In vitro and in silico evaluation of 2-(substituted phenyl) oxazolo[4,5-b]pyridine derivatives as potential antibacterial agents

Article abstract of DOI:10.1007/s00044-017-2026-3

Increasing resistance to antibiotics is a major problem worldwide and stimulates the development of new bacterial inhibitors. Series of oxazolo[4,5-b]pyridine synthesized in our lab was examined to screen them as potential antimicrobial agents. The antimi

Full text of DOI:10.1007/s00044-017-2026-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2026-3
ORIGINAL RESEARCH
In vitro and in silico evaluation of 2-(substituted phenyl) oxazolo
[4,5-b]pyridine derivatives as potential antibacterial agents
Gagandeep Kour Reen1 Ashok Kumar1 Pratibha Sharma1
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Received: 16 April 2017 / Accepted: 5 August 2017
© Springer Science+Business Media, LLC 2017
Abstract Increasing resistance to antibiotics is a major
problem worldwide and stimulates the development of new
bacterial inhibitors. Series of oxazolo[4,5-b]pyridine syn-
thesized in our lab was examined to screen them as potential
antimicrobial agents. The antimicrobial analyses of the
synthesized compound were based on minimum inhibitory
concentration determination, against four strains of bacteria.
The results showed that the synthesized compounds have
elicited good activity profile against methicillin-resistant
Staphylococcus aureus (S. aureus), the bacterium that is
largely responsible for hospital-acquired infections. More-
over, synthesized compounds were also docked against
enterotoxin protein of S. aureus which belongs to Staphy-
lococcal enterotoxin type A(SEA). In vitro and in silico
studies revealed that compounds 3d, 3g, and 3h have
demonstrated significant antibacterial activity in comparison
to the standard control drug ampicillin and streptomycin.
Introduction
The increasing incidence of bacterial resistance towards a
large number of antibacterial agents viz., glycopeptides,
sulfonamides, β-lactams, nitroimidazoles, quinolones, tet-
racyclins, chloramphenicol, and macrolides, is becoming a
major threat of concern now a days (Davies 1996; Chu et al.
1996). One of the most important mechanisms of the
resistance against third generation antibiotics is the pro-
duction of extended spectrum β-lactamases (Murthy et al.
2012). These enzymes are responsible for hydrolyzing and
inactivating the β-lactam rings in the antibiotics like peni-
cillins, cephalosporins, and aztreonam. Strategies to over-
come such resistance require rigorous efforts towards
recategorization of existing classes of antibiotics, mod-
ification of the target, and the innovative approaches to
development of new antimicrobial agents (Vondenhoff and
Aerschot 2011). In this regard nitrogen containing hetero-
cycles, especially those containing pyridine rings have
received a great deal of attention in the literature due to their
role as active pharmacophores, which are found to be
associated with a number of diverse pharmacological
properties such as antimicrobial (Patel et al. 2010), antiviral
(Bernardino et al. 2007), anticonvulsant (Paronikyan et al.
2002), antifungal (Duchowicz et al. 2007), and anticancer
activities (Mohamed et al. 2012).
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Keywords Antibacterial Docking Oxazolo[4,5-b]
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pyridine Staphylococcus enterotoxin A
In particular oxazolo[4,5-b]pyridines, are well known to
the chemists, owing to their inherent wide spectrum of
antimicrobial potential, herbicidal, antihelmintic, anti-
oxidant and antitumoral properties (Mikko et al. 2007;
Arisoy et al. 2008; Ertan et al. 2009; Ouyang et al. 2012;
Khizhan et al. 2011).
Hence, encouraged by these findings it was thought
worthwhile to develop more powerful antibacterial
agents comprising of pyridine skeleton in their core
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-2026-3) contains supplementary material,
which is available to authorized users.
* Pratibha Sharma
drpratibhasharma@yahoo.com
1
School of Chemical Sciences, Devi Ahilya Vishwavidyalaya,
Takshashila Campus, Indore, MP 452 001, India

Med Chem Res
armamentarium. Therefore, in continuation of our work on
the synthesis of a variety of heterocycles and evaluation of
their biological potential (Sharma et al. 2004, 2009, 2010,
2012), we have tried to rationalize the investigation of
antibacterial potential of the compounds synthesized in our
lab (Sharma et al. 2015). The screening was carried out
against a representative panel of two Gram-positive and two
Gram-negative bacterial strains by estimating their potential
in terms of minimum inhibitory concentration (MIC).
Furthermore, in view to gain an insight about the
structure–activity relationship (SAR) of the docked com-
pound at the active site of the enzyme, a docking simulation
of synthesized oxazolopyridines was also performed con-
sidering SEA as the protein receptor of S. aureus belonging
to the family of Staphylococcal enterotoxins (SEs) (Pinchuk
et al. 2010). However, out of a number of protein receptors
derived from twenty distinct SEs, SEA is the most common
one related to the food poisoning. SEs are potent gastro
intestinal toxins that can resist heat treatment, low pH and
proteolytic enzymes, and thereby retained in the GIT after
ingestion (Argudin et al. 2010).
ambient conditions. Progress of the reaction was con-
tinuously monitored by TLC (n-hexane and ethyl acetate in
2:1). After the completion of process as marked by TLC, the
solid was recovered by rotatory evaporator. The beauty of
the process lies in the easy recovery of the catalyst by
adding excess of chloroform. The product was washed with
dichloromethane (2 × 5 mL) and then recrystallized from
acetonitrile.
2-Phenyloxazolo[4,5-b] pyridine (3a) This compound was
obtained from 2-amino-3-hydroxypyridine and benzoic acid
(2a) from the previously described procedure as off-white
solid (90%); M.P.: 132–134 °C; IR (υ, cm−1): 3052, 1678,
1
1595, 1554, 1490, 1260, 1065, 662; H NMR (400 MHz,
DMSO-d6) δ 7.93–8.03 (m, 2H, Ar–H), 7.68 (d, 1H, J =
7.2 Hz, Pyr–H), 7.51–7.60 (m, 2H, Ar–H), 7.41–7.52 (m,
1H, Ar–H), 7.02 (d, 1H, J = 7.2 Hz, Pyr–H), 6.45 (t, 1H,
J = 6.9 Hz, Pyr–H); 13C NMR (100 MHz, DMSO-d6) δ
159.5 (C-2), 156.8 (C-3a), 143.2 (C-5), 138.4 (C-7a), 136.1
(C-10,12), 127.6 (C-11), 122.6 (C-9,13), 119.3 (C-8), 116.2
(C-6), 114.5 (C-7); HRESIMS m/z [M + H]+: 197.067
(calcd. 196.205); anal. calcd. for C21H15N: C, 73.51, H,
4.34, N, 13.98; found C, 73.58, H, 4.45, N, 13.84 (Sharma
et al. 2015).
Experimental
Materials and methods
2-(2-Chlorophenyl)oxazolo[4,5-b]pyridine
(3b) This
compound was obtained from 2-amino-3-hydroxypyridine
and 2-chlorobenzoic acid (2b) from the previously descri-
bed procedure as off white solid (89%); M.P. 98–103 °C; IR
(υ, cm−1): 3061, 1670, 1627, 1579, 1540, 1258, 1050,
All reagents were purchased from Sigma Aldrich, India, and
used without further purification. The reactions were per-
formed in an aerobic atmosphere without any specific pre-
cautions. Melting points were determined in open capillary
tubes on a Veego melting-point apparatus and are uncor-
rected. Fourier transform infrared (FTIR) spectra were
recorded within the range of 4000–400 cm−1 using FTIR
1
1030, 756; H NMR (400 MHz, DMSO-d6) δ 7.72 (d, 1H,
J = 7.2 Hz, Pyr–H), 7.33–7.56 (m, 4H, Ar–H), 7.09 (d, 1H,
J = 7.2 Hz, Pyr–H), 6.54 (t, 1H, J = 6.8 Hz, Pyr–H); 13C
NMR (100 MHz, DMSO-d6) δ 159.2 (C-2), 154.3 (C-3a),
148.4 (C-5), 142.2 (C-7a), 132.5 (C-8), 130.5 (C-9), 129.1
(C-10), 128.3 (C-13), 125.8 (C-11,12), 120.8 (C-6), 118.6
(C-7); HRESIMS m/z [M + H]+: 231.029 (calcd. 230.650);
anal. calcd. for C12H7ClN2O: C, 62.49, H, 3.06, N, 12.15;
found C, 62.42, H, 3.08, N, 12.17.
1
STD10 Perkin-Elmer. The H and 13C nuclear magnetic
resonance (NMR) spectra of the synthesized compounds
were recorded at 400 and 100 MHz respectively, using
Bruker Avance II 400 NMR spectrometer in dimethylsulf-
oxide (DMSO) solvent, and the chemical shifts were
expressed in parts per million. Spin multiplicities are
described as s (singlet), d (doublet), t (triplet), q (quartet),
and m (multiplet). Mass analysis was performed on
quadruple-time-of-flight mass spectrometer (MICRO-
MASS) using electrospray ionization (ESI) in positive
mode. Thin-layer chromatography (TLC) is performed
using precoated aluminum sheets with silica gel 60 F254.
2-o-Tolyloxazolo[4,5-b]pyridine (3c) This compound was
obtained from 2-amino-3-hydroxypyridine and 2-
methylbenzoic acid (2c) from the previously described
procedure as white solid (90%); M.P.: 66–69 °C [Lit.
64–66 °C] (Clark et al. 1978); IR (υ, cm−1): 3046, 2969,
1
2854, 1685, 1600, 1510, 1490, 1264, 1080, 755; H NMR
(400 MHz, DMSO-d6) δ 8.12 (dd, 1H, J = 5.0, 1.6 Hz,
Pyr–H), 7.83 (dd, 1H, J = 8.2, 1.6 Hz, Pyr–H), 7.44 (t, 1H,
J = 7.9 Hz, Pyr–H), 7.29–7.18 (m, 4H, Ar–H), 2.38 (s, 3H,
CH3); 13C NMR (100 MHz, DMSO-d6) δ 160.3 (C-2),
155.4 (C-3a), 148.9 (C-5), 143.4 (C-7a), 137.4 (C-8), 136.5
(C-9), 129.9 (C-10), 128.5 (C-11), 127.2 (C-13), 126.1 (C-
12), 122.4 (C-6), 117.8 (C-7), 18.1 (C, CH3); HRESIMS
General procedure for synthesis of 2-(substituted phenyl)
oxazolo[4,5-b]pyridine
To the mixture of benzoic acid (2 mmol) and 2-amino-3-
hydroxypyridine (1 mmol) in 3 mL methanol, HClO4·SiO2
nanoparticles (5 mol%) were added under stirring under

Med Chem Res
m/z [M + H]+: 211.079 (calcd. 210.231); anal. calcd. for
C13H10N2O: C, 74.27, H, 4.79, N, 13.33; found C, 74.29,
H, 4.82, N, 13.35.
2-(3-Nitrophenyl)oxazolo[4,5-b]pyridine (3g) This com-
pound was obtained from 2-amino-3-hydroxypyridine and
3-nitrobenzoic acid (2g) from the previously described
procedure as white solid (86%); M.P.: 200–203 °C [Lit.
199–201 °C] (Clark et al. 1978); IR (υ, cm−1): 3039, 1690,
1
2-(2-Methoxyphenyl)oxazolo[4,5-b]pyridine
(3d) This
1592, 1519, 1484, 1360, 1348, 1260, 1077, 768; H NMR
compound was obtained from 2-amino-3-hydroxypyridine
and 2-methoxybenzoic acid (2d) from the previously
described procedure as off white solid (92%): M.P.:
110–112 °C; IR (υ, cm−1): 3050, 2962, 2845, 1680, 1596,
1518, 1490, 1244, 1085, 760; 1H NMR (400 MHz, DMSO-
d6) δ 7.95 (d, 1H, J = 6.4 Hz, Pyr–H), 7.69 (d, 1H, J = 6.4
Hz, Pyr–H), 7.357–7.562 (m, 4H, Ar–H), 6.483 (t, 1H, J =
6.2 Hz, Pyr–H), 3.80 (s, 3H, OCH3); 13C NMR (100 MHz,
DMSO-d6) δ 159.3 (C-2), 157.3 (C-9), 154.8 (C-3a), 148.5
(C-5), 142.6 (C-7a), 129.4 (C-11,13), 123.2 (C-6), 121.2
(C-12), 118.9 (C-7), 115.2 (C-10), 111.6 (C-8), 62.2 (C,
OCH3); HRESIMS m/z [M + H]+: 227.077 (calcd.
226.231); anal. calcd. for C13H10N2O2: C, 69.02, H, 4.46,
N, 12.38, found C, 69.05, H, 4.50, N, 12.35.
(400 MHz, DMSO-d6) δ 8.55 (dd, 1H, J = 4.8, 1.4 Hz,
Pyr–H), 8.41(s, 1H, Ar–H), 8.09–7.98 (m, 3H, Ar–H), 7.78
(dd, 1H, J = 7.8, 1.4 Hz, Pyr–H), 7.42 (t, 1H, J = 7.6 Hz,
Pyr–H); 13C NMR (100 MHz, DMSO-d6) δ 159.4 (C-2),
154.6 (C-3a), 149.2 (C-10), 148.4 (C-5), 143.2 (C-7a),
134.1 (C-13), 129.8 (C-12), 127.3 (C-8), 122.3 (C-6,9),
121.5 (C-11), 118.6 (C-7); HRESIMS m/z [M + H]+:
242.028 (calcd. 241.202); anal. calcd. for C12H7N3O3: C,
59.75, H, 2.93, N, 17.42; found C, 59.79, H, 2.95, N, 17.45.
2-(3-Methoxyphenyl)oxazolo[4,5-b]pyridine
(3h) This
compound was obtained from 2-amino-3-hydroxypyridine
and 3-methoxybenzoic acid (2h) from the previously
described procedure as white solid (92%); M.P.: 115–118 °
C; IR (υ, cm−1): 3038, 2958, 2855, 1684, 1590, 1517, 1497,
1260, 1082, 755; 1H NMR (400 MHz, DMSO-d6) δ 7.90 (d,
1H, J = 8.4 Hz, Pyr–H), 7.56 (d, 1H, J = 8.4 Hz, Pyr–H),
7.29–7.36 (m, 3H, Ar–H), 7.16 (t, 1H, J = 9.2 Hz, Pyr–H),
6.59 (s, 1H, Ar–H), 3.84 (s, 3H, OCH3); 13C NMR (100
MHz, DMSO-d6) δ 167.8 (C-10), 161.5 (C-2), 154.8 (C-3a),
148.1 (C-5), 142.8 (C-7a), 130.7 (C-12), 127.1 (C-8), 119.2
(C-6), 118.8 (C-7,13), 114.5 (C-11), 111.3 (C-9), 62.2 (C,
OCH3); HRESIMS m/z [M + H]+: 227.074 (calcd.
226.231); anal. calcd. for C13H10N2O2, C, 69.02, H, 4.46,
N, 12.38; Found C, 69.01, H, 4.48, N, 12.38.
2-(3-Bromophenyl)oxazolo[4,5-b]pyridine (3e) This com-
pound was obtained from 2-amino-3-hydroxypyridine and
3-bromobenzoic acid (2e) from the previously described
procedure as pale yellow solid (88%); M.P.: 156–160 °C;
IR (υ, cm−1): 3047, 1693, 1595, 1522, 1486, 1225, 1062,
1
965, 755; H NMR (400 MHz, DMSO-d6) δ 7.62 (s, 1H,
Ar–H), 7.58 (d, 1H, J = 6.8 Hz, Pyr–H), 7.39 (d, 1H, J =
6.8 Hz, Pyr–H), 7.082–7.196 (m, 3H, Ar–H) 6.58 (t, 1H, J
= 7 Hz, Pyr–H); 13C NMR (100 MHz, DMSO-d6) δ 167.3
(C-2), 154.3 (C-3a), 148.3 (C-5), 142.4 (C-7a), 133.8 (C-9),
131.6 (C-11,12), 128.3 (C-8), 126.3 (C-13), 122.7 (C-6),
119.9 (C-10), 118.8 (C-7); HRESIMS m/z [M + H]+:
276.092 (calcd. 275.101); anal. calcd. for C12H7BrN2O: C,
52.39, H, 2.56, N, 10.18; Found C, 52.42, H, 2.58, N,
10.20.
2-(4-Bromophenyl)oxazolo[4,5-b]pyridine (3i) This com-
pound was obtained from 2-amino-3-hydroxypyridine and
4-bromobenzoic acid (2i) from the previously described
procedure as light yellow solid (90%); M.P.: 172–175 °C;
IR (υ, cm−1): 3061, 1684, 1582, 1516, 1485, 1268, 1086,
1
780; H NMR (400 MHz, DMSO-d6) δ 8.54 (dd, 1H, J =
2-(3-Chlorophenyl)oxazolo[4,5-b]pyridine (3f) This com-
pound was obtained from 2-amino-3-hydroxypyridine and
3-chlorobenzoic acid (2f) from the previously described
procedure as white solid (90%); M.P.: 148–151 °C; IR (υ,
cm−1): 3045, 1684, 1591, 1517, 1492, 1263, 1082, 1055,
5.2, 1.5 Hz, Pyr–H), 7.82 (dd, 1H, J = 8.1, 1.5 Hz, Pyr–H),
7.42 (t, 1H, J = 7.9 Hz, Pyr–H), 7.24–7.36 (m, 4H, Ar–H);
13C NMR (100 MHz, DMSO-d6) δ 158.3 (C-2), 155.2 (C-
3a), 147.8 (C-5), 143.2 (C-7a), 131.8 (C-10,12), 129.4 (C-
9,13), 124.8 (C-8), 123.5 (C-11), 122.2 (C-6), 117.8 (C-7);
HRESIMS m/z [M + H]+: 276.098 (calcd. 275.101); anal.
calcd. for C12H7BrN2O, C, 52.39, H, 2.56, N, 10.18; found
C, 52.42, H, 2.54, N, 10.21.
1
772; H NMR (400 MHz, DMSO-d6) δ 7.561–7.642 (m,
3H, Ar–H), 7.42 (s, 1H, Ar–H), 7.36 (d, 1H, J = 8.4 Hz,
Pyr–H), 7.11 (d, 1H, J = 8.4 Hz, Pyr–H), 6.65 (t, 1H, J =
6.2 Hz, Pyr–H); 13C NMR (100 MHz, DMSO-d6) δ 167.3
(C-2), 154.5 (C-3a), 148.7 (C-5), 142.4 (C-7a), 134.5 (C-9),
129.1 (C-11,12), 128.2 (C-8), 127.8 (C-13), 125.1 (C-6),
119.4 (C-10), 118.6 (C-7); HRESIMS m/z [M + H]+:
231.024 (calcd. 230.65); anal. calcd. for C12H7ClN2O, C,
62.49, H, 3.06, N, 12.15; Found C, 62.45, H, 3.10, N,
12.14.
2-(4-(Trifluoromethyl)phenyl)oxazolo[4,5-b]pyridine (3j)
This compound was obtained from 2-amino-3-
hydroxypyridine and 4-trifluoromethylbenzoic acid (2j)
from the previously described procedure as white solid
(86%); M.P. 162–164 °C; IR (υ, cm−1): 3055, 1693, 1596,
1
1509, 1490, 1265, 1150, 1082, 776; H NMR (400 MHz,

Med Chem Res
DMSO-d6) δ 7.96 (d, 2H, J = 8.4 Hz, Ar–H), 7.88 (d, 1H, J
= 9.6 Hz, Pyr–H), 7.37 (d, 2H, J = 8.4 Hz, Ar–H), 7.15 (d,
1H, J = 9.6 Hz, Pyr–H), 6.72 (t, 1H, J = 7.2 Hz, Pyr–H);
13C NMR (100 MHz, DMSO-d6) δ 167.6 (C-2), 154.6 (C-
3a), 148.8 (C-5), 142.4 (C-7a), 133.5 (C-11), 131.9 (C-8),
127.7 (C-9,13), 125.5 (C-10,12), 124.7 (C, CF3), 119.6
(6C), 118.1 (7C); HRESIMS m/z [M + H]+: 265.059
(calcd. 264.203); anal. calcd. for C13H7F3N2O, C, 59.10, H,
2.67, N, 10.60; found C, 59.09, H, 2.69, N, 10.55.
2-(4-Methoxyphenyl)oxazolo[4,5-b]pyridine
(3n) This
compound was obtained from 2-amino-3-hydroxypyridine
and 4-methoxybenzoic acid (2n) from the previously
described procedure as white solid (94%); M.P.: 178–180 °
C; IR (υ, cm−1): 3047, 2968, 2850, 1691, 1588, 1503, 1492,
1267, 1060, 750; 1H NMR (400 MHz, DMSO-d6) δ 7.95 (d,
2H, J = 8.8 Hz, Ar–H), 7.83 (d, 1H, J = 7.2 Hz, Pyr–H),
7.31 (d, 1H, J = 7.2 Hz, Pyr–H), 7.05 (d, 2H, J = 8.8 Hz,
Ar–H), 6.613–6.645 (t, 1H, J = 6.4 Hz, Pyr–H), 3.86 (s, 3H,
OCH3); 13C NMR (100 MHz, DMSO-d6) δ 167.6 (C-11),
160.5 (C-2), 154.5 (C-3a), 148.1 (C-5), 142.8 (C-7a),
122.3 (C-9,13), 119.2 (C-6), 118.6 (C-8), 115.7 (C-7),
114.5 (C-10,12), 63.2 (C, OCH3); HRESIMS m/z
[M + H]+: 227.082 (calcd. 226.231); anal. calcd. for
C13H10N2O2: C, 69.02, H, 4.46, N, 12.38; found C, 68.99,
H, 4.51, N, 12.35.
2-(4-Chlorophenyl)oxazolo[4,5-b]pyridine
(3k) This
compound was obtained from 2-amino-3-hydroxypyridine
and 4-chlorobenzoic acid (2k) from the previously descri-
bed procedure as off white solid (87%): M.P.: 165–168 °C;
IR (υ, cm−1): 3057, 1689, 1585, 1519, 1488, 1270, 1090,
1056, 768; 1H NMR (400 MHz, DMSO-d6) δ 7.86 (d, 1H, J
= 8.4 Hz, Pyr–H), 7.58 (d, 2H, J = 9.6 Hz, Ar–H), 7.39 (d,
2H, J = 9.6 Hz, Ar–H), 7.22 (d, 1H, J = 8.4 Hz, Pyr–H),
6.68 (t, 1H, J = 7 Hz, Pyr–H); 13C NMR (100 MHz,
DMSO-d6) δ 167.9 (C-2), 154.6 (C-3a), 148.5 (C-5), 142.1
(C-7a), 134.3 (C-11), 129.7 (C-10,12), 128.1 (C-9,13),
119.9 (C-6), 118.3 (C-7a); HRESIMS m/z [M + H]+:
231.030 (calcd. 230.65); anal. calcd. for C12H7ClN2O: C,
62.49, H, 3.06, N, 12.15; found C, 62.48, H, 3.11, N, 12.17.
In vitro antibacterial procedure
All the test compounds were assayed in vitro for anti-
bacterial activity against methicillin-resistant S. aureus, B.
subtilis, E. coli, and P. diminuta using nutrient agar as
growth medium. The MIC was determined by using two-
fold serial dilution method. Stock solutions of test com-
pounds were prepared in dimethyl formamide (DMF) at a
concentration of 800 µg/mL, suspension containing 107
CFU/mL of bacteria was prepared from broth culture.
Bacterial plates were prepared in triplicate and incubated at
37 °C within 16–24 h. Ampicillin and streptomycin were
tested under the similar conditions as reference drugs. In
order to ensure that the solvent had no effect on bacterial
growth, a control test was also performed containing broth
supplemented with only DMF at the same dilution used in
our experiment. The MIC values were obtained from the
lowest concentration of the test compound which was
required to inhibit the bacterial growth. The MIC values
were expressed in µg/mL.
2-(4-Methylphenyl)oxazolo[4,5-b]pyridine (3l) This com-
pound was obtained from 2-amino-3-hydroxypyridine and
4-methylbenzoic acid (2l) from the previously described
procedure as off white solid; yield: 96%; M.P. 138–140 °C;
IR (υ, cm−1): 3043, 2974, 2857, 1689, 1597, 1508, 1493,
1267, 1078, 759; 1H NMR (400 MHz, DMSO-d6) δ
7.87–7.90 (m, 3H, Pyr–H), 7.21–7.34 (m, 4H, Ar–H), 2.24
(s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 167.5 (C-
2), 154.3 (C-3a), 148.8 (C-5), 142.5 (C-7a), 131.4 (C-11),
127.5 (C-10,12), 126.3 (C-9,13), 119.7 (C-6), 118.7 (C-7),
29.3 (C, CH3); HRESIMS m/z [M + H]+: 211.085 (calcd.
210.231); anal. calcd. for C13H10N2O: C, 74.27, H, 4.79, N,
13.33; found C, 74.25, H, 4.83, N, 4.77.
2-(4-Nitrophenyl)oxazolo[4,5-b]pyridine (3m) This com-
pound was obtained from 2-amino-3-hydroxypyridine and
4-nitrobenzoic acid (2m) from the previously described
procedure as off white solid (88%); M.P.: 217–220 °C; IR
(υ, cm−1): 3042, 1680, 1596, 1512, 1482, 1365, 1352,
Molecular docking studies
The X-ray crystal structure of SEA protein of methicillin-
resistant S. aureus (PDB ID: 1esf) was downloaded from
the RCSB Protein Data Bank (http://www.rcsb.org/). The
protein was imported in MVD, and missing bond orders,
hybridization states, and angles were then assigned.
To obtain better potential binding sites in the protein,
a maximum of five cavities were detected using parameters
such as molecular surface (expanded van der Waals),
maximum number of cavities (n = 5), minimum cavity
volume (10), probe size (1.20), maximum number of ray
checks (n = 16), minimum number of ray hits (n = 12), and
grid resolution (0.80).
1
1271, 1074, 789; H NMR (400 MHz, DMSO-d6) δ 8.66
(dd, 1H, J = 5.0, 1.3 Hz, Pyr–H), 8.11–7.98 (m, 4H, Ar–H),
7.81(dd, 1H, J = 8.2, 1.3 Hz, Pyr–H), 7.45 (t, 1H, J = 7.8
Hz, Pyr–H); 13C NMR (100 MHz, DMSO-d6) δ 160.6 (C-
2), 154.5 (C-3a), 148.3 (C-11), 147.6 (C-5), 143.2 (C-7a),
133.1 (C-8), 128.2 (C-9,13), 122.4 (C-6), 121.8 (C-10,12),
118.6 (C-7); HRESIMS m/z [M + H]+: 242.012 (calcd.
241.202); anal. calcd. for C12H7N3O3: C, 59.75, H, 2.93, N,
17.42; found C, 59.72, H, 2.90, N, 17.46.

Med Chem Res
All docking calculations were carried out using the grid-
based Moldock score (GRID) function with a grid resolu-
tion of 0.30 Å. The binding site on the receptor was defined
by cavity of volume 50.688 Å3 and surface area of 200.96
Å2 with a radius of 15 Å. The Moldock optimization search
algorithm with a maximum of ten runs was used through the
calculations, with all other parameters kept as defaults. Each
time five poses were obtained and the best pose was
selected based on the scoring function such as the Rerank
score, Moldock score and interaction energy (Thomsen and
Christensen 2006). This pose was then converted to ligand
and the ligand energy inspector was employed to determine
ligand–protein interaction.
two Gram-negative bacterial strains, Escherichia coli
ATCC 13067 and Pseudomonas diminuta MTCC 3361.
In the present investigation, 2-(phenyl)oxazolo[4,5-b]-
pyridine derivatives were tested against the chosen bacterial
strains using standard two-fold serial dilution method and
reported in terms of MIC (Table 1). The in vitro anti-
bacterial activity results revealed that the synthesized
compounds 3d, 3g, and 3h were found to be highly active
(MIC: 1.56–25 µg/mL), whereas 3b, 3c, 3e, 3f, and 3m
were moderately active (MIC: 6.25–50 µg/mL) as compared
to the standard drug ampicillin and streptomycin. A sys-
tematic perusal of the data depicted in Table 1, reveals that
in comparison to Gram-negative bacteria, Gram-positive
bacteria are more sensitive to synthesized oxazolo[4,5-b]
pyridine analogs. Compounds 3d, 3g, and 3h showed strong
Results and discussion
Chemistry
Table 1 MIC values of oxazolo[4,5-b]pyridine derivatives
MIC in µg/mL
2-(Substituted phenyl) oxazolo[4,5-b]pyridine derivatives
were synthesized as per the procedural follow up developed
in our lab (Sharma et al. 2015). Syntheses and character-
ization details of some of the new derivatives (3c, 3g, 3i,
and 3m) are being reported here. The compounds were
prepared using a one pot synthetic strategy (Scheme 1) in
quantitative yield by the reaction between 2-amino-3-
hydroxy pyridine (1) and substituted benzoic acids (2) in
the presence of silica-supported perchloric acid. The reac-
tion was performed under ambient conditions using
methanol as the solvent. The silica-supported catalyst has
high mechanical and thermal stability, low toxicity, good
recyclability, and ease of handling (Khan et al. 2006).
Structures of the synthesized compounds were confirmed by
Compounds Gram-positive bacteria
Gram-negative bacteria
S. aureus
ATCC
2943
B. subtilis
ATCC
6633
E. coli
ATCC
13067
P. diminuta
MTCC 3361
3a
3b
3c
3d
3e
3f
100
6.25
6.25
3.125
12.5
6.25
1.56
3.125
50
100
25
100
50
>100
25
12.5
6.25
12.5
25
25
25
25
12.5
25
50
50
50
3g
3h
3i
3.125
6.25
50
12.5
12.5
100
50
6.25
12.5
100
50
1
elemental, infrared (IR), H NMR, 13C NMR, and mass
3j
50
50
spectral analyzes.
3k
3l
25
100
50
100
>100
50
50
50
>100
25
Biology
3m
3n
A
12.5
25
25
50
50
100
25
All the synthesized derivatives were screened in vitro for
their antibacterial activities against two Gram-positive
bacterial strains, methicillin-resistant Staphylococcus aur-
eus (MRSA) ATCC 2943, Bacillus subtilis ATCC 6633 and
6.25
12.5
12.5
25
50
S
50
25
A ampicillin, S streptomycin
Scheme 1 Synthesis of 2-
(phenyl)oxazolo[4,5-b]pyridine
derivatives using HClO4·SiO2 as
supported catalyst

Med Chem Res
activity against methicillin-resistant S. aureus (MIC:
1.56–3.125 µg/mL) as compared to control drugs (MIC:
6.25 and 12.5 µg/mL). These compounds are also active
against other strains of bacteria but their MIC values are
more in comparison to methicillin-resistant S. aureus.
Antibacterial profile of the tested compounds elicits the
following progression (Fig. 1):
P. diminuta < E. coli < B. subtilis < S. aureus (MRSA)
In Fig. 1, MIC values greater than 100 have not been
incorporated for compounds 3a and 3l against gram-
negative bacteria.
rationalize the obtained biological data and explain the
possible molecular interactions that might take place. Fur-
thermore, to comprehend the deepness of the ligand–protein
molecular interaction, the ligand energy inspector was
employed. The molecular docking results of test compounds
and reference drug with the protein receptor in terms of
Moldock score, Rerank score, interaction and hydrogen
bonding energies are depicted in Table 2. The docking
studies have revealed that out of fourteen derivatives three
were found to have favorable Rerank score and docking
Further the close inspection of screening results revealed
that substitution in the aromatic ring attached at C-2 posi-
tion of oxazolo[4,5-b]pyridine nucleus exerted significant
influence on the investigated biological activity. Interest-
ingly, it was noticed that in vitro antibacterial activity of the
compounds 3a-n is found to be increased when groups such
as –NO2, –OMe and –Cl and –Br are present in meta and
ortho position (Fig. 2).
Table 2 Docking scores of the synthesized derivatives and control
drug
Compounds MolDock
scorea
Rerank
Score
Interactionb H bondc
3a
3b
3c
3d
3e
3f
−109.06
−116.73
−116.90
−117.31
−114.69
−115.06
−122.65
−119.10
−113.30
−115.34
−113.41
−113.51
−116.99
−113.47
−91.16
−96.50
−97.61
−98.66
−95.42
−95.67
−103.20
−101.02
−92.90
−93.54
−93.06
−93.89
−94.38
−92.33
−100.23
−107.76
−115.05
−114.38
−118.43
−112.35
−112.40
−121.43
−117.10
−110.99
−114.32
−111.14
−111.21
−115.34
−114.01
−122.00
−10.54
−10.58
−10.58
−9.72
Molecular docking studies
−10.59
−10.61
−8.17
Molecular modeling, vis-à-vis molecular docking involves
the prediction of ligand confirmation and orientation in the
binding pocket of receptor. In the present study, docking
simulations were carried out using Molegro Virtual Docker
(MVD) 4.0. All fourteen derivatives along with the standard
drug (ampicillin) were docked with protein receptor SEA of
most sensitive bacteria, S. aureus (MRSA) in order to
3g
3h
3i
−10.13
−10.55
−5.30
3j
3k
3l
−10.55
−10.55
−10.01
−9.11
3m
3n
Ampicillin −121.98
−5.79
100
90
a Moldock score is derived from the PLP scoring functions with a new
hydrogen bonding term and new charge schemes (Pathak et al. 2013)
80
70
b
The total interaction energy between the best pose and the protein
S. aureus
60
(kJ/mol)
B. subꢀlis
50
c
Hydrogen bonding energy (kJ/mol)
E. coli
40
P. diminuta
30
20
10
0
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n
Compounds
Fig. 1 Graphical representation depicting antibacterial activity of the
synthesized moieties
7
9
10
1
7a
3a
O
R
11
8
6
5
2
N
3
N
4
12
13
Fig. 3 Secondary structure of protein along with the binding cavity
(green wireframe) (color figure online)
Fig. 2 Compounds 3a–n

Med Chem Res
Fig. 4 Binding interactions of ampicillin with the protein (PDB ID:
1esf)
score compared to ampicillin. The ligand–protein interac-
tion is considered based on the Rerank score, which is
defined as a linear combination of E-inter which is of van
der Waals, steric, electrostatic, hydrogen bonding, energy
between the ligand and the protein, and E-intra which is of
van der Waals, hydrogen bonding, torsional, electrostatic,
sp2–sp2 energy of the ligand weighted by pre-defined
coefficients (Pathak et al. 2013).
Out of these three compounds 3g has highest affinity
with Rerank score of −103.20 kJ/mol followed by
3h and 3d exhibiting significant binding affinities. In
compound 3a with least binding affinity (−91.16 kJ/mol)
there is no substitution in the aromatic ring, suggesting
that the presence of substitutions on aromatic ring enhances
the ligand–protein interactions, which can be further
enhanced by the presence of aromatic ring with substituents
at ortho and meta positions. Moreover, the docking results
were in good correlation with in vitro antibacterial activity
of these compounds. The most important amino acid
components present in the close proximity to the binding
cavity of size 50.688 Å3 are depicted in Fig. 3, where
arrows represent β sheets and α helices are represented by
helical lines.
Fig. 5 Ligand–protein (PDB ID: 1esf) interactions of compounds 3d
(a), 3g (b) and 3h (c) at the binding cavity
Conclusion
The snapshots of the ligand–protein molecular interac-
tion illustrating the binding mode of the reference drug,
compounds 3d, 3g, and 3h are depicted in Figs. 4, 5,
respectively. Ampicillin showed molecular interaction with
Cys 96, Thr104, Gln95, and Gln19 (Fig. 4).
Compound 3d interacted with Asn102, Thr104, Gly98,
and Gly99 (Fig. 5a), 3g showed interaction with Asn102,
Thr104, Gln95, and Gln19 (Fig. 5b) and 3h showed
molecular interaction with Gly98, Gly99, Thr104, and
Asn102 (Fig. 5c). Green colored dashed lines represent
hydrogen bonds. Furthermore, ligand-protein interaction
analyzes for 3d, 3g, 3h, and reference drug, which repre-
sents the interacting residues, interaction energy and inter-
action distances are shown in Table 3.
In the present studies, synthesized oxazolo pyridine deri-
vatives were evaluated for their antibacterial potency
against four different strains of bacteria. Three derivatives
(3d, 3g, and 3h) possessed strong antibacterial activity
against all the four strains with best activity profile against
S. aureus (MRSA) and five compounds (3b, 3c, 3e, 3f, and
3m) were found to be moderately active against all the
tested strains of bacteria. The compounds were further
screened for molecular docking simulation against the S.
aureus (MRSA) protein (PDB ID: 1esf) and observed that
three compounds were found to exhibit favorable
ligand–protein binding affinities compared to standard drug
(ampicillin and streptomycin).

Med Chem Res
Table 3 Molecular interaction
analysis
Compound
Ampicillin
Interaction
Interaction energy
Interaction distance (Å)
Cys96(O)–N(8)
Thr104(O)–N(11)
Gln95(N)–O(15)
Gln19(N)–N(13)
Asn102(N)–N(2)
Thr104(N)–N(2)
Thr104(O)–N(2)
Asn102(N)–N(6)
Gln95(N)–O(17)
Gln19(N)–N(15)
Gly98(N)–N(2)
Gly99(N)–N(2)
Thr104(O)–N(2)
Asn102(N)–N(6)
Thr104(N)–N(6)
Thr104(O)–N(6)
Asn102(N)–O(8)
Asn102(N)–O(8)
Asn102(N)–N(6)
Thr104(O)–N(6)
Thr104(N)–N(6)
Thr104(O)–N(2)
Gly98(N)–N(2)
Gly99(N)–N(2)
−0.53
−1.59
−2.31
−1.35
−0.74
−0.22
−2.05
−1.26
−2.50
−1.40
−1.41
−1.52
−2.50
−0.34
−1.73
−0.16
−2.49
−2.46
−0.03
−0.06
−1.74
−2.50
−1.52
−1.41
3.28
3.14
2.58
3.22
3.33
3.50
3.10
3.15
2.66
2.91
3.17
3.17
2.97
3.47
3.06
3.50
3.10
2.95
3.58
3.58
3.11
3.05
3.02
3.23
3g
3h
3d
Acknowledgements This work is supported by the financial assis-
tance received from UGC-SAP-DRS-I [No. F. 540/13/DRS-I/2016
(SAP-I)] and G.K.R is thankful to UGC-MANF (F1-17.1/2017-18/
MANF-2017-18-MAD-73866 /(SA-III/Website)) for Junior Research
Fellowship. The authors are thankful to Sophisticated Analytical
Instrumentation Facility Punjab University, Chandigarh, for providing
experimental results.
1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives. Med
Chem Res 16:352–369
Chu DTW, Plattner JJ, Katz L (1996) New directions in antibacterial
research. J Med Chem 39:3853–3874
Clark RL, Pessolano AA, Witzel B, Lanza T, Shen TY, Van Arman
CG, Risley EA (1978) 2- (Substituted phenyl) oxazolo[4,5-b]
pyridines and 2-(substituted phentl) oxazolo[5,4-b] pyridines as
non acidic anti-inflammatory agents. J Med Chem 21:1158–1162
Davies J (1996) Bacteria on the rampage. Nature 383:219–220
Duchowicz PR, Vitale MG, Castro EA, Fernandez M, Caballero J
(2007) QSAR analysis for heterocyclic antifungals. Bioorg Med
Chem 15:2680–2689
Ertan T, Yildiz I, Tekiner-Gulbas B, Bolelli K, Temiz- Arpaci O,
Ozkan S, Kaynak F, Yalçin I, Aki E (2009) Synthesis, biological
evaluation and 2D-QSAR analysis of benzoxazoles as anti-
microbial agents. Eur J Med Chem 44:501–510
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
Khan AT, Choudhary LH, Ghosh S (2006) Silica supported perchloric
acid (HClO4-SiO2): A highly efficient and reusable catalyst for
geminal diacylation of aldehydes under solvent-free conditions. J
Mol Cata A 255:230–235
References
Argudin MA, Mendoza MC, Rodicio MR (2010) Food poisoning and
staphylococcus aureus enterotoxins. Toxins 2:1751–1773
Arisoy M, Temiz-Arpaci O, Yildiz I, Kaynak-Onurdag F, Aki E,
Yalçin I, Abbasoglu U (2008) Synthesis, antimicrobial activity
and QSAR studies of 2,5-disubstituted benzoxazoles. SAR
QSAR Environ Res 19:589–612
Bernardino AMR, De Azevedo AR, Pinheiro LCD, Borges JC, Car-
valho VL, Miranda MD, De Meneses MDF, Nascimento M,
Ferreira D, Rebello MA (2007) Synthesis and antiviral activity of
new 4-(phenylamino)/4-[(methylpyridin-2-yl)amino]-1-phenyl-
Khizhan EI, Khizhan AI, Nikolaevskii AN, Kniga OP, Ivleva TN,
Tikhonova GA (2011) Antioxidant activity of 2-methyl-1,3-
benzoxazol-6-ol. Russ J Gen Chem 81:122–127
Mikko J, Ki M, Koskinen AMP (2007) A rapid method for the pre-
paration of 2-substituted oxazolo[4,5-b]pyridines using micro-
wave assisted direct condensation reactions. Tetrahedron Lett
48:2295–2298
Mohamed MS, Awad YED, Hallouty E, Salwa M, Araby ME (2012)
Design, synthesis and cancer cell line activities of pyrazolo[3,4-b]
pyridine derivatives. Open J Med Chem 2:78–88

Med Chem Res
Murthy YLN, Rajack A, Taraka Ramji M, Jeson babu J, Praveen Ch,
Aruna Lakshmi K (2012) Design, solvent free synthesis and
antimicrobial evaluation of 1,4 DHP. Bioorg Med Chem Lett
22:6016–6023
Reen GK, Dudhe P, Ahuja M, Kumar A, Sharma P (2015) Acid-
catalyzed, silica supported, one-pot benzoylation route to syn-
thesize 2-(substituted phenyl) oxazolo[4,5-b] pyridines under
ambient conditions. Synth Commun 45:1986–1994
Ouyang L, Huang Y, Zhao Y, He G, Xie Y, Liu J, He J, Liu B, Wei Y
(2012) Preparation, antibacterial evaluation and preliminary
structure–activity relationship (SAR) study of benzothiazol- and
benzoxazol-2-amine derivatives. Bioorg Med Chem Lett
22:3044–3049
Paronikyan EG, Noravyan AS, Dzhagatspany IA, Nazaryan IM, Par-
onikyan RG (2002) Synthesis and anticonvulsant activity of
isothiazolo[5,4-b]pyrano(thiopyrano)[4,3-d]pyridine and iso-
thiazolo[4,5-b]-2,7-naphthyridine derivatives. Pharm Chem J
36:465–467
Sharma P, Rane N, Gurram VK (2004) Synthesis and QSAR studies of
pyrimido[4,5-d]pyrimidine-2,5-dione derivatives as potential
antimicrobial agents. Bioorg Med Chem Lett 14:4185–4190
Sharma P, Kumar A, Upadhyay S, Sahu V, Singh J (2009) Synthesis
and QSAR modeling of 2-acetyl-2-ethoxycarbonyl-1-[4(4’-ary-
lazo)-phenyl]-N,N-dimethyl aminophenyl aziridines as potential
antibacterial agents. Eur J Med Chem 44:251–259
Sharma P, Kumar A, Upadhyay S, Singh J, Sahu V (2010) A novel
approach to the synthesis of 1,2,3-triazoles and their SAR studies.
Med Chem Res 19:589–602
Patel NB, Agravat SN, Shaikh FM (2010) Synthesis and antimicrobial
activity of new pyridine derivatives-I. Med Chem Res
20:1033–1041
Pathak R, Kumar R, Gautam HK (2013) Cross-species induction and
enhancement of antimicrobial properties in response to gamma
irradiation in exiguobacterium species HKG 126. Indian J
Microbiol 53:130–136
Sharma P, Kumar A, Kumari P, Singh J, Kaushik MP (2012) QSAR
modeling of synthesized 3-(1,3-benzothiazol-2-yl)2-phenyl qui-
nazolin-4(3H)-ones as potent antibacterial agents. Med Chem Res
21:1136–1148
Thomsen R, Christensen MH (2006) MolDock: A new technique for
high accuracy molecular docking. J Med Chem 49:3315–21
Vondenhoff GHM, Aerschot AV (2011) Aminoacyl-tRNA synthetase
Pinchuk IV, Beswick EJ, Reyes VE (2010) Staphylococcal ene-
trotoxins. Toxins 2:2177–2197
inhibitors as potential antibiotics. Eur J Med Chem 46:
5227–5236