Antitubercular and antimicrobial activity of nh4vo3 promoted 1,4-dihydropyridine incorporated 1,3,4-Trisubstituted pyrazole

Article abstract of DOI:10.2174/1570180813666161103145224

Background: A new series of pyrazole containing 1,4-dihydropyridine derivatives 5a-i and 6a-i were synthesized from substituted acetylated aryls and substituted phenylhydrazine by the multistep reaction. Method: The target compounds 1,4-dihydropyridine de

Full text of DOI:10.2174/1570180813666161103145224

699
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Letters in Drug Design & Discovery, 2017, 14, 699-711
RESEARCH ARTICLE
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Antitubercular and Antimicrobial Activity of NH4VO3 Promoted 1,4-
Dihydropyridine Incorporated 1,3,4-trisubstituted Pyrazole
BENTHAM
S
CIENCE
Nandam Harikrishnaa, Arun M. Isloora,*, K. Anandab, Tanya Parishc, Joazaizulfazli Jamalisd,
Hazem A. Ghabboure and Hoong-Kun Funf
aDepartment of Chemistry, Medicinal Chemistry Laboratory, National Institute of Technology Karnataka, Surathkal,
b
Mangalore 575025, India; Biological Sciences, Poornaprajna Institute of Scientific Research, Bangalore 562110,
c
India; Drug Discovery, Infectious Disease Research Institute (IDRI), 1616 Eastlake, Avenue E, Suite 400, Seattle, WA
d
98102, United States; Department of Chemistry, NMR Instrumentation, Universiti Teknologi Malaysia, 81310, UTM
e
Skudai, Johor, Malaysia; Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O.
f
Box. 2457 - Riyadh 11451, Kingdom of Saudi Arabia; X-ray Crystallography, School of Physics, Universiti Sains
Malaysia, Penang 11800, Malaysia
Abstract: Background: A new series of pyrazole containing 1,4-dihydropyridine derivatives 5a-i and
6a-i were synthesized from substituted acetylated aryls and substituted phenylhydrazine by the multi-
step reaction.
Method: The target compounds 1,4-dihydropyridine derivatives were obtained from green synthesis
of 1,3-disubstituted phenyl-1H-pyrazole-4-carbaldehydes 4a-i with ethyl acetoacetate and methyl
acetoacetate at higher temperatures in the presence of ammonium acetate and the catalytic amount of
ammonium metavanadate (NH4VO3). The role of ammonium metavanadate was increases rate of the
reaction and obtained high yields.
A R T I C L E H I S T O R Yꢀ
Received: August 05, 2016
Revised: October 28, 2016
Accepted: October 28, 2016
Result: Structures of newly synthesized 1,4-dihydropyridine moiety containing pyrazole derivatives
were confirmed by FT-IR, NMR and Mass spectral studies. The structure of compound 5b was
confirmed by S-XRD study. Further, these compounds were tested for in-vitro antitubercular and
antimicrobial studies. Compounds 5a, 5b, 5i, 6a, 6b, 6g, 6h, and 6i were found to be active against all
the bacterial microorganisms.
DOI:
10.2174/1570180813666161103145224
Conclusion: The above mentioned compounds have shown lowest MIC ranging between 3.12-12.5
µg/ml against Mycobacterium tuberculosis and MIC values ranging between 7.8- 15.6 µg/ml for
Mycobacterium smegmatis, Staphylococcus aureus and Pseudomonas aeruginosa. For the control of
life threatening diseases such as tuberculosis, these eight compounds may be strongly promising
synthetic compounds.
Keywords: 1,3,4-Trisubstituted pyrazole, 1,4-dihydropyridine, multistep reaction, ammonium metavanadate, antitubercular
study, antimicrobial studies.
1. INTRODUCTION
mainly due to its improper justification and prolonged
treatment method for tuberculosis. In the recent years, the
use of potent drugs is limited due to their adverse effects. To
overcome certain limitations, researchers have to put their
considerable efforts to minimize these adverse effects that
are directed to the discovery of high potency antimicrobial
and antitubercular agents with reduced or no systemic
adverse effects.
The development of drug resistance in microorganisms is
becoming a major limitation to the clinical use of
antimicrobial and antitubercular agents. The treatment of
antibacterial and antifungal diseases still remains an
important scientific challenge in the field of antimicrobial
therapy [1]. As per WHO reports, Tuberculosis is one of the
deadly diseases and affecting throughout the world and
causing about 1.3 million deaths every year [2, 3]. This is
Recently, multicomponent reactions have gained much
importance by organic chemists due to the shortening of the
reaction time and increased efficacy of the reaction.
Hantzsch developed a four-component reaction to synthesize
1,4-dihydropyridines in 1882 [4]. 1,4-Dihydropyridine
derivatives are playing important role in medicinal chemistry
*Address correspondence to this author at the Department of Chemistry,
Medicinal Chemistry Laboratory, National Institute of Technology
Karnataka, Surathkal, Mangalore 575025, India; Tel: +91-824-2474000;
Fax: +91-824-2474033; E-mail: isloor@yahoo.com
1875-628/17 $58.00+.00
©2017 Bentham Science Publishers

700 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 6
Harikrishna et al.
due to their wide variety of biological activities [5]. The
development of potent and effective antimicrobial agent is
most important to overcome the emerging multi-drug
resistance strains of bacteria and fungi (MRSA) [6].
of biological activities of antitubercular [21], antimicrobial
[22, 23], cardiodepressant [24] and antioxidant [25]
activities. In view of the drawbacks of presently available
drugs as well as the immediate requirement of new potent
antitubercular drugs, we are hereby reporting the preparation
of pyrazole containing 1,4-dihydropyridine derivatives with
the hope of increasing its biological activity. These newly
synthesized derivatives were screened for their antimicrobial
and antitubercular properties. Some of the commercially
available and newly reported potent compounds are
represented in Fig. (1).
The compounds with multi heterocyclic rings play an
important role in developing potent pharmaceutical ingredients.
1,4-Dihydropyridines are nitrogen containing heterocycles
with superfluous calcium channel blocking properties [7] in
the treatment of cardiovascular disorders [8]. 1,4-Dihydro-
pyridine based drugs like Nifedipine, Amlodipine, Felodipine,
Nimodipine, Nitrendipine, Nisoldipine, Nicardipine, Isradipine
[9] exhibit selectivity for cerebral vasculature. Recently,
1,4-dihydropyridine derivatives were found to possess
antihypertensive and antiarrhythmic [10], antitubercular [11],
antitumor [12], antimicrobial [13], antithrombotic [14],
antidiabetic [15] and hypnotic [16] activities.
2. RESULTS AND DISCUSSION
2.1. Chemistry
1,3-Disubstituted phenyl-1H-pyrazole-4-carbaldehydes
4a-i were obtained from suitable hydrazone derivatives 3,
which in turn was obtained by the reaction of substituted
five/six membered acetylated aryls 1 with substituted
phenylhydrazines 2. Vilsmeier-Haack reaction [26] was
performed to get 1,3-disubstituted phenyl-1H-pyrazole-4-
carbaldehydes 4a-i by the cyclization of hydrazone
derivative 3 with DMF/POCl3. The versatile Hantzsch
reaction was used to synthesize the target 1,4-dihydro-
pyridine derivatives 5a-i and 6a-i from 1,3-disubstituted
phenyl-1H-pyrazole-4-carbaldehydes 4a-i reacting with ethyl
acetoacetate or methyl acetoacetate and ammonium acetate
Pyrazole and its derivatives are very well known in
medicinal chemistry because of their broad spectrum of
therapeutic and pharmacological activities such as anticancer
[17], anti-tuberculosis [18] and antiviral [19] activities. The
pyrazole ring is the core moiety in leading drugs such as
Celecoxib, Crizotinib, Fezolamine, Sildenafil, Rimonabant.
However, most of the drugs are having adverse effects. The
effects can be gastro intestinal (GI) ulceration, depression,
increased the risk of serious adverse cardiovascular
thrombotic events [20]. The combination of these two active
dihydropyridine-pyrazole core moieties display wide range
N
O
N
Cl
H3C
OOC
H3C
COOCH3
CH3
H3COOC
H3C
COOC2H5
H3C
N
H
N
H
CH2OCH2CH2NH2
Isradipin
eCalcium antagonist
Amlodipine
Calcium antagonist
O
NO2
O
C2H5OCH2COOC
H3C
COOCH3
H3COOC
H3C
COOCH3
N
H
N
H
CH3
N
H
CH3
1,4-DHP
Aranidipine
Calcium antagonist
Oxodipine
Ca channel blocker
Cl
NO2
Cl
O
O
H3COOC
H3C
COOCH3
COOCH3
C3H7
O
O
N
H
CH3
H3C
N
H
CH3
Nifedipine
Calcium antagonist
Clevidipine
Antihypertensive
Fig. (1). Commercial drugs containing 1,4-Dihydropyridine moiety.

Antitubercular and Antimicrobial Activity of NH4VO3 Promoted
Letters in Drug Design & Discovery, 2017, Vol. 14, No. 6 701
X
X
O
Ar
N
AcOH
DMF
X
N
Ar
10% NaOH
NNH
POCl3
OHC
NHNH2 HCl
4a-i
Ar
1
2
X
3a-i
N
N
5a-i
Ar
C2H5OOC
COOC2H5
EAA
EtOH
N
H
Ar
X
N
NH4OAc
5% NH4VO3
N
X
OHC
4a-i
N
N
MAA
Ar
6a-i
MeOH
H3COOC
COOCH3
N
H
Where Ar= 4-CH3C6H4, 4-OCH3C6H4, 4-ClC6H4, 4-BrC6H4, 3-BrC6H4, Biphenyl, 2-Thiophene,
X= H, Cl
Scheme 1. Synthetic route for the pyrazole containing 1,4-dihydropyridine derivatives.
1
as FT-IR, 13C NMR, H NMR and mass spectral analysis.
FT-IR data of the compound 5a showed a sharp peak at 3329
cm-1 is corresponds to the -NH group on the 1,4-
dihydropyridine ring. An absorption peak at 3102 cm-1 was
because of the -C-H aromatic stretching and absorption peak
at 1688 cm-1 is due to the -C=O stretching of the ester group,
which confirmed the synthesized compound 5a. The proton
NMR spectrum of 5a showed a triplet of six protons at δ
1.05-1.09 which was attributed to the CH3 protons of the
ester group. A singlet of six protons attributed at δ 2.23
which is due to the CH3 group on 1,4-dihydropyridine. The
characteristic peak of the 1,4-dihydropyridine-4H proton was
obtained at δ 5.28 as a singlet. The -NH proton of 1,4-
in alcohol media in the presence of catalytic amount of
ammonium metavanadate. Ammonium metavanadate is the
most common, non-expensive and an environmentally
friendly inorganic acid in pharmaceutical industry. It is a
water soluble, mild and efficient catalyst. Ammonium
metavanadate contains different +5, +4, +3 and +2 oxidative
states that are dependent on pH of the reaction mass. It is
used to form very stable [VO2(H2O)4]+ complex [27]. The
methodology used for this catalyst is an effective formation
of vanadium complex easily with the aldehyde Moreover, it
is a very good dehydrating agent in the reaction mechanism
[28]. Normally Hantzsch dihydropyridine takes more than 3 h
for reaction conversion and also the yield of final compounds
is not more than 70% [21]. The use of ammonium
metavanadate helps to increase the reaction rate and the yield
achieved was 95%. The reaction pathway and mechanism
have been described in Scheme 1. All newly synthesized
compounds were confirmed by different spectral techniques
such as, FT-IR, NMR, mass spectral study and C, H, N
elemental analyses.
1
dihydropyridine attributed at δ 5.44. The detailed H NMR
resonance data was presented in the experimental section.
The mass spectral studies of the compound 5a showed the
presence of the molecular ion peak at m/z = 519.9 (M+1).
This data confirmed the molecular formula of the compound
as C29H30ClN3O4. The physical data of the target 1,4-
dihydropyridine derivatives are presented in Table 2.
The physical data of the synthesized 1,3-disubstituted
phenyl-1H-pyrazole-4-carbaldehydes 4a-i are presented in
Table 1.
2.2. X-ray Diffraction
To understand the molecular interaction, single crystal X-
ray diffraction study was performed for compound 5b and
the same has been shown in Fig. (2) (CCDC No. 1031058). A
Formation of 1,4-dihydropyridine derivatives 5a-i and
6a-i was confirmed by various spectroscopic techniques such

702 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 6
Harikrishna et al.
Table 1. Physical properties of the synthesized compounds 4a-i.
Syn. Comp.
Ar
X
M. Wt
M. F
M. p in °C
Yield in %
4a
4b
4c
4d
4e
4f
4-CH3C6H4
4-OCH3C6H4
4-ClC6H4
Cl
Cl
Cl
Cl
H
296.5
312.75
317.17
361.62
327.18
361.62
358.82
254.31
288.75
C17H13ClN2O
C17H13ClN2O2
C16H10Cl2N2O
C16H10BrClN2O
C16H11BrN2O
C16H10BrClN2O
C22H15ClN2O
C14H10N2OS
133-134
142-144
177-179
156-157
93-94
90
89
85
79
65
72
76
81
82
4-BrC6H4
3-BrC6H4
3-BrC6H4
Cl
Cl
H
147-148
207-209
96-97
4g
4h
4i
Biphenyl
2-Thiophene
2-Thiophene
Cl
C14H9ClN2OS
140-141
Table 2. Physical properties of the target compounds 5a-i & 6a-i.
Comp.
Ar
X
M. wt
M.F
M. p (°C)
Yield (%)
5a
5b
5c
5d
5e
5f
4-CH3C6H4
4-OCH3C6H4
4-ClC6H4
Cl
Cl
Cl
Cl
H
520.02
536.02
540.44
584.88
550.44
584.88
582.08
477.57
512.02
491.96
507.96
512.38
556.83
522.39
556.83
554.04
449.52
483.96
C29H30ClN3O4
237-238
240-241
228-230
238-240
198-199
147-148
172-174
195-196
140-141
243-244
235-236
234-235
248-248
232-232
188-189
200-201
227-228
215-216
92
95
83
83
81
87
89
84
86
89
88
81
80
80
82
88
86
86
C29H30ClN3O5
C28H27Cl2N3O4
C28H27BrClN3O4
C28H28BrN3O4
C28H27BrClN3O4
C34H32ClN3O4
C26H27N3O4S
4-BrC6H4
3-BrC6H4
3-BrC6H4
Cl
Cl
H
5g
5h
5i
Biphenyl
2-Thiophene
2-Thiophene
4-CH3C6H4
4-OCH3C6H4
4-ClC6H4
Cl
Cl
Cl
Cl
Cl
H
C26H26ClN3O4S
C27H26ClN3O4
C27H26ClN3O4
C26H23Cl2N3O4
C26H23BrClN3O4
C26H24BrN3O4
C26H23BrClN3O4
C32H28ClN3O4
C24H23N3O4S
6a
6b
6c
6d
6e
6f
4-BrC6H4
3-BrC6H4
3-BrC6H4
Cl
Cl
H
6g
6h
6i
Biphenyl
2-Thiophene
2-Thiophene
Cl
C24H22ClN3O4S
single crystal was obtained by slow evaporation of solution
from ethyl acetate. A good, shiny crystal was obtained with a
suitable size for XRD analysis. The three dimensional
structure of compound 5b (CCDC No. 1031058) was
determined by X-ray crystallography. Collected the required
data on a D8 Venture area diffractometer equipped with
graphite monochromatic MoK\α radiation (λ = 0.71073 Å) at
100 K. Data reduction and cell refinement were done by
Bruker SAINT (Bruker, 2009); program used to solve
structure and refine structure is SHELXS-97. Correction of
multi-scan absorption was applied by use of SADABS
software [29]. The details of data collection and structure
refinement are listed in Table 3. All bond angles and bond
lengths are in normal ranges [30]. The molecular structure of
compound 5b is composed of the 1,4-dihydropyridine ring
(C16-C18/N3/C19/C20) and pyrazole ring (C7-C9/N1/N2)
which are nearly perpendicular. The planes between two
rings make angles 87.27 (2) ° in compound 5b.

Antitubercular and Antimicrobial Activity of NH4VO3 Promoted
Letters in Drug Design & Discovery, 2017, Vol. 14, No. 6 703
of inhibition against Mycobacterium smegmatis. Among all
the compounds 6h, 6i have shown the highest inhibition
against Mycobacterium smegmatis, which is because of the
existence of a thiophene ring at the 3rd position on the
pyrazole skeleton. In compound 6b, the presence of 4-
methoxyphenyl substituent at the 3rd position on pyrazole has
resulted in increased activity. The compounds 5a, 5b, 5h, 5i
and 6a shown the moderate inhibition zone.
C4
CI1
C29
C3
C6
C27
C5
N 2
C13
C28
C14
C2
05
C15
C10
N1
C12
C1
C11
03
C9
04
C26
C20
C7
C8
C25
C16
C19
02
C23
The inhibition of Gram positive bacteria Staphylococcus
aureus was also shown highest MIC value for 5a, 5b, 5h, 5i,
6a, 6b, 6h and 6i compounds (around 35-40 mm). However,
it was slightly lesser than Mycobacterium smegmatis. The
compounds 6h, 6i have shown reasonably good inhibition
because of a thiophene ring present at the 3rd position of the
pyrazole skeleton. The compounds 5b, 6b shown better
inhibition due to electron donating system present at the 3rd
position of the pyrazole ring. Antifungal activity against
Candida albicans was at the range of 25-30 mm diameter
zone of inhibition for these eight 5a, 5b, 5h, 5i, 6a, 6b, 6h
and 6i compounds. Among all the compounds, the presence
of methyl ester on 1,4-dihydropyridine showed the better
inhibition when compared with 1,4-dihydropyridine contains
ethyl ester group against all bacteria and fungi. The zone of
inhibition was presented in Table 4.
N3
C17
C18
C22
C21
C24
01
Fig. (2). ORTEP diagram of compound 5b.
Table 3. Details of data collection and structure refinement of
compound 5b.
Compound
5b
Molecular formula
Molecular weight
Temperature (K)
Wavelength (A)
Crystal system, Space group
Crystal
C29H29ClN3O5
535.00
100
Further, Minimum Inhibitory Concentration (MIC) was
determined for three bacteria Mycobacterium smegmatis, a
Gram negative Pseudomonas aeruginosa, a Gram positive
Staphylococcus aureus, and a pathogenic fungi Candida
albicans were presented in Table 5. Out of 18 compounds,
5a, 5b, 5h, 5i, 6a, 6b, 6h and 6i have shown excellent to
moderate MIC values in the range of 3.12 to 31.25 µg/mL
for all the tested bacterial and fungal strains. Compounds 6a,
6b, 6h and 6i showed best MIC value of 7.8 µg/mL against
Gram positive Staphylococcus aureus bacteria. Structure-
activity relationship of 6a and 6b derivatives can be
explained due to the presence of electron donating (4-
methylphenyl and 4-methoxyphenyl) group present at 3H-
pyrazole and also these two derivatives were active against
Gram negative Pseudomonas aeruginosa. Compounds 6h
and 6i showed least MIC due to thiophene group present at
3H-pyrazole has enhanced the activity. Some of the
compounds showed moderate activity with the MIC value of
15.6 µg/mL against Mycobacterium smegmatis. In case of
fungal activity, compounds (5a, 5b, 5h, 5i, 6a, 6b, 6h and 6i)
with electron donating and thiophene groups present at 3rd
position of pyrazole showed significant activity when
compared to other compounds with MIC value of 31.25
µg/mL. Antibacterial standard Ciprofloxacin and antifungal
standard Fluconazole was used as standards for comparison.
Mo Kα radiation, λ = 0.71073 Å
Monoclinic, Cc
Colorless, needle
15.963 (3)
a (Å)
b (Å)
23.594 (4)
c (Å)
7.7722 (12)
β (°)
114.956 (5)°
2653.9 (8)
Volume (Å3)
Z, Dcalc (Mg m−3)
4, 1.339
F (000)
1124
Crystal size (mm)
θ Range for data (°)
Reflections collected/unique
parameters
0.84 × 0.37 × 0.21
2.8 – 30.6
35973/7097 [Rint= 0.103]
348
Goodness-of-fit on F2
1.01
Final R indices [I > 2s(I)]
R1 = 0.0475, wR2 = 0.1052
The MIC of the newly synthesized compounds against
Mycobacterium tuberculosis H37Rv strain has been represented
in Fig. (3). All the compounds were tested against pathogenic
strain Mycobacterium tuberculosis H37Rv by serial dilution
method at various concentrations ranging from 100 to 0.78
µg/mL. Anti-tubercular standard Ethambutol (EMB), Strepto-
mycin (STP) and Pyrazinamide (PZA) were used as positive
control for the comparison. Out of 18 synthesized compounds,
8 compounds have showed the MIC ranging between 12.5
µg/mL to 3.12 µg/mL. Among all synthesized derivatives,
compounds 6a, 6b, and 6i showed lowest MIC value of
2.3. Biological Study Results
The preliminary results of in-vitro antimicrobial activities
of newly prepared compounds 5a-i and 6a-i were determined by
well plate diffusion method against Mycobacterium smegmatis
(MTCC 994), Staphylococcus aureus (MTCC3160) and
Candida albicans (MTCC 7253). Among the 18 target
compounds, 5a, 5b, 5h, 5i, 6a, 6b, 6h and 6i showed
significant antibacterial activity with more than 40 mm zone

704 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 6
Harikrishna et al.
Table 4. Antimicrobial screening of the synthesized compounds (5a-i and 6a-i) by zone inhibition method.
Zone of Inhibition in mm; Mean SD
Synthesized
Molecules
M. smegmatis
S. aureus
C. albicans
50 µL
12.5 µL
50 µL
12.5 µL
50 µL
12.5 µL
5a
5b
43.67 0.58
43.00 1.00
28.67 1.15
26.67 0.58
28.67 0.58
25.67 0.58
23.33 0.58
45.00 1.00
44.67 2.31
46.33 1.53
54.00 1.00
17.67 0.58
20.00 1.00
22.33 0.58
21.00 1.00
22.67 0.58
56.00 2.00
53.00 1.00
--
40.33 0.58
39.67 1.53
26.00 1.00
22.67 0.58
26.67 0.58
22.00 1.00
17.67 0.58
41.67 0.58
41.33 0.58
44.00 1.00
43.67 0.58
14.00 1.00
15.00 1.00
19.67 0.58
16.00 0.00
17.00 0.00
49.67 0.58
48.67 1.15
--
34.67 1.15
39.33 1.15
25.67 1.53
29.33 0.58
28.67 0.58
26.67 0.58
23.67 0.58
36.67 0.58
34.67 0.58
39.00 1.00
39.33 0.58
18.67 0.58
20.33 1.15
24.33 0.58
22.33 0.58
22.33 0.58
41.67 0.58
39.67 0.58
--
29.67 1.53
33.00 1.00
23.33 0.58
21.33 0.58
25.67 0.58
23.00 1.00
18.33 0.58
31.33 1.15
28.67 0.58
27.00 1.00
33.33 1.15
12.33 0.58
13.33 0.58
17.33 0.58
13.33 1.15
16.33 0.58
28.33 0.58
30.33 0.58
--
27.00 1.00
28.00 1.00
11.67 0.58
19.33 0.58
11.83 0.29
10.50 0.50
10.67 0.58
28.00 1.00
28.67 0.58
29.33 0.58
31.67 0.58
10.67 0.58
11.67 0.58
11.33 0.58
9.67 0.29
11.67 0.58
27.00 0.29
29.67 0.58
--
24.33 0.58
25.67 0.58
10.33 0.58
10.67 0.58
10.67 0.58
7.33 0.58
8.50 0.50
24.33 0.58
25.67 0.58
27.00 1.00
27.33 1.15
7.33 0.58
7.67 0.58
7.33 0.29
7.00 0.00
9.00 0.00
26.33 0.58
26.67 0.58
--
5c
5d
5e
5f
5g
5h
5i
6a
6b
6c
6d
6e
6f
6g
6h
6i
Control
INN
FLZ
46.67 0.58
--
41.33 0.58
--
33.33 0.58
--
29.33 0.58
--
--
--
30.27 1.55
25.67 0.58
INN; Ciprofloxacin (antibacterial standard); FLZ; Fluconazole (antifungal standard); --: inhibition was not detected; Control; DMSO
Table 5. Antimicrobial screening studies of synthesized molecules 5a-i and 6a-i (in MIC).
Minimum Inhibition Concentration in µg/mL
Synthesized Molecules
S. aureus
P. aeruginosa
M. smegmatis
C. albicans
5a
5b
5c
5d
5e
5f
31.25
15.6
62.5
62.5
62.5
62.5
125
15.6
15.6
62.5
62.5
62.5
62.5
62.5
15.6
15.6
15.6
62.5
62.5
62.5
62.5
62.5
15.6
31.25
31.25
62.5
62.5
62.5
62.5
5g
5h
62.5
31.25
31.25
(Table 5) contd….

Antitubercular and Antimicrobial Activity of NH4VO3 Promoted
Letters in Drug Design & Discovery, 2017, Vol. 14, No. 6 705
Minimum Inhibition Concentration in µg/mL
Synthesized Molecules
S. aureus
P. aeruginosa
M. smegmatis
C. albicans
5i
6a
15.6
7.8
15.6
7.8
15.6
31.25
31.25
62.5
62.5
62.5
62.5
62.5
31.25
31.25
3.12
--
31.25
31.25
31.25
62.5
62.5
62.5
62.5
62.5
31.25
31.25
--
6b
7.8
7.8
6c
62.5
62.5
62.5
62.5
62.5
7.8
62.5
62.5
62.5
62.5
31.25
15.6
15.6
3.12
--
6d
6e
6f
6g
6h
6i
7.8
INN
FLZ
Control
3.12
--
3.12
--
--
--
--
INN; Ciprofloxacin (antibacterial standard); FLZ; Fluconazole (antifungal standard); --: inhibition was not detected; Control; DMSO.
Fig. (3). MIC value against Mycobacterium tuberculosis H37Rv strain of synthesized compounds 5a-i and 6a-i.
3.12 µg/mL and it was equipotent with tested standard anti-
TB drug Ethambutol. The enhanced activity of compound 6a,
6b were due to electron donating group (4-methylphenyl and 4-
methoxyphenyl) at 3H-pyrazole core moiety and the presence
of methyl ester at the 3rd and 5th position of 1,4-dihydro-
pyridine. In a compound 6i, the increased activity was
because of thiophene group at 3H-pyrazole skeleton and methyl
ester at the 3rd and 5th positions of 1,4-dihydropyridine skeleton.

706 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 6
Harikrishna et al.
The second lowest MIC value 6.25 µg/mL obtained for
compounds 5i and 6i which is due to the presence of
thiophene group at 3H-pyrazole with ethyl ester or methyl
ester at third and fifth position on 1,4-dihydropyridine. Other
compounds 5a, 5b, and 6g showed moderate activity with
MIC value 12.5 µg/mL against Mycobacterium tuberculosis.
CCD detector. Elemental analysis was recorded on a Flash
EA 1112 CHNS-O analyzer. In-process reaction monitoring
was checked by TLC on Merck F254 readymade plates.
4.2. General Method for the Preparation of 1,3-
disubstituted phenyl-1H-pyrazole-4-carbaldehydes (4a-i)
Some of the earlier publications were studied the
tuberculosis and antimicrobial activity of dihydropyridine
containing pyrazole derivatives [21-23] with similar results of
present study. In the present study, 4-chlorophenyl substitution
on pyrazole core moiety may be plays a key role to enhance
the antibacterial and antitubercular activity.
A suspension of acetophenone (12.0 g, 0.1 mol) (1a) and
4-chlorophenylhydrazine hydrochloride (18.0 g, 0.1 mol)
(2a) in acetic acid (60 ml) was stirred at R.T for 1 h. The
hydrazone intermediate (3a) was filtered and cyclization
followed by formylation to give 1-(4-chlorophenyl)-3-
phenyl-1H-pyrazole-4-carbaldehyde 4a. Same procedure has
been used to synthesize compounds 4b-i. Yield: 65-90%.
3. CONCLUSION
4.2.1. 3-(3-Bromophenyl)-1-(4-chlorophenyl)-1H-pyrazole-
Catalytically promoted 1,4-dihydropyridine bearing pyrazole
derivatives 5a-i and 6a-i were synthesized, characterized by
FT-IR, proton NMR, 13C NMR and mass spectral study. All
target compounds were tested for their in-vitro antibacterial
and antifungal activities against Mycobacterium smegmatis,
Pseudomonas aeruginosa, Staphylococcus aureus, and a
fungi Candida albicans. Antitubercular activity of compounds
was tested against tuberculosis causing Mycobacterium
tuberculosis H37Rv. Among the investigated samples, 5a, 5b,
5i, 6a, 6b, 6h and 6i have showed excellent to moderate
(MIC value between 3.12-31.25 µg/mL) antibacterial, antifungal
and antitubercular activity. The activity of the compounds
5h, 5i, 6h and 6i against all tested microorganisms were due
to thiophene substitution at 3H-pyrazole. The enhanced
activity of compounds 5a, 5b, 6a and 6b is because of the
existence of electron donating group at 3H-pyrazole.
4-carbaldehyde (4f)
IR (KBr vmax cm-1): 3129 (CH-aromatic str.), 1684 (C=O
1
str.), 1494 (C=C str.), 1217 (C-O str.); H NMR (500 MHz,
CDCl3, ppm): δ 7.38 (t, J = 7.9 Hz, 1H, ArH), 7.50 (d, J =
8.9 Hz, 2H, ArH), 7.60 (d, J = 8.2 Hz, 1H, ArH), 7.74 (d, J =
8.9 Hz, 2H, ArH), 7.77 (d, J = 7.8 Hz, 1H, ArH), 8.02 (s, 1H,
ArH), 8.50 (s, 1H, pyrazole-5H), 10.04 (s, 1H, -CHO); 13C
NMR (100 MHz, CDCl3): δ 184.3, 153.0, 137.4, 133.9,
133.1, 132.4, 131.7, 131.6, 130.2, 129.9, 127.6, 122.8, 122.8,
120.9; MS: m/z = 361.00 (M+11), Mass Calcd. for
C16H10BrClN2O.
4.2.2. 3-Biphenyl-4-yl-1-(4-chlorophenyl)-1H-pyrazole-4-
carbaldehyde (4g)
1H NMR (500 MHz, CDCl3, ppm): δ 7.39 (t, J = 8.7 Hz,
1H, ArH), 7.48 (m, 4H, ArH), 7.65 (d, J = 7.2 Hz, 2H, ArH),
7.75 (m, 4H, ArH), 7.90 (d, J = 8.1 Hz, 1H, ArH), 8.52 (s,
1H, pyrazole-5H), 10.10 (s, 1H, -CHO); 13C NMR (100
MHz, CDCl3): δ 185.0, 154.6, 142.4, 140.5, 133.8, 131.3,
130.2, 130.0, 129.5, 129.0, 127.9, 127.6, 127.3, 123.0, 121.0
Mass Calcd. for C22H15ClN2O.
The relationship between the biological activity and
structure of the synthesized compounds can be well
explained based on SAR studies. Probably in this case the
nature of the heterocyclic ring is not only the reason for
activity. Moreover, the presence of different substituents
causes a certain change of activity. One can conclude that,
by merging of heterocyclic rings namely pyrazole and
pyridine with different substitutions has increased the
antimicrobial activity and hence they could be ideally suited
for further modifications to identify potent antimicrobial and
antitubercular compounds.
4.2.3. 1-Phenyl-3-thiophen-2-yl-1H-pyrazole-4-carbaldehyde
(4h)
1H NMR (500 MHz, CDCl3, ppm): δ 7.15 (t, J = 4.4 Hz,
1H, ArH), 7.38 (t, J = 7.4 Hz, 1H, ArH), 7.42 (d, J = 5.0 Hz,
1H, ArH), 7.49 (t, J = 7.9 Hz, 2H, ArH), 7.76 (d, J = 8.1 Hz,
2H, ArH), 7.89 (d, J = 3.5 Hz, 1H, ArH), 8.47 (s, 1H,
pyrazole-5H), 10.13 (s, 1H, -CHO); 13C NMR (100 MHz,
CDCl3): δ 183.9, 148.1, 138.7, 133.4, 132.4, 129.6, 128.6,
128.0, 127.7, 127.2, 122.0, 119.6.
4. EXPERIMENTAL PART
4.1. Instrumental Analysis
All the required chemicals were procured from Sigma-
Aldrich, Merck and Spectrochem-India. The infrared spectra
were recorded using Shimadzu FT-IR (4000-400 cm-1)
spectrophotometer by KBr pellet method. Melting points
were checked by open capillary tube method and were
uncorrected. Bruker (300, 400, 500 MHz) spectrometer was
used to record 1H NMR and 13C NMR spectra (CDCl3,
DMSO-d6) using Tetramethylsilane as the internal standard
and chemical shift values were shown in δ (ppm) scales.
LC-MS Applied Biosystems (MDS SCIEX-API 4000)
spectrometer was used to detect mass spectrum. Single
crystal XRD analysis for compound 5b was recorded on
Bruker APEX-II diffractometer equipped with dual system
4.3. General Method for the Preparation of the
Compounds (5a-i)
A reaction mixture consisting of compound 4a (1.48 g,
0.005 mol), ethylacetoacetate (1.27 g, 0.01 mol), ammonium
metavanadate (0.075 g, 5% w/w) and ammonium acetate
(0.39 g, 0.005 mol) in ethyl alcohol (10 ml) were mixed in a
RB flask. The reaction content was refluxed for 1 h. The
completion of the chemical reaction was confirmed by TLC
[hexane: ethylacetate (9:1)]; and the mass was quenched
into DM water and stirred to get the solid. Obtained solid
was filtered, washed with pre-chilled ethyl alcohol. The
compound was dried in vacuum oven and used for further

Antitubercular and Antimicrobial Activity of NH4VO3 Promoted
Letters in Drug Design & Discovery, 2017, Vol. 14, No. 6 707
1
analysis. The same procedure has been used to synthesize
compounds 5b-i. The structural elucidation of the synthesized
compounds 5a-i were presented below.
(C=C str.), 1209 (C-O str.); H NMR (300 MHz, CDCl3,
ppm): δ 1.07 (t, J = 7.05 Hz, 6H, CH3), 2.26 (s, 6H, CH3),
3.79 (m, 2H, CH2), 4.02 (m, 2H, CH2), 5.26 (s, 1H,
dihydropyridine-4H), 5.55 (s, 1H, NH), 7.35 (d, J = 8.7 Hz,
2H, ArH), 7.54 (d, J = 8.4 Hz, 2H, ArH), 7.59 (d, J = 8.7
Hz, 2H, ArH), 7.70 (s, 1H, pyrazole-5H), 7.77 (d, J = 8.4
Hz, 2H, ArH); 13C NMR (100 MHz, CDCl3): δ 167.7, 145.3,
143.4, 140.1, 136.4, 129.7, 129.4, 127.9, 127.3, 126.5, 126.3,
125.0, 119.0, 105.1, 60.0, 30.0, 19.8, 14.4; MS: m/z = 583.8
(M+1), Anal. Calcd. for C28H27BrClN3O4; C, 57.50; H, 4.65;
N, 7.18; found: C, 57.51; H, 4.66; N, 7.21.
4.3.1. 4-[1-(4-Chlorophenyl)-3-p-tolyl-1H-pyrazol-4-yl]-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylicacid
diethylester (5a)
IR (KBr vmax cm-1): 3329 (N-H str.), 3102 (CH-aromatic
str.), 2980 (C-H str.), 1688 (C=O str.), 1645 (C=N str.), 1535
1
(C=C str.), 1209 (C-O str.); H NMR (300 MHz, CDCl3,
ppm): δ 1.07 (t, J = 7.05 Hz, 6H, CH3), 2.23 (s, 6H, CH3),
2.39 (s, 3H, CH3), 3.77 (m, 2H, CH2), 4.01 (m, 2H, CH2),
5.28 (s, 1H, dihydropyridine-4H), 5.44 (s, 1H, NH), 7.21 (d,
J = 7.8 Hz, 2H, ArH), 7.37 (d, J = 9.0 Hz, 2H, ArH), 7.60
(d, J = 8.7 Hz, 2H, ArH), 7.69 (s, 1H, pyrazole-5H), 7.72 (d,
J= 9.3 Hz, 2H, ArH); 13C NMR (100 MHz, CDCl3): δ 167.5,
151.6, 143.3, 138.7, 137.2, 131.7, 131.2, 129.2, 129.0, 128.8,
128.6, 126.9, 119.8, 104.4, 59.7, 29.7, 21.3, 19.5, 14.3; MS:
m/z = 519.9 (M+11), Anal. Calcd. for C29H30ClN3O4; C,
66.98; H, 5.81; N, 8.08; found: C, 67.01; H, 5.80; N, 8.09.
4.3.5. 4-[3-(3-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl]-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylicacid
diethylester (5e)
IR (KBr vmax cm-1): 3298 (N-H str.), 3115 (CH-aromatic
str.), 2969(C-H str.), 1678 (C=O str.), 1597 (C=N str.), 1523
1
(C=C str.), 1223 (C-O str.); H NMR (400 MHz, CDCl3,
ppm): δ 1.08 (t, J = 6.9 Hz, 6H, CH3), 2.24 (s, 6H, CH3),
3.80 (m, 2H, CH2), 4.05 (m, 2H, CH2), 5.26 (s, 1H,
dihydropyridine-4H), 5.52 (s, 1H, NH), 7.25 (t, J = 7.2 Hz,
2H, ArH), 7.33 (d, J = 7.8 Hz, 2H, ArH), 7.56 (d, J = 7.5
Hz, 1H, ArH), 7.61 (d, J = 7.8 Hz, 2H, ArH), 7.70 (s, 1H,
pyrazole-5H), 7.78 (d, J = 7.0 Hz, 1H, ArH), 7.99 (d, 1H,
ArH); MS: m/z = 550.3 (M+1).
4.3.2. 4-[1-(4-Chlorophenyl)-3-(4-methoxyphenyl)-1H-
pyrazol-4-yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylicacid diethylester (5b)
IR (KBr vmax cm-1): 3329 (N-H str.), 3084 (CH-aromatic
str.), 2980 (C-H str.), 1688 (C=O str.), 1643 (C=N str.), 1528
4.3.6. 4-[3-(3-Bromophenyl)-1-(4-chlorophenyl)-1H-pyrazol-
4-yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylicacid
diethylester (5f)
1
(C=C str.), 1207 (C-O str.); H NMR (300 MHz, CDCl3,
ppm): δ 1.08 (t, J = 7.05 Hz, 6H, CH3 on ester), 2.24 (s, 6H,
CH3), 3.80 (m, 2H, CH2), 3.85 (s, 3H, OCH3), 4.02 (m, 2H,
CH2), 5.26 (s, 1H, dihydropyridine-4H), 5.44 (s, 1H, NH),
6.94 (d, J = 8.7 Hz, 2H, ArH), 7.34 (d, J = 9.0 Hz, 2H,
ArH), 7.60 (d, J = 9.0 Hz, 2H, ArH), 7.69 (s, 1H, pyrazole-
5H), 7.74 (d, J = 8.7 Hz, 2H, ArH); 13C NMR (100 MHz,
CDCl3): δ 167.5, 159.3, 151.3, 143.2, 138.7, 131.1, 130.1,
129.3, 128.9, 127.3, 126.8, 119.8, 113.4, 104.4, 59.8, 55.4,
29.8, 19.6, 14.3; MS: m/z= 535.9 (M+1), Anal. Calcd. for
C29H30ClN3O5; C, 64.98; H, 5.64; N, 7.84; found: C, 65.01;
H, 5.64; N, 7.84.
IR (KBr vmax cm-1): 3306 (N-H str.), 3100 (CH-aromatic
str.), 2948 (C-H str.), 1698 (C=O str.), 1644 (C=N str.), 1495
1
(C=C str.), 1213 (C-O str.), 725 (C-Clstr.); H NMR (400
MHz, CDCl3, ppm): δ 1.10 (t, J = 6.6 Hz, 6H, CH3), 2.24 (s,
6H, CH3), 3.84 (m, 2H, CH2), 4.05 (m, 2H, CH2), 5.26 (s,
1H, dihydropyridine-4H), 5.50 (s, 1H, NH), 7.29 (t, J = 6.6
Hz, 1H,ArH), 7.37 (d, J = 8.4 Hz, 2H, ArH), 7.48 (d, J = 7.6
Hz, 1H, ArH), 7.60 (d, J = 7.6 Hz, 2H, ArH), 7.72 (s, 1H,
pyrazole-5H), 7.76 (d, J = 7.2 Hz, 1H, ArH), 7.99 (s, 1H,
ArH); MS: m/z = 584.2 (M+1), Anal. Calcd. for C28H27
BrClN3O4; C, 57.50; H, 4.65; N, 7.18; found: C, 57.53; H,
4.70; N, 7.21.
4.3.3.
4-[1,3-Bis(4-chlorophenyl)-1H-pyrazol-4-yl]-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylicacid
diethylester (5c)
IR (KBr vmax cm-1): 3323 (N-H str.), 3100 (CH-aromatic
4.3.7. 4-(3-Biphenyl-4-yl)-1-phenyl-1H-pyrazol-4-yl)-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylicacid
diethylester (5g)
str.), 2980 (C-H str.), 1690 (C=O str.), 1643 (C=N str.), 1533
1
(C=C str.), 1209 (C-O str.); H NMR (300 MHz, CDCl3,
IR (KBr vmax cm-1): 3362 (N-H str.), 3065 (CH-aromatic
ppm): δ 1.07 (t, J = 7.2 Hz, 6H, CH3), 2.26 (s, 6H, CH3),
3.79 (m, 2H, CH2), 4.02 (m, 2H, CH2), 5.26 (s, 1H,
dihydropyridine-4H), 5.52 (s, 1H, NH), 7.39 (m, 4H, ArH),
7.59 (d, J = 8.7 Hz, 2H, ArH), 7.70 (s, 1H, pyrazole-5H),
7.83 (d, J = 8.4 Hz, 2H, ArH); 13C NMR (100 MHz, CDCl3):
δ 167.4, 150.1, 143.3, 138.5, 133.6, 133.1, 131.5, 130.2, 129.3,
129.3, 128.1, 127.2, 119.9, 104.5, 59.8, 29.7, 19.6, 14.3; MS:
m/z = 539.8 (M+1), Anal. Calcd. for C28H27Cl2N3O4; C,
62.23; H, 5.04; N, 7.78; found: C, 62.24; H, 5.05; N, 7.80.
str.), 2978 (C-H str.), 1688 (C=O str.), 1651 (C=N str.), 1530
1
(C=C str.), 1206 (C-O str.); H NMR (400 MHz, CDCl3,
ppm): δ 1.07 (t, J = 6.8 Hz, 6H, CH3), 2.25 (s, 6H, CH3),
3.79 (m, 2H, CH2), 4.04 (m, 2H, CH2), 5.34 (s, 1H,
dihydropyridine-4H), 5.56 (s, 1H, NH), 7.36 (d, J = 7.6 Hz,
3H,ArH), 7.47 (t, J = 7.4 Hz, 2H, ArH), 7.64 (m, 4H, ArH),
7.66 (d, J = 8.8 Hz, 2H, ArH), 7.72 (s, 1H, pyrazole-5H),
7.94 (d, J = 7.2 Hz, 1H, ArH); 13C NMR (100 MHz, CDCl3):
δ 167.6, 151.0, 143.4, 141.1, 140.4, 138.6, 133.7, 131.3,
129.4, 129.3, 129.3, 128.9, 127.3, 127.1, 127.0, 126.7, 119.9,
104.4, 59.8, 29.8, 19.5, 14.3; MS: m/z = 583.1 (M+1), Anal.
Calcd. for C34H32ClN3O4; C, 70.15; H, 5.54; N, 7.22; found:
C, 70.18; H, 5.55; N, 7.22.
4.3.4. 4-[3-(4-Bromophenyl)-1-(4-chlorophenyl)-1H-pyrazol-
4-yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylicacid
diethylester (5d)
IR (KBr vmax cm-1): 3325 (N-H str.), 3100 (CH-aromatic
str.), 2980 (C-H str.), 1690 (C=O str.), 1643 (C=N str.), 1533

708 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 6
Harikrishna et al.
4.3.8. 2,6-Dimethyl-4-(1-phenyl-3-thiophene-2-yl-1H-pyrazol-
4-yl)-1,4-dihydropyridine-3,5-dicarboxylicacid diethylester
(5h)
6H, CH3), 5.26 (s, 1H, dihydropyridine-4H), 5.58 (s, 1H,
NH), 7.25 (d, 2H, J = 6.8 Hz, ArH), 7.35 (d, 2H, J = 7.6 Hz,
ArH), 7.60 (d, J = 7.6 Hz, 2H, ArH), 7.65 (s, 1H, pyrazole-
5H), 7.72 (d, J = 7.2 Hz, 2H, ArH); 13C NMR (100 MHz,
CDCl3): δ 167.8, 151.4, 143.5, 138.7, 137.1, 131.8, 131.2,
129.3, 129.2, 128.7, 128.5, 126.9, 119.9, 104.4, 50.7, 29.5,
21.3, 19.4; MS: m/z = 492.2 (M+1), Anal. Calcd. for
C27H26ClN3O4; C, 65.92; H, 5.33; N, 8.54; found: C, 65.92;
H, 5.34; N, 8.59.
IR (KBr vmax cm-1): 3337 (N-H str.), 3102 (CH-aromatic
str.), 2978 (C-H str.), 1678 (C=O str.), 1614 (C=N str.), 1532
1
(C=C str.), 1204 (C-O str.); H NMR (400 MHz, CDCl3,
ppm): δ 1.04 (t, J = 7.0 Hz, 6H, CH3), 2.31 (s, 6H, CH3),
3.87 (m, 2H, CH2), 4.04 (m, 2H, CH2), 5.34 (s, 1H,
dihydropyridine-4H), 5.55 (s, 1H, NH), 7.11 (m, 1H, ArH),
7.23 (t, J = 7.4 Hz, 1H, ArH), 7.29 (d, J = 4.8 Hz, 1H, ArH),
7.40 (t, J = 7.8 Hz, 2H, ArH), 7.66 (d, J = 7.6 Hz, 2H, ArH),
7.74 (s, 1H, pyrazole-5H), 7.75 (d, J =3.6 Hz,1H, ArH); 13C
NMR (100 MHz, CDCl3): δ 167.7, 145.3, 143.4, 140.0,
136.3, 129.7, 129.4, 127.9, 127.3, 126.4, 126.3, 125.0, 119.0,
105.1, 60.0, 29.9, 19.8, 14.4; MS: m/z= 478.2 (M+1), Anal.
Calcd. for C26H27N3O4S; C, 65.39; H, 5.70; N, 8.80; found:
C, 65.41; H, 5.71; N, 8.81.
4.4.2. 4-[1-(4-Chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-
4-yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylicacid
dimethylester (6b)
IR (KBr vmax cm-1): 3330 (N-H str.), 2904 (C-H str.),
1692 (C=O str.), 1628 (C=N str.), 1542 (C=C str.), 1208 (C-
1
O str.); H NMR (400 MHz, CDCl3, ppm): δ 2.29 (s, 6H,
CH3), 3.37 (s, 6H, CH3), 3.86 (s, 3H, OCH3), 5.24 (s, 1H,
dihydropyridine-4H), 5.56 (s, 1H, NH), 6.99 (d, 2H, J = 7.2
Hz, ArH), 7.35 (d, J = 7.2 Hz, 2H, ArH), 7.60 (d, J = 7.6
Hz, 2H, ArH), 7.65 (s, 1H, pyrazole-5H), 7.77 (d, J = 7.6
Hz, 2H, ArH); 13C NMR (100 MHz, CDCl3): δ 167.8, 159.3,
151.1, 143.5, 138.7, 131.2, 129.8, 129.2, 129.2, 127.3, 126.9,
119.8, 113.5, 104.4, 55.4, 50.7, 29.6, 19.5; MS: m/z = 507.3
(M+1), Anal. Calcd. for C27H26ClN3O5; C, 63.84; H, 5.16; N,
8.27; found: C, 63.83; H, 5.18; N, 8.30.
4.3.9. 4-[1-(4-Chlorophenyl)-3-thiophene-2-yl-1H-pyrazol-
4-yl]-2,6-dimethyl-1,4-dihydro pyridine-3,5-dicarboxylicacid
diethylester (5i)
IR (KBr vmax cm-1): 3331 (N-H str.), 3103 (CH-aromatic
str.), 2976 (C-H str.), 1678 (C=O str.), 1616 (C=N str.), 1537
1
(C=C str.), 1206 (C-O str.); H NMR (400 MHz, CDCl3,
ppm): δ 1.03 (t, J = 7.0 Hz, 6H, CH3), 2.30 (s, 6H, CH3),
3.87 (m, 2H, CH2), 4.02 (m, 2H, CH2), 5.34 (s, 1H,
dihydropyridine-4H), 5.62 (s, 1H, NH), 7.11 (m, 1H, ArH),
7.29 (d, J = 4.4 Hz, 1H, ArH), 7.35 (d, J = 8.0 Hz, 2H,
ArH), 7.56 (d, J = 8.4 Hz, 2H, ArH), 7.70 (s, 1H,pyrazole-
5H), 7.77 (m, 1H, ArH); 13C NMR (100 MHz, CDCl3): δ
167.5, 145.4, 143.2, 138.4, 135.8, 131.4, 129.9, 129.3, 127.6,
127.2, 126.4, 125.0, 119.9, 104.9, 59.9, 29.8, 19.6, 14.2; MS:
m/z = 512.2 (M+1), Anal. Calcd. for C26H26ClN3O4S; C,
60.99; H, 5.12; N, 8.21; found: C, 61.00; H, 5.12; N, 8.21.
4.4.3. 4-[1,3-Bis(4-chlorophenyl)-1H-pyrazol-4-yl]-2,6-dimethyl-
1,4-dihydropyridine-3,5-dicarboxylicacid dimethylester (6c)
IR (KBr vmax cm-1): 3318 (N-H str.), 3104 (CH-aromatic
str.), 2943 (C-H str.), 1697 (C=O str.), 1645 (C=N str.), 1539
1
(C=C str.), 1211 (C-O str.); H NMR (400 MHz, DMSO-d6,
ppm): δ 2.25 (s, 6H, CH3), 3.24 (s, 6H, CH3), 5.09 (s, 1H,
dihydropyridine-4H), 7.50 (d, J = 8.8 Hz, 2H, ArH), 7.56 (d,
J = 8.4 Hz, 2H, ArH), 7.86 (d, J = 8.8 Hz, 4H, ArH), 8.08 (s,
1H, pyrazole-5H), 8.87 (s, 1H, NH); MS: m/z = 512.2
(M+1), Anal. Calcd. for C26H23Cl2N3O4; C, 60.95; H, 4.52;
N, 8.20; found: C, 60.96; H, 4.52; N, 8.23.
4.4. General Method for the Preparation of Compounds
(6a-i)
4.4.4. 4-[3-(4-Bromophenyl)-1-(4-chlorophenyl)-1H-pyrazol-
4-yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylicacid
dimethylester (6d)
A reaction mixture consisting of compound 4a (1.48 g,
0.005 mol), methylacetoacetate (1.08 g, 0.01 mol), ammonium
metavanadate (0.075 g, 5% w/w) and ammonium acetate
(0.39 g, 0.005 mol) in methyl alcohol (10 ml) were mixed in
a round bottom flask. The reaction contents were refluxed
for 1 h. The completion of the chemical reaction was
confirmed by TLC [hexane : ethylacetate (9:1)]. The reaction
mass was quenched into DM water and stirred to get the
solid. Obtained solid was filtered, washed with pre-chilled
methyl alcohol. The compound was used for further analysis
without any purification. The same procedure has been used
to synthesize compounds 6b-i. The structural elucidation
details of the synthesized compounds 6a-i were presented
below.
IR (KBr vmax cm-1): 3323 (N-H str.), 3102 (CH-aromatic
str.), 2943 (C-H str.), 1697 (C=O str.), 1643 (C=N str.), 1539
(C=C str.), 1211 (C-O str.);1H NMR (400 MHz, CDCl3,
ppm): δ 2.31 (s, 6H, CH3), 3.36 (s, 6H, CH3), 5.23 (s, 1H,
dihydropyridine-4H), 5.60 (s, 1H, NH), 7.36 (d, 2H, J = 8.0
Hz, ArH), 7.60 (m, 4H, ArH), 7.66 (s, 1H, pyrazole-5H),
7.77 (d, J = 7.6 Hz, 2H, ArH); 13C NMR (100 MHz, CDCl3):
δ 167.7, 150.0, 143.6, 138.5, 133.7, 131.6, 131.2, 130.2,
129.5, 129.3, 127.3, 121.6, 120.3, 120.0, 104.3, 50.7, 29.5,
19.5; MS: m/z = 556.3 (M+1), Anal. Calcd. for C26H23
BrClN3O4; C, 56.08; H, 4.16; N, 7.55; found: C, 56.11; H,
4.19; N, 7.56.
4.4.1. 4-[1-(4-Chlorophenyl)-3-p-tolyl-1H-pyrazol-4-yl]-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylicacid
dimethylester (6a)
IR (KBr vmax cm-1): 3331 (N-H str.), 3104 (CH-aromatic
str.), 2943 (C-H str.), 1695 (C=O str.), 1647 (C=N str.),
1541(C=C str.), 1213 (C-O str.); 1H NMR (400 MHz,
CDCl3, ppm): δ 2.29 (s, 6H, CH3), 2.41 (s, 3H, CH3), 3.34 (s,
4.4.5. 4-[3-(3-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl]-2,
6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylicacid
dimethylester (6e)
IR (KBr vmax cm-1): 3318 (N-H str.), 3104 (CH-aromatic
str.), 2943 (C-H str.), 1697 (C=O str.), 1643 (C=N str.), 1537

Antitubercular and Antimicrobial Activity of NH4VO3 Promoted
Letters in Drug Design & Discovery, 2017, Vol. 14, No. 6 709
1
(C=C str.), 1211 (C-O str.); H NMR (400 MHz, CDCl3,
MS: m/z = 449.52 (M+1), Anal. Calcd. for C24H23N3O4S; C,
ppm): δ 2.29 (s, 6H, CH3), 3.40 (s, 6H, CH3), 5.24 (s, 1H,
dihydropyridine-4H), 5.66 (s, 1H, NH), 7.25 (t, 1H, J =6.8
Hz, ArH), 7.33 (t, 1H, J = 7.6 Hz, ArH), 7.41 (t, 2H, J = 7.6
Hz, ArH), 7.49 (d, 1H, J = 8.0 Hz, ArH), 7.65 (d, 2H, J =
7.6 Hz, ArH), 7.71 (s, 1H, pyrazole-5H), 7.79 (d, J = 7.6 Hz,
1H, ArH), 8.04 (s, 1H, ArH); 13C NMR (100 MHz, CDCl3):
δ 167.7, 149.5, 143.8, 139.9, 137.0, 131.7, 130.3, 129.7,
129.3, 129.0, 127.4, 127.3, 126.3, 122.0, 119.0, 104.1, 50.7,
29.5, 19.4; MS: m/z = 522.1 (M+1), Anal. Calcd. for
C26H24BrN3O4; C, 59.78; H, 4.63; N, 8.04; found: C, 60.00;
H, 4.64; N, 8.09.
64.13; H, 5.16; N, 9.35; found: C, 64.15; H, 5.20; N, 9.41.
4.4.9. 4-[1-(4-Chlorophenyl)-3-thiophene-2-yl-1H-pyrazol-
4-yl]-2,6-dimethyl-1,4-dihydro pyridine-3,5-dicarboxylicacid
dimethylester (6i)
IR (KBr vmax cm-1): 3341 (N-H str.), 3105 (CH-aromatic
str.), 2943 (C-H str.), 1678 (C=O str.), 1614 (C=N str.), 1535
1
(C=C str.), 1215 (C-O str.); H NMR (400 MHz, CDCl3,
ppm): δ 2.33 (s, 6H, CH3), 3.40 (s, 6H, CH3), 5.31 (s, 1H,
dihydropyridine-4H), 5.64 (s, 1H, NH), 7.15 (m, 1H, ArH),
7.33 (d, J = 5.2 Hz, 1H, ArH), 7.35 (d, J = 8.0 Hz, 2H,
ArH), 7.60 (d, J = 7.6 Hz, 2H, ArH), 7.67 (m, 1H, ArH &
1H, pyrazole-5H); MS: m/z = 485.2 (M+1), Anal. Calcd. for
C24H22ClN3O4S; C, 59.56; H, 4.58; N, 8.68; found: C, 59.61;
H, 4.61; N, 8.72.
4.4.6. 4-[3-(3-Bromophenyl)-1-(4-chlorophenyl)-1H-pyrazol-
4-yl]-2,6-dimethyl-1,4-dihydro pyridine-3,5-dicarboxylicacid
dimethylester (6f)
IR (KBr vmax cm-1): 3323 (N-H str.), 3121(CH-aromatic
str.), 2945 (C-H str.), 1684 (C=O str.), 1597 (C=N str.), 1501
5. ANTIMICROBIAL STUDIES
1
(C=C str.), 1225 (C-O str.); H NMR (400 MHz, CDCl3,
ppm): δ 2.30 (s, 6H, CH3), 3.40 (s, 6H, CH3), 5.23 (s, 1H,
dihydropyridine-4H), 5.60 (s, 1H, NH), 7.33 (t, J = 8.0 Hz,
1H, ArH), 7.37 (d, 2H, J = 7.6 Hz, ArH), 7.50 (d, J = 8.0
Hz, 1H, ArH), 7.60 (d, J = 7.2 Hz, 2H, ArH), 7.68 (s, 1H,
pyrazole-5H), 7.77 (d, 1H, J = 8.0 Hz, ArH), 8.02 (s, 1H,
ArH); 13C NMR (100 MHz, CDCl3): δ 167.6, 149.9, 143.8,
138.5, 136.8, 131.7, 131.6, 130.4, 129.7, 129.4, 129.3, 127.3,
122.0, 120.1, 120.0, 104.1, 50.8, 29.5, 19.5; MS: m/z= 557.9
(M+1), Anal. Calcd. for C26H23BrClN3O4; C, 56.08; H, 4.16;
N, 7.55; found: C, 56.11; H, 4.20; N, 7.59.
Preliminary antimicrobial activities of newly prepared
molecules 5a-i and 6a-i were determined by the zone
inhibition method in nutrient agar media [26]. In-vitro
antibacterial activity was tested against Gram positive
bacteria Staphylococcus aureus (MTCC 3160) and
Mycobacterium smegmatis (MTCC 994). In addition to this,
the antifungal study was tested against Candida albicans
(MTCC 7253). The standard protocol method was followed
for testing antimicrobial activity as mentioned in earlier
publication [26]. Working solution prepared at
a
concentration of 40 mg/mL and used for the test. 50 µL and
12.5 µL of the test compound was transferred to separate
agar wells in triplicates. The plates were incubated for 12 h
at room temperature (~33°C) and noted inhibition zone by
measuring the diameter of the zone of inhibition in
millimeter. DMSO used as negative control. Ciprofloxacin
(INN) was used as antibacterial standard and Fluconazole
(FLZ) was used as antifungal standard.
4.4.7. 4-[3-Biphenyl-4-yl-1-(4-chlorophenyl)-1H-pyrazol-4-
yl]-2,6--dimethyl-1,4-dihydropyridine-3,5-dicarboxylicacid
dimethylester (6g)
IR (KBr vmax cm-1): 3312 (N-H str.), 3101 (CH-aromatic
str.), 2947 (C-H str.), 1698 (C=O str.), 1649 (C=N str.), 1537
1
(C=C str.), 1217 (C-O str.); H NMR (400 MHz, CDCl3,
ppm): δ 2.34 (s, 6H, CH3), 3.39 (s, 6H, CH3), 5.35 (s, 1H,
dihydropyridine-4H), 5.62 (s, 1H, NH), 7.40 (d, J = 8.0 Hz,
3H, ArH), 7.50 (t, J = 7.6 Hz, 2H, ArH), 7.71 (m, 7H, ArH),
7.96 (d, J = 8.0 Hz, 2H, ArH); 13C NMR (100 MHz, CDCl3):
δ 167.3, 149.4, 143.7, 138.6, 136.3, 134.5, 133.5, 132.6,
131.6, 129.4, 128.8, 127.9, 126.2, 126.0, 120.0, 101.9, 59.8,
30.5, 19.5; MS: m/z = 555.3 (M+1), Anal. Calcd. for
C32H28ClN3O4; C, 69.37; H, 5.09; N, 7.58; found: C, 69.40;
H, 5.09; N, 7.60.
The preliminary antimicrobial activity by the zone of
inhibition method confirmed the compounds activity and
further MIC was measured using three bacterial strains M.
smegmatis, P. aeruginosa (MTCC 2640) and S. aureus and a
fungal strain Candida albicans in 96 well microplate using
both plating and Resazurin reduction method. All the
compounds were dissolved in DMSO and 10 mg/ml stock
solution was prepared and followed the standard protocol
procedure as an earlier publication to obtain MIC value [26].
4.4.8. 2,6-Dimethyl-4-(1-phenyl-3-thiophene-2-yl-1H-pyrazol-
4-yl)-1,4-dihydropyridine-3,5-dicarboxylicacid
dimethylester (6h)
6. ANTITUBERCULAR ACTIVITY – MICROPLATE
ALAMAR BLUE ASSAY (MABA) METHOD
IR (KBr vmax cm-1): 3343 (N-H str.), 3107 (CH-aromatic
Anti-tubercular activity for the target compounds 5a-i
and 6a-i was determined by Middle brook 7H9 broth against
pathogenic Mycobacterium tuberculosis of H37Rv strain
(ATCC-27294). The MIC of each compound was identified
by Microplate Alamar Blue Assay method (MABA) [31] and
was defined as the least concentration of the drug, which
inhibits ≤ 99 % of bacterial population present at the
beginning of the assay. 200 µl of sterile de-ionized water
was taken to all sterile 96 wells plate. 100 µl of the
Middlebrook 7H9 broth and serial dilution of compounds
str.), 2945 (C-H str.), 1694 (C=O str.), 1641 (C=N str.), 1533
1
(C=C str.), 1215 (C-O str.); H NMR (400 MHz, CDCl3,
ppm): δ 2.33 (s, 6H, CH3), 3.41 (s, 6H, CH3), 5.32 (s, 1H,
dihydropyridine-4H), 5.60 (s, 1H, NH), 7.14 (t, 1H, ArH),
7.29 (d, J = 6.0 Hz, 1H, ArH), 7.32 (d, J = 4.8 Hz, 1H,
ArH), 7.41 (t, J = 8.0 Hz, 2H, ArH), 7.67 (m, 3H, ArH), 7.71
(s, 1H, pyrazole-5H); 13C NMR (100 MHz, CDCl3): δ 168.0,
145.3, 143.7, 140.1, 136.3, 129.9, 129.7, 129.5, 129.4, 127.9,
127.3, 126.4, 126.1, 125.1, 119.1, 104.8, 50.9, 29.9, 19.6;

710 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 6
Harikrishna et al.
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After that, 25 µl of Alamar Blue reagent + 10 % Tween-80
(1:1) was added to the each well plate and further incubated
for one more day. A blue color in the well was represented
as no bacterial growth, and pink color well was interpreted
as bacterial growth. Ethambutol (EMB), Streptomycin (STP)
and Pyrazinamide (PZA) were used as anti-TB standard
drugs.
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CONFLICT OF INTEREST
The authors confirm that this article content has no
conflict of interest.
ACKNOWLEDGEMENTS
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Zheng, L.; Yin, X.J.; Yang, C.L.; Li, Y.; Yin, S.F. Synthesis and
sedative-hypnotic activity of helicid derivatives containing a 1, 4-
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Harikrishna. N thank, Dr. T. Arulmoli former Vice
President of R&D dept. of Sequent Scientific Limited,
Mangalore for the support and encouragement. AMI thank,
Director, NIT-Karnataka, India for providing the research
facilities. Hazem A. Ghabbour would like to extend their
sincere appreciation to the Deanship of Scientific Research
at King Saud University for its funding of this research
through the Research Group Project no. PRG-1436-38.
Joazaizulfazli Jamalis wish to thank the Ministry of Higher
Education (MOHE) of Malaysia for funding this research
under the Research University Grant (RUP) with vote no
05H95 and Fundamental Research Grant Scheme (FRGS) of
vote numbers 4F448.
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