In-vitro Anti-cancer assay and apoptotic cell pathway of newly synthesized benzoxazole-N-heterocyclic hybrids as potent tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103382

A series of benzoxazole-N-heterocyclic hybrids have been synthesized by a one-pot strategy. Molecular docking study revealed that such compounds have the ability to inhibit enzyme protein tyrosine kinase. The findings of this work have been the successful synthesis of benzoxazole scaffolds, featuring hybrids of benzoxazole with quinoline and quinoxaline respectively. The molecular docking studies have showed these compounds to be inhibitors of tyrosine kinase enzyme which triggers growth of cancer cells. The cytotoxicity study of compounds 4a-f showed better potency against breast cancer cell lines MCF-7 and MDA-MB-231 in contrast to oral and lung cancer cell lines KB and A549. The tyrosine kinase activity was measured using Universal Tyrosine Kinase Assay kit using horseradish peroxide (HRP)-conjugated anti-phosphotyrosine kinase solution as a substrate. The compounds 4c exhibited maximum inhibition in the activity of enzyme tyrosine kinase with IC₅₀ value 0.10 ± 0.16 μM, than other compounds which were studied and thus proved to be inhibitors of enzyme tyrosine kinase. The selective index of all four compounds was found out to be greater than two, indicating the non-toxic behaviour, i.e. good anti-cancer activity. Further, fluorescence microscopic study helped to characterize the mode of cell death, which was found to be late apoptosis as indicated by the orange fluorescence. The SAR analysis has also been carried out.

Full text of DOI:10.1016/j.bioorg.2019.103382

Journal Pre-proofs
In-vitro Anti-cancer Assay and Apoptotic Cell Pathway of Newly Synthesized
Benzoxazole-N-Heterocyclic Hybrids as Potent Tyrosine Kinase Inhibitors
Sulaksha Desai, Vidya Desai, Sunil Shingade
PII:
S0045-2068(19)30988-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.103382
YBIOO 103382
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
20 June 2019
30 September 2019
21 October 2019
Please cite this article as: S. Desai, V. Desai, S. Shingade, In-vitro Anti-cancer Assay and Apoptotic Cell
Pathway of Newly Synthesized Benzoxazole-N-Heterocyclic Hybrids as Potent Tyrosine Kinase Inhibitors,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103382
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In-vitro Anti-cancer Assay and Apoptotic Cell Pathway of Newly Synthesized
Benzoxazole-N-Heterocyclic Hybrids as Potent Tyrosine Kinase Inhibitors
Sulaksha Desaia,b, Vidya Desaia,b and Sunil Shingadec
aDepartment of Chemistry, Dnyanprassarak Mandal’s College and Research Centre, Assagao, Bardez-
Goa. 403507, India.
bDepartment of Chemistry, Goa-University, Taleigao-Goa, India.
cDepartment of Pharmaceutical Chemistry, PES’s Rajaram and Tarabai Bandekars College of Pharmacy,
Pharmagudi, Ponda-Goa
Abstract: A series of benzoxazole-N-heterocyclic hybrids have been synthesized by a one-pot strategy.
Molecular docking study revealed that such compounds have the ability to inhibit enzyme protein tyrosine
kinase. The findings of this work have been the successful synthesis of benzoxazole scaffolds, featuring
hybrids of benzoxazole with quinoline and quinoxaline respectively. The molecular docking studies have
showed these compounds to be inhibitors of tyrosine kinase enzyme which triggers growth of cancer cells.
The cytotoxicity study of compounds 4a-f showed better potency against breast cancer cell lines MCF-7 and
MDA-MB-231 in contrast to oral and lung cancer cell lines KB and A549. The tyrosine kinase activity was
measured using Universal Tyrosine Kinase Assay kit using horseradish peroxide (HRP)-conjugated anti-
phosphotyrosine kinase solution as a substrate. The compounds 4c exhibited maximum inhibition in the
activity of enzyme tyrosine kinase with IC50 value 0.10±0.16µM, than other compounds which were studied
and thus proved to be inhibitors of enzyme tyrosine kinase. The selective index of all four compounds was
found out to be greater than two, indicating the non-toxic behaviour, i.e. good anti-cancer activity. Further,
fluorescence microscopic study helped to characterize the mode of cell death, which was found to be late
apoptosis as indicated by the orange fluorescence. The SAR analysis has also been carried out.
Keywords: Benzoxazole, Heterocyclic hybrids, Cytotoxicity, Selective index, Tyrosine Kinase, Apoptosis.
1. Introduction
 Corresponding author. Tel.: +91-9822180792; fax: +0-083-2268683; e-mail: desai_vidya@ymail.com

Cancer is affecting millions of lives all around the world.1, 2 With the sophisticated and different changing
lifestyle, the growth of cancer disease has erupted rapidly. The most common type of cancers includes lung
cancer, oral cancer3 and breast cancer. Nowadays, due to chemotherapeutic treatment most of them lead to
metastasis, disease to liver, bone, lungs and bronchitis. Several types of breast cancer carcinoma have been
reported that has been responsible for painstaking deaths all round the world. In view of this there is great
need to search for newer breast cancer drug targets. Moreover, the scope to develop novel therapy strategies
becomes the need and a challenging task. From cancer drug target study basal like subtypes triple negative
breast cancer (TNBC) constitutes to about 80% of basal like tumors and is responsible for 10-15% breast
carcinoma and also shows lack of expression of both hormone receptor (Progesterone and Estrogen) and
HER2-receptor over expression.4-5 A drug target for such cancer becomes evident as TNBC and basal like
cancers are considered to be in the lead as an aggressive disease with greater chances of metastasis. The
therapy for such conditions namely, Trastuzumab (Fig. 1. I) cuts the risk of recurrence to half-compared to
chemotherapy alone. Trastuzumab is given through veins in every 3 weeks for 1 year.6-9 Scaffold containing
heterocyclic conjugates such as 3-pyrimidinylazaindole analogue, (Fig 1. II) a potent inhibitor of triple
negative breast cancer which cuts the risk to upto 90% of tumor growth inhibition as compared to the other
leads.10
Human protein kinase forms a large family of enzymes commonly known as human Kinome encoded by
about 1.7% of different types of human genes.11 In this group of enzymes, tyrosine kinase has been the first
protein kinase which has been well identified, described with respect to its location in the cell. Keeping in
view the structural features, small molecule kinase inhibitors can be of great help to design and develop
targeted therapies against breast cancer. The advantage of such inhibitors is high selectivity, better efficacy
and specific action on protein kinase receptors.
In the designing of small drug like molecules, molecular hybridisation of heterocyclic compounds could be
challenging task to gain potency in acting against tyrosine kinase enzymes. Thus, our focus was on
benzoxazole as a core unit as it forms an important class of bicyclic heterocycles having wide spectrum of
biological applications.12 Benzoxazole ring as a core pharmacophoric unit is found in various class of natural
and synthestic compounds showing varied biological properties and has been studied for their antibacterial
and anti-fungal,13, 14 anticancer15, 16 and HIV-1 reverse transcriptase inhibitors.17 It constitutes subunits of
commercially available drug molecules such as; Flunoxaprofen which is a non-steroidal anti-inflammatory
drug,18 NSC693638- an anticancer agent,19 L-697,661 a reverse transcriptase inhibitor, 20 Nataxazole, an anti-
tumor agent21 related to anti-tumor drugs like UK-1 and AJ19561.22 Fig. 2 The synthesis of benzoxazolyl-
carbohydrazide derivatives and their in-silico docking and in-vitro anti-cancer study has been also reported.23

Combretastin A-4 and benzoxazole analogues have been reported to exhibit more potent anti-cancer activity
then the standard drug against A-549 and MCF-7 cell lines.24
O
O
N
NH
O
HO
NH2
NH
O
F
N
H
O
O
O
O
N
N
O
NH
O
N
H
II
I
O
N
O
S
Fig.1. Representative Structures of breast cancer drugs: I. Trastuzumab, sold under the brand name Herceptin among others, is a
monoclinical antibody, used to treat HER2 positive breast cancer; II. 3-Pyrimidinylazaindole, a potent inhibitor of triple negative
breast cancer.
O
OH
O
N
N
O
N
O
N
O
F
O
Nataxazole
Flunoxaprofen
O
Cl
Cl
NH
O
HN
Me
O
N
NH
N
N
Et
NSC 693638
L-697,661
Fig. 2 Bioactive Compounds containing Benzoxazole moiety: flunoxaprofen-NSAIDs, L-697,661 a reverse transcriptase inhibitor,
Nataxazole an anti-tumor, NSC693638- an anticancer agent
Nitrogen-based heterocycles are an important class of compounds in pharmaceutical and agrochemical
industries. Their structural diversity has made them attractive targets in the synthesis of alkaloids and
important precursors to many biologically important active compounds. They have emerged as integral parts
of various drugs known in day to day life. Some of the nitrogen heterocycles of biological importance includes:
quinoxaline, indoles, quinolines, pyridines etc. Designing of heterocyclic hybrids25 has led to newer
breakthrough in the need for treatment of cancer and so it was visualized that heterocyclic hybrids of N-
heterocycles with benzoxazole would have a promising effect on the biological activity. Thus, in continuation
to our work on designing heterocyclic molecules of biological importance using ecofriendly stategies26-29
herein, we present the synthesis of benzoxazoles using silica chloride. This methodology was further extended
towards synthesis of six novel heterocyclic scaffolds. The molecular docking studies and biological evaluation
has also been discussed, revealing our compounds to have the best anti-cancer activity. Development of solid
acid catalysed30-33 synthesis of benzoxazoles and their action as inhibitors of protein kinase enzyme has been
the focus of the work.

2.Results and Discussion:
2.1. Chemistry
The target benzoxazole was synthesized from condensation of 2-aminophenol derivatives with
differently substituted aromatic aldehydes via Schiff base intermediate using previously prepared silica
chloride34 under solvent free conditions. The optimization of the reaction conditions revealed that catalyst
not in mole ratio but in 1 equivalent and temperature of about 120oC gave the cyclised product in short time
of about 3-4 hours. Consequently, a wide variety of 2-aryl benzoxazole derivatives having different functional
groups was obtained successfully in good yields in the range of 70-80%. (Scheme 1, Table 1)
R'
O
R'
NH2
CHO
a
b
N
+
2
OH
R
R
Scheme 1. One-pot synthesis of benzoxazole derivatives (2a-r) Reagents and conditions: a) 1mmole of the 2-aminophenol and
substituted benzaldehyde, 1eq silica chloride, b) 120oC, solvent-free
Table 1.Synthesis of benzoxazole derivatives 2a-ra
Comp
R
R’ Yieldb
Melting Pointc
o C
2
a
b
c
d
e
f
g
h
i
2,4- Cl
4- Cl
4- Br
3-NO2
2-NO2
4-OMe
4-NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
75%
75%
71%
79%
72%
77%
79%
78%
70%
79%
72%
75%
125-127 (127)
144-148 (148-150)
150-152(157-158)
99-102(103-105)
97-100(98-102)
114-116 (113-114)
256-260 (268)
98-100 (101)
2-Cl
4-Me
3-Cl
4-OH, 5-NO2
68-72(73)
j
k
l
104-106 (101-103)
120-122 (122-124)
170-174 (178)
m
2-OH, 5-NO2
H
71%
188-192 (188)
n
o
p
4-F
3-Br
4-NMe2
H
H
H
79%
72%
70%
90-92 (92-95)
133-137 (136-138)
179-182(182-183)
q
5-Br, 4-OH,
3-OMe
H
78%
187-190 (190-192)
r
4-OH-3,5- Br
H
70%
166-169 (168-170)
aReaction conditions: Schiff base(1mmole), silica chloride 1equivalent
bIsolated yields
c Determined using thiels tube paraffin method

However, our focus was on designing of novel heterocyclic scaffolds with benzoxazole as the core unit.
Our previous work on quinoxalinyl chalcones depicted good anti-tubercular and anti-cancer activity.35 As a
continuation to the work on designing of heterocyclic hybrids, an attempt has been made to prepare the new
substituted benzoxazole derivatives bearing heterocyclic moiety. Six new benzoxazole conjugates 4a-f has
been prepared by the same strategy under solvent free conditions from commercially available aldehydes,
previously synthesised quinoxaline-2-carbaldehyde36 and 2-chloro-3-formyl quinoline37. (Scheme 2) All the
six benzoxazole analogues were obtained in good yields. (Table 2)
OH
Silica chloride
1 eq
O
N
R
+
Ar'
Ar'- CHO
R
NH2
Heat, 120oC
3a-d
4a-f
R= 4-H,
= 4-Br
3a, Ar=4-nitrobenzaldehyde
4a, R'=5-Br, Ar=4-Nitroophenyl
4b, R'=5-Br, Ar=4-Bromophenyl
4c, R'=5-Br, Ar= quinoxalinyl
4d, R'=H, Ar=quinoxalinyl
3b, Ar=4-Bromobenzaldehyde
3c, Ar= quinoxaline-2-carbaldehyde
3d, Ar= 2-chloro-3-formylquinoline
4e, R'=H, Ar=2-chloroquinolinyl
4f, R'=5-Br, Ar=2-chloroquinolinyl
Scheme 2 Synthesis of novel 2-Arylbenzoxazole scaffolds 4a-f
Table 2. One-pot Synthesis of benzoxazolyl scaffolds 4.
Entry Compound Time Yielda
4
Melting Pointb
oC
1
2
3
4
5
6
a
b
c
d
e
f
2.5 h
2.5 h
3h
3h
3h
80%
72%
74%
80%
75%
76%
158-162
228-232
160-164
189-192
102-108
170-175
3h
aIsolated yields
bDetermined using thiels tube paraffin method.
1
Their structures were confirmed by HNMR, 13CNMR, and Melting points. The IR spectra showed
disappearance of OH and C=O peak, C=C peak at 1605cm-1, C=N peak at 1677cm-1 and peak at 1301cm-1
attributed to C-O peak. The 1HNMR spectra of the compounds 2a-r showed an average spectrum in which
appeared two doublets at δ 8.20ppm (Ar-H, 3’ & 5’position) and δ 7.51ppm (Ar-H; 2’ & 6’) . In addition,
protons of the fused benzene ring appeared as three multiplets at δ7.79-7.75ppm (–CH=C-N-), δ 7.57-
7.61ppm for C5-H, δ7.36-7.39ppm for C7-H and C6-H; 13C NMR spectra of compounds revealed the
presence of C=N at the range of 164.01 C-Cl at the range of 150.3. The structures of N-heterocyclic hybrids
1
13
4a-f were confirmed by HNMR, CNMR, and HRMS data. The calculated M+H ion peak matched with
that of the observed M+H ion peak, which confirmed the structure.

2.2.
Biological Evaluation
2.2.1. Virtual Screening
The promising approach to cancer therapy has been the targeted therapies which lead to beneficial clinical
effects. Tyrosine kinase is an important target due to its role in modulation of growth factor signalling, it
causes increase in tumor cell proliferation and growth, induces anti-apoptotic effects and promotes
angiogenesis and metastasis. Because of all these effects, receptor tyrosine kinase has been a key target for
cancer therapy. Tyrosine kinases are enzymes which catalyses the transfer of γ- phosphate group from
adenosine triphosphate to target proteins. They play an important role in diverse normal cellular regulatory
processes.38 It is characterised by immunoglobulin-like sequences in their amino-terminal extracellular
domains, a lipophilic transmembrane segment and an intracellular carboxyl-terminal domain which includes
its catalytic site. Ligand binding induces dimerisation of these tyrosine kinase receptor and results in
autophosphorylation of their cytoplasmic domains and activation in activity of tyrosine kinase. The 2-
aminobenzoxazole and benzimidazole derivatives and pyrazolyl-benzoxazole derivatives have been proved
to be active tyrosine kinase inhibitors due to strong binding affinity with enzyme.39, 40 Hence, we chose
enzyme tyrosine kinase as a target for the molecular docking study of the synthesised benzoxazole-N-
heterocylic hybrids.
The crystal structure of the epidermal growth factor receptor tyrosine kinase domain with 4-
anilinoquinazoline inhibitor erlotinib (PDB ID: 1M17) was used for the docking studies, obtained from
Protein Data Bank. The protein file was prepared by the removal of water molecules, addition of polar
hydrogens and removal of other bound ligands. The synthesized benzoxazole-N-heterocyclic conjugates 2a-r
and 4a-f were virtually screened for its anti-cancer activity against tyrosine kinase domain with 4-
anilinoquinazoline inhibitor erlotinib as target enzyme. (Table 3) The molecular docking study revealed that
compounds 4a, 4c and 4d acts as good inhibitors of tyrosine kinase due to characteristic features. (Fig.3)
However, compounds 2a-r were also found to be active with the target PDB ID: 1M17. As depicted in Fig.
4, Compound 4a makes four hydrogen bond interactions at the active site of the enzyme. Three interaction
with the nitro group present on phenyl ring; of which one interactions raised from H-bond interaction between
nitrogen atom of the NO2 group with Thr766 (1.55 Ao) and two interaction arised from H-bond interaction
between oxygen atom of nitro group and CYS751 (1.52 Ao) and Thr830 (1.52 Ao). The remaining one arose
from oxazole ring of benzoxazole moiety. The oxygen atom interacts with Met769 (1.55 Ao).

O
O
Br
N
N
N
O
a
O
Br
O
N
N
N
N
N
b
c
Fig. 3. Structural features of compounds responsible for the cytotoxicity: a) Compound 4a; b) Compound 4c; c) Compound 4d
Compound 4c makes two hydrogen bond interactions at the active site of the enzyme. One interactions
arose from nitrogen atom of oxazole ring, where the nitrogen forms H-interaction with Thr766 (1.52 Ao).
while the other is raised from H-bond interaction of nitrogen atom of quinoxaline ring with Met769 (1.55
Ao). Compound 4d makes three hydrogen bond interactions at the active site of the enzyme. The interaction
arising from nitrogen atom of the quinoxaline, shows H-bonding which interaction with Met769 (1.55 Ao).
The remaining two interactions are raised from nitrogen and oxygen atom of oxazole ring with Thr766 (1.52
Ao). (Fig. 4)

A
B
D
C
Fig 4. Molecular docking data: the compounds docked in best of its conformation into the binding site of 1M17. (A) Binding mode
of compound 4a forming four hydrogen bond interaction. (B) Binding mode of compound 4c forming two hydrogen bond
interaction. (C) Binding mode of compound 4d forming three hydrogen bond interaction. (D) Binding interaction of ligand AQ4-
1M17 forming 10 hydrogen bond interaction.
Table 3. Molegro docking score for benzoxazole derivatives 2a-r and 4a-f
Code
MolDock
Score
Rerank
Score
Protein
H
bond
Heavy
atom
Kcal/mol
Docking
Score
count
B2a
B2b
-86.5224
-80.5605
-64.8155
-61.4823
-90.5300
-86.9539
2
1
-5.08955
-5.03503
-86.0269
-79.6663
19
19
17
17
17
17
17
16
16
16
16
16
19
B2c
B2d
B2e
B2f
B2g
B2h
B2i
-80.2853
-79.8709
-96.1274
-80.6788
-92.6515
-81.1186
-80.4031
-73.0934
-79.7784
-90.1899
-97.8936
-62.0941
-62.0032
-66.7314
-61.6469
-72.1225
-66.7681
-54.6825
-56.8369
-61.9172
-71.7397
-71.8246
-86.7351
-101.419
-99.1471
-86.0643
-104.053
-89.5901
-89.813
-79.4259
-85.919
-102.058
-101.476
3
3
5
2
2
3
3
0
1
3
3
-5.01783
-5.1842
-3.7073
-4.13724
-4.8123
-4.09693
-3.77365
-3.5958
-4.01128
-5.0177
-5.1523
-79.4664
-97.9248
-97.9981
-83.9878
-86.6232
-76.124
-71.5362
-73.2504
-79.2865
-100.482
-96.9324
B2j
B2k
B2l
B2m

B2n
B2o
B2p
B2q
B2r
B4a
B4b
B4c
B4d
B4e
B4f
-75.3016
-78.559
-83.1269
-81.1726
-79.7915
-58.9796
-62.499
-60.3583
-45.0394
-58.6621
-81.593
-88.8241
-87.3281
-100.153
-88.1242
0
0
1
3
1
4
1
2
3
1
1
-3.71931
-3.86762
-3.11882
-4.2722
-3.59923
-4.2037
-4.9468
-4.7468
-4.6325
-3.71632
-3.24967
-80.8615
-79.1946
-87.9088
-78.8718
-86.9973
-81.4409
-93.4702
-89.4702
-91.9065
-95.1771
-91.2059
19
19
17
19
19
17
17
17
17
17
16
-79.8709
-80.3296
-90.1899
-92.6515
-89.4501
-89.7198
-62.0032
-63.3705
-71.7397
-72.1225
-64.1618
-70.4287
-101.419
-87.2282
-102.058
-104.053
-99.8916
-97.4172
2.2.2. Anticancer Evaluation
On the basis of enzymatic inhibitory potency against 1M17, compounds 4a-f was selectively used as
candidates for exploring the mechanisms of anti-cancer. The anti-proliferative activity has been carried out
against MDA MB-231 (ER-negative) & MCF-7 (Breast Cancer), A549 (Lung Cancer), KB (Oral Cancer)
and HEK293 (Normal Human Kidney Cells) by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay method.41 Using graph Pad Prism Version5.1, the IC50 of compounds has been calculated by
taking a percentage of Inhibition Tyrosine Kinase Enzyme at six different concentrations of treatment.
All the six benzoxazole-N-conjugates 4a-f exhibited cell proliferation with IC50 values in the range of
0.42 to 14.39µM. As shown in Table 4, the samples showed moderate to good activity comparable to that of
standard drugs, especially compound 4a, 4c and 4d showed higher anti-proliferation activity with
IC50=0.56±0.07, 0.58±0.05µM; IC50=0.53±0.02, 0.50±0.08µM and IC50=0.73±0.02, 0.60±0.06µM against
MDA-MB-231(TNBC-triple negative breast cancer) and MCF-7 (human breast cancer) cell lines. Compound
4b, 4e and 4f showed moderate activity with IC50 values 1.09±0.05, 0.98±0.04 and 0.85±0.01µM against
MDA-MB-231 and also against MCF-7 cell lines with the IC50 values 1.10±0.03, 0.94±0.04 and
0.91±0.02µM respectively. The higher anti-proliferation against KB (oral cancer) cell line with IC50 value
of 0.71±0.12 was exhibited by compound 4b. Compound 4a, 4d and 4e showed comparable anti-proliferation
against KB cell line with IC50 value 0.82±0.08, 0.86±0.10 and 0.85±0.04μM respectively. Compound 4c and
4f exhibited moderate anti-proliferation effect. Considering the anti-proliferation effect against A549 (lung
cancer), compound 4a showed good anti-proliferation with the IC50 value 0.70±0.11μM. Compound 4c, 4d,
4e and 4f showed moderate anti-proliferation against A549 (Lung cancer) cell line with IC50 value 0.8μM as
compared to the other samples. The results were compatible with the docking studies.
Table 4. IC50 values (µM±S.E.) of compounds 4a, 4b, 4c, 4d, 4e and 4f against four different cell lines and
normal human cell lines.
IC50 μM

MDA MB-
231a
MCF-7b
A549c
KBd
HEK293e
Comp
4a
4b
4c
4d
4e
0.56±0.07
1.09±0.05
0.53±0.02
0.73±0.02
0.98±0.04
0.85±0.01
0.3±0.02
0.58±0.05
1.10±0.03
0.50±0.08
0.60±0.06
0.94±0.03
0.91±0.02
-
0.70±0.11
0.97±0.09
0.82±0.20
0.87±0.08
0.89±0.11
0.83±0.06
-
0.82±0.08
0.71±0.12
0.90±0.05
0.86±0.10
0.85±0.04
0.98±0.03
-
10.11±0.19
3.27±0.20
14.39±0.28
8.39±0.19
6.04±0.23
7.21±0.05
-
4f
aMDA-
cell line- is
MB-231
an
Paclitaxelf
epithelial, human breast cancer cell line established from pleural effusion of a 51-year old Caucasian female with metastatic
memmary adenocarcinoma.
bMCF-7 is a human breast cancer cell line with estrogen, progesterone and glucocorticoid receptors. It is derived from pleural
effusion of 69-year old Caucasian metastatic breast cancer in 1970
cA549 ia a human cancer cell line, derived from the removal and culturing of cancerous lung tissues in the explanted tumor of 58-
year old Caucasian metastatic male.
dKB is derived from an epidermal carcinoma of the mouth, KB cells contain human papilloma virus18 (HPV-18) sequence;
eHEK293 –derived from Human embryonic kidney293 cell lines grown in tissue culture
fPaclitaxel- used as standard drug
4a
120
100
80
60
40
20
0
4a
4b
4c
4d
4e
4f
4a
4b
4c
4d
4e
4f
120
100
80
60
40
20
0
120
100
80
60
40
20
0
4b
4c
4d
4e
4f
31.5 62.5 125 250 500 600
18.75 37.5 75 150 300 600
18.75 37.5 75 150 300 600
Concentration (µg/mL)
Concentration in µg/mL
Concentration in µg/mL
a
b
c
120
100
80
60
40
20
0
4a
4b
4c
4d
4e
4f
120
100
80
60
40
20
0
4a
4b
4c
4d
4e
18.75 37.5 75
150 300 600
18.75 37.5 75
150 300 600
Concentration in µg/mL
Concentration in µg/mL
d
e
Fig. 5. Graphical representation of cell viability exhibited by 4a, 4b, 4c, 4d, 4e and 4f for five different cell lines: a)MDA-MB-
231, b) MCF-7, c) A549, d)KB, e)HEK293
Following this, the in-vitro cytotoxic activity of all the tested compounds was analyzed against normal HEK-
293 cell lines by MTT colorimetric assay.41 The colorimetric results revealed that none of the six evaluated
compounds exhibited any significant toxicity effect on normal HEK-293 cells. Selectivity index (SI) reveals
the differential activity of a pure compound. Higher SI value is attributed to less toxicity and more selectivity.
Whereas, a compound with SI value <2 indicates cytotoxicity of the pure compound. As shown in Table 5
all samples were proved to be non-toxic on the normal tumor cells. The effect of benzoxazole-N-heterocyclic
hybrids 4a-f on viability of MDA MB-231 (ER-negative), MCF-7 (Breast Cancer), A549 (Lung Cancer), KB

(Oral Cancer) and HEK293 (Normal Human Kidney Cells) was investigated and represented graphically as
variation in percentage of viability with respect to time and concentration. (Fig. 5)
Table 5. Selective index (SI) f of compounds
Comp
SIf-MDA
MB-231
9.55
2.18
13.86
7.66
SIf-MCF-
7
9.53
2.17
13.89
7.79
5.10
SIf-
A549
9.41
2.30
13.57
7.52
5.15
6.38
SIf-
KB
4a
4b
4c
4d
4e
4f
9.29
2.56
13.49
7.52
5.19
6.23
5.06
6.36
6.30
fSI-Selective index, calculated as (IC50)HEK293 - (IC50) of the respective cell lines
2.2.3. Enzyme Inhibition studies
The effect of benzoxazole-N-heterocyclic hybrids 4a-f on tyrosine kinase activity in-vitro was carried
out using Fetal Bovine Serum (FBS) cell culture treated with indicated amounts of benzoxazole-N-
heterocyclic hybrids for indicated time. The whole cell extracts were extracted and incubated with tyrosine
kinase substrate for the specified time mentioned in the procedure. The plot of relative rate of inhibition in
activity of enzyme tyrosine kinase with respect to different concentration of compounds 4a-f (Fig. 6)
indicates that compounds 4a-f induced a dose dependent decrease in the enzyme activity. (Table 6) It was
summarized from the results of the enzyme inhibition studies that, compound 4c exhibited higher inhibition
in activity of enzyme Tyrosine kinase with the IC50 value of 0.10±0.16 µM, compounds 4a, 4d and 4e also
exhibited good inhibitory action on the activity with IC50 value in the range 0.31-0.43µM. Compound 4b was
found to be least active in inhibiting the growth of enzyme with IC50 value 0.86±0.42 µM. With decrease in
the concentration of compounds 4a-f, the rate at which the enzyme grow was decreased, with compound 4c
exhibiting the highest inhibition in the activity of enzyme. All compounds demonstrated good rate of
inhibition of tyrosine kinase enzyme at highest concentration but as the concentration decreased the effect of
inhibition was found to decrease.

Fig.6. Graphical representation enzyme Tyrosine kinase inhibition by compounds 4a-f: Plot of concentration in
µL/mL v/s rate of inhibition
Table 6. Enzyme inhibition of Tyrosine kinase enzyme by compounds 4a-f
Compound code
IC50 valuea
0.31±0.11
0.86±0.42
0.10±0.16
0.35±0.17
0.43±0.21
0.56±0.24
4a
4b
4c
4d
4e
4f
aIC50 values measured in µM
2.2.4. Cell Morphology studies by Fluorescence Microscopy:
Fluorescence microscopy study was employed to study the mode of cell death induced by
synthesized benzoxazole-N-heterocyclic hybrids 4a, 4c and 4d in comparison to the standard cis-
platin by virtue of acridine orange/ ethidium bromide staining. The mode of cell death, whether
early or late apoptosis can be characterized based on the fluorescence properties. Acridine orange
was taken up by both viable and apoptotic (dead) cells by emitting green fluorescence. Cells with
disrupted membrane integrity were stained by ethidium bromide, i.e. late apoptotic cells and
necrotic cells. Late aoptotic cells have orange to red nuclei with condensed and fragmented
chromatin, and early apoptotic cells show green fluorescence nuclei. Necrotic cells have uniform

orange to red nuclei with organised structure. Thus, from the results (Fig. 7) it was visualised that,
viable cells without treating with synthesised compound 4a, 4c, 4d showed green fluorescent
nucleus with acridine orange Fig. 7a, Whereas, late apoptotic cells when treated with compounds
4a, 4c and 4d exhibited yellow orange fluorescence with nuclear membrane babbling. Fig. 7b-d;
thus signifies apoptotic mode of cell death.
Further confirmation of the apoptotic mode of cell death was done by using DAPI staining
method, wherein; cells treated with compounds and the viable cells were stained with 4’, 6’-
diamine-2’-phenylindole dihydrochloride (DAPI). Untreated cells showed intact nucleus Fig. 8a
whereas, treated cells showed nuclear condensation and nuclear fragmentation. Fig. 8b-d.
a
c
b
d
Fig. 7. Fluorescence microscopy images of cells stained with acridine orange/ ethidium bromide, a. viable cells, b. Cells treated with compound
4a, c. cells treated with compound 4c, d. cells treated with compound 4d

Fig 8. Fluorescence microscopy images of cells stained with DAPI, showing a) viable cells, b) Cells treated with
compound 4a, c) cells treated with compound 4c, d) cells treated with compound 4d.
2.2.5. SAR studies
The following Structure activity relationship (SAR) analysis has been executed based on the
results of the cytotoxicity studies of benzoxazole-N-heterocyclic hybrids 4a-f against five different
cancer cell lines. All the compounds were found to show good cytotoxicity effect against MDA-
MB- 231 and MCF-7 breast cancer cell lines as compared to other cell lines. Compound 4a, 4c
and 4d exhibited good anti-cancer activity due to strong interaction with the enzyme active site
and their characteristic structural features. Fig. 3 & 9. As in compound 4a, the presence of electron
withdrawing group (i.e. Nitro) onto the phenyl ring and also the presence of bromo substituent on
the benzoxazole moiety enhance the cytotoxicity effect. Replacement of the nitro group by halogen
(i.e. Br) as in compound 4b, has moderate effect on cytotoxicity. In case of compound 4c, replacing
the phenyl ring with quinoxaline ring and also the presence of bromo substituent on the
benzoxazole ring exhibits higher effect on cytotoxicity, so also compound 4d with quinoxaline
moiety exhibited similar effects. Quinoline moiety was found to lower the cytotoxicity effect as in
compound 4e and 4f.

Fig. 9. Structural representation of SAR from cytotoxicity study of benzoxazole hybrids 4a-f
3. Conclusion:
In conclusion, a newly designed synthesis of benzoxazole has been achieved in an ecofriendly
reaction conditions. Such benzoxazole hybrids 2a-r and 4a-f has been evaluated as anti-
proliferative agents, having virtually screened as good inhibitors of enzyme protein tyrosine
kinase, the key enzyme that triggers triple negative breast cancer. Comparative cell cytotoxicity
studies against five different cancer cell lines MCF-7, MDA-MB-231, KB, A549 and HEK-293
revealed that all six benzoxazole hybrids 4a-f showed good inhibitory potency against all cell lines.
It was visualized from the anti-cancer activity results that, compounds 4a, 4c and 4d demonstrated
excellent inhibitory potency against MDA-MB-231 and MCF-7 breast cancer cell lines with the
IC50 value in the range of 0.50-0.73µM as compared to standard drug Paclitaxel (IC50; 0.030±0.02
µM). Compounds 4a-f was also found to be potent against A549 (lung cancer) with IC50 values in
the range of 0.70 to 0.97 µM.
Comparing the anti-cancer potency against all five cancer cell lines, the target compounds 4a, 4c
and 4d exhibited better anti-proliferation effect against MDA-MB-231 and MCF-7 cell lines.
Cytotoxicity against HEK-293; human embryonic kidney cell line was also evaluated and
selectivity was determined. The selective index value in the range of 2.17-13.89 was displayed by
the compounds screened. The docking results revealed that compounds 4a, 4c and 4d had good
interaction with the active site of enzyme tyrosine kinase. Further, enzyme inhibition study of
compounds 4a-f proved that, all compounds inhibits the activity of enzyme tyrosine kinase with
IC50 value in the range of 0.10-0.86µM, with maximum inhibition being exhibited by compound

4c with IC50 value 0.10±0.16µM. The results were found to be compatible with the docking studies,
thus demonstrating that compound 4a, 4c and 4d act as potent inhibitors of tyrosine kinase.
Accordingly, a mode of cell death was studies using double staining and DAPI method thus
signified the mode of cell death to be late apoptosis. SAR analysis reveals the influence of
benzoxazole N-heterocyclic hybrids in defining its cytotoxicity and anticancer activity.
On contrary, the in-vitro anti-tubercular screening42 of the compounds 2a-r against
Mycobacterium tuberculosis H37RV strain exhibited excellent anti-tubercular activity with the
MIC value ranging from 1.6-25 µg/mL compatible with the standard drugs Pyrazinamide,
Streptomycin and Ciprofloxacin, however, compounds 4a-f were active only upto MIC value of
50µg/mL, thus indicating that these compounds are selective inhibitors of tyrosine kinase enzyme.
(Fig. 10)
Antitubercular evaluation
60
Series1
50
40
30
20
10
0
Compounds
Fig. 10. In-vitro anti-tubercular evaluation against mycobacterium tuberculosis by Alamar Blue Dye Assay method

4. Experimental Section
4.1.Chemistry
4.1.1. General methods
All the chemicals were purchased from Avra synthesis; Lobachemie, Finar. Apparatus has been
purchased from J-Sil and Agarwal. Melting points were determined by using thiels tube method
using open capillaries and have been uncorrected. IR was recorded on SHIMADZU FTIR affinity-
1. The NMR spectra were measured in CDCl3 or DMSO at RT on a Brucker AV-II 400
spectrometer. δ is given in ppm relative to tetramethylsilane as an internal reference. Thin layer
chromatography was performed using DDC-fertigfolien ALUGRAMRXtra SIL G/UV254
(Macherey-Nagel GmbH & Co.KG) Compounds were visualized by illumination under UV light
(254nm) or by use of phosphomolybdic acid stain followed by heating. Melting points were
determined using an open capillary tube method and were uncorrected. Chemical shifts are
expressed in parts per million relative to tetramethylsilane, which was used as internal standard,
coupling constant (J) are in hertz (Hz), and signals are designated as follows: s, singlet; d, doublet;
t, triplet; q, quartet; m, multiplet; dt, doublet of triplet; ddd, double of doublet of doublet, dq,
doublet of quartet. All solvents were dried prior to use and stored over 4Ao molecular sieves.
4.1.2. Synthesis of benzoxazole derivatives 2a-r and 4a-f (Scheme 1 & 2)
To the flask containing a mixture of substituted benzaldehyde (1mmole) and 2-aminophenol
(1mmole) was added silica chloride (1eq) and was heated on a sand bath at 120oC, TLC was taken
after every 1 hour. After 4 hours, TLC showed appearance of new spot. The product was isolated
by first separating out the catalyst by filtration using organic solvent; the organic layer was dried
using anhydrous sodium sulfate and evaporated under vacuum. The solid thus obtained was

recrystallized using petroleum ether and its % yield and melting points were determined. The
results are tabulated in Table 1 and Table 2.
4.1.2.1. 2-(2’, 4’-dichlorophenyl)-1, 3-benzoxazole (2a)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 75%, m. p.: 125-127oC; IR (KBr, cm-1): 3010, 1605 (C=C), 1677 (C=N),
1
1030; HNMR (400MHz, δ, ppm, CDCl3): 7.37-8.15( m, 4H, Ar-H), 8.0 ( s, 1H, J= 6.26Hz, 3’-
13
H), 7.62 (dd, 1H, J=6.26, 1.8Hz, 5’-H), 7.24 (dd, 1H, J= 1.8 Hz, 6’-H); C NMR (100 MHz, δ,
ppm, CDCl3): 161.81, 150.1, 141.125, 135.66, 133.89, 129.54, 128.90, 128.12, 127.88, 124.98,
124.11, 119.859, 110.372.
4.1.2.2. 2-(4’-chlorophenyl)-1, 3-benzoxazole (2b)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 75%, m. p.: 144-148oC; IR (KBr, cm-1): 3015, 1657 (C=N), 1607 (C=C),
1
1035 (C-O), 780; HNMR (400MHz, δ, ppm, CDCl3): 7.27-8.07( m, 4H, Ar-H), 7.92 (ddd, 2H,
J=8.2, 1.8Hz, 3’ & 5’-H), 7.814 (ddd, 1H, J= 1.6, 8.4 Hz, 2’ & 6’-H); 13C NMR (100 MHz, δ, ppm,
CDCl3): 164.81, 151.1, 140.125, 135.70, 129.35, 129.35, 128.88, 128.88, 127.57, 124.92, 124.15,
119.859, 110.383.
4.1.2.3. 2-(4’-bromophenyl)-1, 3-benzoxazole (2c)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 71%, m. p.: 150-152oC; IR (KBr, cm-1): 3011, 1640 (C=N), 1600 (C=C),
1550, 1034 (C-O), 730; 1HNMR (400MHz, δ, ppm, CDCl3): 7.42-8.02( m, 4H, Ar-H), 7.83 (ddd,
2H, J=8.5, 1.76Hz, 3’ & 5’-H), 7.77 (ddd, 1H, J= 1.58, 8.36 Hz, 2’ & 6’-H); 13C NMR (100 MHz,
δ, ppm, CDCl3): 162.81, 150.8, 142.125, 136.70, 132.35, 132.35, 129.88, 129.88, 127.67, 124.98,
124.20, 119.859, 112.
4.1.2.4.
2-(3’-Nitrophenyl)-1, 3-benzoxazole (2d)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 79%, m. p.: 99-100oC; IR (KBr) (max, cm-1): 3010, 1639 (C=N), 1599
(C=C), 1450, 1350, 1036; 1HNMR (400MHz, δ, ppm, CDCl3): 8.88 ( ddd, 1H, J= 1.6, 1.0, 1.56Hz,
2’- H), 8.58 (dt, 1H, J=1.8, 8.6, 1.56 Hz, 4’-H), 7.58-8.2( m, 4H, Ar-H), 8.18 (dt, 1H, J=1.6, 1.8,

8.0Hz, 6’-H), 7.68 (ddd, 1H, J= 7.96, 8.02, 1.02 Hz, 5’-H); 13C NMR (100 MHz, δ, ppm, CDCl3):
163.81, 151.3, 142.125, 140.27, 127.29, 127.15, 125.22, 124.99, 124.11, 119.89, 117.30, 116.65,
110.372.
4.1.2.5.
2-(2’-Nitrophenyl)-1, 3-benzoxazole (2e)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 72%, m. p.: 98-103oC; IR (KBr, cm-1): 3011, 1632 (C=C), 1677 (C=N),
1550, 1380, 1030; 1HNMR (400MHz, δ, ppm, CDCl3): 8.78 ( ddd, 1H, J= 1.0, 1.78, 7.9Hz, 3’- H),
8.02 (dd, 1H, J=, 8.6, 7.56 Hz, 4’-H), 7.58-8.2( m, 4H, Ar-H), 8.18 (dt, 1H, J=1.6, 8.0Hz, 6’-H),
8.23 (ddd, 1H, J= 7.96, 8.02, 1.02 Hz, 5’-H); 13C NMR (100 MHz, δ, ppm, CDCl3): 162.98, 153.3,
142.125, 140.27, 127.29, 127.15, 125.22, 124.99, 124.11, 119.89, 117.30, 116.65, 110.372.
4.1.2.6.
2-(2’-Chlorophenyl)-1, 3-benzoxazole (2f)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 70%, m. p.: 68-72oC; IR (KBr, cm-1): 3010, 1657 (C=N), 1607 (C=C), 1035
1
(C-O), 780; HNMR (400MHz, δ, ppm CDCl3): 7.32-8.17( m, 4H, Ar-H), 7.9 (dt, 2H, J=8.2,
1.8Hz, 3’-H), 7.80 (dt, 1H, J= 1.6, 8.4 Hz, 5’-H); 13C NMR (100 MHz, δ, ppm, CDCl3): 163.81,
150.3, 140.25, 135.80, 129.85, 129.15, 128.78, 128.18, 127.77, 125.02, 124.75, 118.89, 113.383.
4.1.2.7. 2-(4’-Nitrophenyl)-1, 3-benzoxazole (2g)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 79%, m. p.: 256-260oC; IR (KBr, cm-1): 3010, 1639 (C=N), 1599 (C=C),
1450, 1350, 1036; 1HNMR (400MHz, δ, ppm CDCl3): 8.98 ( dd, 2H, J= 1.6, 8.2Hz, 3’ & 5’- H),
13
8.56 (dd, 2H, J=8.3, 1.7Hz, 2’ & 6’-H), 7.58-8.2( m, 4H, Ar-H); C NMR (100 MHz, δ, ppm,
CDCl3):164.81, 152.3, 142.125, 140.27, 131.21, 131.21, 125.99, 125.11, 120.89, 118.30, 117.65,
117.65, 113.372.
4.1.2.8. 2-Phenyl-1, 3-benzoxazole (2h)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 78%, m. p.: 98-100oC; IR (KBr, cm-1): 3010, 1605 (C=C), 1550 (C=N),

1030; 1HNMR (400MHz, δ, ppm CDCl3): 7.37-8.15( m, 4H, Ar-H), 7.17-7.80( m, 4H, Ar’-H); 13C
(100 MHz, δ, ppm, CDCl3):162.81, 150.1, 141.125, 128.90, 128.90, 128.88, 127.99, 127.99, 127.1,
124.98, 124.11, 119.859, 110.372.
4.1.2.9. 2-(4’-Methoxyphenyl)-1, 3-benzoxazole (2i)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 77%, m. p.: 114-116oC; IR (KBr, cm-1): 3010, 1620 (C=N), 1590 (C=C),
1210, 1030; 1HNMR (400MHz, δ, ppm CDCl3): 7.29-7.90 ( m, 4H, Ar-H), 7.89 ( dd, 2H, J= 1.75,
8.8 Hz, 2’ & 6’- H), 7.34 (dd, 2H, J=8.8, 1.75Hz, 3’ & 5’-H), 3.84 (s, 3H, OMe); 13C NMR (100
MHz, δ, ppm, CDCl3): 164.399, 160.42, 150.11, 128.92, 128.92, 127.43, 124.89, 124.211, 119.85,
114.783, 114.783, 110.347, 55.89.
4.1.2.10.
2-(4’-Methylphenyl)-1, 3-benzoxazole (2j)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 79%, m. p.:104-106oC; IR (KBr) (max, cm-1): 3030, 1605 (C=N), 1580
(C=C), 1030; 1HNMR (400MHz, δ, ppm, CDCl3): 7.30-8.08( m, 4H, Ar-H), 7.84 ( dd, 2H, J= 1.5,
13
7.8 Hz, 2’ & 6’- H), 7.52 (dd, 2H, J=7.88, 1.5Hz, 3’ & 5’-H), 2.34 (s, 3H, Me); C NMR (100
MHz, CDCl3) (δ, ppm): 161.81, 150.1, 141.125, 135.66, 133.89, 129.54, 128.90, 128.12, 127.88,
124.98, 124.11, 119.859, 110.372.
4.1.2.11.2-(3’-Chlorophenyl)-1, 3-benzoxazole (2k)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 72%, m. p.: 120-122oC; IR (KBr) (max, cm-1): 3020, 1635 (C=N), 1600
(C=C), 1030, 760; 1HNMR (400MHz, δ, ppm, CDCl3): 7.88 ( dt, 1H, J= 1.6, 1.0, 1.56Hz, 2’- H),
7.82 (dd, 1H, J= 8.6, 1.56 Hz, 4’-H), 7.46-8.10 ( m, 4H, Ar-H), 7.78 (dt, 1H, J=1.6, 1.8Hz, 6’-H),
7.68 (dd, 1H, J= 7.86, 8.2 Hz, 5’-H); 13C NMR (100MHz, δ, ppm, CDCl3):162.81, 151.1, 140.125,
138.66, 135.89, 127.54, 125.90, 124.88, 124.12, 123.98, 120.11, 119.59, 110.72.
4.1.2.12. 2-(4’-Hydroxy, 5’-nitrophenyl)-1, 3-benzoxazole (2l)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 75%, m. p.:170-174oC; IR (KBr, cm-1): 3200, 3010, 1630 (C=N), 1600
(C=C), 1550, 1350, 1030; 1HNMR (400MHz, δ, ppm, CDCl3):8.8 (dd, 1H, J=1.65, 2.8Hz, 6’-H),

8.25 (dd, 1H, J=1.6, 7.9Hz, 2’-H), 7.52-8.02 ( m, 4H, Ar-H), 7.35 (dd, 1H, J=7.9,2.6 Hz, 3’-H);
13CNMR (100MHz, δ, ppm, CDCl3): 161.81, 150.1, 141.125, 135.66, 133.89, 129.54, 128.90,
128.12, 127.88, 124.98, 124.11, 119.859, 110.372.
4.1.2.13.2-(2’-Hydroxy, 5’-nitrophenyl)-1, 3-benzoxazole (2m)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 71%, m. p.: 188-192oC; IR (KBr, cm-1): 3210, 1635 (C=N), 1600 (C=C),
1580,1359, 1030; 1HNMR (400MHz, δ, ppm, CDCl3): 8.79 (dd, 1H, J=3.11, 1.7 Hz, 6’-H), 8.34
(dd, 1H, J=1.7, 7.6Hz, 3’-H), 7.52-8.02 ( m, 4H, Ar-H), 7.38 (dd, 1H, J=8.6,1.4 Hz, 4’-H);13C
NMR (100MHz, δ, ppm, CDCl3): 161.81, 150.1, 141.125, 135.66, 133.89, 129.54, 128.90, 128.12,
127.88, 124.98, 124.11, 119.859, 110.372.
4.1.2.14.2-(4’-Fluorophenyl)-1, 3-benzoxazole (2n)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 79%, m. p.: 90-92oC; IR (KBr, cm-1): 3010, 1660 (C=N), 1610 (C=C), 1030
1
1000; HNMR (400MHz, δ, ppm, CDCl3): 7.30-8.05( m, 4H, Ar-H), 7.83 ( dd, 2H, J= 1.2, 8.12
Hz, 2’ & 6’- H), 7.34 (dd, 2H, J= 8.12, 1.02 Hz, 3’ & 5’-H); 13C NMR (100MHz, δ, ppm, CDCl3):
164.4, 163.35, 150.11, 141.125, 133.4, 133.4, 127.5, 124.98, 124.33, 119.85, 117.8, 117.98,
110.76.
4.1.2.15.
2-(3’-Bromophenyl)-1, 3-benzoxazole (2o)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 72%, m. p.: 133-137oC; IR (KBr, cm-1): 3010, 1655 (C=N), 1610 (C=C),
1030, 760; 1HNMR (400MHz, δ, ppm, CDCl3):7.42-8.1 ( m, 4H, Ar-H), 7.77 ( dt, 1H, J= 1.5Hz,
2’- H), 7.64 (dd, 1H, J=1.5, 8.1Hz, 4’-H), 7.57 (dt, 1H, J= 1.5, 1.4, 7.8 Hz, 6’-H), 7.51 (dd, 1H, J=
8.0, 7.8 Hz, 5’-H) ; 13C NMR (100MHz, δ, ppm, CDCl3):163.67, 150.11, 141.125, 132.5, 130.89,
130.11, 129.89, 127.99, 125.76, 124.98, 124.311, 119.03, 110.9.
4.1.2.16.
2-(4’-N-dimethylaminophenyl)-1, 3-benzoxazole (2p)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 70%, m. p.: 179-182oC; IR (KBr, cm-1): 3100, 3010, 1605 (C=N), 1590

(C=C), 1030; 1HNMR (400MHz, δ, ppm, CDCl3):7.2-7.8 ( m, 4H, Ar-H), 7.8 ( dd, 2H, J= 1.45,
8.8 Hz, 2’ & 6’- H), 6.75 (dd, 2H, J= 8.8, 1.5 Hz, 3’ & 5’-H), 2.89 (s, 6H, Me); 13C NMR (100MHz,
δ, ppm, CDCl3):: 164.4, 151.22, 150.11, 141.125, 131.3, 131.3, 127.87, 124.90, 124.311, 119.85,
111.3, 111.3,110.2, 40.5.
4.1.2.17.
2-(5’-Bromo-4’-hydroxy-3’-methoxyphenyl)-1,3-benzoxazole (2q)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 78%, m. p.: 187-192oC; IR (KBr, cm-1): 3250, 3010, 1610 (C=N), 1590
(C=C), 1030, 780; 1HNMR (400MHz, δ, ppm, CDCl3): 7.16-7.88 ( m, 4H, Ar-H), 7.53 ( d, 1H, J=
13
2.0Hz, 2’- H), 7.32 (d, 1H, J=2.0Hz, 6’-H), 4.5 (brs, 1H, OH), 3.7(s, 1H, OMe); C NMR (100
MHz, δ, ppm, CDCl3): 163.67, 150.1, 149.8, 148.7, 141.25, 130.87, 125.96, 124.93, 124.112,
119.89, 110.49, 110.3, 110.25, 55.21.
4.1.2.18.
2-(3’,5’-Dibromo-4’-hydroxyphenyl)-1,3-benzoxazole (2r)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 70%, m. p.: 166-169oC; IR (KBr, cm-1): 3300, 3010, 1630 (C=N), 1600
1
(C=C), 1030, 780; HNMR (400MHz, δ, ppm, CDCl3): 7.16-7.89 (m, 4H, Ar-H), 7.96 (d, 2H,
13
J=2.0Hz, 2’ & 6’-H), 4.5 (brs, 1H, OH); C NMR (100MHz, δ, ppm, CDCl3):163.67, 152.11,
150.4, 141.3, 130.71, 130.71, 111.08, 111.08, 110.37, 124.97, 124.211, 125.52.
4.1.2.19.
5-Bromo-2-(4’-nitrophenyl)-1, 3-benzoxazole (4a)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 80%, m. p.: 228-232oC; IR (KBr, cm-1): 3010, 1650 (C=N), 1600 (C=C),
1550, 1380, 1030; 1HNMR (400MHz, δ, ppm, CDCl3): 8.28 (dd, 2H, J=8.8Hz, 3’ & 5’-H), 7.99(
d, 1H, J= 8.6Hz, 7- H), 7.97 (dd, 1H, J=8.8Hz, 2’ & 6’-H), 7.82 (d, 1H, 4-H), 7.53 (dd, 1H,
J=8.6Hz, 6-H); 13C NMR (100MHz, δ, ppm, CDCl3):164.04, 150.03, 142.53, 131.3, 131.3, 129.37,
128.62, 124.27, 119.66, 118.03, 117.3, 117.3, 114.7. HRMS: (M+H) Obs: 317.196; Cal: 317.218.
4.1.2.20.5-Bromo-2-(4’-bromophenyl)-1, 3-benzoxazole (4b)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 72%, m. p.: 158-162oC; IR (KBr, cm-1): 3010, 1655 (C=N), 1620 (C=C),
1030, 780, 760; 1HNMR (400MHz, δ, ppm, CDCl3):8.06 (s, 1H, J=2.7Hz, 4-H), 7.87(dd, 1H, J=

2.7, 8.3Hz, 7- H), 7.83 (dd, 1H, J=8.6Hz, 2’ & 6’-H), 7.80 (dd, 2H, J=8.6Hz, 3’ & 5’-H), 7.28 (dd,
13
1H, J=7.9Hz, 6-H); C NMR (100MHz, δ, ppm, CDCl3):164.04, 149.63, 141.23, 132.6, 132.6,
129.37, 128.62, 128.62, 119.66, 118.03, 117.87, 117.2, 114.7. HRMS: (M+H) Obs: 350.90
(100.0%); Cal: 350.89.
4.1.2.21.2-(5-Bromophenyl-1, 3-benzoxazol-2yl)quinoxaline (4c)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 80%, m. p.: 189-192oC; IR (KBr, cm-1): 3010, 1665 (C=N), 1617 (C=C),
1030, 780; 1HNMR (400MHz, δ, ppm, CDCl3): 9.3 (s, 1H, 3-H), 8.47 (ddt, 1H, J= 8.2, 1.9, 3.5 Hz,
5-H), 8.36 ( dt, 1H, J= 3.5, 1.9, 8.4Hz, 8- H), 8.06 (dt, 1H, J=8.4, 7.9, 6-H), 7.87 (dd, 1H, J= 8.4,
7.3Hz, 7’-H), 7.(dd, 1H, J= 7.7Hz, 7-H), 7.89 (dd, 1H, J= 8.5Hz, 6’-H), 7.72 (dt, 1H, J=7.9Hz, 5’-
H); 13C NMR (100MHz, δ, ppm, CDCl3):161.5, 152.01, 149.7, 145.8, 142.35, 141.9, 141.78,
132.52, 129.28, 128.56, 128.08, 119.43, 117.84. HRMS: (M+H)+Obs: 326.26; Cal: 326.25.
4.1.2.22.
2- (1, 3-benzoxazol-2-yl) quinoxaline (4d)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 74%, m. p.: 160-164oC; IR (KBr, cm-1): 3010, 1650 (C=N), 1607 (C=C),
1030; 1HNMR (400MHz, δ, ppm, CDCl3):9.4 (s, 1H, 3-H), 8.47 (ddt, 1H, J= 8.12, 3,48, 1.96 Hz),
8.35 ( dd, 1H, J= 8.4Hz, 8- H), 8.1 (dd, 1H, J= 8.6Hz), 8.03 (dd, 1H, J=8.0, 6-H), 7.99 (dd, 1H, J=
8.4, 4.5Hz, 7’-H), 7.96 (dd, 1H, J= 7.7Hz, 7-H), 7.89 (dd, 1H, J= 8.5Hz, 6’-H), 7.72 (dt, 1H,
J=7.9Hz, 5’-H) ; 13C NMR (100MHz, δ, ppm, CDCl3): 161.4, 152.04, 148.58, 145.64, 142.29,
141.53, 141.01, 131.10, 128.8, 127.97, 125.44, 124.3, 121.14, 115.41, 111.54. HRMS:
(M+H)+Obs: 247.08; Cal: 247.07.
4.1.2.23.
3-(1, 3-benzoxazol-2yl) - 3-chloroquinoline (4e)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 75%, m. p.: 102-108oC; IR (KBr, cm-1): 3010, 1665 (C=N), 1637 (C=C),
1030, 830; 1HNMR (400MHz, δ, ppm, CDCl3): 7.37-8.15( m, 4H, Ar-H), 8.0 ( s, 1H, J= 6.26Hz,
3’- H), 7.62 (dd, 1H, J=6.26, 1.8Hz, 5’-H), 7.24 (dd, 1H, J= 1.8 Hz, 6’-H); 13C NMR (100MHz, δ,
ppm, CDCl3): 161.81, 150.1, 141.125, 135.66, 133.89, 129.54, 128.90, 128.12, 127.88, 124.98,
124.11, 119.859, 110.372. HRMS: (M+H)+Obs: 280.05; Cal: 280.04.

4.1.2.24.3-(5-Bromophenyl- 1, 3-benzoxazol-2yl) - 3-chloroquinoline (4f)
This compound was prepared according to general procedure and it was obtained as buff
coloured solid; yield: 76%, m. p.: 170-175oC; IR (KBr, cm-1): 3010, 1660 (C=N), 1620 (C=C),
1030, 830; 1HNMR (400MHz, δ, ppm, CDCl3): 7.37-8.15 ( m, 4H, Ar-H), 8.0 ( s, 1H, J= 6.26 Hz,
3’- H), 7.62 (dd, 1H, J=6.26, 1.8Hz, 5’-H), 7.24 (dd, 1H, J=1.8 Hz, 6’-H); 13C NMR (100MHz, δ,
ppm, CDCl3):161.81, 150.1, 141.125, 135.66, 133.89, 129.54, 128.90, 128.12, 127.88, 124.98,
124.11, 119.859, 110.372. HRMS: (M+H)+Obs: 294.07; Cal: 294.06
4.2. Biological Evaluation
4.2.1. Virtual screening: Protein Structure Preparation
The molecular docking study was performed using Molegro Virtual Docker (MVD-2013, 6.0).
The crystal structure of the enoyl- ACP reductase (InhA) complexed with an isonicotinic-acyl-
NADH inhibitor and epidermal growth factor receptor tyrosine kinase domain with 4-
anilinoquinazoline inhibitor erlotinib (PDB ID: 1M17) were downloaded from Protein Data Bank
(PDB ID: 1ZID). Molecular docking studies of the synthesized compounds/ligands were
performed in order to understand the various interactions between the ligand and enzyme active
site in detail. The molecular docking study was performed for the target compounds by using
MVD-2013 (Version: 6.0).
4.2.2. Molecular Docking Study
The synthesized compounds were built using Chemdraw 11.0. The 2D structures were then
converted into energy minimized 3D structures and were saved as MDL Molfile (.mol2). The
coordinate files and crystal structures of enoyl-acpreductase (InhA, PDB ID: 1ZID) and tyrosine
kinase (PDB ID: 1M17) were obtained from the RCSB PDB website. The protein files were
prepared by the removal of water molecules, addition of polar hydrogens and removal of other
bound ligands. In the present study, the binding sites were selected based on the amino acid
residues, which are involved in binding with isonicotinic-acyl-NADH inhibitor of InhA and
erlotinib as obtained from protein data bank, which would be considered as the probable best

accurate regions as they are solved by experimental crystallographic data. The docking protocol
was carried out for the synthesized compounds as listed in Table 4 using MVD-2013 (6.0) software
using the standard operating procedures.
4.2.3. Anti-cancer Evaluation:
4.2.3.1. General procedure for evaluation of anti-cancer inhibitory activity
The anti-cancer activity of the compounds has been accessed by MTT assay. Initially, MTT
stock solution has been prepared by taking 5mg in 1 mL of PBS. MCF-7 cell line has been used
for the study. The cell line was maintained in 96 wells micro titer plate containing MEM media
supplemented with 10% heat inactivated fetal calf serum(FCS), containing 5% mixture of
Gentamicin (10µg), Penicillin (100units/mL) and streptomycin (100µg/mL) in presence of 5%
CO2 at 37oC for 48-72 hours. In-vitro growth inhibition effect of test compounds was assessed by
colorimetric or spectrophotometric determination of conversion of MTT into Formazan blue by
living cells. Initially the supernatant from the plate was removed and fresh MEM solution was
added and was treated with different concentrations of extract or compound appropriately diluted
with DMSO. In our study, 10, 20, 25, 30 and 50µL (10mg/mL prepared in DMSO) of stock
solutions were added to respective wells containing 100µL of the medium. Thereby making the
final concentrations as 10, 20, 25, 30 and 50µg/mL. After 48hrs of incubation at 37oC in a
humidified atmosphere of 5% CO2, stock solution of 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl
tetrazolium bromide was added to each well (20µL, 5mg per mL in sterile phosphate buffered
saline) for further 4 hour incubation. The supernatant carefully aspirated, the precipitated crystals
of Formazan blue were solubilised by adding DMSO and optical density was measured at
wavelength of 570 nm by using LISA plus. The results were represented as mean of five readings
upto the concentration at which the OD of treated cells was reduced by 50% with respect to the
untreated control. During this assay, reduction of yellow 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyl tetrazolium bromide was measured by mitochondrial succinate dehydrogenase.
Formula: Surviving cells= Mean OD of test compound x100
Mean OD at control
4.2.3.2. Enzyme inhibition studies

Cell Treatment- The cells were seeded at a density of approximately 1×105cells/well in a 96-
well flat-bottom micro plate and maintained at 37ºC in 95% humidity and 5% CO2 for overnight.
Cells were treated with different concentration of test samples. Then cells were incubated for
another 24 hours. The cells in well were washed twice with phosphate buffer solution, and 1 ml of
extraction buffer was added. Using cell scraper cells were recovered carefully and centrifuge the
cells at 4ºC for 10 min at 10,000 rpm. Collect the supernatant and store as further analysis.
Tyrosine Kinase Assay- Collected supernatant was diluted 25 times with kinase reacting solution
provided along with kit. The diluted control was added and treated to sample in each well in
duplicate. Then 10 µl of 40 mM ATP-2Na solution was added into each well and mixed well.
Further, incubated for 30 min at 37ºC.Then the samples were removed, the wells were washed 3
times with wash buffer. 100µl of blocking solution was added into each well and incubated for 30
min at 37ºC. The blocking solution was discarded and then 50 µl of Anti-phosphotyrosine - HRP
solution was added into each well and incubatedt for 30 min at 37ºC. Now, the antibody solution
was discarded and then each well was washed 4 times with washing buffer. Further, 100 µl of HRP
substrate solution (TMBZ) was added into each well. Incubated for 30 min at 37ºC. Finally, 100
µl of stop solution was added into each well in the same order as HRP substrate solution. The
absorbance is measured at 450 nm with a plate reader.
% Inhibition = 1- (Abs of sample /Abs of control) x 100
4.3. Cell morphology studies by Fluorescence microscopy
4.3.1.1. Double staining (Acridine orange-Ethidium bromide):
The cells were seeded at a density of approximately 1x104cells/well in a 24 well flat bottom
micro plate containing cover slips and maintained at 37oC in CO2 incubator for overnight. More
than the IC50 of synthesised compounds was treated at 72 hrs. After the incubation, cells were
washed with PBS and fixed with 4% paraformaldehyde for 30min. 20 µL of dye mixture was
incubated for half an hour, examined under fluorescent microscope.
4.3.1.2. DAPI:
The cells were seeded at a density of approximately 1X105cells/well in a 12 well flat bottom
micro plate containing cover slips and maintained at 37oC in CO2 incubator for overnight. More

than the IC50 of synthesised compounds was treated at 72 hrs. After the incubation, cells were
washed with PBS and fixed with 4% paraformaldehyde for 30min. 20 µL of dye mixture was
incubated for 20min, examined under fluorescent microscope
5. Acknowledgements
This research did not receive any specific grants from funding agencies in the public,
commercial, or not-for-profit sector. The authors acknowledge Department of Science and
Technology, Goa. We are grateful to the Department of Chemistry, Dnyanprassarak Mandal’s
College and Research Centre for Laboratory Facilities. Authors also thank Sophisticated
Analytical Instrumentation Facilities Chandigarh, India for spectroscopic data and Maratha
Mandal’s NGH Institute of Dental Sciences and Research Centre for bioactivity results.
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