Discovery of new antitubercular agents by combining pyrazoline and benzoxazole pharmacophores: Design, synthesis and insights into the binding interactions

Article abstract of DOI:10.1007/s00044-013-0815-x

The present study is aimed at combining two well-known pharmacophores (pyrazoline and benzoxazole nucleus) to design and synthesize a series of new benzoxazole-based pyrazoline derivatives. In vitro antitubercular evaluation against Mycobacterium tuberculosis H₃₇Rv and multidrug-resistant TB (MDR-TB) strains showed that most of the target compounds displayed potent activity (MIC ～0.625-25 μg/mL). Few compounds were found to be better than isoniazid against MDR-TB (MIC = 3.25 μg/mL). In order to gain insights into the plausible binding motifs, the target compounds were docked into enoyl-acyl carrier protein (ACP) reductase, a molecular target of isoniazid. Many compounds were successfully docked into the active site of enoyl-ACP reductase and all the docked compounds occupied the same hydrophobic binding pocket and interacted mostly by dispersion interactions with the neighboring residues Met103, Met155, Tyr158, Met199, Ile202, Ile215, and Leu218. Contribution of the three pharmacophoric fragments (pyrazoline, benzoxazole and aryl ring) towards protein-ligand binding was evaluated at semi-empirical quantum mechanics level. The interaction energies suggested that most of the binding was governed by the benzoxazole moiety followed by pyrazoline and aryl rings. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0815-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2218–2228
DOI 10.1007/s00044-013-0815-x
ORIGINAL RESEARCH
Discovery of new antitubercular agents by combining pyrazoline
and benzoxazole pharmacophores: design, synthesis and insights
into the binding interactions
•
Dharmarajsinh N. Rana Mahesh T. Chhabria
•
•
Nisha K. Shah Pathik S. Brahmkshatriya
Received: 3 January 2013 / Accepted: 26 September 2013 / Published online: 15 October 2013
Ó Springer Science+Business Media New York 2013
Abstract The present study is aimed at combining two
well-known pharmacophores (pyrazoline and benzoxazole
nucleus) to design and synthesize a series of new benz-
oxazole-based pyrazoline derivatives. In vitro antituber-
cular evaluation against Mycobacterium tuberculosis
H37Rv and multidrug-resistant TB (MDR-TB) strains
showed that most of the target compounds displayed potent
activity (MIC *0.625–25 lg/mL). Few compounds were
found to be better than isoniazid against MDR-TB
(MIC = 3.25 lg/mL). In order to gain insights into the
plausible binding motifs, the target compounds were
docked into enoyl-acyl carrier protein (ACP) reductase, a
molecular target of isoniazid. Many compounds were
successfully docked into the active site of enoyl-ACP
reductase and all the docked compounds occupied the same
hydrophobic binding pocket and interacted mostly by dis-
persion interactions with the neighboring residues Met103,
Met155, Tyr158, Met199, Ile202, Ile215, and Leu218.
Contribution of the three pharmacophoric fragments (pyr-
azoline, benzoxazole and aryl ring) towards protein–ligand
binding was evaluated at semi-empirical quantum mechanics
level. The interaction energies suggested that most of the
binding was governed by the benzoxazole moiety followed
by pyrazoline and aryl rings.
Keywords Antitubercular ꢀ Benzoxazole ꢀ
Interaction energy ꢀ Molecular docking ꢀ
Pharmacophore ꢀ Pyrazoline
Introduction
The designation of tuberculosis as a global public health
crisis by the World Health Organization in the mid-1990s
has underscored severe challenges facing the research
community globally. It has been estimated by the World
Health Organization (WHO) that almost one-third of the
world’s population, around two billion people, are infected
with the disease (Dye et al., 2009). Every year, more than
eight million people develop an active form of the disease,
which subsequently claims the lives of nearly two million.
In 2002, the WHO estimated that if the worldwide spread
of tuberculosis was left unchecked, then the disease would
be responsible for approximately 36 million more deaths
by the year 2020. Further, the emergence of multidrug-
resistant TB (MDR-TB), a form of TB that does not
respond to the first-line TB drugs and extensively drug-
resistant TB (XDR-TB), an MDR-TB with resistance to
fluoroquinolones and aminoglycosides, have become a
serious threat to TB control and its treatment. On top of
this, the recent cases of totally drug-resistant tuberculosis
have raised alarming concerns on the existing drug regimen
implying urgent need to discover newer antitubercular
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0815-x) contains supplementary
material, which is available to authorized users.
D. N. Rana ꢀ N. K. Shah (&)
Department of Chemistry, Gujarat University, Ahmedabad 380
009, Gujarat, India
e-mail: nishchem2004@yahoo.co.in
M. T. Chhabria
Department of Pharmaceutical Chemistry, L. M. College of
Pharmacy, Ahmedabad 380 009, Gujarat, India
P. S. Brahmkshatriya (&)
Center for Biomolecules and Complex Systems, Institute
of Organic Chemistry and Biochemistry, Academy of Sciences
´
of the Czech Republic, Flemingovo nam. 2, 166 10 Prague 6,
Czech Republic
e-mail: pathik26@yahoo.com
123

Med Chem Res (2014) 23:2218–2228
2219
agents with newer molecular mechanisms (Udwadia et al.,
2012).
To gain insights into the plausible binding interactions,
we carried out docking of the designed compounds into the
crystal structure of M. tuberculosis enoyl reductase (InhA)
(PDB Code 2H7I) (He et al., 2006). We also investigated
contribution of the three fragments/pharmacophores of the
target compounds (pyrazoline core, benzoxazole moiety,
and aryl ring) towards protein binding.
The benzoxazole scaffold has provided the basis for the
design of biologically relevant molecules with broad ther-
apeutic importance. Several benzoxazole derivatives have
been reported to possess antitubercular activity (Fig. 1,
right pane) (Vinsova et al., 2006; Temiz-Arpaci et al.,
ˇ´
2008; Kocı et al., 2002). Compounds with N–N bond are of
primary importance due to the known fact that living
organism finds it difficult to construct N–N bonds which
limits the natural abundance of compounds having such
bonds. Pyrazoline and their derivatives, a class of com-
pounds containing the N–N bond, exhibit a wide range of
biological activities (Ahsan et al., 2012; Siddiqui et al.,
2011; Bandgar et al., 2012; Abdel-Wahab et al., 2012)
including antimycobacterial activity (Fig. 1, left pane)
(Manna and Agrawal, 2010; Shaharyar et al., 2006; Sharma
et al., 2011). In light of these facts, we thought of incor-
porating the two scaffolds to come out with chemically
new antimycobacterial agents (Fig. 1, compound 5a–5h
and 6a–6h). The synthesized compounds were then sub-
jected to antimycobacterial screening against Mycobacte-
rium tuberculosis H37Rv and MDR-TB strains. We also
evaluated compounds 4a–4h, which are immediate pre-
cursors of compounds 5a–5h and 6a–6h.
Materials and methods
Chemistry
All chemicals and reagents used in the study were of
analytical grade and were used directly. Melting points
were determined in an open glass capillary and are
uncorrected. Thin layer chromatography (TLC) was per-
formed on Silica Gel G (Merck) and spots were visualized
under UV radiation, exposure to iodine vapors and with
various spray reagents. IR spectra were recorded on FT-IR
Spectrometer Shimadzu 8201 PC instrument in KBr pel-
1
lets. H NMR and 13C NMR spectra were recorded on
400 MHz BRUKER ULTRASHIELD using DMSO-d6 as
solvent and chemical shift values were expressed in d ppm
downfield from the internal standard tetramethylsilane.
Fig. 1 Structures of some
N
representative pyrazoline,
benzoxazole derivatives, and
designed compounds.
Pyrazoline and benzoxazole
moieties are encircled
O
N
N
O
O
N
R
R
R = OH OCH N(CH
,
,
,
)
3 2
H N3
l
O2 C
,
COO
,
R = H, X, CF3, OH, NO2, SCH3
O
N
R1
R2
O
N
O
H
C
S
n
(
)
2
N
N
R
N
N
X
R2
R3
O
R = H, NO2, CN, CSNH2
R1
n = 0,1; R1 = aryl
2 = aryl, R3 = H, 4-OH
5a-5h; X = NH2
6a-6h; X = SH
R
=
R1 H, Cl, OCH3
R2 = substituted phenyl
O
N
S
R1
H
HN
R2
N
N
H
R3
H
H
O
H
O
=
=
=
R1
R2
R3
CH3, OCH3
aryl, heteroaryl
2-CH3, 3-CH3
123

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Med Chem Res (2014) 23:2218–2228
1
General procedure for synthesis of 1-aryl-3-(4-hydroxy-3-
N), 1514 (C=C), 1241 (C–N); H NMR (DMSO-d6): d 7.
686–7.626 (m, 2H), 7.364–7.348 (m, 3H), 6.183 (d, 2H,
methoxy-5-nitrophenyl)-2-propenone (2a–2h)
0
00
J = 2.12 Hz), 5.357 (q, 1H, JHH = 11.64 Hz, JHH = 4.
16 Hz), 3.741 (s, 3H), 3.694–3.620 (m, 2H), 2.315 (s, 3H);
13C NMR (DMSO-d6): d 21.65, 40.01, 42.19, 55.47, 59.50,
77.86, 78.18, 78.38, 78.51, 98.54, 104.60, 126.20, 128.32,
129.78, 131.10, 131.68, 133.12, 136.33, 147.27, 153.56,
167.46; MS (m/z); 326.2 (M?1).
To a solution of 4-hydroxy-3-substituted-5-nitrobenzalde-
hyde (1a–1c, 50.8 mmol) and acetophenone (50.8 mmol)
in methanol (75 mL) was added 25 % aqueous NaOH
solution (75 mL) slowly while maintaining the temperature
below 25 °C. The resulting reaction mixture was stirred for
20 h at room temperature. The reaction mixture was then
acidified with 5 N HCl solution and the resulting orange-
yellow precipitates were filtered off to afford 1-aryl-3-(4-
hydroxy-3-substituted-5-nitrophenyl)-2-propenone (2a–2h)
in good yield (75–90 %).
1-(5-(3-Amino-4-hydroxy-5-methoxyphenyl)-3-(2,4-dichlo-
rophenyl)-4,5-dihydropyrazol-1-yl)ethanone (4b) Yield,
64.6 %; mp 156–158 °C; IR (KBr): 3450–3210 (O–H),
3315 (N–H), 3037 (Ar–H), 2918 (aliphatic C–H), 1678 (C=
O), 1593 (C=N), 1533 (C=C), 1138 (C–N); 1H NMR
(DMSO-d6): d 7.711 (d, 1H, Jab = 8.52 Hz), 7.461 (d, 1H,
Jbc = 1.9 Hz), 7.348 (dd, 1H, Jab = 8.52 Hz, Jbc = 1.
9 Hz), 6.201 (d, 2H, J = 10.56 Hz), 5.341 (dd, 1H,
General procedure for synthesis of 1-(5-(4-hydroxy-3-
substituted-5-nitrophenyl)-3-aryl-4,5-dihydropyrazol-1-
yl)ethanone (3a–3h)
0
JHH = 11.64 Hz, JHH = 4.2 Hz), 3.861 (q, 1H, JHH =
00
0
0
00
To
a solution of 1-aryl-3-(4-hydroxy-3-substituted-5-
12.16 Hz, JH H = 18.44 Hz), 3.738 (s, 3H), 3.200 (dd, 1H,
JHH = 4.28 Hz, JH H = 18.24 Hz), 2.275 (s, 3H); 13C
NMR (DMSO-d6): d 21.68, 44.80, 55.45, 59.76, 98.12,
104.81, 127.17, 128.99, 130.10, 131.30, 131.66, 132.78,
132.83, 135.02, 136.54, 147.41, 151.62, 167.63; MS (m/z);
394.4 (M?1, 100 %), 396.2 (34 %), 398.2 m/z (7 %).
00
0
00
nitrophenyl)-2-propenone (2a–2h, 16.7 mmol) and glacial
acetic acid (50 mL) was added hydrazine hydrate (80 %
w/v) (33.4 mmol) solution drop wise. After completion of
addition, the reaction mixture was refluxed for 3 h. The
reaction mixture was then poured into ice-water. Resulting
yellow residues were filtered off and dried to get the crude
product which was further purified by recrystallization
from ethanol to afford 1-(5-(4-hydroxy-3-substituted-5-
nitrophenyl)-3-aryl-4,5-dihydropyrazol-1-yl)ethanone (3a–
3h) in good yield (55–85 %).
1-(5-(3-Amino-4-hydroxy-5-methoxyphenyl)-3-(4-fluoro-
phenyl)-4,5-dihydropyrazol-1-yl)ethanone (4c) Yield, 47.
8 %; mp 143–145 °C; IR (KBr): 3498–3228 (O–H), 3334
(N–H), 3042 (Ar–H), 2947 (aliphatic C–H), 1682 (C=O),
1
1602 (C=N), 1545 (C=C), 1227 (C–N); H NMR (DMSO-
General procedure for synthesis of 1-(5-(3-amino-4-
hydroxy-5-substituted-phenyl)-3-aryl-4,5-dihydropyrazol-
1-yl)ethanone (4a–4h)
d6): 7.976 (broad s, 1H), 7.799–7.764 (sextet, 2H), 7.185 (t,
00
2H), 6.168 (s, 2H), 5.393 (dd, 1H, JHH = 4.4 Hz, JHH
0
=
11.7 Hz), 4.583 (broad s, 1H), 3.780 (s, 3H), 3.765 (q, 1H,
0
00
0
JH H = 17.84 Hz, JHH = 11.78 Hz), 3.128 (dd, 1H,
JH H = 17.8 Hz, JHH = 4.48 Hz), 2.345 (s, 3H); 13C
NMR (DMSO-d6): d 21.65, 42.29, 55.47, 59.61, 98.37,
104.58, 115.32, 115.53, 127.63, 128.38, 131.62, 133.08,
136.52, 147.33, 152.66, 161.90, 164.38, 167.33; MS (m/z);
344.2 (M?1).
0
00
00
To a solution of 1-(5-(4-hydroxy-3-substituted-5-nitrophenyl)-
3-aryl-4,5-dihydropyrazol-1-yl)ethanone (3a–3h, 7.04 mmol)
in methanol (25 mL) was added sodium dithionite (6.13 g,
35.2 mmol) portion wise at 55–60 °C to avoid vigorous
frothing. The color of the reaction mixture slowly turned
orange to colorless with the progress of the reaction. The
colorless reaction mixture was further refluxed for additional
30 min to ensure the completion of reaction after which the
reaction mixture was poured into ice-water. The resulting
off-white residues were filtered off and dried to get the crude
product which was further recrystallized from isopropyl
alcohol to yield 1-(5-(3-amino-4-hydroxy-5-substituted-
phenyl)-3-aryl-4,5-dihydropyrazol-1-yl)ethanone (4a–4h)
as pure product.
1-(5-(3-Amino-4-hydroxy-5-methoxyphenyl)-3-(2-bromo-
phenyl)-4,5-dihydropyrazol-1-yl)ethanone (4d) Yield, 43.
5 %; mp 147–149 °C; IR (KBr): 3519–3218 (O–H), 3364
(N–H), 3071 (Ar–H), 2943 (aliphatic C–H), 1677 (C=O),
1
1599 (C=N), 1519 (C=C), 1123 (C–N); H NMR (DMSO-
d6): d 10.316 (s, 1H), 7.787 (q, 2H, J = 4.32 Hz, J = 6.
00 Hz), 7.463 (t, 2H), 7.332 (d, 1H, J = 1.96 Hz), 7.100
00
(d, 1H, J = 1.96 Hz), 5.580 (dd, 1H, JHH = 5.2 Hz,
0
JHH = 12.4 Hz), 4.083 (q, 1H, JH H = 18.39 Hz, JHH =
0
00
0
0
00
1-(5-(3-Amino-4-hydroxy-5-methoxyphenyl)-3-phenyl-4,5-
dihydropyrazol-1-yl)ethanone (4a) Yield, 60.8 %; mp
137–139 °C; IR (KBr): 3500–3213 (O–H), 3360 (N–H),
3054 (Ar–H), 2924 (aliphatic C–H), 1684 (C=O), 1599 (C=
12.51 Hz), 3.894 (s, 3H), 3.234 (dd, 1H, JH H = 18.47 Hz,
JHH = 5.13 Hz), 2.374 (s, 3H); 13C NMR (DMSO-d6): d
21.56, 56.28, 58.78, 59.66, 99.86, 104.38, 112.24, 114.32,
00
126.29, 128.34, 130.05, 130.60, 134.63, 144.88, 153.69,
123

Med Chem Res (2014) 23:2218–2228
2221
00
1H), 6.663 (s, 1H), 5.559 (dd, 1H, JHH = 4.82 Hz,
160.31, 168.10; MS (m/z); 404.9 (M?1, 98 %), 407.1
0
JHH = 11.62 Hz), 3.843 (q, 1H, JH H = 17.93 Hz, JHH =
0
00
0
(100 %).
0
00
00
11.69 Hz), 3.463 (dd, 1H, JH H = 17.91 Hz, JHH = 4.
79 Hz), 2.391 (s, 3H); 13C NMR (DMSO-d6): d 21.86, 58.
72, 60.13, 105.23, 113.15, 115.12, 126.97, 128.69, 129.22,
130.41, 131.81, 132.40, 133.09, 134.63, 144.54, 154.47,
168.18; MS (m/z); 408.2 (M?1, 100 %), 410.0 (99 %).
1-(5-(3-Amino-4-hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-
1-yl)ethanone (4e) Yield, 58.9 %; mp 86–88 °C; IR
(KBr): 3522–3186 (O–H), 3311 (N–H), 3062 (Ar–H), 2912
(aliphatic C–H), 1676 (C=O), 1598 (C=N), 1510 (C=C),
1180 (C–N); 1H NMR (DMSO-d6): d 7.681–7.623 (m, 2H),
7.380–7.350 (m, 3H), 6.676 (d, 1H, Jab = 7.92 Hz), 6.591
(dd, 1H, dd, 1H, Jab = 7.91 Hz, Jbc = 2.01 Hz), 6.487 (d,
General procedure for synthesis of 1-(5-(2-amino-7-
substituted-benzoxazol-5-yl)-3-aryl-4,5-dihydro-1H-
pyrazol-1-yl)ethanone (5a–5h)
00
1H, d, 1H, Jbc = 1.99 Hz), 5.388 (q, 1H, JHH = 3.92 Hz,
JHH = 11.35 Hz), 3.878–3.633 (m, 2H), 2.327 (s, 3H); 13
C
0
NMR (DMSO-d6): d 24.62, 43.11, 59.63, 72.28, 107.94,
113.76, 123.47, 126.07, 129.25, 132.81, 134.04, 136.37,
138.14, 146.16, 154.12, 168.10; MS (m/z); 296.3 (M?1).
To a suspension of 1-(5-(3-amino-4-hydroxy-5-substituted-
phenyl)-3-aryl-4,5-dihydropyrazol-1-yl)ethanone (4a–4h,
1.54 mmol) in 1:1 mixture of methanol and water (20 mL)
was added a solution of CNBr (0.18 g, 1.54 mmol) in
methanol (5 mL) drop wise. The resulting solution was
then stirred for the next 30 min. The resulting reaction
mixture was then neutralized to pH *8 using sodium
bicarbonate and the reaction mixture was stirred for
30 min. The reaction mixture was then poured into ice-
water and the resulting off-white residues were filtered off,
dried, and recrystallized from ethanol to yield 1-(5-(2-
amino-7-substituted-benzoxazol-5-yl)-3-aryl-4,5-dihydro-
1H-pyrazol-1-yl)ethanone (5a–5h).
1-(5-(3-Amino-4-hydroxyphenyl)-3-(2-bromophenyl)-4,5-
dihydropyrazol-1-yl)ethanone (4f) Yield, 55.6 %; mp
95–97 °C; IR (KBr): 3512–3225 (O–H), 3305 (N–H), 3044
(Ar–H), 2932 (aliphatic C–H), 1658 (C=O), 1610 (C=N),
1542 (C=C), 1215 (C–N); 1H NMR (DMSO-d6): d 7.
775–7.751 (m, 2H), 7.454–7.432 (m, 2H), 6.721 (d, 1H,
Jab = 8.13 Hz), 6.661 (dd, 1H, dd, 1H, Jab = 8.19 Hz,
Jbc = 1.93 Hz), 6.538 (d, 1H, d, 1H, Jbc = 2.04 Hz), 5.589
00
(dd, 1H, JHH = 5.7 Hz, JHH = 12.66 Hz), 4.185 (q, 1H,
0
0
00
0
JH H = 18.78 Hz, JHH = 12.66 Hz), 3.486 (dd, 1H,
JH H = 18.71 Hz, JHH = 5.72 Hz), 2.372 (s, 3H); 13C
NMR (DMSO-d6): d 21.73, 59.24, 59.94, 99.62, 105.84,
113.30, 115.71, 126.35, 128.34, 130.65, 132.54, 135.67,
144.93, 155.42, 160.38, 168.23; MS (m/z); 374.1 (M?1,
100 %), 376.1 (95 %).
1-(5-(2-Amino-7-methoxybenzoxazol-5-yl)-3-phenyl-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (5a) Yield, 65.0 %;
mp 115–117 °C; IR (KBr): 3474 (N–H), 3079 (Ar–H),
2935 (aliphatic C–H), 1712 (C–O), 1632 (C=O), 1615 (C=
0
00
00
1
N), 1468 (C=C), 1203 (C–N); H NMR (DMSO-d6): d 7.
477–7.460 (m, 2H), 7.152–7.136 (m, 3H), 6.785 (s, 2H), 6.
00
353 (s, 1H), 6.203 (s, 1H), 5.283 (dd, 1H, JHH = 11.
1-(5-(3-Amino-4-hydroxy-5-chlorophenyl)-3-phenyl-4,5-di-
hydropyrazol-1-yl)ethanone (4g) Yield, 72.7 %; mp
103–105 °C; IR (KBr): 3478–3247 (O–H), 3289 (N–H),
3080 (Ar–H), 2910 (aliphatic C–H), 1611 (C=O), 1591 (C=
0
74 Hz, JHH = 4.4 Hz), 3.623 (s, 3H), 3.571 (q, 1H,
0
JH H = 17.8 Hz, JHH = 11.6 Hz), 2.893 (dd, 1H, JH -
00
0
0
= 17.78 Hz, JHH = 4.52 Hz), 2.090 (s, 3H); 13C NMR
00
H00
1
N), 1499 (C=C), 1182 (C–N); H NMR (DMSO-d6): d 7.
692–7.635 (m, 2H), 7.396–7.315 (m, 3H), 6.719 (s, 1H), 6.
(DMSO-d6): d 21.62, 42.41, 55.79, 59.72, 102.07, 104.77,
126.27, 128.36, 129.88, 130.99, 135.15, 138.37, 142.66,
00
541 (s, 1H), 5.595 (dd, 1H, JHH = 5.2 Hz, JHH = 12.
0
145.41, 153.63, 162.90, 167.55; MS (m/z): 351.2 (M?1).
0
4 Hz), 3.905 (q, 1H, JH H = 18.39 Hz, JHH = 12.51 Hz),
00
0
0
00
00
3.201 (dd, 1H, JH H = 18.47 Hz, JHH = 5.13 Hz), 2.388
(s, 3H); 13C NMR (DMSO-d6): d 21.59, 40.17, 42.16, 60.
41, 80.14, 78.62, 104.94, 126.81, 129.55, 130.47, 131.73,
132.26, 133.12, 137.09, 147.34, 154.16, 167.83; MS (m/z);
330.2 (M?1).
1-(5-(2-Amino-7-methoxybenzoxazol-5-yl)-3-(2,4-dichloro-
phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (5b) Yield,
75.3 %; mp 139–141 °C; IR (KBr): 3459 (N–H), 3062
(Ar–H), 2947 (aliphatic C–H), 1696 (C–O), 1627 (C=O),
1
1603 (C=N), 1484 (C=C), 1145 (C–N); H NMR (DMSO-
d6): d 7.897 (broad s, 2H), 7.717 (d, 1H, J = 8.44 Hz), 7.
459 (s, 1H), 7.345 (d, 1H, J = 8.48 Hz), 6.603 (s, 1H), 6.
1-(5-(3-Amino-4-hydroxy-5-chlorophenyl)-3-(2-bromophe-
nyl)-4,5-dihydropyrazol-1-yl)ethanone (4h) Yield, 57.
9 %; mp 112–114 °C; IR (KBr): 3556–3311 (O–H), 3317
(N–H), 3077 (Ar–H), 2931 (aliphatic C–H), 1653 (C=O),
00
435 (s, 1H), 5.506 (dd, 1H, JHH = 4.04 Hz, JHH = 11.
0
0
88 Hz), 3.939 (q, 1H, JH H = 18.83 Hz, JHH = 11.
00
0
0
00
00
88 Hz), 3.230 (dd, 1H, JH H = 18.24 Hz, JHH = 4.
32 Hz), 2.289 (s, 3H); 13C NMR (DMSO-d6): d 21.62, 44.
98, 55.75, 59.98, 101.93, 104.89, 127.13, 128.84, 130.09,
1
1596 (C=N), 1507 (C=C), 1201 (C–N); H NMR (DMSO-
d6): d 7.831–7.795 (m, 2H), 7.414–7.398 (m, 2H), 6.732 (s,
123

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Med Chem Res (2014) 23:2218–2228
131.20, 132.89, 135.17, 135.24, 137.95, 142.71, 145.38,
151.66, 162.91, 167.85; MS (m/z): 419.3 (M?1, 100 %),
421.1 (30 %), 423.0 (12 %).
844–7.790 (m, 3H), 7.753–7.531 (m, 4H), 7.475 (d, 1H,
J = 1.64 Hz), 7.351 (d, 1H, J = 1.73 Hz), 5.559 (dd, 1H,
00
0
JHH = 3.70 Hz, JHH = 11.45 Hz), 3.709–3.547 (m, 2H),
2.361 (s, 3H); 13C NMR (DMSO-d6): d 23.41, 69.37, 98.
14, 103.32, 105.42, 109.51, 117.86, 120.45, 126.35, 129.
03, 131.12, 132.09, 140.33, 154.03, 164.24, 167.91; MS
(m/z); 399.2 (M?1), 401.1 (90 %).
1-(5-(2-Amino-7-methoxybenzoxazol-5-yl)-3-(4-fluoro-
phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone
(5c)
Yield, 65.3 %; mp 132–134 °C; IR (KBr): 3512 (N–H),
3103 (Ar–H), 2955 (aliphatic C–H), 1687 (C–O), 1662 (C=
O), 1547 (C=N), 1461 (C=C), 1213 (C–N); 1H NMR
(DMSO-d6): d 7.841 (sextet, 2H), 7.328 (s, 2H), 7.276 (t,
2H), 6.556 (d, 1H, J = 0.92 Hz), 6.509 (d, 1H, J = 1.
1-(5-(2-Amino-7-chlorobenzoxazol-5-yl)-3-phenyl-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (5g) Yield, 55.0 %;
mp 97–99 °C; IR (KBr): 3528 (N–H), 3067 (Ar–H), 2976
(aliphatic C–H), 1720 (C–O), 1631 (C=O), 1563 (C=N),
1498 (C=C), 1239 (C–N); 1H NMR (DMSO-d6): d 7.
667–7.632 (m, 2H), 7.587 (s, 2H), 7.296–7.252 (m, 3H), 6.
00
0
08 Hz), 5.547 (dd, 1H, JHH = 11.72 Hz, JHH = 4.52 Hz),
00
0
3.868 (s, 3H), 3.850 (q, 1H, JH H = 17.88 Hz, JHH = 11.
00
0
0
00
00
72 Hz), 3.175 (dd, 1H, JH H = 18.02 Hz, JHH = 4.
64 Hz), 2.316 (s, 3H); 13C NMR (DMSO-d6): d 21.56, 42.
43, 55.76, 59.82, 87.04, 99.48, 102.06, 104.83, 115.26,
115.48, 128.31, 130.04, 131.08, 138.17, 152.74, 164.27,
168.16; MS (m/z): 369.4 (M?1).
569 (s, 1H), 6.429 (s, 1H), 5.372 (dd, 1H, dd, 1H, JHH
=
0
4.36 Hz, JHH = 11.40 Hz), 3.962 (q, 1H, JH H = 17.
0
00
0
0
00
48 Hz, JHH = 11.37 Hz), 3.140 (dd, 1H, JH H = 17.
52 Hz, JHH = 4.29 Hz), 2.428 (s, 3H); 13C NMR (DMSO-
00
d6): d 23.66, 70.35, 102.28, 102.97, 105.16, 129.42, 132.70,
137.63, 143.69, 153.88, 165.54, 168.31; MS (m/z); 354.9
(M?1, 100 %), 356.8 (30 %).
1-(5-(2-Amino-7-methoxybenzoxazol-5-yl)-3-(2-bromophe-
nyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (5d) Yield,
75.6 %; mp 141–143 °C; IR (KBr): 3571 (N–H), 3068
(Ar–H), 2956 (aliphatic C–H), 1707 (C–O), 1671 (C=O),
1-(5-(2-Amino-7-chlorobenzoxazol-5-yl)-3-(2-bromophe-
nyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (5h) Yield,
69.0 %; mp 121–123 °C; IR (KBr): 3501 (N–H), 3106
(Ar–H), 2934 (aliphatic C–H), 1736 (C–O), 1627 (C=O),
1
1626 (C=N), 1495 (C=C), 1178 (C–N); H NMR (DMSO-
d6): d 7.904-7.819 (m, 2H), 7.731–7.622 (m, 2H), 7.473 (s,
2H), 7.331 (d, 2H, J = 1.11 Hz), 7.173 (d, 2H, J = 1.
1
1572 (C=N), 1453 (C=C), 1193 (C–N); H NMR (DMSO-
00
09 Hz), 5.848 (dd, 1H, JHH = 5.2 Hz, JHH = 12.4 Hz), 4.
0
d6): d 7.861–7.835 (m, 2H), 7.562 (s, 2H), 7.426–7.386 (m,
2H), 6.761 (s, 1H), 6.594 (s, 1H), 5.396 (dd, 1H, dd, 1H,
0
208 (q, 1H, JH H = 18.39 Hz, JHH = 12.51 Hz), 3.931 (s,
00
0
0
00
00
3H), 3.339 (dd, 1H, JH H = 18.47 Hz, JHH = 5.13 Hz),
2.418 (s, 3H); 13C NMR (DMSO-d6): d 23.54, 58.12, 70.
23, 98.31, 102.20, 105.63, 118.07, 121.26, 126.71, 128.92,
131.35, 131.94, 140.15, 153.22, 164.31, 168.42; MS (m/z);
429.1 (M?1, 100 %), 431.1 (95 %).
00
JHH = 4.92 Hz, JHH = 11.86 Hz), 3.868 (q, 1H, JH H =
0
0
00
0
0
00
17.97 Hz, JHH = 11.86 Hz), 3.833 (dd, 1H, JH H = 17.
96 Hz, JHH = 4.90 Hz), 2.335 (s, 3H); 13C NMR (DMSO-
00
d6): d 23.73, 60.11, 61.47, 106.11, 113.75, 116.01, 127.30,
128.71, 129.54, 131.18, 132.24, 133.49, 134.10, 136.15,
145.42, 154.49, 165.27, 168.63; MS (m/z); 432.3(M?1,
100 %), 434.1 (98 %).
1-(5-(2-Aminobenzoxazol-5-yl)-3-phenyl-4,5-dihydro-1H-
pyrazol-1-yl)ethanone (5e) Yield, 66.4 %; mp 93–95 °C;
IR (KBr): 3532 (N–H), 3097 (Ar–H), 2953 (aliphatic C–H),
1721 (C–O), 1626 (C=O), 1550 (C=N), 1534 (C=C), 1088
General procedure for synthesis of 1-(5-(2-mercapto-7-
substituted-benzoxazol-5-yl)-3-aryl-4,5-dihydro-1H-
pyrazol-1-yl)ethanone (6a–6h)
1
(C–N); H NMR (DMSO-d6): d 7.709–7.686 (m, 2H), 7.
594–7.488 (m, 2H), 7.527 (d, 1H, Jbc = 2.17 Hz), 7.424 (s,
2H), 7.315–7.227 (m, 3H), 7.279 (dd, 1H, Jab = 8.13 Hz,
To a solution of potassium hydroxide (1.54 mmol) in
methanol (15 mL) was added carbon disulfide (0.1 mL,
1.54 mmol) drop wise. The reaction mixture turned to light
yellow upon addition of carbon disulphide. To the resulting
00
0
=
Jbc = 2.21 Hz), 5.365 (dd, 1H, JHH = 4.12 Hz, JHH
11.27 Hz), 3.715–3.675 (m, 2H), 2.372 (s, 3H); 13C NMR
(DMSO-d6): d 23.45, 69.32, 100.92, 102.64, 104.59, 126.
30, 129.13, 131.81, 133.87, 144.39, 153.71, 165.02, 168.
64; MS (m/z); 321.1 (M?1).
solution,
1-(5-(3-amino-4-hydroxy-5-substituted-phenyl)-3-
phenyl-4,5-dihydropyrazol-1-yl)ethanone (4a–4h, 1.54 mmol)
was added and the reaction mixture was refluxed for the
3 h. The reaction mixture was acidified with 2 N HCl
solution. The resulting off-white residues were filtered off,
dried, and recrystallized from ethanol to afford 1-(5-(2-
mercapto-7-substituted-benzoxazol-5-yl)-3-aryl-4,5-dihy-
dro-1H-pyrazol-1-yl)ethanone (6a–6h).
1-(5-(2-Aminobenzoxazol-5-yl)-3-(2-bromophenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (5f) Yield, 58.3 %;
mp 106–108 °C; IR (KBr): 3496 (N–H), 3087 (Ar–H),
2941 (aliphatic C–H), 1763 (C–O), 1625 (C=O), 1549 (C=
1
N), 1515 (C=C), 1191 (C–N); H NMR (DMSO-d6): d 7.
123

Med Chem Res (2014) 23:2218–2228
2223
1-(5-(2-Mercapto-7-methoxybenzoxazol-5-yl)-3-phenyl-
4,5-dihydro-1H-pyrazol-1-yl)ethanone (6a) Yield, 73.
0 %; mp 207–209 °C; IR (KBr): 3366 (S–H), 3010 (Ar–H),
2928 (aliphatic C–H), 1719 (C–O), 1659 (C=O), 1577 (C=
d6): d 13.795 (s, 1H), 7.447–7.405 (m, 1H), 7.274–7.224
(m, 3H), 7.117 (d, 1H, J = 0.92 Hz), 7.071–7.048 (m, 1H),
00
5.863 (dd, 1H, JHH = 4.96 Hz, JHH = 11.96 Hz), 4.019
0
0
(s, 3H), 3.964 (q, 1H, JH H = 17.88 Hz, JHH = 12.0 Hz),
00
0
1
0
00
00
N), 1445 (C=C), 1199 (C–N); H NMR (DMSO-d6): d 13.
554 (s, 1H), 7.730–7.706 (m, 2H), 7.414–7.390 (m, 3H), 6.
3.120 (dd, 1H, JH H = 17.88 Hz, JHH = 5.0 Hz), 2.423
(s, 3H); 13C NMR (DMSO-d6): d 21.73, 58.43, 71.20, 98.
65, 102.56, 107.05, 118.58, 121.32, 129.25, 131.32, 133.
74, 140.17, 141.79, 153.49, 165.56, 180.42; MS (m/z); 446.
0 (M?1, 100 %), 448.2 (95 %).
684 (d, 1H, J = 1.28 Hz), 6.546 (d, 1H, J = 1.24 Hz), 5.
00
561 (dd, 1H, JHH = 4.92 Hz, JHH = 11.86 Hz), 3.857 (q,
0
0
00
0
1H, JHH = 17.96 Hz, JH H = 11.92 Hz), 3.148 (dd, 1H,
JHH = 5.04 Hz, JH H = 17.98 Hz), 2.325 (s, 3H); 13C
NMR (DMSO-d6): d 21.60, 42.18, 56.23, 59.45, 99.38,
105.09, 126.34, 128.41, 130.05, 130.75, 132.88, 135.92,
140.54, 142.82, 153.70, 167.74, 180.16; MS (m/z): 368.4
(M?1).
00
00
0
1-(5-(2-Mercaptobenzoxazol-5-yl)-3-phenyl-4,5-dihydro-
1H-pyrazol-1-yl)ethanone (6e) Yield, 73.6 %; mp
123–125 °C; IR (KBr): 3392 (S–H), 3022 (Ar–H), 2923
(aliphatic C–H), 1732 (C–O), 1677 (C=O), 1582 (C=N),
1
1440 (C=C), 1276 (C–N); H NMR (DMSO-d6): d 13.807
(s, 1H), 7.748–7.532 (m, 4H), 7.446 (d, 1H, Jbc = 2.
1-(5-(2-Mercapto-7-methoxybenzoxazol-5-yl)-3-(2,4-
dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone
(6b) Yield, 75.9 %; mp 212–214 °C; IR (KBr): 3347 (S–
H), 3076 (Ar–H), 2939 (aliphatic C–H), 1739 (C–O), 1651
00
17 Hz), 7.364–7.251 (m, 3H), 5.606 (dd, 1H, JHH = 5.
0
04 Hz, JHH = 12.49 Hz), 3.824 (q, 2H, JH H = 18.21 Hz,
0
00
0
0
00
JHH = 12.42 Hz), 3.190 (dd, 1H, JH H = 18.25 Hz,
1
JHH = 5.06 Hz), 2.354 (s, 3H); 13C NMR (DMSO-d6): d
00
(C=O), 1542 (C=N), 1435 (C=C), 1205 (C–N); H NMR
(DMSO-d6): d 13.615 (s, 1H), 7.769 (d, 1H, Jab = 8.
48 Hz), 7.537 (d, 1H, Jbc = 2.08), 7.419 (dd, 1H, Jab = 8.
46 Hz, Jbc = 2.12 Hz), 6.643 (d, 1H, J = 1.23 Hz), 6.708
23.31, 69.76, 100.45, 105.71, 108.28, 126.10, 126.13, 129.
44, 132.07, 134.21, 149.16, 153.82, 166.72, 180.51; MS
(m/z); 338.1 (M?1).
00
(d, 1H, J = 1.21 Hz), 5.600 (dd, 1H, JHH = 4.84 Hz,
0
JHH = 11.92 Hz), 4.01–3.916 (m, 4H), 3.309 (dd, 1H,
1-(5-(2-Mercaptobenzoxazol-5-yl)-3-(2-bromophenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (6f) Yield, 59.1 %;
mp 149–151 °C; IR (KBr): 3370 (S–H), 3015 (Ar–H),
2931 (aliphatic C–H), 1723 (C–O), 1662 (C=O), 1502 (C=
JH H = 18.24 Hz, JHH = 4.96 Hz), 2.352 (s, 3H); 13C
NMR (DMSO-d6): d 21.60, 44.74, 56.19, 59.67, 99.61,
104.86, 127.19, 128.63, 130.12, 131.27, 132.87, 132.91,
135.31, 136.01, 140.11, 142.89, 151.72, 168.01, 180.19;
MS (m/z): 436.7 (M?1, 100 %), 438.0 (37 %), 440.1
(10 %).
0
00
00
1
N), 1446 (C=C), 1262 (C–N); H NMR (DMSO-d6): d 13.
812 (s, 1H), 7.596 (dd, 1H, J = 1.87 Hz, J = 8.40 Hz), 7.
574–7.571 (d, 1H, J = 8.43 Hz), 7.190–7.064 (m, 5H), 5.
00
816 (dd, 1H, JHH = 4.92 Hz, JHH = 12.16 Hz), 3.997 (q,
0
0
00
0
1-(5-(2-Mercapto-7-methoxybenzoxazol-5-yl)-3-(4-fluoro-
phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (6c) Yield,
80.2 %; mp 200–202 °C; IR (KBr): 3359 (S–H), 3025
(Ar–H), 2948 (aliphatic C–H), 1748 (C–O), 1672 (C=O),
2H, JH H = 18.24 Hz, JHH = 12.21 Hz), 3.050 (dd, 1H,
JH H = 18.27 Hz, JHH = 5.02 Hz), 2.373 (s, 3H); 13C
NMR (DMSO-d6): d 23.47, 69.58, 104.29, 109.63, 117.83,
121.31, 126.50, 129.26, 130.44, 132.12, 134.14, 148.76,
155.19, 165.23, 180.86; MS (m/z); 415.4 (M?1, 100 %),
417.4 (90 %).
0
00
00
1
1580 (C=N), 1448 (C=C), 1254 (C–N); H NMR (DMSO-
d6): d 13.841 (s, 1H), 7.638 (dd, 1H, J = 8.48 Hz, J = 1.
48 Hz), 7.572 (s, 1H), 7.448–7.418 (m, 2H), 7.274-7.251
00
(m, 2H), 7.076–7.053 (m, 1H), 5.848 (dd, 1H, JHH = 4.
1-(5-(2-Mercapto-7-chlorobenzoxazol-5-yl)-3-phenyl-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (6g) Yield, 74.8 %;
mp 122–124 °C; IR (KBr): 3345 (S–H), 3060 (Ar–H),
2930 (aliphatic C–H), 1731 (C–O), 1701 (C=O), 1533 (C=
0
0
00
96 Hz, JHH = 11.92 Hz), 3.991 (q, 1H, JH H = 17.88 Hz,
0
0
00
JHH = 11.96 Hz), 3.109 (dd, 1H, JH H = 17.88 Hz,
JHH = 4.96 Hz), 2.406 (s, 3H); 13C NMR (DMSO-d6): d
00
1
21.62, 42.26, 56.25, 59.55, 99.45, 105.09, 115.43, 115.65,
127.30, 128.60, 132.84, 135.87, 140.58, 142.83, 152.85,
167.62, 180.14; MS (m/z): 385.9 (M?1).
N), 1442 (C=C), 1230 (C–N); H NMR (DMSO-d6): d 13.
798 (s, 1H), 7.570–7.568 (d, 1H), 7.512–7.445 (m, 2H), 7.
314–7.312 (d, 1H), 7.292–6.195 (m, 3H), 5.873–5.831 (dd,
00
0
1H, dd, 1H, JHH = 4.86 Hz, JHH = 12.55 Hz), 3.974–3.
0
892 (q, 1H, JH H = 18.10 Hz, JHH = 12.53 Hz), 3.216–3.
00
0
1-(5-(2-Mercapto-7-methoxybenzoxazol-5-yl)-3-(2-bromo-
phenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (6d) Yield,
76.5 %; mp 218–220 °C; IR (KBr): 3334 (S–H), 3028
(Ar–H), 2932 (aliphatic C–H), 1755 (C–O), 1684 (C=O),
0
00
00
159 (dd, 1H, JH H = 18.13 Hz, JHH = 4.91 Hz), 2.426 (s,
3H); 13C NMR (DMSO-d6): d 23.54, 69.75, 118.04, 121.67,
123.05, 129.35, 130.44, 132.79, 134.53, 137.85, 143.71, 149.
21, 153.79, 165.68, 168.46; MS (m/z); 371.8 (M?1).
1
1581 (C=N), 1442 (C=C), 1247 (C–N); H NMR (DMSO-
123

2224
Med Chem Res (2014) 23:2218–2228
1-(5-(2-Mercapto-7-chlorobenzoxazol-5-yl)-3-(2-bromophe-
nyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (6h) Yield, 77.
0 %; mp 136–138 °C; IR (KBr): 3382 (S–H), 3018 (Ar–H),
2934 (aliphatic C–H), 1751 (C–O), 1658 (C=O), 1576 (C=
medium containing isoniazid. The medium containing
DMSO (control) was inoculated with the test organism for
positive and negative controls. Medium without any test
compound/DMSO was also inoculated with the test
organism to check whether the media supports the growth
of the tubercle bacilli or not. The inoculated bottles were
incubated at 37 °C for 6 weeks, at the end of which read-
ings were taken. Bacterial counts were measured and
compared with the standard drugs and controls (vehicle-
treated).
1
N), 1439 (C=C), 1235 (C–N); H NMR (DMSO-d6): d 13.
774 (s, 1H), 7.398 (d, 1H, J = 8.84 Hz), 7.353 (s, 1H), 7.
263 (t, 2H), 7.183 (d, 1H, J = 7.8 Hz), 7.115 (d, 1H, J =
00
1.24 Hz), 5.579 (dd, 1H, JHH = 4.8 Hz, JHH = 11.
0
0
92 Hz), 4.033 (s, 3H), 3.888 (q, 1H, JH H = 17.92 Hz,
00
0
0
00
JHH = 12.0 Hz), 3.218 (dd, 1H, JH H = 17.92 Hz,
JHH = 4.88 Hz), 2.394 (s, 3H); 13C NMR (DMSO-d6): d
00
23.73, 67.47, 110.11, 119.89, 121.23, 125.00, 127.37, 129.
73, 131.05, 132.92, 135.56, 140.18, 144.82, 155.71, 165.
32, 180.68; MS (m/z); 450.2 (M?1, 100 %), 452.1 (99 %).
Docking studies
The complex was subjected to preparation steps using the
Protein Preparation Wizard in Maestro using the default
˚
settings. First, the waters beyond 5 A from the ligand were
Antitubercular screening
removed, bond orders assigned and hydrogens added. Next,
the orientation of amide (Asn and Gln), hydroxyl (Ser, Thr,
and Tyr), and thiol groups (Cys), and the protonation and
tautomeric state of the His residues were optimized using
the exhaustive sampling option. A grid box of 20 9 20 9
The in vitro antitubercular activity was carried out by
measuring the growth of M. tuberculosis (H37RV) using
Lowensteine–Jensen medium (L. J. medium) (Stover et al.,
2000). Briefly, eggs were broken aseptically to obtain
200 mL of egg solution. The solution was filtered through a
sterile muslin cloth into a sterile conical flask containing
glass beads. Sterilized mineral salt solution (120 mL)
(consisting of 4.0 g potassium phosphate, 0.4 g of mag-
nesium sulfate, 1.6 g magnesium citrate, 6.0 g of aspara-
gine, 20 mL of glycerol, distilled water makeup up to
1,000 mL) and 4 mL of sterilized malachite green solution
(2.0 %) were added to the 200 mL of egg solution. The
contents were mixed well to form a uniform medium.
Compounds (10 mg) were dissolved in 10.0 mL of dime-
thyl sulfoxide (DMSO) and were diluted with DMSO to
make 250 and 10 mg/mL stock solutions. An aliquot
(0.8 mL) of each concentration was transferred into dif-
ferent McCartney bottles. To this, 7.2 mL of L. J. medium
was added and mixed well. Isoniazid was considered as a
reference standard for the comparison of antitubercular
activity. The drug was dissolved in DMSO and diluted as
described above. The bottles were incubated at 75–80 °C
for 3 days for solidification and sterilization.
3
˚
20 A for the receptor was generated with a default inner
3
box of 10 9 10 9 10 A , which was centered on the cor-
˚
responding ligand. The default parameters were used, and
no constraints were included. Docking calculations were
performed using the Glide Extra Precision (XP) algorithm
(Friesner et al., 2006). In the protocol, Glide was set to
determine the five best poses per ligand.
Interaction energies of the ligand fragments
with enoyl-ACP reductase
The inhibitor structures were fragmented into three seg-
ments: pyrazoline ring, benzoxazole ring, and aryl ring.
The separated side chains and the main chains were capped
by hydrogens. PM6-D3H4X method developed in our
laboratory (Brahmkshatriya et al., 2013) in implicit solvent
model was used to calculate interaction energies.
Results and discussion
Procedure for inoculation
Chemistry
A sweep from multidrug-resistant H37Rv strain of M.
tuberculosis culture was transferred with the help of 22
S.W. nichrome wire loop of 3-mm external diameter into a
sterile bijou bottle containing six 3-mm glass beads and
4 mL of sterile distilled water. Each loop of culture
delivered approximately 4 mg of bacilli cells. The bottle
was shaken with the help of vertex mixture for 2 min. The
suspension was inoculated on the surface of each L. J.
medium containing test compounds using 27 S.W.G
nichrome wire loop of 3 mm external diameter and L. J.
The title compounds (4a–4h, 5a–5h, and 6a–6h) were
synthesized in good yields by multistep chemical synthesis
as outlined in the Fig. 2.
Aldol condensation of 4-hydroxy-3-nitro-5-substituted-
benzaldehyde (1a–1c) with substituted acetophenones in
presence of sodium hydroxide afforded corresponding
chalcone derivatives (2a–2h). Cyclization of these chal-
cone derivatives (2a–2h) was carried out by reaction with
hydrazine hydrate in glacial acetic acid to yield corresponding
123

Med Chem Res (2014) 23:2218–2228
2225
Fig. 2 Pathway for the
synthesis of the title
O
O
R1
R1
OH
(a)
compounds. a Substituted
acetophenones, 25 % aqueous
NaOH solution, methanol, 24 h,
rt; b hydrazine hydrate, glacial
acetic acid, 3 h, reflux; c sodium
dithionite, methanol, 30 min,
reflux; d cyanogen bromide,
sodium bicarbonate, methanol–
water, 1 h, rt; e carbon
OH
R3
R2
N
O2
NO2
(2a-2h)
(1a-1c)
(b)
O
disulphide, methanol, 3 h, reflux
O
CH3
N
CH3
NH2
N
N
(c)
N
R1
R3
R1
R3
R2
R2
(4a-4h)
OH
OH
NO2
(3a-3h)
(e)
(d)
O
O
CH3
N
N
CH3
N
N
R1
R3
R1
R3
R2
(6a-6h)
R2
O
O
N
N
SH
NH2
(5a-5h)
pyrazoline derivatives (3a–3h) in good yields. Reduction
of nitro group by sodium dithionite afforded respective
o-aminophenol derivatives (4a–4h). Cyclization of these
o-aminophenol derivatives was brought by two different
ways: (a) treatment with cyanogen bromide in methanol–
water mixture at room temperature to yield corresponding
2-aminobenzoxazole derivatives (5a–5h); and (b) treatment
with carbon disulphide and potassium hydroxide in ethanol
at reflux temperature to afford 2-mercaptobenzoxazole
derivatives (6a–6h).
against M. tuberculosis H37Rv, compound 4d was found to
be more potent than isoniazid against MDR-TB strain.
Docking study
In order to gain better insights into the plausible binding
motifs of the target compounds, we carried out molecular
docking. For this, we chose the crystal structure of M.
tuberculosis enoyl reductase (InhA) (PDB Code 2H7I) (He
et al., 2006). We have shown earlier that Glide (Friesner
et al., 2006) performs better over other programs for the
present target (Mohan et al., 2012). We showed in that
paper that Glide performed better in finding the docked
geometry closer to experimental geometry (root mean
Antitubercular screening
Synthesized compounds (4a–4h, 5a–5h, and 6a–6h) were
screened for their antitubercular activity against M. tuber-
culosis H37Rv and MDR-TB strains. Table 1 shows the
results of the biological screening. Isoniazid was also
screened as a standard drug for their antitubercular activity
against both H37Rv and MDR-TB strains under similar
experimental conditions. It was encouraging to see that
majority of the compounds displayed satisfactory MIC
values against M. tuberculosis H37Rv and MDR-TB strains.
Compounds 4f and 5a were found to be potent as good as
isoniazid against M. tuberculosis H37Rv, while 4d was
found to be potent against MDR-TB stain. Although none
of the compounds was found to be potent than isoniazid
˚
square deviation = 0.47 A for the native ligand of 2H7I).
The docking studies revealed that most of the target com-
pounds occupied the hydrophobic cavity of enoyl-ACP
reductase. A visual analysis of the crystal structure of
enoyl-ACP reductase (PDB Code 2H7I) suggests that chief
interactions are of dispersion type. The ligand is less
exposed to solvent and buried deep inside a hydrophobic
pocket made up of Tyr158, Ile215, Met103, and Met199
residues. Most of the target compounds showed consistent
orientations of the pyrazoline, aryl, and benzoxazole moi-
eties within the active site (Fig. S1, Supplementary Mate-
rial). Figure S1a shows detailed interactions of the most
123

2226
Med Chem Res (2014) 23:2218–2228
Table 1 Structures of the synthesized target compounds and their in vitro antitubercular activity
Compound
no.
R1
R2
R3
X
MIC (lg/mL)
H37Rv
MDR-
TB
4a
4b
4c
–OCH3
–OCH3
–OCH3
–OCH3
–H
H
H
–
25
25
Cl
Cl
F
–
12.5
12.5
12.5
1.25
0.625
3.25
12.5
0.625
12.5
6.25
12.5
25
12.5
25
H
–
4d
4e
Br
–H
–Br
–H
–Br
H
H
–
3.25
25
–H
–H
–H
–H
H
–
4f
–H
–
12.5
25
4g
4h
5a
5b
5c
–Cl
–
–Cl
–
25
–OCH3
–OCH3
–OCH3
–OCH3
–H
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–SH
–SH
–SH
–SH
–SH
–SH
–SH
–SH
–
6.25
12.5
25
Cl
Cl
F
H
5d
5e
Br
–H
–Br
–H
–Br
H
H
25
–H
–H
–H
–H
H
25
5f
–H
25
12.5
25
5g
5h
6a
6b
6c
–Cl
25
–Cl
3.25
6.25
6.25
12.5
12.5
25
25
–OCH3
–OCH3
–OCH3
–OCH3
–H
12.5
12.5
6.25
25
Cl
Cl
F
H
6d
6e
Br
–H
–Br
–H
–Br
–
H
–H
–H
–H
–H
–
25
6f
–H
12.5
1.25
6.25
0.625
6.25
25
6g
6h
Isoniazid
–Cl
–Cl
12.5
6.25
–
potent compound 5a with the neighboring residues. It was
encouraging to see that most of the ligands retained all the
dispersion interactions found in the crystal structure (PDB
Code 2H7I) with some additional interactions with residues
Ile202, Met155, and Leu218. However, it should also be
noted that for some compounds, we could not obtain sat-
isfactory docking poses. Also, it was noteworthy that Gli-
de_Score did not correlate with the biological activity
(r2 = 0.04) which is understandable because of the sim-
plicity of the molecular mechanics-based scoring function.
Interaction energies of the ligand fragments
with enoyl-ACP reductase
In order to investigate the pharmacophoric contribution of
the target compounds, we investigated contribution of the
123

Med Chem Res (2014) 23:2218–2228
2227
Table 2 Glide_Score of the target compounds and their interaction energies using PM6-D3H4X method (in kcal/mol)
Comp. no.
pMIC
Glide score
Total interaction
energy
Substituted
phenyl
Acetyl
pyrazoline
(Mercapto/amino)
benzoxazole
4a
7.11
7.50
7.44
8.37
8.01
7.51
8.75
7.11
7.20
8.44
–
-7.22
-4.64
-6.23
-7.1
-0.20
-1.67
-1.25
-3.84
-3.90
-0.41
-7.87
-3.27
-1.22
–
8.53
6.44
4.19
6.51
7.96
6.94
3.30
5.22
5.29
–
1.54
-1.54
-3.49
-3.26
-1.25
3.56
-10.27
-6.57
-1.95
-7.09
-10.61
-10.90
-14.67
-8.23
-9.76
–
4b
4c
4e
4g
-7.13
-5.66
-5.78
-6.87
-3.2
4h
5a
-4.60
-0.25
3.25
5e
5f
Isoniazid
-3.59
-8.01
–
1-Cyclohexyl-5-oxo-N-
phenypyrrolidine-3-
carboxamide (2H7I-ligand)
–
–
–
–
three fragments of the target compounds (pyrazoline core,
benzoxazole moiety, and aryl ring) towards protein bind-
ing. We have shown earlier that the semi-empirical quan-
tum mechanical scoring method with corrections for
hydrogen bonding, dispersion and halogen bond, called
PM6-D3H4X, developed in our laboratory, accurately
describes all protein–ligand noncovalent interactions
(Brahmkshatriya et al., 2013). Thus, in consistent with our
earlier study (Brahmkshatriya et al., 2013), we carried out
fragmentation of the ligands and measured the interaction
of energy of these ligand subsystems with the neighboring
residues using PM6-D3H4X method. Results clearly
showed that benzoxazole moiety is found to be a critical
pharmacophore as it interacted favorably with Met103,
Phe149, Ala157, Tyr158, Ile202, and Ile215 (blue region,
Fig. S1b). Pyrazoline ring interacted with Ala198 and
Met199 by dispersion interactions (yellow region,
Fig. S1b). However, the interaction energies were much
lower than those for benzoxazole moiety. It was surprising
to find that the aryl ring faces towards solvent and does not
find any interacting partner (Fig. S1b) and hence, the
interaction energies were found to be against the binding.
This indicates that the fifth position on the pyrazoline ring
need not be substituted with aryl ring and can be replaced
with smaller substituents such as alkyl. Glide score values
of the ligands and the interaction energies of the ligand
fragments are summarized in Table 2 and Fig. S2, Sup-
plementary Material. It was very encouraging to see that
the total interaction energy (sum of all contributing inter-
action energies of the fragments) well correlated with the
pMIC values of the compounds (r2 = 0.70, Fig. 3). A very
good predictive index (PI) (Pearlman and Charifson, 2001)
of 0.83 indicates good rank ordering of the compounds
based on the predicted interaction energy. Comparison of
Fig. 3 The correlation of total interaction energy (in kcal/mol) with
pMIC
the docking pose of isoniazid was quite surprising. The
designed compounds interacted favorably with the active
site residues than isoniazid which is reflected by their better
score values. We think that this might be due to better fit of
the target compounds in the active site cavity as compared
to isoniazid (Fig. S1c, d). However, it should also be noted
that the poor score of isoniazid might be due to two well-
known issues with the docking: sampling and force field-
based scoring function. We understand that the Glide_
Score is a crude estimate of the binding interactions as it
depends on the underlying force field (OPLS in Glide) and
better chemical description (e.g. quantum mechanics-based
scoring function) is necessary.
Conclusion
In the present study, a series of 1-(5-(2-amino-7-sub-
stituted-benzoxazol-5-yl)-3-aryl-4,5-dihydro-1H-pyrazol-
123

2228
Med Chem Res (2014) 23:2218–2228
ˇ
Brahmkshatriya PS, Dobes P, Fanfrlık J, Rezac J, Paruch K,
Bronowska A, Lepsık M, Hobza P (2013) Quantum mechanical
´
´ˇ
1-yl)ethanones and 1-(5-(2-mercapto-7-substituted-benzox-
azol-5-yl)-3-aryl-4,5-dihydro-1H-pyrazol-1-yl)ethanones
were designed by combining the two pharmacophoric
motifs (pyrazoline and benzoxazole) for antitubercular
activity. Synthesized target compounds and their interme-
diate aminophenol derivatives were evaluated for their
ability to inhibit M. tuberculosis H37Rv and MDR-TB
strains in vitro. The biological screening provided inter-
esting results where some of the compounds displayed
significant antitubercular activity with a few analogs
showing better activity than isoniazid against MDR-TB
strains. In order to have insights into the nature of binding
of the target compounds with the molecular target of
tuberculosis (enoyl-ACP reductase), molecular docking
was carried out. Docking results suggest that the com-
pounds chiefly interact with the protein by dispersion
interactions. The interaction energies of the ligand frag-
ments (the three key cores) provided useful information
about the key pharmacophores suggesting importance of
benzoxazole core over the other two ring nucleuses. The
present study provides a useful protocol to combine two
pharmacophoric motifs in one compound to design potent
antitubercular agents.
ˇ´
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¨
Dye C, Lonnroth K, Jaramillo E, Williams BG, Raviglione M (2009)
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He X, Alian A, Stroud R, de Montellano PRO (2006) Pyrrolidine
carboxamides as a novel class of inhibitors of enoyl acyl carrier
protein reductase from mycobacterium tuberculosis. J Med
Chem 49:6308–6323
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U (2002) Heterocyclic benzoxazole derivatives with antimyco-
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