Development of benzo[d]oxazol-2(3H)-ones derivatives as novel inhibitors of Mycobacterium tuberculosis InhA

Article abstract of DOI:10.1016/j.bmc.2014.08.031

A series of twenty seven substituted 2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetamide derivatives were designed based on our earlier reported Mycobacterium tuberculosis (MTB) enoyl-acyl carrier protein reductase (InhA) lead. Compounds were evaluated for MTB InhA inhibition study, in vitro activity against drug-sensitive and -resistant MTB strains, and cytotoxicity against RAW 264.7 cell line. Among the compounds tested, 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(5-nitrothiazol-2-yl)acetamide (30) was found to be the most promising compound with IC₅₀ of 5.12 ± 0.44 μM against MTB InhA, inhibited drug sensitive MTB with MIC 17.11 μM and was non-cytotoxic at 100 μM. The interaction with protein and enhancement of protein stability in complex with compound 30 was further confirmed biophysically by differential scanning fluorimetry.

Full text of DOI:10.1016/j.bmc.2014.08.031

Accepted Manuscript
Development of benzo[d]oxazol-2(3H)-ones derivatives as novel inhibitors of
Mycobacterium tuberculosis InhA
Ganesh S. Pedgaonkar, Jonnalagadda Padma Sridevi, Variam Ullas Jean Kumar,
Shalini Saxena, Parthiban Brindha Devi, Janupally Renuka, Perumal
Yogeeswari, Dharmarajan Sriram
PII:
S0968-0896(14)00626-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.08.031
BMC 11779
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 June 2014
21 August 2014
26 August 2014
Please cite this article as: Pedgaonkar, G.S., Sridevi, J.P., Kumar, V.U.J., Saxena, S., Devi, P.B., Renuka, J.,
Yogeeswari, P., Sriram, D., Development of benzo[d]oxazol-2(3H)-ones derivatives as novel inhibitors of
Mycobacterium tuberculosis InhA, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.
2014.08.031
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Development of benzo[d]oxazol-2(3H)-ones derivatives as novel inhibitors of
Mycobacterium tuberculosis InhA
Ganesh S Pedgaonkar, Jonnalagadda Padma Sridevi, Variam Ullas Jean Kumar, Shalini Saxena,
Parthiban Brindha Devi, Janupally Renuka, Perumal Yogeeswari, Dharmarajan Sriram*
Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus,
Shameerpet, R.R. District, Hyderabad-500078, India.
Abstract
A series of twenty seven substituted 2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetamide derivatives
were designed based on our earlier reported Mycobacterium tuberculosis (MTB) enoyl-acyl
carrier protein reductase (InhA) lead. Compounds were evaluated for MTB InhA inhibition
study, in vitro activity against drug-sensitive and -resistant MTB strains, and cytotoxicity against
RAW 264.7 cell line. Among the compounds tested, 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-
N-(5-nitrothiazol-2-yl)acetamide (30) was found to be the most promising compound with IC50
of 5.12±0.44 µM against MTB InhA, inhibited drug sensitive MTB with MIC 17.11 µM and was
non-cytotoxic at 100 µM. The interaction with protein and enhancement of protein stability in
complex with compound 30 was further confirmed biophysically by differential scanning
fluorimetry.
Key words: Tuberculosis, InhA, Enoyl acyl carrier protein reductase, Cytotoxicity
*For correspondence: D. Sriram, Chair Professor, Department of Pharmacy,
Birla Institute of Technology & Science-Pilani, Hyderabad Campus,
Jawaharnagar, Hyderabad- 500 078. INDIA
Telephone: +91-40663030506; Fax: +91-4066303998
Email: dsriram@hyderabad.bits-pilani.ac.in

1. Introduction
Today's tuberculosis (TB) drug regimen takes too long to be effective and requires too many
medications. Treatment of drug sensitive disease requires 6-9 months whereas treatment of drug-
resistant TB is even lengthier, taking 18-24 months or longer. Second-line drugs are also much
more toxic and considerably more expensive than the standard first-line anti-TB regimen.
Furthermore, current first-line treatment regimens are not compatible with the common
antiretroviral therapies used to treat HIV/AIDS. Therefore, new drugs are needed which will be
effective in treating active, resistant and latent TB infections, and will also be compatible with
antiretroviral therapy. Among the most attractive molecular targets to design novel anti-
tubercular agents are the fatty acid synthase (FAS) pathway enzymes.1 The 2-trans-enoyl-acyl
carrier protein (ACP) reductase (E.C.1.3.1.9) or MTB InhA, the fourth enzyme of the type II
fatty acid synthase system (FAS II), is one of the key enzymes involved in the elongation cycle
of fatty acids in MTB. InhA is essential to the mycolic acid biosynthesis in mycobacterium.2 Its
inactivation induces accumulation of saturated fatty acids, leading to cell wall alterations, lysis
and consequently to death of the organism. Isoniazid, a first-line drug currently used to treat TB,
is activated within the mycobacterial cell by KatG catalase. The activated molecule was thought
to suppress mycolic acid biosynthesis through the inhibition of InhA, making it one of the best
validated targets for the treatment of TB.3 Isoniazid resistance is mediated through mutations in
KatG that deters activation of the drug.4 Therefore one should be able to attain same clinical
efficacy as isoniazid and avoid much of the current resistance to isoniazid by bypassing the
requirement for KatG activation and directly inhibiting InhA. Earlier we reported design of novel
leads of MTB InhA inhibitors.5 In the course of our studies on one of the lead N-(2,5-
dichlorophenyl)-2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetamide (UPS 14) (Figure 1), we

attempted to synthesize analogues around the lead to establish their biological activities and
reported herein.
2. Results and discussion
2.1. Design and chemistry
In our earlier work we had generated and validated quantitative pharmacophore models using
known set of reported MTB InhA inhibitors. The validated pharmacophore model was used as a
query to screen an in-house/commercial database of 400,000 compounds and retrieved 25,000
hits. These hits were further ranked based on its shape and feature similarity with potent MTB
InhA inhibitor using rapid overlay of chemical structures (ROCS) and subsequent hits were
subjected for docking. Based on the pharmacophore, ROCS model and docking interactions, 32
compounds were selected finally and assayed for InhA inhibitory activity.5 One of the lead UPS
14 showed MTB InhA IC50 of 22.12±0.8 µM. Based on this and inputs from protein-ligand
interactions observed in the structure of InhA with lead molecules, further modifications (and
combinations thereof) were explored as a ligand expansion step. The lead molecule UPS 14
enabled us convenient structure-activity relationship (SAR) investigations at both 6th and N-1
positions of benzo[d]oxazol-2(3H)-one nucleus. Investigation of benzoxazolone pharmacophores
has recently generated significant interest from a medicinal chemistry point of view and their
synthesis has been well explored in the literature. The synthetic pathway used to achieve the lead
modifications is delineated in Schemes 1 and 2. A library of 27 derivatives was synthesized
(compound 8-25 and 28-36, Table 1) and evaluated for their ability to inhibit InhA enzyme as
step towards the derivation of SAR and hit optimization.
Construction of the target molecule was achieved by first treating the commercially available 2-
aminophenol (4) with 1,1’-carbonyldiimidazole (CDI) in refluxing tetrahydrofuran to afford the
corresponding benzoxazolone (5) in good yield. This benzoxazolone (5) was further alkylated at

the N-1 position with tert-butyl bromoacetate in presence of sodium hydride as base to give the
corresponding acetate (6) in quantitative yield, which when subjected to acidic hydrolysis using
trifluoroacetic acid (TFA) afforded the corresponding acid (7). The final library was developed
by condensing this key intermediate acid derivative (7) with various substituted amines.
Selection of amino substituents at N-1 position was based on our previous research experience in
tuberculosis, in an effort to improve the anti-tubercular potency of these molecules. The final
coupling was achieved by using EDC/HOBT as coupling agents. Though the reaction went
smoothly for aromatic amines giving the desired products in good yield, a similar conversion
was not observed in the case of heterocyclic amines especially with 2-amino-5-nitrothiazole, 2-
aminobenzothiazole and 2-amino-6-nitrobenzothiazole, probably due to their low
nucleophillicity and solubility issues. Hence, an alternative strategy was designed as depicted in
Scheme 2, wherein the corresponding amine was first treated with chloroacetyl chloride in
presence of triethylamine as base to give the corresponding 2-chloro-N-
(aryl/heteroaryl)acetamide. This on further alkylation with the corresponding benzoxazolone
derivative gave the desired amide derivative in good yields and purity. Both analytical and
13
spectral data (1H NMR, C NMR and mass spectra) of all the synthesized compounds were in
full agreement with the proposed structures.
2.2 MTB InhA inhibition assay and SAR
Recombinant MTB InhA was expressed in E. coli and subsequently purified according to a
previously reported procedure.6 The synthetic compounds were initially evaluated in vitro for the
MTB InhA inhibition at 10 µM6 and the results are presented in Table 1. Most of the synthesized
compounds (except 16) showed percentage inhibition ranging from 26.12 to 82.43 at 10 µM.
Five compounds (19, 29-30, 32-33) showed more than 55 % inhibition and were subsequently
tested for their IC50s. Dose-response curves were plotted for these potent inhibitors using

GraphPad Prism software (GraphPad Software Inc., La Jolla, CA) by taking log (inhibitor
concentration) on the x-axis and response (% inhibition) on the y-axis as shown in Figure 1,
Supplementary data. Compound 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(5-nitrothiazol-
2-yl)acetamide (30) was found to be the most potent compound with IC50 of 5.12±0.44 µM.
To study the SAR, we carried out docking studies of all the synthesized compounds together
with lead compound UPS 14 for comparison. The crystal structure of MTB InhA complexed
with reference inhibitor 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide
(PDB:2H7M) having resolution of 1.62 Å was selected and docking results obtained with Glide,
version 5.7, Schrodinger, LLC, New York, NY, 2012. Analysis of the crystal structure of 2H7M
revealed that the reference inhibitor in the InhA active site formed hydrogen-bonding network
between Tyr158, enzyme active site residues, and the NAD+ cofactor that probably served as the
key feature that governed the orientation of the compound within the active site. Dual hydrogen
bonding network was involved with the oxygen atom on the pyrrolidine carbonyl group, InhA
catalytic residue Tyr158, and the NAD+. This hydrogen bonding network seemed to be a
conserved feature among all the InhA-inhibitor complexes identified so far.7 The reference 1-
cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide was re-docked with the
active site residues of the MTB InhA to validate the active site cavity. The ligand exhibited
highest Glide score of -8.02 kcal/mol and was found in the vicinity of amino acids Tyr158,
Phe149, Met199, Ile215, Pro156, Leu218, Met155, Ala211, Ile202, Met103, Leu207, Ala157,
Met161, Phe97, Met98, Gly96, Gly104 and Lys165 residues. The re-docking results showed that
the compound exhibited similar interactions as that of the original crystal structure with RMSD
of 0.87 Å suggesting reliability of the docking method in terms of. The superimposition of Glide
docked conformation of co-crystal with co-crystal of 2H7M is shown in Figure 2.

The binding mode of lead compound UPS14 with InhA enzyme was analysed in more detail.
The binding orientation of lead compound N-(2,5-dichlorophenyl)-2-(2-oxobenzo[d]oxazol-
3(2H)-yl)acetamide (UPS14) within the InhA binding pocket is represented in Figure 3. The
predicted bound conformation of the lead compound UPS14 showed that the carbonyl oxygen
atom of benzo[d]oxazol-2(3H)-one ring formed hydrogen bond with the side chain of the
Tyr158. The 2,5-dichlorophenyl ring attached to benzo[d]oxazol-2(3H)-one ring through an
acetamide linker was surrounded by hydrophobic amino acids such as Phe149, Met199, Leu218,
Ile215, Pro156, Ile202, Met103 and Ala157. The compound was very well fit into the active site
cavity of the protein with a docking score of -7.52 kcal/mol.
Our SAR studies started from various substituted aryl/heteroaryl ring systems attached to
benzo[d]oxazol-2(3H)-one ring through an acetamide linker. The effect of various
aryl/heteroaryl substitutions at the R2 position was explored. As shown in Table 1, the activity of
most of the compounds exhibited % inhibition in the range of 26.12 to 82.43 % at 10 μM against
InhA which was better than the lead compound UPS14 (19.5 % at 10 μM).
The first subset of compounds (8, 11, 14, 17, 20, 23, 28, 31 and 34), with H atom at R1 with
various substituted aryl/heteroaryl groups at R2 position is shown in Table 1. Phenyl substitution
at the R2 position (compound 8) displayed good InhA inhibitory activity. Compound with
substitution at 2nd and 5th position of the phenyl ring (2-chloro-5-(trifluoromethyl)-2-phenyl
group) (compound 11) displayed moderate InhA inhibitory activity, similar was the introduction
of 2-pyridyl group at R2 position (compound 14). The compounds substituted with 2-
pyridylmethyl, 2-benzothiazolyl, 6-nitro-2-benzothiazoyl and 5-nitro-2-thiazolyl group
(respective compounds 17, 28, 31, and 34) at R2 position resulted in satisfactory in vitro InhA
inhibitory activity in the range of 42.15 to 48.15 % inhibition at 10 μM, whereas substitution
with 2-thiophenylmethyl ring (compound 23) led to decreased potency compared to other

compounds from this subset. This was well supported by the interaction profile of all the
molecules in the docking studies. The docking score of all the compounds from this subset was
found to be in the range of -6.87 to -7.59 kcal/mol. All compounds, except compound 14, were
found to be associated with hydrogen bonding interactions with the side chain of Tyr158 and
NAD+. In most of the compounds the carbonyl oxygen atom on benzo[d]oxazol-2(3H)-one ring
interacted with the side chain of Tyr158. Eventually, SAR from this subset of compounds
revealed compound 28 as the most selective inhibitor of InhA and compound 23 to be
comparatively least active. The bound conformation of compound 28 showed carbonyl oxygen
atom of benzo[d]oxazol-2(3H)-one ring forming hydrogen bond with the side chain of Tyr158
and ribose hydroxyl group of NAD+, which was also observed in the crystal ligand and also well
correlated with the in vitro InhA inhibitory activity (Figure 2, Supplementary data). On the
other hand binding analysis of compound 23 in the active site of protein revealed that the
compound failed to produce any hydrogen bonding interaction with the side chain of Tyr158
residue of the protein and thus the inactivity of the compound could be correlated (Figure 3,
Supplementary data).
In the second subset of compounds (9, 12, 15, 18, 21, 24, 29, 32 and 35), the effect of chloro
substitution on the benzo[d]oxazol-2(3H)-one ring at R1 position was investigated to understand
the effect of similar aryl/heteroaryl substituent’s at R2 position as that of the previous set of
compounds. SAR study revealed that, chloro group at R1 position increased hydrophobicity of
the compounds due to hydrophobic interactions with the active site which was crucial for
inhibition. This subset of compounds displayed more inhibitory potency than the previous subset.
Here, all the compounds showed satisfactory in vitro InhA inhibitory activity in the range of
28.16 to 72.60 % inhibition at 10 μM concentrations. The compounds 12, 15 and 35 with 2-
chloro-5-(trifluoromethyl)-2-phenyl, 2-pyridyl and 6-nitro-2-benzothiazoyl groups respectively

as R2 substituents, showed good InhA inhibitory activity, whereas compound 29 with 5-nitro-2-
thiazolyl group as R2 substituent was more active than compound 32 with 2-benzothiazolyl
group as R2 substituent. It was observed that phenyl substitution at R2 (9) was somewhat less
favoured than the other substitutions. Additionally, 2-furylmethyl, 2-pyridylmethyl and 2-
thiophenylmethyl ring substitutions at R2 position (respective compounds 21, 18 and 24) also
displayed good activity in InhA enzyme inhibition studies. In this subset, compounds 29 and 32
were demonstrated to be effective InhA inhibitors with good activity. Both the compounds
displayed docking scores of -7.12 and -7.073 kcal/mol respectively. The predicted binding pose
of the most active compounds (29 and 32) suggested that the observed potency may be due to the
extensive hydrophobic interactions predicted to be formed with the side chains of Met199,
Leu218, Met155, Pro156, Ala157, Ile202, Met103, Phe149 and Met161 along with hydrogen
bonding interactions with side chain of Tyr158 as well as with the ribose hydroxyl group of
NAD+ (Figure 4). Ligand binding analysis of one of the less active derivative from this subset
(9) showed hydrophobic interactions between the phenyl ring and some hydrophobic amino acid
residues. It also displayed hydrogen bonding interaction between carbonyl oxygen atom of 6-
chlorobenzo[d]oxazol-2(3H)-one ring and side chain of Tyr158 amino acid residue. However,
the orientation in the active site cavity of InhA pushed the chloro group away from the cavity,
which might be the reason for its lesser enzyme inhibition (Figure 4, Supplementary data).
In the third subset of compounds (10, 13, 16, 19, 22, 25, 30, 33 and 36), R1 consisted of nitro
group and R2 was substituted with various aryl/heteroaryl groups similar to previous two subsets
as shown in Table 1. The compounds 30, 33 and 19 with 5-nitro-2-thiazolyl, 2-benzothiazolyl
and 2-pyridylmethyl substitution at R2 respectively, emerged as most promising compounds from
this subset showing satisfactory in vitro InhA inhibitory activity in the range of 60.80 to 82.43 %
inhibition at 10 μM with IC50s of 5.12 to 8.23 μM. This was well supported by their interaction

profile in docking studies where docking score of these compounds were found in the range of -
7.11 to -7.48 kcal/mol. Closer analysis of compounds 30, 33 and 19 in the binding site revealed
that the carbonyl oxygen atom on 6-nitrobenzo[d]oxazol-2(3H)-one ring was involved in
hydrogen bonding interactions with side chain of Tyr158 residue as well as with ribose hydroxyl
group of NAD+ (Figure 5). In this subset of compounds, 2-pyridyl at R2 position (16) showed
least activity among the whole series of compounds with 15.63 % inhibition at 10 μM
concentration. In silico analysis of this compound indicated that the molecule oriented in a
different manner than that of other active derivatives and failed to demonstrate any interaction
with Tyr158 residue as well as with NAD+ resulting in less enzyme inhibition potency (Figure 5,
Supplementary data).
From the docking results, it was evident that the formation of hydrogen bonds with side chain of
Tyr158 and hydroxyl group of NAD+ along with hydrophobic interactions with the active site
were predicted to be the most crucial factors affecting the inhibitory potency of these
compounds. The overall trend in enzyme inhibition activity showed that substitution at R1
position with nitro group was more favoured than chloro group whereas H substitution was least
favoured. In case of R2 position, substitutions with heteroaryl groups such as 5-nitro-2-thiazolyl,
2-benzothiazolyl, 6-nitro-2-benzothiazolyl, 2-methylpyridyl, 2-furylmethyl and
2-
thiophenylmethyl group were more favoured than with 2-chloro-5-(trifluoromethyl)-2-phenyl,
phenyl and 2-pyridyl group.
2.3 In vitro MTB activity studies
All the synthesized compounds were also screened for their in vitro anti-tubercular activity
against MTB H37Rv (ATCC27294) using agar dilution method8 with drug concentrations from
50 µg/mL to 0.78 µg/mL in duplicates. The minimum inhibitory concentration (MIC) was
determined for each compound which was measured as the minimum concentration of compound

required to completely inhibit the bacterial growth. Isoniazid, ethambutol, rifampicin and
ofloxacin were used as reference compounds for comparison. The MIC values of the synthesized
compounds along with the standard drug for comparison are presented in Table 1. All the
synthesized compounds showed activity against MTB with MIC ranging from 15.04 to 82.58
µM. Six compounds (25, 29-32 and 36) inhibited MTB with MIC of <20 µM. Compound 30
which showed IC50 of 5.12±0.44 µM in the MTB InhA enzyme also exhibited MTB MIC of
17.11 µM. In general, compounds which possessed (sub)-thiazolyl and –benzothiazolyl showed
good MICs. Some of the molecules showed good potency in the enzyme inhibition study but
failed to exhibit equally potent inhibition of MTB, and this seem to be a major challenge in the
arena of TB drug discovery. Among other parameters, efflux mechanisms contributed in a major
way to intrinsic resistance to drugs.9 Efflux pumps of MTB belong to the ABC (ATP-binding
cassette) transporters and major facilitator superfamily (MFS) proteins,10 or antibiotic-modifying
and -degrading enzymes,11 and SMR (small multidrug resistance) family. Efflux-mediated drug
resistance in MTB could be due to one or more efflux pumps working alone or in coordination.
This was possible because of the redundancy of their functions, which overlapped extensively.12
In the current work, we carried out MTB screening of the synthesized compounds in the presence
of reported efflux pump inhibitors verapamil (50 µg/mL) and piperine (8 µg/mL).13 In most of
the cases MIC decreased 2 to 10 fold when compared to the absence of an efflux pump inhibitor.
Compound 30 showed MTB MIC of 8.55 µM in the presence of verapamil and piperine and its
MTB InhA IC50 was very well correlated. Encouraged by their promising in vitro anti-tubercular
activity, the more potent analogues were further evaluated for their in vitro potency against the
clinical isolate of XDR-TB strain (strain resistant to Isoniazid, Rifampicin, Ofloxacin and
Kanamycin). All the compounds displayed mycobacterial kill against the XDR strain and five
compounds (20, 25, 29-30 and 36) showed XDR-TB MIC of <20 µM (Table 1).

2.4. Cytotoxicity study
All the compounds were further tested for cytotoxicity in a RAW 264.7 cell line (mouse
leukemic monocyte macrophage) at 50 and 100 µM. We selected macrophage cell line to test
toxicity as it was known that MTB reside inside the macrophages and drug molecules should not
possess any toxicity against these macrophages. After 72 h of exposure, viability of the cells was
assessed on the basis of cellular conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) into a formazan product using the Promega Cell Titer 96
non-radioactive cell proliferation assay.14 Most of the tested compounds were not cytotoxic (less
than 50% inhibition at 50 µM) to RAW 264.7 cells and their percentage growth inhibitions are
reported in Table 1. The most active MTB InhA inhibitor (30) showed lesser toxicity of 20.24
and 43.79 % inhibition at 50 and 100 µM drug concentrations respectively.
2.5. Biophysical characterization
One of the active compounds from this series of chemical class of molecules was further
investigated using a biophysical technique, differential scanning fluorimetry (DSF). The ability
of the compounds to stabilize the catalytic domain of the InhA protein was assessed utilizing the
DSF technique by which the thermal stability of the catalytic domain of InhA native protein and
of the protein bound with the ligand was measured.15 Complex with compound 30 was heated
stepwise from 25 to 95 °C in steps of 0.1 °C rise in the presence of a fluorescent dye (sypro
orange), whose fluorescence increased as it interacted with hydrophobic residues of the InhA
protein. As the protein was denatured, the amino acid residues became exposed to the dye. A
right side positive shift of Tm in comparison to native protein meant higher stabilization of the
protein-ligand complex, which was a consequence of the inhibitor binding. In our study,
compound 30 showed significant positive Tm shift of 1.9 °C confirming the stability of the

protein-ligand complex as shown in Figure 6 which depicts the curves obtained in the DSF
experiment for the MTB InhA protein (pink) and protein-compound 30 complex (green).
3. Conclusion
In present work, we reported synthesis and screening results of twenty seven substituted 2-(2-
oxobenzo[d]oxazol-3(2H)-yl)acetamide derivatives against MTB InhA as well as drug sensitive
and resistant MTB strains. Most of the synthesized compounds showed better InhA inhibition as
compared to lead molecules, and 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(5-nitrothiazol-2-
yl)acetamide (compound 30) was found to be the most active compound with IC50 of 5.12±0.44
µM against MTB InhA, inhibited drug sensitive MTB with MIC of 17.11 µM and was non-
cytotoxic at 100 µM.
4. Experimental
4.1. Chemistry
4.1.1. General
The melting points were determined by capillary melting point apparatus and were not corrected.
Homogeneity of the compounds was monitored by thin layer chromatography (TLC) on silica
1
gel 60 F254 coated on aluminium plates, visualized by UV light and KMnO4 treatment. All H
13
and C NMR spectra were recorded on a Bruker AM-300 (300.12 MHz, 75.12 MHz) NMR
spectrometer, Bruker BioSpin Corp, Germany. Chemical shifts were reported in ppm (δ) with
reference to the internal standard TMS. Molecular weights of the synthesized compounds were
checked by LCMS 6100B series Agilent Technology. Elemental analyses were carried out on an
automatic Flash EA 1112 Series, CHN Analyser (Thermo). All commercially available
chemicals and solvents were used without further purification.
4.1.2. General procedure for the synthesis of 6-substituted benzo[d]oxazol-2(3H)-ones (5a-c)

To a stirred solution of appropriate aminophenol (4a-c) (1 mmol) in dry tetrahydrofurane (THF)
(20 mL) was added 1,1’-carbonyldiimidazole (CDI) (1.1 mmol) at room temperature (rt). The
solution was refluxed for about 4 h (monitored by TLC & LCMS for completion), and solvent
evaporated under reduced pressure.16 The residue was further diluted with water (20 mL) and
ethyl acetate (20 mL) and the layers separated. The organic layer was washed with 2 N
hydrochloric acid (15 mL), water (10 mL), dried over anhydrous sodium sulphate and evaporated
under reduced pressure. The residue was purified by silica gel column chromatography using
hexane: ethyl acetate as eluent to give the corresponding benzoxazolone (5a-c) in good yield.
4.1.2.1. Benzo[d]oxazol-2(3H)-one (5a)
The compound was synthesized according to the general procedure using 2-aminophenol (4a)
(2.0 g, 18.32 mmol) and CDI (3.2 g, 20.15 mmol) to afford 5a (1.9 g, 76.7 %) as white solid.
M.p: 139-141 °C. 1H NMR (DMSO-d6): δH 8.18 (s, 1H), 7.47 – 6.91 (m, 4H). 13C NMR (DMSO-
d6): δC 155.3, 144.6, 129.8, 125.4, 124.6, 110.1, 109.6. ESI-MS m/z 136 (M+H)+. Anal. Calcd.
for C7H5NO2: C, 62.22; H, 3.73; N, 10.37; Found: C, 62.25; H, 3.70; N, 10.40.
4.1.2.2. 6-Chlorobenzo[d]oxazol-2(3H)-one (5b)
The compound was synthesized according to the general procedure using 2-amino-5-
chlorophenol (4b) (2.0 g, 13.93 mmol) and CDI (2.4 g, 15.32 mmol) to afford 5b (1.8 g, 78.4 %)
as white solid. M.p: 193-195 °C. 1H NMR (DMSO-d6): δH 8.25 (s, 1H), 7.83 – 7.41 (m, 3H). 13C
NMR (DMSO-d6): δC 155.6, 140.3, 131.7, 128.5, 125.9, 123.2, 122.8. ESI-MS m/z 168 (M-H)-.
Anal. Calcd. for C7H4ClNO2: C, 49.58; H, 2.38; N, 8.26; Found: C, 49.61; H, 2.40; N, 8.29.
4.1.2.3. 6-Nitrobenzo[d]oxazol-2(3H)-one (5c)
The compound was synthesized according to the general procedure using 2-amino-5-nitrophenol
(4c) (2.0 g, 12.97 mmol) and CDI (2.3 g, 14.27 mmol) to afford 5c (1.8 g, 79.3 %) as pale yellow
1
13
solid. M.p: 250-252 °C. H NMR (DMSO-d6): δH 8.29 (s, 1H), 8.31 – 8.01 (m, 3H). C NMR

(DMSO-d6): δC 155.7, 144.0, 140.1, 136.6, 121.1, 117.5, 115.1. ESI-MS m/z 179 (M-H)-. Anal.
Calcd. for C7H4N2O4: C, 46.68; H, 2.24; N, 15.55; Found: C, 46.65; H, 2.21; N, 15.58.
4.1.3. General procedure for the synthesis of 6-substituted tert-butyl 2-(2-
oxobenzo[d]oxazol-3(2H)-yl)acetate (6a-c)
To a well stirred suspension of sodium hydride (60% dispersion in mineral oil, 1.2 mmol) in
N,N-dimethylformamide (DMF) (10 mL ) at 0 °C was added drop wise a solution of the
corresponding benzoxazolone (5a-c) (1 mmol) in DMF (10 mL), followed by tert-butyl
bromoacetate (1.1 mmol). The solution was slowly warmed to rt and stirred at rt for about 1 h
(monitored by TLC & LCMS for completion), quenched with saturated ammonium chloride
solution (15 mL) and concentrated under reduced pressure. The residue was partitioned between
ethyl acetate (20 mL) and water (10 mL) and the organic layer was dried over anhydrous sodium
sulphate and concentrated under reduced pressure. The crude product was purified by silica gel
column chromatography using hexane: ethyl acetate as eluent to give the corresponding product
(6a-c).
4.1.3.1. tert-butyl 2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetate (6a)
The compound was synthesized according to the general procedure using benzo[d]oxazol-2(3H)-
one (5a) (1.5 g, 11.10 mmol) and tert-butyl bromoacetate (2.3 g, 12.21 mmol) to afford 6a (1.9
g, 69.4 %) as white solid. M.p: 150-152 °C. 1H NMR (DMSO-d6): δH 8.15 – 7.42 (m, 4H), 4.27
13
(s, 2H), 1.39 (s, 9H). C NMR (DMSO-d6): δC 169.7, 154.8, 139.2, 132.5, 125.7, 124.6, 116.3,
116.0, 83.1, 53.6, 28.2. ESI-MS m/z 250 (M+H)+. Anal. Calcd. for C13H15NO4: C, 62.64; H,
6.07; N, 5.62; Found: C, 62.58; H, 6.04; N, 5.65.
4.1.3.2. tert-butyl 2-(6-chloro-2-oxobenzo[d]oxazol-3(2H)-yl)acetate (6b)
The compound was synthesized according to the general procedure using 6-
chlorobenzo[d]oxazol-2(3H)-one (5b) (1.5 g, 8.84 mmol) and tert-butyl bromoacetate (1.8 g,

1
9.73 mmol) to afford 6b (1.37 g, 82.1 %) as white solid. M.p: 197-199 °C. H NMR (DMSO-
13
d6): δH 7.87 – 7.31 (m, 3H), 4.23 (s, 2H), 1.34 (m, 9H). C NMR (DMSO-d6): δC 169.6, 154.7,
140.5, 131.7, 130.1, 125.9, 123.6, 122.8, 82.3, 53.2, 28.5. ESI-MS m/z 284 (M+H)+. Anal. Calcd.
for C13H14ClNO4: C, 55.04; H, 4.97; N, 4.94; Found: C, 55.07; H, 4.99; N, 4.96.
4.1.3.3. tert-butyl 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)acetate (6c)
The compound was synthesized according to the general procedure using 6-nitrobenzo[d]oxazol-
2(3H)-one (5c) (3.0 g, 16.65 mmol) and tert-butyl bromoacetate (3.5 g, 18.32 mmol) to afford 6c
1
(2.1 g, 68.6 %) as white solid. M.p: 182-184 °C. H NMR (DMSO-d6): δH 8.28 – 7.98 (m, 3H),
13
4.31 (s, 2H), 1.37 (s, 9H). C NMR (DMSO-d6): δC 169.5, 154.5, 144.2, 140.3, 138.6, 123.1,
121.2, 117.5, 82.0, 53.6, 28.4. ESI-MS m/z 295 (M+H)+. Anal. Calcd. for C13H14N2O6: C, 53.06;
H, 4.80; N, 9.52; Found: C, 53.09; H, 4.78; N, 9.49.
4.1.4. General procedure for the synthesis of 6-substituted 2-(2-oxobenzo[d]oxazol-3(2H)-
yl)acetic acids (7a-c)
Trifluoroacetic acid (TFA) (2 mmol) was added drop wise over 5 min to a stirred solution of
corresponding 6-substituted tert-butyl 2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetate (6a-c) (1 mmol)
in anhydrous dichloromethane (DCM) (15 mL) under nitrogen at 0 °C. The solution was slowly
warmed to the rt and stirred at rt for about 4 h (monitored by TLC & LCMS for completion) and
concentrated under reduced pressure. The residue was dissolved in toluene and concentrated
under reduced pressure (twice), and finally triturated with ether to give a corresponding product
(7a-c).
4.1.4.1. 2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7a)
The compound was synthesized according to the general procedure using tert-butyl 2-(2-
oxobenzo[d]oxazol-3(2H)-yl)acetate (6a) (1.5 g, 6.0 mmol), and TFA (1.3 g, 12.0 mmol) to

1
afford 7a (0.9 g, 83.1 %) as white solid. M.p: 226-228 °C. H NMR (DMSO-d6): δH 11.17 (s,
13
1H), 7.82 – 7.18 (m, 4H), 4.12 (s, 2H). C NMR (DMSO-d6): δC 176.6, 154.7, 139.2, 132.5,
125.9, 124.8, 116.4, 116.1, 56.1. ESI-MS m/z 192 (M-H)-. Anal. Calcd. for C9H7NO4: C, 55.96;
H, 3.65; N, 7.25; Found: C, 56.00; H, 3.67; N, 7.23.
4.1.4.2. 2-(6-chloro-2-oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7b)
The compound was synthesized according to the general procedure using tert-butyl 2-(6-chloro-
2-oxobenzo[d]oxazol-3(2H)-yl)acetate (6b) (1.35 g, 4.75 mmol), and TFA (1.08 g, 9.51 mmol)
1
to afford 7b (0.91 g, 87.7 %) as white solid. M.p: 268-270 °C. H NMR (DMSO-d6): δH 11.20
(s, 1H), 8.14 – 7.31 (m, 3H), 4.14 (s, 2H). 13C NMR (DMSO-d6): δC 176.2, 154.8, 140.1, 131.6,
130.4, 126.0, 123.6, 122.7, 56.3. ESI-MS m/z 226 (M-H)-. Anal. Calcd. for C9H6ClNO4: C,
47.49; H, 2.66; N, 6.15; Found: C, 47.52; H, 2.65; N, 6.13.
4.1.4.3. 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7c)
The compound was synthesized according to the general procedure using tert-butyl 2-(6-nitro-2-
oxobenzo[d]oxazol-3(2H)-yl)acetate (6c) (1.5 g, 5.09 mmol), and TFA (1.16 g, 10.19 mmol) to
1
afford 7c (0.95 g, 78.7 %) as white solid. M.p: 261-263 °C. H NMR (DMSO-d6): δH 11.22 (s,
13
1H), 8.31 – 7.98 (m, 3H), 4.17 (s, 2H). C NMR (DMSO-d6): δC 176.3, 154.6, 144.2, 140.0,
138.5, 123.2, 121.1, 117.7, 55.4. ESI-MS m/z 237 (M-H)-. Anal. Calcd. for C9H6N2O6: C, 45.39;
H, 2.54; N, 11.76; Found: C, 45.42; H, 2.52; N, 11.78.
4.1.5. General procedure for the synthesis of substituted 2-(2-oxobenzo[d]oxazol-3(2H)-
yl)acetamides (8-25)
A solution of corresponding 2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7a-c) (1 mmol),
substituted amine (1.2 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (1.2
mmol), N-hydroxybenzotriazole (HOBT) (1.2 mmol), and triethylamine (TEA) (2.2 mmol) were

stirred for 6 h at rt in DCM (4 mL) (monitored by TLC & LCMS for completion). The reaction
mixture was further diluted with water (10 mL) and DCM (10 mL), and the layers separated. The
organic layer was dried over anhydrous sodium sulphate and concentrated under reduced
pressure. The crude product was purified by silica gel column chromatography using hexane:
ethyl acetate as eluent to give the corresponding product (8-25).
4.1.5.1. 2-(2-oxobenzo[d]oxazol-3(2H)-yl)-N-phenylacetamide (8)
The compound was synthesized according to the general procedure using 2-(2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7a) (0.1 g, 0.51 mmol), aniline (0.057 g, 0.62 mmol),
EDC (0.12 g, 0.62 mmol) and HOBT (0.083 g, 0.62 mmol) to afford 8 (0.092 g, 66.6 %) as white
1
solid. M.p: 190-191 °C. H NMR (DMSO-d6): δH 10.25 (s, 1H), 8.16 – 7.04 (m, 9H), 4.76 (s,
13
2H). C NMR (DMSO-d6): δC 169.5, 155.6, 139.0, 138.7, 132.1, 128.3, 128.0, 125.7, 124.6,
121.2, 116.4, 115.9, 54.1. ESI-MS m/z 269 (M+H)+. Anal. Calcd. for C15H12N2O3: C, 67.16;
H,4.51; N, 10.44; Found: C, 67.12; H, 4.52; N, 10.42.
4.1.5.2. 2-(6-chloro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-phenylacetamide (9)
The compound was synthesized according to the general procedure using 2-(6-chloro-2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7b) (0.1 g, 0.43 mmol), aniline (0.049 g, 0.52 mmol),
EDC (0.10 g, 0.52 mmol) and HOBT (0.070 g, 0.52 mmol) to afford 9 (0.097 g, 73.2 %) as white
1
solid. M.p: 181-183 °C. H NMR (DMSO-d6): δH 10.26 (s, 1H), 8.22 - 7.05 (m, 8H), 4.77 (s,
13
2H). C NMR (DMSO-d6): δC 169.6, 154.8, 140.1, 138.7, 131.3, 130.6, 128.7, 128.3, 125.4,
123.5, 122.7, 121.6, 54.2. ESI-MS m/z 301 (M-H)-. Anal. Calcd. for C15H11ClN2O3: C, 59.52; H,
3.66; N, 9.25; Found: C, 59.55; H, 3.64; N, 9.28.
4.1.5.3. 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-phenylacetamide (10)

The compound was synthesized according to the general procedure using 2-(6-nitro-2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7c) (0.1 g, 0.41 mmol), aniline (0.046 g, 0.50 mmol),
EDC (0.098 g, 0.50 mmol) and HOBT (0.067 g, 0.50 mmol) to afford 10 (0.096 g, 73.3 %) as
pale yellow solid. M.p: 222-223 °C. 1H NMR (DMSO-d6): δH 10.28 (s, 1H), 7.92 – 7.06 (m, 8H),
13
4.83 (s, 2H). C NMR (DMSO-d6): δC 169.6, 154.9, 143.9, 140.2, 138.6, 138.4, 129.0, 128.2,
122.8, 121.7, 120.9, 117.7, 54.4. ESI-MS m/z 312 (M-H)-. Anal. Calcd. for C15H11N3O5: C,
57.51; H, 3.54; N, 13.41; Found: C, 57.55; H, 3.52; N, 13.44.
4.1.5.4. N-(2-chloro-5-(trifluoromethyl)phenyl)-2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetamide
(11)
The compound was synthesized according to the general procedure using 2-(2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7a) (0.1 g, 0.51 mmol), 2-chloro-5-
(trifluoromethyl)aniline (0.12 g, 0.62 mmol), EDC (0.12 g, 0.62 mmol) and HOBT (0.083 g, 0.62
1
mmol) to afford 11 (0.13 g, 69.6 %) as pale yellow solid. M.p: 157-159 °C. H NMR (DMSO-
13
d6): δH 9.97 (s, 1H), 8.10 – 7.19 (m, 7H), 4.82 (s, 2H). C NMR (DMSO-d6): δC 169.7, 168.6,
139.1, 137.9, 132.2, 129.5, 129.3, 125.9, 125.7, 124.8, 124.3, 122.2, 118.7, 116.6, 116.1, 54.2.
ESI-MS m/z 369 (M-H)-. Anal. Calcd. for C16H10ClF3N2O3: C, 51.84; H, 2.72; N, 7.56; Found:
C, 51.87; H, 2.70; N, 7.53.
4.1.5.5. 2-(6-chloro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(2-chloro-5-
(trifluoromethyl)phenyl)acetamide (12)
The compound was synthesized according to the general procedure using 2-(6-chloro-2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7b) (0.1 g, 0.43 mmol), 2-chloro-5-
(trifluoromethyl)aniline (0.10 g, 0.52 mmol), EDC (0.10 g, 0.52 mmol) and HOBT (0.070 g, 0.52
1
mmol) to afford 12 (0.13 g, 74.3 %) as white solid. M.p: 139-140 °C. H NMR (DMSO-d6): δH

13
9.98 (s, 1H), 8.11 – 7.19 (m, 6H), 4.82 (s, 2H). C NMR (DMSO-d6): δC 169.5, 154.0, 140.8,
137.3, 131.7, 130.5, 129.6, 129.4, 125.9, 125.6, 124.4, 123.7, 122.8, 122.2, 118.9, 54.5. ESI-MS
m/z 404 (M-H)-. Anal. Calcd. for C16H9Cl2F3N2O3: C, 47.43; H, 2.24; N, 6.91; Found: C, 47.45;
H, 2.25; N, 6.94.
4.1.5.6. N-(2-chloro-5-(trifluoromethyl)phenyl)-2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-
yl)acetamide (13)
The compound was synthesized according to the general procedure using 2-(6-nitro-2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7c) (0.1 g, 0.41 mmol), 2-chloro-5-
(trifluoromethyl)aniline (0.097 g, 0.50 mmol), EDC (0.098 g, 0.50 mmol) and HOBT (0.067 g,
1
0.50 mmol) to afford 13 (0.13 g, 78.5 %) as pale yellow solid. M.p: 127-129 °C. H NMR
(DMSO-d6): δH 9.98 (s, 1H), 8.12 – 7.19 (m, 6H), 4.83 (s, 2H). 13C NMR (DMSO-d6): δC 169.4,
154.6, 143.9, 140.1, 138.5, 137.7, 129.5, 129.3, 126.1, 124.3, 122.9, 122.4, 121.0, 118.8, 117.7,
54.2. ESI-MS m/z 414 (M-H)-. Anal. Calcd. for C16H9ClF3N3O5: C, 46.23; H, 2.18; N, 10.11;
Found: C, 46.19; H, 2.16; N, 10.14.
4.1.5.7. 2-(2-oxobenzo[d]oxazol-3(2H)-yl)-N-(pyridin-2-yl)acetamide (14)
The compound was synthesized according to the general procedure using 2-(2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7a) (0.1 g, 0.51 mmol), pyridin-2-amine (0.058 g, 0.62
mmol), EDC (0.12 g, 0.62 mmol) and HOBT (0.083 g, 0.62 mmol) to afford 14 (0.094 g, 68.2
%) as white solid. M.p: 221-223 °C. 1H NMR (DMSO-d6): δH 10.42 (s, 1H), 8.67 – 7.02 (m, 8H),
13
4.87 (s, 2H). C NMR (DMSO-d6): δC 169.5, 154.7, 151.8, 146.7, 139.1, 138.5, 132.1, 127.6,
124.8, 124.3, 116.5, 116.1, 115.8, 52.4. ESI-MS m/z 270 (M+H)+. Anal. Calcd. for C14H11N3O3:
C, 62.45; H, 4.12; N, 15.61; Found: C, 62.42; H, 4.10; N, 15.59.

4.1.5.8. 2-(6-chloro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(pyridin-2-yl)acetamide (15)
The compound was synthesized according to the general procedure using 2-(6-chloro-2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7b) (0.1 g, 0.43 mmol), pyridin-2-amine (0.048 g, 0.52
mmol), EDC (0.10 g, 0.52 mmol) and HOBT (0.070 g, 0.52 mmol) to afford 15 (0.086 g, 65.3
%) as white solid. M.p: 243-245 °C. 1H NMR (DMSO-d6): δH 10.44 (s, 1H), 8.67 – 7.10 (m, 7H),
13
4.88 (s, 2H). C NMR (DMSO-d6): δC 169.6, 154.8, 151.6, 146.6, 140.2, 138.5, 131.4, 130.4,
125.7, 124.3, 123.6, 122.7, 116.6, 52.3. ESI-MS m/z 302 (M-H)-. Anal. Calcd. for C14H10ClN3O3:
C, 55.37; H, 3.32; N, 13.84; Found: C, 55.34; H, 3.35; N, 13.87.
4.1.5.9. 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(pyridin-2-yl)acetamide (16)
The compound was synthesized according to the general procedure using 2-(6-nitro-2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7c) (0.1 g, 0.41 mmol), pyridin-2-amine (0.047 g, 0.50
mmol), EDC (0.098 g, 0.50 mmol) and HOBT (0.067 g, 0.50 mmol) to afford 16 (0.088 g, 67.1
1
%) as pale yellow solid. M.p: 253-255 °C. H NMR (DMSO-d6): δH 10.51 (s, 1H), 8.68 – 7.11
(m, 7H), 4.89 (s, 2H). 13C NMR (DMSO-d6): δC 168.9, 154.9, 151.8, 146.7, 144.0, 139.9, 138.8,
138.4, 124.6, 122.9, 121.2, 117.2, 115.9, 52.6. ESI-MS m/z 313 (M-H)-. Anal. Calcd. for
C14H10N4O5: C, 53.51; H, 3.21; N, 17.83; Found: C, 53.48; H, 3.18; N, 17.86.
4.1.5.10. 2-(2-oxobenzo[d]oxazol-3(2H)-yl)-N-(pyridin-2-ylmethyl)acetamide (17)
The compound was synthesized according to the general procedure using 2-(2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7a) (0.1 g, 0.51 mmol), pyridin-2-ylmethanamine
(0.067 g, 0.62 mmol), EDC (0.12 g, 0.62 mmol) and HOBT (0.083 g, 0.62 mmol) to afford 17
(0.095 g, 65.2 %) as white solid. M.p: 162-164 °C. 1H NMR (DMSO-d6): δH 9.921 (s, 1H), 8.37
13
– 7.01 (m, 8H), 4.75 (s, 2H), 4.54 (s, 2H). C NMR (DMSO-d6): δC 169.6, 155.8, 154.2, 148.5,

139.5, 139.2, 132.0, 125.9, 124.8, 124.2, 121.1, 116.4, 116.1, 54.3, 45.2. ESI-MS m/z 284
(M+H)+. Anal. Calcd. for C15H13N3O3: C, 63.60; H, 4.63; N, 14.83; Found: C, 63.57; H, 4.60; N,
14.81.
4.1.5.11. 2-(6-chloro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(pyridin-2-ylmethyl)acetamide (18)
The compound was synthesized according to the general procedure using 2-(6-chloro-2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7b) (0.1 g, 0.43 mmol), pyridin-2-ylmethanamine
(0.056 g, 0.52 mmol), EDC (0.10 g, 0.52 mmol) and HOBT (0.070 g, 0.52 mmol) to afford 18
(0.089 g, 64.4 %) as white solid. M.p: 182-184 °C. 1H NMR (DMSO-d6): δH 9.92 (s, 1H), 8.41 –
13
7.01 (m, 7H), 4.81 (s, 2H), 4.55 (s, 2H). C NMR (DMSO-d6): δC 169.7, 156.2, 154.3, 148.8,
140.7, 139.9, 131.8, 130.1, 125.7, 124.3, 126.3, 122.2, 121.1, 54.4, 45.5. ESI-MS m/z 316 (M-H)-
. Anal. Calcd. for C15H12ClN3O3: C, 56.70; H, 3.81; N, 13.23; Found: C, 56.66; H, 3.78; N,
13.20.
4.1.5.12. 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(pyridin-2-ylmethyl)acetamide (19)
The compound was synthesized according to the general procedure using 2-(6-nitro-2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7c) (0.1 g, 0.41 mmol), pyridin-2-ylmethanamine
(0.054 g, 0.50 mmol), EDC (0.098 g, 0.50 mmol) and HOBT (0.067 g, 0.50 mmol) to afford 19
(0.092 g, 66.7 %) as white solid. M.p: 187-189 °C. 1H NMR (DMSO-d6): δH 9.92 (s, 1H), 8.42 –
13
7.02 (m, 7H), 4.83 (s, 2H), 4.55 (s, 2H). C NMR (DMSO-d6): δC 169.5, 156.3, 154.9, 148.3,
144.2, 139.9, 139.6, 138.4, 124.5, 123.1, 121.1, 120.8, 117.7, 54.4, 45.4. ESI-MS m/z 327 (M-H)-
. Anal. Calcd. for C15H12N4O5: C, 54.88; H, 3.68; N, 17.07; Found: C, 54.87; H, 3.70; N, 17.10.
4.1.5.13. N-(furan-2-ylmethyl)-2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetamide (20)

The compound was synthesized according to the general procedure using 2-(2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7a) (0.1 g, 0.51 mmol), furan-2-ylmethanamine (0.060
g, 0.62 mmol), EDC (0.12 g, 0.62 mmol) and HOBT (0.083 g, 0.62 mmol) to afford 20 (0.101 g,
72.2 %) as white solid. M.p: 110-112 °C. 1H NMR (DMSO-d6): δH 9.76 (s, 1H), 7.45 – 6.79 (m,
13
7H), 4.79 (s, 2H), 4.33 (s, 2H). C NMR (DMSO-d6): δC 169.3, 154.2, 149.3, 142.5, 132.1,
130.3, 124.5, 122.6, 119.2, 116.1, 110.4, 108.6, 51.3, 35.0. ESI-MS m/z 273 (M+H)+. Anal.
Calcd. for C14H12N2O4: C, 61.76; H, 4.44; N, 10.29; Found: C, 61.73; H, 4.42; N, 10.26.
4.1.5.14. 2-(6-chloro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(furan-2-ylmethyl)acetamide (21)
The compound was synthesized according to the general procedure using 2-(6-chloro-2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7b) (0.1 g, 0.43 mmol), furan-2-ylmethanamine (0.050
g, 0.52 mmol), EDC (0.10 g, 0.52 mmol) and HOBT (0.070 g, 0.52 mmol) to afford 21 (0.098 g,
73.4 %) as white solid. M.p: 142-144 °C. 1H NMR (DMSO-d6): δH 10.42 (s, 1H), 7.60 – 6.36 (m,
13
6H), 4.62 (s, 2H), 4.33 (s, 2H). C NMR (DMSO-d6): δC 169.5, 154.6, 154.1, 149.2, 142.6,
132.3, 130.0, 122.7, 119.0, 116.3, 110.6, 108.3, 51.5, 34.9. ESI-MS m/z 305 (M-H)-. Anal.
Calcd. for C14H11ClN2O4: C, 54.83; H, 3.62; N, 9.13; Found: C, 54.79; H, 3.60; N, 9.15.
4.1.5.15. N-(furan-2-ylmethyl)-2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)acetamide (22)
The compound was synthesized according to the general procedure using 2-(6-nitro-2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7c) (0.1 g, 0.41 mmol), furan-2-ylmethanamine (0.048
g, 0.50 mmol), EDC (0.098 g, 0.50 mmol) and HOBT (0.067 g, 0.50 mmol) to afford 22 (0.097
g, 73.2 %) as pale yellow solid. M.p: 165-167 °C. 1H NMR (DMSO-d6): δH 11.04 (s, 1H), 7.75 –
13
6.80 (m, 6H), 4.81 (s, 2H), 4.33 (s, 2H). C NMR (DMSO-d6): δC 169.7, 154.5, 144.2, 149.2,
142.4, 132.2, 130.0, 122.5, 119.2, 116.4, 110.7, 108.5, 51.2, 34.9. ESI-MS m/z 316 (M-H)-. Anal.
Calcd. for C14H11N3O6: C, 53.00; H, 3.49; N, 13.24; Found: C, 52.97; H, 3.46; N, 13.22.

4.1.5.16. 2-(2-oxobenzo[d]oxazol-3(2H)-yl)-N-(thiophen-2-ylmethyl)acetamide (23)
The compound was synthesized according to the general procedure using 2-(2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7a) (0.1 g, 0.51 mmol), thiophen-2-ylmethanamine
(0.070 g, 0.62 mmol), EDC (0.12 g, 0.62 mmol) and HOBT (0.083 g, 0.62 mmol) to afford 23
(0.111 g, 74.4 %) as white solid. M.p: 128-130 °C. 1H NMR (DMSO-d6): δH 9.87 (s, 1H), 7.47 –
13
6.81 (m, 7H), 4.79 (s, 2H), 4.34 (s, 2H). C NMR (DMSO-d6): δC 169.6, 154.7, 153.2, 138.2,
128.8, 128.7, 127.2, 126.8, 126.3, 123.3, 119.1, 116.6, 51.6, 36.3. ESI-MS m/z 287 (M-H)-. Anal.
Calcd. for C14H12N2O3S: C, 58.32; H, 4.20; N, 9.72; Found: C, 58.35; H, 4.18; N, 9.73.
4.1.5.17. 2-(6-chloro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(thiophen-2-ylmethyl)acetamide (24)
The compound was synthesized according to the general procedure using 2-(6-chloro-2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7b) (0.1 g, 0.43 mmol), thiophen-2-ylmethanamine
(0.058 g, 0.52 mmol), EDC (0.10 g, 0.52 mmol) and HOBT (0.070 g, 0.52 mmol) to afford 24
(0.102 g, 72.3 %) as white solid. M.p: 166-168 °C. 1H NMR (DMSO-d6): δH 10.32 (s, 1H), 7.52
13
– 6.24 (m, 6H), 4.79 (s, 2H), 4.35 (s, 2H). C NMR (DMSO-d6): δC 169.4, 154.6, 153.0, 139.6,
139.4, 129.5, 128.3, 126.7, 126.0, 124.5, 120.4, 115.6, 51.4, 36.3. ESI-MS m/z 321 (M-H)-. Anal.
Calcd. for C14H11ClN2O3S: C, 52.10; H, 3.44; N, 8.68; Found: C, 52.13; H, 3.42; N, 8.66.
4.1.5.18. 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(thiophen-2-ylmethyl)acetamide (25)
The compound was synthesized according to the general procedure using 2-(6-nitro-2-
oxobenzo[d]oxazol-3(2H)-yl)acetic acid (7c) (0.1 g, 0.41 mmol), thiophen-2-ylmethanamine
(0.056 g, 0.50 mmol), EDC (0.098 g, 0.50 mmol) and HOBT (0.067 g, 0.50 mmol) to afford 25
(0.104 g, 74.7 %) as white solid. M.p: 220-222 °C. 1H NMR (DMSO-d6): δH 11.06 (s, 1H), 7.76
13
– 6.97 (m, 6H), 4.81 (s, 2H), 4.50 (s, 2H). C NMR (DMSO-d6): δC 169.2, 154.4, 152.5, 146.1,

137.9, 130.2, 128.0, 127.4, 126.8, 126.2, 114.2, 111.1, 51.2, 36.4. ESI-MS m/z 332 (M-H)-. Anal.
Calcd. for C14H11N3O5S: C, 50.45; H, 3.33; N, 12.61; Found: C, 50.42; H, 3.35; N, 12.64.
4.1.6. General procedure for the synthesis of 2-chloro-N-(aryl/heteroaryl)acetamides (27a-
c)
Chloroacetyl chloride (2 mmol) was added drop wise to a well stirred suspension of
corresponding amine (26) (1 mmol) and TEA (3 mmol) in DMF (15 mL) at 0 °C. The reaction
mixture was then slowly warmed to rt and stirred at rt for about 3 h (monitored by TLC & LCMS
for completion). The solvent was then evaporated under reduced pressure and the residue was
further diluted with water (20 mL) and ethyl acetate (30 mL), and the layers separated. The
aqueous layer was re-extracted with ethyl acetate (2 × 30 mL) and the combined organic layer
was washed with brine (20 mL), dried over anhydrous sodium sulphate and evaporated under
reduced pressure. The residue was then purified by silica column chromatography using hexane:
ethyl acetate as eluent to give corresponding product (27a-c).
4.1.6.1. 2-chloro-N-(5-nitrothiazol-2-yl)acetamide (27a)
The compound was synthesized according to the general procedure using chloroacetyl chloride
(1.55 g, 13.77 mmol) and 5-nitrothiazol-2-amine (1.0 g, 6.88 mmol) to afford 27a (1.03 g, 67.7
1
%) as pale yellow solid. M.p: 252-254 °C. H NMR (DMSO-d6): δH 9.94 (s, 1H), 8.34 (s, 1H),
4.45 (s, 2H). 13C NMR (DMSO-d6): δC 169.4, 166.3, 148.1, 138.6, 43.1. ESI-MS m/z 220 (M-H)-
. Anal. Calcd. for C5H4ClN3O3S: C, 27.10; H, 1.82; N, 18.96; Found: C, 27.13; H, 1.79; N,
18.99.
4.1.6.2. N-(benzo[d]thiazol-2-yl)-2-chloroacetamide (27b)

The compound was synthesized according to the general procedure using chloroacetyl chloride
(1.50 g, 13.31 mmol) and benzo[d]thiazol-2-amine (1.0 g, 6.65 mmol) to afford 27b (0.92 g, 66.2
1
%) as pale yellow solid. M.p: 276-278 °C. H NMR (DMSO-d6): δH 11.78 (s, 1H), 8.21 – 7.63
(m, 4H), 4.39 (s, 2H). 13C NMR (DMSO-d6): δC 173.7, 168.8, 153.4, 130.9, 125.5, 124.7, 121.5,
118.4, 42.9. ESI-MS m/z 225 (M-H)-. Anal. Calcd. for C9H7ClN2OS: C, 47.69; H, 3.11; N, 12.36;
Found: C, 47.72; H, 3.13; N, 12.39.
4.1.6.3. 2-chloro-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide (27c)
The compound was synthesized according to the general procedure using chloroacetyl chloride
(1.15 g, 10.24 mmol) and 6-nitrobenzo[d]thiazol-2-amine (1.0 g, 5.12 mmol) to afford 27c (0.92
g, 66.2 %) as pale yellow solid. M.p: 273-275 °C. 1H NMR (DMSO-d6): δH 12.62 (s 1H), 9.11 (s,
13
1H), 8.34 – 7.97 (m, 2H), 4.55 (s, 2H). C NMR (DMSO-d6): δC 173.9, 168.5, 159.6, 144.8,
131.6, 121.5, 119.2, 117.1, 43.0. ESI-MS m/z 270 (M-H)-. Anal. Calcd. for C9H6ClN3O3S: C,
39.79; H, 2.23; N, 15.47; Found: C, 39.76; H, 2.22; N, 15.50.
4.1.7. General procedure for the synthesis of substituted 2-(2-oxobenzo[d]oxazol-3(2H)-
yl)acetamides (28-36)
To a solution of corresponding 2-chloro-N-(aryl/heteroaryl)acetamides (27a-c) (1.2 mmol) in
DMF (3 mL) was added anhydrous potassium carbonate (1.5 mmol) and corresponding
benzo[d]oxazol-2(3H)-one (5a-c) (1 mmol). The reaction mixture was heated at 60 °C for 3 h
(monitored by TLC & LCMS for completion). The residue was further diluted with water (10
mL) and ethyl acetate (20 mL) and the layers separated. The aqueous layer was re-extracted with
ethyl acetate (2 x 15 mL) and the combined organic layer was washed with brine (15 mL), dried
over anhydrous sodium sulphate and evaporated under reduced pressure and the residue was

purified by silica column chromatography using hexane: ethyl acetate as eluent to give the
desired product (28-36).
4.1.7.1. N-(5-nitrothiazol-2-yl)-2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetamide (28)
The compound was synthesized according to the general procedure using 2-chloro-N-(5-
nitrothiazol-2-yl)acetamide (27a) (0.137 g, 0.62 mmol) and benzo[d]oxazol-2(3H)-one (5a)
(0.1g, 0.51 mmol) to afford 28 (0.11 g, 72.2 %) as pale yellow solid. M.p: 193-195 °C. 1H NMR
(DMSO-d6): δH 13.60 (s, 1H), 8.67 (s, 1H), 7.41 – 7.15 (m, 4H), 4.95 (s, 2H). 13C NMR (DMSO-
d6): δC 169.5, 162.8, 154.4, 145.7, 138.8, 135.5, 132.5, 125.8, 124.7, 116.6, 116.1, 53.2. ESI-MS
m/z 319 (M-H)-. Anal. Calcd. for C12H8N4O5S: C, 45.00; H, 2.52; N, 17.49; Found: C, 45.04; H,
2.49; N, 17.46.
4.1.7.2. 2-(6-chloro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(5-nitrothiazol-2-yl)acetamide (29)
The compound was synthesized according to the general procedure using 2-chloro-N-(5-
nitrothiazol-2-yl)acetamide (27a) (0.156 g, 0.70 mmol) and 6-chlorobenzo[d]oxazol-2(3H)-one
(5b) (0.1g, 0.58 mmol) to afford 29 (0.148 g, 70.9 %) as pale yellow solid. M.p: 268-270 °C. 1H
13
NMR (DMSO-d6): δH 13.51 (s, 1H), 8.65 (s, 1H), 7.89 – 7.12 (m, 3H), 4.81 (s, 2H). C NMR
(DMSO-d6): δC 169.6, 162.6, 154.7, 147.1, 140.0, 135.6, 131.5, 130.4, 125.9, 123.5, 122.8, 53.4.
ESI-MS m/z 353 (M-H)-. Anal. Calcd. for C12H7ClN4O5S: C, 40.63; H, 1.99; N, 15.79; Found: C,
40.60; H, 2.01; N, 15.81.
4.1.7.3. 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(5-nitrothiazol-2-yl)acetamide (30)
The compound was synthesized according to the general procedure using 2-chloro-N-(5-
nitrothiazol-2-yl)acetamide (27a) (0.147 g, 0.66 mmol) and 6-nitrobenzo[d]oxazol-2(3H)-one
(5c) (0.1g, 0.55 mmol) to afford 30 (0.149 g, 73.6 %) as pale yellow solid. M.p: 273-275 °C. 1H

13
NMR (DMSO-d6): δH 13.50 (s, 1H), 8.55 (s, 1H), 7.89 – 7.19 (m, 3H), 4.83 (s, 2H). C NMR
(DMSO-d6): δC 169.6, 162.8, 154.2, 147.1, 143.3, 140.2, 138.5, 135.6, 123.2, 121.3, 117.7, 53.2.
ESI-MS m/z 364 (M-H)-. Anal. Calcd. for C12H7N5O7S: C, 39.46; H, 1.93; N, 19.17; Found: C,
39.49; H, 1.94; N, 19.20.
4.1.7.4. N-(benzo[d]thiazol-2-yl)-2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetamide (31)
The compound was synthesized according to the general procedure using N-(benzo[d]thiazol-2-
yl)-2-chloroacetamide (27b) (0.140 g, 0.62 mmol) and benzo[d]oxazol-2(3H)-one (5a) (0.1g,
0.51 mmol) to afford 31 (0.11 g, 67.9 %) as white solid. M.p: 250-252 °C. 1H NMR (DMSO-d6):
13
δH 10.68 (s, 1H), 7.85 – 7.37 (m, 8H), 4.79 (s, 2H). C NMR (DMSO-d6): δC 173.5, 168.6,
154.7, 153.3, 139.2, 132.4, 130.9, 125.8, 125.5, 124.7, 124.5, 121.9, 118.7, 116.6, 116.0, 54.2.
ESI-MS m/z 326 (M+H)+. Anal. Calcd. for C16H11N3O3S: C, 59.07; H, 3.41; N, 12.92; Found: C,
59.04; H, 3.38; N, 12.89.
4.1.7.5. N-(benzo[d]thiazol-2-yl)-2-(6-chloro-2-oxobenzo[d]oxazol-3(2H)-yl)acetamide (32)
The compound was synthesized according to the general procedure using N-(benzo[d]thiazol-2-
yl)-2-chloroacetamide (27b) (0.158 g, 0.70 mmol) and 6-chlorobenzo[d]oxazol-2(3H)-one (5b)
1
(0.1g, 0.58 mmol) to afford 32 (0.108 g, 68.4 %) as white solid. M.p: 267-269 °C. H NMR
(DMSO-d6): δH 10.73 (s, 1H), 8.02 – 7.34 (m, 7H), 4.81 (s, 2H). 13C NMR (DMSO-d6): δC 173.4,
168.5, 154.2, 153.5, 140.8, 131.7, 130.8, 130.5, 125.9, 125.5, 124.3, 123.5, 122.8, 121.9, 118.8,
54.4. ESI-MS m/z 358 (M-H)-. Anal. Calcd. for C16H10ClN3O3S: C, 53.41; H, 2.80; N, 11.68;
Found: C, 53.39; H, 2.78; N, 11.71.
4.1.7.6. N-(benzo[d]thiazol-2-yl)-2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)acetamide (33)

The compound was synthesized according to the general procedure using N-(benzo[d]thiazol-2-
yl)-2-chloroacetamide (27b) (0.135 g, 0.66 mmol) and 6-nitrobenzo[d]oxazol-2(3H)-one (5c)
1
(0.1g, 0.55 mmol) to afford 33 (0.105 g, 67.6 %) as pale yellow solid. M.p: 256-258 °C. H
NMR (DMSO-d6): δH 10.75 (s, 1H), 8.12 – 7.41 (m, 7H), 4.81 (s, 2H). 13C NMR (DMSO-d6): δC
173.7, 168.7, 154.8, 153.1, 144.2, 139.8, 138.6, 130.9, 125.5, 124.9, 123.3, 121.9, 121.0, 118.4,
117.7, 54.2. ESI-MS m/z 369 (M-H)-. Anal. Calcd. for C16H10N4O5S: C, 51.89; H, 2.72; N,
15.13; Found: C, 51.86; H, 2.70; N, 15.16.
4.1.7.7. N-(6-nitrobenzo[d]thiazol-2-yl)-2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetamide (34)
The compound was synthesized according to the general procedure using 2-chloro-N-(6-
nitrobenzo[d]thiazol-2-yl)acetamide (27c) (0.168 g, 0.62 mmol) and benzo[d]oxazol-2(3H)-one
1
(5a) (0.1g, 0.51 mmol) to afford 34 (0.124 g, 64.8 %) as pale yellow solid. M.p: >280 °C. H
13
NMR (DMSO-d6): δH 13.08 (s, 1H), 8.46 (s, 1H), 8.23 – 6.99 (m, 6H), 4.78 (s, 2H). C NMR
(DMSO-d6): δC 173.5, 168.6, 159.5, 154.7, 144.5, 139.2, 132.5, 131.5, 125.8, 124.4, 121.1,
119.6, 117.4, 116.4, 116.1, 54.4. ESI-MS m/z 369 (M-H)-. Anal. Calcd. for C16H10N4O5S: C,
51.89; H, 2.72; N, 15.13; Found: C, 51.86; H, 2.74; N, 15.10.
4.1.7.8. 2-(6-chloro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(6-nitrobenzo[d]thiazol-2-
yl)acetamide (35)
The compound was synthesized according to the general procedure using 2-chloro-N-(6-
nitrobenzo[d]thiazol-2-yl)acetamide (27c) (0.190 g, 0.70 mmol) and 6-chlorobenzo[d]oxazol-
2(3H)-one (5b) (0.1g, 0.58 mmol) to afford 35 (0.156 g, 65.7 %) as pale yellow solid. M.p: 269-
1
271 °C. H NMR (DMSO-d6): δH 13.00 (s, 1H), 8.46 (s, 1H), 8.24 – 7.11 (m, 5H), 4.89 (s, 2H).
13C NMR (DMSO-d6): δC 173.3, 168.7, 160.1, 154.6, 144.7, 140.4, 131.7, 131.5, 130.6, 125.7,

123.3, 122.5, 121.7, 119.4, 117.7, 54.5. ESI-MS m/z 403 (M-H)-. Anal. Calcd. for
C16H9ClN4O5S: C, 47.47; H, 2.24; N, 13.84; Found: C, 47.44; H, 2.22; N, 13.82.
4.1.7.9. 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide
(36)
The compound was synthesized according to the general procedure using 2-chloro-N-(6-
nitrobenzo[d]thiazol-2-yl)acetamide (27c) (0.179 g, 0.66 mmol) and 6-nitrobenzo[d]oxazol-
2(3H)-one (5c) (0.1g, 0.55 mmol) to afford 36 (0.157 g, 68.1 %) as pale yellow solid. M.p: 265-
1
267 °C. H NMR (DMSO-d6): δH 13.02 (s, 1H), 8.46 (s, 1H), 8.24 – 7.82 (m, 5H), 4.89 (s, 2H).
13C NMR (DMSO-d6): δC 174.0, 168.8, 160.0, 154.9, 144.9, 144.3, 140.2, 138.6, 131.7, 123.1,
121.4, 121.2, 119.4, 117.7, 117.5, 54.6. ESI-MS m/z 414 (M-H)-. Anal. Calcd. for C16H9N5O7S:
C, 46.27; H, 2.18; N, 16.86; Found: C, 46.24; H, 2.17; N, 16.84.
4.2. Biological activity
4.2.1. InhA protein expression, isolation and purification
MTB inhA gene that encodes InhA was cloned and transformed into E. coli BL21 (DE3) cells.6
The transformed colonies were grown in LB broth at 37 ºC with constant aeration, in the
presence of 100 μg/ml of ampicillin. Later, the transformed colonies were grown at 37 ºC to
reach an optical density of 0.6 (A595). Exponentially growing cultures were induced with 0.1
mM IPTG (isopropyl-β-D-thiogalactopyranoside) and further the cells were grown at 18 ºC for
12-16 h. Cells were harvested and lysed by sonication in lysis buffer containing 50 mM Tris-
HCl, 200 mM NaCl, 0.05 % β-mercaptoethanol, 0.1 mM PMSF and 8 % glycerol, pH-7.5. The
cell lysate containing His 6-fusion protein was equilibrated with Ni-NTA affinity resins for 3-4
h, and washed with wash buffer containing 50 mM Tris-HCl, 200 mM NaCl, 20 mM imidazole