Synthesis, biological evaluation, and molecular docking studies of novel pyrazole, pyrazoline-clubbed pyridine as potential antimicrobial agents

Article abstract of DOI:10.1002/jhet.4208

We have prepared 15 hybrid pyrazole, pyrazoline-clubbed pyridine–containing compounds (5a-o) and tested for their antibacterial and antifungal activities for the development of potential antimicrobial agents. The structures of this novel series were chara

Full text of DOI:10.1002/jhet.4208

Synthesis, biological evaluation and molecular docking studies of novel
pyrazole, pyrazoline clubbed pyridine as potential antimicrobial agents
Nisheeth C. Desai*a, Darshita V. Vajaa, Jahnvi D. Monaparaa, Vijjulatha Mangab &
Tamalapakula Vanib
aDivision of Medicinal Chemistry,
Department of Chemistry (DST-FIST Sponsored), Mahatma Gandhi Campus,
Maharaja Krishnakumarsinhji Bhavnagar University,
Bhavnagar 364 002, India.
bMolecular Modeling and Medicinal Chemistry Group, Department of Chemistry, University
College of Science, Osmania University, Hyderabad-500007, Telangana, India.
E-mail: dnisheeth@rediffmail.com
Abstract
We have prepared fifteen hybrid pyrazole, pyrazoline clubbed pyridine containing
compounds (5a-o) and tested for their antibacterial and antifungal activities for the
development of potential antimicrobial agents. The structures of this novel series were
1
characterized by various spectral techniques like IR, H NMR, 13C NMR, LC-MS, and
elemental analysis. The synthesized compounds 5d, 5e, 5i, 5k, 5m and 5o exhibited
significant antimicrobial activity in the comparison of standard drugs. Molecular docking
studies that have been carried out to emphasize the binding orientations of these molecules
were in good compliance with crystal structure interactions. The predicted drug-likeness
(ADME) properties were found to be in the acceptable range.
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Graphical Abstract
KEYWORDS
antimicrobial activity, molecular docking, pyrazole, pyrazoline, pyridine
1 | INTRODUCTION
An increase in antimicrobial resistance including antibiotic resistance is one of the major
global threats.[1] The development of methicillin-resistant Staphylococcus aureus (MRSA)
and vancomycin-resistant Staphylococcus aureus (VRSA) have spread the resistance to the
available antibiotics.[2] The development of multidrug-resistant (MDR) superbugs,
extensively drug-resistant (XDR) and totally drug-resistant (TDR) strains have caused the
need of developing new effective antimicrobials. Pyrazole, pyrazoline and pyridine
heterocycles themselves possessed different biological activities and are parent heterocycles
of various commercially available drugs. Pyrazole, first isolated from watermelon seeds is an
active heterocycle used as an antimicrobial,[3] antiviral,[4] anti-tubercular,[5] anticancer,[6]
analgesic and anti-inflammatory[7] agents. Pyrazoline is dihydropyrazole possessed
anticancer,[8] anti-inflammatory[9] and antimalarial[10] activity, etc. Pyridine is also an
important heterocycle in medicinal chemistry and is associated with several pharmaceutical
activities.[11-18] Figure 1 represents the concept of drug design based on commercially
available drugs isoniazid, fezolamine and dipyrone heterocycles.
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It is well-known that the activity of a drug is attained through its interaction with the active
site of the target protein. Molecular docking is one of the best methods to study
intermolecular interactions of a protein-ligand complex. The preferred geometry and
orientation of a ligand that interacts with protein were assessed employing the docking score
in Schrodinger. The binding free energies were the ΔG for an ideal hydrogen bond, ionic,
aromatic, or lipophilic interactions were calculated by using the following equation.
ΔGbindMM-GB/SA = ΔEintramolecular + ΔGsolvation - TΔSconf + EvdW + Eelectrostatic + Eprotein
FIGURE 1 Concept of design based on the commercially available drugs containing
pyrazole, pyrazoline and pyridine heterocycles
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SCHEME 1 Synthetic pathway for compounds 5a-o
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
The synthetic route for the titled compounds (3'-(4-nitrophenyl)-1'-phenyl-5-(aryl)-3,4-
dihydro-1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridin-4-yl)methanones (5a-o) was achieved as
described in Scheme 1. The formation of chalcones (4a-o) was achieved by Aldol
condensation of 3-(4-nitrophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3) and substituted
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acetophenones. The reaction of these chalcones (4a-o) with isoniazid forms titled compounds
(3'-(4-nitrophenyl)-1'-phenyl-5-(aryl)-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridine-4-
yl)methanones (5a-o).
A series of (3'-(4-nitrophenyl)-1'-phenyl-5-(aryl)-3,4-dihydro-1'H,2H-[3,4'-bipyrazol]-2-
yl)(pyridine-4-yl)methanones (5a-o) were synthesized and structures of these products were
confirmed based on various spectroscopy analysis. In the IR spectra of compound 5k, a
characteristic absorption band at 1647 cm-1 represent the presence of >C=O group in the
structure from isoniazid ring. Different vibrations at 3142, 3047 and 3244 cm-1 confirm the
presence of aromatic ring by -C-H- stretching. Different vibrations at 1504 and 1598 cm-1
confirm the presence of heterocyclic rings by >C=C< and >C=N- stretching respectively. An
absorption band appeared at 1448 cm-1 correspond to -C-N< stretching vibrations. The
presence of fluoro and nitro groups were confirmed by -C-F- and –N=O stretching at 1072
1
and 1465 cm-1 respectively. The H NMR spectra of compound 5k represent the presence of
twenty-one protons in the structure by signals at different values. Multiple signals at δ =7.40-
8.90 ppm confirm the presence of a total of seventeen aromatic hydrogens of phenyl ring
while a singlet peak observed at δ = 7.70 ppm was due to one proton of pyrazole ring. Two
doublets at δ = 3.55 and δ = 3.90 ppm confirm the presence of two hydrogens at C3 of
pyrazoline ring while a singlet at δ = 6.12 ppm correspond to hydrogen at C4 of pyrazoline
13
ring. The C NMR spectra of compound 5k confirm structure by different peaks. A signal
that appeared at δ = 167.2 ppm was due to carbon of the carbonyl group. Presence of
pyrazole ring confirmed by different signals at δ = 117.3, 123.1 and 149.5 ppm while
different chemical shifts at δ = 149.8, 140.4 and 121.7 ppm found due to carbons of the
pyridine ring. The presence of pyrazoline ring was confirmed by signals at δ = 39.1, 61.2 and
151.2 ppm. In mass spectra of compound 5k, m/z = 533.60 showed propinquity with
proposed molecular weight. The characteristic spectra were given as supporting information.
2.2 | Biological evaluation
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2.2.1 | Discussion on antimicrobial activity
The results of the antimicrobial activity of synthesized compounds (5a-o) were represented in
Table 1. Compound 5m (2,4-dichloro) showed significant antibacterial activity against
different bacterial strains. Compound 5m (2,4-dichloro) showed excellent activity (MIC=
12.5 μg/mL) against P. aeruginosa and very good activity (MIC = 25 μg/mL) against S.
pyogenes. Compounds 5e (3-OH) and 5k (4-F) displayed good activity (MIC = 50 μg/mL)
against S. aureus and E. coli respectively. Compounds 5c (4-Br) and 5n (2-Br-4-Cl) were
also active against S. aureus and E. coli, respectively with MIC = 62.5 μg/mL. The remaining
derivatives of this series possess moderate antibacterial activity against tested Gram-positive
and Gram-negative strains of bacteria. Ampicillin and chloramphenicol were used as standard
drugs for antibacterial screening. Compounds 5i (4-CH3) and 5o (4-OH-3-OCH3) showed
significant antifungal activity against different fungal strains. Compound 5i (4-CH3) showed
excellent activity (MIC = 12.5 μg/mL) against A. niger while compound 5o (4-OH-3-OCH3)
showed excellent activity (MIC = 12.5 μg/mL) against C. albicans and A. clavatus.
Compound 5d (2-OH) displayed good activity (MIC = 50 μg/mL) against A. niger. The
remaining derivatives of this series possess moderate antifungal activity against tested fungal
strains. Nystatin and griseofulvin were used as standard drugs for antifungal screening.
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Entry
5a
Pyrazole, pyrazoline clubbed
pyridine derivatives-structural
formula
Molecular
Formula
Yield M. P. (ºC)
(%)
Minimum bactericidal
concentrations (MBC) in μg/mL
Minimum fungicidal
concentrations (MFC) in
μg/mL
E. c.
P. a.
S. a.
S. p.
C. a.
A. n.
A. c.
C30H22N6O3
68
159-161
100
500
500
500
250
>1000
>1000
C30H21BrN6O3
75
145-148
125
100
100
250
1000
500
500
5b
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C30H21BrN6O3
C30H22N6O4
C30H22N6O4
70
68
65
210-212
170-172
202-204
100
250
100
250
500
500
62.5
100
50
100
125
500
1000
>1000
>1000
>1000
>1000
5c
5d
5e
500
50
100
>1000
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C31H24N6O4
C31H24N6O4
C31H24N6O4
65
66
69
191-193
177-179
152-154
125
100
100
250
250
500
125
500
500
500
500
250
100
>1000
500
1000
>1000
1000
1000
5f
5g
5h
100
1000
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C31H24N6O3
C30H21ClN6O3
C30H21FN6O3
71
75
73
207-209
124-126
130-132
100
500
50
500
500
500
250
100
200
250
200
200
>1000
12.5
500
500
>1000
5i
500
500
5j
500
500
5k
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C30H20F2N6O3
C30H20Cl2N6O3
C30H20BrClN6O3
68
69
70
191-193
204-206
212-214
100
250
62.5
200
12.5
100
250
500
500
250
200
1000
500
5l
5m
5n
25
250
500
500
500
1000
1000
1000
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C31H24N6O5
71
198-200
100
500
125
500
12.5
>1000
12.5
5o
100
50
-
100
50
-
250
50
-
100
50
-
-
-
-
Ampicillin
-
-
-
Chloramphenicol
Nystatin
100
500
100
100
100
100
-
-
-
-
Griseofulvin
Abbreviations: E. c. - Escherichia coli, P. a. - Pseudomonas aeruginosa, S. a. - Staphylococcus aureus, S. p. - Streptococcus pyogenes; C.a. - Candida
albicans, A. n.- Aspergillus niger, A. c. – Aspergillus clavatus.
TABLE 1 Antimicrobial screening results of compounds 5a-o with structural and molecular formula
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2.3 | Molecular docking studies
The standard drugs selected for antimicrobial activity preferably ampicillin is an anti-
staphylococcus agent that can withstand acid attack acts by inhibition of cell wall synthesis,
while chloramphenicol is used in treating streptococcal infections acts by inhibiting the
production of proteins.[19] The best-preferred orientation of the standard drugs has been
characterized by molecular docking studies. Further validation of our docking has been
carried out and their root mean square deviation (RMSD) values were found to be 0.648 Å
(ampicillin) and 0.243 Å (chloramphenicol). The carboxylic acid group attached to β-lactam
of standard ampicillin has formed two H-bond interactions with the amine group of Ser 41
and Arg 110 in the protein 3KP3 with a dock score of -8.253 kcal/mol, while the benzyl
amine group has interacted with carboxy terminal of Glu 39 with single H- bond as depicted
in Figure 2. Similarly, the nitro group of chloramphenicol has formed two hydrogen bond
interactions with the amine functionality of Lys 74 and Arg 70 with a dock score of -5.554
kcal/mol, in the protein 4EJV as shown in Figure 3. Their binding energies were -87.984
kcal/mol and -67.278 kcal/mol for ampicillin and chloramphenicol respectively. All the
synthesized compounds were docked into the active site of ampicillin and chloramphenicol
their dock scores with binding free energies in both proteins were tabulated in Table 2.
The molecular docking studies reveal that all the synthesized molecules were suitably
oriented in the active site of proteins displaying H-bond and п-п stacking interactions with
amino acids Glu 39, Arg 110, Ser 41 in protein 3KP3, and Lys 74, Arg 70 in 4EV protein
which were identical to that of standard ampicillin and chloramphenicol as depicted in Figure
4 and Figure 5. Among the synthesized compounds, the compounds 5k (50 μg/mL), 5n (62.5
μg/mL) were effective against E. coli, while 5e (50 μg/mL), 5m (25 μg/mL) were effective
against P. aeruginosa and 5o (12.5 μg/mL) the only compound that is effective against fungal
strains such as C. albicans and A. clavatus. The minimum inhibitory bacterial concentrations
for the above-mentioned compounds were found to be lesser than the standard drugs
inhibitory concentration. As far as activity is concerned high electronegative group such as
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fluorine at para position as in compound 5k (4-F) aid in retaining hydrophobic nature
contributing for antimicrobial activity, while electron donor group (-OH group) that is meta
directing in 5e (3-OH) compound is found to be essential feature for treating pseudomonol
infections. Both electron donating groups at meta (-OCH3 group) and para (-OH group)
positions in compound 5o (4-OH-3-OCH3) aid in exhibiting antifungal activity.
FIGURE 2 Dock pose image of the standard drug ampicillin with its ligand interaction
diagram displaying hydrogen bond interactions with Serine 41, Glu 39, Arg 110 in the 3KP3
protein
FIGURE 3 The dock pose image of standard chloramphenicol showing hydrogen bond
interaction with Lys 74 and one п-п stacking interaction with Arg 70 in the protein 4EJV
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FIGURE 4 Dock pose image of compound 5k in the proteins 3KP3 (a) displaying H-Bond
interaction with Gln 61 and two п-п stacking interaction with His 42, while 4EJV (b) forming
H-bond with Lys 74 and water
FIGURE 5 Dock pose image of compound 5l in the proteins 3KP3 (a) displaying H-Bond
interaction with His 42 and Arg 110, while 4EJV (b) forming H-bond with Lys 74 and Arg 70
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TABLE 2 The molecular docking scores with binding energies for the synthesized
compounds 5a-o in both proteins 3KP3 and 4EJV
Sr.
Compounds
Antimicrobial Dock scores
Antimicrobial binding energies
No.
3HUN
(kcal/mol)
-6.444
-7.838
-6.294
-7.565
-6.574
-7.844
-5.909
-6.133
-7.771
-7.176
-6.072
-6.454
-8.555
-7.658
-8.427
-8.253
-
4EJV
(kcal/mol)
-6.935
-7.801
-5.249
-4.539
-7.462
-6.260
-5.701
-8.304
-5.391
-7.743
-6.924
-4.194
-2.377
-3.809
-3.482
-
3HUN
(kcal/mol)
-56.832
-85.728
-55.726
-71.540
-49.423
-78.894
-61.146
-76.290
-74.117
-63.541
-54.642
-60.383
-89.343
-57.3296
-88.0809
-87.984
-
4EJV
(kcal/mol)
-51.111
-51.958
-52.106
-58.862
-55.606
-62.456
-63.483
-54.691
-57.374
-58.363
-58.097
-49.184
-51.135
-64.120
-62.073
-
1
2
5a
5b
3
5c
4
5d
5
5e
6
5f
7
5g
8
5h
9
5i
10
11
12
13
14
15
16
5j
5k
5l
5m
5n
5o
Ampicillin
17 Chloramphenicol
-5.554
-67.278
3 | CONCLUSION
A novel series of titled compounds based on pyrazole, pyrazoline clubbed-pyridine (5a-o)
were prepared and tested for their antimicrobial potency. The antimicrobial screening results
revealed the potency of synthesized compounds 5d, 5e, 5i, 5k, 5m and 5o as antimicrobial
agents against various bacterial and fungal species. Further, it is observed that the presence of
electron-donating groups in compounds such as 2-OH, 3-OH, 4-CH3 and 4-OH-3-OCH3
augmented antifungal and antibacterial potency while the presence of electron-withdrawing
groups in compounds such as 4-F and 2,4-dichloro showed an increment in the antibacterial
potency. Molecular docking studies and ADME properties calculations were effective in
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compliance with the antimicrobial activity that supports our explanation for antibacterial
activity.
4 | EXPERIMENTAL SECTION
4.1 | Synthesis
4.1.1 | Synthesis of 3-(4-nitrophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3)
3-(4-Nitrophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3) was prepared in two steps as
described under:
4.1.1.1 | Synthesis of 1-(1-(4-nitrophenyl)ethylidene)-2-phenylhydrazine (2)
To a solution of p-nitroacetophenone (1) in ethanol (95 %, 30 mL), glacial acetic acid (1 mL),
and phenylhydrazine (0.01 mol) were added and the reaction mixture was warmed for an
hour. The precipitates formed were filtered and washed with 95 % ethanol and recrystallized
from methanol.
4.1.1.2 | Synthesis of 3-(4-nitrophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3) from
compound (2)
A solution of compound (2) (0.11 mol) in dimethylformamide (5 mL) was added dropwise to
previously cooled mixture of dimethylformamide (0.35 mol) and phosphorus oxychloride
(0.35 mol) which was then allowed to attain room temperature and refluxed at 70–80 °C for 5
h. After cooling at room temperature, the mixture was treated with a cold saturated K2CO3
solution. The precipitate was filtered, washed with water and recrystallized from ethanol
(95%).
Yield: 65 %; m.p.:173-175 ºC; Anal. calcd. for C16H11N3O3: C, 65.53; H, 3.78; N, 14.33;
Found: C, 65.55; H, 3.80; N, 14.37 %.
4.1.2 | Synthesis of 3-(3-(4-nitrophenyl)-1-aryl-1H-pyrazol-4-yl)-1-phenylprop-2-en-1-
ones (4a-o)
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A mixture of 3-(4-nitrophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3) (0.01 mol) and
substituted acetophenones (0.01 mol) were stirred in methanolic potassium hydroxide at room
temperature for 4 hours. The product generated was then filtered, washed with water and
recrystallized from 95 % ethanol.
Yield (4a): 71%; m.p.: 190-192 °C; Anal. calcd. for C24H17N3O3: C, 72.90; H, 4.33; N, 10.63;
Found: C, 72.95; H, 4.36; N, 10.68 %.
4.1.3 | General preparation of of (3'-(4-nitrophenyl)-1'-phenyl-5-(aryl)-3,4-dihydro-
1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridine-4-yl)methanones (5a-o)
3-(3-(4-Nitrophenyl)-1-aryl-1H-pyrazol-4-yl)-1-phenylprop-2-en-1-ones (4a-o) (0.001 mol)
and isoniazid (0.002 mol) were taken in a round bottom flask containing 20 mL glacial acetic
acid and refluxed for 28 hours above 140 ºC using a drop of H2SO4 as a catalyst. The reaction
mixture was then poured onto crushed ice. The separated product was filtered, washed with
water and recrystallized from 95 % ethanol. All compounds of series were prepared using the
same method.
Characterization
of
(3'-(4-nitrophenyl)-1',5-diphenyl-3,4-dihydro-1'H,2H-[3,4'-
bipyrazol]-2-yl)(pyridin-4-yl)methanone (5a)
Yellow; Yield: 68 %; m.p.: 159-161 ºC; IR (KBr)νmax: 3245 (C-H stretching, aromatic ring),
3144, 3049 (C-H stretching, -CH2-), 1649 (>C=O stretching), 1592 (>C=N- stretching,
aromatic ring), 1500 (>C=C< stretching, aromatic ring), 1450 (C-N< stretching), 1465 (-NO2
stretching); 1H NMR (500 MHz, DMSO-d6, δppm): 7.72 (s, 1H, =CH-N), 7.44-8.92 (m, 18H,
Ar-H), 6.16 (s, 1H, >CH-), 3.93 (dd, 1H, Hb-C-Ha-C), 3.50 (dd, 1H, Hb-C-Ha-C); 13C NMR
(125 MHz, DMSO-d6, δppm): 39.2, 61.8, 103.7, 104.7, 119.5 (2), 123.4 (2), 124.2 (2), 126.0,
127.1, 128.3 (2), 129.4, 129.6, 129.7, 129.9 (2), 131.0, 133.2, 139.4, 141.3, 143.4, 143.7,
145.3, 147.6, 151.3 (2), 165.1; LCMS: m/z = 515.10 [M+] (36 %); Anal. calcd. for
C30H22N6O3: C, 70.03; H, 4.31; N, 16.33; Found: C, 70.10; H, 4.39; N, 16.39 %.
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Characterization
of
(5-(3-bromophenyl)-3'-(4-nitrophenyl)-1'-phenyl-3,4-dihydro-
1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridin-4-yl)methanone (5b)
Cremes yellow; Yield: 75 %; m.p.: 145-148 ºC; IR (KBr)νmax: 3250 (C-H stretching,
aromatic ring), 3148, 3059 (C-H stretching, -CH2-), 1665 (>C=O stretching), 1580 (>C=N-
stretching, aromatic ring), 1505 (>C=C< stretching, aromatic ring), 1453 (C-N< stretching),
1425 (-NO2 stretching), 540 (C-Br stretching); 1H NMR (500 MHz, DMSO-d6, δppm): 7.65
(s, 1H, =CH-N), 7.40-8.96 (m, 17H, Ar-H), 6.14 (s, 1H, >CH-), 3.96 (dd, 1H, Hb-C-Ha-C),
3.57 (dd, 1H, Hb-C-Ha-C); 13C NMR (125 MHz, DMSO-d6, δppm): 39.4, 61.6, 117.2, 119.1
(2), 121.6 (2), 123.7 (2), 124.6 (2), 126.3 (3), 126.9, 127.5, 129.7 (2), 129.9, 133.4, 136.2,
139.7, 139.9, 140.5, 147.8, 149.6 (2), 149.4, 151.0, 167.6; LCMS: m/z =595.03 [M+] (39 %);
Anal. calcd. for C30H21BrN6O3: C, 60.72; H, 3.57; N, 14.16; Found: C, 60.78; H, 3.59; N,
14.20 %.
Characterization
of
(5-(4-bromophenyl)-3'-(4-nitrophenyl)-1'-phenyl-3,4-dihydro-
1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridin-4-yl)methanone (5c)
Cremes yellow; Yield: 70 %; m.p.: 210-212 ºC; IR (KBr)νmax: 3255 (C-H stretching,
aromatic ring), 3141, 3051 (C-H stretching, -CH2-), 1661 (>C=O stretching), 1578 (>C=N-
stretching, aromatic ring), 1505 (>C=C< stretching, aromatic ring), 1456 (C-N< stretching),
1421 (-NO2 stretching), 545 (C-Br stretching); 1H NMR (500 MHz, DMSO-d6, δppm): 7.64
(s, 1H, =CH-N), 7.38-8.91 (m, 17H, Ar-H), 6.10 (s, 1H, >CH-), 3.91 (dd, 1H, Hb-C-Ha-C),
3.50 (dd, 1H, Hb-C-Ha-C);13C NMR (125 MHz, DMSO-d6, δppm): 39.5, 61.4, 117.4, 119.7,
121.0 (2), 123.7 (2), 124.8 (2), 125.7, 126.1 (3), 128.5 (2), 129.6 (2), 131.9 (2), 135.7, 139.4,
139.8, 140.0, 147.5, 149.6, 149.8 (2), 151.4, 167.2; LCMS: m/z =595.01 [M+] (41 %); Anal.
calcd. For C30H21BrN6O3: C, 60.72; H, 3.57; N, 14.16; Found: C, 60.78; H, 3.66; N, 14.23 %.
Characterization of (5-(2-hydroxyphenyl)-3'-(4-nitrophenyl)-1'-phenyl-3,4-dihydro-
1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridin-4-yl)methanone (5d)
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Yellow; Yield: 68 %; m.p.: 170-172 ºC; IR (KBr)νmax: 3545 (-OH stretching, aromatic ring),
3240 (C-H stretching, aromatic ring), 3135, 3032 (C-H stretching, -CH2-), 1645 (>C=O
stretching), 1599 (>C=N- stretching, aromatic ring), 1500 (>C=C< stretching, aromatic ring),
1457 (C-N< stretching), 1464 (-NO2 stretching); 1H NMR (500 MHz, DMSO-d6, δppm): 7.63
(s, 1H, =CH-N), 7.40-8.92 (m, 17H, Ar-H), 6.13 (s, 1H, >CH-), 5.37 (s, 1H, -OH), 3.92 (dd,
13
1H, Hb-C-Ha-C), 3.54 (dd, 1H, Hb-C-Ha-C); C NMR (125 MHz, DMSO-d6, δppm): 40.7,
61.7, 117.8, 117.9, 118.4, 119.6 (2), 121.4, 121.8 (2), 123.5 (2), 124.9 (2), 126.3 (2), 129.5
(2), 132.6, 132.7, 139.4, 139.7, 140.5, 147.8, 149.6 (2), 149.5, 151.4, 162.7, 167.9; LCMS:
m/z = 531.20 [M+] (61 %); Anal. calcd. for C30H22N6O4: C, 67.92; H, 4.18; N, 15.84; Found:
C, 67.96; H, 4.22; N, 15.89 %.
Characterization of (5-(3-hydroxyphenyl)-3'-(4-nitrophenyl)-1'-phenyl-3,4-dihydro-
1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridin-4-yl)methanone (5e)
Pale Yellow; Yield: 65 %; m.p.: 202-204 ºC; IR (KBr)νmax: 3545 (-OH stretching, aromatic
ring), 3245 (C-H stretching, aromatic ring), 3130, 3020 (C-H stretching, -CH2-), 1647 (>C=O
stretching), 1600 (>C=N- stretching, aromatic ring), 1502 (>C=C< stretching, aromatic ring),
1460 (C-N< stretching), 1460 (-NO2 stretching); 1H NMR (500 MHz, DMSO-d6, δppm): 7.59
(s, 1H, =CH-N), 7.38-8.94 (m, 17H, Ar-H), 6.15 (s, 1H, >CH-), 5.30 (s, 1H, -OH), 3.91 (dd,
1H, Hb-C-Ha-C), 3.57 (dd, 1H, Hb-C-Ha-C); 13C NMR (125 MHz, DMSO-d6, δppm): 39.5,
61.7, 114.8, 117.8, 118.7, 119.5 (2), 120.0, 121.4 (2), 123.0 (2), 124.9 (2), 126.5 (3), 129.7
(2), 130.7, 135.2, 139.7, 139.9, 140.8, 147.6, 149.8 (2), 149.4, 151.7, 158.6, 167.3; LCMS:
m/z = 531.15 [M+] (47 %); Anal. calcd. for C30H22N6O4: C, 67.92; H, 4.18; N, 15.84; Found:
C, 67.99; H, 4.22; N, 15.89 %.
Characterization of (5-(2-methoxyphenyl)-3'-(4-nitrophenyl)-1'-phenyl-3,4-dihydro-
1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridin-4-yl)methanone (5f)
Pale yellow; Yield: 65%; m.p.: 191-193 ºC; IR (KBr)νmax: 2830 (C-H stretching, -OCH3),
3232 (C-H stretching, aromatic ring), 3130, 3020 (C-H stretching, -CH2-), 1636 (>C=O
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stretching), 1610 (>C=N- stretching, aromatic ring), 1502 (>C=C< stretching, aromatic ring),
1475 (C-N< stretching), 1455 (-NO2 stretching); 1H NMR (500 MHz, DMSO-d6, δppm): 7.55
(s, 1H, =CH-N), 7.34-8.90 (m, 17H, Ar-H), 6.10 (s, 1H, >CH-), 3.82 (s, 3H, -OCH3), 3.91
13
(dd, 1H, Hb-C-Ha-C), 3.51 (dd, 1H, Hb-C-Ha-C); C NMR (125 MHz, DMSO-d6, δppm):
40.6, 55.4, 61.8, 111.6, 117.2 (2), 119.6 (2), 121.5, 121.6 (2), 123.6, 124.6 (2), 126.6 (3),
129.6 (2), 131.4, 132.1, 139.5, 139.7, 140.6, 147.8, 149.6 (2), 149.4, 151.6, 160.4, 167.3;
LCMS: m/z = 545.20 [M+] (32%); Anal. calcd. for C31H24N6O4: C, 68.37; H, 4.44; N, 15.43;
Found: C, 68.45; H, 4.49; N, 15.48 %.
Characterization of (5-(3-methoxyphenyl)-3'-(4-nitrophenyl)-1'-phenyl-3,4-dihydro-
1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridin-4-yl)methanone (5g)
Pale yellow; Yield: 66 %; m.p.: 177-179 ºC; IR (KBr)νmax: 2833 (C-H stretching, -OCH3),
3232 (C-H stretching, aromatic ring), 3129, 3022 (C-H stretching, -CH2-), 1633 (>C=O
stretching), 1600 (>C=N- stretching, aromatic ring), 1509 (>C=C< stretching, aromatic ring),
1470 (C-N< stretching), 1455 (-NO2 stretching); 1H NMR (500 MHz, DMSO-d6, δppm): 7.52
(s, 1H, =CH-N), 7.28-8.88 (m, 17H, Ar-H), 6.12 (s, 1H, >CH-), 3.87 (s, 3H, -OCH3), 3.94
13
(dd, 1H, Hb-C-Ha-C), 3.57(dd, 1H, Hb-C-Ha-C); C NMR (125 MHz, DMSO-d6, δppm):
39.4, 55.1, 61.6, 113.4, 116.3, 117.6, 119.4 (2), 120.6, 121.6 (2), 123.4, 124.9 (2), 126.6 (3),
129.4 (2), 129.1, 135.7, 139.4, 139.8, 140.7, 147.8, 149.4 (2), 149.9, 151.8, 160.7, 167.6;
LCMS: m/z = 545.18 [M+] (32%); Anal. calcd. for C31H24N6O4: C, 68.37; H, 4.44; N, 15.43;
Found: C, 68.42; H, 4.50; N, 15.49 %.
Characterization of (5-(4-methoxyphenyl)-3'-(4-nitrophenyl)-1'-phenyl-3,4-dihydro-
1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridin-4-yl)methanone (5h)
Yellow; Yield: 69 %; m.p.: 152-154 ºC; IR (KBr)νmax: 2835 (C-H stretching, -OCH3), 3232
(C-H stretching, aromatic ring), 3125, 3022 (C-H stretching, -CH2-), 1629 (>C=O stretching),
1601 (>C=N- stretching, aromatic ring), 1504 (>C=C< stretching, aromatic ring), 1464 (C-
1
N< stretching), 1451 (-NO2 stretching); H NMR (500 MHz, DMSO-d6, ppm): 7.45 (s, 1H,
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=CH-N), 7.21-8.86 (m, 17H, Ar-H), 6.10 (s, 1H, >CH-), 3.84 (s, 3H, -OCH3), 3.85 (dd, 1H,
Hb-C-Ha-C), 3.52 (dd, 1H, Hb-C-Ha-C); 13C NMR (125 MHz, DMSO-d6, ppm): 39.4, 55.1,
61.7, 114.7 (2), 117.3, 119.7 (2), 121.8 (2), 123.7, 124.8 (2), 126.9 (3), 128.1 (3), 129.4 (2),
139.1, 139.4, 140.6, 147.3, 149.8 (2), 149.0, 151.2, 162.7, 167.8; LCMS: m/z =545.15 [M+]
(34 %); Anal. calcd. for C31H24N6O4: C, 68.37; H, 4.44; N, 15.43; Found: C, 68.45; H, 4.52;
N, 15.56 %.
Characterization of (3'-(4-nitrophenyl)-1'-phenyl-5-(p-tolyl)-3,4-dihydro-1'H,2H-[3,4'-
bipyrazol]-2-yl)(pyridin-4-yl)methanone (5i)
Yellow; Yield: 71 %; m.p.: 207-209 ºC; IR (KBr)νmax: 3220 (C-H stretching, aromatic ring),
3115, 3017 (C-H stretching, -CH2-and -CH3), 1614 (>C=O stretching), 1595 (>C=N-
stretching, aromatic ring), 1500 (>C=C< stretching, aromatic ring), 1445 (C-N< stretching),
1
1447 (-NO2 stretching), 1373 (-C-H stretching, -C-CH3); H NMR (500 MHz, DMSO-d6,
δppm): 7.35 (s, 1H, =CH-N), 7.18-8.80 (m, 17H, Ar-H), 6.05 (s, 1H, >CH-), 3.74 (dd, 1H,
Hb-C-Ha-C), 3.50 (dd, 1H, Hb-C-Ha-C), 2.35 (s, 3H, -CH3); 13C NMR (125 MHz, DMSO-d6,
δppm): 21.4, 39.7, 61.4, 117.5, 119.2 (2), 121.5 (2), 123.0, 124.6 (2), 126.2 (3), 127.4 (2),
129.2 (2), 129.9 (2), 133.3, 139.4, 139.5, 140.6, 140.9, 147.0, 149.3 (2), 149.4, 151.3, 167.9;
LCMS: m/z = 529.20 [M+] (38 %); Anal. calcd. for C31H24N6O3: C, 70.44; H, 4.58; N, 15.90;
Found: C, 70.49; H, 4.63; N, 15.96 %.
Characterization
of
(5-(4-chlorophenyl)-3'-(4-nitrophenyl)-1'-phenyl-3,4-dihydro-
1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridin-4-yl)methanone (5j)
Pale yellow; Yield: 75 %; m.p.: 124-126 ºC; IR (KBr)νmax: 3214 (C-H stretching, aromatic
ring), 3115, 3020 (C-H stretching, -CH2-), 1620 (>C=O stretching), 1590 (>C=N- stretching,
aromatic ring), 1499 (>C=C< stretching, aromatic ring), 1440 (C-N< stretching), 1435 (-NO2
stretching), 762 (C-Cl stretching); 1H NMR (500 MHz, DMSO-d6, δppm): 7.20 (s, 1H, =CH-
N), 7.12-8.72 (m, 17H, Ar-H), 6.15 (s, 1H, >CH-), 3.72 (dd, 1H, Hb-C-Ha-C), 3.49 (dd, 1H,
Hb-C-Ha-C); 13C NMR (125 MHz, DMSO-d6, δppm): 39.4, 61.5, 117.2, 119.5 (2), 121.3 (2),
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123.0, 124.7 (2), 126.6 (3), 128.2 (2), 128.3 (2), 129.7 (2), 134.0, 136.1, 139.1, 139.7, 140.0,
147.2, 149.5 (2), 149.6, 151.3, 167.0; LCMS: m/z =549.10 [M+] (70 %); Anal. calcd. for
C30H21ClN6O3: C, 65.63; H, 3.86; N, 15.31; Found: C, 65.70; H, 3.93; N, 15.36 %.
Characterization
of
(5-(4-fluorophenyl)-3'-(4-nitrophenyl)-1'-phenyl-3,4-dihydro-
1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridin-4-yl)methanone (5k)
Light Yellow; Yield: 73 %; m.p.: 130-132 ºC; IR (KBr)νmax: 3224 (C-H stretching, aromatic
ring), 3142, 3047 (C-H stretching, -CH2-), 1647 (>C=O stretching), 1598 (>C=N- stretching,
aromatic ring), 1504 (>C=C< stretching, aromatic ring), 1448 (C-N< stretching), 1465 (-NO2
stretching), 1072 (C-F stretching); 1H NMR (500 MHz, DMSO-d6, δppm): 7.70 (s, 1H, =CH-
N), 7.40-8.90 (m, 17H, Ar-H), 6.12 (s, 1H, >CH-), 3.90 (dd, 1H, Hb-C-Ha-C), 3.55(dd, 1H,
Hb-C-Ha-C); 13C NMR (125 MHz, DMSO-d6, δppm): 39.1, 61.2, 115.2 (2), 117.3, 119.4 (2),
121.7 (2), 123.1, 124.3 (2), 126.3 (3), 129.3 (2),129.5 (2), 132.1, 139.3, 139.5, 140.4, 147.6,
149.5, 149.8 (2), 151.2, 165.3, 167.2; LCMS: m/z = 533.60 [M+] (37 %); Anal. calcd. for
C30H21FN6O3: C, 67.66; H, 3.97; N, 15.78; Found: C, 67.72; H, 4.05; N, 15.83 %.
Characterization of (5-(2,4-difluorophenyl)-3'-(4-nitrophenyl)-1'-phenyl-3,4-dihydro-
1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridin-4-yl)methanone (5l)
Light Yellow; Yield: 68 %; m.p.: 191-193 ºC; IR (KBr)νmax: 3229 (C-H stretching, aromatic
ring), 3143, 3045 (C-H stretching, -CH2-), 1642 (>C=O stretching), 1596 (>C=N- stretching,
aromatic ring), 1504 (>C=C< stretching, aromatic ring), 1450 (C-N< stretching), 1468 (-NO2
stretching), 1075 (C-F stretching); 1H NMR (500 MHz, DMSO-d6, δppm): 7.74 (s, 1H, =CH-
N), 7.41-8.85 (m, 16H, Ar-H), 6.09 (s, 1H, >CH-), 3.95 (dd, 1H, Hb-C-Ha-C), 3.57 (dd, 1H,
Hb-C-Ha-C); 13C NMR (125 MHz, DMSO-d6, δppm): 39.4, 61.3, 111.2, 112.0, 113.0, 117.0,
119.5 (2), 121.7 (2), 123.1, 124.7 (2), 126.3 (3), 129.6 (2), 132.1, 139.2, 139.7, 140.1, 147.8,
149.3 (2), 149.2, 151.3, 161.5, 163.8, 167.7; LCMS: m/z =551.20 [M+] (52 %); Anal. calcd.
for C30H20F2N6O3: C, 65.45; H, 3.66; N, 15.27; Found: C, 65.52; H, 3.70; N, 15.32 %.
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Characterization of (5-(2,4-dichlorophenyl)-3'-(4-nitrophenyl)-1'-phenyl-3,4-dihydro-
1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridin-4-yl)methanone (5m)
Pale yellow; Yield: 69 %; m.p.: 204-206 ºC; IR (KBr)νmax: 3223 (C-H stretching, aromatic
ring), 3130, 3030 (C-H stretching, -CH2-), 1625 (>C=O stretching), 1509 (>C=N- stretching,
aromatic ring), 1460 (>C=C< stretching, aromatic ring), 1450 (C-N< stretching), 1435 (-NO2
stretching), 755 (C-Cl stretching); 1H NMR (500 MHz, DMSO-d6, δppm): 7.77 (s, 1H, =CH-
N), 7.33-8.75 (m, 16H, Ar-H), 6.12 (s, 1H, >CH-), 3.99 (dd, 1H, Hb-C-Ha-C), 3.54 (dd, 1H,
Hb-C-Ha-C); 13C NMR (125 MHz, DMSO-d6, δppm): 39.1, 61.5, 117.2, 119.2 (2), 121.1 (2),
123.0, 124.2 (2), 126.3 (3), 126.6, 128.1, 129.6, 129.9 (2), 133.5, 134.6, 135.8, 139.4, 139.9,
140.7, 147.9, 149.6 (2), 149.6, 151.3, 167.8; LCMS: m/z = 583.07 [M+] (72 %); Anal. calcd.
for C30H20Cl2N6O3: C, 61.76; H, 3.46; N, 14.40; Found: C, 61.83; H, 3.52; N, 14.50 %.
Characterization
of
(5-(2-bromo-4-chlorophenyl)-3'-(4-nitrophenyl)-1'-phenyl-3,4-
dihydro-1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridin-4-yl)methanone (5n)
Pale yellow; Yield: 70 %; m.p.:212-214 ºC; IR (KBr)νmax: 3230 (C-H stretching, aromatic
ring), 3121, 3018 (C-H stretching, -CH2-), 1620 (>C=O stretching), 1505 (>C=N- stretching,
aromatic ring), 1452 (>C=C< stretching, aromatic ring), 1440 (C-N< stretching), 1425 (-NO2
1
stretching), 765 (C-Cl stretching), 545 (C-Br stretching); H NMR (500 MHz, DMSO-d6,
δppm): 7.64 (s, 1H, =CH-N), 7.27-8.50 (m, 16H, Ar-H), 6.11 (s, 1H, >CH-), 3.94 (dd,1H,
13
Hb-C-Ha-C), 3.51 (dd,1H, Hb-C-Ha-C); C NMR (125 MHz, DMSO-d6, δppm): 39.5, 61.2,
117.6, 119.8 (2), 121.9 (2), 123.6 (2), 124.9 (2), 126.9 (3), 127.5, 129.3 (2), 130.7, 132.1,
133.8, 137.5, 139.5, 139.6, 140.8, 147.6, 149.6 (2), 149.8, 151.0, 167.3; LCMS: m/z = 626.80
[M+] (32%); Anal. calcd. for C30H20BrClN6O3: C, 57.39; H, 3.21; N, 13.38; Found: C, 57.45;
H, 3.25; N, 14.49 %.
Characterization of (5-(4-hydroxy-3-methoxyphenyl)-3'-(4-nitrophenyl)-1'-phenyl-3,4-
dihydro-1'H,2H-[3,4'-bipyrazol]-2-yl)(pyridin-4-yl)methanone (5o)
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Light yellow; Yield: 71 %; m.p.: 198-200 ºC; IR (KBr)νmax: 3541, (-OH stretching, aromatic
ring), 3234 (C-H stretching, aromatic ring), 3123, 3019 (C-H stretching, -CH2-), 2825 (C-H
stretching, -OCH3), 1623 (>C=O stretching), 1508 (>C=N- stretching, aromatic ring), 1454
(>C=C< stretching, aromatic ring), 1444 (C-N< stretching), 1428 (-NO2 stretching); 1H NMR
(500 MHz, DMSO-d6, δppm): 7.52 (s, 1H, =CH-N), 7.27-8.52 (m, 16H, Ar-H), 6.15 (s, 1H,
>CH-), 5.32 (s, 1H, -OH), 3.91 (dd,1H, Hb-C-Ha-C), 3.89 (s, 3H, -OCH3), 3.54 (dd,1H, Hb-
13
C-Ha-C); C NMR (125 MHz, DMSO-d6, δppm): 39.5, 56.3, 61.2, 117.5 (2), 119.6 (2),
121.8 (2), 123.3 (2), 124.5 (2), 126.6 (2), 127.4 (2), 129.5 (2), 130.6, 132.4, 133.8, 137.6,
139.6, 140.5, 147.0, 149.5, 149.6 (2), 151.0, 167.6; LCMS: m/z =561.20 [M+] (53 %); Anal.
calcd. for C31H24N6O5: C, 66.42; H, 4.32; N, 14.99; Found: C, 66.49; H, 4.36; N, 14.93 %.
5 | MATERIALS AND METHODS
5.1 | Antimicrobial screening
For the antimicrobial screening of the compounds (5a-o), we have used standard MTCC
strains of bacteria and fungi. The newly synthesized product was tested according to the
Broth dilution method.[20] In the present protocol, various bacteria like Staphylococcus
aureus, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa were used.
Ampicillin and chloramphenicol used as standard drugs for antibacterial activity. The fungal
strains like Aspergillus niger, Candida albicans and Aspergillus clavatus used for the
antifungal activity. The details were given in supporting information.
5.2 | Methodology for molecular docking
The tentative drug molecular binding interactions into the preferred active site of the receptor
were assessed by means of molecular docking studies. The proteins in which the standard
drugs used for activity studies that have been crystallized were selected for molecular
docking. Since there were no available antifungal proteins with Nystatin and Griseofulvin as
co-crystal structures, molecular docking studies were therefore carried on antimicrobial
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proteins. Therefore the proteins, Staphylococcus epidermidis in complex with ampicillin
(PDB ID:3KP3)[21] and Staphylococcus epidermidis TcaR in complex with chloramphenicol
(PDB ID:4EJV)[22] has been retrieved from the protein data bank to assess essential binding
free energies along with its interaction into the antimicrobial proteins. Grid was centered on
the respective crystal structures and all the synthesized molecules were docked into the active
site using Glide docking in Schrodinger.[23] The essential drugs like properties were evaluated
for the synthesized molecules using QikProp.[24]
ACKNOWLEDGEMENTS
One of the authors Prof. N C Desai is thankful to the University Grant Commission, New
Delhi for awarding BSR Faculty-Fellowship 2019 (No. F 18-1/2011 (BSR)). Miss Jahnvi
Monapara is grateful to DST INSPIRE PROGRAM for the award of INSPIRE Fellowship
[No. IF180817]. Authors are thankful to the University Grants Commission, New Delhi and
Department of Science and Technology, New Delhi for financial support under the NON-
SAP and DST-FIST programs respectively.
SUPPORTING INFORMATION
Supporting information associated with this article can be found, in the online version.
CONFLICT OF INTEREST
The authors confirm that there is no conflict of interest.
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