Synthesis and molecular docking study of pyrazole clubbed oxazole as antibacterial agents

Article abstract of DOI:10.1007/s11164-020-04286-6

Abstract: We have developed a simple synthetic protocol for the preparation of novel 3-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-arylisoxazoles. The structure of synthesized compounds was elucidated by spectral techniques like FT-IR, ¹H-NMR, ¹³C-NMR, and mass. The novel bioactive compounds 3a-t were evaluated for in vitro antibacterial activity on several bacterial species. Compounds 3c (–4–NO₂), 3o (–4–F), and 3r (–3,4–Cl₂) exhibited good in vitro antibacterial activity. Furthermore, molecular docking on?DNA gyrase subunit b could shed some light on the mechanism of action which can serve as a guide for lead optimization. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11164-020-04286-6

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-020-04286-6
Synthesis and molecular docking study of pyrazole clubbed
oxazole as antibacterial agents
Nisheeth C. Desai1 · Darshita V. Vaja1 · Surbhi B. Joshi1 · Vijay M. Khedkar2
Received: 20 July 2020 / Accepted: 26 September 2020
© Springer Nature B.V. 2020
Abstract
We have developed a simple synthetic protocol for the preparation of novel
3-(3-(4-fuorophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-arylisoxazoles. The structure
1
of synthesized compounds was elucidated by spectral techniques like FT-IR, H-
NMR, 13C-NMR, and mass. The novel bioactive compounds 3a-t were evaluated for
in vitro antibacterial activity on several bacterial species. Compounds 3c (–4–NO2),
3o (–4–F), and 3r (–3,4–Cl2) exhibited good in vitro antibacterial activity. Further-
more, molecular docking on DNA gyrase subunit b could shed some light on the
mechanism of action which can serve as a guide for lead optimization.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1116
4-020-04286-6) contains supplementary material, which is available to authorized users.
*
Nisheeth C. Desai
dnisheeth@redifmail.com
1
Division of Medicinal Chemistry, Department of Chemistry (DST-FIST Sponsored & UGC
NON-SAP), Maharaja Krishnakumarsinhji Bhavnagar University, Mahatma Gandhi Campus,
Bhavnagar 364002, India
2
Department of Pharmaceutical Chemistry, School of Pharmacy, Vishwakarma University, Pune,
Maharashtra 411 048, India
Vol.:(0123456789)
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N. C. Desai et al.
Graphic abstract
Keywords Pyrazole · Isoxazole · Antibacterial activity · Molecular modelling ·
Docking
Introduction
The inactivation of antibiotics because of the resistance towards the existing anti-
microbial agents is one of the major challenges posed to the scientifc community.
The mortality and morbidity data, published by WHO, support this fact. Antibi-
otic resistance is now a growing threat to mankind [1]. Chemists are trying to fnd
the solution to this problem [2, 3]. To fnd a solution to this pressing problem, our
research group has extensively synthesized antimicrobial scafolds with a novel
mechanism of action against these pathogens [4–7]. Azoles are known for their
antibacterial property. Selma SARAÇ and several other workers have synthesized
and developed azole-based antimicrobial compounds that showed good-to-excellent
activity [8–12]. In continuation, we have incorporated pyrazole and isoxazole as a
building block of our targeted compounds and studied their antibacterial efciency.
Synthesis of isoxazoles goes back to 1883 when it was explained by Claisen in 1888
[13] utilising β-diketones and hydroxylamine. It ultimately gave 3,5-disubstituted
isoxazoles. However, the formation of unsymmetrical 1,3-diketones can result in
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Synthesis and molecular docking study of pyrazole clubbed…
mixtures of isomeric isoxazoles. Numerous research groups have developed isoxa-
zoles through 1,3-dipolar cycloaddition or 3+2 cycloaddition. The major disadvan-
tages of this method are low yield, side reactions, and poor regioselectivity [14].
With this objective, we synthesized isoxazoles in good yield from easily available
reactants with simple workup. Furthermore, considering the signifcant place occu-
pied by the heterocyclic structural unit among pharmaceutically important synthetic
and natural materials, detailed antimicrobial profling was carried out for these isox-
azoles. Molecular docking study against crucial target DNA gyrase subunit b could
provide valuable insights into the binding mode and afnity of these isoxazole ana-
logs. The drug design concept on the basis of commercially available drugs contain-
ing pyrazole and isoxazole scafolds is given in Fig. 1.
Experimental
Synthesis of 3-(4-fuorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (1) was
achieved by reported method [15].
3-(3-(4-fuorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1-phenylprop-2-en-1-ones
(2a-t) was prepared by Shaharyar et al. (2010) method [16].
Preparation of 3‑(3‑(4‑fuorophenyl)‑1‑phenyl‑1H‑pyrazol‑4‑yl)‑5‑arylisoxazoles
(3a‑t)
A mixture of 3-(3-(4-fuorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1-arylprop-2-en-1-
ones (2a-t) and hydroxylamine hydrochloride (0.03 mol) was refuxed in the ethanol
(30 mL) at 120 °C for 15 h in the presence of NaOH which is used as a catalyst.
Once the reaction mixture is cooled, it was poured into crushed ice. The obtained
crude product was crystallized in CH3CH2OH (95%) to produce the fnal com-
pounds (3a-t).
Fig. 1 Drug design concept on the basis of commercially available drugs containing pyrazole and isoxa-
zole scafold
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N. C. Desai et al.
Physical constants and characterization of 3‑(3‑(4‑fuorophenyl)‑1‑phenyl‑1H‑
pyrazol‑4‑yl)‑5‑phenylisoxazole (3a)
1
Yield 65%; m.p. 158–160 ºC; IR: 1095, 1450, 1598, 1544, 1649, 2848, 3055; H
NMR (DMSO-d6, 400 MHz): δ=6.70 (s, 1H,=CH–isoxazole), 7.20–8.30 (m, 14H,
Ar–H), 8.60 (s, 1H,=CH–N<pyrazole); 13C NMR (100 MHz, DMSO-d6): δ=98.2,
103.5, 116.2 (2), 119.8 (2), 125.4 (2), 126.2, 126.3, 128.3, 128.4, 129.3 (2), 129.4
(2), 130.5 (2), 131.4, 143.7, 145.2, 162.2, 162.7, 139.4; LCMS: m/z 381.2 (60.1%).
Anal. Calcd. For C24H16FN3O: C, 75.58; H, 4.23; N, 11.02. Found: C, 75.78; H,
4.29; N, 11.15.
Physical constant and characterization of 3‑(3‑(4‑fuorophenyl)‑1‑phenyl‑1H‑
pyrazol‑4‑yl)‑5‑(3‑nitrophenyl)isoxazole (3b)
Yield 62%; mp 145–147 ºC; IR: 1060, 1435, 1523, 1570, 1565, 1690, 2865, 3030;
1H NMR (DMSO-d6, 400 MHz): δ=6.80 (s, 1H,=CH–isoxazole), 7.00–8.30 (m,
13H, Ar–H–), 8.70 (s, 1H,=CH–N<pyrazole); 13C NMR (100 MHz, DMSO-d6):
δ=98.0, 103.5, 116.5 (2), 119.4 (2), 122.7, 123.7, 126.7, 128.4, 129.0 (2), 130.2
(2), 130.8, 131.5, 131.7, 131.9, 143.0, 145.3, 148.9, 162.7, 162.7, 169.0; LCMS: m/z
426.1 (100.0%). Anal. Cacld. For C24H15FN4O3: C, 67.60; H, 3.55; N, 13.14. Found:
C, 67.67; H, 3.65; N, 13.19.
Physical constant and characterization of 3‑(3‑(4‑fuorophenyl)‑1‑phenyl‑1H‑
pyrazol‑4‑yl)‑5‑(4‑nitrophenyl)isoxazole (3c)
Yield 69%; mp 148–150 ºC; IR: 1025, 1400, 1550, 1545, 1585, 1660, 2828, 3045;
1H NMR (DMSO-d6, 400 MHz): δ=6.75 (s, 1H, = CH– isoxazole), 7.29–8.15
(m, 13H, Ar–H-), 8.63 (s, 1H,=CH–N<pyrazole); 13C NMR (100 MHz, DMSO-
d6): δ=98.1, 103.3, 116.8 (2), 119.4 (2), 124.4 (2), 124.9 (2), 126.1, 129.1, 129.8,
130.7 (2), 131.9, 132.2, 143.8, 145.2, 147.4, 162.1, 162.8, 169.1; LCMS: m/z 426.1
(100.0%). Anal. Cacld. For C24H15FN4O3: C, 67.60; H, 3.55; N, 13.14. Found: C,
67.70; H, 3.75; N, 13.23.
Physical constant and characterization of 5‑(3‑bromophenyl)‑3‑(3‑
(4‑fuorophenyl)‑1‑phenyl‑1H‑pyrazol‑4‑yl)isoxazole (3d)
Yield 62%; mp 151–153 ºC; IR: 540, 1010, 1460, 1545, 1600, 1670, 2820, 3010;
1H NMR (DMSO-d6, 400 MHz): δ=6.72 (s, 1H,=CH–isoxazole), 7.19–8.25 (m,
13H, Ar–H–), 8.45 (s, 1H,=CH–N<pyrazole); 13C NMR (100 MHz, DMSO-d6):
δ=98.4, 103.0, 116.8 (2), 119.1 (2), 122.4, 124.9, 126.9, 128.0, 128.8, 129.3 (2),
130.4 (2), 131.2, 131.9, 132.0, 133.0, 132.2, 133.8, 143.9, 145.1, 162.0, 162.8,
169.6; LCMS: m/z 462.0 (26.6%). Anal. Cacld. For C24H15BrFN3O: C, 62.62; H,
3.28; N, 9.13; Found: C, 62.70; H, 3.36; N, 9.23.
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Synthesis and molecular docking study of pyrazole clubbed…
Physical constant and characterization of 5‑(4‑bromophenyl)‑3‑(3‑
(4‑fuorophenyl)‑1‑phenyl‑1H‑pyrazol‑4‑yl)isoxazole (3e)
Yield 70%; mp 171–173 ºC; IR: 520, 1008, 1422, 1523, 1510, 1645, 2800, 3036; 1H
NMR (DMSO-d6, 400 MHz): δ=6.65 (s, 1H,=CH- isoxazole), 7.35–8.30 (m, 13H,
Ar–H-), 8.69 (s, 1H,=CH–N<pyrazole); 13C NMR (100 MHz, DMSO-d6): δ=99.0,
103.1, 116.9 (2), 119.5 (2), 123.7, 125.1, 126.7, 127.6 (2), 128.8, 129.8 (2), 130.1
(2), 131.3, 132.9 (2), 143.9, 145.8, 162.0, 162.9, 139.6; LCMS: m/z 459.0 (99.0%);
Anal. Cacld. For C24H15BrFN3O: C, 62.62; H, 3.28; N, 9.13; Found: C, 62.69; H,
3.35; N, 9.28.
Physical constant and characterization of 2‑(3‑(3‑(4‑fuorophenyl)‑1‑phenyl‑1H‑
pyrazol‑4‑yl)isoxazol‑5‑yl)phenol (3f)
Yield 69%; mp 175–177 ºC; IR: 1060, 1430, 1536, 1555, 1652, 2835, 3040, 3250;
1H NMR (DMSO-d6, 400 MHz): δ=5.40 (s, 1H,–OH), 6.77 (s, 1H,=CH–isoxazole),
7.02–8.20 (m, 13H, Ar–H–), 8.50 (s, 1H,=CH–N<pyrazole); 13C NMR (100 MHz,
DMSO-d6): δ=98.1, 103.4, 116.2, 116.8 (2), 117.8, 119.3 (2), 121.1, 126.5, 126.9,
128.4, 129.6 (2), 130.3, 130.8, 131.8, 143.1, 145.9, 155.1, 162.8, 162.9, 169.0;
LCMS: m/z 397.1 (90.0%). Anal. Cacld. For C24H16FN3O2: C, 72.54; H, 4.06; N,
10.57. Found: C, 72.60; H, 3.40; N, 9.35.
Physical constant and characterization of 3‑(3‑(3‑(4‑fuorophenyl)‑1‑phenyl‑1H‑
pyrazol‑4‑yl)isoxazol‑5‑yl)phenol (3 g)
Yield 68%; mp 185–187 ºC; IR: 1020, 1406, 1535, 1558, 1660, 2810, 3010, 3300;
1H NMR (DMSO-d6, 400 MHz): δ=5.46 (s, 1H, –OH), 6.64 (s, 1H,=CH–isoxa-
zole), 6.86–8.20 (m, 13H, Ar–H–), 8.45 (s, 1H,=CH–N<pyrazole); 13C NMR
(100 MHz, DMSO-d6): δ=98.6, 103.9, 114.9, 115.1, 116.8 (2), 117.4, 119.4 (2),
126.7, 128.9, 129.1 (2), 130.2 (3), 131.7, 131.5, 143.7, 145.3, 157.1, 162.0, 162.7,
169.5; LCMS: m/z 398.1 (27.3%). Anal. Cacld. For C24H16FN3O2: C, 72.54; H,
4.06; N, 10.57. Found: C, 72.59; H, 4.10, N, 10.63.
Physical constant and characterization of 4‑(3‑(3‑(4‑fuorophenyl)‑1‑phenyl‑1H‑
pyrazol‑4‑yl)isoxazol‑5‑yl)phenol (3 h)
Yield 65%; mp 188–190 ºC; IR: 1030, 1412, 1545, 1569, 1695, 2860, 3020, 3350;
1H NMR (DMSO-d6, 400 MHz): δ=5.30 (s, 1H, –OH), 6.52 (s, 1H,=CH–isoxa-
zole), 6.60–8.00 (m, 13H, Ar–H–), 8.25 (s, 1H,=CH–N<pyrazole); 13C NMR
(100 MHz, DMSO-d6): δ=98.4, 103.9, 116.1 (2), 116.8 (2), 119.1, 119.6 (2), 126.7,
127.3 (2), 128.4, 129.3 (2), 130.9 (2), 131.7, 143.8, 145.9, 158.3, 162.3, 162.8,
169.6; LCMS: m/z 398.1 (30.5%). Anal. Cacld. For C24H16FN3O2: C, 72.54; H,
4.06; N, 10.57. Found: C, 72.69; H, 4.09; N, 10.60.
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N. C. Desai et al.
Physical constant and characterization of 3‑(3‑(4‑fuorophenyl)‑1‑phenyl‑1H‑
pyrazol‑4‑yl)‑5‑(2‑methoxyphenyl)isoxazole (3i)
Yield 67%; mp 196–198 ºC; IR: 1000, 1396, 1501, 1510, 1640, 2820, 2835, 3029;
1H NMR (DMSO-d6, 400 MHz): δ=3.30 (s, 3H, –OCH3), 6.40 (s, 1H,=CH- isox-
azole), 7.05–8.10 (m, 13H, Ar–H–), 8.21 (s, 1H,=CH–N<pyrazole); 13C NMR
(100 MHz, DMSO-d6): δ=56.3, 98.3, 103.7, 114.2, 116.2, 116.9 (2), 119.9, 121.4,
126.1, 126.3, 128.2, 129.3, 129.6 (2), 130.9 (2), 131.9, 143.9, 145.0, 155.9, 162.6,
162.8, 169.6; LCMS: m/z 411.1 (100.0%). Anal. Calcd. For C25H18FN3O2: C, 72.98;
H, 4.41; N, 10.21. Found: C, 73.02; H, 4.49; N, 10.26.
Physical constant and characterization of 3‑(3‑(4‑fuorophenyl)‑1‑phenyl‑1H‑
pyrazol‑4‑yl)‑5‑(3‑methoxyphenyl)isoxazole (3j)
Yield 70%; mp 205–207 ºC; IR: 1015, 1396, 1532, 1508, 1630, 2815, 2825, 3036;
1H NMR (DMSO-d6, 400 MHz): δ=3.26 (s, 3H, –OCH3), 6.47 (s, 1H,=CH–isox-
azole), 7.87–8.12 (m, 13H, Ar–H–), 8.26 (s, 1H,=CH–N<pyrazole); 13C NMR
(100 MHz, DMSO-d6): δ=55.9, 98.7, 103.1, 111.8, 114.8, 116.9 (2), 117.9, 119.3
(2), 126.9, 129.1 (2), 130.8, 130.7 (2), 131.1, 131.9, 143.9, 145.7, 161.9, 162.7,
162.8, 169.3; LCMS: m/z 411.1 (45.0%). Anal. Calcd. For C25H18FN3O2: C, 72.98;
H, 4.41; N, 10.21. Found: C, 73.15; H, 4.55; N, 10.36.
Physical constant and characterization of 3‑(3‑(4‑fuorophenyl)‑1‑phenyl‑1H‑
pyrazol‑4‑yl)‑5‑(4‑methoxyphenyl)isoxazole (3 k)
Yield 72%; mp 200–202 ºC; IR: 1040, 1339, 1549, 1515, 1638, 2839, 2828, 3059;
1H NMR (DMSO-d6, 400 MHz): δ=3.30 (s, 3H, –OCH3), 6.53 (s, 1H,=CH–isox-
azole), 7.87–8.12 (m, 13H, Ar–H–), 8.24 (s, 1H,=CH–N<pyrazole); 13C NMR
(100 MHz, DMSO-d6): δ=55.1, 98.4, 103.1, 114.9 (2), 116.6 (2), 118.1, 119.7
(2), 126.8, 127.7 (2), 128.4, 129.1 (2), 130.8 (2), 131.1, 143.3, 145.1, 160.6, 162.9,
162.6, 169.7; LCMS: m/z 412.1 (28.4%). Anal. Cacld. For C25H18FN3O2: C, 72.98;
H, 4.41; N, 10.21. Found: C, 73.09; H, 4.57; N, 10.28.
Physical constant and characterization of 3‑(3‑(4‑fuorophenyl)‑1‑phenyl‑1H‑
pyrazol‑4‑yl)‑5‑(p‑tolyl)isoxazole (3 l)
Yield 70%; mp 204–206 ºC; IR: 1032, 1345, 1310, 1540, 1512, 1625, 2830, 3040;
1H NMR (DMSO-d6, 400 MHz): δ = 2.30 (s, 3H, –CH3), 6.49 (s, 1H,=CH–isoxa-
zole), 7.36–7.69 (m, 13H, Ar–H–), 8.14 (s, 1H, = CH–N < pyrazole); 13C NMR
(100 MHz, DMSO-d6): δ = 21.8, 98.6, 103.9, 116.8 (2), 119.5 (2), 123.7, 124.3
(2), 126.0, 128.6, 129.6 (2), 129.7 (2), 130.1 (2), 131.8, 131.2, 143.1, 145.3,
162.7, 162.9, 169.0; LCMS: m/z 396.1 (28.8%). Anal. Cacld. For C25H18FN3O: C,
75.93; H, 4.59; N, 10.63. Found: C, 75.99; H, 4.63; N, 10.66.
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Synthesis and molecular docking study of pyrazole clubbed…
Physical constant and characterization of 5‑(2‑chlorophenyl)‑3‑(3‑
(4‑fuorophenyl)‑1‑phenyl‑1H‑pyrazol‑4‑yl)isoxazole (3 m)
Yield 71%; mp 209–211 ºC; IR: 835, 1045, 1306, 1568, 1522, 1636, 2839, 3060;
1H NMR (DMSO-d6, 400 MHz): δ = 6.49 (s, 1H,=CH–isoxazole), 7.30–8.00 (m,
13H, Ar–H–), 8.05 (s, 1H,=CH–N < pyrazole); 13C NMR (100 MHz, DMSO-
d6): δ = 98.1, 103.2, 116.8 (2), 119.6 (2), 126.3, 127.1, 128.6, 129.9, 129.7 (2),
130.7, 130.9 (2), 131.8, 132.9, 136.7, 143.2, 145.9, 162.0, 162.8, 169.6; LCMS:
m/z 404.1 (35.5%). Anal. Cacld. For C24H15ClFN3O: C, 68.58; H, 3.50; N, 10.43.
Found: C, 68.63; H, 3.56; N, 10.49.
Physical constant and characterization of 5‑(4‑chlorophenyl)‑3‑(3‑
(4‑fuorophenyl)‑1‑phenyl‑1H‑pyrazol‑4‑yl)isoxazole (3n)
Yield 68%; mp 156–158 ºC; IR: 850, 1059, 1296, 1563, 1536, 1670, 2852, 3075;
1H NMR (DMSO-d6, 400 MHz): δ = 6.41 (s, 1H, = CH–isoxazole), 7.24–8.10 (m,
13H, Ar–H–), 8.12 (s, 1H, = CH–N < pyrazole); 13C NMR (100 MHz, DMSO-
d6): δ = 98.7, 103.9, 116.7 (2), 119.6 (2), 124.1, 124.9 (2), 126.0, 128.3, 129.1
(4), 130.1 (2), 131.9, 134.0, 143.0, 145.0, 162.0, 162.9, 169.0; LCMS: m/z 417.1
(35.7%); Anal. Cacld. For C24H15ClFN3O: C, 69.32; H, 3.64; N, 10.10. Found: C,
69.36; H, 3.74; N, 10.16.
Physical constant and characterization of 5‑(4‑fuorophenyl)‑3‑(3‑
(4‑fuorophenyl)‑1‑phenyl‑1H‑pyrazol‑4‑yl)isoxazole (3o)
1
Yield 75%; mp 135–137 ºC; IR: 1025, 1260, 1540, 1505, 1605, 2830, 3070; H
NMR (DMSO-d6, 400 MHz): δ = 6.31 (s, 1H, = CH–isoxazole), 7.03–8.06 (m,
13H, Ar–H–), 8.30 (s, 1H,=CH–N < pyrazole); 13C NMR (100 MHz, DMSO-
d6): δ = 97.8, 103.4, 116.5 (4), 119.1 (2), 122.9, 126.0, 127.3 (2), 128.3, 129.6
(2), 130.0 (2), 131.1, 143.2, 145.9, 162.5, 162.9 (2), 169.0; LCMS: m/z 400.1
(27.3%). Anal. Cacld. For C24H15F2N3O: C, 72.17; H, 3.79; N, 10.52. Found: C,
72.29; H, 3.82; N, 10.59.
Physical constant and characterization of 5‑(2,4‑difuorophenyl)‑3‑(3‑
(4‑fuorophenyl)‑1‑phenyl‑1H‑pyrazol‑4‑yl)isoxazole (3p)
1
Yield 73%; mp 151–153 ºC; IR: 1035, 1269, 1549, 1509, 1608, 2830, 3045; H
NMR (DMSO-d6, 400 MHz): δ = 6.21 (s, 1H, = CH–isoxazole), 6.80–8.06 (m,
12H, Ar–H–), 8.50 (s, 1H,=CH–N < pyrazole); 13C NMR (100 MHz, DMSO-d6):
δ = 98.0, 102.1, 103.2, 116.0, 116.1 (2), 119.3, 119.6 (2), 126.0, 128.6, 129.6 (2),
130.1, 130.3 (2), 131.0, 143.9, 145.3, 159.3, 161.1, 162.7, 162.9, 169.2; LCMS:
m/z 418.1 (25.3%). Anal. Cacld. For C24H14F3N3O: C, 69.06; H, 3.38; N, 10.07.
Found: C, 69.10; H, 3.42; N, 10.09.
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N. C. Desai et al.
Physical constant and characterization of 5‑(2,4‑dichlorophenyl)‑3‑(3‑
(4‑fuorophenyl)‑1‑phenyl‑1H‑pyrazol‑4‑yl)isoxazole (3q)
Yield 64%; mp 161–163 ºC; IR: 812, 1053, 1212, 1598, 1523, 1623, 2839, 3053;
1H NMR (DMSO-d6, 400 MHz): δ = 6.21 (s, 1H, = CH- isoxazole), 7.80–8.30 (m,
12H, Ar–H–), 8.69 (s, 1H,=CH–N < pyrazole); 13C NMR (100 MHz, DMSO-d6):
δ = 98.0, 103.0, 116.3 (2), 119.3 (2), 126.4, 127.6, 128.1, 129.1 (2), 130.1, 130.8
(2), 130.8, 131.2, 133.8, 135.0, 135.1, 143.2, 145.3, 162.3, 162.9, 169.4; LCMS:
m/z 450.1 (26.2%). Anal. Cacld. For C24H14Cl2FN3O: C, 64.02; H, 3.13; N, 9.33.
Found: C, 64.12; H, 3.18; N, 9.38.
Physical constant and characterization of 5‑(3,4‑dichlorophenyl)‑3‑(3‑
(4‑fuorophenyl)‑1‑phenyl‑1H‑pyrazol‑4‑yl)isoxazole (3r)
Yield 68%; mp 207–209 ºC; IR: 855, 1049, 1219, 1590, 1512, 1623, 2850, 3053;
1H NMR (DMSO-d6, 400 MHz): δ = 6.28 (s, 1H, = CH–isoxazole), 7.75–8.15 (m,
12H, Ar–H–), 8.42 (s, 1H,=CH–N < pyrazole); 13C NMR (100 MHz, DMSO-d6):
δ = 98.3, 103.1, 116.0, 119.3 (2), 123.1, 126.3, 128.1, 128.8, 129.1 (2), 130.7 (2),
130.9, 131.0, 132.6, 133.8, 143.1, 145.3, 162.9, 162.8, 169.0; LCMS: m/z 451.0
(63.9%). Anal. Cacld. For C24H14Cl2FN3O: C, 64.02; H, 3.13; N, 9.33. Found: C,
64.10; H, 3.19; N, 9.39.
Physical constant and characterization of 5‑(2‑bromo‑4‑chlorophenyl)‑3‑(3‑
(4‑fuorophenyl)‑1‑phenyl‑1H‑pyrazol‑4‑yl)isoxazole (3 s)
Yield 76%; mp 213–215 ºC; IR: 510, 836, 1070, 1249, 1585, 1550, 1678,
2878, 3085; 1H NMR (DMSO-d6, 400 MHz): δ = 6.36 (s, 1H,=CH–isoxa-
zole), 7.52–8.29 (m, 12H, Ar–H–), 8.50 (s, 1H,=CH–N < pyrazole); 13C NMR
(100 MHz, DMSO-d6): δ = 98.1, 103.1, 116.1 (2), 119.3 (2), 121.0, 126.3, 128.6,
128.7, 129.7 (2), 130.1 (2), 130.3, 131.8, 131.9, 135.5, 137.1, 143.3, 145.3, 162.8,
162.7, 169.5; LCMS: m/z 493.0 (75.2%). Anal. Cacld. For C24H14BrClFN3O: C,
58.26; H, 2.85; N, 8.49. Found: C, 58.32; H, 2.96; N, 8.53.
Physical constant and characterization of 4‑(3‑(3‑(4‑fuorophenyl)‑1‑phenyl‑1H‑
pyrazol‑4‑yl)isoxazol‑5‑yl)‑3‑methoxyphenol (3t)
Yield 70%; mp 217–219 ºC; IR: 1064, 1248, 1522, 1536, 1645, 2835, 2010,
1
3074, 3350; H NMR (DMSO-d6, 400 MHz): δ=3.86 (s, 3H,–OCH3), 5.39 (s,
1H,–OH), 6.78 (s, 1H,=CH–isoxazole), 6.35–8.25 (m, 12H, Ar–H–), 8.60 (s,
1H,=CH–N<pyrazole); 13C NMR (100 MHz, DMSO-d6): δ=56.3, 98.5, 103.1,
105.3, 107.3, 108.6, 116.5 (2), 119.1 (2), 126.0, 127.0, 128.6, 129.6 (2), 130.1 (2),
131.1, 143.2, 145.9, 154.6, 158.3, 162.0, 162.8, 169.7; LCMS: m/z 428.1 (28.5%).
Anal. Cacld. For C25H18FN3O3: C, 70.25; H, 4.24; N, 9.83. Found: C, 70.29; H,
4.30; N, 9.93.
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Synthesis and molecular docking study of pyrazole clubbed…
Results and discussion
Chemistry
The need of the hour is to develop simple yet efective methodologies in organic
reactions. Organic chemists are constantly working to fnd newer routes for the
synthesis of bioactive molecules. In continuation, a mixture of 3-(4-fuorophenyl)-
1-phenyl-1H-pyrazole-4-carbaldehyde (1) and derivatives of acetophenone, in the
same ratio, was stirred in methanolic potassium hydroxide solution for 4 h at room
temperature. With the help of the Claisen–Schmidt condensation reaction, chalcones
were produced (2a-t). Then, chalcones and hydroxylamine hydrochloride (0.03 mol)
were refuxed for 15 h at 120○C. This simple condensation reaction produced fnal
compounds (3a-t).
Characterization of newly synthesized compounds of the series was carried out
by IR, NMR, and mass spectra, and the data with a detailed discussion are given in
the experimental section.
IR spectrum of compound 3c (molecular formula C24H15FN4O3, M.W. 426.41
gm/mol) shows an absorption band at 1025 cm−1 due to halogen group -C-F stretch-
ing. Compound 3c has a stretching vibration at 1400 cm−1 indicating the presence
of>C–N stretching corresponding to cyano group. 1550 cm−1 confrms the presence
of the nitro group at the para position. Stretching vibration at 1545 and 1585 cm−1 is
an indication of the presence of –C=N, –C=C stretching of the aromatic ring. The
absorption band at 1660 cm−1 indicates the presence of –C=O stretching due to
isoxazole ring.>C–H stretching vibrations at 2828 and 3045 cm−1 show the pres-
ence of aromatic ring.
1
The H-NMR spectra of the fnal compound 3c showed that the presence of
singlet peak at δ=8.63 ppm was due to one proton of pyrazole ring (C-12). One
proton attached at isoxazole ring (C-2) gave a singlet peak at δ=6.75 ppm. In
7.29–8.15 ppm range, multiplet peaks appeared due to thirteen protons of the aro-
matic ring.
The chemical shifts of the compound 3c have carbons that ranged from
δ=169.1–98.1 ppm. Carbon (C-9) attached to the nitro (Ar–NO2) group has a chem-
ical shift at δ=147.4 ppm. The carbon-21 attached with fuoro group has a chemical
shift value at δ=162.1 ppm. The carbons of the pyrazole ring of carbon C-12, C-13,
and C-14 showed chemical shifts at δ=131.9, 103.3, and 145.2 ppm, respectively.
The carbons of isoxazole ring, C-1, C-2, and C-3, confrmed with the value of the
chemical shift at d=162.8, 98.1, and 169.1 ppm, respectively. Aromatic carbons
showed a chemical shift value between 116.8 and 143.8.
Discussion on antibacterial activity
With the intention of developing potential antibacterial agents, we synthesized
twenty hybrid bioactive molecules. These compounds were screened for their anti-
bacterial activity against gram-positive and gram-negative bacteria by Broth dilu-
tion method. For comparison of activity, we used ampicillin and chloramphenicol as
1 3

N. C. Desai et al.
standard drugs. Details of bacterial strains, MTCC numbers, and antibacterial activ-
ity data are depicted in Table 1. Some of the compounds showed minimum inhibi-
tory concentration (MIC) value less than the standard drug, while some compounds
showed similar values with the standard drug. Compound 3c displayed excellent
activity towards S. pyogenes at MIC 12.5 μg/mL. Compound 3r found very good
towards S. aureus, while compound 3o showed good results towards E. coli and S.
aureus.
Molecular docking
With the increasing number and accuracy of X-ray crystal structures of target pro-
teins, molecular docking has emerged as an important tool for the synthetic elabo-
ration of novel therapeutics based on chemical scafolds [17]. Therefore, to eluci-
date the plausible mechanism by which these novel isoxazole analogs could exhibit
the antibacterial activity and to understand the SAR based on experimental fnd-
ings, molecular docking against DNA gyrase subunit b (PDB ID:1KZN) was per-
formed using the standard protocol implemented in GLIDE (Grid-Based Ligand
Docking with Energetics) module of the Small Drug Discovery Suite (Schrödinger,
LLC, New York, NY) [18–20]. DNA gyrase, encoded by the gyrB gene, catalyses
the ATP-dependent negative supercoiling of double-stranded closed-circular DNA.
Inhibition of this type II topoisomerase enzyme blocks the relaxation of supercoiled
DNA, required for transcription and replication, resulting in disruption of DNA syn-
thesis and, consequently, cell cycle arrest. Being exclusive to prokaryotes and essen-
tial across bacterial species for the survival of the organism qualifes this enzyme as
an attractive target for antibacterial drugs.
Molecular docking showed that the docked conformations of all the heterocy-
clic analogues (3a-3t) could bind within the active site of DNA gyrase with varying
afnities (Table 1) at coordinates proximity to the native structure (Chlorobiocin,
docking score: −9.902) through the formation of various bonded and non-bonded
interactions. An investigation into the per-residue interactions was also performed
to identify the most signifcant thermodynamic interactions. This analysis is elabo-
rated for the most active compound 3c and summarized in Table 2 for other active
compounds (3o and 3r). The lowest energy docked conformation of 3c (Fig. 2a) was
observed to be snugly ftting into the active site of DNA gyrase with a signifcantly
higher binding afnity (Glide score: −8.698, Glide energy: −50.489 kcal/mol). The
compound is seen to be stabilized within the active site through an extensive net-
work of favourable van der Waals (steric) interactions with Arg136, Pro79, Gly77,
Arg76, and Glu50 through the 4-nitro phenylisoxazoles scafold, while the other half
of the molecule, i.e. 3-(4-fuorophenyl)-1-phenyl-1H-pyrazol-4-yl scafold, engaged
in a similar type of favourable interactions with Val167, Thr165, Val120, Ile90,
Ile78, Val71, Ala47, Asn46, and Val43 residues. Furthermore, the higher binding
afnity of 3c is also attributed to signifcant electrostatic (Columbic) interactions
with Arg136, Gly77, Asp73, Glu50, Asp49, and Asn46 residues of the active site.
This balanced network of steric and electrostatic interactions with the active site
residues was observed for other active molecules (3o—Fig. 2b and 3r—Fig. 2c) as
1 3

Synthesis and molecular docking study of pyrazole clubbed…
Table 1 Results of biological activities of compounds (3a-t)
Sr. No
–R
Minimum inhibitory
Glide docking score
concentrations(MIC) in µg/mL
E. c
P. a
S. a
S. p
3a
3b
3c
3d
3e
3f
3 g
3 h
3i
3j
3 k
3 l
3 m
3n
3o
3p
–H
250
250
500
500
250
200
125
200
250
100
100
200
125
200
50
125
200
500
500
200
100
50
250
100
125
200
250
200
125
200
250
100
250
250
250
100
100
200
250
500
250
250
250
50
250
100
62.5
100
200
200
250
250
100
200
250
200
250
250
50
200
250
25
125
200
100
50
250
500
12.5
200
250
500
250
100
250
200
250
250
200
250
500
250
100
200
200
250
–
−6.177
−7.627
−8.698
−7.499
−6.353
−6.548
−7.024
−7.553
−7.442
−7.655
−7.483
−6.478
−6.983
−7.558
−8.584
−6.957
−7.493
−8.565
−6.864
−6.464
–
–3–NO2
–4–NO2
–3–Br
–4–Br
–2–OH
–3–OH
–4–OH
–2–OCH3
–3–OCH3
–4–OCH3
–4–CH3
–2–Cl
–4–Cl
–4–F
–2,4-F2
–2,4–Cl2
–3,4–Cl2
–2–Br–4–Cl
–4–OH–3–OCH3
100
3q
3r
3 s
3t
Ampicillin
Chloramphenicol
50
–
–
E. c.—Escherichia coli (MTCC-443), P. a.—Pseudomonas aeruginosa (MTCC-1688), S. a.—Staphylo-
coccus aureus (MTCC-96), S. p.—Streptococcus pyogenes (MTCC-442)
well. Furthermore, all three actives, i.e. 3c, 3o, and 3r, exhibited a prominent
pi–pi (π–π) stacking interactions with Arg76 residue (3c: 2.492 Å, 3o: 2.496 Å, 3r:
2.66 Å) through the aromatic ring attached to the isoxazole heterocycle which serves
as “anchor” for binding of the ligand to the active site of an enzyme and facilitates
the steric and electrostatic interactions. Overall this in silico binding study suggests
that these novel heterocycles possess an excellent afnity for the DNA gyrase quali-
fying them for structure-based optimization to improve the afnity and selectivity.
Glide docking scores of ampicillin and chloramphenicol are −4.426 and −5.769,
respectively (Scheme 1).
Structure–activity relationship
An overview of the SAR showed that the antimicrobial activity is highly impacted
by the variation of substituents on the aromatic ring of compounds (3a-t). The
1 3

N. C. Desai et al.
1 3

Synthesis and molecular docking study of pyrazole clubbed…
Fig. 2 a Binding mode of 3c into the active site of DNA gyrase subunit b. (On right side, green lines
signify pi–pi stacking interactions.) b Binding mode of 3o into the active site of DNA gyrase subunit b
(on right side: green lines signify pi–pi stacking interactions). c Binding mode of 3r into the active site
of DNA gyrase subunit b (on right side, green lines signify pi–pi stacking interactions). (Color fgure
online)
introduction of electron-withdrawing group at aromatic ring increased antibacterial
potency of the compounds. Nitro group at para position, containing compound 3c ,
showed maximum efcacy against S. pyogenes, i.e. inhibition at MIC 12.5 μg/mL
which is also supported by the signifcantly higher binding afnity (Glide docking
1 3

N. C. Desai et al.
score -8.698). Substitution of halogen (Fluoro) at 4th position (compound 3o) of
the substituted benzene ring also produced very good activity against E. coli and S.
aureus (MIC 50 μg/mL and a docking score of −8.584). Furthermore, di-halogen
substitution (3r:–3,4–Cl2 at meta and para position) also incremented the activity
moderately against S. aureus at MIC 25 μg/mL (docking score of −8.565).
Conclusion
Preparation of 3-(3-(4-fuorophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-arylisoxazoles
was associated with the achievement of fne yield. Results of biological activ-
ity revealed that the electronic environment has a noteworthy impact on the anti-
bacterial activity of the fnal compounds. Out of twenty reported bioactive com-
pounds, 3c (–4–NO2), 3o (–4–F), and 3r (–3,4–Cl2) displayed in vitro antibacterial
activity. Antibacterial screening revealed that electron-withdrawing groups present
in the product have potent activity towards bacterial strains. Furthermore, in sil-
ico binding afnity data against DNA gyrase could provide an insight into the bind-
ing mode and the associated thermodynamic interactions. The outcome will help in
identifying more potent analogs in the coming times.
Scheme 1 Synthetic pathway of reported compounds (3a-t)
1 3

Synthesis and molecular docking study of pyrazole clubbed…
Acknowledgement One of the authors Prof. Nisheeth C Desai is thankful to UGC for awarding BSR
faculty fellowship-2019 (No. F 18-1/2011 (BSR)) and fnancial assistant. The authors are grateful to the
UGC, New Delhi, and Department of Science and Technology, New Delhi (DST-FIST-SR/FST/CSI-
212/2010), for fnancial support under the NON-SAP and DST-FIST programs, respectively. The authors
are also thankful to Schrödinger Inc. for providing the GLIDE program to carry out the molecular dock-
ing. Authors are also thankful to Priyanka Desai, founder of iScribblers for the English editing portion of
the manuscript.
Compliance with ethical standards
Confict of interest The authors declare that they have no confict of interest.
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