H23N7: C, 75.44; H, 4.70; N, 19.87. Found: C, 75.31; H, 4.83; N,
19.81.
aminonicotinonitrile (8ael, 9aeh) under conventional conditions
6.4.4. 6-([1,1'-Biphenyl]-4-yl)-2-amino-4-(3-methyl-5-(4-methyl-
1H-imidazol-1-yl)-1-phenyl-1H-pyrazol-4-yl) nicotinonitrile (8d)
m.p. 233e234 ꢁC; IR (KBr, nmax, cmꢀ1): 3449 & 3339 (asym. &
sym. stretching of eNH2), 3015 (Ar CeH), 2209 (C^N stretching),
The substituted 5-(1H-imidazol/4-methyl-1-yl)-3-methyl-1-
phenyl-1H-pyrazole-4-carbaldehyde 3aeb (5 mmol), malononi-
trile 4 (5 mmol), aromatic/heterocyclic methyl ketone 6aef/7aed
(5 mmol), ammonium acetate 5 (40 mmol) and absolute alcohol
(10 mL) were charged in a 50 mL round bottom flask. The reaction
mixture was refluxed for 1.5e3 h. Progress of the reaction was
monitored by the TLC. After the completion of reaction, the reaction
mixture was cooled to room temperature and stirred for 0.5 h. The
resulting solid was collected by filtration and washed well with
absolute alcohol to obtain the pure solid sample of product 8ael
and 9aeh.
1370 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 2.11 (s, 3H,
CH3), 2.50 (s, 3H, CH3), 7.09e7.72 (m, 19H, AreH þ NH2); 13C NMR
(100 MHz, DMSO-d6) d: 12.6 (CH3), 13.9 (CH3), 88.4 (CeCN), 106.3,
114.0, 115.6, 122.1, 122.9, 123.4, 125.4, 125.9, 126.4, 127.0, 127.9,
128.0, 128.2, 128.5, 129.0, 131.4, 133.6, 137.0, 139.5, 139.9, 141.0.6,
142.0, 142.7, 145.8, 153.4, 157.2, 160.2 (27C, AreC); ESI-MS (m/z):
508.5 (Mþ); Anal. Calcd. (%) for C32H25N7: C, 75.72; H, 4.96; N, 19.32.
Found: C, 75.59; H, 5.06; N, 19.43.
6.4. General procedure for the synthesis of substituted 4-(5-(1H-
imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2-
aminonicotinonitrile (8ael, 9aeh) under ultrasound irradiation
6.4.5. 4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-
yl)-2-amino-6-(4-chlorophenyl)nicotinonitrile (8e)
m.p. 254e255 ꢁC; IR (KBr, nmax, cmꢀ1); 3422 & 3336 (asym. &
sym. stretching of eNH2), 2999 (Ar CeH), 2192 (C^N stretching),
The substituted 5-(1H-imidazol/4-methyl-1-yl)-3-methyl-1-
phenyl-1H-pyrazole-4-carbaldehyde 3aeb (5 mmol), malononi-
trile 4 (5 mmol), aromatic/heterocyclic methyl ketone 6aef/7aed
(5 mmol), ammonium acetate 5 (40 mmol) and absolute alcohol
(10 mL) were charged in a 50 mL round bottom flask. The reaction
mixture was irradiated by ultrasound at room temperature for
required minutes. Progress of the reaction was monitored by the
TLC. The resulting solid was collected by filtration and washed well
with absolute alcohol to obtain the pure solid sample of product
8ael and 9aeh.
1372 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 2.20 (s, 3H,
CH3), 7.13e7.82 (m, 15H, AreH þ NH2); 13C NMR (100 MHz, DMSO-
d6) d: 12.8 (CH3), 89.5 (CeCN), 108.2, 115.4, 116.9, 117.2, 118.9, 123.4,
126.6, 127.5, 128.2, 128.9, 129.5, 129.8, 130.2, 131.2, 136.5, 139.1,
140.0, 142.7, 147.5, 155.8, 160.2, 161.4 (22C, AreC); ESI-MS (m/z):
452.8 (Mþ), 453.6 (Mþ2); Anal. Calcd. (%) for C25H18ClN7: C, 66.44;
H, 4.01; N, 21.70. Found: C, 66.29; H, 3.91; N, 21.64.
6.4.6. 2-Amino-6-(4-chlorophenyl)-4-(3-methyl-5-(4-methyl-1H-
imidazol-1-yl)-1-phenyl-1H-pyrazol-4-yl)nicotinonitrile (8f)
m.p. 260e261 ꢁC; IR (KBr, nmax, cmꢀ1); 3449 & 3354 (asym. &
sym. stretching of eNH2), 3012 (Ar CeH), 2199 (C^N stretching),
6.4.1. 4-(5-(1H-imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-
yl)-2-amino-6-phenylnicotinonitrile (8a)
1373 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 2.10 (s, 3H,
m.p. 221e222 ꢁC; IR (KBr, nmax, cmꢀ1): 3435 & 3356 (asym. &
sym. stretching of eNH2), 2951 (Ar CeH), 2189 (C^N stretching),
CH3), 2.50 (s, 3H, CH3), 6.98e7.93 (m, 14H, AreH þ NH2); 13C NMR
(100 MHz, DMSO-d6) d: 12.9 (CH3), 14.0 (CH3), 89.7 (CeCN), 107.4,
1376 (eCH3 stretching); 1H NMR
d
ppm (DMSO-d6):
d
2.11 (s, 3H,
114.1, 115.9, 116.1, 117.8, 123.5, 126.6, 127.0, 128.1, 128.5, 128.9, 129.5,
131.2, 135.4, 138.9, 140.6, 141.7, 146.4, 153.2, 158.2, 160.9 (21C,
AreC); ESI-MS (m/z): 466.7 (Mþ), 467.7 (Mþ2); Anal. Calcd. (%) for
CH3), 7.14e8.22 (m, 16H, AreH þ NH2); 13C NMR (100 MHz, DMSO-
d6) d: 13.2 (CH3), 89.7 (CeCN), 110.3, 116.5, 116.9, 117.7, 123.2, 124.2,
125.2, 127.5, 128.2, 128.4, 128.5, 128.9, 129.1, 130.2, 131.5, 139.4,
140.3, 140.8, 147.4, 156.3, 159.2, 161.2 (22C, AreC); ESI-MS (m/z):
418.2 (Mþ); Anal. Calcd. (%) for C25H19N7: C, 71.93; H, 4.59; N, 23.49.
Found: C, 71.80; H, 4.61; N, 23.66.
C26H20ClN7: C, 67.02; H, 4.33; N, 21.04. Found: C, 66.89; H, 4.23; N,
21.21.
6.4.7. 4-(5-(1H-imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-
yl)-2-amino-6-(4-bromophenyl)nicotinonitrile (8g)
6.4.2. 2-Amino-4-(3-methyl-5-(4-methyl-1H-imidazol-1-yl)-1-
phenyl-1H-pyrazol-4-yl)-6-phenylnicotinonitrile (8b)
m.p. 269e270 ꢁC; IR (KBr, nmax, cmꢀ1); 3460 & 3345 (asym. &
sym. stretching of eNH2), 3001 (Ar CeH), 2212 (C^N stretching),
m.p. 223e224 ꢁC; IR (KBr, nmax, cmꢀ1): 3459 & 3335 (asym. &
sym. stretching of eNH2), 2992 (Ar CeH), 2179 (C^N stretching),
1376 (eCH3 stretching): 1H NMR
d ppm (DMSO-d6): d 2.08 (s, 3H,
CH3), 7.27e8.24 (m, 15H, AreH þ NH2); 13C NMR (100 MHz, DMSO-

662
P.N. Kalaria et al. / European Journal of Medicinal Chemistry 83 (2014) 655e664
d6)
d
: 12.1 (CH3), 88.9 (CeCN), 107.4, 115.4, 115.9, 116.4, 117.8, 122.3,
Anal. Calcd. (%) for C27H23N7O: C, 70.27; H, 5.02; N, 21.24. Found: C,
70.42; H, 4.99; N, 21.31.
125.6, 127.5, 128.1, 128.8, 129.9, 130.0, 130.8, 132.4, 135.5, 138.9,
141.1, 142.9, 148.9, 156.7, 159.6, 162.3 (22C, AreC); ESI-MS (m/z):
497.10 (Mþ), 497.9 (Mþ2); Anal. Calcd. (%) for C25H18BrN7: C, 60.49;
H, 3.66; N, 19.75. Found: C, 60.57; H, 3.65; N, 19.65.
6.4.13. 4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-
yl)-2-amino-6-(furan-2-yl) nicotinonitrile (9a)
m.p. 211e212 ꢁC; IR (KBr, nmax, cmꢀ1); 3462 & 3330 (asym. &
sym. stretching of eNH2), 2970 (Ar CeH), 2212 (C^N stretching),
6.4.8. 2-Amino-6-(4-bromophenyl)-4-(3-methyl-5-(4-methyl-1H-
imidazol-1-yl)-1-phenyl-1H-pyrazol-4-yl)nicotinonitrile (8h)
m.p. 276e277 ꢁC; IR (KBr, nmax, cmꢀ1); 3430 & 3337 (asym. &
sym. stretching of eNH2), 2993 (Ar CeH), 2203 (C^N stretching),
1375 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 2.21 (s, 3H,
CH3), 7.47e8.19 (m, 14H, AreH þ NH2); 13C NMR (100 MHz, DMSO-
d6) d: 12.9 (CH3), 90.0 (CeCN), 99.8, 107.5, 114.3, 115.6, 116.0, 117.2,
1368 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 2.10 (s, 3H,
122.4, 125.6, 127.9, 128.3, 128.7, 128.9, 129.6, 138.4, 141.2, 142.6,
147.5, 149.7, 151.5, 162.3 (20C, AreC); ESI-MS (m/z): 408.1 (Mþ);
Anal. Calcd. (%) for C23H17N7O: C, 67.80; H, 4.21; N, 24.06. Found: C,
67.90; H, 4.31; N, 23.96.
CH3), 2.43 (s, 3H, CH3), 7.31e8.19 (m, 14H, AreH þ NH2); 13C NMR
(100 MHz, DMSO-d6) d: 13.0 (CH3), 13.9 (CH3), 89.9 (CeCN), 107.5,
113.5, 114.7, 115.1, 117.8, 123.5, 125.6, 127.2, 128.0, 128.6, 129.0, 129.9,
130.4, 134.3, 137.2, 139.5, 142.9, 145.8, 155.2, 159.2, 161.2 (21C,
AreC); ESI-MS (m/z): 511.2 (Mþ), 512.1 (Mþ2); Anal. Calcd. (%) for
C
26H20BrN7: C, 61.18; H, 3.95; N, 19.21. Found: C, 61.11; H, 4.05; N,
6.4.14. 2-Amino-6-(furan-2-yl)-4-(3-methyl-5-(4-methyl-1H-
imidazol-1-yl)-1-phenyl-1H-pyrazol-4-yl) nicotinonitrile (9b)
m.p. 218e219 ꢁC; IR (KBr, nmax, cmꢀ1); 3423 & 3349 (asym. &
sym. stretching of eNH2), 3012 (Ar CeH), 2190 (C^N stretching),
19.37.
6.4.9. 4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-
yl)-2-amino-6-(4-fluorophenyl) nicotinonitrile (8i)
1369 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 2.21 (s, 3H,
m.p. 243e244 ꢁC; IR (KBr, nmax, cmꢀ1); 3449 & 3312 (asym. &
sym. stretching of eNH2), 2989 (Ar CeH), 2189 (C^N stretching),
CH3), 2.51 (s, 3H, CH3), 7.22e7.98 (m, 13H, AreH þ NH2); 13C NMR
(100 MHz, DMSO-d6) d: 12.9 (CH3), 13.8 (CH3), 89.2 (CeCN), 99.1,
1373 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 2.09 (s, 3H,
106.1, 113.2, 114.5, 117.1, 117.9, 123.4, 126.1, 126.9, 128.5, 128.9, 129.4,
130.5, 137.1, 140.1, 143.4, 145.6, 151.9, 161.8 (19C, AreC); ESI-MS (m/
z): 422.2 (Mþ); Anal. Calcd. (%) for C24H19N7O: C, 68.40; H, 4.54; N,
23.26. Found: C, 68.42; H, 4.54; N, 23.36.
CH3), 7.08e7.82 (m, 15H, AreH þ NH2); 13C NMR (100 MHz, DMSO-
d6) d: 13.1 (CH3), 90.0 (CeCN), 109.4, 116.3, 116.9, 118.0, 118.9, 124.4,
125.6, 127.5, 128.0, 128.8, 129.9, 130.8, 130.9, 132.2, 137.9, 140.1,
141.0, 142.7, 148.5, 156.8, 161.9, 163.3 (22C, AreC); ESI-MS (m/z):
436.2 (Mþ); Anal. Calcd. (%) for C25H18FN7: C, 68.95; H, 4.17; N,
22.52. Found: C, 69.09; H, 4.29; N, 22.36.
6.4.15. 4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-
yl)-2-amino-6-(thiophen-2-yl) nicotinonitrile (9c)
m.p. 246e247 ꢁC; IR (KBr, nmax, cmꢀ1); 3441 & 3317 (asym. &
sym. stretching of eNH2), 3001 (Ar CeH), 2210 (C^N stretching),
6.4.10. 2-Amino-6-(4-fluorophenyl)-4-(3-methyl-5-(4-methyl-1H-
imidazol-1-yl)-1-phenyl-1H-pyrazol-4-yl) nicotinonitrile (8j)
m.p. 239e240 ꢁC; IR (KBr, nmax, cmꢀ1); 3451 & 3341 (asym. &
sym. stretching of eNH2), 3004 (Ar CeH), 2211 (C^N stretching),
1372 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 2.22 (s, 3H,
CH3), 7.09e8.07 (m, 14H, AreH þ NH2); 13C NMR (100 MHz, DMSO-
1373 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 2.08 (s, 3H,
d6) d: 12.5 (CH3), 88.9 (CeCN), 100.1, 106.4, 114.5, 115.3, 116.9, 117.6,
CH3), 2.47 (s, 3H, CH3), 6.93e8.06 (m, 14H, AreH þ NH2); 13C NMR
122.5, 125.9, 128.0, 128.4, 128.8, 129.3, 129.8, 138.5, 141.2, 143.5,
144.6, 148.4, 150.6, 161.9 (20C, AreC); ESI-MS (m/z): 424.5 (Mþ);
Anal. Calcd. (%) for C23H17N7S: C, 65.23; H, 4.05; N, 23.15. Found: C,
65.17; H, 3.97; N, 23.34.
(100 MHz, DMSO-d6) d: 13.2 (CH3), 14.1 (CH3), 88.9 (CeCN), 107.3,
112.5, 113.7, 114.1, 117.8, 122.9, 124.5, 126.2, 127.9, 128.5, 129.3, 130.1,
130.9, 133.1, 136.1, 138.4, 141.5, 144.8, 156.2, 159.9, 162.9 (21C,
AreC); ESI-MS (m/z): 450.4 (Mþ); Anal. Calcd. (%) for C26H20FN7: C,
69.48; H, 4.48; N, 21.81. Found: C, 69.64; H, 4.54; N, 21.74.
6.4.16. 2-Amino-4-(3-methyl-5-(4-methyl-1H-imidazol-1-yl)-1-
phenyl-1H-pyrazol-4-yl)6-(thiophen-2-yl)nicotinonitrile (9d)
m.p. 252e253 ꢁC; IR (KBr, nmax, cmꢀ1); 3456 & 3342 (asym. &
sym. stretching of eNH2), 2981 (Ar CeH), 2181 (C^N stretching),
6.4.11. 4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-
yl)-2amino-6-(4-methoxyphenyl)nicotinonitrile (8k)
m.p. 212e213 ꢁC; IR (KBr, nmax, cmꢀ1); 3423 & 3313 (asym. &
sym. stretching of eNH2), 3003 (Ar CeH), 2184 (C^N stretching),
1376 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 2.16 (s, 3H,
CH3), 2.52 (s, 3H, CH3), 6.94e7.94 (m, 13H, AreH þ NH2); 13C NMR
1375 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 2.09 (s, 3H,
(100 MHz, DMSO-d6) d: 13.1 (CH3), 13.9 (CH3), 89.3 (CeCN), 100.5,
CH3), 3.82 (s, 3H, OCH3), 7.32e8.16 (m, 15H, AreH þ NH2); 13C NMR
104.2, 112.2, 113.9, 116.1, 116.9, 123.4, 125.1, 126.8, 127.3, 129.1, 129.8,
130.4, 136.8, 140.1, 143.9,146.4, 150.2, 160.7 (19C, AreC); ESI-MS (m/
z): 438.3 (Mþ); Anal. Calcd. (%) for C24H19N7S: C, 65.88; H, 4.38; N,
22.41. Found: C, 66.02; H, 4.29; N, 22.62.
(100 MHz, DMSO-d6) d: 12.5 (CH3), 53.5 (OCH3), 89.0 (CeCN), 109.1,
111.4,112.9,115.6,116.9,122.3,126.6,127.9,128.0,128.5,128.7,128.9,
129.8, 131.2, 132.8, 136.9, 139.5, 140.7, 147.9, 156.0, 161.2, 162.4 (22C,
AreC); ESI-MS (m/z): 448.2 (Mþ); Anal. Calcd. (%) for C26H21N7O: C,
69.78; H, 4.73; N, 21.91. Found: C, 69.91; H, 4.82; N, 22.04.
6.4.17. 4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-
yl)2-amino-6-(2-oxo-2H-chromen-3-yl)nicotinonitrile (9e)
6.4.12. 2-Amino-6-(4-methoxyphenyl)-4-(3-methyl-5-(4-methyl-
1H-imidazol-1-yl)-1-phenyl-1H-pyrazol-4-yl) nicotinonitrile (8l)
m.p. 234e235 ꢁC; IR (KBr, nmax, cmꢀ1); 3447 & 3342 (asym. &
sym. stretching of eNH2), 2985 (Ar CeH), 2200 (C^N stretching),
m.p. 205e206 ꢁC; IR (KBr, nmax, cmꢀ1); 3421 & 3333 (asym. &
sym. stretching of eNH2), 3012 (Ar CeH), 2291 (C^N stretching),
1370 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 2.09 (s, 3H,
1372 (eCH3 stretching); 1H NMR
d
ppm (DMSO-d6):
d
2.04 (s, 3H,
CH3), 7.16e8.06 (m, 16H, AreH þ NH2); 13C NMR (100 MHz, DMSO-
CH3), 2.35 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 7.24e8.18 (m, 14H,
d6) d: 12.7 (CH3), 88.5 (CeCN), 104.2, 115.6, 116.0, 116.4, 116.9, 121.4,
AreH þ NH2); 13C NMR (100 MHz, DMSO-d6)
d
: 12.8 (CH3), 54.6
122.9, 124.1, 126.8, 127.5, 127.9, 128.3, 128.7, 128.9, 129.5, 130.9,
131.3, 137.9, 140.2, 141.4, 145.5, 152.6, 156.9, 161.9 (24C, AreC); ESI-
MS (m/z): 486.4 (Mþ); Anal. Calcd. (%) for C28H19N7O2: C, 69.27; H,
3.94; N, 20.20. Found: C, 69.41; H, 4.10; N, 20.03.
(OCH3), 88.9 (CeCN), 108.1, 111.4, 112.9, 114.7, 115.8, 121.3, 125.9,
126.0, 127.6, 128.1, 128.6, 129.3, 130.9, 133.4, 136.9, 139.6, 141.8,
145.6, 155.0, 162.1, 162.6 (21C, AreC); ESI-MS (m/z): 462.3 (Mþ);

P.N. Kalaria et al. / European Journal of Medicinal Chemistry 83 (2014) 655e664
663
6.4.18. 2-Amino-4-(3-methyl-5-(4-methyl-1H-imidazol-1-yl)-1-
phenyl-1H-pyrazol-4-yl)-6-(2-oxo-2H-chromen-3-yl)nicotinonitrile
(9f)
tubes showing no visible growth (same as the control tube) were
subcultured and incubated overnight at 37 ꢁC. The amount of
growth from the control tube before incubation was compared. In
this study Ampicillin was used as the standard antibacterial drugs
while Griseofulvin was used as standard antifungal drugs. The
outcomes are summarized in Table 2.
m.p. 208e210 ꢁC; IR (KBr, nmax, cmꢀ1); 3413 & 3312 (asym. &
sym. stretching of eNH2), 3005 (Ar CeH), 2199 (C^N stretching),
1373 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 2.12 (s, 3H,
CH3), 2.51 (s, 3H, CH3), 7.28e7.73 (m, 15H, AreH þ NH2); 13C NMR
(100 MHz, DMSO-d6) d: 12.9 (CH3), 88.4 (CeCN), 105.1, 114.9, 115.4,
7.2. In vitro antituberculosis assay
116.0, 116.8, 122.0, 123.3, 124.1, 125.9, 127.2, 127.8, 128.3, 128.4,
128.8, 129.0, 131.2, 132.4, 136.3, 140.5, 141.9, 145.5, 156.1, 162.8 (23C,
AreC); ESI-MS (m/z): 500.2 (Mþ); Anal. Calcd. (%) for C29H21N7O2: C,
69.73; H, 4.24; N, 19.63. Found: C, 69.83; H, 4. 25; N, 19.53.
Drug susceptibility and determination of antituberculosis ac-
tivity was carried out by adding 250 mg/mL dilution of each com-
pound to LowensteineeJensen medium and then media was
uncontaminated by inspissations method. A culture of M. tubercu-
losis H37Rv growing on LowensteineeJensen medium was har-
vested in 0.85% saline in bijou bottle. The stock solutions of the title
6.4.19. 4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-
yl)-2-amino-6-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)
nicotinonitrile (9g)
compounds were prepared in DMSO i.e. 250 mg/mL. These tubes
m.p. 229e230 ꢁC; IR (KBr, nmax, cmꢀ1); 3449 & 3329 (asym. &
sym. stretching of eNH2), 2973 (Ar CeH), 2212 (C^N stretching),
were then incubated at 37 ꢁC for 1 day followed by streaking of M.
tuberculosis H37Rv (5 ꢂ 10ꢀ4 bacilli per tube). The growth of bacilli
was seen after two weeks, three weeks and finally after four weeks
of incubation. The tubes having the compounds were compared
with control tubes where medium alone was incubated with M.
tuberculosis H37Rv. The concentration at which complete inhibition
of colonies occurred was taken as active concentration of the tested
compound. The standard drugs isoniazid and rifampicin were used
for comparison purpose. The results are summarized in Table 3.
1374 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 1.67 (s, 6H,
2CH3), 2.17 (s, 3H, CH3), 7.33e8.19 (m, 14H, AreH þ NH2); 13C NMR
(100 MHz, DMSO-d6)
d: 12.7 (CH3), 23.7, 23.8 (2C, CH3), 89.6
(CeCN), 108.1, 111.3, 115.9, 116.4, 117.2, 119.4, 122.5, 123.6, 127.3,
128.0, 128.4, 128.8, 129.0, 129.5, 130.4, 138.5, 140.4, 141.9, 142.9,
147.8, 155.8, 161.5, 162.9 (23C, AreC); ESI-MS (m/z): 490.1 (Mþ);
Anal. Calcd. (%) for C28H23N7O2: C, 68.70; H, 4.74; N, 20.03. Found: C,
68.83; H, 4.68; N, 19.93.
7.3. In vitro antioxidant assay
6.4.20. 2-Amino-6-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)-4-(3-
methyl-5-(4-methyl-1H-imidazol-1-yl)-1-phenyl-1H-pyrazol-4-yl)
nicotinonitrile (9h)
FRAP assay measure a reduction power of all the synthesized
compounds, converting ferric tripyridyl triazine (Fe (III)-TPTZ)
complex into a blue colour ferrous tripyridyl triazine (Fe (II)-TPTZ)
complex at 593 nm [45]. Fe(II)-TPTZ(2,4,6-tripyridyl-s-triazine)
reagent was prepared by mixing a 10.0 mL TPTZ solution (0.155 g
TPTZ was dissolved in 100 mL 40 mM HCl), 10 mL FeCl3$6H2O so-
lution (0.324 g FeCl3 was dissolved in 100 mL distilled water) and
100 mL acetate buffer (0.187 g sodium acetate and 1.6 mL acetic acid
dissolved in double distilled water to make 100 mL) at pH 3.6. A
mixture of 200.0 mL sample solution and 3 mL of Fe(II)TPTZ reagent
was incubated at 37 ꢁC for 25 min. The absorbance of colour
complex Fe(II)TPTZ was measured at 593 nm using ascorbic acid as
standard. The results were expressed as ascorbic equivalent (mmol/
100 g compound) summarized in Table 5.
m.p. 235e236 ꢁC; IR (KBr, nmax, cmꢀ1); 3430 & 3321 (asym. &
sym. stretching of eNH2), 3000 (Ar CeH), 2197 (C^N stretching),
1368 (eCH3 stretching); 1H NMR
d ppm (DMSO-d6): d 1.66 (s, 6H,
2CH3), 2.18 (s, 3H, CH3), 2.55 (s, 3H, CH3), 7.32e8.18 (m, 13H,
AreH þ NH2); 13C NMR (100 MHz, DMSO-d6)
d: 12.7 (CH3), 13.9
(CH3), 23.9, 24.0 (2C, CH3), 88.9 (CeCN), 107.2, 110.4, 114.8, 115.3,
116.1, 119.4, 122.8, 123.5, 126.8, 127.4, 128.3, 128.6, 128.9, 129.3,
130.4,137.1,140.7,142.0,143.5,146.9,154.7,160.6,161.7 (22C, AreC);
ESI-MS (m/z): 504.4 (Mþ); Anal. Calcd. (%) for C29H25N7O2: C, 69.17;
H, 5.00; N, 19.47. Found: C, 69.35; H, 5.13; N, 19.29.
7. Biological evaluation
7.1. In vitro antimicrobial assay
Acknowledgements
MuellereHinton broth was used as nutrient medium to grow
and dilute the compound suspension for the test bacteria. 2% DMSO
in water was used as the diluent to get the desired concentration of
compounds to test upon standard bacterial stains. Sabouraud
Dextrose broth was used for fungal nutrition. Inoculum size for test
stain was adjusted to 108 CFU mLꢀ1 by comparing the turbidity.
Serial dilutions were prepared in primary and secondary screening.
Each synthesized compound and the standard drugs were diluted
SPS and PNK acknowledge UGC, New Delhi for providing
research fellowships. The authors thank the Head, Department of
Chemistry, Sardar Patel University for providing research facilities
and SICART, Vallabh Vidyanagar for elemental analysis at conces-
sional rates. We are also thankful to DST, New Delhi for the assis-
tance in general and for the PURSE central facility for mass
spectrometry sponsored under PURSE program grant vide sanction
letter DO. No. SR/59/Z-23/2010/43. We are also thankful to Micro-
care Laboratory, Surat for antimicrobial and antituberculosis
activity.
obtaining 2000
which were found to be active in primary screening (i.e. 500, 250
and 200 g/mL concentrations) were further screened in their
second set of dilution at 100, 50, 25 and 12.5 g/mL concentration
against all microorganisms. 10 L suspensions were further inoc-
mg/mL concentration as a stock solution. The drugs
m
m
Appendix A. Supplementary data
m
ulated on appropriate media and growth was noted after 24 and
48 h. The tubes were then put overnight for incubation at 37 ꢁC. The
control tube containing no antibiotic was instantaneously sub-
cultured by spreading a loopful consistently over an area of plate of
medium fitting for the growth of the test organism. The maximum
dilution preventing appearance of turbidity after spot subculture
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.06.071.
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