Convenient method for the synthesis of some novel chiral methyl 2-(2-oxo-2H-benzo[e][1,3]oxazin-3(4H)-yl)propanoate derivatives and biological evaluation of their antioxidant, cytotoxic, and molecular docking properties

Article abstract of DOI:10.1002/jhet.4196

Ten chiral methyl 2-(2-oxo-2H-benzo[e][1,3]oxazin-3(4H)-yl)propanoate derivatives 6a-6j have been synthesized from optically pure amino methyl phenol 5 and 4-nitrophenyl chloroformate. These derivatives 6a-6j are characterized by ¹H NMR, ¹³C NMR, FT-IR, and HRMS spectral techniques. Optical purity of these derivatives was confirmed by chiral HPLC method. Ten synthesized ester derivatives 6a-6j were screened for their in vitro antioxidant activity. Among the compounds 6b-d and 6h-j have exhibited comparable antioxidant activity with ascorbic acid as a standard. Compounds 6a and 6e-g have shown moderate antioxidant activity. Further, the in vitro cytotoxicity of these compounds were studied through MTT cell proliferation assay in addition the effect on LDH leakage and NO release. Among the derivatives, 6j showed extremely best activity and the IC₅₀ value (12.54 ± 0.71 μM) is very close to doxorubicin (7.2 ± 0.58 μM) as a standard. Compounds 6b, 6h, and 6i showed better inhibition next to compound 6j on the viability of HepG2 cells with an IC₅₀ value (μM) of 56.02 ± 1.4, 41.76 ± 0.58, and 38.17 ± 0.34, respectively. Also, molecular docking studies have been carried out with STAT-3 (PDB ID: 1BG1) and BCL-2 (PDB ID: 4AQ3) proteins against the four active compounds 6b, 6h, 6i, and 6j. The binding energies of the tested compounds were in the range of ?7.76 to ?8.41 kcal/mol, which is very close to doxorubicin (?8.53 kcal/mol) as a standard. These molecular docking results are in good agreement with the in vitro studies.

Full text of DOI:10.1002/jhet.4196

Convenient Method for the Synthesis of Some Novel Chiral
Methyl 2-(2-oxo-2H-benzo[e][1,3]oxazin-3(4H)-yl)propanoate
Derivatives and Biological Evaluation of their Antioxidant,
Cytotoxic and Molecular Docking Properties
Matam Sivakumara, Kaliyan Prabakarana, Selvaraj Loganathana Muthu Seenivasa
Perumala* Lohanathan Bharathi Priyab, Viswanadha Vijaya Padmab*, Himesh Makalac,
Venkatasubramanian Ulaganathanc*
a
Department of Chemistry, The Gandhigram Rural Institute-Deemed to be University, Gandhigram, Dindigul district,
Tamilnadu- 624 302, India. Corresponding author: E. mail: mspchem99@gmail.com,
m.seenivasaperumal@ruraluniv.ac.in
Department of Biotechnology, Bharathiar University, Coimbatore, Tamilnadu, India.
b
c
Department of Biotechnology, School of Chemical and Biotechnology, SASTRA Deemed University, Tanjavur district,
Tamilnadu, India.
Received: ((will be filled in by the editorial staff))
Abstract: Ten chiral methyl 2-(2-oxo-2H-benzo[e][1,3]oxazin-
Keywords: Aminomethyl phenol; Benzoxazolones;
Antioxidant activity; Cytotoxicity; Molecular docking
3(4H)-yl)propanoate derivatives 6a-6j have been synthesized from
optically pure amino methyl phenol
5
and 4-Nitrophenyl
chloroformate. These derivatives 6a-6j are characterized by 1H NMR,
13C NMR, FT-IR, and HRMS spectral techniques. Optical purity of
these derivatives were confirmed by chiral HPLC method. Ten
synthesized ester derivatives 6a-6j were screened for their in vitro
antioxidant activity. Among the compounds 6b-d and 6h-j have
exhibited comparable antioxidant activity with ascorbic acid as a
standard. Compounds 6a, 6e-g have shown moderate antioxidant
activity. Further, the in vitro cytotoxicity of these compounds were
studied through MTT cell proliferation assay in addition the effect on
LDH leakage and NO release. Among the derivatives 6j showed
extremely best activity and the IC50 value (12.54 ± 0.71 µM) is very
close to doxorubicin (7.2 ± 0.58 µM) as a standard. Compounds 6b,
6h, and 6i showed better inhibition next to compound 6j on the
viability of HepG2 cells with an IC50 value (µM) of 56.02 ± 1.4, 41.76
± 0.58, and 38.17 ± 0.34 respectively. Also, molecular docking
studies have been carried out with STAT-3 (PDB ID: 1BG1) and
BCL-2 (PDB ID: 4AQ3) proteins against the four active compounds
6b, 6h, 6i and 6j. The binding energies of the tested compounds
were in the range of -7.76 to -8.41 kcal/mol, which is very close to
doxorubicin (-8.53 kcal/mol) as a standard. These molecular docking
results are in good agreement with the in vitro studies.
cerebrovascular diseases,29 antimicrobial agents30 anti-
Introduction
fungal31 and acetylcholinesterase inhibitors32 (Figure
1).These derivatives are used for the treatment of several
In several clinical pharmaceuticals, benzoxazolone
derivatives are used as an important pharmacophore and
also these compounds possess a major role in numerous
biological activities. In the medicinal world drug
designing and developing purpose these derivatives are
used as an advanced skeleton and best unit cell. In 1998
FDA had approved Efavirenz (Sustiva), it is an anti HIV
drug and non-nucleoside reverse transcriptase
inhibitor.1-2 Hence, in the recent years many chemist
focused and developed these derivatives to enhance the
efficiency of anti HIV drug activity compared to
Efavirenz.3-6 Benzoxazolone derivatives exhibits
important biological activities like neurological,7-9
cardiovascular diseases27,
control of hypertension
33-34
and offered good oral pharmacological development in
rats. It is also used to cure some of the skin diseases.35
It is an active anti-depressant drug and anti-reserpine
activity in number of animal species.36-39 2-oxo-2H-1,3-
benzoxazine-3(4H)-acetamide, it is a new psychotropic
agent in clinical studies and it possess a good relevant
anti-depressant activity.40 Hence, we have interested to
develop the convenient method for the synthesis of
chiral methyl 2-(2-oxo-2H- benzo[e] [1,3]oxazin-3(4H)-
yl)propanoate derivatives and studied their biological
activity. Since liver cancer (hepatocellular carcinoma) is
the most common and growing challenges of global
health with increasing frequency in developing
countries,41 we chose HepG2 cells as a hepatocellular
allergy,10,
autoimmune,11
anti-inflammatory,12-15
psychiatric,16 anti-tumor,17 metabolic disorders,18
cytotoxicity activities,19 antimalarial activity,20-21 anti
AIDS,22-26
cardiovascular,27
anti-cancer,28
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which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1002/jhet.4196
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carcinoma model to test the antitumor activity of
synthesized target compounds. HepG2 cells are liver
cancer cells and has been used in cancer research for
toxicity testing and target identification/validation of
several natural and synthetic compounds.42
benzo[e][1,3]oxazin-3(4H)-yl)propanoate 6, we report
herein the synthesis of chiral methyl 2-(2-oxo-2H-
benzo[e][1,3]oxazin-3(4H)-yl)propanoate derivatives 6
starting from corresponding optically pure amino acid 1.
Initially, the condensation of amino esters 2 with
salicylaldehyde 3 to form imine 4, followed by
reduction with NaBH(OAc)3 to form chiral aminomethyl
phenol 5 and cyclization by using commercially
available, cheap and easy handling of 4-nitrophenyl
chloroformate as a carbonyl source. The reaction
proceeded smoothly to afford chiral methyl 2-(2-oxo-
2H-benzo[e][1,3]oxazin-3(4H)-yl)propanoate
derivatives 6 in good yields (Scheme 1)
Synthesis of benzo [e][1, 3]oxazin ester derivatives 6
The chiral aminomethyl ester hydrochloride 2 was
synthesized from commercially available optically pure
amino acid 1 by well-known procedure reported in
earlier43 and the desired products were obtained in good
yields (92-98%). These chiral aminomethyl ester
hydrochlorides 2 were treated with salicyaldehyde3 and
triethylamine at room temperature in the presence of
molecular sieves 4Å to give imines 4 with the yields of
Figure 1. Some examples of biologically active
Benzoxazolones.
Results and Discussion
To pursue our interest in the synthesis and biological
activity
of
chiral
methyl
2-(2-oxo-2H-
Scheme 1. S General scheme of synthesis of compound 6: a) SOCl2, MeOH, reflux for 3 h; b) TEA, DCM/ MS 4Ao, RT for 3 h;
c) NaBH(OAc)3, DCM/AcOH at 0-5oC for 5 h; d) 4-Nitrophenyl chloroformate/ DCE/ THF/TEA, reflux for 5 h
82-90%. Further, the imines 4 have been reduced by
single solvent. When the reaction was carried out using
o
using NaBH(OAc)3 in DCM/AcOH at 0-5 C to give
compound 5j, 4-nitrophenyl chloroformate in DCE (6
o
aminomethyl phenols 5 in 88-94% yield. Then the chiral
mL) in the presence of TEA at 80 C for 8 h gave 84%
methyl
2-(2-oxo-2H-benzo[e][1,3]oxazin-3(4H)-
yield of desired product 6j (Table 1, entry 4). When we
performed the reaction under the same condition except
solvent CH3CN gave 76% yield of 6j (Table 1, entry 5).
While replacing the solvent such as THF and Ethyl
yl)propanoate derivatives, 6 was synthesized via
cyclization of corresponding amino methylphenols 5
with 4-nitrophenyl chloroformate as a carbonyl source
in a single step (step d). Efforts were undertaken
towards the cyclisation of aminomethyl phenol 5 with 4-
nitrophenyl chloroformate. We have examined the
reaction of aminomethyl phenol 5j with 4-nitrophenyl
chloroformate in dichloromethane in the presence of
triethylamine at room temperature for 22 h to give only
o
acetate instead CH3CN at 70 C for 8 h gave 6j with
86% and 74% yields respectively (Table 1, entry 3 & 9).
In order to increase the yield, the reaction was carried
out using mixture of solvents DCM: CH3CN (6 mL; 1:1)
for 6 h at 65 oC gave 84% yield of 6j (Table 1, entry 6).
When we carried out the reaction using DCE:CH3CN at
75 oC for 5 h gave 6j with 87% yield (Table 1, entry 7).
The reaction was performed by using DCE:THF (6 mL;
45% yield of compound 6j (Table 1, entry 1). The same
o
reaction was repeated at 40 C for 8 h to obtain 73%
o
yield of compound 6j (Table 1, entry 2). In order to
optimize the reaction conditions, we have performed the
reaction in various solvents and different temperatures.
The results are summarized in table 1. The performance
of the reactions were better in mixture of solvents than
1:1) as solvent mixture at 80 C for 5 h gave desired
product 6j with 91% yield (Table 1, entry 8).
Table 1. Optimization of the reaction condition with various
solvents
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cheaper than DBU. Hence, TEA is chosen as a base and
DCE: THF as the solvent for these reactions.
Table 2. Optimization of reaction condition with various base
Entrya Solvent
Temp. (oC) Time Yield
Entry a Base
Time (h)
Yield (%)b
78
(h)
(%)b
1
2
3
4
5
6
DIPEA
8
8
5
8
5
8
8
1
2
3
4
5
6c
DCM
DCM
THF
27
40
70
80
80
65
22
8
45
73
86
84
76
84
DMAP
TEA
69
91
8
DABCO
DBU
58
DCE
8
91
CH3CN
8
K2CO3
NaOH
Trace
Trace
DCM:
6
7
CH3CN
a)
All the reactions were carried out using compound 5j (1.19
7c
DCE:
75
5
87
mmol), 4-Nitrophenyl chloroformate (1.31 mmol) in
DCE:THF (6 mL; 1:1) in the presence of different bases (2
eq) at 80 oC for 5 h. b) Yields are of isolated product.
CH3CN
8c
DCE:THF 80
5
8
8
8
91
74
80
83
In general the organic bases such as DIPEA, DMAP,
TEA, DABCO and DBU would give better yields than
inorganic bases like K2CO3 and NaOH. Probably, the
solubility of organic bases were better than inorganic
bases in DCE:THF. With optimized conditions in hand,
we have examined the reaction with different
9
EA
70
60
70
10c
11c
a)
EA:DCM
EA:THF
aminomethyl
phenols
5
with
4-nitrophenyl
o
chloroformate in DCE:THF at 80 C for 5 h in the
presence of TEA. The reactions were carried out with
optically pure form aminomethyl phenols 5 with 4-
nitrophenyl chloroformate and the desired products 6
were obtained in 81-91% yields with >98% ee. The
results are shown in Figure 2. All the synthesised
aminomethyl phenols gave good yields on cyclization
with 4-nitrophenyl chloroformate. The main advantage
of 4-nitrophenyl chloroformate is good replacement for
highly carcinogenic phosgene and costly CDI reagents.
According to above observations, the plausible reaction
mechanism was proposed. As shown in Figure 3, the
reaction using 4-nitrophenyl chloroformate is initiated
by base.The base abstract the proton present in the
hydroxyl group 5, followed by the nucleophilic
phenolate attacks the acyl group present in compound 8,
to form the adduct 9, which eliminate the 4-nitro phenol
to give the intermediate 10. Finally, amino group of
intermediate 10 attacked on the acyl group in an
intramolecular fashion gave the desired product 6
All the reactions were carried out using compound 5j (1.19
mmol), 4-nitrophenyl chloroformate (1.31 mmol) and TEA
(2.38 mmol) in an appropriate solvent (6 mL), temperature
b)
and time mentioned in the table.
Yields are of isolated
c)
product. All the reactions were carried out using compound
5j (1.19 mmol), 4-Nitrophenyl chloroformate (1.31 mmol)
and TEA (2.38 mmol) in a mixture of solvent ( 6 mL; 1:1),
temperature and time mentioned in the table.
Once we have optimized the solvents, we have screened
the reaction with different bases to achieve good yields.
The obtained results are summarized in the table
2.When we performed the reactions with TEA and DBU
as bases afforded 91% of yield within 5 h (Table 2,
entry 3 & 5). Though the both bases would gave same
yield, partial racemisation was observed when DBU
used as base. When we used TEA as base, good yield
was obtained without racemisation and also TEA is
without
racemization.
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Figure 2. Synthesis of methyl 2-(2-oxo-2H-benzo[e][1,3]oxazin-3(4H)-yl)propanoate derivatives 6.
Figure 3. Plausible mechanism
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Biology
increases, the radical scavenging activity increases
and lower IC50 value reflects better protective action.
From results, it may be claimed that compounds 6b-d
and 6h-j were able to reduce the stable free radical
DPPH to the yellow-colored diphenylpicrylhydrazine
revealing good free radical scavenging activity as
compared with ascorbic acid as a standard.
Antioxidant activity
On reduction of the radicals of DPPH with
appropriate reducing agents, result of which forming
the corresponding hydrazine and electrons become
paired off. Therefore, the solution loses its colour
stoichiometrically subjecting on the number of
electrons taken up. Substances capable of giving
electrons/hydrogen atoms are able to convert DPPH
(purple colour) into their non- radical form 1, 1-
diphenyl-2- picrylhydrazine (yellow colour), this
Cytotoxic studies
In order to determine the anticancer properties of the
synthesised compounds and to exhibit the potential
differences in the anticancer activity between the
derivatives 6a-6j, MTT cell proliferation assay was
carried in human hepatoma HepG2 cells, which aims
in the measurement of mitochondrial dehydrogenase
activity as an indication of cell viability after an
exposure to the compounds 6a-6j between the
concentration range of 1 and 100 µM incubated for
48 h time period. Of all the benzoxazolone
derivatives 6a-6j, compounds 6b, 6h, 6i and 6j
showed better inhibition on the viability of HepG2
cells with an IC50 value (µM) of 56.02 ± 1.4, 41.76 ±
0.58, 38.17 ± 0.34 and 12.54 ± 0.71 respectively
(Table 4 & Figure 4). Among these, compound 6j
showed a highly significant effect on the inhibitory
action on HepG2 cell viability. Anticancer drug,
doxorubicin (DOX) was used as the positive control.
reaction
which
can
be
followed
spectrophotometrically.44
All the synthesized 2-oxo-2H-benzo[e][1,3]oxazin-
3(4H)-yl ester derivatives 6a-6j were subjected for
evaluation of their free radical scavenging activity
with ascorbic acid as standard compound. The IC50
value was calculated for each individual compound as
well as ascorbic acid, results were summarized in
table 3 and shown in supplementary information
figures 1-11. It was observed that as the concentration
of the test compound increases, the scavenging effect
also increases. All the synthesized compounds
showed good radical scavenging activities. The IC50
value of compounds 6b-d and 6h-j were 0.121x10-3,
0.119x10-3, 0.122x10-3 and 0.122x10-3, 0.122x10-3,
0.122x10-3 respectively which were comparable with
IC50 value (0.103x10-3) of standard ascorbic acid. The
IC50 values of compounds 6a, 6e-g were slightly
higher than the standard (table 3). DPPH radical
easily accepts an electron or hydrogen radical to form
a stable diamagnetic molecule.
Table 4. Cytotoxicity of compounds 6a-6j on HepG2
cells (MTT cell viability assay
)
IC50
Entry
Compound
concentration
(µM ± SE)
1
84.11 ± 0.52
56.02 ± 1.4
6a
6b
6c
6d
6e
6f
Table 3. Anti-oxidant activity study for compounds 6a-6j
2
IC50 Value
Entry
Compound
(1×10-3) M
0.103
0.282
0.121
0.119
0.122
0.341
0.157
0.392
0.122
0.122
0.122
3
241.27 ± 0.68
129.68 ± 1.13
164.17 ± 0.72
246.35 ± 0.66
181.41 ± 1.06
41.76 ± 0.58
38.17 ± 0.34
12.54 ± 0.71
7.2 ± 0.58
1
Ascorbic acid
4
2
6a
6b
6c
6d
6e
6f
5
3
4
6
5
7
6g
6h
6i
6
8
7
8
6g
6h
6i
9
9
10
11
6j
10
11
6j
Doxorubicin
Compound 6b showed significantly higher activity
compared to the compound 6a. This may be due to
the presence of isopropyl group present in the α-
position of the amino ester. Among the compounds
Free radical scavenging activity of the chiral methyl
2-(2-oxo-2H-benzo[e][1,3]oxazin-3(4H)-
yl)propanoate
derivatives
are
concentration
dependent, as the concentration of the test sample
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factors.46 Exposure of HepG2 cells with the IC50
value of compound 6j showed a significant leakage
on LDH and NO release comparable to the standard
anticancer drug doxorubicin (Table 5 and 6).
6h-6j, compound 6j exhibited a significant decrease
in cell viability compared to compound 6h. Possible
ways in the significant exhibition of inhibition on cell
viability of these compound on HepG2 cells may be
due to the presence of tert-butyl group present in the
α-position of the amino ester, which may increase the
bulkiness of the molecule, may allow to bind a
specific site of the protein. Also, the IC50 value of
compound 6j on HepG2 cells was nearer to the
standard drug doxorubicin. In general, among the
compounds 6a, 6b, 6j increase the bulkiness of in the
α-position of the amino ester, shows increase in the
inhibitory activity shown in table 4.
Table 5. Effect of Compounds 6a-6j on Lactate
Dehydrogenase (LDH) leakage
.
Entry
Compound
LDH (in %)
26.87 ± 0.95
21.29 ± 0.91
17.8 ± 1.02
25.36 ± 0.67
36.78 ± 1.03
23.45 ± 1.6
24.23 ± 0.82
27.58 ± 2.22
42.15 ± 1.02
55.48 ± 0.99
59.13 ± 0.87
1
6a
6b
6c
6d
6e
6f
2
3
4
5
6
7
6g
6h
6i
8
9
10
11
6j
Doxorubicin
Figure 4. Cell viability assay: HepG2 cells were exposed
to the synthesized compounds (between 1 and 100 µM) for
48 hours and their IC50 values were obtained using a dose
response curve by nonlinear regression using a curve
fitting program, Graph Pad Prism Software Version 5.0.
Treatment with doxorubicin (DOX) served as a positive
control. Each bar represents mean ± SEM from three
independent experiments performed in triplicates.
Table 6. Effect of Compound 6a-6j on Nitric oxide
(NO) release
NO (nM of nitrite
released)
Compound
Compound
Effect of compounds on LDH leakage and NO
release
1
2
3
4
5
6
7
8
9
10
71.24 ± 1.13
65.16 ± 0.83
32.14 ± 1.69
63.45 ± 1.33
104.8 ± 1.5
62.35 ± 2.28
61.25 ± 2.39
49.56 ± 1.6
142.1 ± 3.45
164.13 ± 2.5
6a
6b
6c
6d
6e
6f
To examine the cytotoxic effect on the IC50 value of
these compounds 6a-6j on HepG2 cell line, assay on
lactate dehydrogenase (LDH) leakage and nitric
oxide release into the extracellular medium was
performed. Both LDH and NO serves as an excellent
marker for the determination of cellular toxicity
despite the reports on other cytotoxic enzyme
markers since, their use has been limited by the
presence of low amount in many cells and requires an
elaborate kinetic assay to measure most enzyme
activities. Treatment with anticancer/cytotoxic
compounds leads to the release of cytoplasmic LDH
into the culture medium upon loss of membrane
integrity resulting due to cell death signalling.45
Hence LDH activity has been used as an indicator of
cell membrane integrity and serves as a general
means to assess cytotoxicity resulting from exposure
of chemical compounds or environmental toxic
6g
6h
6i
6j
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multifunctional gaseous molecule and a highly
reactive free radical NO plays a crucial role in
regulating several physiological and pathological
processes including vascular functions (angiogenesis,
blood flow, vascular permeability, leukocyte–
endothelial interaction, platelet aggregation and
microlymphatic flow), neurological functions and,
concentration dependently regulate cytotoxic
functions (cytostasis and cytolysis).48 NO produced
endogenously from L-Arginine by the action of NO
synthase (NOS) exists in three isoforms. Of these,
two isoforms exhibit a constitutive expression and the
third isoform exhibits an inducible expression (iNOS)
which is expressed in response to stress. Under
oxidative stress conditions, increased expression of
iNOS was reported leading to increased nitric oxide
accumulation. Effects of NO in tumours rest on the
activity and localization of NOS isoforms,
concentration and duration of NO exposure, and
cellular sensitivity to NO.49,50 Our present results
evidences the correlation between the concentration
dependent decline in HepG2 cell viability to NO
release upon exposure to the synthesized compounds
6a-6j. A significantly higher concentration of nitrite,
i.e., 164.13 nM in condition of HepG2 cells exposed
to compound 6j shows its higher potency compared
to other compounds (Figure 6).
11
181.24 ± 1.24
Doxorubicin
This indicating that a significant decline in cell
viability with the IC50 of compound 6j compared to
other compounds might have occurred by a
significant loss in membrane integrity evident by the
increased LDH activity and nitrite content in the
culture medium compared to other compounds
(Figure 5 and Figure 6) with 48 h of incubation
period.
Figure 5. LDH leakage: Exposure of HepG2 cells to the
synthesized compounds resulted in high levels of LDH
leakage. The bar represents mean ± SEM from three
independent experiments performed in triplicates.
Molecular Docking studies
The docking studies were performed for four
potential compounds 6b, 6h, 6i and 6j against the
target proteins apoptosis regulator STAT-3 (PDB
ID: 1BG1) and BCL-2 (PDB ID: 4AQ3). The
result indicates that these derivatives have higher
binding energies for both the target protein apoptosis
regulator STAT-3 and BCL-2. In the STAT-3, k370
and T440 residues forms two hydrogen bonds with
the compound 6b (D…A=3.2Å and 2.49Å)
respectively. C251 also forms a hydrogen bond with
the compound 6h (D…A=3.07Å).
Figure 6. Nitric oxide assay: Results shown are nmoles of
nitrite released by HepG2 cells upon exposure to the
synthesized compounds. Each bar represents mean ± SEM
from three independent experiments performed in
triplicates.
Mechanistic action of these compounds needs further
elucidation to delineate the differential action of each
specific compound in HepG2 cells. One possible
mechanism behind the cytotoxic action of these
compounds may be the induction of oxidative stress
evident from the increased NO release measured in
Table 7. Binding energies obtained from molecular
docking studies for the synthesized compounds 6a-6j.
Binding
Protein
Signal
and activator of
transcription 3
Ligand name energy
(kcal/mol)
transducer
-7.99
-8.42
-7.57
-8.76
6b
6h
6i
or
6j
STAT-3 (PDB ID:
1BG1)
-8.97
Doxorubicin
-8.15
-8.02
6b
6h
Apoptosis Regulator
BCL-2 (PDB ID:
terms of the stable oxidation product nitrite.47 Being a
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compounds. In general, ligand molecules were
surrounded by 9 residues: F63, Y67, M74, V92, E95,
L96, G104, R105 and A108. The binding energies of
the tested compounds are in the range of -7.76 to -
8.41 kcal/mol. The binding energy for the control
molecule (doxorubicin) has -8.53 kcal/mol (Table 7).
The root mean square deviation (RMSD) of standard
compound doxorubicin was determined by docking
and re-docking was found to be 0.59Å. The binding
regions of tested inhibitors were shown in the Figure
8. This represents that the docking procedure will
4AQ3).
-7.76
-8.41
-8.53
6i
6j
Doxorubicin
In common, ligand molecules were surrounded by 14
residues: W243, K244, R245, Q247, Q248, V322,
V323, Q326, T456, H457, S458, L459, N485 and
P487. The binding energies of the tested compounds
are in the range of -7.57 to -8.76 kcal/mol. The
binding energy for the control molecule
(Doxorubicin) has -8.97 kcal/mol (Table 7). The root
mean square deviation51 (RMSD) of standard
compound doxorubicin was determined by docking
and re-docking was found to be 0.36 Å. The binding
regions of tested inhibitors were shown in the Figure
7. In the BCL-2, the active site residues D70 and
R123 forms direct hydrogen bonds with the tested
constantly
reproduce
the
receptor
bound
conformation with present ligand molecules. In
general, all the four 6b, 6h, 6i and 6j ligand
molecules were showed promising results against
both STAT-3 and BCL-2 proteins. These molecular
docking results were in good agreement with the in
vitro cytotoxic studies. Hence, they may act as
potential inhibitors.
Figure 7. (a – d) The Ligplot images representing the amino acids within 4 Å region of STAT-3 and their corresponding
interactions with the inhibitors. e) The cartoon representation of STAT-3 protein with the potential inhibitors (sticks) 2D
image showing the inhibitors interacting with the residues of STAT-3 protein.
This article is protected by copyright. All rights reserved.

Figure 8. (a – d) The Ligplot images representing the amino acids within 4 Å region of BCL-2 and their corresponding
interactions with the inhibitors. e) The cartoon representation of BCL-2 protein with the potential inhibitors (sticks).
A. C. McCreary, Eur. Neuropsychopharmacol.
Conclusions
2010, 20, 582.
In conclusions, we have developed convenient
method for synthesis of novel chiral methyl 2-(2-oxo-
2H-benzo[e][1,3]oxazin-3(4H)-yl)propanoate
[4] O. Diouf, P. Carato, P. Depreux, J. P. Bonte, D.
H. Caignard, B. G. Lemaitre, M. C. Rettori, C.
Belzung, D. Lesieur, Bioorg. Med. Chem. Lett.
1997, 7, 2579.
derivatives by using 4-nitrophenyl chloroformate as a
carbonyl source. This method provides several
advantages such as inexpensive reagents, avoid toxic
phosgene, easy synthetic procedure and good yields.
It is expected to be useful synthetic procedure for the
synthesis of wide variety of drug-like molecules
contains benzoxazinone derivatives 6. All the
synthesized compounds 6a-j were screened for
antioxidant activity and cytotoxicity studies. Among
the synthesized compound 6c exhibits an excellent
antioxidant activity and 6j exhibits an excellent
cytotoxicity. The four 6b, 6h, 6i and 6j active
compounds were screened for molecular docking
studies and the results were in good agreement with
the in vitro cytotoxic studies. Hence, these derivatives
may act as potential inhibitors.
[5] P. Kowalski, M. J. Mokrosz, Z. Majka, T.
Kowalska, B. Duszynska, J. Heterocyclic. Chem.
2000, 37, 187.
[6] S. M. Ceccarelli, G. Jaeschke, B. Buettelmann, J.
Huwyler, S. Kolczewski, J. U. Peters, E. Prinssen,
R. Porter, W. Spooren, E. Vieira, Bioorg. Med.
Chem. Lett. 2007, 17, 1302.
Supplementary information
[7] T. G. Gant, S. Sarshar, 3H-benzooxazol-2-one
modulators of D2 receptor and/or 5-HT1A
receptor, US20100119622A1, 2010.
The Supplementary information file containing
general procedure for the preparation of
aminomethylester hydrochlorides derivatives 2,
aminomethyl phenol 5 and methyl 2-(2-oxo-2H-
benzo[e][1,3]oxazin-3(4H)-yl)propanoate derivatives
6. Characterization data for compounds 2, 5, 6 and
Copies of NMR, Mass spectras and Chiral HPLC
chromatograms.
[8] D. J. Johnson, I. T. Forbes, S. P. Watson, V.
Garzya, G. I. Stevenson, G. R. Walker, H. S.
Mudhar, S. T. Flynn, P. A. Wyman, P. W. Smith,
G. S. Murkitt, A. J. Lucas, C. R. Mookherjee, J.
M. Watson, J. E. Gartlon, A. M. Bradford, F.
Brown, Bioorg. Med. Chem. Lett.2010, 20, 5434.
Acknowledgements
We are thankful to the SERB, New Delhi, UGC-BSR, New Delhi
for a research grant. K. Prabakaran thank SERB, New Delhi for
financial support. We are also grateful to DST, New Delhi for
support under “DST-FIST” and UGC-SAP (DRS-II) program.
[9] R. Pahwa, K. E. Lyons, Curr. Opin. Neurol. 2014,
27, 442.
References
[10]D. Gottschling, G. Dahmann, H. Doods, A.
Dreyer, S. G. Mueller, K. Rudolf, G. Schaenzle,
D. Stenkamp, Patent WO 2009034028, 2009.
[1] J. Poupaert, P. Carato, E. Colacino, Curr. Med.
Chem. 2005, 12, 877.
[11]Z. Soyer, M. Bas, A. Pabuccuoglu, V.
Pabuccuoglu, Arch. Pharm. Chem. Life Sci. 2005,
338, 405.
[2] J. P. Bonte, M. C. Piancastelli, I. Lesieur, J. C.
Lamar, M. Beaughard, G. Dureng, Eur. J. Med.
Chem.1990, 25, 361.
[12]A. A. Hare, L. Leng, S. Gandavadi, X. Du, Z.
Cournia, R. Bucala, W. L. Jorgensen, Bioorg.
Med. Chem. Lett. 2010, 20, 5811.
[3] C. A. Jones, L. C. Johnston, M. J. Jackson, L. A.
Smith, G. V. Scharrenburg, S. Rose, P. G. Jenner,
This article is protected by copyright. All rights reserved.

[13]L. Tang, W. H. Ma, Y. L. Ma, S. R. Ban, X. E.
Feng, Q.S. Li, Bioorg. Med. Chem. Lett. 2013, 23,
2380.
M. Klabe, S. E. Viitanen, G. L. Trainor, S. P.
Seitz, Bioorg. Med. Chem. Lett. 1999, 9, 2805.
[24]B. L. Deng, M. D. Cullen, Z. Zhou, T. L.
Hartman, R. W. J. Buckheit, C. Pannecouque, E.
D. Clercq, P. E. Fanwick, M. Cushman, Bioorg.
Med. Chem. 2006, 14, 2366.
[14]E. Laurini, D. Zampieri, M. G. Mamolo, L. Vio,
C. Zanette, C. Florio, P. Posocco, M. Fermeglia,
S. Pricl, Bioorg. Med. Chem. Lett. 2010, 20, 2954.
[15](a) A. H. Abdelazeem, A.G. Safi El-Din, M. T.
El-Saadi, S. M. El-Moghazy, N. H. Amin.
Research J. Pharm. and Tech. 2020, 13, 4255. (b)
G. Eren, S. Unlu, M. T. Nunez, L. Labeaga, F.
Ledo, A. Entrena, E. Banoglu, G. Costantino, M.
F. Sahin, Bioorg. Med. Chem. 2010, 18, 6367.
[25]M. Patel, R. J. J. McHugh, B. C. Cordova, R. M.
Klabe, S. E. Viitanen, G. L. Trainor, S. S. Koo,
Bioorg. Med. Chem.Lett. 1999, 9, 3221-3226.
[26]T. Sakamoto, M. D. Cullen, T. L. Hartman, K. M.
Watson, R. W. Buckheit, C. Pannecouque, E. D.
Clercq, M. Cushman, J. Med. Chem. 2007, 50,
3314.
[16]P. Zhang, A. M. K. Pennell, J .J. K. Wright, L. Li,
E. J. Sullivan, W. Chen, K. L. Greenman, Patent
WO 2007/022257, 2007.
[27]G. Q. Shi, J. F. Dropinski, B. M. McKeever, S.
Xu, J. W. Becker, J. P. Berger, K. L. MacNaul, A.
Elbrecht, G. Zhou, T. W. Doebber, P. Wang, Y. S.
Chao, M. Forrest, J. V. Heck, D. E. Moller, A. B.
Jones, J. Med. Chem. 2005, 48, 4457.
[17]P. D. St-Coeur, D. Ferguson, P. J. Morin, M.
Touaibia, Arch. Pharm. Chem. Life Sci. 2013,
346, 91.
[18]P. Szymanski, A. Janik, E. Zurek, E. Mikiciuk-
[28]M. Delot, P. Carato, C. Furman, A. Lemoine, N.
Lebegue, P. Berthelot, S. Yous, Synthesis 2009,
22, 3819.
Olasik. Pharmazie, 2011, 66, 399.
[19]N. Kaushal, M. J. Robson, H. Vinnakota, S.
Narayanan, B. A. Avery, C. R. McCurdy, R. R.
Matsumoto, AAPS. J. 2011, 13, 336.
[29]Q. Y. Hu, J. Papillon, Patent, WO 2010130773,
2010.
[20]F. E. Saenz, A. N. LaCrue, R. M. Cross, J. R.
Maignan, K. O. Udenze, R. Manetsch, D. E. Kyle,
Antimicrob. agents chemother. 2013, 57, 6187.
[30]J. Shavel, J. G. Bobowski, US Patent, 3446804,
1969.
[31]H. Bentoumi, N. Chettibi, A. Djrourou, N. E.
Aouf, M. Liacha,. Sci. Bull. B Chem. Mater. Sci.
UPB 2020, 82, 7.
[21]Y. Zhang, J. A. Clark, M. C. Connelly, F. Zhu, J.
Min, W. A. Guiguemde, A. Pradhan, L. Iyer, A.
Furimsky, J. Gow, T. Parman, F. E. Mazouni, M.
A. Phillips, D. E. Kyle, J. Mirsalis, R. K. Guy, J.
Med. Chem. 2012, 55, 4205.
[32]H. Sugimoto, M. Yonaga, N. Karibe, Y. Iimura,
S. Nagato, A. Sasaki, Y. Yamanishi, H. Ogura, T.
Kosasa, K. Uchikoshi, K. Yamatsu, EP0468187,
1990.
[22]M. He, H. Zhang, X. Yao, M. Eckart, E. Zuo, M.
Yang, Chem. Biol. Drug Des. 2010, 76, 174.
[33](a) A. Sala, F. Benedini, R. Cereda, P. D. Soldato,
European Patent, 0490183A1, 1991. (b) A. Sala,
[23]M. Patel, S. S. Ko, R. J. J. McHugh, J. A.
Markwalder, A. S. Srivastava, B. C. Cordova, R.
This article is protected by copyright. All rights reserved.

F. Benedini, R. Cereda, P. D. Soldato,
Håkansson, P. R. Hansen, J. Svenson, Sci. Rep.
2020, 10, 13206. (b) G. Li, J. Zhou, M. Sun, J.
Cen, J. Xu, J. Funct. Foods, 2020, 74, 104196. (c)
J. Tan, P. Li, H. Xue, Q. Li, Biotechnol. Lett.
2020, 42, 2453.
EP0490183B1, 1991.
[34]G. Bertolini, A. Sala, Curr. Opin.Ther.Patents,
1993, 3, 1499.
[35]F. Benedini, G. Bertolini, R. Cereda, G. Dona, G.
Gromo, S. Levi, J. Mizrahi, A. Sala, J. Med.
Chem. 1995, 38, 130.
[43](a) S. M. Jones, J. E. Urch, M. Kaiser, R. Brun, J.
L. Harwood, C. Berry, I. H. Gilbert, J. Med.
Chem. 2005, 48, 5932. (b) Q. H. Song, W. J.
Tang, X. M. Hei, H. B. Wang, Q. X. Guo, S. Q.
Yu, Eur. J. Org. Chem. 2005, 6, 1097.
[36]S. Cecchini, P. Petri, R. Ardito, S. R. Bareggi, A.
Torriti, J. Int. Med. Res. 1978, 6, 388.
[44]M. Z. Gul, F. Ahmad, A. K. Kondapi, I. A.
Qureshi, I. A. Ghazi. BMC Complement. Altern.
Med. 2013, 13, 1.
[37]L. Conti, L. Bonollo, A. Martini, P. Fornaro, A.
Guerrini. C. Romerio, E. Scillieri, S. Soverini. B.
M. Stramb and G. B. Cassano, Curr. Ther. Res.
1980,27, 458.
[45]R. Baskaran, L. B. Priya, P. Kalaiselvi, P.
Poornima, C. Y. Huang, V. V. Padma, Biomed.
Pharmacother. 2017, 93, 730.
[38]G. K. Suchowsky and L. Pegrassi, Arzneim.
Forsch. 1969, 19,643.
[46]L. B. Priya, R. Baskaran, P. Elangovan, V.
Dhivya, C. Y. Huang, V. V. Padma, Biomed.
Pharmacother. 2017, 87, 280.
[39]G. K. Suchowsky, L. Pegrassi, A. Moretti and A.
Bonsignori, Arch. Int. Phurmacodyn. 1969, 182,
332.
[47]H. Vahora, M. A. Khan, U. Alalami, A. Hussain,
[40]R. BYCK, Drugs and the treatment of psychiatric
disorders in, the pharmacologic basis of
therapeutics (5th ed.), edited by L. S. Goodman,
A. Gilman New York, MacMillan Publishing Co.
Inc., 1975, pp. 152-200.
J. Cancer. Prev, 2016, 21, 1.
[48]N. Rodriguez, J. A. R. Revilla, M. A. F. Ibanez,
J. C. Carretero, Chem. Sci. 2013, 4, 175.
[49]T. Mosmann, J. Immunol. Methods, 1983, 65, 55.
[41](a) D. Anwanwan, S. K. Singh, S. Singh, V.
Saikam, R. Singh, BBA - Reviews on Cancer,
2019,
[50]D. J. Stuehr, M. A. Marletta, J. Immunol. 1987,
139, 518.
https://doi.org/10.1016/j.bbcan.2019.188314. (b)
X. Huang, X. Zhang, X. Bai, T. Liang, Cell Mol.
Immunol. 2020, 17, 677. (c) L. Lin, L. Yan, Y.
Liu, C. Qu, J. Ni and H. Li, Liver Cancer 2020, 9,
563.
[51]G. M. Morris, D. S. Goodsell, R. S. Halliday, R
Huey, W. E. Hart, R. K. Belew, A. J. Olson, J.
Comput. Chem. 1998, 19, 1639.
[42](a) I. Greco, N. Molchanova, E. Holmedal, H.
Jenssen, B. D. Hummel, J. L. Watts, J.
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ARTICLE
Convenient Method for the Synthesis of Some
Novel
Chiral
Methyl
2-(2-oxo-2H-
benzo[e][1,3]oxazin-3(4H)-yl)propanoate
Derivatives and Biological Evaluation of their
Antioxidant, Cytotoxic and Molecular Docking
Properites
J. Heterocyclic Chem. Year, Volume, Page – Page
Matam Sivakumara, Kaliyan Prabakarana, Selvaraj
Loganathana, Muthu Seenivasa Perumala*
Lohanathan Bharathi Priyab, Viswanadha Vijaya
Padmab*, Himesh Makalac, Venkatasubramanian
Ulaganathanc*
This article is protected by copyright. All rights reserved.