Non-peptide-based new class of platelet aggregation inhibitors: Design, synthesis, bioevaluation, SAR, and in silico studies

Article abstract of DOI:10.1002/ardp.201700349

A series of 2-oxo-2-phenylethylidene linked 2-oxo-benzo[1,4]oxazine analogues 17a–x and 18a–o, incorporated with a variety of electron-withdrawing as well as electron-donating groups at ring A and ring C, were synthesized under greener conditions in excel

Full text of DOI:10.1002/ardp.201700349

Received: 8 November 2017
Revised: 7 February 2018
Accepted: 13 February 2018
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DOI: 10.1002/ardp.201700349
FULL PAPER
Non-peptide-based new class of platelet aggregation
inhibitors: Design, synthesis, bioevaluation, SAR, and
in silico studies
Pradeep K. Jaiswal1
Vashundhra Sharma1
Surendra Kumar2
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Manas Mathur3
Ajit K. Swami3
Dharmendra K. Yadav2,4
Sandeep Chaudhary1
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1 Laboratory of Organic and Medicinal
Chemistry, Department of Chemistry,
Malaviya National Institute of Technology,
Jaipur, India
Abstract
A series of 2-oxo-2-phenylethylidene linked 2-oxo-benzo[1,4]oxazine analogues
17a–x and 18a–o, incorporated with a variety of electron-withdrawing as well as
electron-donating groups at ring A and ring C, were synthesized under greener
conditions in excellent yields (up to 98%). These analogues 17a–x and 18a–o were
evaluated for their arachidonic acid (AA)-induced platelet aggregation inhibitory
activities in comparison with the standard reference aspirin (IC50 = 21.34 1.09 µg/
mL). Among all the screened compounds, eight analogues, 17i, 17x, 18f, 18g, 18h, 18i,
18l, and 18o, were identified as promising platelet aggregation inhibitors as compared
to aspirin. In addition, cytotoxic studies in 3T3 fibroblast cell lines by MTT assay of the
promising compounds (17i, 17x, 18f–18i, 18l, and 18o) were also performed and the
compounds were found to be non-toxic in nature. Furthermore, the results on the AA-
induced platelet aggregation inhibitory activities of these compounds (17i, 17x,
18f–18i, 18l, and 18o) were validated via in silico molecular docking simulation studies.
To the best of our knowledge, this is the first report of the identification of non-
peptide-based functionalized 2-oxo-benzo[1,4]oxazines as platelet aggregation
inhibitors.
2 College of Pharmacy, Gachon University of
Medicine and Science, Incheon City, Korea
3 Department of Advance Molecular
Microbiology, Seminal Applied Sciences Pvt.
Ltd., Jaipur, India
4 Department of Biochemistry, All India
Institute of Medical Sciences (AIIMS),
Jodhpur, Rajasthan, India
Correspondence
Prof. Sandeep Chaudhary, Laboratory of
Organic and Medicinal Chemistry, Department
of Chemistry, Malaviya National Institute of
Technology, Jawaharlal Nehru Marg,
Jaipur-302017, India.
Email: schaudhary.chy@mnit.ac.in
Dr. Dharmendra K. Yadav, College of
Pharmacy, Gachon University of Medicine and
Science, Incheon City-21936, Korea.
Email: dharmendra30oct@gmail.com
Funding information
K E Y W O R D S
National Research Foundation of Korea (NRF),
Grant number: 2017R1C1B2003380; SERB,
New Delhi for Fast Track Scheme for Young
Scientist, Grant number: CS-037/2013; DST-
RFBR Indo-Russian Joint Research Project,
Grant number: INT/RUS/RFBR/P-169; CSIR-
EMR Grant, New Delhi, Grant number: 02
(0189)/14/EMR-II
2-oxo-benzo[1,4]oxazine, cytotoxicity, in silico molecular docking, platelet aggregation
inhibitors, structure−activity relationship
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1
INTRODUCTION
diseases, such as ischemic stroke, myocardial infarction, angina
pectoris, and other cardiovascular diseases; and is responsible for
morbidity and mortality in developing countries.[1,2] Primarily, platelet
aggregation is a vital process in hemostasis formation which is being
activated by the enzyme thrombin and transforms the fibrinogen
(insoluble) into fibrin (soluble). It was firstly reported by Coller[3] (1960)
Platelet aggregation, which causes thrombosis, represents one of the
most important causes of cerebral stroke and arterial thromboembotic
Pradeep K. Jaiswal and Vashundhra Sharma contributed equally to this paper.
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Arch Pharm Chem Life Sci. 2018;e1700349.
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© 2018 Deutsche Pharmazeutische Gesellschaft
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that the platelet aggregation inhibitors might be the rationale for
antithrombotic therapy. Anti-thrombotic therapy, which represents
the treatment of thrombin platelet and thrombosis, are divided into
three categories: (1) anti-platelet drugs (such as aspirin, ticlopidine, and
indomethacin)[4]; (2) anti-coagulants or thrombin inhibiting drugs (such
as heparin and warfarin)[5]; and (3) thrombolytic drugs (such as fibrin
receptor antagonists).[6] Based on the mode of action, type 1 category,
i.e., platelet aggregation inhibitors can be further sub-divided into
three classes: (1) agents affecting arachidonic acid metabolism; (2)
platelet activation factor (PAF) receptor antagonists; and (3) agents
affecting ADP-dependent platelet aggregation pathway.[7]
anti-microbial,[15c] antifungal,[15d] COX-2 inhibitor,[15e] rennin inhib-
itor,[15f] and non-steroidal progesterone receptor agonists.[15g] Re-
cently, Zuo and co-workers reported benz[1,4]oxazine-3-one
derivatives as an efficient GPIIb/IIIa receptor antagonists and displayed
promising platelet aggregation inhibiting activity.[8,16] Dudley et al.[17]
reported that benz[1,4]oxazine-3-one derivatives are remarkable
inhibitors of Xa factor. Jakobson et al.[18] identified 2-aryl substituted
benz[1,3]oxazine-4-one class of compounds as inhibitors of the tissue
factor/factor VIIa-induced activation of factor Xa and in situ activates
the fibrin clot formation. Moreover, Kikelj et al.[19] and Ilaš et al.[20] had
also identified benz[1,4]oxazines as glycoprotein IIb/IIIa antagonists.
Furtherstructure–activityrelationship(SAR)studyonbenz[1,4]oxazine-
3-ones revealed them to show promising thrombin inhibitory and
fibrinogen receptor antagonist activity.[21]
The most clinically used anti-platelet drugs are either phosphodi-
esterase inhibitor/ADP receptor antagonists, or cyclooxygenase (COX)
inhibitor as well as GpIIb/IIIa receptor antagonist.[8] Arachidonic acid
(AA) is a fatty acid, which is liberated from the activated platelets and is
converted into a potent inducer of platelet aggregation by the enzyme
cyclooxyenase.[9] On the other hand, AA is also a precursor for
thromboxane A2 (TBXA2) synthesis, which can stimulate platelet
aggregation after conversion to prostaglandin G1 and H2.[10] Thus, in
the initial step, prostaglandin-endoperoxide synthase 1 (PTGS1; which
is also known as cyclooxygenase-1) catalyses the transformation of AA
into cyclic endoperoxide PG G2 and H2 and after that this PGG2 and
PGH2 are converted into TBXA2 by TBXA synthase in platelets.[10]
Therefore, the inhibition of cyclooxygenase-1 is an important target in
the identification of novel platelet aggregation inhibitors.[11] As a
result, several non-peptide mimics such as aspirin 1, tirofiban 2,
ticlopidine 3, clopidogrel 4, sulfinpyrazone 5, etc. have been developed
as a potent inhibitors of platelet aggregation[12] (Figure 1).
Recent literature revealed that 2-phenyl-4-quinolones have been
reported to show platelet aggregation inhibitory activity via COX-1
inhibitors. Moreover, several similar bioactive classes of compounds
were reported to show promising platelet aggregation inhibitory
activity such as functionalized 1,3-benzoxazines 6,[22a–c] 2-morpholino
substituted benzoxazines 7,[22d] benz[1,4]oxazine-3-ones 8,[16]
functionalized quinolin-4(1H)-ones 9,[22e,f] substituted coumarins 10,
etc.[22g] (Figure 2).
It has been well documented that 3-oxo-benz[1,4]oxazines
analogues 8 displayed good ADP, collagen, and PAF-induced
platelet aggregation inhibitory activity. However, the AA-induced
platelet aggregation inhibiting activities of 3-oxo-benz[1,4]oxazines
were less explored. Therefore, in our endeavor in search for novel
bioactive heterocycles as new antiplatelet agents, we designed
prototype 11, i.e., functionalized 2-oxo-benzo[1,4]oxazines incor-
porating subunits of 5–10 (Figure 2), and assessed their AA-induced
platelet aggregation inhibiting activities with the anticipation that
the 2-oxo-benz[1,4]-oxazines scaffold would also show promising
inhibitory activity. So far, literature report revealed that there is no
report available showing 2-oxo-benz[1,4]oxazines as platelet
aggregation inhibitory activity.
Herein, we report the ultrasonic-assisted synthesis,[23] platelet
aggregation inhibitory activity, SAR, and cytotoxic studies of a series of
functionalized 2-oxo-benzo[1,4]oxazines 17a–x and 18a–o, respec-
tively. We also report the validation of our activity results via in silico
molecular docking simulation studies. Although compounds 17a–x,
18a,b, and 18i have been prepared earlier by other routes[24]; however,
for the first time, compound 17b,c, 17g, 17q, 17s,t, 17v, 18a,b, and 18i
have been prepared via “on water” ultrasound-assisted methodology.
Interestingly, the antiplatelet aggregation activities of all the synthe-
sized compounds (17a–x and 18a–o) have never been reported. To the
best of our knowledge, for the first time, 2-oxo-benzo[1,4]oxazines,
17a–x and 18a–o, have been identified as new class of AA-induced
platelet aggregation inhibitors. In this study, aspirin was used as
standard reference. In addition, the cytotoxic studies of active
compounds (17i, 17x, 18f–18i, 18l, and 18o) using 3T3 fibroblast
cell lines in MTT assay were also performed. We also report the
validation of results via in silico molecular docking simulation studies of
active compounds 17i, 17x, 18f–18i, 18l, and 18o.
In spite of the efficacy of platelet aggregation inhibitory effect,
several antiplatelet agents have been withdrawn from the market as
these are associated with serious drawbacks such as adverse side-
effects, increased mortality rate, high toxicity, etc.[13] Therefore, the
identification of potent and safe platelet aggregation inhibitors is of
great interest to synthetic medicinal chemists.[14]
Benzoxazine class of compounds are endowed with a wide range of
biological activities[15] such as anti-tumor,[15a] anti-inflammatory,[15b]
FIGURE 1 Structures of some clinically used platelet aggregation
inhibitors

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FIGURE 2 Design strategy for the target functionalized 3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one as a new
class of potential platelet aggregation inhibitors
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2
RESULTS AND DISCUSSION
synthesized compounds were confirmed by their spectral analysis
(1H NMR, 13C NMR, FT-IR, and HRMS) (Scheme 1).
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2.1 Chemistry
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Recently, we reported two highly efficient, one pot, green methodol-
ogies for the synthesis of functionalized 2-oxo-benzo[1,4]oxazines
under mild conditions.[23a,b] The “on water” ultrasonic-assisted[23a]
methodology was utilized in the synthesis of a series of functionalized
2-oxo-benzo[1,4]oxazines 17a–x and 18a–o upto 98% yields. The
required starting material for the synthesis of prototype 11 i.e., diketo-
acids 15a–g were synthesized in excellent yields (upto 92%) via base-
catalyzed reaction of acetophenone 12a–g with dimethyl oxalate 13 in
toluene under refluxing condition for 6 h followed by the hydrolysis of
resulting diketoesters 14a–g with LiOH.H2O in MeOH/THF/
H2O (7:2:1; v/v) as solvents. While exploring target prototype 11,
we had observed that the reaction of nitro/alkyl/halide-substituted
2,4-dioxo-4-phenylbutanoic acid 15a–g with nitro/alkyl/halide-
substituted 2-aminophenol 16a–f or 16e–g in water furnished desired
pure functionalized 2-oxo-benzo[1,4]oxazines 17a–x and 18a–o in
excellent yields (up to 98%), respectively. The structures of all the
2.2 Biological evaluation
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2.2.1 Platelet aggregation inhibitory activity
evaluation
The generic structure of 2-oxo-benzo[1,4]oxazines is a bicyclic rings A
& B having pendant substituted 2-oxo-phenylidene ring C (Table 1).
Initially, we prepared 24 2-oxo-benzo[1,4]oxazines 17a–x having
various substituents at rings A and C, and evaluated for their
arachidonic acid (AA) induced-antiplatelet aggregation inhibitory
activities. In this study, the standard reference drug used was aspirin
which showed IC50 values of 21.34 1.09 µg/mL. As depicted from
Table 1, out of all compounds, compounds such as 17i, 17m, 17s, and
17x having halogen atom at ring C showed promising inhibitory
activities in comparison to the standard reference except compound
17k. The model compound 17a, which has no substitutions at ring A or

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SCHEME 1 Ultrasound-assisted one-pot green synthesis of 2-oxo-benzo [1, 4]oxazines analogues (17a–x and 18a–o)a
ring C, displayed more than two times lesser activity in comparison to
the standard reference. Then, we synthesized compounds 17b–x
having various electron-withdrawing groups (EWG) as well as
electron-donating groups (EDG) at ring A or ring C to interpret SAR
studies with the anticipation that these substituents might cause an
increase in the platelet aggregation inhibitory activity (Table 1).
Among all screened compounds, EDG (such as CH3, Cl, Br, etc.) at
ringA linkedwithEDG at ringC (compounds17b–h and 17j–o; entry 2–8
and entry 10–15; Table 1)displayed lesseractivity than aspirin. Whereas,
the compound 17i (IC50 = 22.87 0.26 μg/mL; entry 9; Table 1), which
has 3-methyl-6-bromo substituent at ring A and bromo substituent at
ring B displayed comparable platelet aggregation inhibitory activity
compared to aspirin. The compounds, which have EWG on 4- or
5-position at ring A (17p–w; entries 16–23; Table 1) exhibited lesser
inhibitory activity than both drugs aspirin, except compound 17x
(IC50 = 19.74 0.21 μg/mL; entry 24; Table 1), which has NO2 EWG on
5-position at ring A, displayed better platelet aggregation inhibitory
activity than standard drug aspirin. The promising compounds, 17i and
17x, found in our preliminary results prompted us to prepare further
functionalized 2-oxo-benzo[1,4]-oxazines having similar variations.
Based on the structure of active compounds, 17i and 17x, and the
preliminary biological results, we observed that the compounds having
bromo substituent at ring C on 4-position and EDG (such as CH3 and
Br) on 3- and 6-position at ring A as well as EWG (such as NO2) on
5-position at ring A were found to show potent platelet aggregation
inhibitory antiplatelet aggregation activity (Table 2). The standard
reference aspirin was analyzed again along with 18a–o.
As it can be seen from Table 2, all the 15 compounds 18a–o
exhibited moderate to excellent anti-platelet aggregatory activities
having IC50 values in the range of 17.96 0.18–98.75 1.46 µg/mL in
comparison to the reference drug aspirin (IC50 = 21.59 0.63).
Compounds having EWG such as NO2 at 5-position on ring A
(18a–d; entry 1–4) having halo or methoxy group on ring C displayed
lesser platelet aggregation inhibitory activity than aspirin. Whereas
6-Br and 4-Me groups at ring A along with F-, Cl-, OMe or CH3-group
on ring C i.e., compound 18e–h (entry 5–8) were found to show either
comparable platelet aggregation inhibiting activity (18g and 18h; entry
7–8) or greater inhibitory activity (18f; entry 6) than aspirin. Moreover,
while the compounds having EWG such as NO2 at 3-position of ring A
along with either no substitution (18i; entry 9) or EDG (such as Cl or Br)
at 4-position of ring C (18l and 18o; entry 12 and 15) displayed greater
inhibitory activity than aspirin; compounds having CH3-, OCH3-, or
F-groups exhibited lesser activity than aspirin. It is also noted that the
compound having NO2 EWG at ring A and 2,4-dichloro substituent on
ring C (18m; entry 13) further decreases the inhibitory activity in
comparison to the monochloro-substitution at ring A (18l; entry 12).
The SAR study indicates that the compounds with two EDG (i.e.,
3-methyl-6-bromo substituted) at ring A along with X-group (X = Br,
Cl) or OMe-group at ring C (i.e., 17i and 18f–h) displayed either equal
inhibitory activity (as in 17i and 18g,h) or greater (18f) aggregation
inhibitory activity than aspirin. In addition, the NO2 EWG at
3-position (18i, 18l, and 18o) or 5-position (17x) on ring A along
with either no substitution (18i) or X-group (X = Br, Cl) at 4-position
inhibitory activity. Therefore,
a new series of functionalized
2-oxo-benzo[1,4]oxazines (18a–o; Scheme 1) incorporating different
EWG and EDG at rings A and C were synthesized and assessed for their

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TABLE 1 In vitro AA-induced platelet aggregation inhibitory activity[22f] of a series of functionalized 2-oxobenzo[1,4]oxazines (17a–x)
Antiplatelet activitya,b
S. no.
1
Comp. no.
17a
17b
17c
R1
H
R2
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
–
R3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
–
R4
R5
H
R6
H
H
H
H
H
H
H
Br
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
–
(IC50 in μg/mL)
H
48.25 0.71
94.67 1.41
85.21 1.16
525 3.41
2
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Cl
H
3
CH3
OCH3
Cl
H
4
17d
17e
17f
H
5
H
95.4 1.43
6
F
H
93.37 1.38
467 2.89
7
17g
17h
17i
Br
H
8
H
H
87.32 0.96
22.87 0.26
92.94 1.38
33.97 0.54
531 3.44
9
Br
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
17j
H
H
17k
17l
CH3
Cl
Cl
H
Cl
H
17m
17n
17o
17p
17q
17r
F
Cl
H
30.11 0.37
91.23 1.36
473.45 2.47
455.38 2.28
91.23 1.36
88.23 1.28
28.12 0.22
87.03 1.21
469.21 2.31
89.11 1.27
84.21 1.13
19.74 0.21
21.34 1.09
Br
Cl
H
OCH3
H
Cl
H
NO2
NO2
NO2
NO2
NO2
NO2
NO2
H
H
CH3
OCH3
Cl
H
H
17s
H
17t
Cl
H
17u
17v
17w
17x
Aspirin
F
H
Br
H
H
NO2
NO2
–
Br
H
–
–
The bold values indicate promising anti-platelet activity of compounds.
aPlatelets were incubated along with either a tested compound or 0.5% DMSO at 37°C for 60 s, then AA (100 µM) was added to accelerate the aggregation.
Aspirin was used as positive control. Values are expressed as mean SE from three to six separations.
bThe data represent mean of three independent determinations.
on ring C (18l and 18o) exhibited greater platelet aggregation
The result showed that these compounds were non-toxic to 3T3
fibroblast cell lines even at 250 µg/mL concentration and conse-
quently displayed permissible values of cell viability (Figure 3).
inhibitory activity than aspirin.
2.2.2 Cytoxicity evaluation[25]
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2.3 In silico molecular docking studies[26]
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Out of 39 compounds, 8 compounds i.e., 17i, 17x, 18f, 18g, 18h, 18i,
18l, and 18o (showing promising or greater potency) were selected for
their cytotoxic studies using 3T3 fibroblast cell lines in MTT assay.[25]
To study the binding modes of the eight molecules (17i, 17x, 18f, 18g,
18h, 18i, 18l, and 18o) in the cyclooxygenase-1 (COX-1) enzyme, first

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TABLE 2 In vitro AA-induced platelet aggregation inhibitory activity of a new series of functionalized 2-oxo-benzo[1,4]oxazines (18a–o)
a,b
Antiplatelet activity
S. no.
1
Comp. no.
18a
R1
R2
H
H
Cl
H
H
H
Cl
H
H
H
H
H
Cl
H
H
–
R3
R4
H
R5
NO2
NO2
NO2
NO2
H
R6
H
H
H
H
Br
Br
Br
Br
H
H
H
H
H
H
H
–
(IC50 in μg/mL)
OCH3
Cl
H
82.35 1.11
28.12 0.22
98.75 1.46
41.11 0.47
45.97 0.65
19.83 0.22
20.08 0.24
21.47 0.24
16.96 0.18
29.87 0.30
36.54 0.35
19.19 0.22
41.35 0.53
44.08 0.64
17.96 0.18
21.59 0.63
2
18b
H
H
3
18c
Cl
H
H
4
18d
F
H
H
5
18e
CH3
OCH3
Cl
H
CH3
CH3
CH3
CH3
H
6
18f
H
H
7
18g
H
H
8
18h
F
H
H
9
18i
H
NO2
NO2
NO2
NO2
NO2
NO2
NO2
–
H
10
11
12
13
14
15
16
18j
CH3
OCH3
Cl
H
H
18k
H
H
18l
H
H
18m
18n
Cl
H
H
F
H
H
18o
Aspirin
Br
H
H
–
–
–
The bold values indicate promising anti-platelet activity of compounds.
aPlatelets were incubated along with either a tested compound or 0.5% DMSO at 37°C for 60 s, then AA (100 µM) was added to accelerate the aggregation.
Aspirin was used as positive control. Values are expressed as mean SE from three to six separations.
bThe data represent mean of three independent determination.
we performed molecular docking study with reference standard
compound aspirin on COX-1 domain (PDB ID: 2OYE) using Surflex-
Dock. After running Surflex-Dock, the conformers were ranked in a
molecular spread sheet based on their docking scores, and the best
docked score conformers were selected, and speculated concerning
the detailed binding mode in the active site.[26e,f] A docking score
represents binding affinity, which include hydrophobic, repulsive,
entropic, polar, and solvation. The docking results for 18i and 18o
against the antiplatelet target COX-1 showed a high binding affinity
docking score indicated by a total score of 5.5546 and 5.7941,
respectively, forms a H-bond of length 1.7 and 2.3 Å to the side chain
of nucleophilic (polar, hydrophobic) residue i.e., Ser-530. In the docking
pose of compound 18i and 18o-COX-1 complex, the chemical nature
of binding site residues within
a radius of 4 Å was aromatic
(hydrophobic), for example, Phe-381, Phe-518, Trp-387, Tyr-385,
Tyr-348; nucleophilic (polar, hydrophobic) residue Ser-353; hydro-
phobic, for example, Val-349, Ala-527, Gly-526, Ile-523, Leu-352, and
Met-522; as
a result, the bound compound showed a strong
hydrophobic interaction with COX-1, thus leading to more stability
and activity in this compound (predicted interactions of 18i (4A) and
18o (4B) with antiplatelet target enzyme COX-1, as given in
Figure 4A and B).
On the other hand, docking results for compound 18g and 18l
against the target protein COX-1 showed a high binding affinity
docking score indicated by a total score of 4.5281 and 4.4766,
respectively, forms a salt bridge of length 4.6 Å to the polar,
hydrophobic, positive-charged residue Arginine-120. In the docking
pose of the complex, the chemical nature of binding site residues
FIGURE 3 Percentage cell viability test

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FIGURE 4 (A and B) Predicted interactions of 18i (A) and 18o (B) with antiplatelet target enzyme COX-1 (PDB: 2OYE) with a docking total
score of 5.5546 and 5.7941, respectively, revealing H-bonds of length 1.7 and 2.3, respectively, to the binding site pocket residues Ser-530.
(C and D) Predicted interactions of 18g (C) and 18l (D) with antiplateles target enzyme COX-1 (PDB: 2OYE) with a docking total score of
4.5281 and 4.4766, respectively, revealing a salt bridge of length 4.6, to the binding site pocket residues Arg-120
within a radius of 4 Å was acidic (polar, negative charged), for example,
Glu-524; hydrophobic, for example, Val-116, Val-349, Ala-527, Ile-89,
Ile-523, Leu-93, Leu-531, Leu-359, Leu-352, and Met-113; nucleo-
philic (polar, hydrophobic) residue Ser-530, Ser-353; and aromatic
(hydrophobic), for example, Phe-518, Tyr-355; as a result, the bound
compound showed a strong hydrophobic interaction with COX-1, thus
leading to more stability and activity in this compound (predicted
interactions of 18g (4C) and 18l (4D) with antiplatelet target enzyme
COX-1, as shown in Figure 4C and D).
(hydrophobic), for example, Tyr-355; hydrophobic, for example, Leu-
359, Leu-531, Leu-352, Leu-93, Met-113, Ala-527, Ile-523, Ile-89,
Val-349, Val-116, as a result, the bound compound showed a strong
hydrophobic interaction with COX-1, thus leading to more stability and
significant activity in this compound (predicted interactions of 17i (5A)
and 17x (5B) with antiplatelet target enzyme COX-1, as shown in
Figure 5A and B).
Moreover, the docking results for compound 18f and 18h against
the target protein COX-1 showed a high binding affinity docking score
indicated by a total score of 5.6388 and 4.5653, respectively.
Compound 18f formed a π–π stacking with of Tyr-385, ─Cl atom of
pyridine ring formed π–π stacking with ─OH group of Tyr-385
(pyridine-Cl. . .OH, Tyr-385, 5.3 Å), and Tyr-355 ─F atom of pyridine
ring ─OH group of Tyr-355 (pyridine-F...OH, Tyr-355, 5.4 Å). In the
docking pose of the complex, the chemical nature of binding site
residues within a radius of 4 Å was hydrophobic, for example, Leu-352,
Leu-359, Leu-531, Ala-527, Val-349, Val-116, Ile-523; polar amide
type, for example, His-90; aromatic (hydrophobic), for example, Tyr-
355; nucleophilic (polar, hydrophobic) residue that is, Ser-353, Ser-
530; positive charged), for example, Arg-120; as a result, the bound
compound showed a strong hydrophobic interaction with COX-1, thus
Likewise, docking results for compound 17i and 17x against the
target protein COX-1 showed a high binding affinity docking score
indicated by a total score of 4.9098 and 4.9098, respectively, forms a
H-bond of length 1.8 and 2.7 Å to the side chain of hydrophobic
aromatic residue that is, Phen-518 and nucleophilic (polar, hydropho-
bic) residue that is, Ser-553. On the other hand, docking results for 17x
formed a π-cation interaction with side chain of Tyr-385, ─NH+ atom
of pyridine ring formed π-cation with ─H atom of OH group of Tyr-385
(pyridine-NH+. . .OH, Tyr-385, 3.9 Å). In the docking pose of the
complex, the chemical nature of binding site residues within a radius of
4 Å was basic (polar, hydrophobic, positive charged), for example, Arg-
120; acidic (polar, negative charged), for example, Glu-524; aromatic

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JAISWAL ET AL.
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FIGURE 5 (A and B) Predicted interactions of 17i (A) and 17x (B) with antiplatelet target enzyme COX-1 (PDB: 2OYE) with a docking total
score of 4.9098 and 4.9098, respectively, revealing H-bonds of length 1.8 and 2.7, to the binding site pocket residues Phen-518 and Ser-553,
respectively. (C and D) Predicted interactions of 18f (C) and 18h (D) with antiplatelet target enzyme COX-1 (PDB: 2OYE) with a docking total
score of 5.6388 and 4.5653, respectively, revealing π–π stacking of length 5.3 and 5.4, to the binding site pocket residues Tyr-385 and Tyr-
355, respectively
leading to more stability and significant activity in this compound
(predicted interactions of 18f (5C) and 18h (5D) with antiplatelet target
enzyme COX-1, as shown in Figure 5C and D).
binding site pocket residues of the COX-1 depicting a low binding
affinity. Succinctly, this infer us that these class of
2-oxo-benzo[1,4]oxazines showed better antiplatelet activity
due to greater binding affinity as compared to aspirin.
Furthermore, the docking result for the reference drug aspirin with
the antiplatelet target protein COX-1 showed a low binding affinity
docking score, indicated by a low total score of 4.4422 and forms a
H-bond of length 1.8 Å to the side chain of basic (polar, hydrophobic,
positive charged), for example, Arg-120 (predicted interactions of
aspirin with antiplatelet target enzyme COX-1, as shown in Figure 6).
Thus, the docking procedure of Surflex-Dock software (Sybyl-X 2.1) in
reproducing the experimental binding affinity seems reliable, and
therefore predicted as true positive.
|
2.4 Pharmacokinetic and toxicity study
These ADME descriptors were calculated for 2-oxo-2-phenyl-
ethylidene linked 2-oxo-benzo[1,4]oxazine analogues and compared
with standard ranges. All the analogues possessed a good number of
hydrogen bond donors and acceptors.[26c,d] These derivatives were
designed to increase the binding of the drug with the receptor
through hydrogen bonding. These derivatives were found to follow
Lipinski's rule of 5, affording drug likeness to the designed
compounds. Polar surface area was calculated to estimate the
ability of the compounds to permeate cell membranes. Lipophilicity
was (ratio of octanol solubility to water solubility) measured through
log P, which has been implicated in blood brain barrier penetration
and permeability prediction. The excretion of drugs depends
on molecular weight and log P-values.[26e–h] Succinctly, the
After docking analysis, it has been observed that the most
active 2-oxo-benzo[1,4]oxazines, 18i (IC50 = 16.96 0.18) and
18o (IC50 = 17.96 0.18), showed strong interaction at Ser-530
binding site pocket residues (docking total score of 5.5546 and
5.7941, respectively) of the antiplatelet target enzyme COX-1
depicting
reference
a
strong binding affinity. However, the standard
aspirin (IC50 = 21.59 0.63; docking total
score = 4.4422) showed slightly weaker interaction at Arg-120

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FIGURE 6 Predicted interactions of aspirin with antiplatelet target
enzyme COX-1 (PDB: 2OYE) with a docking total score of 4.4422,
revealing a H-bond of length 1.8, to the binding site pocket residues
Arg-120
FIGURE 7 Plot of polar surface area (PSA) versus ALogP for
2-oxo-2-phenylethylidene linked 2-oxo-benzo[1,4]oxazine analogues
17i, 17x, 18f–i, 18l, and 18o. The 95 and 99% confidence
limit ellipses corresponding to the blood–brain barrier (BBB)
and intestinal absorption are shown separately
2-oxo-2-phenylethylidene linked 2-oxo-benzo[1,4]oxazine ana-
logues showed significant antiplatelet activity having some pharma-
cokinetic (PK) limitations.
|
3
CONCLUSION
We have successfully designed and synthesized
a series of
The calculated TPSA (topological polar surface area) values of
these compounds were within acceptable limits. The distribution of
compounds in the human body was described by the predicted
blood–brain barrier coefficient (logBB), apparent Caco-2 perme-
ability, log Kp for skin permeability, volume of distribution and
plasma protein binding (log Khsa for serum protein binding).[27f–h]
All compounds show good aqueous solubility (Supporting Informa-
tion Table S1). Functionalized antiplatelet active and promising
analogues of 2-oxo-benzo[1,4]oxazines (i.e., 17i, 17x, 18f–18i, 18l,
and 18o) were also analyzed for permeability compliance, a key
determinant factor in ADMET studies or prior to clinical trials, with
the help of human skin and human jejunal effective permeability
parameters, along with apparent Madin-Darby Canine Kidney
Cells-On-Sheet (MDCK COS) permeability, and permeability
through rabbit cornea as well as permeability through human
skin. MDCK permeability was in acceptable limit. Moreover,
ADMET results of predicted all analogues revealed liver high
intrinsic passive uptake capacity, which is considered safe in sense
of pharmacology studies. Also, calculated the brain/blood partition
coefficient was detected (in logarithm), whereas the percent
unbound to blood plasma proteins was detected under acceptable
limit. The calculated values for these ADME parameters showed
close similarity between the analogues and that of the reference
drug aspirin and lie within the standard range of values exhibited by
95% of known drugs shown in Figure 7.
functionalized 2-oxo-phenylidene linked functionalized 2-oxo-benz-
[1,4]oxazine analogues 17a–x and 18a–o, whose activities as platelet
aggregation inhibitory activity as well as their SAR and cytotoxic
studies followed by validation of results via in silico molecular
docking simulation studies, were further investigated. Compounds
17a–x and 18a–o possessed moderate to good AA induced platelet
aggregation inhibitory activities as compared to standard drug
aspirin. Among all the tested compounds, three compounds (17i,
18g, and 18h) exhibited comparable platelet aggregation inhibition
activity to aspirin, whereas five compounds (17x, 18f, 18i, 18l, and
18o) showed greater aggregation inhibitory activity than aspirin. The
cytotoxicity of these compounds were found to be non-toxic in
nature in 3T3 fibroblast cell line in MTT assay. Moreover, the in
silico molecular docking simulation studies were also performed to
validate the platelet aggregation inhibitory activity of active
compounds. To the best of our knowledge, this is the first report
of the identification, SAR and in silico molecular docking studies of 2-
oxo-benz[1,4]oxazines as a novel AA-induced platelet aggregation
inhibitors. These findings serve functionalized 2-oxo-benz[1,4]-
oxazines as a promising scaffold to develop as hopeful inhibitors
and aiming to improve the activity in order to get more potent and
active platelet aggregation inhibitors. Further structural modifica-
tions are currently in progress in our laboratory and will be updated
in other reports.

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4
EXPERIMENTAL
To a solution of 14a–g (1.00 mmol, 1 eq.) in MeOH/THF/
H2O (15 mL, 7:2:1) was added LiOH.H2O (1.30 mmol, 1.3 eq.) at
0°C, and stirred for 4–6 h at room temperature (depending upon the
substrate used). The progresses of the reaction were monitored by TLC
using hexane/ethyl acetate (30:70) as an eluent. After completion of
the reaction, the reaction mixture was quenched by 3 N HCl and
extracted with ethyl acetate (3 × 50 mL), washed with distilled water
(50 mL), then with brine (3 × 20 mL). The combined organic layer was
dried over anhyd. Na2SO4 and concentrated under reduced pressure.
The resultant crude products were further purified by recrystallization
using EtOAc/hexane (v/v = 10:90), which furnished the pure diketo-
acids 15a–g in good yields (86–97%). Compounds 15a–g were used for
next step without any further purification.
|
4.1 Chemistry
|
4.1.1 General
All glass apparatus were completely oven dried prior to use. Melting
points were taken in open capillaries using complab melting point
apparatus and are presented uncorrected. Ultrasonic irradiation was
performed in a Elmasonic S 30 (H) ultrasonic water bath cleaner and
the reaction vessel was positioned in the maximum energy area in
the cleaner and the removal or addition of water was used to control
the temperature of the water bath. Infrared spectra were recorded
on a Perkin-Elmer FT-IR Spectrum 2 spectrophotometer. 1H NMR
and 13C NMR spectra were recorded on ECS 400 MHz (JEOL) NMR
spectrometer using CDCl3, and CD3SOCD3 as well as mixture of
(CDCl3 + CD3SOCD3) as solvent and tetramethylsilane as internal
reference. Electrospray ionization mass spectrometry (ESI-MS) and
HRMS were recorded on Xevo G2-S Q-TOF (Waters, USA)
spectrometer. Column chromatography was performed over Merck
silica gel (particle size: 60–120 mesh) procured from Qualigens™
(India), flash silica gel (particle size: 230–400 mesh). All chemicals
and reagents were obtained from Sigma–Aldrich (USA), Merck
(India), or Spectrochem (India) and were used without further
purification.
|
4.1.3 General procedure for the synthesis of
functionalized benzo[1,4]oxazin-2-ones (17a–x and
18a–o)
To a solution of the compounds 15a–g (0.20 mmol; 1 eq.) in water
(2.0 mL) was added compounds 16a–f (0.20 mmol; 1 eq.) or 16e–g
(0.20 mmol; 1 eq.) and the reaction mixture was irradiated under
ultrasonic sonicator at 80°C temperature for 75–120 min (depending
upon the substrate employed). The progress of the reaction was
monitored by TLC (9:1 hexane/ethyl acetate as an eluent). Then, the
reaction mixture was extracted with ethyl acetate (3 × 10 mL), washed
with distilled water (20 mL), then with brine (3 × 10 mL). The combined
organic layer was dried over anhyd. Na2SO4 and concentrated under
reduced pressure. The resultant crude products were further purified
either by recrystallization using EtOAc/hexane (v/v = 10:90) or by flash
column chromatography technique over silica gel (using 9:1 to 7:3
hexane/ethyl acetate as an eluent), which furnished the 2-oxo-benzo-
[1,4]oxazine derivatives (17a–x and 18a–o) in 78–98% yield range.
Copies of the 1H and 13C spectral data of the novel and active
compounds,
a table showing the pharmacokinetic and toxicity
properties of the 2-oxo-benzo[1,4]oxazin-2-ones 17i, 17x, 18f–i,
18l, and 18o, and statistical analysis calculations for the platelet
aggregation inhibitory activity can be found in the Supporting
Information. The InChI codes of the investigated compounds together
with some biological activity data are also provided as Supporting
Information.
(Z)-3-(2-Oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b]-
[1,4]-oxazin-2-one (17a)
|
4.1.2 General procedure for the synthesis of the
Yellowish solid; yield: 53.2 mg (98%), Rf (EtOAc/hexane;
20:80) = 0.85; m.p. 185–186°C; FT-IR (KBr, νmax/cm−1) 3434, 1754,
1614, 1594, 1270, 1113; 1H NMR (400 MHz, CDCl3) δ 8.00 (d,
J = 7.4 Hz, 2H), 7.56–7.47 (m, 3H), 7.21–7.06 (m, 5H); 13C NMR
(100 MHz, CDCl3) δ 191.6, 156.3, 141.3, 139.1, 138.3, 132.8, 128.8,
127.7, 126.0, 124.0, 123.8, 117.2, 116.0, 94.7; HRMS (ESI) calcd. for
starting compounds, i.e., the substituted diketo-acids
(15a–g)
Substituted acetophenones 12a–g (1.00 mmol, 1 eq.) were taken in
toluene (40 mL) and NaH (1.20 mmol, 1.2 eq.) was added cautiously.
After stirring the reaction mixture at 0°C for 30 min, dimethyl oxalate
13 (1.00 mmol, 1 eq.) was added and heated at 80°C for 5–6 h
(depending upon the substrate used). The progresses of the reaction
were monitored by TLC using 9:1 hexane/ethyl acetate as an eluent.
After completion of the reaction, the reaction mixture was quenched
with distilled water (2 mL) and extracted with ethyl acetate (3 × 50 mL),
washed with distilled water (50 mL), then with brine (3 × 20 mL). The
combined organic layer was dried over anhyd. Na2SO4 and concen-
trated under reduced pressure. The resultant crude products were
purified by recrystallization using EtOAc/hexane (v/v = 20:80), which
afforded the pure desired diketo-esters 14a–g in 72–94% yields.
Compounds 14a–g were used for next step without any further
purification.
C
16H11NO3 [M+H]+: 266.0739. Found: 266.0744.
(Z)-6-Methyl-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-
benzo[b][1,4]oxazin-2-one (17b)
Yellow solid; yield: 51.73 mg (93%), Rf (EtOAc/hexane;
20:80) = 0.90; m.p. 157–158°C; FT-IR (KBr, νmax/cm−1) 3436, 1750,
1618, 1572, 1123, 740; 1H NMR (400 MHz, CDCl3) δ 8.00–7.98 (m,
2H), 7.56–7.45 (m, 3H), 7.07–7.02 (m, 2H), 6.88 (d, J = 8.5 Hz, 2H), 2.34
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 191.5, 156.5, 139.4, 139.2,
138.4, 136.1, 132.7, 128.8, 127.7, 124.8, 123.4, 116.8, 116.2, 94.5,
21.08; HRMS (ESI) calcd. for C17H13NO3 [M+H]+: 280.0895. Found:
280.0899.

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(Z)-6-Methyl-3-(2-oxo-2-(p-tolyl)ethylidene)-3,4-dihydro-2H-
benzo[b][1,4]oxazin-2-one (17c)
94.0, 21.1; HRMS (ESI) calcd. for C17H12BrNO3 [M+H]+: 358.0001.
Found: 358.0007.
Yellow solid; yield: 56.08 mg (96%), Rf (EtOAc/hexane;
20:80) = 0.80; m.p. 162–164°C; FT-IR (KBr, νmax/cm−1) 3434, 1762,
1602, 1313, 1047; 1H NMR (400 MHz, CDCl3) δ 7.90 (d, J = 8.1 Hz,
2H), 7.27 (d, J = 8.0 Hz, 2H), 7.06 (d, J = 8.1 Hz, 1H), 7.02 (s, 1H), 6.88 (d,
J = 9.4 Hz, 2H), 2.42 (s, 3H), 2.35 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
191.3, 156.6, 143.5, 139.3, 139.0, 136.0, 135.8, 129.5, 127.8, 124.6,
123.5, 116.8, 116.1, 94.6, 21.7, 21.0; HRMS (ESI) calcd. for
(Z)-8-Bromo-3-(2-(4-fluorophenyl)-2-oxoethylidene)-6-methyl-
3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (17h)
Yellowish solid; yield: 67.6 mg (90%); Rf (EtOAc/hexane;
20:80) = 0.80; m.p. 230–232°C; FT-IR (KBr, νmax/cm−1) 3417, 1771,
1631, 1285, 1159; 1H NMR (400 MHz, CDCl3) δ 8.03–7.99 (m, 2H),
7.17–7.13 (m, 3H), 6.99 (s, 1H), 6.84 (brs, 1H), 2.34 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 190.1, 167.0, 164.5, 155.6, 138.7, 136.9, 136.6,
134.5, 134.4, 130.4, 130.3, 128.3, 124.6, 116.1, 115.9, 115.5, 110.2,
94.8, 20.9; HRMS (ESI) calcd. for C17H11BrFNO3 [M+2]+: 376.9906.
Found: 376.9909.
C
18H15NO3 [M+H]+: 294.1052. Found: 294.1055.
(Z)-3-[2-(4-Methoxy-phenyl)-2-oxo-ethylidene]-6-methyl-3,4-
dihydro-benzo[1,4]oxazin-2-one (17d)
Yellowish solid; yield: 57.7 mg (93%), Rf (EtOAc/hexane;
20:80) = 0.75; m.p. 180–182°C; FT-IR (KBr, νmax/cm−1) 3452, 1755,
1625, 1581; 1H NMR (400 MHz, CDCl3) δ 7.99 (d, J = 8.8 Hz, 2H), 7.06
(d,J = 8.0 Hz, 1H),7.00(s,1H), 6.96(d, J = 9.4 Hz, 2H), 6.88–6.86(m,2H),
3.88 (s, 3H), 2.34 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 190.4, 163.4,
156.8, 139.3, 138.7, 136.0, 131.3, 129.9, 124.4, 123.6, 116.8, 116.0,
114.0, 94.5, 55.6, 21.1; HRMS (ESI) calcd. for C18H15NO4 [M+H]+:
310.1001. Found: 310.1009.
(Z)-8-Bromo-3-(2-(4-bromophenyl)-2-oxoethylidene)-6-methyl-
3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (17i)
Yellowish solid; yield: 77.6 mg (89%); Rf (EtOAc/hexane;
20:80) = 0.90; m.p. 259–260°C; FT-IR (KBr, νmax/cm−1) 3417, 1768,
1629, 1562, 1283, 1138; 1H NMR (400 MHz, CDCl3) δ 7.86 (d,
J = 8.8 Hz, 2H), 7.62 (d, J = 8.4 Hz, 2H), 7.16 (s, 1H), 7.00 (s, 1H), 6.86 (s,
1H), 2.35 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 190.4, 155.6, 139.0,
137.0, 136.9, 136.7, 132.2, 129.3, 128.5, 128.1, 124.5, 115.6, 110.3,
94.8, 20.9; HRMS (ESI) calcd. for C17H11Br2NO3 [M+2]+: 436.9106.
Found: 436.9100.
(Z)-3-[2-(2,4-Dichloro-phenyl)-2-oxo-ethylidene]-6-methyl-3,4-
dihydro-benzo[1,4]oxazin-2-one (17e)
Yellowish solid; yield: 65.4 mg (94%), Rf (EtOAc/hexane;
20:80) = 0.80; m.p. 142–145°C; FT-IR (KBr, νmax/cm−1) 3436, 2913,
1755, 1618, 1570, 1083; 1H NMR (400 MHz, CDCl3) δ 7.53 (d,
J = 8.3 Hz, 1H), 7.45 (d, J = 2.0 Hz, 1H), 7.33–7.30 (m, 1H), 7.10–7.08
(m, 1H), 6.94–6.92 (m, 2H), 6.73 (s, 1H), 2.36 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 191.6, 155.9, 139.5, 139.2, 137.5, 137.2, 136.2,
132.5, 130.7, 130.6, 127.4, 125.4, 123.0, 117.0, 116.4, 98.0, 21.1;
HRMS (ESI) calcd. for C17H11Cl2NO3 [M+2]+: 349.0116. Found:
349.0112.
(Z)-6-Chloro-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-
benzo[b][1,4]oxazin-2-one (17j)
Yellowish solid; yield: 57.2 mg (95%), Rf (EtOAc/hexane;
20:80) = 0.80; m.p. 185–187°C; FT-IR (KBr, νmax/cm−1) 3434, 1761,
1555, 1622, 1174; 1H NMR (400 MHz, CDCl3) δ 8.00–7.98 (m, 2H),
7.58–7.55 (m, 1H), 7.50–7.47 (m, 2H), 7.13–7.03 (m, 4H); 13C NMR
(100 MHz, CDCl3) δ 191.8, 155.8, 139.8, 138.4, 138.1, 133.0, 131.1,
128.9, 127.8, 124.8, 123.8, 118.3, 115.8, 95.7; HRMS (ESI) calcd. for
C
16H10ClNO3 [M+2]+: 301.7085. Found: 301.7089.
(Z)-3-(2-(4-Fluorophenyl)-2-oxoethylidene)-6-methyl-3,4-
dihydro-2H-benzo[1,4]oxazin-2-one (17f)
(Z)-6-Chloro-3-(2-oxo-2-(p-tolyl)ethylidene)-3,4-dihydro-2H-
benzo[b][1,4]oxazin-2-one (17k)
Yellowish solid; yield: 53.7 mg (90%); Rf (EtOAc/hexane;
20:80) = 0.85; m.p. 145–147°C; FT-IR (KBr, νmax/cm−1) 3433, 2930,
1770, 1624, 1596, 1128; 1H NMR (400 MHz, CDCl3) δ 8.02 (dd, J = 5.6,
8.8 Hz, 2H), 7.17–7.07 (m, 3H), 6.98–6.90 (m, 3H), 2.36 (s, 1H);
13C NMR (100 MHz, CDCl3) δ 190.0, 166.9, 156.4, 139.3, 136.1,
134.7, 130.3, 130.2, 124.9, 123.3, 116.9, 116.2, 115.9, 115.8, 94.1,
21.1; HRMS (ESI) calcd. for C17H12FNO3 [M+H]+: 298.0801. Found:
298.0807.
Yellowish solid; yield: 60.7 mg (95%); Rf (EtOAc/hexane;
20:80) = 0.90; m.p. 160–162°C; FT-IR (KBr, νmax/cm−1
) 3434,
2925, 1624, 1766, 1494, 1178; 1H NMR (400 MHz, CDCl3) δ
7.94–7.92 (m, 2H), 7.30 (d, J = 8.0 Hz, 2H), 7.14–7.04 (m, 4H), 2.44
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 191.6, 156.0, 144.0, 139.8,
138.2, 135.6, 131.2, 129.7, 128.0, 125.0, 123.7, 118.3, 115.8, 95.9,
21.8; HRMS (ESI) calcd. for C17H12ClNO3 [M+H]+: 314.0506. Found:
314.0509.
(Z)-3-(2-(4-Bromophenyl)-2-oxoethylidene)-6-methyl-3,4-
dihydro-2H-benzo[b][1,4]oxazin-2-one (17g)
(Z)-6-Chloro-3-(2-(4-chlorophenyl)-2-oxoethylidene)-3,4-
dihydro-2H-benzo[b][1,4]oxazin-2-one (17l)
Yellow solid; yield: 66.48 mg (93%); Rf (EtOAc/hexane;
20:80) = 0.85; m.p. 179–181°C; FT-IR (KBr, νmax/cm−1) 3435, 2923,
1763, 1624, 1543, 1052; 1H NMR (400 MHz, CDCl3) δ 7.87–7.84 (m,
2H), 7.63–7.59 (m, 2H), 7.08 (d, J = 9.2 Hz, 1H), 6.96 (s, 1H), 6.92–6.90
(m, 2H), 2.35 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 190.2, 156.3, 139.5,
139.4, 137.1, 136.2, 132.0, 129.2, 127.7, 125.1, 123.2, 117.0, 116.3,
Yellowish solid; yield: 65.2 mg (95%); Rf (EtOAc/hexane;
20:80) = 0.90; m.p. 182–185°C; FT-IR (KBr, νmax/cm−1) 3434, 1761,
1631, 1586, 1088; 1H NMR (400 MHz, CDCl3) δ 7.93 (d, J = 8.2 Hz,
2H), 7.45 (d, J = 8.1 Hz, 2H), 7.13–7.00 (m, 4H); 13C NMR (100 MHz,
CDCl3) δ 190.4, 155.7, 139.8, 139.4, 138.7, 136.3, 131.2, 129.2, 129.1,

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JAISWAL ET AL.
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124.6, 124.0, 118.3, 115.9, 95.3; HRMS (ESI) calcd. for C16H9Cl2NO3
[M+2]+: 334.9959. Found: 334.9956.
(Z)-3-(2-(4-Methoxyphenyl)-2-oxoethylidene)-6-nitro-3,4-
dihydro-2H-benzo[b][1,4]oxazin-2-one (17r)
Yellowish solid; yield: 60.4 mg (86%); Rf (EtOAc/hexane;
20:80) = 0.75; m.p. 195–197°C; FT-IR (KBr, νmax/cm−1) 3435, 2926,
1599, 1758, 1633, 1594; 1H NMR (400 MHz, CDCl3) δ 8.02–7.93 (m,
4H), 7.29 (d, J = 8.9 Hz, 1H), 7.10 (s, 1H), 6.98 (d, J = 8.7 Hz, 2H), 3.89 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 190.7, 164.0, 155.3, 145.2, 144.9,
137.0, 130.7, 130.3, 124.9, 118.6, 117.8, 114.2, 111.2, 97.0, 55.7;
(Z)-6-Chloro-3-(2-(4-fluorophenyl)-2-oxoethylidene)-3,4-
dihydro-2H-benzo[b][1,4]oxazin-2-one (17m)
Yellowish solid; yield: 57.8 mg (93%); Rf (EtOAc/hexane;
20:80) = 0.80; m.p. 155–157°C; FT-IR (KBr, νmax/cm−1) 3434, 1754,
1634, 1601, 1495, 1226, 1160; 1H NMR (400 MHz, CDCl3) δ 8.06–
8.02 (m, 2H), 7.20–7.13 (m, 4H), 7.08–7.04 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ 190.4, 155.8, 139.8, 138.6, 131.3, 130.5, 130.4,
124.8, 123.9, 118.4, 116.2, 115.9, 115.8, 95.4; HRMS (ESI) calcd. for
HRMS (ESI) calcd. for
341.0692.
C
17H12N2O6 [M+H]+: 341.0695. Found:
C
16H9ClFNO3 [M+H]+: 318.0255. Found: 318.0259.
(Z)-3-(2-(4-Chlorophenyl)-2-oxoethylidene)-6-nitro-3,4-dihydro-
2H-benzo[b][1,4]oxazin-2-one (17s)
(Z)-3-(2-(4-Bromophenyl)-2-oxoethylidene)-6-chloro-3,4-
dihydro-2H-benzo[b][1,4]oxazin-2-one (17n)
Yellow solid; yield: 55.88 mg (81%); Rf (EtOAc/hexane;
20:80) = 0.70; m.p. 239–240°C; FT-IR (KBr, νmax/cm−1) 3435, 2924,
1622, 1525, 1272; 1H NMR (400 MHz, DMSO-d6) δ 8.73 (s, 1H), 8.03
(d, J = 7.2 Hz, 2H), 7.91 (d, J = 9.1 Hz), 7.59 (d, J = 7.2 Hz, 2H), 7.42 (d,
J = 9.0 Hz, 1H), 6.90 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 188.9,
156.0, 146.0, 144.7, 139.8, 138.2, 137.1, 129.9, 129.6, 125.8, 119.0,
117.8, 113.1, 94.4; HRMS (ESI) calcd. for C16H9ClN2O5 [M+2H]+:
346.0200. Found: 346.0204.
Yellowish solid; yield: 71.5 mg (92%); Rf (EtOAc/hexane;
20:80) = 0.80; m.p. 175–177°C; FT-IR (KBr, νmax/cm−1) 3436, 2924,
1755, 1632, 1583, 1007; 1H NMR (400 MHz, CDCl3) δ 7.85 (d,
J = 7.9 Hz, 2H), 7.62 (d, J = 7.9 Hz, 2H), 7.13–7.00 (m, 4H); 13C NMR
(100 MHz, CDCl3) δ 190.5, 155.6, 139.8, 138.8, 136.8, 132.1, 131.2,
129.3, 128.1, 124.6, 124.0, 118.3, 115.9, 95.2; HRMS (ESI) calcd. for
C
16H9BrClNO3 [M+2]+: 377.9454. Found: 377.9458.
(Z)-3-(2-(2,4-Dichlorophenyl)-2-oxoethylidene)-6-nitro-3,4-
dihydro-2H-benzo[b][1,4]oxazin-2-one (17t)
(Z)-6-Chloro-3-(2-(4-methoxyphenyl)-2-oxoethylidene)-3,4-
dihydro-2H-benzo[b][1,4]oxazin-2-one (17o)
Yellow solid; yield: 64.05 mg (84%), Rf (EtOAc/hexane;
20:80) = 0.75; m.p. 185–187°C; FT-IR (KBr, νmax/cm−1) 3588, 2930,
1769, 1585, 1685, 1108; 1H NMR (400 MHz, CDCl3) δ 8.05–7.99 (m,
2H), 7.55 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.37–7.33 (m, 2H),
6.89 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 192.4, 154.5, 145.2, 145.1,
138.0, 137.6, 136.9, 132.7, 131.0, 130.8, 127.6, 124.2, 119.4, 118.1,
Yellowish solid; yield: 62.6 mg (90%); Rf (EtOAc/hexane;
20:80) = 0.80; m.p. 178–180°C; FT-IR (KBr, νmax/cm−1) 3434, 1764,
1628, 1594, 1018; 1H NMR (400 MHz, CDCl3) δ 7.99–7.97 (m, 2H),
7.10–7.06 (m, 2H), 7.02–6.99 (m, 2H), 6.97–6.94 (m, 2H), 3.87 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 190.5, 163.7, 156.1, 139.6, 137.8, 131.1,
130.9, 130.1, 125.0, 123.4, 118.2, 115.6, 114.1, 95.7, 55.6; HRMS (ESI)
calcd. for C17H12ClNO4 [M+2]+: 331.7345. Found: 331.7349.
111.7, 100.4; HRMS (ESI) calcd. for
379.9810. Found: 379.9815.
C
16H8Cl2N2O5 [M+2H]+:
(Z)-6-Nitro-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-
benzo[b][1,4]oxazin-2-one (17p)
(Z)-3-(2-(4-Fluorophenyl)-2-oxoethylidene)-6-nitro-3,4-dihydro-
2H-benzo[b][1,4]oxazin-2-one (17u)
Yellowish solid; yield: 57.2 mg (89%); Rf (EtOAc/hexane;
20:80) = 0.85; m.p. 198–200°C; FT-IR (KBr, νmax/cm−1) 3436, 2928,
1762, 1625, 1581, 1142; 1H NMR (400 MHz, CDCl3) δ 8.03–7.96 (m,
4H), 7.61–7.49 (m, 3H), 7.31 (d, J = 8.8 Hz, 1H), 7.15 (s, 1H); 13C NMR
(100 MHz, CDCl3) δ 192.1, 155.1, 145.2, 144.9, 137.7, 137.6, 133.4,
128.9, 127.9, 124.7, 118.9, 118.0, 111.5, 96.9; HRMS (ESI) calcd. for
Yellowish solid; yield: 62.2 mg (94%); Rf (EtOAc/hexane;
20:80) = 0.75; m.p. >250°C; FT-IR (KBr, νmax/cm−1) 3435, 3107,
1759, 1622, 1594, 1156; 1H NMR (400 MHz, DMSO-d6) δ 8.73 (s, 1H),
8.11 (d, J = 5.2 Hz, 2H), 7.92 (d, J = 6.4 Hz, 1H), 7.44–7.36 (m, 3H), 6.92
(s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 188.7, 156.0, 145.9, 144.6,
139.5, 135.1, 130.9, 125.8, 118.9, 117.7, 116.6, 116.4, 113.0, 94.5;
C
16H10N2O5 [M+H]+: 311.0590. Found: 311.0593.
HRMS (ESI) calcd. for
329.0490.
C
16H9FN2O5 [M+H]+: 329.0495. Found:
(Z)-6-Nitro-3-(2-oxo-2-(p-tolyl)ethylidene)-3,4-dihydro-2H-
benzo[b][1,4]oxazin-2-one (17q)
(Z)-3-(2-(4-Bromophenyl)-2-oxoethylidene)-6-nitro-3,4-dihydro-
2H-benzo[b][1,4]oxazin-2-one (17v)
Yellow solid; yield: 53.99 mg (83%); Rf (EtOAc/hexane;
20:80) = 0.75; m.p. 220–223°C; FT-IR (KBr, νmax/cm−1) 3433, 1760,
1624, 1524, 1109; 1H NMR (400 MHz, DMSO-d6) δ 8.73 (d, J = 2.4 Hz,
1H), 7.96–7.92 (m, 3H), 7.46–7.37 (m, 3H), 6.95 (s, 1H), 2.40 (s, 3H);
13C NMR (100 MHz, DMSO-d6) 189.9, 155.7, 145.9, 143.8, 139.2,
138.5, 135.2, 130.1, 128.1, 119.5, 118.9, 117.7, 112.6, 94.8, 21.7;
Yellow solid; yield: 60.92 mg (78%); Rf (EtOAc/hexane;
20:80) = 0.70; m.p. 195–197°C; FT-IR (KBr, νmax/cm−1) 3435, 2925,
1759, 1525, 1023; 1H NMR (400 MHz, CDCl3) δ 8.03–7.97 (m, 2H),
7.89–7.86 (m, 2H), 7.66–7.63 (m, 2H), 7.32 (d, J = 9.2 Hz, 1H), 7.07 (s,
1H); 13C NMR (100 MHz, CDCl3) δ 190.8, 154.9, 145.3, 145.0, 138.0,
136.5, 132.3, 129.4, 128.6, 124.5, 119.2, 118.0, 111.6, 96.4; HRMS
(ESI) calcd. for C16H9BrN2O5 [M+2H]+: 389.9695. Found: 389.9699.
HRMS (ESI) calcd. for
325.0741.
C
17H12N2O5 [M+H]+: 325.0746. Found:

JAISWAL ET AL.
13 of 17
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(Z)-7-Nitro-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-
benzo[b][1,4]oxazin-2-one (17w)
(Z)-3-(2-(4-Fluorophenyl)-2-oxoethylidene)-7-nitro-3,4-dihydro-
2H-benzo[b][1,4]oxazin-2-one (18d)
Yellowish solid; yield: 57.5 mg (89%); Rf (EtOAc/hexane;
Yellowish solid; yield: 52.7 mg (80%); Rf (EtOAc/hexane;
20:80) = 0.70; m.p. 240–242°C; FT-IR (KBr, νmax/cm−1
)
3436,
20:80) = 0.70; m.p. 235–237°C; FT-IR (KBr, νmax/cm−1
) 3411,
1763, 1622, 1596, 1268; 1H NMR (400 MHz, CDCl3) δ 8.06–8.00
(m, 4H), 7.83–7.81 (m, 1H), 7.62 (t, J = 7.3 Hz, 1H), 7.54 (t, J = 7.5 Hz,
2H), 6.99 (s, 1H); 13C NMR (100 MHz, CDCl3) 190.7, 156.1, 142.2,
141.1, 139.2, 138.3, 133.6, 131.3, 129.6, 128.0, 121.4, 117.4, 112.6,
96.0; HRMS (ESI) calcd. for C16H10N2O5 [M+H]+: 311.0590. Found:
311.0595.
3090, 1773, 1626, 1516, 1473; 1H NMR (400 MHz, DMSO-d6) δ
8.18–8.08 (m, 4H), 7.88–7.86 (m, 1H), 7.42–7.38 (m, 2H), 7.02 (s,
1H); 13C NMR (100 MHz, DMSO-d6) 188.7, 163.7, 155.5, 141.8,
140.6, 138.7, 134.4, 130.7, 130.6, 130.5, 120.9, 116.9, 116.2, 115.9,
112.0, 95.3; HRMS (ESI) calcd. for C16H9FN2O5 [M+H]+: 329.0495.
Found: 329.0499.
(Z)-3-(2-(4-Bromophenyl)-2-oxoethylidene)-7-nitro-3,4-dihydro-
2H-benzo[b][1,4]oxazin-2-one (17x)
(Z)-8-Bromo-6-methyl-3-(2-oxo-2-(p-tolyl)ethylidene)-3,4-
dihydro-2H-benzo[b][1,4]oxazin-2-one (18e)
Yellowish solid; yield: 69.6 mg (87%); Rf (EtOAc/hexane;
20:80) = 0.80; m.p. 230–232°C; FT-IR (KBr, νmax/cm−1) 3435, 3093,
1769, 1619, 1521; 1H NMR (400 MHz, DMSO-d6) δ 8.09–7.96 (m, 4H),
7.83–7.44 (m, 3H), 7.01 (d, J = 4.8 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6) δ 188.9, 154.7, 141.8, 140.1, 137.9, 136.6, 131.5, 130.0,
129.1, 126.6, 120.7, 116.5, 111.4, 95.7; HRMS (ESI) calcd. for
Yellowish solid; yield: 70.7 mg (95%); Rf (EtOAc/hexane;
20:80) = 0.90; m.p. 219–220°C; FT-IR (KBr, νmax/cm−1
) 3417,
1764, 1630, 1602, 1281, 1182; 1H NMR (400 MHz, CDCl3) δ 7.86
(d, 2H, J = 8.4 Hz, Ar-H), 7.24 (s, 1H, Ar-H), 7.08 (s, 1H, Ar-H), 7.00

(s, 1H, Ar-H), δ 6.80 (s, 1H, C CH), 2.39 (s, 3H, CH3), 2.30 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 191.4, 155.8, 143.9, 138.3, 136.8,
136.5, 135.6, 129.6, 128.0, 127.9, 124.8, 115.4, 110.1, 95.3, 21.8,
20.9; HRMS (ESI) calcd. for C18H14BrNO3 [M+2]+: 373.0157. Found:
373.0152.
C
16H9BrN2O5 [M+2]+: 389.9695. Found: 389.9691.
(Z)-3-(2-(4-Methoxyphenyl)-2-oxoethylidene)-7-nitro-3,4-
dihydro-2H-benzo[b][1,4]oxazin-2-one (18a)
Yellowish solid; yield: 60.8 mg (92%); Rf (EtOAc/hexane;
20:80) = 0.70; m.p. 218–220°C; FT-IR (KBr, νmax/cm−1) 3437, 2927,
2854, 1632, 1517; 1H NMR (400 MHz, DMSO-d6) δ 8.07–8.02 (m, 4H),
7.73 (d, J = 10.8 Hz, 1H), 7.09 (d, J = 8.8 Hz, 2H), 7.02 (s, 1H), 3.89 (s,
3H); 13C NMR (100 MHz, DMSO-d6) δ 189.1, 163.4, 155.8, 141.7,
140.6, 138.1, 131.1, 130.7, 130.1, 121.2, 116.8, 114.5, 112.2, 95.9,
55.8; HRMS (ESI) calcd. for C17H12N2O6 [M+H]+: 341.0695. Found:
341.0699.
(Z)-8-Bromo-3-(2-(4-methoxyphenyl)-2-oxoethylidene)-6-
methyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (18f)
Yellowish solid; yield: 70.5 mg (91%); Rf (EtOAc/hexane;
20:80) = 0.80; m.p. 189–190°C; FT-IR (KBr, νmax/cm−1) 3446, 2926,
1757, 1623, 1598, 1107; 1H NMR (400 MHz, CDCl3) δ 7.92 (d,
J = 8.0 Hz, 2H), 7.03 (s, 1H), 6.90 (m, 3H), 6.74 (s, 1H), 3.84 (s, 3H), 2.28
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 190.3, 163.5, 155.8, 137.9,
136.7, 136.4, 130.9, 130.0, 127.8, 124.8, 115.3, 114.0, 110.0, 95.1,
55.6, 20.9; HRMS (ESI) calcd. for C18H14BrNO4 [M+2]+: 389.0106.
Found: 389.0109.
(Z)-3-(2-(4-Chlorophenyl)-2-oxoethylidene)-7-nitro-3,4-dihydro-
2H-benzo[b][1,4]oxazin-2-one (18b)
Yellowish solid; yield: 63.2 mg (90%); Rf (EtOAc/hexane;
20:80) = 0.80; m.p. 205–207°C; FT-IR (KBr, νmax/cm−1) 3432, 2925,
2860, 1633, 1525, 1776, 1075; 1H NMR (400 MHz, DMSO-d6) δ 8.08
(d, J = 8.4 Hz, 4H), 7.89–7.87 (m, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.01 (s,
1H); 13C NMR (100 MHz, DMSO-d6) δ 188.9, 155.4, 141.9, 140.7,
138.9, 136.5, 130.7, 129.8, 129.5, 129.2, 120.9, 117.0, 112.1, 95.2;
HRMS (ESI) calcd. for C16H9ClN2O5 [M+H]+: 345.0200. Found:
345.0207.
(Z)-8-Bromo-3-(2-(2,4-dichlorophenyl)-2-oxoethylidene)-6-
methyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (18g)
Yellowish solid; yield: 79.7 mg (94%); Rf (EtOAc/hexane;
20:80) = 0.85; m.p. 224–226°C; FT-IR (KBr, νmax/cm−1) 3417, 1763,
1626, 1561, 1294, 1127; 1H NMR (400 MHz, CDCl3) δ 7.54 (d,
J = 8.4 Hz, 1H), 7.47 (s, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.18 (s, 1H), 6.88
(s, 1H), 6.79 (s, 1H), 2.35 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 191.9,
155.2, 138.7, 137.5, 137.3, 137.0, 136.8, 132.7, 130.8, 130.7, 128.8,
127.5, 124.3, 115.7, 110.3, 98.8, 20.9; HRMS (ESI) calcd. for
(Z)-3-(2-(2,4-Dichlorophenyl)-2-oxoethylidene)-7-nitro-3,4-
dihydro-2H-benzo[b][1,4]oxazin-2-one (18c)
C
17H10BrCl2NO3 [M+2]+: 426.9221. Found: 426.9227.
Yellowish solid; yield: 64.6 mg (85%); Rf (EtOAc/hexane;
20:80) = 0.70; m.p. 208–210°C; FT-IR (KBr, νmax/cm−1) 3433, 3088,
1770, 1620, 1522, 1470, 1072; 1H NMR (400 MHz, DMSO-d6) δ 12.39
(s, 1H), 8.09–8.08 (m, 2H), 7.95–7.93 (s, 1H), 7.78–7.58 (m, 3H), 6.62 (s,
1H); 13C NMR (100 MHz, DMSO-d6) δ 190.3, 155.3, 142.2, 140.8,
138.5, 137.7, 136.2, 131.2, 131.0, 130.5, 130.1, 127.9, 120.8, 117.3,
112.1, 98.8; HRMS (ESI) calcd. for C16H8Cl2N2O5 [M+H]+: 378.9810.
Found: 378.9818.
(Z)-8-Bromo-3-(2-(4-fluorophenyl)-2-oxoethylidene)-6-methyl-
3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (18h)
Yellowish solid; yield: 67.6 mg (90%); Rf (EtOAc/hexane;
20:80) = 0.80; m.p. 230–232°C; FT-IR (KBr, νmax/cm−1) 3417, 1771,
1631, 1285, 1159; 1H NMR (400 MHz, CDCl3) δ 8.03–7.99 (m, 2H),
7.17–7.13 (m, 3H), 6.99 (s, 1H), 6.84 (brs, 1H), 2.34 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 190.1, 167.0, 164.5, 155.6, 138.7, 136.9, 136.6,
134.5, 134.4, 130.4, 130.3, 128.3, 124.6, 116.1, 115.9, 115.5, 110.2,

14 of 17
JAISWAL ET AL.
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94.8, 20.9; HRMS (ESI) calcd. for C17H11BrFNO3 [M+2]+: 376.9906.
Found: 376.9909.
J = 8.4 Hz, 1H), 6.78 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 190.3,
153.7, 142.4, 136.9, 136.2, 133.4, 131.1, 130.4, 129.5, 129.4, 127.2,
127.1, 122.3, 121.7, 121.4, 100.4; HRMS (ESI) calcd. for
(Z)-5-Nitro-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-
benzo[b][1,4]oxazin-2-one (18i)
C
16H8Cl2N2O5 [M+2]+: 379.9810. Found: 379.9815.
Yellowish solid; yield: 55.7 mg (88%); Rf (EtOAc/hexane; 20:80) = 0.80;
m.p. 210–212°C; FT-IR (neat) 3054, 1766, 1622, 1584, 1461, 1041;
1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 8.8 Hz, 1H), 8.08 (d,
J = 7.2 Hz, 2H), 7.59 (t, J = 7.6 Hz, 1H), 7.52–7.47 (m, 3H), 7.29 (s, 1H),
7.15 (t, J = 8.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 191.5, 155.1,
142.6, 137.9, 135.5, 134.0, 133.5, 128.9, 128.3, 123.0, 122.8, 122.6,
121.6, 99.7; HRMS (ESI) calcd. for C16H10N2O5 [M+H]+: 311.0590.
Found: 311.0596.
(Z)-3-(2-(4-Fluorophenyl)-2-oxoethylidene)-5-nitro-3,4-dihydro-
2H-benzo[b][1,4]oxazin-2-one (18n)
Yellowish solid; yield: 58.6 mg (89%); Rf (EtOAc/hexane;
20:80) = 0.75; m.p. 269–271°C; FT-IR (KBr, νmax/cm−1) 3417, 1760,
1628, 1595, 1514, 1279, 1256, 1237, 1159; 1H NMR (400 MHz,
DMSO-d6) δ 8.21–8.14 (m, 3H), 7.71 (d, J = 7.6 Hz, 1H), 7.41–7.37 (m,
2H), 7.31–7.27 (m, 1H), 7.11 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ
189.8, 166.9, 164.4, 155.2, 143.4, 137.8, 135.1, 134.2, 131.2, 131.1,
123.4, 122.8, 122.3, 116.5, 97.73, 97.60; HRMS (ESI) calcd. for
C16H9FN2O5 [M+H]+: 329.0495. Found: 329.0498.
(Z)-5-Nitro-3-(2-oxo-2-(p-tolyl)ethylidene)-3,4-dihydro-2H-
benzo[b][1,4]oxazin-2-one (18j)
Brownish solid; yield: 51.5 mg (79%); Rf (EtOAc/hexane;
20:80) = 0.70; m.p. 230–232°C; FT-IR (KBr, νmax/cm−1) 3442, 2925,
1771, 1629, 1525, 1178; 1H NMR (400 MHz, DMSO-d6) δ 8.13 (d,
J = 8.0 Hz, 1H), 7.99 (d, J = 8.0 Hz, 2H), 7.67 (t, J = 8.4 Hz, 1H), 7.37 (d,
J = 8.0 Hz, 2H), 7.27 (t, J = 8.8 Hz, 1H), 7.10 (s, 1H), 2.39 (s, 3H);
13C NMR (100 MHz, DMSO-d6) δ 189.9, 154.3, 143.2, 142.4, 136.5,
134.9, 133.2, 129.1, 127.3, 126.2, 122.3, 122.0, 121.3, 97.06, 96.9,
20.6; HRMS (ESI) calcd. for C17H12N2O5 [M+H]+: 325.0746. Found:
325.0742.
(Z)-3-(2-(4-Bromophenyl)-2-oxoethylidene)-5-nitro-3,4-dihydro-
2H-benzo[b][1,4]oxazin-2-one (18o)
Yellowish solid; yield: 64.1 mg (82%); Rf (EtOAc/hexane;
20:80) = 0.75; m.p. 232–234°C; FT-IR (KBr, νmax/cm−1) 3442, 3098,
1766, 1620, 1583, 1067; 1H NMR (400 MHz, DMSO-d6) δ 8.13 (d,
J = 8.4 Hz, 1H), 7.99 (d, J = 7.6 Hz, 2H), 7.74 (d, J = 8.0 Hz, 2H), 7.65 (d,
J = 8.0 Hz, 1H), 7.29 (t, J = 8.4 Hz, 1H), 7.09 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) δ 189.1, 154.1, 142.5, 137.1, 136.4, 133.3,
131.5, 129.2, 126.7, 122.4, 121.8, 121.5, 121.4, 96.4; HRMS (ESI)
calcd. for C16H9BrN2O5 [M+2]+: 389.9695. Found: 389.9699.
(Z)-3-(2-(4-Methoxyphenyl)-2-oxoethylidene)-5-nitro-3,4-
dihydro-2H-benzo[b][1,4]oxazin-2-one (18k)
|
4.2 Biological activity assays
Yellowish solid; yield: 55.3 mg (83%); Rf (EtOAc/hexane;
20:80) = 0.65; m.p. 244–246°C; FT-IR (KBr, νmax/cm−1) 3417, 1764,
1631, 1598, 1524, 1257, 1168, 1319; 1H NMR (400 MHz, DMSO-d6) δ
8.14–8.08 (m, 3H), 7.68 (d, J = 8.0 Hz, 1H), 7.25 (t, J = 8.4 Hz, 1H),
7.10–7.08 (m, 3H), 3.87 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ
188.8, 163.1, 154.4, 142.3, 136.1, 133.1, 130.3, 129.6, 122.2, 121.1,
114.0, 113.8, 97.2, 96.9, 55.2; HRMS (ESI) calcd. for C17H12N2O6
[M+H]+: 341.0695. Found: 341.0692.
|
4.2.1 Platelet aggregation inhibitory activity
evaluation
All synthesized 2-oxo-2-phenylethylidene linked 2-oxo-benzo[1,4]-
oxazine analogues (17a–x and 18a–o) were dissolved in DMSO before
testing. In order to eliminate the effects of the solvent on aggregation,
the final concentration of DMSO was fixed at 0.5%. Arachidonic acid
(AA), EDTA (disodium salt), bovine serum albumin, and dimethyl
sulfoxide (DMSO) were purchased from Sigma Chemical Co.
Blood was collected from the rabbit marginal ear vein (several
studies established that rabbit platelets are surrogate to human
platelets both in vitro as well as in vivo)[25] and was mixed with EDTA
to a final concentration of 6 mM. It was centrifuged for 10 min at
90 g at room temperature, and the supernatant was obtained as
platelet-rich plasma. The latter was further centrifuged at 500×g for
10 min. The platelet pellets were washed with Tyrode's solution
(Ca+2-free) containing 2 mM EDTA, 0.1 and 3.5 mg/mL bovine serum
albumin, and centrifuged at 500×g for 10 min. Then, the pellets were
washed with Tyrode's solution without EDTA. After centrifugation
under the same conditions, the platelet pellets were finally
suspended in Tyrode's solution of the following composition (mM):
NaCl (136.8), KCl (2.8), NaHCO3 (11.9), MgCl2 (2.1), NaH2-PO4
(0.33), CaCl2 (1.0), and glucose (11.2) containing bovine serum
albumin (0.35%).
(Z)-3-(2-(4-Chlorophenyl)-2-oxoethylidene)-5-nitro-3,4-dihydro-
2H-benzo[b][1,4]oxazin-2-one (18l)
Yellowish solid; yield: 59.4 mg (86%); Rf (EtOAc/hexane;
20:80) = 0.75; m.p. 255–257°C; FT-IR (KBr, νmax/cm−1) 3416, 1767,
1629, 1588, 1316, 1258, 1092; 1H NMR (400 MHz, DMSO-d6) δ 8.17–
8.06 (m, 3H), 7.64–7.53 (m, 3H), 7.25 (t, J = 8.4 Hz, 1H), 7.10 (s, 1H);
13C NMR (100 MHz, DMSO-d6) δ 188.9, 153.9, 142.2, 138.1, 136.3,
135.9, 133.3, 128.8, 128.3, 122.1, 121.6, 121.4, 121.1, 97.2; HRMS
(ESI) calcd. for C16H9ClN2O5 [M+2]+: 346.0200. Found: 346.0204.
(Z)-3-(2-(2,4-Dichlorophenyl)-2-oxoethylidene)-5-nitro-3,4-
dihydro-2H-benzo[b][1,4]oxazin-2-one (18m)
Yellowish solid; yield: 61.7 mg (81%); Rf (EtOAc/hexane;
20:80) = 0.75; m.p. 252–254°C; FT-IR (KBr, νmax/cm−1) 3417, 1762,
1630, 1518, 1384, 1285, 1116; 1H NMR (400 MHz, DMSO-d6) δ 8.18–
8.14 (m, 1H), 7.69–7.64 (m, 3H), 7.50 (d, J = 7.6 Hz, 1H), 7.29 (t,

JAISWAL ET AL.
15 of 17
|
|
Aggregation was measured by a turbidimetric method using a
4.3.1 Molecular docking
Lumi-aggregometer (Chrono-Log Corp., Havertown, PA). All glassware
was siliconized. Three minutes before the addition of the aggregation
inducer, the platelet suspension was stirred at 1200 rpm. The
percentage of aggregation was calculated as follows (abs. = absor-
bance):
Molecular modeling studies of 2-oxo-2-phenylethylidene linked 2-
oxo-benzo[1,4]oxazine analogues 17i, 17x, 18f–i, 18l, and 18o were
carried out using the molecular modeling software Sybyl-X 2.1 (Tripos
International). Drawing of structures and simple geometry optimiza-
tion were performed with ChemBio-Office suite Ultra v12.0 (2012)
(Cambridge Soft Corp). The binding affinity of all compounds were
predicted with the platelet aggregation inhibitor target enzyme
cyclooxygenase-1 (COX-1). Energy minimization was done using the
Tripos force field with a distance-dependent dielectric and the Powell
gradient minimization algorithm, with a convergence criterion of
0.001 kcal/mol for the determination of conformations with the most
favorable (lowest energy). Aromatase at 3.5 Å resolution (PDB:
2OYE)[27a] was selected. The crystal structure of COX-1 enzyme of
sheep (it has been well established that COX-1 of sheep is almost
similar to that of COX-1 of human)[27b,c] in complex with indomethacin
obtained from the PDB. Hydrogen atoms were added to the protein
with the protonation 3D tool in Sybyl. Partial atomic charges were
assigned using the Gasteiger-Hückel method in Sybyl. All 2D
structures were converted to 3D structures using the program
Concord v4.0 and the maximum number of iterations performed in
the energy minimization was set to 2000. Further geometry
optimization was done with the MOPAC-6 package using the semi-
empirical PM3 Hamiltonian method.[27d–g]
%Aggregation
Abs: of platelet suspensionꢀFinal abs: after aggregation
¼
ꢁ 100
Abs: of platelet suspensionꢀabs: of Tyrode solution
Percent aggregation was expressed assuming the absorbance of
platelet suspension as 0% aggregation and the absorbance of platelet-
free Tyrode's solution as 100% aggregation. For each compound IC50
values were calculated by SigmaPlot.
4.2.2 Cytotoxicity MTT assay[26]
|
The cytotoxic effect of the 17i, 17x, 18f–18i, 18l, and 18o on cells
were detected in vitro using the mitochondrial cytotoxic test. Cell
viability was evaluated using thiazolyl blue tetrazolium bromide
(MTT), which indicates the metabolic activity of cells. The experi-
ment was performed in 96-well microplates. The cells were seeded
at a density of 3.5 × 103 3T3 fibroblast cells per well. Samples were
dissolved in DMSO (stock solution 10 mM) and subsequently diluted
in medium to the final concentration of 25–250 µM (concentration
of DMSO 0.5%) and after 24 h they were added to the cells.
Microplates were cultivated for 72 h in thermostat at 37°C and 5%
CO2 atmosphere. After incubation thiazolyl blue tetrazolium
bromide (3.33 mg/mL phosphate buffered saline, pH = 7.4) was
pipetted to each well and left to incubate for further 2 h. Then the
medium with MTT solution was removed. Formazan crystals in viable
cells were dissolved in the lysis solution (4 mM HCl and 0.1%
Nonidet P40 in ethanol). Microplates were shaken 15 min at
1500 rpm. Absorbance was measured at 540 nm and reference
wavelength at 740 nm. Experiments were performed in triplicates
and percent viability was calculated by dividing the OD obtained in
treatment group by OD of the untreated cell control multiplied by
hundred. Inhibition activity was expressed as percentages of control
with DMSO.
|
4.3.2 Prediction of in silico pharmacokinetic and
toxicity parameters
Pharmacokinetic (PK) properties depend on the chemical properties of
drugs, which determine their absorption, distribution, metabolism,
excretion, and toxicity (ADMET) properties, which are the key
descriptors for the human therapeutic use of any compound.
Predictive ADMET mathematical models were derived with different
PK parameters, namely, aqueous solubility, blood–brain barrier
penetration, cytochrome P-450 2D6 inhibition, hepatotoxicity, human
intestinal absorption, and plasma protein binding. Predictions from
these models were contrasted with known rules for appropriate
ADMET characteristics for 2-oxo-2-phenylethylidene linked 2-oxo-
benzo[1,4]oxazine analogues 17i, 17x, 18f–i, 18l, and 18o. Some
properties correlate well with PK, e.g, primary determinant of
fractional absorption can be represented by polar surface area (PSA)
(cut-off ≤140 Å2) and low molecular weight (MW) for absorption.[27h–j]
For secondary determination of fractional absorption (passive
membrane transport), the sum of H-bond donors and acceptors
(cut-off ≤12) was used. The number of rotatable bonds were used as a
measure of flexibility (cut-off ≤10) and bioavailability. Drug distribu-
tion depends on a number of factors, such as permeability (indicated by
apparent Caco-2 and MDCK permeability, log Kp for skin permeabil-
ity), blood-brain barrier (log BB), the volume of distribution and plasma
protein binding (log Khsa for serum protein binding).[27d–h] These
ADME descriptors were calculated and compared with standard
ranges. The octanol-water partition coefficient (logP) has been
|
4.3 In silico molecular docking simulation studies
Molecular docking studies were performed via constructing 3D model
of the structures using Surflex-Dock module in Sybyl-X 2.1 (Tripos
International). The X-ray crystallographic structure of the ACC (PDB
ID: 2OYE)[27a] solved at 2.85 Å resolution was retrieved from the PDB,
and modified for docking calculations. Co-crystallized ligand indo-
methacin was removed from the binding site, water molecules were
removed, ─H atoms were added, and side chains were fixed during
protein preparation. Protein structure minimization was performed by
applying Tripos force field, and partial atomic charges were calculated
by Gasteiger-Huckel method.[27b–d]

16 of 17
JAISWAL ET AL.
|
implicated in BB penetration and permeability studies. The excretion of
drugs from the body depends on logP and MW. Likewise, rapid renal
clearance is associated with hydrophilicity and small MW. In the liver,
drug metabolism is associated with hydrophobicity and large MW.
Higher lipophilicity leads to poor absorption and increase in metabolic
processes. ADME descriptors for 90% of orally active compounds
follow Lipinski's rule of 5. These ADME parameters were calculated
through Qikprop v3.2 (Schrödinger, LLC, USA, 2015 and Discovery
Studio 3.5). The recommended toxicity screening models for
carcinogenicity are developmental toxicity, mutagenicity, and skin
irritancy or sensitization, and these were calculated with the
DSTOPKAT module. These predictions are useful for the optimization
of therapeutic ratios of lead compounds and assessment of their
potential safety. These predictions also help in evaluating intermedi-
ates, metabolites and pollutants, along with setting dose range for
animal assays.
[9] K. Jurk, B. E. Kehrel, Semin. Thromb. Hemost. 2005, 31, 381.
[10] a) K. Sangkuhl, A. R. Shuldiner, T. E. Klein, R. B. Altman,
Pharmacogenet. Genom. 2011, 21, 516; b) J. M. Stassen, J. Arnout,
H. Deckmyn, Curr. Med. Chem. 2004, 11, 2245; c) C. Patrono,
N. Engl. J. Med. 1994, 330, 1287.
[11] a) O. Yasuda, Y. Takemura, H. Kawamoto, H. Rakugi, Cell. Mol. Life Sci.
2008, 65, 354; b) D. R. Phillips, I. F. Charo, L. V. Parise, L. A. Fitzgerald,
Blood 1988, 71, 831.
[12] a) J. A. Zablocki, M. Miyano, R. B. Garland, D. Pireh, L. Schretzman,
S. N. Rao, R. J. Lindmark, M. V. Toth, M. E. Zupec, D. E. McMackins,
S. P. Adams, M. Miyano, C. S. Markos, M. N. Milton, S. Paulson, M.
Herin, P. Jacqmin, N. S. Nicholson, S. G. Panzer-Knodle, N. F. Haas,
J. D. Page, J. A. Szalony, B. B. Taite, A. K. Salyers, L. W. King, J. G.
Campion, L. P. Feigen, J. Med. Chem. 1993, 36, 1811; b) D. F. Kong,
R. M. Califf, D. P. Miller, D. J. Moliterno, H. D. White, R. A. Harrington,
J. E. Tcheng, A. M. Lincoff, V. Hasselblad, E. J. Topol, Circulation 1998,
98, 2829.
[13] a) J. R. Leclerc, Crit. Care Med. 2002, 30, S332; b) M. J. Quinn, T. V.
Byzova, J. Qin, E. J. Topol, E. F. Plow, Arterioscler. Thromb. Vasc. Biol.
2003, 23, 945.
[14] a) J. Ruef, H. A. Katus, Expert Opin. Investig. Drugs 2003, 12, 781; b)
A. J. Lopez Farre, J. Tamargo, P. J. Mateos-Caceres, L. Azcona, C.
Macaya, Pharma. Res. 2010, 27, 2365.
ACKNOWLEDGMENTS
[15] a) B. C. Das, A. V. Madhukumar, J. Anguiano, S. Mani, Bioorg. Med.
Chem. Lett. 2009, 19, 4204; b) H. Otsuka, Y. Hirai, T. Nagao, K.
Yamasaki, J. Nat. Prod. 1988, 51, 74; c) B. P. Mathew, A. Kumar, S.
Sharma, P. K. Shukla, M. Nath, Eur. J. Med. Chem. 2010, 45, 1502; d)
H. B. Borate, S. R. Maujan, S. P. Sawargave, M. A. Chandavarkar, S. R.
Vaiude, V. A. Joshi, R. D. Wakharkar, R. Iyer, R. G. Kelkar, S. P. Chavan,
S. S. Kunte, Bioorg. Med. Chem. Lett. 2010, 20, 722; e) G. J. Reddy, S.
Sailaja, K. S. Rao, P. Reddanna, D. B. Reddy, Indian J. Chem., Sect. B:
Org. Chem. Incl. Med. Chem. 2005, 44B, 204; f) N. A. Powell, F. L. Ciske,
C. Cai, D. D. Holsworth, K. Mennen, C. A. Van Huis, M. Jalaie, J. Day,
M. Mastronardi, P. McConnell, I. Mochalkin, E. Zhang, M. J. Ryan, J.
Bryant, W. Collard, S. Ferreira, C. Gu, R. Collins, J. J. Edmunds, Bioorg.
Med. Chem. 2007, 15, 5912; g) P. Zhang, E. A. Terefenko, A. Fensome,
Z. Zhang, Y. Zhu, J. Cohen, R. Winneker, J. Wrobel, J. Yardley, Bioorg.
Med. Chem. Lett. 2002, 12, 787.
S.C. acknowledges SERB, New Delhi for Fast Track Scheme for young
scientist (Grant no. CS-037/2013); DST, New Delhi for DST-RFBR
Indo-Russian Joint Research Project (INT/RUS/RFBR/P-169); and
CSIR, New Delhi for CSIR-EMR Grant [02(0189)/14/EMR-II]. V.S.
thanks MNIT, Jaipur for providing financial assistance in the form of
fellowship. P.K.J acknowledges CSIR, New Delhi for providing RA
fellowship. D.K.Y acknowledges the National Research Foundation of
Korea (NRF), which is funded by the Ministry of Education, Science,
and Technology (No.: 2017R1C1B2003380) at the Gachon University,
Incheon City, Korea. Materials Research Centre (MRC), MNIT, Jaipur is
gratefully acknowledged for providing analytical facilities.
[16] a) X. Tian, L.-Y. Wang, S. Xia, Z.-B. Li, X.-H. Liu, Y. Yuan, L. Fang, H.
Zuo, Bioorg. Med. Chem. Lett. 2012, 22, 204; b) S. Xia, J-Q. Liu, X-H.
Wang, Y. Tian, Y. Wang, J-H. Wang, L. Fang, H. Zuo, Bioorg. Med.
Chem. Lett. 2014, 24, 1479.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
[17] D. A. Dudley, A. M. Bunker, L. G. Chi, W. L. Cody, D. R. Holland, D. P.
Ignasiak, N. Janiczek-Dolphin, T. B. McClanahan, T. E. Mertz, L. S.
Narasimhan, S. T. Rapundalo, J. A. Trautschold, C. A. Van Huis, J. J.
Edmunds, J. Med. Chem. 2000, 43, 4063.
ORCID
[18] a) P. Jakobsen, A. M. Horneman, E. Persson, Bioorg. Med. Chem. 2000,
8, 2803; b) P. Jakobsen, B. R. Pedersen, E. Persson, Bioorg. Med. Chem.
2000, 8, 2095.
Sandeep Chaudhary
http://orcid.org/0000-0002-1444-3895
[19] M. Anderluh, J. Cesar, P. Stefanic, D. Kikelj, D. Janes, J. Murn, K.
Nadrah, M. Tominc, E. Addicks, A. Giannis, M. Stegnar, M. S. Dolenc,
Eur. J. Med. Chem. 2005, 40, 25.
REFERENCES
̌
[1] A. D. Lopez, C. D. Mathers, M. Ezzati, D. T. Jamison, C. J. Murray,
Lancet 2006, 367, 1747.
[20] J. Ilaš, Z. Jakopin, T. Borštnar, M. Stegnar, D. Kikelj, J. Med. Chem.
2008, 51, 5617.
[2] a) E. Falk, Circulation 1985, 71, 699; b) B. S. Coller, N. Engl. J. Med.
1990, 322, 33.
[21] P. Š. Anderluh, M. Anderluh, J. Ilaš, J. Mravljak, M. S. Dolenc, M.
Stegnar, D. Kikelj, J. Med. Chem. 2005, 48, 3110.
[3] B. S. Coller, Circulation 1995, 92, 2373.
[22] a) S. K. Ihmaid, J. M. A. Al-Rawi, C. J. Bradley, M. J. Angove, M. N.
Robertson, Eur. J. Med. Chem. 2012, 57, 85; b) S. K. Ihmaid, J. M. A.
Al-Rawi, C. J. Bradley, Eur. J. Med. Chem. 2010, 45, 4934; c) S. K.
Ihmaid, J. M. A. Al-Rawi, C. J. Bradley, M. J. Angove, M. N. Robertson,
R. L. Clark, Bioorg. Med. Chem. 2011, 19, 3983; d) K. M. Pritchard,
J. M. A. Al-Rawi, C. J. Bradley, Eur. J. Med. Chem. 2007, 42, 1200; e) G.
Roma, M. D. Braccio, G. Grossi, D. Piras, G. Leoncini, D. Bruzzese,
M. G. Signorello, P. Fossa, L. Mosti, J. Med. Chem. 2007, 50, 2886; f)
T.-C. Ko, M.-J. Hour, J.-C. Lien, C.-M. Teng, K.-H. Lee, S.-C. Kuo, L.-J.
[4] E. Van De Graaff, S. R. Steinhubl, Ann. Med. 2000, 32, 561.
[5] a) A. Schwienhorst, Cell. Mol. Life Sci. 2006, 63, 2773; b) S. Srivastava,
L. N. Goswami, D. K. Dikshit, Med. Res. Rev. 2005, 25, 66.
[6] H. U. Stilz, B. Jablonka, M. Just, J. Knolle, E. F. Paulus, G. Zoller, J. Med.
Chem. 1996, 39(11), 2118.
[7] W. F. Urmey, J. Rowlingson, Reg. Anesth. Pain Med. 1998, 23, 146.
[8] S. A. Andronati, T. L. Karaseva, A. A. Krysko, Curr. Med. Chem. 2004,
11, 1183.

JAISWAL ET AL.
17 of 17
|
Huang, Bioorg. Med. Chem. Lett. 2001, 11, 279; g) C. Kontogiorgis, O.
499; e) D. K. Yadav, F. Khan, J. Chemometrics 2013, 27, 21; f) D. K.
Yadav, R. Rai, N. Kumar, S. Singh, S. Misra, P. Sharma., P. Shaw, H.
Pérez-Sánchez, R. L. Mancera, E. H. Choi, M. H. Kim, R. Pratap, Sci.
Rep. 2016, 6, 38128; g) D. K. Yadav, S. Dhawan, A. Chauhan, T.
Qidwai, P. Sharma, R. S. Bhakuni, O. P. Dhawan, F. Khan, Curr. Drug
Targ. 2014, 15, 753; h) D. K. Yadav, K. Kalani, A. K. Singh, F. Khan, S. K.
Srivastava, A. B. Pant, Curr. Med. Chem. 2014, 21, 1160; i) D. K. Yadav,
Saloni, P. Sharma, S. Misra, H. Singh, R. L. Mancera, K. Kim, C. Jang,
M. H. Kim, H. Pérez-Sánchez, E. H. Choi, S. Kumar, Arch. Pharm. Res.
2017, https://doi.org/10.1007/s12272-017-0945-7; j) D. K. Yadav,
S. Kumar, Saloni, H. Singh, M. H. Kim, P. Sharma, S. Misra, F. Khan,
Drug. Des. Devel. Ther. 2017, 11, 1859.
Nicolotti, G. F. Mangiatordi, M. Tognolini, F. Karalaki, C. Giorgio, A.
Patsilinakos, A. Carotti, D. Hadjipavlou-Litina, E. Barocelli, J. Enzyme
Inhib. Med. Chem. 2015, 30(6), 925.
[23] a) P. K. Jaiswal, V. Sharma, J. Prikhodko, I. V. Mashevskaya, S.
Chaudhary, Tetrahedron Lett. 2017, 58(22), 2077; b) R. Sharma, L.
Yadav, J. Lal, P. K. Jaiswal, M. Mathur, A. K. Swami, S. Chaudhary,
Bioorg. Med. Chem. Lett. 2017, 27(18), 4393.
[24] a) I. V. Mashevskaya, I. A. Tolmacheva, E. V. Voronova, T. F.
Odegova, G. A. Aleksandrova, A. F. Goleneva, S. V. Kol'tsova, A. N.
Maslivets, Pharm. Chem. J. 2002, 36(2), 33; b) Y. Iwanami, T. Seki, T.
Inagaki, Bull. Chem. Soc. Jpn. 1971, 44(5), 1316; c) V. L. Gein, N. A.
Rassudikhina, N. V. Shepelina, M. I. Vakhrin, E. B. Babushkina, E. V.
Voronina, Pharm. Chem. J. 2008, 42(9), 529; d) M. Xia, B. Wu, G.
Xiang, J. Fluor. Chem. 2008, 129(5), 402; e) E. E. Stepanova, Z. G.
Aliev, A. N. Maslivets, Russ. J. Org. Chem. 2013, 49, 1762 and
references cited therein.
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article.
[25] M. A. Packham, M. L. Rand, R. L. Kinlough-Rathbone, Camp. Biochem.
Physiol. 1992, 103A (I), 35.
[26] M. Danihelová, M. Veverka, E. Šturdík, S. Jantová, Interdiscip. Toxicol.
2013, 6, 209.
[27] a) A. L. Lourenço, M. S. Saito, L. E. Dorneles, G. M. Viana, P. C. Sathler,
L. C. Aguiar, M. de Pádula, T. F. Domingos, A. G. Fraga, C. R. Rodrigues,
V. P. de Sousa, H. C. Castro, L. M. Cabral, Molecules 2015, 20, 7174; b)
J. K. Gierse, J. J. McDonald, S. D. Hauser, S. H. Rangwala, C. M.
Koboldt, K. Seibert, J Biol. Chem. 1996, 271(26), 15810; c) R. G. Huff,
E. Bayram, H. Tan, S. T. Knutson, M. H. Knaggs, A. B. Richon, P.
Santago, II, Chem. Biodivers. 2005, 2, 1533; d) D. K. Yadav, I. Ahmad,
A. Shukla, F. Khan, A. S. Negi, A. Gupta, J. Chemometrics 2014, 28,
How to cite this article: Jaiswal PK, Sharma V, Kumar S,
et al. Non-peptide-based new class of platelet aggregation
inhibitors: Design, synthesis, bioevaluation, SAR, and in silico
studies. Arch Pharm Chem Life Sci. 2018;1–17.
https://doi.org/10.1002/ardp.201700349