Identification and development of benzoxazole derivatives as novel bacterial glutamate racemase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.12.088

In the present study, we attempted to develop novel class of Mycobacterium tuberculosis (Mtb) inhibitors by exploring the pharmaceutically underexploited enzyme targets which are majorly involved in cell wall biosynthesis of mycobacteria. For this purpose

Full text of DOI:10.1016/j.ejmech.2017.12.088

Accepted Manuscript
Identification and development of benzoxazole derivatives as novel bacterial
glutamate racemase inhibitors
Prasanthi Malapati, Vagolu Siva Krishna, Radhika Nallangi, Rudraraju Reshma
Srilakshmi, Dharmarajan Sriram
PII:
S0223-5234(17)31119-4
10.1016/j.ejmech.2017.12.088
EJMECH 10066
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 October 2017
Revised Date: 27 December 2017
Accepted Date: 27 December 2017
Please cite this article as: P. Malapati, V.S. Krishna, R. Nallangi, R.R. Srilakshmi, D. Sriram,
Identification and development of benzoxazole derivatives as novel bacterial glutamate racemase
inhibitors, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.088.
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ACCEPTED MANUSCRIPT
Identification and development of benzoxazole derivatives
as novel bacterial glutamate racemase inhibitors
Prasanthi Malapati, Vagolu Siva Krishna, Radhika Nallangi, Rudraraju Reshma Srilakshmi,
Dharmarajan Sriram*
We identified lead compound for M. tuberculosis glutamate racemase by thermal shift assay. We
further synthesize further compounds and tested against various model of M. tuberculosis
together with docking studies.

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SHORT COMMUNICATIONS
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Identification and development of benzoxazole derivatives as novel bacterial
glutamate racemase inhibitors
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Prasanthi Malapati, Vagolu Siva Krishna, Radhika Nallangi, Rudraraju Reshma Srilakshmi,
Dharmarajan Sriram*
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Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus,
Shameerpet, Jawaharnagar, RangaReddy District, Hyderabad 500 078, India
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Abstract
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In the present study, we attempted to develop novel class of Mycobacterium tuberculosis (Mtb)
inhibitors by exploring the pharmaceutically underexploited enzyme targets which are majorly
involved in cell wall biosynthesis of mycobacteria. For this purpose glutamate racemase was
selected which racemizes D-glutamate from L-glutamate, a key step in peptidoglycan synthesis.
Furthermore, enzyme is neither expressed nor its product, D-glutamate is produced in mammals,
and hence inhibiting this enzyme will have no vulnerable effect in host organism. A library of
our in-house compounds were screened against glutamate racemase using a biophysical
technique; thermal shift assay and further by enzyme inhibition assay to identify Lead 1
molecule. Lead 1 optimization and expansion resulted in twenty four compounds. Among the
synthesized compounds twelve compounds shown good enzyme inhibition than Lead 1 (IC50
20.07±0.29 µM). Among all the compounds; compound 22 (IC50 1.1±0.52 µM) showed potent
non-competitive mode of inhibition in enzyme assay. Further showed good susceptibility (in
replicating bacteria) of MIC 8.72 µM and bactericidal time dependant kill on dormant culture. It

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also exhibited significant activity in Mtb nutrient starvation model (2.5) and Mtb biofilm model
(2.4) and in vivo M. marinum infected Zebra fish model studies (3.6) reduction at logarithmic
scale.
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Key words: Tuberculosis, glutamate racemase, racemization, benzoxazole, thermal shift assay
screening
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Contact for correspondence Tel.: +91 40663030506; fax: +91 4066303998. E-mail address:
dsriram@hyderabad.bits-pilani.ac.in
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Identification and development of Benzoxazole derivatives
as bacterial glutamate racemase inhibitors
Prasanthi Malapati, Vagolu Siva Krishna, Radhika Nallangi, Rudraraju Reshma Srilakshmi,
Dharmarajan Sriram*
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1. Introduction
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Tuberculosis (TB) is considered to be one of the deadly diseases in world. It’s an air borne
disease primarily infecting lungs caused by Mycobacterium tuberculosis (Mtb). Despite of
potent drugs and treatment regimen available, emergence of drug tolerant strains led to the report
of millions of deaths and new cases every year [30, 31]. As soon as the Mtb enters the body
bacteria will be phagocytized by macrophages initiating the host immune system [1]. If the
virulence of the bacteria is high Mtb can survive and replicate in host spreading to other organs
through lymphatic system [2, 3]. Cellwall contributes to a major part for the virulence nature of
Mtb. Synthetic mechanism of Mtb cellwall is complex and has been divided into three phases I,
II, III characterized by synthesis of peptidoglycan, lipid lined intermediates and polymerization
respectively. Majority of active drugs reported act on cellwall target phase III [4]. Pertaining to
the development of tolerance to these drugs there is an urgency to look for alternative valid
targets against Mtb. Phase I in cellwall formation was under exploited in drug research. Insertion
of D-glutamate is very essential for the peptidoglycan formation; glutamate racemase (GR) is
responsible for catalyzing racemization of L-glutamate (L-glu) to D-glutamate (D-glu). GR is an
important enzyme conserved in most of the bacterial species depicting the importance of the
enzyme for survivability of bacteria. There were reports for GR inhibitors against various
bacteria naming few like 8-benzyl pteridine-6,7-diones, pyridodiazepine amines acting against
Staphylococcus aureus and Helicobacter pylori respectively [4, 5].
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In this regard this co-factor independent enzyme can be exploited as a potent target in Mtb. We
have attempted to develop the inhibitors against GR by designing, synthesizing followed by valid
screening and evaluation of compounds resulting in active drug like molecules targeting GR.

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Designing of compounds started with the identification of lead compound from our in-house
database using one of the biophysical technique thermal shift assay.
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Results and Discussion
1.1. Lead identification by fluorescence thermal shift screening:
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Ligand screening using biophysical techniques has gained popularity in current research owing
to their broad range of applicability and functional relevance [6]. The fluorescence thermal shift
(FTS) assay is a technique that improves the outcome of novel drug pipeline and provides a fast
and reliable platform for screening and further identifying ligands in protein function or drug
discovery domain[20]. In this study, we have screened our in-house ligand library of 650
molecules with structural diversity. We conducted the assay with purified Mtb glutamate
racemase enzyme with substrate D-glu in bound and free forms against test compounds. Melt
curve of protein in its free state has shown Tm at ~43.6 oC and in complex with D-glu at ~44.8oC.
Screened compounds showed a varied range
∆
Tm of which lead 1 compound has shown Tm at
2.0 oC with protein bound to
~46.8 oC, which is showing a 3.2 oC with unbound protein and
∆
∆
D-glu. This result shows the competence of compound lead 1 in stabilizing the protein from rest
of the library and showing more chances for considering it as a lead compound.
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Recent reports have stated that obtaining pure form of recombinant Mtb GR enzyme with good
catalytic activity wasn’t successful even by trying with different optimal purification procedures
[8]. Therefore we have considered performing activity assay on same enzyme from different
bacteria having more similarities with Mtb in terms of genetic makeup. Following the recent
literature, we have considered Bacillus subtilis (Bsb) having 40% and 56% of respective
sequence identity and similarity with Mtb and carried out the enzyme inhibitory assay for library

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compounds screened by FTS method [9]. Compound Lead 1 has shown an IC50 of 20.7±0.29
µM against Bsb glutamate racemase (Fig.1). In both FTS and inhibitory assay compound Lead 1
has shown good activity, hence it was taken as a lead molecule and further optimized through
structural modifications using chemical procedures.
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1.2. Chemistry:
The target molecules were synthesized by two step synthetic protocol (Scheme 1); in first
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step 4-substituted-2-aminophenols (1a-b) were treated with 3-aminobenzoicacid (2) to produce
corresponding substituted cyclized compounds 3a-b (3-(benzo[d]oxazol-2-yl)aniline). In next
step, the primary amine group of phenyl ring was reacted with various substituted
arylisocyanates using ethanol as solvent to produce N, N-substituted urea derivatives (4-16); but
using similar conditions for the preparation of N, N-substituted thiourea derivatives, the
reactions were not successful, then we employed K2CO3 as base and DMF/xylene as solvents to
produce N, N-substituted thiourea derivatives (17-28). All the analytical data (1H NMR, 13C
NMR, and mass spectra) of synthesized compounds (both intermediates and finals) were on par
with designed structures. A total twenty four compounds were synthesized as represented in
(Table 1).
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Enzyme activity assay:
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Enzyme activity analysis was performed for the twenty seven compounds synthesized during hit
expansion. The recombinant gene from Bsb was cloned, expressed and purified as described in
experimental section, using pET28a+plasmid and BL21 cells as expression vector followed by
purification and confirmation through SDS-PAGE. A reaction mixture containing glutamate
racemase, D-glu and test compounds was incubated and the product resulted (L-glu) was reacted

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with L-Glutamate dehydrogenase (GDH) and NAD+ where the inhibitory activity was
determined by employing an coupled assay by measuring the absorption readings of NADH
formed after GDH mediated conversion from NAD+ at λ340. The IC50 values of compounds were
shown in Table 1. A total of thirteen compounds have shown activity less than 25 µM, of which
except two compounds rest were active below 10 µM concentration. Compounds 16, 19, 21 and
22 have shown activity <5 µM. Most of the compounds showing good activity have thiourea
substitution, out of twelve compounds belonging to thiourea group, eight compounds have
shown less than 10 µM activity and one has shown activity of 22.8±0.42 µM. Most potent of the
series, compound 22 showed an IC50 of 1.1±0.52 µM which is less inhibitory concentration than
lead compound (IC50 of 20.7±0.29 µM) by twenty times. The log dose response curve of most
potent compound 22 of series was shown in (Fig. 2).
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Molecular docking and dynamic studies:
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Molecular docking is one technique which gives the all the possible interactions of ligand
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with the protein and suggests the possibility of a compound to accept as an inhibitor. In this
study we have performed the inhibitory activity on glutamate racemase of Bsb, stillthere is
requirement for further validation of results. We performed all the docking analysis using
Schrodinger software. We have selected substrate (D-glu) bound glutamate racemase crystal
structures of both organisms from protein data bank namely PDB ID: 5HJ7 (Mtb) and 1ZUW
(Bsb), the protein alignment of both proteins has an root mean square deviation (rmsd) 1.33 Å.
We tried docking of energy minimized ligands into substrate binding site but was unsuccessful,
hence we have tried docking in allosteric sites (assuming the inhibitors act through allosteric site)
generated using an in-built program called SiteMap. SiteMap is considered to be a tool that helps
in interpreting the additional important sites of the protein which help in increasing the

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probability of finding the drugs in drug research areas. In the present study, five sites were
identified in the each crystal structures of proteins. Among all generated sites considering the
site-scores, site 1 in both the proteins was selected for further docking studies. Site 1 of Mtb has
the site-score 0.8203 and was surrounded by hydrophobic residues namely Pro43, Pro46, Pro146,
Val149 and Leu250 and few polar amino acid residues Glu 153, Ser13, Thr39, Thr119 and
His187. In Bsb site 1 having site-score 0.6982 was boarded by hydrophobic residues Pro41,
Pro44, Pro146, Val149 and Cys40 and also by polar residues like Thr37, Thr118 and Gln245
respectively. The allosteric sites considered in both the bacteria are lined by the identical
residues (Supplementary Fig. 1). Therefore the compounds show similar kind of action in both
the bacteria binding in these sites.
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Compounds were docked into allosteric sites of both the crystal structures. We have
superimposed both the proteins bound with ligand 22 using superimposition module in
Schrodinger shown in Fig. 3. In this study, we have studied the docking orientation of most
active compound 22 in both the crystal structures. The binding orientation showed a two H-bond
interactions with allosteric residue Glu153 in both the crystal structures with NH moiety of
compound (Fig. 4). Along with previous interactions, the orientation was stabilized by
hydrophobic and polar interactions. The compound was very well fit into the cavity of both
proteins retaining similar kind of interaction with docking scores of -5.075 kcal/mol and -3.457
kcal/mol respectively in Bsb and Mtb. Hence we infer that there established a correlation of
inhibitory activity of compound 22 in both the organisms.
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Molecular dynamics simulations help in understanding the structure and function of
macromolecules at basic level. Simulations provide every minute detail about every
particle/atom movement with respect to time. This will help in answering the questions arising

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on deviation and fluctuation pattern of protein. [We have subjected the Compound 22 bound in
5HJ7 and 1ZUW protein complexes to a 10 ns simulation using Desmond. The deviation and
fluctuation patterns were measured in terms of rmsd and rmsf (root mean square fluctuation).
Rmsd of protein (Cᾳ) and ligand obtained during 10 ns simulation for the 1ZUW crystal structure
were within average of ~1.21 Å and ~0.9 Å and for 5HJ7 both Cᾳ and ligand were within an
average of ~1.35 Å and ~0.82 Å respectively (Supplementary Fig. 2). Rmsf analysis inferred
that proteins and ligand at their binding site have shown negligible fluctuations with ~1.36 Å and
1.41 Å respectively for 5HJ7 and 1ZUW (Supplementary Fig. 3).
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Thermal shift assay:
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FTS is known majorly for establishing the stability data of a protein. Based on the principle
involved, this tool can also be used to characterize the compound or ligand and its effects on
protein by studying the shift in melt temperature (∆Tm) in the presence of different ligands. A
positive shift in (Tm) in comparison with protein in its free state (unbound) indicates the stability
enhancement of protein and a negative shift indicates the destabilization of protein. Based on this
principle, mode of inhibition can be categorized into three [22]. First is competitive mode of
inhibition, where the inhibitor induces a negative shift in Tm. Second, uncompetitive inhibition
showing a positive
∆Tm only in the presence of substrate-bound state and in the absence of
substrate no change is recorded. Third is the non-competitive way of inhibition, here a positive
shift in melt temperature will be seen irrespective of substrate existence in protein [22].
We have analysed inhibitor characterization in Mtb glutamate racemase. The thermal shift
analysis of active compound 22 shows a clear non-competitive mode of inhibition. The protein
unbound form showed Tm ~43.7 oC and bound form at ~45.1 oC respectively. The compound 22
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showed Tm ~49.8 C with unbound protein and Tm ~51.9 C with protein bound with substrate

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(Fig. 5). These melt temperature shifts (
∆
Tm) were higher compared to that of lead 1 (reported
above). This infers that the protein and protein-substrate complex were more stabilized in the
presence of compound 22 when compared to lead 1.
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1.6. Mtb susceptibility testing:
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Determining whole cell activity against Mtb is an important factor in drug development research
against tuberculosis. Microplate Alamar Blue Assay (MABA) is an economic and expeditious
assay determining the drug susceptibility against replicating Mtb in quantitative terms [23].
MABA assay was performed for all the synthesized compounds to determine the minimum
inhibitory concentration (MIC) of each compound when tested at concentrations varying from 50
µ
g/mL to 0.78
Ethambutol and Rifampicin) were represented in Table 1. Compounds 17, 18, 19, 22 and 23
having thiourea substitution, have shown inhibition below 10 M. Of which four compounds
(17, 18, 19 and 23) were more active than standard drug Ethambutol (MIC 7.89 M).
µg/mL. MIC values of the test compounds and the standard drugs (Isoniazid,
µ
µ
Compounds 17, 18, 23 have shown more whole cell inhibition compared to that of enzyme
inhibition. To our assumption, this conjugate effect might be due to an additional pathway
inhibition. Few compounds in spite of having good activity profile in enzyme assay failed to
inhibit the whole organism, where the reason could be the efflux of compound by the complex
cellwall of bacteria. Efflux mechanism of bacteria is the major hurdle for drug susceptibility
mediated by major facilitator superfamily (MFS) proteins or antibiotic-modifying, degrading
enzymes and transporters [26]. Reports were available stating that combining efflux pump
inhibitors verapamil and piperine has increased the intake of drug by the bacteria than in their
absence [28, 29]. Assuming that the failure of compounds in replicating bacteria might be due to
efflux of drug, the compounds 4, 6, 13, 14, 16, 20, 21, 22 and 24 active in enzyme assay were

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tested for their susceptibility in the presence of verapamil and piperine. Compounds 16, 20, 21
and 22 have shown drastic increase in activity against Mtb than as individual drugs (Table 1).
The remaining compounds haven’t showed any difference in their activities and the reason might
be the lack of permeability of compounds across the complex lipid bacterial cellwall. The most
active molecule in enzyme assay, compound 22 (IC50 of 1.1±0.52
8.72 M in the absence of efflux pump inhibitors and 2.01 M with a difference of four times in
the presence of efflux pump inhibitors.
Mycobacterium tuberculosis nutrient starved dormancy model:
µM) has shown an MIC of
µ
µ
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Mtb attains dormancy and phenotypically develop drug resistance when it undergoes stress
within the host. Inability of currently available and developing drugs to kill persistent Mtb is a
major hindrance to find therapeutic possibility to diminish tuberculosis (TB). An in vitro stress
model that generates dormancy in Mtb cells is required to reveal the essential metabolics that
allow bacteria to undergo into latent phase in turn help in identification and screening of novel
drugs to kill persistent bacteria. Nutrient starvation is one such model which creates the
dormancy in bacteria by starving culture in phosphate buffer saline (PBS) for 6 weeks. The
dormant culture was tested with selected compounds at concentration of 10 µg/mL [14]. Based
on enzyme and MABA assay activities, compounds 17, 18, 19 and 22 were selected to test
against dormant assays. Rifampicin (RIF), Isoniazid (INH) and Moxifloxacin (MOXI) were used
as reference compounds. The comparative inhibitory activity plots in nutrient starvation model
were shown in Fig. 6. Compounds 17, 18 and 22 have shown a similar inhibition with a log
reduction of 2.5 (with control), 1.4 (with INH), 0.8 (with RIF) and 0.3 with (MOXI). Compound
19 has shown a difference of 0.2 log reduction when compared to other test compounds. Test

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compounds have shown similar inhibition with less variation with good activities in active and
dormant model.
1.8. Mtb time-Kill Kinetics determination:
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It’s essential to study the pharmacodynamics of novel drug molecules by analyzing the kill of
drug molecule at different concentrations against time. This aids in determining the
concentration-dependent and time-dependent bactericidal activities of drug molecules. Minimum
bactericidal concentration (MBC) is defined as the concentration at which inhibitor shows three
fold inhibition of bacteria, incubated for 21 days. To determine MBC, the compound at different
concentrations (decided according to MIC value) was incubated for 21 days and recorded the
readings at different time points (0, 7, 15 and 21 days). A compound is said to be bactericidal if
MBC is within 4 folds of its MIC and bacteriostatic if it’s more than four folds [15]. Bactericidal
compounds exhibit two types of kill namely time dependant and concentration dependant. We
have used starved culture (depleted of nutrients for 2 weeks) for assay. Compounds 17, 18, 19
and 22 were tested for determining their kill kinetics. Compounds 19 and 22 have shown
bactericidal mode of inhibition with their MBC values 27.47 µM and 25.38 µM respectively. We
have represented the kinetics study of compound 22 in Fig. 7. It shows a time dependant kill,
where one can observe the kill increased with time irrespective of concentration.
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1.9. Mtb biofilm assay:
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Mtb forms organized biofilm containing mycolic-acid-rich extracellular matrix. Assessment of
Mtb biofilm cultures to available potent antibiotics (INH and RIF) resulted in the survival of
cells confirming that these biofilms harbor the drug-tolerant cells [24]. Compounds 17, 18, 19
and 22 were tested for their activity on biofilm at concentration of10 µg/mL. Reference drugs

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INH, RIF and MOXI have shown a log reduction of 1.2, 1.5 and 2.0 respectively compared to
control. Test compounds 17, 18 have shown 0.8 and compounds 19 and 22 have shown log
reduction of 0.4 and 2.4 respectively (Fig. 8). These values infer that compound 22 is active in
both active and dormant forms of bacteria.
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1.10. In-vivo activity testing assay by Mycobacterium marinum infected adult zebrafish:
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Identification of novel antimicrobial compounds can be accelerated using the zebrafish model,
especially in tuberculosis. Activity and dosage of antibiotics in zebrafish closely be like
characteristics in humans. This model has emerged to be the most economic and reliable model
for researches to study the in vivo activity of new drug compounds [25]. In this study, zebrafish
is infected with Mycobacterium marinum (belongs to same family genetically). Active molecule
22 at in vitro level was tested for in vivo inhibitory activity using adult zebra fish model.
Standard compounds INH and MOXI have shown reductions of 2.5 and 2.4 in log scale and test
compound 22 has shown 3.4 log reduction with control, which is more than standards (Fig. 9).
1.11. Cytotoxicity determination:
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Toxicity testing of new compounds is very much essential for drug development process, can be
observed by in vivo and in vitro models. In this study, we have tested toxicity using in vitro
technique employing cell lines by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
Bromide) assay. RAW 246.7 cell lines were used for experimental studies [18]. All the
compounds were tested at a concentration of 25 µM and the percentage inhibition were found to
be in different ranges as shown in table 1. Hence most of the compounds are devoid of effects on
metabolism.
2. Conclusion

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In the current scenario persistence, resistance and co-infection are the foremost drawbacks that
hinder the tuberculosis treatment regimen. In present work, we have focused on combating
persistence and resistance by targeting a novel enzyme vital for the bacterial survival. We have
we have identified and optimized through hit expansion. Compounds which showed good in
vitro and in vivo activities belong to thiourea family. Compound 22 (1-(4-chlorophenyl)-3-(3-(5-
methylbenzo[d]oxazol-2-yl)phenyl)thiourea) was proven to be the most potent molecule of series
in both in vitro and in vivo evaluation studies. This data was supported by molecular docking and
simulation studies, both the compounds retained similar kind of interactions with amino acid
residues. There were some compounds which are active in vitro but were unsuccessful on whole
cell organism, one of the drawback can be the strong efflux nature of cell wall. Further, there
were few compounds showing very food whole organism inhibition than enzyme inhibition,
these might be targeting an additional pathway for inhibition. These compounds need further
studies to answer all questions in drug discovery. Further optimization of compounds can results
in still better compounds for treating tuberculosis.
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3. Experimental Section
3.1. Thermal shift assay:
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Thermal stability assays were performed as reported in literature [7]. The reaction was carried
out in 0.2 mL PCR tubes (Bio-Rad) containing a 25 µL mixture of Mtb glutamate racemase
(0.2 mg/mL concentration), test compounds (diluted in DMSO < 2 %v/v), 2 mM D-glutamate
and buffer (100 mM Tris HCl, 100 mM NaCl, pH 8.0) and 6.25x SYPRO orange dye (Sigma).
The reaction was using real-time PCR thermal cycler (Bio-Rad). The protocol was set with
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heating from 20 to 80 C with a gradient of 0.02 C/s. All the reactions were carried in
duplicates. Changes in fluorescence of SYPRO orange dye were monitored, to determine the

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unfolding pattern of protein. Tm values were obtained from the minima of the first derivative
(−dF/dt) plots of unfolding protein curves using an in-built function in Bio-Rad prime PCR
software.
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3.2. Chemistry :
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Reagents obtained from commercial sources were used directly without further purification.
Reactions were carried out under inert atmosphere of nitrogen or argon. All the reactions were
monitored by thin layer chromatography (TLC) on silica gel 40 F254 (Merck, Darmstadt,
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Germany) coated on aluminium plates. H and C NMR spectra were recorded on a Bruker
AM-400 NMR spectrometer, Bruker BioSpin Corp., Germany. Chemical shifts are in parts per
million (ppm) using tetramethylsilane as internal standard. Temperatures are reported in degrees
Celsius and are uncorrected. Compounds were analysed for C, H, N and analytical results
obtained were within ±0.4% of the calculated values for the formula shown. Molecular weights
of the synthesised compounds were checked by (Shimadzu, LCMS-2020) ESI-MS method.
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3.2.1. General procedure for the preparation of 3a-b: 3-amiobenzoicacid (2) (1.0 equiv),
compound 1a-b (1.1 equiv) and PPA (10 volumes) mixture was allowed to reflux at 185 °C for
6 hours. After the completion of reaction, the contents were cooled and neutralized with cold
6N NaOH solution. The precipitate formed was filtered and further continued for heating in
DMF at 60 °C for 15 minutes, and contents were transferred into crushed ice. The solid was
filtered and washed with cold ethanol and diethyl ether to get the corresponding recrystallized
compound 3a-b respectively.
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3.2.2. Synthesis of 3-(benzo[d]oxazol-2-yl)aniline
(3a): The compound was synthesized
according to above general procedure using 2-aminophenol (1 g, 9.17 mmol), 3-

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amiobenzoicacid (0.9 g, 6.84 mmol) and PPA (10 mL) to obtain 3-(benzo[d]oxazol-2-yl)aniline
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(1.6 g,73%) as pale yellow solid. H NMR (400 MHz, CDCl3):
δ
8.29 (s, 1H), 8.11-7.89 (m,
163.3, 151.3 (2C), 143.6,
3H), 7.58-7.39 (m, 4H), 6.87 (s, 2H). 13C NMR (100 MHz, CDCl3):
δ
132.7, 128.6, 124.9 (2C), 119.5 (3C), 116.3, 109.9. MS(ESI) m/z 211 [M+H]+. Anal calcd for
C13H10N2O: C, 74.27; H, 4.79; N, 13.33; Found: C, 74.34; H, 4.78; N, 13.35.
3.2.3. Synthesis of 3-(5-methylbenzo[d]oxazol-2-yl)aniline
(3b): The compound was
synthesized according to above general procedure using 2-amino-4-methylphenol (1g,
8.13mmol), 3-amiobenzoicacid (0.9 g, 6.84 mmol) and PPA (10 mL) to obtain 3-(5-
1
methylbenzo[d]oxazol-2-yl)aniline (1.8 g,71%) as white solid. H NMR (400 MHz, CDCl3):
δ
13
8.42 (s, 1H), 8.22 (s, 1H), 7.92-7.52 (m, 5H), 6.94 (s, 2H), 2.45 (s, 3H). C NMR (100 MHz,
CDCl3):
δ 162.9, 149.2, 145.6, 140.8, 135.2, 132.7, 127.6 (2C), 118.5 (3C), 113.8, 108.7, 22.3.
MS(ESI) m/z 225 [M+H]+. Anal calcd for C14H12N2O: C, 74.98; H, 5.39; N, 12.49; Found: C,
74.82; H, 5.38; N, 12.47.
3.2.4. General procedure for the preparation of compounds 4-15: To the mixture of
compounds 3a-b (1.0 equiv), DIPEA (2.0 equiv) was added with the corresponding
arylisocyanate (1.25 equiv) in DMF. After the reaction has completed, mixture was added to
EtOAc followed by washing with water, Brine solution and distilled under reduced pressure to
obtain product. The crude product was purified by column chromatography using
EtOAc/hexane as eluent to obtain pure compounds 4-15.
3.2.5. Synthesis of 1-(3-(5-methylbenzo[d]oxazol-2-yl)phenyl)-3-phenylurea
(4): The
compound was synthesized according to above general procedure using 3-(5-
methylbenzo[d]oxazol-2-yl)aniline (1.5 g, 6.69 mmol), DIPEA (1. 8 g, 12.62 mmol) and
isocyanatobenzene (1.2 g, 10.05 mmol) to yield 1-(3-(5-methylbenzo[d]xazol-2-yl)phenyl)-3-

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phenylurea (1.8 g, 77%) as grey solid. Mp: 240-243 C. H NMR (400 MHz, CDCl3): δ 10.87
(s, 2H), 8.31 (s, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.84–7.72 (m, 4H), 7.63 (d, J = 7.2 Hz, 2H),
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7.58–7.49 (m, 4H), 2.39 (s, 3H); C NMR (100 MHz, CDCl3):
δ 174.6, 160.2, 144.9, 141.4,
140.6, 135.2, 135.0, 133.3, 132.4(2C), 130.8, 129.4, 127.8(2C), 125.9, 125.6, 125.2, 124.6,
120.3, 116.3, 22.9. MS(ESI) m/z 344 [M+H]+. Anal calcd for C21H17N3O2: C, 73.45; H, 4.99; N,
12.24% Found C, 73.58; H, 4.98; N, 12.26%.
3.2.6. Synthesis of 1-(4-chlorophenyl)-3-(3-(5-methylbenzo[d]oxazol-2-yl)phenyl)urea
(5):
The compound was synthesized according to above general procedure using 3-(5-
methylbenzo[d]oxazol-2-yl)aniline (1.5 g, 6.69 mmol), DIPEA (1.8 g, 12.62 mmol) and 1-
chloro-4-isocyanatobenzene (1.3 g, 8.51 mmol) to yield 1-(4-chlorophenyl)-3-(3-(5-
o
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methylbenzo[d]oxazol-2-yl)phenyl)urea (1.9 g, 73%) as white solid. M.p:181-183 C. H NMR
(400 MHz, DMSO-d6):
δ
9.34 (s, 2H), 8.52 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.8 (d, J = 8.4 Hz,
13
1H), 7.75 (t, J = 8.0 Hz, 1H), 7.71–7.62 (m, 5H), 7.43 (d, J = 8.0 Hz, 2H), 3.82 (s, 3H); C
NMR (100 MHz, DMSO-d6):
δ 176.3, 163.3, 154.3, 146.4, 139.6, 137.5, 135.6, 134.8, 133.2,
132.0(2C), 131.8, 131.3, 129.9(2C), 128.6, 127.4, 126.0, 120.4, 113.8, 60.4, 23.2. MS(ESI) m/z
379 [M+H]+. Anal calcd for C21H16ClN3O2: C, 70.76; H, 5.13; N, 11.25 Found C, 70.88; H,
5.14; N, 11.23.
3.2.7. Synthesis of 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(4-methoxyphenyl)urea
(6): The
compound was synthesized according to above general procedure using 3-(benzo[d]oxazol-2-
yl)aniline (1.3 g, 6.19 mmol), DIPEA (1.8 g, 12.62 mmol) and 1-isocyanato-4-methoxybenzene
(1.2 g, 8.02 mmol) to yield 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(4-methoxyphenyl)urea (1.9 g,
86%) as grey solid. M.p:228-230 oC. H NMR (400 MHz, DMSO-d6):
δ 10.25 (s, 2H), 8.40 (s,
1
1H), 7.89 (d, J = 8.0 Hz, 2H), 7.76–7.63 (m, 3H), 7.56–7.48 (m, 4H), 7.18 (t, J = 7.6 Hz, 2H),

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3.96 (s, 3H); 13C NMR (100 MHz, DMSO-d6):
δ
172.4, 162.4, 150.2, 145.8, 141.3, 138.2,
136.4, 135.1, 134.6, 134.1(2C), 133.2, 131.4, 128.2(2C), 126.4(2C), 125.3, 124.2, 119.4, 114.6.
MS(ESI) m/z 360 [M+H]+. Anal calcd for C21H17N3O3: C, 70.18; H, 4.77; N, 11.69 Found C,
70.28; H, 4.76; N, 11.66.
3.2.8. Synthesis of 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(4-nitrophenyl)urea (7): The
compound was synthesized according to above general procedure using 3-(benzo[d]oxazol-2-
yl)aniline (1.3 g, 6.19 mmol), DIPEA (1.8 g, 12.62 mmol) and 1-isocyanato-4-nitrobenzene
(1.3 g, 7.98 mmol) to yield 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(4-nitrophenyl)urea (1.9 g,
o
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84%) as yellow solid. M.p:250-252 C. H NMR (400 MHz, DMSO-d6):
δ 10.08 (s, 2H), 8.45
(s, 1H), 8.19 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 8.0 Hz, 2H), 7.88 (d, J = 7.6 Hz, 1H), 7.82 (d, J =
13
8.0 Hz, 2H), 7.78–7.71 (m, 3H), 7.69 (t, J = 7.6 Hz, 1H); C NMR (100 MHz, DMSO-d6):
δ
167.4, 156.3, 152.3, 146.6, 143.5, 139.3, 136.4, 134.8(2C), 133.2, 132.8(2C), 131.2, 130.5,
130.0, 128.4, 126.2, 125.9, 120.9, 116.7. MS(ESI) m/z 375 [M+H]+. Anal calcd for C20H14N4O4:
C, 64.17; H, 3.77; N, 14.97 Found C, 64.28; H, 3.78; N, 14.95.
3.2.9. Synthesis of 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-benzylurea (8): The compound was
synthesized according to above general procedure using 3-(benzo[d]oxazol-2-yl)aniline (1.3 g,
6.19 mmol), DIPEA (1.8 g, 12.62 mmol) and (isocyanatomethyl)benzene (1.1 g, 9.6 mmol) to
yield 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-benzylurea (2.0 g, 88%) as brown solid. M.p:243-
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245 C. H NMR (400 MHz, CDCl3):
δ 10.27 (s, 2H), 8.48 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H),
7.88 (t, J = 7.6 Hz, 2H), 7.72 (d, J = 7.6 Hz, 1H), 7.68–7.56 (m, 3H), 7.42–7.26 (m, 5H), 5.08
(s, 2H); 13C NMR (100 MHz, CDCl3):
δ
183.9, 163.4, 154.3, 144.3, 142.4, 137.9, 135.6, 135.1,
134.3(2C), 129.4, 128.7(2C), 127.1, 126.2, 125.4, 125.0, 124.3, 120.4, 113.8, 62.6. MS(ESI)

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m/z 360 [M+H]+. Anal calcd for C21H17N3O2: C, 73.45; H, 4.99; N, 12.24 Found C, 73.52; H,
4.98; N, 12.21.
3.2.10.
Synthesis
of
1-(4-methoxyphenyl)-3-(3-(5-methylbenzo[d]oxazol-2-
yl)phenyl)urea (9): The compound was synthesized according to above general procedure using
3-(5-methylbenzo[d]oxazol-2-yl)aniline (1.5 g, 6.69 mmol), DIPEA (1.8 g, 12.62 mmol) and 1-
isocyanato-4-methoxybenzene (1.5 g, 9.1 mmol) to yield 1-(4-methoxyphenyl)-3-(3-(5-
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methylbenzo[d]oxazol-2-yl)phenyl)urea (2.1 g, 87%) as grey solid. M.p:228-230 C. H NMR
(400 MHz, DMSO-d6): 9.25 (s, 2H), 8.44 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 8.4
δ
Hz, 1H), 7.72 (t, J = 8.0 Hz, 1H), 7.67–7.60 (m, 5H), 7.21 (d, J = 7.6 Hz, 2H), 3.94 (s, 3H), 2.46
13
(s, 3H); C NMR (100 MHz, DMSO-d6):
δ 176.3, 163.3, 154.3, 146.4, 139.6, 137.5, 135.6,
134.8, 133.2, 132.0(2C), 131.8, 131.3, 129.9(2C), 128.6, 127.4, 126.0, 120.4, 113.8, 60.4, 23.2.
MS(ESI) m/z 374 [M+H]+. Anal calcd for C22H19N3O3: C, 70.76; H, 5.13; N, 11.25 Found C,
70.88; H, 5.14; N, 11.28.
3.2.11.
Synthesis of 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(4-chlorophenyl)urea (10):
The compound was synthesized according to above general procedure using 3-(benzo[d]oxazol-
2-yl)aniline (1.3 g, 6.19 mmol), DIPEA (1.8 g, 12.62 mmol) and 1-chloro-4-isocyanatobenzene
(1.3 g, 8.51 mmol) to yield 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(4-chlorophenyl)urea (1,9 g,
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85%) as white solid. M.p:175-177 C. H NMR (400 MHz, CDCl3):
δ 10.29 (s, 2H), 8.46 (s,
1H), 7.94 (t, J = 8.0 Hz, 2H), 7.82 (d, J = 7.6 Hz, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.69–7.60 (m,
3H), 7.56 (d, J = 7.6 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H); 13C NMR (100 MHz, CDCl3):
δ
174.4,
160.5, 148.7, 144.9, 137.5, 136.4, 135.3, 134.9, 134.3, 133.2(2C), 132.7, 131.6, 130.2(2C),
127.3, 125.2(2C), 120.6, 117.0. MS(ESI) m/z 364 [M+H]+. Anal calcd for C20H14ClN3O2: C,
66.03; H, 3.88; N, 11.55 Found C, 66.18; H, 3.89; N, 11.53.

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3.2.12.
Synthesis of 1-(4-fluorophenyl)-3-(3-(5-methylbenzo[d]oxazol-2-yl)phenyl)urea
(11): The compound was synthesized according to above general procedure using 3-(5-
methylbenzo[d]oxazol-2-yl)aniline (1.5 g, 6.69 mmol), DIPEA (1.8 g, 12.62 mmol) and 1-
fluoro-4-isocyanatobenzene (1.2g, 9.2 mmol) to yield 1-(4-fluorophenyl)-3-(3-(5-
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methylbenzo[d]oxazol-2-yl)phenyl)urea (2.0 g, 88%) as pale brown solid. M.p:169-171 C. H
NMR (400 MHz, DMSO-d6):
δ 10.71 (s, 2H), 8.46 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.74–7.62
(m, 3H), 7.56 (d, J = 7.6 Hz, 1H), 7.54–7.46 (m, 3H), 7.36 (d, J = 8.0 Hz, 2H), 2.43 (s, 3H); 13C
NMR (100 MHz, DMSO-d6):
δ 174.3, 160.4, 144.7, 143.4, 139.6, 137.3, 136.2, 134.6, 133.0,
132.6(2C), 131.8, 128.5, 127.4(2C), 126.8, 125.2, 123.9, 120.8, 116.9, 22.4. MS(ESI) m/z 362
[M+H]+. Anal calcd for C21H16FN3O2: C, 69.80; H, 4.46; N, 11.63 Found C, 69.88; H, 4.45; N,
11.65.
3.2.13.
Synthesis of 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-phenylurea
(12): The
compound was synthesized according to above general procedure using 3-(benzo[d]oxazol-2-
yl)aniline (1.3 g, 6.19 mmol), DIPEA (1.8 g, 12.62 mmol) and isocyanatobenzene (1.2 g, 10.05
mmol) to yield 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-phenylurea (1.7 g, 83%) as grey solid.
M.p:210-212 oC. 1H NMR (400 MHz, DMSO-d6):
δ 10.26 (s, 2H), 8.46 (s, 1H), 7.96 (d, J = 8.0
Hz, 1H), 7.84 (t, J = 8.0 Hz, 2H), 7.72 (d, J = 7.6 Hz, 1H), 7.63–7.56 (m, 3H), 7.48–7.36 (m,
13
4H), 7.22 (t, J = 8.0 Hz, 1H); C NMR (100 MHz, DMSO-d6):
δ 176.4, 162.9, 154.2, 144.7,
142.6, 142.4, 138.5, 137.6, 136.0, 133.9(2C), 133.0, 130.6, 128.3, 127.8(2C), 126.3, 120.3,
119.3, 115.9. MS(ESI) m/z 330 [M+H]+. Anal calcd for C20H15N3O2: C, 72.94; H, 4.59; N,
12.76 Found C, 72.81; H, 4.58; N, 12.78.
3.2.14.
Synthesis of 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(4-fluorophenyl)urea (13):
The compound was synthesized according to above general procedure using 3-(benzo[d]oxazol-

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2-yl)aniline (1.3 g, 6.19 mmol), DIPEA (1.8 g, 12.62 mmol) and fluoro-4-isocyanatobenzene
(1.0g, 8.7 mmol) to yield 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(4-fluorophenyl)urea (1.8 g,
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81%) as grey solid. M.p:180-182 C. H NMR (400 MHz, DMSO-d6):
δ
9.00 (s, 1H), 8.73 (s,
13
1H), 8.12 (s, 1H), 8.84–8.73 (m, 3H), 7.54–7.36 (m, 6H), 7.12 (t, J = 8.0 Hz, 2H); C NMR
(100 MHz, DMSO-d6):
δ 178.6,156.8, 152.1, 144.2, 143.1, 136.5, 131.2, 127.9, 127.4, 126.1,
123.1, 122.7, 122.4 (3C), 120.5, 115.4, 113.2 (3C), 111.2 . MS(ESI) m/z 348 [M+H]+. Anal
calcd for C20H14FN3O2: C, 69.16; H, 4.06; N, 12.10 Found C, 69.24; H, 4.07; N, 12.08.
3.2.15.
Synthesis of 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(p-tolyl)urea
(14): The
compound was synthesized according to above general procedure using 3-(benzo[d]oxazol-2-
yl)aniline (1.3 g, 6.19 mmol), DIPEA (1.8 g, 12.62 mmol) and 1-isocyanato-4-methylbenzene
(1.2 g, 9.0 mmol) to yield 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(p-tolyl)urea (1.9 g, 88%) as
white solid. M.p:164-166 oC. 1H NMR (400 MHz, DMSO-d6):
δ 9.21 (s, 2H), 8.41 (s, 1H), 7.89
(d, J = 8.0 Hz, 1H), 7.81–7.72 (m, 2H), 7.67–7.56 (m, 3H), 7.54–7.45 (m, 3H), 7.33 (t, J = 8.0
13
Hz, 2H), 2.46 (s, 3H); C NMR (100 MHz, DMSO-d6):
δ 174.6, 162.6, 154.0, 146.5, 136.4,
135.3, 134.8, 134.0, 133.4, 132.3(2C), 130.5, 129.4, 128.2(2C), 126.8, 126.4, 124.5, 121.3,
117.4, 22.6. MS(ESI) m/z 344 [M+H]+. Anal calcd for C21H17N3O2: C, 73.45; H, 4.99; N, 12.24
Found C, 73.56; H, 4.98; N, 12.26.
3.2.16.
Synthesis of 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-isopropylurea
(15): The
compound was synthesized according to above general procedure using 3-(benzo[d]oxazol-2-
yl)aniline (1.3 g, 6.19 mmol), DIPEA (1.8 g, 12.62 mmol) and 2-isocyanatopropane (0.7 g, 8.2
mmol) to yield 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-isopropylurea (1.5 g, 78%) as pale red
o
solid. M.p:174-176 C. 1H NMR (400 MHz, DMSO-d6):
δ 10.17 (s, 1H), 9.43 (s, 1H), 8.36 (s,
1H), 7.84 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.67–7.56 (m, 3H), 7.36 (t, J = 8.0 Hz,

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1H), 3.85–3.81 (m, 1H), 1.33 (d, J = 8.8 Hz, 6H); C NMR (100 MHz, DMSO-d6):
δ
171.9,
160.2, 154.4, 143.4, 136.4, 135.3, 135.0, 133.3, 129.3, 127.6, 126.1, 125.4, 118.6, 115.3, 54.6,
24.6(2C). MS(ESI) m/z 296 [M+H]+. Anal calcd for C17H17N3O2: C, 69.14; H, 5.80; N, 14.23
Found C, 69.26; H, 5.81; N, 14.25.
3.2.17.
General procedure for the preparation of compounds 16-27: To a solution of
compounds 3a-b (1.0 equiv) and the corresponding arylisothiocyanate (1.3 equiv) in
DMF:xylene (4:1), potassium carbonate (2.0 equiv) was added with stirring. The whole reaction
was carried out under microwave irradiation conditions (temperature 110 °C) for about 30
minutes. The contents of flask were poured into the crushed ice for cooling and the resulted
precipitate was filtered and washed twice with ethanol. The crude residue was further purified
by column chromatography using ethylacetate/hexane as eluent to get the corresponding pure
compounds 16-27.
3.2.18.
Synthesis of 1-(3-(Benzo[d]oxazol-2-yl)phenyl)-3-benzylthiourea (16): The
compound was synthesized according to above general procedure using 3-(benzo[d]oxazol-2-
yl)aniline (1.3 g, 6.19 mmol), K2CO3 (2.0 g, 14.1 mmol) and (isothiocyanatomethyl)benzene (
1.2 g, 8.0 mmol) in DMF: xylene (4:1) to get 1-(3-(Benzo[d]oxazol-2-yl)phenyl)-3-
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benzylthiourea (1.9 g, 84%) as pale grey solid. M.p:210-212 C. H NMR (400 MHz, DMSO-
d6): 9.27 (s, 1H), 9.01 (s, 1H), 8.44 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.82 (t, J = 7.6 Hz, 2H),
δ
7.68 (d, J = 8.0 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.42–7.31 (m, 4H),
13
7.12 (t, J = 7.2 Hz, 1H), 5.04 (s, 2H); C NMR (100 MHz, DMSO-d6):
δ 180.9, 166.8, 156.1,
144.3, 138.3, 136.2, 134.5, 134.0, 133.2(2C), 128.3, 127.4(2C), 125.3(2C), 125.1, 124.7, 123.6,
117.3, 112.5, 62.4. MS(ESI) m/z 360 [M+H]+. Anal calcd for C21H17N3OS: C, 70.17; H, 4.77;
N, 11.69%Found C, 70.28; H, 4.76; N, 11.71.

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3.2.19.
Synthesis
of
1-(4-Fluorophenyl)-3-(3-(5-methylbenzo[d]oxazol-2-
yl)phenyl)thiourea
(17: The compound was synthesized according to above general procedure
using 3-(5-methylbenzo[d]oxazol-2-yl)aniline (1.5 g, 6.69 mmol), K2CO3 (2.0 g, 14.1 mmol)
and 1-fluoro-4-isothiocyanatobenzene ( 1.2 g, 7.8 mmol) in DMF: xylene (4:1) to get 1-(4-
Fluorophenyl)-3-(3-(5-methylbenzo[d]oxazol-2-yl)phenyl)thiourea (2.1 g, 81%) as white solid.
M.p:235-237 oC. H NMR (400 MHz, DMSO-d6):
δ 10.27 (s, 2H), 8.49 (s, 1H), 7.84–7.74 (m,
1
3H), 7.62–7.44 (m, 5H), 7.09 (t, J = 7.6 Hz, 2H), 2.42 (s, 3H); 13C NMR (100 MHz, DMSO-d6):
182.4, 164.5, 160.2, 146.1, 142.3, 138.5, 135.6, 133.2, 131.4(2C), 130.9, 130.2, 128.2, 126.9,
δ
125.6(2C), 124.3, 123.1, 118.4, 113.6, 22.5. MS(ESI) m/z 378 [M+H]+. Anal calcd for
C21H16FN3OS: C, 66.83; H, 4.27; F, 5.03; N, 11.13 Found C, 66.90; H, 4.28; N, 11.15.
3.2.20.
Synthesis of 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(4-nitrophenyl)thiourea (18):
The compound was synthesized according to above general procedure using 3-(5-
methylbenzo[d]oxazol-2-yl)aniline (1.5 g, 6.69 mmol), K2CO3 (2.0 g, 14.1 mmol) and 1-
isothiocyanato-4-nitrobenzene ( 1.2 g, 8.0 mmol) in DMF: xylene (4:1) to get 1-(3-
(benzo[d]oxazol-2-yl)phenyl)-3-(4-nitrophenyl)thiourea (2.1 g, 85%) as yellow solid. M.p:236-
238 oC. H NMR (400 MHz, DMSO-d6):
δ
10.07 (s, 2H), 8.48 (s, 1H), 8.05 (d, J = 8.0 Hz, 2H),
1
7.92 (d, J = 7.6 Hz, 1H), 7.84 (t, J = 7.6 Hz, 2H), 7.65–7.58 (m, 3H), 7.48 (d, J = 8.0 Hz, 2H),
13
7.12 (t, J = 8.0 Hz, 1H); C NMR (100 MHz, DMSO-d6):
δ 181.6, 163.4, 153.4, 143.8, 141.3,
139.3, 136.3, 134.5, 133.2, 132.9(2C), 130.4, 129.4, 128.6(2C), 127.2(2C), 125.3, 118.6, 116.1.
MS(ESI) m/z 391 [M+H]+. Anal calcd for C20H14N4O3S: C, 61.53; H, 3.61; N, 14.35 Found C,
61.58; H, 3.60; N, 14.37.
3.2.21.
Synthesis of 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(4-fluorophenyl)thiourea (19):
The compound was synthesized according to above general procedure using 3-(benzo[d]oxazol-

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2-yl)aniline (1.3 g, 6.19 mmol), K2CO3 (2.0 g, 14.1
mmol) and 1-fluoro-4-
isothiocyanatobenzene ( 1.2 g, 7.6 mmol) in DMF: xylene (4:1) to get 1-(3-(benzo[d]oxazol-2-
yl)phenyl)-3-(4-fluorophenyl)thiourea (1.9 g, 84%) as pale red solid. M.p:235-237 oC. H NMR
1
(400 MHz, DMSO-d6):
δ
9.67 (s, 1H), 9.21 (s, 1H), 8.46 (s, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.89
13
(d, J = 7.6 Hz, 1H), 7.72–7.58 (m, 4H), 7.24 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 7.6 Hz, 2H); C
NMR (100 MHz, DMSO-d6):
δ 182.8, 164.2, 156.8, 144.4, 140.4, 138.2, 135.6, 133.9, 133.3,
132.3(2C), 130.2, 129.4, 128.2, 127.3(2C), 125.5, 124.2, 119.3, 115.2. MS(ESI) m/z 364
[M+H]+. Anal calcd for C20H14FN3OS: C, 66.10; H, 3.88; N, 11.56 Found C, 66.18; H, 3.87; N,
11.59.
3.2.22.
Synthesis
of
1-(3-(5-methylbenzo[d]oxazol-2-yl)phenyl)-3-(4-
nitrophenyl)thiourea (20): The compound was synthesized according to above general
procedure using 3-(5-methylbenzo[d]oxazol-2-yl)aniline (1.5 g, 6.69 mmol), K2CO3 (2.0 g,
14.1 mmol) and 1-isothiocyanato-4-nitrobenzene ( 1.1 g, 7.5 mmol) in DMF: xylene (4:1) to
get 1-(3-(5-methylbenzo[d]oxazol-2-yl)phenyl)-3-(4-nitrophenyl)thiourea (2.2 g, 81%) as pale
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yellow solid. M.p:241-243 C. H NMR (400 MHz, DMSO-d6):
δ 9.47 (s, 1H), 9.03 (s, 1H),
8.41 (s, 1H), 8.09 (d, J = 8.0 Hz, 2H), 7.89 (d, J = 7.6 Hz, 1H), 7.87–7.74 (m, 3H), 7.69–7.56
(m, 2H), 7.36 (d, J = 8.0 Hz, 2H), 2.40 (s, 3H); 13C NMR (100 MHz, DMSO-d6):
δ
182.9,
161.9, 150.3, 144.6, 142.0, 138.4, 136.0, 133.9, 133.2, 132.6(2C), 131.4, 128.9, 128.2(2C),
127.6, 126.5, 124.1, 117.9, 114.3, 21.9. MS(ESI) m/z 405 [M+H]+. Anal calcd for C21H16N4O3S:
C, 62.36; H, 3.99; N, 13.85 Found C, 62.48; H, 3.98; N, 13.87.
3.2.23.
Synthesis of 1-(3-(5-methylbenzo[d]oxazol-2-yl)phenyl)-3-phenylthiourea (21):
The compound was synthesized according to above general procedure using 3-(5-
methylbenzo[d]oxazol-2-yl)aniline (1.5 g, 6.69 mmol), K2CO3 (2.0 g, 14.1 mmol) and

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isothiocyanatobenzene ( 1.0 g, 7.4 mmol) in DMF: xylene (4:1) to get 1-(3-(5-
methylbenzo[d]oxazol-2-yl)phenyl)-3-phenylthiourea (1.9 g, 79%) as grey solid. M.p:155-157
1
oC. H NMR (400 MHz, DMSO-d6):
δ 10.26 (s, 2H), 8.41 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H),
7.84–7.76 (m, 3H), 7.62 (d, J = 7.2 Hz, 2H), 7.46–7.35 (m, 3H), 7.22 (d, J = 7.6 Hz, 2H), 2.44
13
(s, 3H); C NMR (100 MHz, DMSO-d6):
δ 183.5, 164.6, 148.3, 143.2, 137.4, 135.9, 135.0,
134.2, 133.8(2C), 130.4, 128.6, 128.2, 126.2(2C), 125.4, 124.3, 123.9, 118.2, 113.7, 22.3.
MS(ESI) m/z 360 [M+H]+. Anal calcd for C21H17N3OS: C, 70.17; H, 4.77; N, 11.69% Found C,
70.28; H, 4.76; N, 11.67%.
3.2.24.
Synthesis of
1-(4-chlorophenyl)-3-(3-(5-methylbenzo[d]oxazol-2-
yl)phenyl)thiourea
(22): The compound was synthesized according to above general procedure
using 3-(5-methylbenzo[d]oxazol-2-yl)aniline (1.5 g, 6.69 mmol), K2CO3 (2.0 g, 14.1 mmol)
and 1-chloro-4-isothiocyanatobenzene ( 1.3 g, 7.6 mmol) in DMF: xylene (4:1) to get 1-(4-
chlorophenyl)-3-(3-(5-methylbenzo[d]oxazol-2-yl)phenyl)thiourea (2.1 g, 80%) as white solid.
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M.p:190-192 C. H NMR (400 MHz, DMSO-d6):
δ 10.45 (s, 2H), 8.48 (s, 1H), 7.96 (s, 1H),
7.89 (t, J = 8.0 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.56–7.48 (m, 4H), 7.26 (d, J = 7.6 Hz, 2H),
13
7.18 (t, J = 7.6 Hz, 1H), 2.41 (s, 3H); C NMR (100 MHz, DMSO-d6):
δ 182.9, 163.9, 149.2,
143.6, 136.8, 135.4, 134.5, 133.2, 132.4(2C), 129.5, 127.8(2C), 127.2, 126.5, 125.3, 124.6,
124.2, 120.3, 116.4, 22.6. MS(ESI) m/z 394 [M+H]+. Anal calcd for C21H16ClN3OS: C, 64.03;
H, 4.09; N, 10.67 Found C, 64.11; H, 4.10; N, 10.65.
3.2.25.
Synthesis of 1-benzyl-3-(3-(5-methylbenzo[d]oxazol-2-yl)phenyl)thiourea (23):
The compound was synthesized according to above general procedure using 3-(5-
methylbenzo[d]oxazol-2-yl)aniline (1.5 g, 6.69 mmol), K2CO3 (2.0 g, 14.1 mmol) and
(isothiocyanatomethyl)benzene ( 1.2 g, 7.9 mmol) in DMF: xylene (4:1) to get 1-benzyl-3-(3-

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(5-methylbenzo[d]oxazol-2-yl)phenyl)thiourea (2.0 g, 79%) as grey solid. M.p:209-211 C. H
NMR (400 MHz, DMSO-d6):
δ 9.46 (s, 1H), 9.21 (s, 1H), 8.34 (s, 1H), 8.21 (s, 1H), 7.83–7.74
(m, 3H), 7.63–7.48 (m, 2H), 7.31–7.24 (m, 5H), 5.01 (s, 2H), 2.43 (s, 3H); 13C NMR (100 MHz,
DMSO-d6):
δ 180.2, 163.7, 146.9, 142.3, 138.6, 134.7, 134.4, 133.8, 133.2(2C), 131.4, 127.6,
126.4, 125.9(2C), 125.3, 124.5, 123.6, 123.0, 119.6, 54.6, 23.1. MS(ESI) m/z 374 [M+H]+. Anal
calcd for C22H19N3OS: C, 70.75; H, 5.13; N, 11.25 Found C, 70.88; H, 5.12; N, 11.27.
3.2.26.
Synthesis of 1-(3-(5-methylbenzo[d]oxazol-2-yl)phenyl)-3-(p-tolyl)thiourea (24):
The compound was synthesized according to above general procedure using 3-(5-
methylbenzo[d]oxazol-2-yl)aniline (1.5 g, 6.69 mmol), K2CO3 (2.0 g, 14.1 mmol) and 1-
isothiocyanato-4-methylbenzene ( 1.2 g, 7.9 mmol) in DMF: xylene (4:1) to get 1-(3-(5-
methylbenzo[d]oxazol-2-yl)phenyl)-3-(p-tolyl)thiourea (2.1 g, 83%) as pale brown solid.
o
M.p:231-233 C. 1H NMR (400 MHz, DMSO-d6):
δ 9.63 (s, 2H), 8.36 (s, 1H), 7.96 (d, J = 8.0
Hz, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.76 (d, J = 8.0 Hz, 1H), 7.64–7.56 (m, 3H), 7.26 (d, J = 8.0
Hz, 2H), 7.18 (d, J = 8.0 Hz, 1H), 2.44 (s, 3H), 2.41 (s, 3H); 13C NMR (100 MHz, DMSO-d6):
δ
179.7, 162.4, 146.4, 144.4, 140.8, 138.6, 135.7, 135.3, 134.0, 133.9(2C), 131.4, 130.5,
129.3(2C), 127.5(2C), 124.8, 117.4, 115.2, 23.4, 22.5. MS(ESI) m/z 374 [M+H]+. Anal calcd for
C22H19N3OS: C, 70.75; H, 5.13; N, 11.25 Found C, 70.83; H, 5.12; N, 11.23.
3.2.27.
The compound was synthesized according to above general procedure using 3-(benzo[d]oxazol-
2-yl)aniline (1.3 g, 6.19 mmol), K2CO3 (2.0 g, 14.1 mmol) and 1-chloro-4-
isothiocyanatobenzene (1.3 g, 7.7 mmol) in DMF: xylene (4:1) to get 1-(3-(benzo[d]oxazol-2-
Synthesis of 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(4-chlorophenyl)thiourea (25):
o
1
yl)phenyl)-3-(4-chlorophenyl)thiourea (1.9 g, 81%) as white solid. M.p:230-232 C. H NMR
(400 MHz, DMSO-d6): 10.23 (s, 2H), 8.39 (s, 1H), 7.90 (t, J = 8.0 Hz, 2H), 7.81 (d, J = 7.6
δ

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Hz, 2H), 7.56 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.21–7.12
13
(m, 3H); C NMR (100 MHz, DMSO-d6):
δ 182.7, 163.5, 144.7, 142.5, 139.8, 137.5, 134.8,
134.3, 134.1, 133.4(2C), 132.2, 131.4, 129.6(2C), 128.1, 126.4(2C), 118.6, 116.4. MS(ESI) m/z
380 [M+H]+. Anal calcd for C20H14ClN3OS: C, 63.24; H, 3.71; N, 11.06 Found C, 63.32; H,
3.70; N, 11.08.
3.2.28.
Synthesis of 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-phenylthiourea (26): The
compound was synthesized according to above general procedure using 3-(benzo[d]oxazol-2-
yl)aniline (1.3 g, 6.19 mmol), K2CO3 (2.0 g, 14.1 mmol) and isothiocyanatobenzene (1.1 g, 8.1
mmol) in DMF: xylene (4:1) to get 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-phenylthiourea (1.7 g,
82%) as white solid. M.p:182-184 oC. 1H NMR (400 MHz, DMSO-d6):
δ 10.01 (s, 2H), 8.43 (s,
1H), 7.93 (d, J = 8.0 Hz, 1H), 7.81 (t, J = 7.6 Hz, 2H), 7.20 (d, J = 7.6 Hz, 1H), 7.54 (t, J = 8.0
13
Hz, 1H), 7.49 (d, J = 8.0 Hz, 2H), 7.45–7.30 (m, 4H), 7.15 (t, J = 8.0 Hz, 1H); C NMR (100
MHz, DMSO-d6):
δ 179.8, 164.6, 152.3, 146.4, 143.4, 140.3, 131.2, 129.2 (2C), 128.3, 127.1,
126.3, 125.1, 124.6, 124.2 (2C), 123.8, 123.2, 120.4, 112.7. MS(ESI) m/z 346 [M+H]+. Anal
calcd for C20H15N3OS: C, 69.54; H, 4.38; N, 12.17 Found C, 69.63; H, 4.37; N, 12.19.
3.2.29.
compound was synthesized according to above general procedure using 3-(benzo[d]oxazol-2-
yl)aniline (1.3 g, 6.19 mmol), K2CO3 (2.0 g, 14.1 mmol) and 1-isothiocyanato-4-
Synthesis of 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(p-tolyl)thiourea (27): The
methylbenzene ( 1.2 g, 7.9 mmol) in DMF: xylene (4:1) to get 1-(3-(benzo[d]oxazol-2-
1
yl)phenyl)-3-(p-tolyl)thiourea (1.9 g, 81%) as pale brown solid. M.p:221-223 oC. H NMR (400
MHz, DMSO-d6):
δ
10.21 (s, 2H), 8.37 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.78–7.64 (m, 3H),
13
7.60–7.47 (m, 5H), 7.27 (t, J = 7.6 Hz, 2H), 2.43 (s, 3H); C NMR (100 MHz, DMSO-d6):
δ
176.4, 163.3, 152.1, 144.7, 138.3, 138.0, 136.7, 136.1, 135.3, 133.3(2C), 132.6, 132.0,

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129.3(2C), 127.2, 126.6, 125.3, 120.4, 116.8, 22.3. MS(ESI) m/z 360 [M+H]+. Anal calcd for
C21H17N3OS: C, 70.17; H, 4.77; N, 11.69 Found C, 70.26; H, 4.76; N, 11.71.
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4.3. In vitro glutamate racemase activity assay
4.3.1. Expression and purification of GR from M. tuberculosis and B. subtilis:
The recombinant genes coding for Mtb H37Rv and Bsb subsp. subtilis 168 glutamate racemase
were PCR amplified using an expression plasmid pET28a+ with N-terminal histidine tags and an
expression vector E.coli BL21 (DE3) cells. Ni±NTA metal-affinity column (His-bind resin,
Novagen) was used for purification of GR by using wash buffer (20 mM TEA, 10 mM MgCl2,
10 % glycerol, 500 mM NaCl, 50 mM imidazole, 1 mM DTT, 1 mg/mL lysozyme, 0.1 mM
phenyl methane sulfonyl fluoride, pH 7.8). The pure enzyme was obtained after running the
column with 500 mM imidazole in wash buffer. Purity of protein was confirmed using SDS-
PAGE. The protein was stored in aliquots at -80oC and single time thawed protein was used for
analysis [8, 9].
4.3.2. Inhibitory activity screening of compounds:
Inhibitory activity testing of synthesized compounds was carried out using a coupled assay, a
100 µL mixture consisting of 100 mM Tris–HCl pH 8.0, 5 mM NAD+, 1 mM D-Glutamate, 10
U/mL L-glutamate dehydrogenase (LDH) (all the chemicals were procured from Sigma) and
enzyme concentration of 1µM Bsb glutamate racemase at different concentrations (50µM to
0.5µM) of compounds monitored at 340 nm using microplate spectrophotometer (Spectromax
M4, Molecular devices). IC50 was determined by measuring the NADH conversion from NAD+
at 340nm [8].
4.4. Molecular docking simulations: