Synthesis and biological evaluation of 1,3,4-trisubstituted pyrazole analogues as anti-mycobacterial agents

Article abstract of DOI:10.1007/s00044-017-1821-1

A series of 1,3,4-trisubstituted pyrazole derivatives (3a–f), (4a–r), (5a–f) and (6a–f) have been synthesized and evaluated for their Mycobacterium tuberculosis (MTB) (H37Rv) inhibitory activity. The structures of newly synthesized compounds were characterized by IR, ¹H NMR, ¹³C NMR and mass spectral analysis. Among the thirty six compounds screened for in vitro anti-mycobacterial activity against MTB, three compounds 3b, 3e and 3f demonstrated significant growth inhibitory activity with minimum inhibitory concentration of 0.78 μg/ml. The selected compounds were further tested for cytotoxic activity against normal human dermal fibroblast cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and exhibited no significant cytotoxicity. Molecular docking simulation studies were carried out in order to better understand the hypothetical binding interaction to the MTB NADH-dependent enoyl–acyl carrier protein reductase.

Full text of DOI:10.1007/s00044-017-1821-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1821-1
ORIGINAL RESEARCH
Synthesis and biological evaluation of 1,3,4-trisubstituted pyrazole
analogues as anti-mycobacterial agents
Shankar G. Alegaon1 Mita B. Hirpara1 K. R. Alagawadi1 S. S. Jalalpure2,3
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V. P. Rasal4 Preeti S. Salve1 V. M. Kumbar3
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Received: 18 June 2016 / Accepted: 17 February 2017
© Springer Science+Business Media New York 2017
Abstract A series of 1,3,4-trisubstituted pyrazole deriva-
tives (3a–f), (4a–r), (5a–f) and (6a–f) have been synthe-
sized and evaluated for their Mycobacterium tuberculosis
(MTB) (H37Rv) inhibitory activity. The structures of newly
synthesized compounds were characterized by IR, 1H
NMR, 13C NMR and mass spectral analysis. Among the
thirty six compounds screened for in vitro anti-
mycobacterial activity against MTB, three compounds 3b,
3e and 3f demonstrated significant growth inhibitory activ-
ity with minimum inhibitory concentration of 0.78 µg/ml.
The selected compounds were further tested for cytotoxic
activity against normal human dermal fibroblast cell lines
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-
bromide (MTT) assay and exhibited no significant cyto-
toxicity. Molecular docking simulation studies were carried
out in order to better understand the hypothetical binding
interaction to the MTB NADH-dependent enoyl–acyl carrier
protein reductase.
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Keywords Thiazole Thiadiazole Pyrazole Anti-
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mycobacterial Docking Drug likeness
Introduction
Tuberculosis (TB) is a highly infectious disease claiming
thousands of lives worldwide every year. It is a widespread
airborne disease caused due to the causative agent bacter-
ium Mycobacterium tuberculosis (MTB). TB has been
emerging as a major global health problem accounting for 9
million new cases and 1.5 million deaths during the year
2013 (WHO 2014). According to WHO, about 4.5 million
prevalent and 3.4 million incident cases reside in the South-
East Asia region, with India alone being associated with
21% of world deaths and 23% of world’s incident cases for
TB (WHO 2015). Various first line drugs like isoniazid
(Nydrazid), rifamipin (Rimactane), etambutol (Myambutol),
pyrazinamide (Pyrazide) have been successfully used as
antitubercular agents. However, resurgence of TB in form
of multi drug resistant TB (MDR-TB), extremely drug
resistant TB (XDR-TB) and totally drug resistant TB (TDR-
TB) has resulted in drug resistant-TB strains which no
longer respond to first-line drugs and are inexorably
spreading among populations globally (Abubakar et al.
2013; Lawn et al. 2013). Thus, chemotherapy of TB has
been a subject of immense investigation owing to the
emergence of resistant TB strains and continued rise in
mortality rates due to this disease.
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1821-1) contains supplementary material,
which is available to authorized users.
* Shankar G. Alegaon
sgalegaon@gmail.com
sgalegaon@klepharm.edu
1
Department of Pharmaceutical Chemistry, KLE University’s
College of Pharmacy, Nehru Nagar, Belagavi 590 010 Karnataka,
India
2
Department of Pharmacognosy KLE University’s College of
Pharmacy, Nehru Nagar, Belagavi 590 010 Karnataka, India
3
Dr. Prabhakar Kore Basic Science Research Center, KLE
University, Nehru Nagar, Belagavi 590010 Karnataka, India
4
Department of Pharmacology and Toxicology, KLE University’s
Pyrazole frameworks constitute a primary class of
heterocyclic compounds grabbing a great portion of
College of Pharmacy, Nehru Nagar, Belagavi 590 010 Karnataka,
India

Med Chem Res
limelight in the research field owing to the wide range
of activities displayed by them. Pyrazole derivatives
have displayed diverse biological activities like antic-
ancer, analgesic, anti-inflammatory, antipyretic, anti-
mycobacterial, antifungal, antibacterial, antioxidant,
antiviral (Koca et al. 2013; Chandra et al. 2013; El-Sayed
et al. 2012; Dannhardt and Kiefer 2001; Alegaon et al.
2014a, c; Faraj Khan et al. 2016; Sullivan et al. 2005;
Havrylyuk et al. 2016; Bandgar et al. 2009). Additionally,
they have also been utilized in the treatment of influenza,
depression, diabetes, Alzheimer’s disease and athero-
sclerosis (Gokhan-Kelekci et al. 2009; Shih et al. 2010;
Shen et al. 2011; Liu et al. 2011). Furthermore, thiazoles
represent a key scaffold in heterocyclic chemistry, occu-
pying a place of prime importance in drug development
due to their capability of exhibiting a wide range of
bioactivities such as antifungal, anti-inflammatory, antic-
ancer, antiparasitic, antiviral, antiulcer, antitubercular,
and hypolipidemic (Helal et al. 2013; El-Messery et al.
2012; Cai et al. 2013; Luzina and Popov 2009; Cohen
et al. 2012; Mokale et al. 2010; Makam et al. 2013;
Karuvalam et al. 2012). In view of the diverse biological
activities of 1,3,4-trisubstituted pyrazole compounds and
in an attempt to search new antitubercular agents with
potent MTB inhibitory activity (Alegaon et al. 2012,
2014a). In the present study, we report the design,
synthesis and the structure relationship of series of 1,3,4-
trisubstituted pyrazole derivatives possessing thiazole,
thiazolidinone and thiadiazole moieties as selective MTB
inhibitors.
Chemistry
General procedure for the synthesis of the compounds (4a–r)
Equimolar amount of (3a–c) 1-((3-(substituted)-1-phenyl-
4,5-dihydro-1H-pyrazol-4-yl) methylene)thiosemicarbazide
(5 mmole) or (3d–f) 1-((3-(substituted)-1-phenyl-4,5-dihy-
dro-1H-pyrazol-4-yl)methylene)4-phenylthiosemicarbazide
(5 mmole), substituted phenacyl bromide (5 mmol) were
dissolved in 20 ml of absolute ethanol and stirred 20 h at
80 °C with catalytic amount of acetic acid. The reaction was
monitored by TLC for completion. Reaction mixture was
cooled to room temperature; the solid precipitated was fil-
tered; washed with water; dried, further washed with sol-
vent ether.
(Z)-4-(4-chlorophenyl)-2-(2-((3-(4-chlorophenyl)-1-phenyl-
1H-pyrazol-4-yl) methylene) hydrazinyl) thiazole (4a)
This compound was prepared according to general proce-
dure and it was obtained as Brown solid; yield 32%; m.p.:
183–185 °C; IR (KBr, cm−1): 3334 (N–H), 1602 (C=C),
1506 (C=N), 1348 (C-S).1H NMR (400 MHz, δ, ppm,
DMSO-d6): 7.39–7.41 (m, 2H, Ar–H), 7.54–7.59 (m, 5H,
Ar–H), 7.70–7.76 (m, 4H, Ar–H), 7.90 (d, 2H, J = 7.6 Hz,
Ar–H), 8.20 (s, 1H, thiazole), 8.25 (s, 1H, C=N), 9.18 (s,
1H, pyrazole), 11.31 (s, 1H, NH). 13C NMR (100 MHz, δ,
ppm, DMSO-d6): 117.3, 118.5, 128.7, 129.6, 129.7, 133.3,
134.6, 150.0, 177.5. MS (ESI) m/z = 491 (M+1).
(Z)-4-(4-bromophenyl)-2-(2-((3-(4-chlorophenyl)-1-phenyl-
1H-pyrazol-4-yl) methylene) hydrazinyl)thiazole (4b)
This compound was prepared according to general proce-
dure and it was obtained as yellow colour solid; yield: 58%,
m.p.: 248–250 °C; IR (KBr, cm−1): 3278 (N–H), 1620
Materials and methods
1
(C=C), 1535 (C=N), 1368 (C–S). H NMR (400 MHz, δ,
Chemicals and instrumentation
ppm, DMSO-d6): 7.38–7.90 (m, 2H, Ar–H), 7.53–7.63 (m,
2H, Ar–H), 7.71–7.76 (m, 2H, Ar–H), 7.78–7.81 (m, 3H,
Ar–H), 7.90 (d, 2H, J = 7.6 Hz, Ar–H), 7.99 (d, 2H,
J = 7.6 Hz, Ar–H), 8.15 (s, 1H, thiazole), 8.19 (s, 1H,
C=N), 9.19 (s, 1H, pyrazole), 11.33 (s, 1H, NH). 13C NMR
(100 MHz, δ, ppm, DMSO-d6): 116.9, 118.5, 118.7, 120.4,
121.8, 126.9, 127.4, 129.1, 129.6, 130.0, 130.5, 131.4,
131.4, 131.5, 131.6, 138.9, 149.4. MS (ESI) m/z = 536
(M+1).
All the chemicals used in this study were purchased from
Aldrich, SD fine and Himedia. Melting points were deter-
mined in open capillary tubes and are uncorrected. The
progress of the reactions was monitored by thin-layer
chromatography (TLC) using Merck precoated silica gel
plates (F 254). The IR spectra were recorded on Shimadzu
FTIR spectrophotometer. NMR spectra were recorded on
AMX-400, Bruker-400 liquid-state NMR spectrometer
using tetramethylsilane (TMS) as the internal standard.
Chemical shifts were recorded as δ (ppm). Mass spectra
were measured on an LCMS-2010A. Spectra facilities were
carried out by Sophisticated Analytical Instruments Facility
(SAIF) division of Indian Institute of Science, Bangalore,
India. Compounds (3a–f, 5a–f, and 6a–f) have been pre-
viously reported by our research group (Alegaon et al.
2014b).
(Z)-2-(2-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)hydrazinyl)-4-(4-nitrophenyl)thiazole (4c) This
compound was prepared according to general procedure and
it was obtained as red colour solid; yield: 57%, m.p.:
249–251 °C; IR (KBr, cm−1): 3327 (N–H), 1597 (C=C)
1543 (C=N), 1344 (C–S), 729 (C–Cl). 1H NMR (400 MHz,
δ, ppm, DMSO-d6): 7.38 (t, 1H, J = 7.4 Hz, Ar–H),
7.53–7.61 (m, 2H, Ar–H), 7.70–7.72 (m, 2H, Ar–H), 7.78

Med Chem Res
(s, 1H, thiazole), 7.86–7.91 (m, 2H, Ar–H), 7.99 (d, 2H, J
= 7.6 Hz, Ar–H), 8.11 (d, 2H, J = 8.8 Hz, Ar–H), 8.29 (d,
2H, J = 8.8 Hz, Ar–H), 8.93 (s, 1H, C=N), 9.19 (s, 1H,
pyrazole), 11.33 (s, 1H, NH). 13C NMR (100 MHz, δ, ppm,
DMSO-d6): 116.0, 118.8, 125.2, 127.2, 128.1, 128.3, 128.8,
129.6, 129.8, 130.7, 133.3, 135.4, 138.7, 138.8, 150.4,
175.2. MS (ESI) m/z = 502 (M+1). C25H17ClN6O2S.
(Z)-4-(4-chlorophenyl)-2-(2-((3-(4-methoxyphenyl)-1-phe-
nyl-1H-pyrazol-4-yl) methylene)hydrazinyl)thiazole (4g)
This compound was prepared according to general proce-
dure and it was obtained as brown colour solid; brown solid,
yield: 45%, m.p.: 236–238 °C; IR (KBr, cm−1): 3346
1
(N–H), 1629 (C=C), 1525 (C=N), 1350 (C–S). H NMR
(400 MHz, δ, ppm, DMSO-d6): 3.84 (s, 3H, OCH3), 7.09
(d, 2H, J = 8.8 Hz, Ar–H), 7.38 (t, 1H, J = 7.6 Hz, Ar–H),
7.54 (d, 2H, J = 8 Hz, Ar–H), 7.58 (d, 2H, J = 8.8 Hz,
Ar–H), 7.74 (d, 2H, J = 8.8 Hz, Ar–H), 7.79 (d, 2H, J =
8.4 Hz, Ar–H), 7.97 (d, 2H, J = 8 Hz, Ar–H), 7.98 (s, 1H,
thiazole), 8.15 (s, 1H, C = N), 8.85 (s, 1H, pyrazole), 11.95
(s, 1H, NH). 13C NMR (100 MHz, δ, ppm, DMSO-d6):
55.3, 116.9, 118.7, 125.4, 127.8, 128.5, 128.8, 129.4,
130.0, 150.3, 175.2. MS (ESI) m/z = 487 (M+1).
(Z)-2-(2-((3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene) hydrazinyl)-4-(4-chlorophenyl) thiazole (4d)
This compound was prepared according to general proce-
dure and it was obtained as yellow colour solid; yield: 32%,
m.p.: 238–240 °C; IR (KBr, cm−1): 3334 (N–H), 1600
1
(C=C), 1545 (C=N), 1352 (C–S). H NMR (400 MHz, δ,
ppm, DMSO-d6): 7.39–7.41 (m, 2H, Ar–H), 7.55–7.59 (m,
6H, Ar–H), 7.72 (d, 2H, J = 8.4 Hz, Ar–H), 7.79 (s, 1H,
Ar–H), 7.91 (d, 2H, J = 8.0 Hz, Ar–H), 8.20 (s, 1H, thia-
zole), 8.27 (s, 1H, C=N), 9.19 (s, 1H, pyrazole), 11.33 (s,
1H, NH). 13C NMR (100 MHz, δ, ppm, DMSO-d6): 117.3,
118.5, 127.0, 127.9, 128.7, 129.6, 129.8, 130.9, 133.3,
134.6, 138.9, 150.0, 177.5. MS (ESI) m/z = 536 (M+1),
537 (M+2).
(Z)-4-(4-bromophenyl)-2-(2-((3-(4-methoxyphenyl)-1-phe-
nyl-1H-pyrazol-4-yl) methylene)hydrazinyl)thiazole (4h)
This compound was prepared according to general proce-
dure and it was obtained as yellow colour solid; yield: 74%,
m.p.: 223–225 °C; IR (KBr, cm−1): 3309 (N–H), 1612
1
(C=C), 1504 (C=N), 1396 (C–S). H NMR (400 MHz, δ,
ppm, DMSO-d6): 3.84 (s, 3H, OCH3), 7.07 (d, 2H, J = 8.8
Hz, Ar–H), 7.36 (t, 1H, J = 7.6 Hz Ar–H), 7.54 (d, 2H, J =
8 Hz, Ar–H), 7.58 (d, 2H, J = 8.8 Hz, Ar–H), 7.72 (d, 2H, J
= 8.8 Hz, Ar–H), 7.78 (d, 2H, J = 8.4 Hz, Ar–H), 7.97 (d,
2H, J = 8 Hz, Ar–H), 7.99 (s, 1H, thiazole), 8.15 (s, 1H, C
= N), 8.85 (s, 1H, pyrazole), 11.95 (s, 1H, NH). 13C NMR
(100 MHz, δ, ppm, DMSO-d6): 118.6, 120.5, 124.3, 127.4,
129.6, 130.5, 131.4, 131.8, 132.3, 134.7, 137.7, 138.2,
149.6, 173.9. MS (ESI) m/z = 531 (M+1).
(Z)-4-(4-bromophenyl)-2-(2-((3-(4-bromophenyl)-1-phe-
nyl-1H-pyrazol-4-yl) methylene) hydrazinyl) thiazole (4e)
This compound was prepared according to general proce-
dure and it was obtained as yellow colour solid; yield: 51%,
m.p.: 243–245 °C; IR (KBr, cm−1): 3309 (N–H), 1628
1
(C=C), 1502 (C=N), 1317 (C–S). H NMR (400 MHz, δ,
ppm, DMSO-d6): 7.36–7.39 (m, 3H, Ar–H), 7.53–7.63 (m,
2H, Ar–H), 7.71–7.73 (m, 2H, Ar–H), 7.78–7.81 (m, 2H,
Ar–H), 7.90 (d, 2H, J = 7.6 Hz, Ar–H), 7.99 (d, 2H, J =
7.6 Hz, Ar–H), 8.20 (s, 1H, thiazole), 8.92 (s, 1H, C=N),
9.19 (s, 1H, pyrazole), 11.74 (s, 1H, NH). 13C NMR (100
MHz, δ, ppm, DMSO-d6): 118.6, 127.4, 129.6, 130.0,
130.5, 131.2, 131.5, 131.7, 135.4, 138.5, 138.9, 140.3,
150.3, 173.2. MS (ESI) m/z = 580 (M+1), 581 (M+2).
(Z)-2-(2-((3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)hydrazinyl)-4-(4-nitrophenyl)thiazole (4i) This
compound was prepared according to general procedure and
it was obtained as yellowish-orange colour solid; yield:
60%, m.p.: 248–250°C; IR (KBr, cm−1): 3327 (N–H), 1631
1
(C=C), 1508 (C=N), 1344 (C–S). H NMR (400 MHz, δ,
ppm, DMSO-d6): 3.84 (s, 3H, OCH3) 7.10 (d, 2H, J = 8.8
Hz, Ar–H), 7.37 (t, 1H, J = 7.6 Hz, Ar–H), 7.54 (m, 2H,
Ar–H), 7.71–7.74 (m, 3H, Ar–H), 7.99 (d, 2H, J = 7.6 Hz,
Ar–H), 8.11 (d, 2H, J = 8.8 Hz, Ar–H), 8.16 (s, 1H, thia-
zole), 8.29 (d, 2H, J = 8.8 Hz, Ar–H), 8.87 (s, 1H, pyr-
azole), 12.07 (s, 1H, NH). 13C NMR (100 MHz, δ, ppm,
DMSO-d6): 55.2, 113.9, 118.6, 124.0, 126.2, 129.5, 129.7,
130.6, 133.8, 137.7, 138.5, 150.1, 175.3. MS (ESI) m/z =
497 (M+1).
(Z)-2-(2-((3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)hydrazinyl)-4-(4-nitrophenyl)thiazole (4f) This
compound was prepared according to general procedure and
it was obtained as orange colour solid; yield: 60%, m.p.:
248–250 °C; IR (KBr, cm−1): 3327 (N–H), 1597 (C=C),
1
1504 (C=N), 1344 (C–S). H NMR (400 MHz, δ, ppm,
DMSO-d6): 7.38 (t, 1H, J = 7.6 Hz, Ar–H), 7.53–7.61 (m,
3H, Ar–H), 7.72 (d, 2H, J = 6.4 Hz, Ar–H), 7.86–7.91 (m,
2H, Ar–H), 7.99 (d, 2H, J = 8.0 Hz, Ar–H), 8.09–8.29 (m,
3H, Ar–H), 8.93 (s, 1H, thiazole), 9.19 (s, 1H, C=N), 11.33
(s, 1H, pyrazole), 12.11 (s, 1H, NH). 13C NMR (100 MHz,
δ, ppm, DMSO-d6): 118.7, 124.0, 126.2, 128.5, 129.6,
130.2, 131.5, 135.6, 137.6, 138.2, 150.3, 172.2. MS (ESI)
m/z = 546 (M+1).
(Z)-4-(4-chlorophenyl)-2-((Z)-((3-(4-chlorophenyl)-1-phe-
nyl-1H-pyrazol-4-yl) methylene) hydrazono)-3-phenyl-2,3-
dihydrothiazole (4j) This compound was prepared
according to general procedure and it was obtained as yel-
low colour solid; yield: 50%, m.p.: 248–250 °C; IR (KBr,

Med Chem Res
cm−1): 3067 (Ar–H), 1598 (C=C), 1504 (C=N), 1350
(C–S). H NMR (400 MHz, δ, ppm, DMSO-d6): 7.22–7.25
135.9, 138.4, 138.9, 151.3, 173.6. MS (ESI) m/z = 611 (M
+1), 612 (M+2).
1
(m, 2H, Ar–H), 7.37–7.41 (m, 4H, Ar–H), 7.55–7.63 (m,
5H, Ar–H), 7.69 (d, 2H, J = 8.4 , thiazole), 9.24 (s, 1H,
C=N), 9.79 (s, 1H, pyrazole), 11.75 (s, 1H, NH). 13C NMR
(100 MHz, δ, ppm, DMSO-d6): 116.9, 118.7, 122.0, 125.2,
127.2, 128.2, 128.3, 129.7, 130.2, 131.3, 131.7, 135.4,
138.7, 138.9, 150.3, 175.2. MS (ESI) m/z = 567 (M+1).
(Z)-4-(4-bromophenyl)-2-((Z)-((3-(4-bromophenyl)-1-phe-
nyl-1H-pyrazol-4-yl) methylene)hydrazono)-3-phenyl-2,3-
dihydrothiazole (4n) This compound was prepared
according to general procedure and it was obtained as yel-
low colour solid; yield: 55%, m.p.: 236–238 °C; IR (KBr,
cm−1): 3140 (N–H), 1598 (C=C), 1546 (C=N), 1352
1
(Z)-4-(4-bromophenyl)-2-((Z)-((3-(4-chlorophenyl)-1-phe-
nyl-1H-pyrazol-4-yl)methylene)hydrazono)-3-phenyl-2,3-
dihydrothiazole (4k) This compound was prepared
according to general procedure and it was obtained as yel-
low colour solid; yield: 54%, m.p.: 230–232 °C; IR (KBr,
cm−1): 3052 (Ar–H), 1624 (C=C), 1546 (C=N), 1323
(C–S). H NMR (400 MHz, δ, ppm, DMSO-d6): 7.21–7.24
(m, 2H, Ar–H), 7.37–7.41 (m, 4H, Ar–H), 7.51–7.61
(m, 5H, Ar–H), 7.70 (d, 2H, J = 8.0 Hz, Ar–H), 7.76 (d, 2H,
J = 8.4 Hz, Ar–H), 7.92 (d, 2H, J = 8.0 Hz, Ar–H), 8.32
(s, 1H, thiazole), 9.25 (s, 1H, C=N), 9.80 (s, 1H, pyrazole),
11.75 (s, 1H, NH). 13C NMR (100 MHz, δ, ppm, DMSO-
d6): 117.0, 118.7, 125.3, 127.2, 128.2, 128.8, 129.6, 129.9,
133.4, 138.7, 138.9, 150.3, 175.2. MS (ESI) m/z = 656 (M
+1), 657 (M+2).
1
(C–S). H NMR (400 MHz, δ, ppm, DMSO-d6): 7.22–7.26
(m, 2H, Ar–H), 7.33–7.41 (m, 4H, Ar–H), 7.49–7.61
(m, 5H, Ar–H), 7.68–7.76 (m, 4H, Ar–H), 7.92 (d, 2H,
J = 7.6 Hz, Ar–H), 8.32 (s, 1H, thiazole), 9.25 (s, 1H,
C=N), 9.79 (s, 1H, pyrazole), 11.75 (s, 1H, NH).13C NMR
(100 MHz, δ, ppm, DMSO-d6): 150.3, 138.9, 138.7, 135.4,
133.4, 130.9, 129.9, 129.6, 128.8, 128.3, 128.2, 127.2,
125.3, 118.7, 117.0. 175.2. MS (ESI) m/z = 612 (M+1),
613 (M+2).
(Z)-2-((Z)-((3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)hydrazono)-4-(4-nitrophenyl)-3-phenyl-2,3-
dihydrothiazole (4o) This compound was prepared
according to general procedure and it was obtained as
brown colour solid; yield: 60%, m.p.: 240–242 °C; IR (KBr,
cm−1): 3060 (Ar–H), 1597 (C=C), 1546 (C=N), 1348
1
(Z)-2-((Z)-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)hydrazono)-4-(4-nitrophenyl)-3-phenyl-2,3-
dihydrothiazole (4l) This compound was prepared
according to general procedure and it was obtained as brown
colour solid; yield: 60%, m.p.: 235–237 °C; IR (KBr, cm−1):
3026 (Ar–H), 1597 (C=C), 1546 (C=N), 1348 (C–S), 729
(C–S), 630 (C–Br). H NMR (400 MHz, δ, ppm, DMSO-
d6): 7.22 (t, 1H, J = 7.4 Hz, Ar–H), 7.39 (m, 3H, Ar–H),
7.55–7.64 (m, 4H, Ar–H), 7.69 (m, 5H, Ar–H), 7.74 (d, 2H,
J = 8.4 Hz, Ar–H), 7.92 (d, 2H, J = 8.0 Hz, Ar–H), 8.31 (s,
1H, thiazole), 9.24 (s, 1H, C=N), 9.79 (s, 1H, pyrazole),
11.75 (s, 1H, NH). 13C NMR (100 MHz, δ, ppm, DMSO-
d6): 116.9, 118.7, 125.2, 128.2, 128.3, 129.6, 130.2, 131.7,
175.2, MS (ESI) m/z = 622 (M+1).
1
(C–Cl). H NMR (400 MHz, δ, ppm, DMSO-d6): 7.21 (d,
2H, J = 7.6Hz, Ar–H), 7.37–7.41 (m, 4H, Ar–H), 7.55–7.62
(m, 5H, Ar-H), 7.69 (d, 2H, J = 8.4 Hz, Ar-H), 7.74 (d, 2H,
J = 8.4 Hz, Ar–H), 7.92 (d, 2H, J = 7.6 Hz, Ar–H), 8.31 (s,
1H, thiazole), 9.24 (s, 1H, C=N), 9.77 (s, 1H, pyrazole),
11.74 (s, 1H, NH). 13C NMR (100 MHz, δ, ppm, DMSO-
d6): 125.3, 128.2, 129.7, 130.2, 131.7, 132.6, 135.1, 138.3,
138.9, 150.7, 171.8. MS (ESI) m/z = 578 (M+1).
(Z)-4-(4-chlorophenyl)-2-((Z)-((3-(4-methoxyphenyl)-1-
phenyl-1H-pyrazol-4-yl) methylene)hydrazono)-3-phenyl-
2,3-dihydrothiazole (4p) This compound was prepared
according to general procedure and it was obtained as yel-
low colour solid; yield: 25%, m.p.: 228–230 °C; IR (KBr,
cm−1): 3040 (Ar–H), 1612 (C=C), 1525 (C=N), 1350
1
(Z)-2-((Z)-((3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)hydrazono)-4-(4-chlorophenyl)-3-phenyl-2,3-
dihydrothiazole (4m) This compound was prepared
according to general procedure and it was obtained as yel-
low colour solid; yield: 46%, m.p.: 238–240 °C; IR (KBr,
cm−1): 3050 (Ar–H), 1597 (C=C), 1546 (C=N), 1323
(C–S). H NMR (400 MHz, δ, ppm, DMSO-d6): 3.76 (s,
3H, OCH3), 6.69 (s, 1H, thiazole), 6.99–7.10 (m, 3H,
Ar–H), 7.35 (m, 2H, Ar–H), 7.42 (d, 2H, J = 8 Hz, Ar–H),
7.56 (d, 2H, J = 7.6 Hz, Ar–H), 7.71–7.78 (m, 3H, Ar–H),
7.89–7.96 (m, 3H, Ar–H), 8.33 (d, 2H, J = 8.4 Hz, Ar–H),
8.57 (s, 1H, C=N), 9.02 (s, 1H, pyrazole), 9.67 (s, 1H, NH).
13C NMR (100 MHz, δ, ppm, DMSO-d6): 55.9, 116.3,
118.8, 126.4, 127.6, 128.5, 128.8, 129.4, 129.6, 149.5,
160.0. MS (ESI) m/z = 563 (M+1).
1
(C–S). H NMR (400 MHz, δ, ppm, DMSO-d6): 7.20 7.24
(m, 3H, Ar–H), 7.37–7.39 (m, 3H, Ar–H), 7.55–7.59 (m,
5H, Ar–H), 7.67 (d, 2H, J = 8.4 Hz, Ar–H), 7.72 (d, 2H, J
= 8.4 Hz, Ar–H), 7.90 (d, 2H, J = 7.6 Hz, Ar–H), 8.31 (s,
1H, thiazole), 9.24 (s, 1H, C=N), 9.77 (s, 1H, pyrazole),
11.74 (s, 1H, NH). 13C NMR (100 MHz, δ, ppm, DMSO-
d6): 116.9, 118.7, 125.3, 128.2, 129.7, 130.2, 131.7, 135.4,
(Z)-4-(4-bromophenyl)-2-((Z)-((3-(4-methoxyphenyl)-1-
phenyl-1H-pyrazol-4-yl) methylene)hydrazono)-3-phenyl-
2,3-dihydrothiazole (4q) This compound was prepared

Med Chem Res
according to general procedure and it was obtained as yel-
low colour solid; yield: 65%, m.p.: 230–232 °C; IR (KBr,
cm−1): 3068 (Ar–H), 1631 (C=C), 1525 (C=N), 1359
to pink. Isoniazid, Pyrazinamide, Ciprofloxacin was used as
the reference agent.
1
(C–S). H NMR (400 MHz, δ, ppm, DMSO-d6): 3.84 (s,
Cytotoxic activity evaluation
3H, OCH3), 7.07 (s, 1H, thiazole), 7.10 (d, 2H, J = 8.8 Hz,
Ar–H), 7.34–7.38 (m, 3H, Ar–H), 7.52–7.56 (m, 4H,
Ar–H), 7.60 (d, 2H, J = 8.4 Hz, Ar–H), 7.74 (d, 2H, J =
8.4 Hz, Ar–H), 7.81 (d, 2H, J = 8.4 Hz, Ar–H), 7.99 (d, 2H,
J = 8.0 Hz, Ar–H), 8.15 (s, 1H, C=N), 8.85 (s, 1H, pyr-
azole), 11.96 (s, 1H, NH). 13C NMR (100 MHz, δ, ppm,
DMSO-d6): 55.2, 113.9, 117.3, 118.5, 125.1, 127.1, 127.4,
129.5, 129.9, 130.1, 131.4, 150.3, 175.7. MS (ESI) m/z =
607 (M+1).
In vitro cytotoxicity was determined using a standard MTT
assay (Mosmann 1983) with protocol appropriate for the
individual test system. Test compounds were prepared prior
to the experiment by dissolving in 0.1% DMSO and diluted
with medium. The cells were then exposed to different
concentrations of the drugs. Cells in the control wells
received the same volume of medium containing 0.1%
DMSO. After 24 h, the medium was removed and cell
cultures were incubated with 100 µM MTT reagent (1 mg/
mL) for 5 h at 37 °C. The suspension was placed on
microvibrator for 10 min and absorbance was recorded by
the ELISA reader.
(Z)-2-((Z)-((3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)methylene)hydrazono)-4-(4-nitrophenyl)-3-phenyl-2,3-
dihydrothiazole (4r) This compound was prepared
according to general procedure and it was obtained as
brown colour solid; yield: 36%, m.p.: 243–245 °C; IR (KBr,
cm−1): 3062 (Ar–H), 1685 (C=C), 1556 (C=N), 1346 (C-
Molecular docking study
The molecular docking study was performed by using
AutoDock tools 1.5.6, running on A8 VISION AMD A8-
4500M APU with Radeon(tm) HD Graphics 1.90 GHz,
RAM Memory 4GB under Windows 8 pro system. The
crystal structure of the MtInhA was downloaded from
Protein Data Bank (PDB ID: 1P44).
1
S). H NMR (400 MHz, δ, ppm, DMSO-d6): 3.76 (s, 3H,
OCH3), 6.62 (s, 1H, thiazole), 6.97–6.99 (m, 3H, Ar–H),
7.34–7.39 (m, 3H, Ar–H), 7.42 (d, 2H, J = 8 Hz, Ar–H),
7.55 (d, 2H, J = 7.6 Hz, Ar–H), 7.71–7.78 (m, 2H, Ar–H),
7.89–7.96 (m, 3H, Ar–H), 8.33 (d, 2H, J = 8.4 Hz, Ar–H),
8.57 (s, 1H, C=N), 9.02 (s, 1H, pyrazole), 9.67 (s, 1H, NH).
13C NMR (100 MHz, δ, ppm, DMSO-d6): 55.3, 116.3,
118.8, 125.4, 126.3, 127.3, 127.4, 128.5, 129.4, 129.6,
130.0, 150.3. 175.7, MS (ESI) m/z = 573 (M+1).
Results and discussion
Chemistry
Antimycobacterial inhibitory activity
The 1,3,4-trisubstituted pyrazole analogues (3a–f), (4a–r),
(5a–f) and (6a–f) were synthesized according to the reaction
sequence presented in Schemes 1 and 2. Some of these
compounds (3a–f, 5a–f and 6a–f) have been previously
reported by our research group (Alegaon et al. 2014b).
Those that have not (4a–r) were synthesized by reaction
sequences which are briefly presented in the following
sections. The aryl hydrazone derivatives (1a–c) were pro-
duced by condensation of the phenyl hydrazine with
appropriate acetophenones. Treatment of the phenyl
hydrazones (1a–c) with Vilsmeir–Haack reagent
(DMF–POCl3) afforded the corresponding 1,3-diphenyl-
1H-pyrazole-4-carbaldehydes (2a–c). Thus obtained 4-
formylpyrazole derivatives (2a–c) were then condensed
with thiosemicarbazide or 4-phenylthiosemicarbazide in
ethanol:chloroform (7:3) in presence of catalytic amount of
concentrated hydrochloric acid under reflux for 10 h, thio-
semicarbazone derivatives (3a–f) were obtained in moder-
ate to good yields. The thiosemicarbazone derivatives
(3a–f) when allowed to react with substituted phenacyl
bromides in refluxed dry ethanol gave target thiazole
The antitubercular activities of newly synthesized com-
pounds have been assessed against Mycobacterium tuber-
culosis H37Rv (ATCC 27294) using the Micro plate Alamar
Blue assay (MABA) method (Franzblau et al. 1998). This
methodology is nontoxic, uses a thermally-stable reagent.
The method is described as follows: 200 µl of sterile deio-
nized water was added to all outer-perimeter wells of
96 sterile well plates to minimize evaporation of the med-
ium in the test wells during incubation. The 96 plates
received 100 µl of the Middlebrook 7H9 broth and succes-
sive dilution of the compounds was made directly on the
plate. The final drug concentrations tested were 100 to
0.195 µg/ml. Plates were covered and sealed with parafilm
and incubated at 37 °C for 5 days. Twenty five micro litre of
a freshly prepared 1:1 mixture of Alamar Blue reagent and
10% tween 80 was then added to the plate and incubated for
24 h. A blue colour in the well was interpreted as no bac-
terial growth, and a pink colour was scored as growth. The
activity is expressed as MIC, i.e., the lowest concentration
of compound which prevented the colour change from blue

Med Chem Res
Scheme 1 Reagents and
conditions: a dry ethanol, glacial
acetic acid (1 mL) reflux 5 h; b
DMF/POCl3, 80 °C; c
thiosemicarbazide or 4-
phenylthiosemicarbazide,
ethanol: chloroform (7:3), reflux
10 h; d substituted phenacyl
bromide, dry ethanol, reflux 24 h
Comp
3a
3b
3c
3d
3e
3f
R 1
Cl
Br
OCH 3
Cl
Br
OCH 3 C6H5
Cl
Cl
R 2
H
H
H
C6H5
C6H5
R 3
-
-
-
-
-
-
Cl
Br
Comp
4c
4d
4e
4f
4g
4h
4i
R 1
Cl
Br
Br
Br
OCH 3
OCH 3
OCH 3
Cl
R 2
H
H
H
H
H
H
H
C6H5
R 3
NO2
Cl
Br
NO2
Cl
Br
NO2
Cl
Comp
4k
4l
4m
4n
4o
4p
4q
4r
R 1
Cl
Cl
Br
Br
Br
R 2
C6H5
C6H5 NO2
C6H5
C6H5
R 3
Br
Cl
Br
C6H5 NO2
OCH 3 C6H5
OCH 3 C6H5
OCH 3 C6H5 NO2
Cl
Br
4a
4b
H
H
4j
Scheme 2 Reagents and
conditions: a ethylbromoacetate,
anhydrous sodium acetate,
glacial acetic acid, reflux 8 h;
b acetic anhydride, reflux 5 h
Comp
R 1
Cl
Br
R 2 Comp
R 1
Cl
Br
R 2 Comp
R 1
Cl
Br
R 2 Comp
R 1
Cl
Br
R 2
C6H5
C6H5
5a
5b
5c
H
H
H
5d
5e
5f
C6H5
C6H5
6a
6b
6c
H
H
H
6d
6e
6f
OCH 3
OCH 3 C6H5
OCH 3
OCH 3 C6H5
derivatives (4a–r). The structures of 1,3,4-trisubstituted
pyrazole derivatives (3a–f and 4a–r) were characterized by
IR, H NMR, and mass spectral analysis. The IR spectrum
tautomerism, while these protons disappeared in com-
pounds (4a–r). 13C NMR spectrum of compounds (4a–r)
revealed signals at δ 105 ppm (thiazole-C5) and 174 ppm
(thiazole-C2). Finally, formation of these thiazole deriva-
tives was also evidenced by mass spectral data.
IR spectra of (6a–i) exhibited a sharp band between 3310
and 3395 cm−1, characteristic for NH stretching. The
stretching band of the C=N appeared between 1550 and
1
of thiosemicarbazone derivatives (3a–f) showed absorption
peak at the range 3395 and 3294 cm−1 due to amino (NH)
1
stretching. However, H NMR spectrum of thiosemicarba-
zone derivatives (3a–c) evidenced characteristic two sing-
lets at the range 7.7 and 8.2 ppm due to en-thiol

Med Chem Res
1585 cm−1, and their proton NMR spectroscopic analysis
revealed diagnostic singlet signal at around δ 8.2 ppm
attributed to H5-thiazole. 13 C NMR spectrum of com-
pounds (6a–l) revealed signals at δ 104.9 (thiazole-C5) and
175.6 (thiazole-C2). Finally, formation of these compounds
was also evidenced by mass spectral data.
positions R1 and R3 have displayed moderate activity at
MIC 12.5 µg/ml. Similar activity has been displayed by
compounds 4p–r which possess electronegative groups (Cl,
Br, NO2) attached at R3 position and electropositive group
(–OCH3) attached at R1 position. The other thiazoles 4a–h
with H substitution at R2 position have all displayed an
activity of >25 µg/ml MIC value irrespective of the sub-
stitutions at positions R1 and R3, except 4i (MIC = 3.12 µg/
ml) which has an electron donating methoxy group at R1
position and electron accepting NO2 group at R3 position.
All the synthesized thiazolidinones 5a–f displayed an MIC
of >25 µg/ml. Amongst the synthesized thiadiazoles com-
pounds, 6c and 6d displayed significant antitubercular
activity at MIC of 3.12 µg/ml. 6c had OCH3 attached to the
R1 position, whereas 6d showed Cl at R1 and phenyl ring at
R2 position. Compounds 6e with Br and 6f with OCH3 at R1
position showed moderate activity at an MIC of 12.5 µg/ml
and compounds 6a with Cl and 6b with Br at R1 position
displayed an MIC of >25 µg/ml. In general, the promising
Mycobacterium tuberculosis inhibitory activity of com-
pounds 3b, 3e and 3f may be due to selectivity towards
antitubercular activity.
Antimycobacterial inhibitory activity
The new 2-(2-((1,3-diphenyl-1H-pyrazol-4-yl) methylene)
hydrazinyl)-4-phenylthiazole (4a–i), and 2-((1,3-diphenyl-
1H-pyrazol-4-yl) methylene) hydrazono-3,4-diphenyl-2,3-
dihydrothiazole (4j–r), along with previously reported
thiosemicarbazone derivatives (3a–f), 4-thiazolidinone
compounds (5a–f) and 1,3,4-thiadiazole derivatives
(6a–f), were evaluated against MTB H37Rv (ATCC 27294)
by using the Microplate Alamar Blue Assay (MABA). The
results of the studies on antitubercular activity of the com-
pounds are as given in Table 1. Twenty two out of thirty six
pyrazole compounds showed moderate to good anti-
mycobacterial activity (MIC range 0.78 to 25 µg/ml) as
compared to the standard pyrazinamide (MIC 50 µg/ml) and
ciprofloxacin (MIC 12.5 µg/ml). From the results it was
observed that the 2-((1,3-diaryl-pyrazol-4-yl) methylene)
hydrazine carbothioamide showed better antimycobacterial
inhibitory activity as compared to the other tested com-
pounds. Amongst all the pyrazoles examined, six com-
pounds, 3a, 3b, 3c, 3d, 3e and 3f inhibited MTB with MIC
ranging 0.78 to 1.56 µg/ml, and five compounds 4i, 4j, 4o,
6c and 6d inhibited MTB with MIC of 3.12 µg/ml. When
compared to the first line anti-TB drug isoniazid (MIC =
1.56 µg/ml), three compounds 3b, 3e and 3f are found to be
more potent than isoniazid, and other three compounds 3a,
3c and 3d are found to be equally active as isoniazid.
Furthermore, thiosemicarbazone 3a, 3b, 3c, 3d, 3e and 3f
compounds exhibited positive drug-likeness score of 0.6,
0.19, 0.3, 0.46, 0.05 and 0.12, respectively, Table 1. In the
pyrazole-thiazole hybrid, compounds 4i, 4j and 4o exhib-
ited significant activity at 3.12 µg/ml. Compounds 4k–n and
4p–r displayed moderate antitubercular activity at 12.5 µg/
ml, compounds 4g and 4h and 3d displayed antitubercular
activity at an MIC of 25 µg/ml, whereas compounds 4a–f
showed activity at >25 µg/ml. All the thiazoles with
3,4-diarylthiazole system (4j–r) like 2-((1,3-diphenyl-pyr-
azol-4-yl) methylene) hydrazono)-3,4-diphenyl-2,3-dihy-
drothiazoles exhibited better antitubercular activity as
compared to 4-arylthiazole containing compounds like 2-(2-
((1,3-diaryl-pyrazol-4-yl) methylene) hydrazinyl)-4-phe-
nylthiazole (4a–i).
Cytotoxicity activity
The selected compounds were further tested for cytotoxic
activity against normal HDF cell lines using MTT assay to
assess the safety of these synthesized compounds.
According to the literature on drug susceptibility of TB, the
antitubercular activity was considered to be specific with
selectivity index >10.35 (Hartkoorn et al. 2007; Subhedar
et al. 2016). From the data it was found that compounds 4g,
(5a–f), 6a, and 6b displayed selectivity index <10 against
MTB. The compounds that showed selectivity index value
more than 10 were categorized non-toxic agents. Com-
pounds (3a–f), (4h–r), 6c, 6d, 6e and 6 f showed selectivity
index higher than >200 against MTB. The cytotoxicity
selectivity index results were potted in Table 1.
Molecular docking study
According to the study of Kuo and coworkers (Kuo et al.
2003) pyrazole analogs (Genz-8575) are active inhibitors of
MTB NADH-dependent enoyl–acyl carrier protein reduc-
tase (MtInhA). Sullivan et al. identified series of pyrazole
compounds as a potent MtInhA inhibitors (Sullivan et al.
2005). In addition (Nayak et al. 2015), Nayak et al. reported
that 1,3,4-trisubstituted pyrazole structure and anti-
mycobacterial activity relationship was well documented
by docking study against MtInhA (PDB ID: 1P44). Based
on these proofs, a molecular docking study was carried out
for the most active compounds against Mycobacterium
tuberculosis NADH-dependent enoyl–acyl carrier protein
The moderate active compounds of the series 4i, 4j and
4o have an electronegative (4-Cl and 4-NO2) substitution at
the para position of phenyl ring (R3 position). Compounds
4k–n with electronegative groups (Cl, Br, NO2) attached at

Med Chem Res
Table 1 Antimycobacterial
activity data, cytotoxicity
activity data, selectivity index
data, and drug-likeness data of
the pyrazole analogues
Comp. R1
R2
R3
MIC (µg/ml) in
MTB H37Rv
IC50 (µg/ml)
HDF cells
Selectivity index
(SI) value
Drug-likenessa
model score
3a
3b
3c
Cl
H
–
1.56
0.78
1.56
1.56
0.78
0.78
>25
>25
399.05
320.56
352.49
343.03
326.74
292.75
ND
>200
>400
>200
>200
>400
>300
ND
0.60
0.19
Br
H
–
OCH3
Cl
H
–
0.30
3d
3e
C6H5
C6H5
–
0.46
Br
–
0.05
3f
OCH3 C6H5
–
0.12
4a
4b
4c
Cl
H
H
H
H
H
H
H
H
H
Cl
Br
0.01
Cl
ND
ND
−0.25
−0.13
−0.25
−0.36
−050
0.32
Cl
NO2 >25
ND
ND
4d
4e
Br
Cl
Br
>25
>25
ND
ND
Br
ND
ND
4f
Br
NO2 >25
ND
ND
4g
4h
4i
OCH3
OCH3
OCH3
Cl
Cl
Br
25
25
130.7
316.8
216.7
306.3
294.7
151.2
209.6
166.2
92.3
5.22
12.67
69.45
98.17
23.57
12.09
16.76
13.29
29.58
20.136
10.72
11.76
6.50
−0.05
−0.39
−0.01
−0.20
−0.20
−0.28
−0.40
−0.59
0.32
NO2 3.12
4j
C6H5 Cl
C6H5 Br
3.12
12.5
4k
4l
Cl
Cl
C6H5 NO2 12.5
4m
4n
4o
4p
4q
4r
Br
C6H5 Cl
C6H5 Br
12.5
12.5
Br
Br
C6H5 NO2 3.12
OCH3 C6H5 Cl
OCH3 C6H5 Br
12.5
12.5
251.7
134.8
147.0
329.21
311.78
334.42
348.36
329.41
363.86
321.17
287.02
315.22
343.03
285.74
341.37
ND
−0.08
−0.51
0.83
OCH3 C6H5 NO2 12.5
5a
5b
5c
Cl
H
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
>25
>25
>25
>25
>25
>25
>25
>25
3.12
3.12
12.5
12.5
1.56
50.0
12.5
Br
H
6.23
0.41
OCH3
Cl
H
6.68
0.38
5d
5e
C6H5
C6H5
6.96
0.30
Br
6.58
−0.10
−0.10
1.03
5f
OCH3 C6H5
7.27
6a
6b
6c
Cl
H
6.42
Br
H
5.74
0.62
OCH3
Cl
H
>100
>100
22.85
27.30
ND
0.49
6d
6e
C6H5
C6H5
0.55
Br
0.14
6f
OCH3 C6H5
0.11
INH
Pyra
Cipro
–
–
–
–
–
–
0.53
ND
ND
−0.15
0.93
ND
ND
MIC minimum inhibitory concentration, IC50 concentration to inhibit 50% growth of cells, Selectivity index
(in vitro) IC50 against vero cells/MIC against Mycobacterium tuberculosis (MTB), INH isoniazid, Pyra
pyrazinamide, Cipro ciprofloxacin
a
Calculated using molsoft (http://molsoft.com/mprop/)
reductase. Molecular docking study was performed by using
AutoDock tools 1.5.6 (AutoDock 2015). The 3D structures
of compounds in pdb form were prepared by using Marvin
6.2.1 ChemAxon (Marvin 2016). The 3D crystal structure
of MtInhA was downloaded from Protein Data Bank (PDB
ID: 1P44). All hetero atoms, water molecules were removed
and hydrogen was added to the protein for correct calcu-
lation of partial atomic charges. The grid box was set at 60,

Med Chem Res
60 and 60 Å (x, y and z) with a spacing 0.375 Å, were
created. The Lamarckin genetic algorithm was applied to
search conformers that possessed lowest binding energy.
Results of molecular docking analysis were obtained as
estimated free energy of binding in kcal/mol. The molecular
docking of the ligands 3a–f reveals that all the inhibitors
compounds are exhibiting the bonding with one or more
amino acids in active pockets. The active compounds
showed docking score in the range of −7.34 to −9.6 kcal/
mol (Table 2). The amino hydrogen atom of semicarbazone
group of compounds 3b, 3c and 3e forms hydrogen bonds
(N–H----O: GLY 96) with the oxygen atom of GLY 96
residue, whereas, The amino hydrogen atom of semi-
carbazone group of compounds 3d and 3f forms hydrogen
bonds (N–H----O: GLY 192) with the oxygen atom of GLY
192 residue, nitrogen atom of compounds 3e and 3f forms
hydrogen bonds (N----H–O: ILE 194) with the hydroxyl
hydrogen atom of ILE 194. Compounds 3a and 3d forms
hydrogen bonds (N----H–N: ILE 194) with the amino
hydrogen atom of ILE 194 also amino hydrogen atom of
compound 3a forms hydrogen bonds (N–H----O: ILE 194)
with the amino hydrogen atom of ILE 194 (Fig. 1). In
general, the molecular docking studies results were essen-
tially in agreement with the antitubercular data in terms of
evaluation of binding energy and binding mode. Further-
more, analysis of molecular parameters was done that
connected with Lipinski’s rule of five and drug likeness
model score (Lipinski et al. 2001; Molinspiration Che-
minformatics 2015; Drug-Likeness 2016). Briefly, this
simple rule is based upon the observation that most of
medicinal agents have a molecular weight (MW) of 500 or
less, a log p not more than 5, number of hydrogen bond
donor atoms of 5 or less and number of hydrogen bond
accepter atoms of 10 or less (N or O) (Table 3). Results
showed that most of the synthesized compounds obey the
Table 2 Binding energy of compounds (3a–f) after docking in to
Mycobacterium tuberculosis NADH-dependent enoyl-acyl carrier
protein reductase
Compound
Binding energy
Hydrogen bonding residues
3a
−7.93
3a: NH---O:ILE 194
3a: N---HN: ILE 194
3b: NH---O: GLY96
3b: NH---O: GLY96
3c: NH---O: GLY96
3c: NH---O: GLY96
3d: NH---O:GLY192
3d: N---HN: ILE194
3e: NH---O: GLY96
3e: N---HO: ILE194
3f: NH---O:GLY192
3f: N---HO: ILE194
3b
3c
3d
3e
3f
−7.57
−7.34
−9.6
−9.45
−8.56
Fig. 1 Docking figures of (3a–f) in Mycobacterium tuberculosis NADH-dependent enoyl–acyl carrier protein reductase

Med Chem Res
Table 3 Molecular parameters
of 1,3,4-trisubstituted pyrazole
derivatives (3a–f), (4a–r),
(5a–f) and (6a–f)
Compound
Rule of 5
TPSA
n-rotb
nON
nOHNH
miLogp
≤5
MW
Rule of 5 violation
–
–
≤10
≤5
≤500
≤1
3a
3b
3c
68.24
68.24
77.47
54.24
54.24
63.47
55.11
55.11
100.93
55.11
55.11
100.93
64.34
64.34
110.17
46.32
46.32
92.14
46.32
46.32
92.14
55.55
55.55
101.38
71.65
72.65
80.88
62.86
62.86
72.09
79.59
79.59
88.31
70.80
70.80
80.04
68.013
68.878
74.569
5
5
6
7
7
8
6
6
7
6
6
7
7
7
8
7
7
8
7
7
8
8
8
9
5
5
6
5
5
6
4
4
5
5
5
6
1
1
3
5
5
6
5
5
6
5
5
8
5
5
8
6
6
9
5
5
8
5
5
8
6
6
9
6
6
7
6
6
7
7
7
8
7
7
8
4
4
6
3
3
3
2
2
2
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
1
1
1
0
0
0
1
1
1
0
0
0
3
2
2
3.88
4.01
3.26
5.31
5.45
4.69
6.64
6.77
5.92
6.77
6.90
6.05
6.02
6.15
5.30
8.46
8.54
7.86
8.54
8.61
7.99
7.96
8.09
7.24
3.68
3.81
3.06
5.09
5.22
4.47
2.69
2.82
2.07
4.63
4.76
4.01
−0.969
−0.711
−0.701
355.85
400.30
351.43
431.95
476.40
427.53
490.42
534.87
500.97
534.87
579.32
545.42
486.00
530.45
496.55
566.52
610.97
577.07
610.97
655.42
621.52
562.10
606.55
572.65
395.87
440.32
391.45
471.97
516.42
467.55
439.92
484.37
435.50
516.02
560.47
511.60
137
0
0
0
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
1
2
0
0
0
0
1
1
1
0
0
0
3d
3e
3f
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
INH
Pyra
Cipro
123
331
TPSA topological polar surface area, n-rotb number of rotatable bonds, nON number of hydrogen bond
acceptors, nOHNH number of hydrogen bond donors, miLogP logarithm of compound partition coefficient
between n-octanol and water, MW molecular weight, INH isoniazid, Pyra pyrazinamide, Cipro ciprofloxacin
Lipinski’s rule and having positive drug score. Overall,
these data suggest that compounds 3a, 3b, 3c, 3f, 5a, 5b, 5c,
6a, 6b and 6c (Positive Drug-score, log p < 5, hydrogen
bond donor <5, hydrogen bond accepter <10, Molecular
weight <500 ) are predicted as drug-like compounds.
Conclusion
In conclusion, a series of 1,3,4-trisubstituted pyrazole
derivatives (3a–f), (4a–r), (5a–f) and (6a–f) have been
synthesized and evaluated for their MTB (H37Rv) inhibitory

Med Chem Res
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1
were characterized by IR, H NMR, 13C NMR and mass
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significant anti-mycobacterial activity with MIC 0.78 µg/ml.
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decent interaction with active site of MTB NADH-
dependent enoyl-acyl carrier protein reductase (MtInhA).
Biological activity data and simulation results strongly
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Acknowledgements The authors are grateful to Principal, KLE
University’s College of Pharmacy, Belagavi, for providing necessary
facilities for the research. The authors are also grateful to NMR
Research center, IISC, Bangalore, India, for providing the spectral
data.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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