Potential antimicrobial agents from triazole-functionalized 2H-benzo[b][1,4]oxazin-3(4H)-ones

Article abstract of DOI:10.1016/j.bmcl.2017.10.061

A series of substituted triazole functionalized 2H-benzo[b][1,4]oxazin-3(4H)-ones were synthesized by employing click chemistry and further characterized based on ¹H NMR, ¹³C NMR, IR and mass spectral studies. All the synthesized derivatives were screened for their in vitro antimicrobial activities. Further, molecular docking studies were accomplished to explore the binding interactions between 1,2,3-triazol-4-yl-2H-benzo[b][1,4]oxazin-3(4H)-one and the active site of Staphylococcus aureus (CrtM) dehydrosqualene synthase (PDB ID: 2ZCS). These docking studies revealed that the synthesized derivatives showed high binding energies and strong H-bond interactions with the dehydrosqualene synthase validating the observed antimicrobial activity data. Based on antimicrobial activity and docking studies, the compounds 9c, 9d and 9e were identified as promising antimicrobial leads.

Full text of DOI:10.1016/j.bmcl.2017.10.061

Accepted Manuscript
Potential antimicrobial agents from triazole-functionalized 2H-benzo[b]
[1,4]oxazin-3(4H)-ones
Rajitha Bollu, Saleha Banu, Rajashaker Bantu, Gopi Reddy A, Lingaiah
Nagarapu, K. Sirisha, C. Ganesh Kumar, Shravan Kumar Gunda, Kamal Shaik
PII:
S0960-894X(17)31061-2
https://doi.org/10.1016/j.bmcl.2017.10.061
BMCL 25390
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 August 2017
13 October 2017
24 October 2017
Please cite this article as: Bollu, R., Banu, S., Bantu, R., Reddy A, G., Nagarapu, L., Sirisha, K., Ganesh Kumar,
C., Kumar Gunda, S., Shaik, K., Potential antimicrobial agents from triazole-functionalized 2H-benzo[b]
[1,4]oxazin-3(4H)-ones, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.
2017.10.061
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www. elsevier.com
Potential antimicrobial agents from triazole-functionalized 2H-benzo[b][1,4]oxazin-
3(4H)-ones
Rajitha Bollua, Saleha Banua, Rajashaker Bantua, Gopi Reddy A, Lingaiah Nagarapua*, K. Sirishab,,
C. Ganesh Kumarb, Shravan Kumar Gundad, Kamal Shaikd
aOrganic Chemistry Division II (CPC), CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500007, Telangana, India
bMedicinal Chemistry and Biotechnology Division (MCB), CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500
007, India
dBioinformatics Wing, Mycology & Plant Pathology Lab, Department of Botany, Osmania University, Hyderabad-500007, India
Fax: + 91 40 27193382; Tel: +91 40 27191509; E-mail: lnagarapuiict@yahoo.com/nagarapu@iict.res.in
AR TIC LE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of substituted triazole functionalized 2H-benzo[b][1,4]oxazin-3(4H)-ones were
synthesized by employing click chemistry and further characterized based on 1H NMR, 13C
NMR, IR and mass spectral studies. All the synthesized derivatives were screened for their
in vitro antimicrobial activities. Further, molecular docking studies were accomplished to
explore the binding interactions between 1,2,3-triazol-4-yl-2H-benzo[b][1,4]oxazin-3(4H)-
one and the active site of Staphylococcus aureus (CrtM) dehydrosqualene synthase (PDB
ID: 2ZCS). These docking studies revealed that the synthesized derivatives showed high
binding energies and strong H-bond interactions with the dehydrosqualene synthase
validating the observed antimicrobial activity data. Based on antimicrobial activity and
docking studies, the compounds 9c, 9d and 9e were identified as promising antimicrobial
leads.
Keywords:
1,4-Benzoxazine,
1,2,3 Triazoles,
Antimicrobial activity,
Molecular docking,
Dehydrosqualene synthase.
2009 Elsevier Ltd. All rights reserved.
Antibiotics were claimed as wonder drugs to cure various
diseases and have saved millions of lives globally which has
considerably improved the public health. Nevertheless, a serious
drug-resistance crisis has evolved in the recent years due to
overuse and abuse of antibiotics, as well as the lack of research
and development towards identifying new drugs by the pharma
industry due to increased cost for drug discovery and reduced
economic incentives and challenging regulatory requirements.1-2
The emergence of bacterial resistance pattern towards various
antimicrobial agents has generated various drug-resistant strains
including methicillin-resistant Staphylococcus aureus (MRSA),
methicillin-resistant S. epidermidis (MRSE), penicillin-resistant
Streptococcus pneumonia (PRSP) and vancomycin-resistant
enterococci (VRE) which has resulted in increased morbidity and
mortality rates and this has mandated a pressing concern for
human health.3-6 Considering these facts, there is an urgent need
to identify new types of antibacterial drugs, and/or to search new
targets or mechanisms to understand the antimicrobial effects.7-14
In this regard, attempts were made to target the golden carotenoid
pigment, staphyloxanthin produced by S. aureus,15 which
functions as a virulence factor for S. aureus. The staphyloxanthin
pigment acts as an antioxidant and protects the microbial cells
against oxidative stress due to host immune defense by reactive
oxygen species and neutrophils.16-17
The chemistry of heterocyclic compounds continues to be
an interesting field to explore for various biological activities. A
large volume of research has been carried out on 2H-1,4-
benzoxazin-3-(4H)-ones from
a
pharmaceutical chemistry
perspective. The 2H-1,4-benzoxazin-3-(4H)-ones and 3,4-
dihydro-2H-1,4-benzoxazines are considered as privileged
scaffolds for the design of biologically active compounds. As
shown in Fig. 1, the benzoxazinone-based compounds form an
important class of benzo-fused heterocycles exhibiting a wide
spectrum of biological activities including antitumor (I),18
antiphlogistic, antipyretic and analgesic effects of 4-aminoalkyl-
2,3-dihydro-1,4-benzoxazin-3-ones (II) which has led to the
proposed use of some of these compounds as efficient
pharmaceuticals19 and indeed, calcium channel blockers based on
substituted 1,4-benzoxazinones (III)20 were claimed for their
functional role as anti-hypertensives, vasodilators, and anti-
ischemic agents. In addition, several 1,4-benzoxazinone
derivatives find use as antifungal drugs (IV, V)21-22 and
broncholytics (VI). 23
(1,2,3)-Triazole moieties are attractive bridging units in
view of their stability to metabolic degradation and are capable of
hydrogen bonding, which is a favourable aspect for binding the
bio-molecular targets and can also improve the solubility.24 Like
azoles, triazoles find use in many antifungal drugs and
fungicides; however, the triazole-based drugs are more selective

for fungi as compared to mammalian cells which are selective to
the azole-based antifungal compounds.25 1,2,3-Triazole is a
privileged scaffold present in diverse bioactive molecules
functioning as anti-cancer (VII), anti-fungal (VIII)26,
antibacterial27-28, anti-allergic29, anti-HIV(IX)30-31, anti-tubercular
(X)32-33 and anti-inflammatory agents.34 The importance of
(1,2,3)-triazole molecules based on their biological activities are
illustrated in Fig. 1.35
Scheme 1. Synthesis of 4-(1H-1,2,3-triazol-4-yl)methyl)-2H-
benzo[b][1,4]oxazin-3(4H)-one derivatives 8a-h and 9a-h
Fig. 1. Structures of 1,4-benzoxazinone-based pharmaceuticals
and some bioactive compounds with (1,2,3)-triazole scaffold.
In view of biological significance of benzoxazinones and
triazoles, it was planned to synthesize benzoxazinone-based
triazole containing substituted functional group derivatives which
were further screened for invitro antimicrobial activities. In this
context, the Cu(I) catalyzed click chemistry approach was
applied to prepare efficiently various 1,2,3-triazole derivatives. In
continuation to our ongoing research activities,36 to discover and
develop new antimicrobial agents, we herein report an efficient
method for the synthesis of novel 1,4-benzoxazine-1,2,3-triazole
hybrids (8a-h and 9a-h) with excellent yields.
Fig. 2. Design strategy for 4-(1H-1,2,3-triazol-4-yl)methyl)-2H-
benzo[b][1,4]oxazin-3(4H)-one hybrids
O
O
O
O
O
N
N
N
O
N
N
O
N
N
O
N
N
N
N
N
N
N
N
N
CH3
A
series of substituted triazole-functionalized 2H-
(8d, 91%)
(8c, 90%)
(8a, 89%)
(8b, 88%)
benzo[b][1,4]oxazin-3(4H)-one derivatives 8a-h and 9a-h were
synthesized in three steps starting from amino phenol using
propargyl bromide and substituted azides as intermediates. The
first synthetic step involved the acylation of substituted
aminophenol (1 and 2) with TEBA, NaHCO3 and chloroacetyl
chloride in chloroform for about 12 h and “in situ” cyclization
afforded substituted benzoxazinones 3 and 4. The key propargyl
benzoxazine intermediates (3 and 4) was prepared from
benzoxazinones (1, 2) by using K2CO3 and catalytic amount of
TBAI, propargyl bromide (5) in DMF solution for about 3 h to
afford the corresponding tertiary amines 6 and 7 under room
temperature conditions with good yields. Finally, using a one-
pot protocol, 1,4-benzoxazin-1,2,3-triazole hybrids 8a-h and 9a-
CH3
Cl
O
N
O
N
O
N
O
N
O
N
O
N
O
N
O
N
N
N
N
N
N
N
N
N
(8h, 86%)
(8g, 81%)
(8e, 93%)
(8f, 95%)
NO2
F
OCH3
Cl
O
N
O
N
O
N
O
Cl
O
Cl
O
N
O
N
Cl
N
O
N
N
N
N
N
N
N
N
N
N
CH3
(9d,89%)
(9c, 85%)
(9a, 83%)
(9b, 90%)
h
were obtained by reacting 4-(prop-2-yn-1-yl)-2H-
benzo[b][1,4]oxazin-3(4H)-ones (6, 7) with various substituted
azides (a-h)37 via copper (I)-catalyzed 1,3-dipolar cycloaddition
(Scheme 1) under room temperature conditions (Fig. 2 and Fig.
3).
Cl
Cl
O
N
O
N
CH3
O
N
O
N
Cl
O
Cl
O
Cl
O
O
N
N
N
N
N
N
N
N
N
N
N
N
(9e, 90%)
(9f, 94%)
(9g, 79%)
(9h, 89%)
F
OCH3
NO2
Fig. 3: 1,4-benzoxazine-1,2,3-triazole hybrides 8a-h and 9a-h
The synthesized hybrids 8a-h and 9a-h were evaluated for
their in vitro antimicrobial activity against Micrococcus luteus
MTCC 2470, Staphylococcus aureus MTCC 96, Staphylococcus

aureus MLS-16 MTCC 2940, Bacillus subtilis MTCC 121,
Escherichia coli MTCC 739, Klebsiella planticola MTCC 530,
Pseudomonas aeruginosa MTCC 2453 and Candida albicans
MTCC 3017, and the results to this regard are tabulated in Table
1. Ciprofloxacin and Miconazole were used as standard controls
for the bacterial and fungal strains, respectively. The compounds
9c, 9d and 9e exhibited promising and broad spectrum
antimicrobial activity against all the tested pathogens except for
Pseudomonas aeruginosa MTCC 2453 with MIC values ranging
from 1.9 to 3.9 µg/mL. Further, the compounds 9c, 9d and 9e
exhibited an MIC value of 7.8 µg/mL against Candida albicans
MTCC 3017. From a structure-activity relationship (SAR)
perspective, it was observed that the synthesized compounds 9c,
while compound 8h showed H-bond interactions with Val268
and Lys273. It was observed that all the screened compounds
showed good binding energy and H-bond interactions with
dehydrosqualene synthase. The docking interaction analysis is
illustrated in Fig. 4. The molecular docking studies revealed that
the compounds 8a-h and 9a-h exhibited strong H-bond
interactions and high binding energies with dehydrosqualene
synthase. The compounds with their docking scores and the
hydrogen bond interactions of these ligands with amino acid
residues in the active site of target protein are provided in Table
3.
9d and 9e has ortho-methyl,
para-methyl and 4-fluoro
substituents, respectively, attached to the chloro-benzoxazinone
scaffold, which exhibited strong electron donating and/or
electron withdrawing properties that plausibly may be
contributing to the antibacterial and anti-Candida activities.
While, the compounds 8c and 8d exhibited antibacterial activity
against Staphylococcus aureus MLS-16 MTCC 2940,
Escherichia coli MTCC 739 and Klebsiella planticola MTCC
530 with MIC values of 3.9 µg/mL. Further, these compounds 8c
and 8d also exhibited antifungal activity against Candida
albicans MTCC 3017 with MIC value of 7.8 µg/mL. Further, the
synthesized compounds 8c and 8d have ortho-methyl and para-
methyl substituents attached to the benzoxazinone scaffold which
may be exhibiting strong electron donating and/or electron
withdrawing properties that may be attributing to the antibacterial
and anti-Candida activities. In addition, the benzoxazinone
derivatives 8b, 8e, 8f, 8g, 9c, 9d, 9e, 9f, 9g and 9h exhibited
promising antifungal activity against Candida albicans MTCC
3017 with MIC values ranging between 7.8 to 31.2 µg/mL.
Furthermore, all the synthesized compounds were evaluated for
the minimum bactericidal concentration (MBC) and the results to
this regard are tabulated in Table 2. In this case too, the
compounds 9c, 9d and 9e were found to be promising and
exhibited broad spectrum antimicrobial activity. Based on these
evaluation studies, it was observed that the compounds 9c, 9d
and 9e were identified as the most promising leads among all the
tested derivatives.
8a
8c
8d
8h
Molecular docking has become a versatile and most
important computational method for the prediction of protein-
ligand interactions37 and is an essential and powerful tool for
rational drug designing.38 Over the past several years using high
throughput protein purification, protein-ligand complex
structures were isolated and were solved using X-ray and NMR
spectroscopic methods. Further significant improvements in the
computational methods for studying ligand interactions with
biological targets have increased in the recent past. In the present
study, the three dimensional crystal structure of the C(30)
9c
9d
carotenoid
dehydrosqualene
synthase
(CrtM)
from
Staphylococcus aureus complexed with bisphosphonate BPH-
700 (PDB ID: 2ZCS) was retrieved from protein databank. All
the lead compounds were docked using Autodock 4.2 software
against the promising antimicrobial target, dehydrosqualene
synthase. For each ligand, the docking score was calculated in
terms of kcal/mol. The docking score and H-bond interactions
were determined for the lead compounds identified in this study.
Among all the screened compounds, 8a, 8c, 8d, 8h, 9c, 9f were
found to be interacting with Lys273 and 9f interacted with
Tyr248. Compound 9f exhibited highest binding energy of -7.04
kcal/mol, showing H-bond interactions with Tyr248 and Lys273,
9e
9f
Fig.4. Docking of all the lead compounds with active site of
Staphylococcus aureus (CrtM) dehydrosqualene synthase (PDB
ID: 2ZCS; deposited on 2007-11-11 and it was released on 2008-
03-11).

Table 1: Antimicrobial activity of the synthesized triazolyl benzoxazine hybrids 8a-h and 9a-h
Test
compounds
Minimum inhibitory concentration (µg/mL)
Micrococcus
luteus MTCC
2470a
Staphyloco Staphyloco Bacillus
Escheric
hia coli
MTCC
739e
Klebsiella Pseudo Candida
planticol monas albicans
a MTCC aerugin MTCC
530f
ccus
aureus
ccus
aureus
subtilis
MTCC
121d
MTCC 96b MLS-16
osa
MTCC
2453g
3017h
MTCC
2940c
8a
8b
8c
8d
8e
8f
8g
8h
9a
9b
9c
9d
9e
9f
9g
-
-
-
-
-
-
-
-
-
3.9
-
-
-
-
-
-
-
-
-
-
-
3.9
3.9
-
-
-
1.9
-
-
-
-
-
-
-
-
-
-
-
-
-
3.9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
7.8
-
3.9
3.9
-
-
-
-
-
-
3.9
3.9
7.8
7.8
-
3.9
-
-
7.8
7.8
3.9
3.9
15.6
-
-
-
-
-
1.9
1.9
1.9
-
-
-
1.9
1.9
3.9
-
-
-
1.9
1.9
1.9
1.9
-
1.9
1.9
1.9
-
-
-
3.9
-
3.9
-
-
-
3.9
3.9
3.9
-
-
-
7.8
7.8
7.8
7.8
7.8
7.8
7.8
9h
-
-
-
-
-
-
-
Miconazole
(Standard)
Ciprofloxacin
(Standard)
0.9
0.9
0.9
0.9
0.9
0.9
0.9
-
Table 2: Minimum Bactericidal / Fungicidal Concentration (MBC / MFC) of the synthesized triazolyl benzoxazine hybrids 8a-h and
9a-h
Test
compounds
Minimum bactericidal / fungicidal concentration (µg/mL)
Micrococcu Staphyloco Staphyloco Bacillus
Escherichi
a coli
Klebsiella Pseudomona Candida
planticol s aeruginosa albicans
a MTCC MTCC
530f 2453g
s luteus
MTCC
2470a
ccus
aureus
ccus
aureus
MTCC 96b MLS-16
subtilis
MTCC
121d
MTCC
739e
MTCC
3017h
MTCC
2940c
8a
8b
8c
8d
8e
8f
8g
-
-
-
-
-
-
-
-
-
7.8
-
-
-
-
-
-
-
-
-
7.8
7.8
-
-
-
3.9
-
-
-
-
-
-
-
-
-
-
-
7.8
-
7.8
7.8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
7.8
7.8
15.6
15.6
-
15.6
15.6
15.6
7.8
7.8
31.2
-
8h
9a
7.8
-
-
-
9b
9c
9d
9e
-
-
-
-
-
7.8
-
7.8
-
-
-
-
3.9
3.9
3.9
-
-
-
3.9
3.9
7.8
-
-
-
3.9
3.9
3.9
3.9
-
3.9
3.9
3.9
-
-
-
7.8
7.8
7.8
-
-
-
15.6
15.6
15.6
15.6
15.6
15.6
7.8
9f
9g
9h
-
-
-
-
-
-
-
-
Miconazole
(Standard)
Ciprofloxacin
(Standard)
0.9
0.9
0.9
0.9
0.9
0.9
0.9
-
Table 3: Molecular docking studies of all lead compounds with the active site of Staphylococcus aureus (CrtM) dehydrosqualene
synthase (PDB ID: 2ZCS)

Test compounds
Interacting amino acids
Binding energy, ∆G (Kcal/Mol)
Dissociation constant (kI) (µM)
Lys273
-6.61
-6.62
-6.81
-6.59
-6.82
-6.86
-6.74
-7.04
14.23
14.15
10.26
14.84
10.06
9.43
8a
8c
8d
8h
9c
9d
9e
9f
Lys273
Lys273
Val268, Lys273
Lys273
Arg45
Arg45
11.42
6.91
Tyr248, Lys273
In conclusion, we have synthesized a series of triazolyl
benzoxazine derivatives 8a-h and 9a-h using simple, three-step
procedure with excellent yields. The entire series of synthesized
derivatives were screened for antimicrobial properties. Further,
docking studies were also performed on these promising derivatives
to validate our wet-lab antimicrobial studies. In general, the
majority of target compounds displayed moderate to promising
activity against the tested pathogenic strains. Based on the
antimicrobial studies, the compounds 9c, 9d and 9e showed
promising antibacterial and antifungal activities against the
pathogenic strains. Docking studies showed that the compounds 8h
and 9f showed H-bond interactions with high binding energies,
while compounds 9c, 9d and 9e showed good H-bond interactions
with moderate binding energies. In light of the above, these studies
provide insights to develop new drug leads to target the virulence
factor, dehydrosqualene synthase (CrtM) of S. aureus.
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Acknowledgements
The authors gratefully acknowledge the financial support through
the project: DST-SERB/EMEQ-078/2013 and the Council of
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Leave this area blank for abstract info.
Potential antimicrobial agents from triazole-
functionalized 2H-benzo[b][1,4]oxazin-3(4H)-ones
Rajitha Bollua, Saleha Banua, Rajashaker Bantua, Gopi Reddy A, Lingaiah Nagarapua*, K. Sirishab,c, C. Ganesh
Kumarb,c*, Shravan Kumar Gundad, Kamal Shaikd.
A series of substituted triazole-functionalized 2H-benzo[b][1,4]oxazin-3(4H)-ones were synthesized and compounds
were screened for their in vitro antimicrobial activity against Gram positive and Gram negative strains using Miconazole
and Ciprofloxacin as standard drugs.
O
9c
MIC and MBC Inhibition
R1
N
O
N
9c, 9d, 9e MIC Inhibition 1.9-7.8 mg/mL
9c, 9d, 9e MBC Inhibition 3.9-15.6 mg/mL
N
N
R2
9d
9e
Molecular docking with
dehydrosqualene synthase (PDB ID: 2ZCS)
(CrtM)
S. aureus
Design Strategy
antimicrobial
In vitro
Supplementary Information
1
Experimental section and copies of the H NMR, 13C NMR
HRMS and IR spectra for some of the important compounds.

Research Highlights
A series of novel triazole-functionalized
•
2H-benzo[b][1,4]oxazin-3(4H)-ones (8a-h
and 9a-h)were efficiently synthesized.
Evaluated for their in vitro antimicrobial
activity; 9c, 9d and 9e showed promising
activity.
•
•
•
Compounds 8a, 8c, 8d, 8h, 9c & 9f showed
good docking with Lys273.
Compound 9f exhibited highest binding
energy of -7.04 kcal/mol,