Rational Design, Synthesis, and Biological Activity of N-(1,4-Benzoxazinone)Acetamide Derivatives as Potent Platelet Aggregation Inhibitors

Article abstract of DOI:10.1002/bkcs.11359

Inappropriate thrombus formation within blood vessels is the leading cause of mortality in the industrialized world. Platelet aggregation activated by thrombin may have close relationship with thrombosis. Based on our studies on the pharmacophoric role of 1,4-benzoxazine-3(4H)-one for desirable platelet aggregation inhibitory activity, we identified N-(4-ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-2-(4-methylpiperazin-1-yl)acetamide (BOAP-AM6) and N-(4-butyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-2-(4-(4-fluorophenyl)piperazin-1-yl)acetamide (BOAP-AM21) as platelet aggregation inhibitors with an IC₅₀ of 8.93 and 8.67 μM, respectively, as potent as the positive control aspirin. A combination of structure–activity relationships studies and molecular modeling revealed that the molecule BOAP-AM6 interacted with the amino acid residue TYR166 and ARG214 in the binding site of GPIIb/IIIa receptor through hydrogen bond and compound BOAP-AM21 acted on the amino acid residue ASN215 and ALA218, both through the same approach as the reported potent molecules 7a and 7b.

Full text of DOI:10.1002/bkcs.11359

Article
DOI: 10.1002/bkcs.11359
BULLETIN OF THE
Y. Xiang et al.
KOREAN CHEMICAL SOCIETY
Rational Design, Synthesis, and Biological Activity of N-(1,4-
Benzoxazinone)Acetamide Derivatives as Potent Platelet Aggregation
Inhibitors
Yi Xiang,† Xiu-Hua Wang,
,k
‡,§,k
Quan Yang, Jia-Lian Tan, Hee-Jae Jang, Hua Zuo, and
†
‡
¶
‡,*
¶
,
*
Dong-Soo Shin
†Department of Thoracic surgery, Fourth People’s Hospital of Sichuan Province, Chengdu 610016,
China
‡
College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China.
*
E-mail: zuohua@swu.edu.cn
§
st
Department of Pharmacy, The 251 Hospital of People’s Liberation Army of China, Zhangjiakou
75000, China
Department of Chemistry, Changwon National University, Changwon 641-773, Korea.
E-mail: dsshin@changwon.ac.kr
Received September 15, 2017, Accepted November 28, 2017
0
¶
*
Inappropriate thrombus formation within blood vessels is the leading cause of mortality in the industrial-
ized world. Platelet aggregation activated by thrombin may have close relationship with thrombosis.
Based on our studies on the pharmacophoric role of 1,4-benzoxazine-3(4H)-one for desirable platelet
aggregation inhibitory activity, we identified N-(4-ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-
2
-(4-methylpiperazin-1-yl)acetamide (BOAP-AM6) and N-(4-butyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]
oxazin-7-yl)-2-(4-(4-fluorophenyl)piperazin-1-yl)acetamide (BOAP-AM21) as platelet aggregation inhibi-
tors with an IC50 of 8.93 and 8.67 μM, respectively, as potent as the positive control aspirin. A combina-
tion of structure–activity relationships studies and molecular modeling revealed that the molecule BOAP-
AM6 interacted with the amino acid residue TYR166 and ARG214 in the binding site of GPIIb/IIIa recep-
tor through hydrogen bond and compound BOAP-AM21 acted on the amino acid residue ASN215 and
ALA218, both through the same approach as the reported potent molecules 7a and 7b.
Keywords: N-(1,4-Benzoxazinone)acetamide, Smiles rearrangement, Platelet aggregation inhibition,
Structure–activity relationships, GPIIb/IIIa receptor
0
agonist is involved in initiation of platelet aggregation (5 -
Introduction
diphosphate [ADP], epinephrine, collagen, platelet-activat-
ing factor (PAF), and others), thus blocking the platelet
GPIIb/IIIa receptor could effectively inhibit platelet
Abnormal coagulation and inappropriate thrombus forma-
tion within blood vessels precipitates many acute cardiovas-
cular disease states. The resulting clinical ramifications of
occlusive thrombus development, including myocardial
infarction, deep-vein thrombosis, pulmonary embolism, and
stroke, annually affect millions of people worldwide and
are the leading causes of mortality and morbidity in the
5–8
aggregation.
Many antiplatelet drugs have been developed according
9–13
to different mechanisms of action.
Based on the ample
evidences that exists for the role of benzoxazinone as
antithrombotic agents, acting as the inhibitors of a factor
Xa and the tissue factor/factor VIIa-induced coagulation,
GPIIb/IIIa receptor antagonist, and thrombin inhibitor and
1
industrialized world. Platelets are key participants in path-
ologic thrombosis due to their capacity to adhere to injured
blood vessels and to accumulate at the place of injury
14–19
2,3
fibrinogen receptor antagonist,
our previous efforts for
although they play a central role in normal hemostasis.
new drug discovery demonstrated that a group of 1,4-ben-
And platelet aggregation, activated by thrombin which prin-
zoxazine-3(4H)-one derivatives were novel antiplatelet
cipally transforms the soluble fibrinogen to the insoluble
20,21
4
agents.
Among them, BOAP-L9u was the most potent
fibrin, may have close relationship with thrombosis. The
compound against platelet aggregation and had an IC50
final obligatory step of the platelet aggregation is that the
fibrinogen binds to the activated platelet fibrinogen recep-
tors (glycoprotein IIb/IIIa; GPIIb/IIIa) regardless of which
value of 9.20 μM in an assay using ADP-induced rabbit
platelet-rich plasma, as potent as the positive control ticlo-
pidine and aspirin. Overall, BOAP-L9u is a promising lead
for continuous study.
k
These authors contributed equally to this work.
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Article
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ISSN (Print) 0253-2964 | (Online) 1229-5949
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substituted-2-chloroacetamide 2 (5.0 mmol, 1.0 equiv) and
2
1
-chloro-4-nitrophenol (5.0 mmol, 1.0 equiv) was heated at
40 C and generated the desired product by slowly adding
ꢀ
NaH (12.5 mmol, 2.5 equiv) in DMF (40 mL). The reaction
was monitored by TLC and pH indicator (control value 8.0).
After completion of the reaction, the solvent was removed
under vacuum and water (40 mL) was added into the resi-
due. The mixture was then extracted with ethyl acetate
(4 × 40 mL). The organic layers were combined, dried over
Figure 1. BOAP-L9u and structural formula for a new class of
N-(1,4-benzoxazinone)acetamides BOAP-AM.
In continuation of our quest to find new structures with
2
2
potent antiplatelet aggregation activity, we have designed
and systematically synthesized a new class of platelet aggre-
gation inhibitor N-(1,4-benzoxazinone)acetamides of struc-
tural type BOAP-AM, which originated from BOAP-L9u in
our lab and was followed by structural modifications
anhydrous MgSO , and evaporated under vacuum to give
4
the crude product. The residue obtained was purified by sil-
ica gel column chromatography (elute; ethyl acetate:petro-
leum = 1:10) to obtain the corresponding compound 3.
Representative Procedure for the Synthesis of 7-Amino-
2H-Benzo[b][1,4]oxazin-3(4H)-ones (4). To a stirred solu-
tionof7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one3(5.0mmol,
1.0 equiv) in methanol (30 mL), 10% Pd/C (0.4 mmol, 0.08
equiv) was added to the reaction mixture at room temperature.
Hydrogen was bubbled into the round bottomed flask con-
necting with a sealing system at atmospheric pressure at about
1 mL/min. The air in the sealing system was replaced by H2
(Figure 1). In this report, we will describe the synthesis and
in vitro activity of this novel series of antiplatelet aggregator,
to further exploit their structure–activity relationship (SAR)
profiles, and their molecular docking study on the plausible
binding way with the active site of GPIIb/IIIa receptor.
Experimental
4–6 times after the system was closed and tested. After hydro-
Chemistry. All of the reagents were obtained from com-
mercial sources. Solvents were dried and purified with
known conventional methods. Melting points (uncorrected)
were determined on a micro melting point apparatus
genation was terminated, the brown solid 4 formed was
isolated by filtration and the filtrate was distilled at vacuum
without further purification.
Representative Procedure for the Synthesis of 2-Chloro-
N-(4-substituted-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)
Acetamide (5). To a magnetically stirred solution of
(
Shanghai Shenguang Instrument Co., Ltd., Shanghai,
1
13
China). H and C NMR spectra (at 400 MHz and
00 MHz, respectively) were recorded in CDCl with tetra-
1
substituted
7-amino-2H-benzo[b][1,4]oxazin-3(4H)-one
3
methylsilane as internal reference on a Bruker Advance
00 FT spectrometer. Chemical shifts were reported in parts
4 (5.0 mmol, 1.0 equiv) and K CO (7.5 mmol, 1.5 equiv)
in CH Cl (30 mL), cooled in an ice bath, the chloroacetyl
2 2
2
3
5
per million. Mass spectra (MS) were measured by the ESI
method on an Agilent 6510 Q-TOF mass spectrometer.
CDCl3 was used as delivered from Adamas Co., Ltd.
chloride (6.0. mmol, 1.2 equiv) was added dropwise
slowly. The reaction mixture was stirred at room tempera-
ture and monitored by TLC. After the reaction was com-
pleted, the solvent was removed under vacuum and ice
water (30 mL) was added into the residue. The mixture
was then extracted with ethyl acetate (3 × 30 mL). The
organic layers were combined, dried over anhydrous
(Shanghai, China). Silica gel (70–230 mesh) was used for
flash column chromatography. All reactions were monitored
by TLC using 0.25 mm silica gel plates with UV indicator
(Shanghai Jiapeng Technology Co., Ltd., Shanghai, China).
Unless otherwise noted, other reagents were obtained from
commercial suppliers and used without further purification.
Representative Procedure for the Synthesis of N-
Substituted-2-Chloroacetamide (2). To a magnetically stir-
red solution of substituted aniline 1 (50.0 mmol, 1.0 equiv)
and K CO (75.0 mmol, 1.5 equiv) in CH Cl (100 mL),
MgSO , and evaporated under vacuum to give the crude
product 5 without further purification.
4
Representative Procedure for the Synthesis of N-(4-
substituted-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
substituted Acetamide BOAP-AM. The reaction mixture
of 2-chloro-N-(4-substituted-2H-benzo[b][1,4]oxazin-3(4H)
one-7-yl)acetamide 5 (5.0 mmol, 1.0 equiv), piperazine/
2
3
2
2
cooled in an ice bath, the chloroacetyl chloride (60.0 mmol,
.2 equiv.) was added dropwise slowly. The reaction mix-
1
morpholine derivative 6 (5.0 mmol, 1.0 equiv) and K CO3
2
ture was stirred at room temperature and monitored by
TLC (iodine as streak reagent). After the reaction was com-
pleted, the solvent was removed under vacuum and ice
water (50 mL) was added into the residue. The mixture
was then extracted with ethyl acetate (3 × 50 mL). The
organic layers were combined, dried over anhydrous
(7.5 mmol, 1.5 equiv) in MeCN (30 mL) was refluxed and
monitored by TLC. When the reaction was accomplished,
the solvents were removed under vacuum. To the obtained
residue, water (30 mL) was added. The mixture was then
extracted by ethyl acetate (3 × 30 mL), and the combined
organic layers were dried over MgSO . Removal of MgSO4
4
MgSO , and evaporated under vacuum to give the crude
product 2 without further purification.
Representative Procedure for the Synthesis of 7-Nitro-2H-
Benzo[b][1,4]oxazin-3(4H)-ones (3). The solution of N-
and evaporation of the solvent at reduced pressure gave the
crude product. The pure product BOAP-AM was obtained
by column chromatography on silica gel (elute; ethyl
acetate:petroleum = 1:4).
4
Bull. Korean Chem. Soc. 2018
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N-(1,4-Benzoxazinone)Acetamides with Antiplatelet Aggregation Activity
KOREAN CHEMICAL SOCIETY
N-(4-Ethyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
4-phenylpiperazin-1-yl)acetamide (BOAP-AM1). Brown
(CH ), 35.3 (CH ), 11.6 (CH ) ppm. HRMS (ESI): Calcd
2 2 3
for [M + H] (C H N O ) requires m/z 409.2234,
23 29 4 3
found m/z 409.2243.
+
(
ꢀ
1
solid; mp 139.6–141.1 C; H NMR (400 MHz, CDCl )
3
δ 9.01 (s, 1H), 7.23–7.15 (m, 4H; ArH), 6.88–6.80 (m,
N-(4-Ethyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
4
H; ArH), 4.50 (s, 2H; CH ), 3.89 (q, J = 7.2 Hz, 2H;
(morpholin-4-yl)acetamide (BOAP-AM5). Light yellow
2
ꢀ
1
CH ), 3.19 (t, J = 4.8 Hz, 4H; CH ), 3.13 (s, 2H; CH ),
solid; mp 132.4–133.7 C; H NMR (400 MHz, CDCl )
2
2
2
3
2
.71 (t, J = 4.8 Hz, 4H; CH ), 1.19 (t, J = 7.0 Hz, 3H;
δ 8.94 (s, 1H), 7.21–7.16 (m, 2H; ArH), 6.87 (d,
2
1
3
CH3); C NMR (100 MHz, CDCl ) δ 167.2 (C O),
J = 8.4 Hz, 1H; ArH), 4.51 (m, 2H; CH ), 3.93–3.88 (m,
3
2
1
(
1
62.6 (C O), 150.1 (C), 144.8 (C), 132.7 (C), 128.3
CH), 123.8 (C), 119.3 (CH), 115.4 (CH), 114.0 (CH),
12.8 (CH), 107.9 (CH), 66.8 (CH ), 61.0 (CH ), 52.7
2H; CH ), 3.72–3.70 (m, 4H; CH ), 3.07–3.06 (m, 2H;
2
2
CH ), 2.55–2.54 (m, 4H; CH ), 1.22–1.18 (m, 3H; CH );
2 2 3
1
3
C NMR (100 MHz, CDCl ) δ 166.9 (C O), 162.4
3
2
2
(
(
CH ), 48.6 (CH ), 35.3 (CH ), 11.6 (CH ) ppm. HRMS
ESI): Calcd. for [M + H] (C H N O ) requires m/z
22 27 4 3
(C O), 144.7 (C), 132.5 (C), 123.7 (C), 113.9 (CH),
112.7 (CH), 107.8 (CH), 66.7 (CH ), 66.0 (CH ), 61.4
2
2
2
3
+
2
2
3
95.2078, found m/z 395.2080.
N-(4-Ethyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
4-(2-fluorophenyl)piperazin-1-yl)acetamide (BOAP-AM2).
(CH ), 52.8 (CH ), 35.1 (CH ), 11.5 (CH ) ppm. HRMS
2 2 2 3
+
(ESI): Calcd for [M + H] (C H N O ) requires m/z
1
6
22
3
4
(
320.1605, found m/z 320.1605.
N-(4-Ethyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
ꢀ
1
Light brown solid; mp 118.0–119.0 C; H NMR
400 MHz, CDCl ) δ 9.03 (s, 1H), 7.22–7.17 (m, 2H; ArH),
(
7
(4-methylpiperazin-1-yl)acetamide (BOAP-AM6). Brown
3
1
.01–6.85 (m, 5H; ArH), 4.49 (s, 2H; CH ), 3.88 (q,
oil; H NMR (400 MHz, CDCl ) δ 9.07 (s, 1H),
2
3
J = 7.2 Hz, 2H; CH ), 3.12 (s, 2H; CH ), 3.10 (t,
7.29–7.27 (m, 2H; ArH), 6.94 (d, J = 9.2 Hz, 1H; ArH),
2
2
J = 4.0 Hz, 4H; CH ), 2.72 (t, J = 4.4 Hz, 4H; CH ), 1.18
4.58 (s, 2H; CH ), 3.97 (m, 2H; CH ), 3.17 (s, 2H; CH ),
2
2
2
2
2
1
3
(
t, J = 7.0 Hz, 3H; CH3); C NMR (100 MHz, CDCl ) δ
2.74 (s, 4H; CH ), 2.65 (s, 4H; CH ), 2.42 (s, 3H; CH ),
2 2 3
3
1
3
1
1
1
1
1
67.1 (C O), 162.4 (C O), 154.7 (d, J = 246.0 Hz; C),
44.6 (C), 138.6 (d, J = 8.6 Hz; C), 132.6 (C), 123.5 (C),
23.4 (d, J = 3.6 Hz; CH), 121.8 (d, J = 7.9 Hz; CH),
18.0 (d, J = 2.8 Hz; CH), 115.2 (d, J = 20.7 Hz; CH);
1.27 (t, J = 6.2 Hz, 3H; CH3); C NMR (100 MHz,
CDCl ) δ 168.1 (C O), 163.5 (C O), 145.6 (C), 133.6
3
(C), 124.6 (C), 114.9 (CH), 113.8 (CH), 108.8 (CH),
67.7 (CH ), 61.6 (CH ), 54.9 (CH ), 52.7 (CH ), 45.5
2
2
2
2
13.8 (CH), 112.7 (CH), 107.8 (CH), 66.6 (CH ), 60.9
(CH ), 36.2 (CH ), 12.5 (CH ) ppm. HRMS (ESI): Calcd
2 3 3
2
+
(
1
CH ), 52.5 (CH ), 49.6 (CH ), 49.6 (CH ), 35.1 (CH ),
for [M + H] (C H N O ) requires m/z 333.1921,
17 25 4 3
2
2
2
2
2
+
1.4 (CH ) ppm. HRMS (ESI): Calcd for [M + H]
found m/z 333.1933.
3
(
C H FN O ) requires m/z 413.1983, found m/z 413.1989.
N-(4-Propyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
2
2
26
4 3
N-(4-Ethyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
(4-phenylpiperazin-1-yl)acetamide (BOAP-AM7). Brown
ꢀ
1
(
4-(4-fluorophenyl)piperazin-1-yl)acetamide (BOAP-AM3).
solid; mp 109.6–109.9 C; H NMR (400 MHz, CDCl ) δ
3
ꢀ
1
Light brown solid; mp 121.4–122.6 C; H NMR
9.01 (s, 1H), 7.24–7.15 (m, 4H; ArH), 6.88–6.81 (m, 4H;
ArH), 4.51 (s, 2H; CH ), 3.80 (t, J = 7.2 Hz, 2H; CH ),
(
400 MHz, CDCl ) δ 9.00 (s, 1H), 7.23–7.15 (m, 2H;
3
2
2
ArH), 6.90–6.78 (m, 5H; ArH), 4.47 (s, 2H; CH ), 3.87 (q,
3.19 (t, J = 4.4 Hz, 4H; CH ), 3.13 (s, 2H; CH ), 2.71 (t,
2 2
2
J = 7.2 Hz, 2H; CH ), 3.11–3.08 (m, 6H; CH ), 2.69 (t,
J = 4.8 Hz, 4H; CH ), 1.61 (m, 2H; CH ), 0.89 (t,
2 2
2
2
13
13
J = 4.6 Hz, 4H; CH ), 1.16 (t, J = 7.0 Hz, 3H; CH );
C
J = 7.4 Hz, 3H; CH3); C NMR (100 MHz, CDCl ) δ
2
3
3
NMR (100 MHz, CDCl ) δ 167.0 (C O), 162.4 (C O),
167.2 (C O), 162.9 (C O), 150.1 (C), 144.9 (C), 132.7
(C), 128.3(CH), 124.0 (C), 119.3 (CH), 115.4 (CH), 114.2
(CH), 112.8 (CH), 107.9 (CH), 66.8 (CH ), 61.0 (CH ),
3
156.3 (d, J = 239.3 Hz; C), 146.5 (d, J = 2.2 Hz; C), 144.6
(
(
C), 132.5 (C), 123.5 (C), 116.9 (d, J = 7.6 Hz, CH), 114.5
d, J = 22.1 Hz, CH), 113.8 (CH), 112.6 (CH), 107.8 (CH),
2
2
52.7 (CH ), 48.6 (CH ), 41.7 (CH ), 19.5 (CH ), 10.3
2
2
2
2
+
6
6.6 (CH ), 60.8 (CH ), 52.4 (CH ), 49.3 (CH ), 35.1
(CH3) ppm. HRMS (ESI): Calcd for [M + H]
(C H N O ) requires m/z 409.2234, found m/z 409.2232.
2
2
2
2
+
(CH ), 11.4 (CH ) ppm. HRMS (ESI): Calcd for [M + H]
2 3
23 29 4 3
(
C H FN O ) requires m/z 4132.1983, found m/z
N-(4-Propyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
2
2
26
4 3
4
13.1980.
N-(4-Ethyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
4-Benzylpiperazin-1-yl)acetamide (BOAP-AM4). Brown
(4-(2-fluorophenyl)piperazin-1-yl)acetamide (BOAP-AM8).
Brown solid; mp 150.0–150.4 C; H NMR (400 MHz,
ꢀ
1
(
CDCl ) δ 9.01 (s, 1H), 7.19–7.16 (m, 2H; ArH), 7.01–6.82
3
ꢀ
1
solid; mp 118.6–121.8 C; H NMR (400 MHz, CDCl )
(m, 5H; ArH), 4.49 (s, 2H; CH ), 3.79 (t, J = 7.6 Hz; 2H;
3
2
δ 9.04 (s, 1H), 7.26–7.18 (m, 7H; ArH), 6.88–6.85 (m,
CH ), 3.12 (s, 2H; CH ), 3.09 (t, J = 4.6 Hz, 4H; CH ),
2
2
2
1
H; ArH), 4.51 (s, 2H; CH ), 3.90 (q, J = 7.2 Hz, 2H;
2.72 (t, J = 4.6 Hz, 4H; CH ), 1.64–1.55 (m, 2H; CH ),
2 2
2
13
CH ), 3.48 (s, 2H; CH ), 3.06 (s, 2H; CH ), 2.52 (m, 8H;
0.88 (t, J = 7.4 Hz, 3H; CH3); C NMR (100 MHz,
CDCl3) 167.3 (C O), 162.9 (C O), 154.9 (d,
2
2
2
1
3
CH ), 1.20 (t, J = 7.2 Hz, 3H; CH );
C NMR
δ
2
3
(
(
(
100 MHz, CDCl ) δ 167.5 (C O), 162.6 (C O), 144.8
C), 136.9 (C), 132.8 (C),128.2 (CH), 127.4 (CH), 126.3
CH), 123.6 (C), 113.9 (CH), 112.8 (CH), 107.9 (CH),
J = 246.1 Hz; C), 144.9 (C), 138.8 (d, J = 8.6 Hz; C),
132.8 (C), 124.0 (C), 123.7 (d, J = 3.6 Hz; CH), 122.1 (d,
J = 7.9 Hz; CH), 118.2 (d, J = 2.9 Hz; CH), 115.4 (d,
J = 20.7 Hz; CH), 114.3 (CH), 112.9 (CH), 108.0 (CH),
3
6
6.8 (CH ), 62.0 (CH ), 60.9 (CH ), 52.6 (CH ), 52.3
2 2 2 2
Bull. Korean Chem. Soc. 2018
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BULLETIN OF THE
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N-(1,4-Benzoxazinone)Acetamides with Antiplatelet Aggregation Activity
KOREAN CHEMICAL SOCIETY
6
6.9 (CH ), 61.1 (CH ), 52.8 (CH ), 49.9 (CH ), 49.9
(C O), 144.9 (C), 132.8 (C), 123.9 (C), 114.3 (CH),
112.8 (CH), 107.9 (CH), 66.8 (CH ), 60.9 (CH ), 54.4
2
2
2
2
(CH ), 41.8 (CH ), 19.5 (CH ), 10.3 (CH ) ppm. HRMS
2 2 2 3
+
2
2
(ESI): Calcd for [M + H] (C H FN O ) requires m/z
(CH ), 52.6 (CH ), 45.2 (CH ), 41.7 (CH ), 19.5 (CH ),
2 2 2 3 2
2
3
28
4
3
+
4
27.2140, found m/z 427.2145.
N-(4-Propyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
4-(4-fluorophenyl)piperazin-1-yl)acetamide (BOAP-AM9).
10.3 (CH ) ppm. HRMS (ESI): Calcd for [M + H]
3
(C H N O ) requires m/z 347.2078, found m/z 347.2074.
1
8 27 4 3
(
N-(4-Isopropyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-
1
Dark brown oil; H NMR (400 MHz, CDCl ) δ 9.00 (s,
2-(4-phenylpiperazin-1-yl)acetamide (BOAP-AM13). Light
3
1
1
4
H), 7.21–7.54 (m, 2H; ArH), 6.92–6.80 (m, 5H; ArH),
.50 (s, 2H; CH ), 3.79 (t, J = 7.4 Hz, 2H; CH ), 3.13–3.11
yellow oil; H NMR (400 MHz, CDCl ) δ 9.02 (s, 1H),
3
7.25–7.14 (m, 4H; ArH), 7.01 (d, J = 8.8 Hz, 1H; ArH),
6.88–6.80 (m, 3H; ArH), 4.71–4.60 (m, 1H; CH), 4.39 (s,
2H; CH ), 3.19 (t, J = 4.8 Hz, 4H; CH ), 3.12 (s, 2H;
2
2
(m, 6H; CH ), 2.72–2.71 (m, 4H; CH ), 1.60 (m, 2H; CH ),
2 2 2
1
3
0
.88 (t, J = 7.4 Hz, 3H; CH3); C NMR (100 MHz,
167.4 (C O), 163.1 (C O), 156.7 (d,
J = 239.3 Hz; C), 146.9 (d, J = 2.2 Hz; C), 145.0 (C),
32.9 (C), 124.2 (C), 117.35 (d, J = 7.7 Hz, CH), 114.94
2
2
CDCl3)
δ
CH ), 2.70 (t, J = 4.8 Hz, 4H; CH ), 1.45 (d, J = 7.0 Hz,
2 2
13
6H; CH3); C NMR (100 MHz, CDCl ) δ 167.1 (C O),
3
1
164.4 (C O), 149.9 (C), 145.9 (C), 132.6 (C), 128.2 (CH),
124.5 (C), 119.2 (CH), 115.4 (CH), 115.2 (CH), 112.5
(CH), 108.0 (CH), 67.6 (CH ), 60.9 (CH ), 52.5 (CH ),
(
(
(
d, J = 22.1 Hz, CH), 114.43 (CH), 113.0 (CH), 108.1
CH), 67.0 (CH ), 61.2 (CH ), 52.8 (CH ), 49.7 (CH ), 41.9
CH ), 19.7 (CH ), 10.5 (CH ) ppm. HRMS (ESI): Calcd
2
2
2
2
2
2
2
48.4 (CH ), 46.3 (CH), 18.9 (CH ) ppm. HRMS (ESI):
2 3
2
2
3
+
+
for [M + H] (C H FN O ) requires m/z 427.2140, found
Calcd for [M + H] (C H N O ) requires m/z 409.2234,
23 29 4 3
23
28
4
3
m/z 427.2136.
found m/z 409.2227.
N-(4-Isopropyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-
2-(4-(2-fluorophenyl)piperazin-1-yl)acetamide (BOAP-
N-(4-Propyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
(
4-benzylpiperazin-1-yl)acetamide (BOAP-AM10). Light
ꢀ
1
1
yellow solid; mp 101.8–102.2 C; H NMR (400 MHz,
AM14). Dark brown oil; H NMR (400 MHz, CDCl ) δ
3
CDCl ) δ 9.02 (s, 1H), 7.24–7.15 (m, 7H; ArH),
9.04 (s, 1H), 7.23–7.18 (m, 2H; ArH), 7.04–6.88 (m, 5H;
3
6
.84–6.81 (m, 1H; ArH), 4.49 (s, 2H; CH ), 3.79 (t,
ArH), 4.70–4.63 (m, 1H; CH), 4.41 (s, 2H; CH ), 3.14 (s,
2
2
J = 7.6 Hz, 2H; CH ), 3.46 (s, 2H; CH ), 3.03 (s, 2H;
2H; CH ), 3.11 (t, J = 4.0 Hz, 4H; CH ), 2.74 (t,
2
2
2
2
13
CH ), 2.55 (s, 4H; CH ), 2.45 (s, 4H; CH ), 1.64–1.59 (m,
J = 4.0 Hz, 4H; CH ), 1.46 (d, J = 7.0 Hz, 6H; CH );
C
2
2
2
2
3
1
3
2
H; CH ), 0.88 (t, J = 7.4 Hz, 3H; CH ); C NMR
NMR (100 MHz, CDCl ) δ 167.2 (C O), 164.5 (C O),
2
3
3
(
(
(
100 MHz, CDCl ) δ 167.8 (C O), 163.1 (C O), 145.1
C), 137.2 (C), 133.0 (C), 128.5 (CH), 127.7 (CH), 126.6
CH), 124.1 (C), 114.5 (CH), 113.0 (CH), 108.1 (CH),
154.8 (d, J = 246.0 Hz; C), 145.9 (C), 138.7 (d,
J = 8.6 Hz; C), 132.7 (C), 124.6 (C), 123.6 (d, J = 3.6 Hz;
CH), 121.9 (d, J = 7.9 Hz; CH), 118.09 (d, J = 2.9 Hz;
CH), 115.4 (d, J = 4.5 Hz; CH), 115.2 (CH), 112.6 (CH),
108.1 (CH), 67.7 (CH ), 61.0 (CH ), 52.7 (CH ), 49.8
3
6
7.0 (CH ), 62.3 (CH ), 61.2 (CH ), 52.9 (CH ), 52.6
2 2 2 2
(
(
CH ), 41.9 (CH ), 19.7 (CH ), 10.5 (CH ) ppm. HRMS
2 2 2 3
2
2
2
+
ESI): Calcd for [M + H] (C H N O ) requires m/z
(CH ), 49.7 (CH ), 46.4 (CH), 18.9 (CH ) ppm. HRMS
2 2 3
2
4
31
4
3
+
4
23.2391, found m/z 423.2391.
N-(4-Propyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
(ESI): Calcd for [M + H] (C H FN O ) requires m/z
23 28 4 3
426. 427.2140, found m/z 427.2148.
N-(4-Isopropyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-
2-(4-(4-fluorophenyl)piperazin-1-yl)acetamide (BOAP-
1
(
morpholin-4-yl)acetamide (BOAP-AM11). Yellow oil; H
NMR (400 MHz, CDCl ) δ 8.97 (s, 1H), 7.25–7.15 (m,
3
1
2
H; ArH), 6.85 (d, J = 8.8 Hz, 1H; ArH), 4.50 (s, 2H;
AM15). Yellow oil; H NMR (400 MHz, CDCl ) δ 9.01 (s,
3
CH ), 3.80 (t, J = 7.6 Hz, 2H; CH ), 3.70 (t, J = 4.6 Hz,
1H), 7.24–6.77 (m, 7H; ArH), 4.64–4.59 (m, 1H; CH),
4.36 (s, 2H; CH ), 3.10–3.08 (m, 6H; CH ), 2.69–2.68 (m,
2
2
4
H; CH ), 3.06 (s, 2H; CH ), 2.55 (t, J = 4.6 Hz, 4H;
2 2
2
2
13
CH ), 1.65–1.56 (m, 2H; CH ), 0.89 (t, J = 7.6 Hz, 3H;
4H; CH ), 1.42 (d, J = 6.8 Hz, 6H; CH ); C NMR
2 3
2
2
1
3
CH3); C NMR (100 MHz, CDCl ) δ 167.0 (C O),
62.8 (C O), 144.7 (C), 132.6 (C), 123.9 (C), 114.2
(100 MHz, CDCl ) δ 168.1 (C O), 165.4 (C O), 157.3
3
3
1
(d, J = 239.2 Hz; C), 147.6 (d, J = 2.2 Hz; C), 146.8 (C),
133.6 (C), 125.5 (C), 118.0 (d, J = 7.6 Hz, CH), 116.36
(CH), 115.6 (d, J = 22.0 Hz, CH), 113.6 (CH), 109.0 (CH),
68.6 (CH ), 61.9 (CH ), 53.5 (CH ), 50.3 (CH ), 47.3
(
CH), 112.8 (CH), 107.9 (CH), 66.7 (CH ), 66.0 (CH ),
2
2
6
1.4 (CH ), 52.9 (CH ), 41.6 (CH ), 19.4 (CH ), 10.2
2 2 2 2
+
(
(
CH3) ppm. HRMS (ESI): Calcd for [M + H]
C H N O ) requires m/z 334.1761, found m/z 334.1769.
2
2
2
2
+
(CH ), 19.8 (CH ) ppm. HRMS (ESI): Calcd for [M + H]
1
7
24
3
4
2
3
N-(4-Propyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
(C H FN O ) requires m/z 427.2140, found m/z
23 28 4 3
(
4-methylpiperazin-1-yl)acetamide (BOAP-AM12). Dark
427.2138.
ꢀ
1
brown solid; mp 99.8–100.5 C; H NMR (400 MHz,
N-(4-Isopropyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-
CDCl ) δ 9.01 (s, 1H), 7.22–7.17 (m, 2H; ArH), 6.85 (d,
2-(4-Benzylpiperazin-1-yl)acetamide (BOAP-AM16). Dark
brown solid; mp 118.2–121.2 C; H NMR (400 MHz,
3
ꢀ
1
J = 9.2 Hz, 1H; ArH), 4.51 (m, 2H; CH ), 3.80 (t,
2
J = 7.3 Hz, 2H; CH ), 3.06 (d, J = 1.2 Hz, 2H; CH ),
CDCl ) δ 9.03 (s, 1H), 7.23–7.16 (m, 7H; ArH), 7.00 (d,
2
2
3
2
3
.58 (s, 4H; CH ), 2.43 (s, 4H; CH ), 2.25 (d, J = 1.2 Hz,
J = 8.4 Hz, 1H; ArH), 4.68–4.61 (m, 1H; CH), 4.39 (s, 2H;
2
2
H; CH ), 1.66–1.57 (m, 2H; CH ), 0.90 (m, 3H; CH );
CH ), 3.46 (s, 2H; CH ), 3.04 (s, 2H; CH ), 2.55 (s, 4H;
3
2
3
2
2
2
1
3
C NMR (100 MHz, CDCl ) δ 167.5 (C O), 162.9
CH ), 2.45 (s, 4H; CH ), 1.44 (d, J = 6.9 Hz, 6H; CH );
2 2 3
3
Bull. Korean Chem. Soc. 2018
© 2018 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
4

BULLETIN OF THE
Article
N-(1,4-Benzoxazinone)Acetamides with Antiplatelet Aggregation Activity
KOREAN CHEMICAL SOCIETY
1
3
13
C NMR (100 MHz, CDCl ) δ 167.5 (C O), 164.5
J = 7.4 Hz, 3H; CH3); C NMR (100 MHz, CDCl ) δ
3
3
(
(
C O), 146.0 (C), 137.0 (C), 132.8 (C), 128.2 (CH), 127.4
CH), 126.3 (CH), 124.5 (C), 115.5 (CH), 112.7 (CH),
167.4 (C O), 163.0 (C O), 155.0 (d, J = 246.1 Hz; C),
145.0 (C), 138.9 (d, J = 8.7 Hz; C), 132.8 (C), 124.1 (C),
123.8 (d, J = 3.6 Hz; CH), 122.2 (d, J = 8.0 Hz; CH), 118.3
(d, J = 2.8 Hz; CH), 115.6 (d, J = 20.7 Hz; CH), 114.4
(CH), 113.0 (CH), 108.1 (CH), 67.0 (CH ), 61.2 (CH ), 52.9
1
08.1 (CH), 67.8 (CH ), 62.0 (CH ), 61.0 (CH ), 52.6
2 2 2
(CH ), 52.3 (CH ), 46.4 (CH), 19.0 (CH ) ppm. HRMS
2 2 3
+
24 31 4 3
(ESI): Calcd for [M + H] (C H N O ) requires m/z
2
2
423.2391, found m/z 423.2383.
(CH ), 50.0 (CH ), 50.0 (CH ), 40.1 (CH ), 28.4 (CH ),
2 2 2 2 2
N-(4-Isopropyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-
-(morpholin-4-yl)acetamide (BOAP-AM17). Yellow oil;
19.3 (CH ), 13.0 (CH ) ppm. HRMS (ESI): Calcd for
2 3
[M + H] (C H FN O ) requires m/z 441.2296, found m/z
24 30 4 3
+
2
1
H NMR (400 MHz, CDCl ) δ 8.95 (s, 1H), 7.22–7.14 (m,
441.2292.
3
2
H; ArH), 7.02 (d, J = 8.8 Hz, 1H; ArH), 4.66 (m, 1H;
N-(4-Butyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
CH), 4.40 (s, 2H; CH ), 3.70 (t, J = 4.6 Hz, 4H; CH ),
(4-(4-fluorophenyl)piperazin-1-yl)acetamide (BOAP-AM21).
Yellow solid; mp 99.8–101.5 C; H NMR (400 MHz,
2
2
ꢀ
1
3
.06 (s, 2H; CH ), 2.55 (t, J = 4.6 Hz, 4H; CH ), 1.45 (d,
2
2
1
3
J = 7.2 Hz, 6H; CH3); C NMR (100 MHz, CDCl ) δ
CDCl ) δ 8.98 (s, 1H), 7.21–7.15 (m, 2H; ArH), 6.91–6.78
3
3
166.7 (C O), 164.2 (C O), 145.6 (C), 132.3 (C), 124.3
(m, 5H; ArH), 4.48 (d, J = 1.6 Hz, 2H; CH ), 3.81 (t,
2
(
(
C), 115.2 (CH), 112.4 (CH), 107.8 (CH), 67.4 (CH ), 65.8
J = 7.4 Hz, 2H; CH ), 3.11–3.09 (m, 6H; CH ), 2.69 (s, 4H;
2
2
2
CH ), 61.1 (CH ), 52.6 (CH ), 46.1 (CH), 18.6 (CH )
CH ), 1.57–1.50 (m, 2H; CH ), 1.34–1.25 (s, 2H; CH ), 0.86
2 2 2
2
2
2
3
+
13
ppm. HRMS (ESI): Calcd for [M + H] (C H N O )
requires m/z 334.1761, found m/z 334.1769.
(t, J = 7.4 Hz, 3H; CH3); C NMR (100 MHz, CDCl ) δ
1
7
24
3
4
3
166.9 (C O), 162.6 (C O), 156.3 (d, J = 239.4 Hz; C),
146.5 (d, J = 2.3 Hz; C), 144.6 (C), 132.4 (C), 123.7 (C),
116.9 (d, J = 7.6 Hz, CH), 114.5 (d, J = 22.1 Hz, CH), 114.0
(CH), 112.5 (CH), 107.7 (CH), 66.6 (CH ), 60.8 (CH ), 52.4
N-(4-Isopropyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-
2
-(4-methylpiperazin-1-yl)acetamide (BOAP-AM18). Yel-
1
low oil; H NMR (400 MHz, CDCl ) δ 9.12 (s, 1H),
7
4
3
2
2
.36–7.23 (m, 2H; ArH), 7.09 (d, J = 8.8 Hz, 1H; ArH),
(CH ), 49.3 (CH ), 39.7 (CH ), 28.0 (CH ), 18.9 (CH ), 12.7
2 2 2 2 2
+
.76–4.69 (m, 1H; CH), 4.47 (s, 2H; CH ), 3.14 (s, 2H;
(CH3) ppm. HRMS (ESI): Calcd for [M + H]
2
CH ), 2.66 (s, 4H; CH ), 2.52 (s, 4H; CH ), 2.33 (s, 3H;
(C H FN O ) requires m/z 441.2296, found m/z 441.2297.
N-(4-Butyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
2
2
2
24 30
4 3
1
3
CH ), 1.53 (d, J = 7.0 Hz, 6H; CH );
C NMR
3
3
(
(
(
4
100 MHz, CDCl ) δ 168.3 (C O), 165.4 (C O), 146.8
(4-benzylpiperazin-1-yl)acetamide (BOAP-AM22). Yel-
3
1
C), 133.6 (C), 125.3 (C), 116.3 (CH), 113.5 (CH), 108.9
low oil; H NMR (400 MHz, CDCl ) δ 9.03 (s, 1H),
3
CH), 68.6 (CH ), 61.7 (CH ), 55.1 (CH ), 53.3 (CH ),
7.24–7.16 (m, 7H; ArH), 6.84 (d, J = 9.2 Hz, 1H; ArH),
4.48 (s, 2H; CH ), 3.82 (t, J = 7.4 Hz, 2H; CH ), 3.46 (s,
2
2
2
2
7.2 (CH), 45.9 (CH ), 19.8 (CH ) ppm. HRMS (ESI):
3 3
2
2
+
Calcd for [M + H] (C H N O ) requires m/z 347.2078,
2H; CH ), 3.04 (s, 2H; CH ), 2.55 (s, 4H; CH ), 2.45 (s,
2 2 2
1
8
27
4
3
found m/z 347.2083.
N-(4-Butyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
4H; CH ), 1.58–1.50 (m, 2H; CH ), 1.31 (m, 2H; CH ),
2 2 2
0.87 (t, J = 7.4 Hz, 3H; CH3); C NMR (100 MHz,
1
3
(
4-phenylpiperazin-1-yl)acetamide (BOAP-AM19). Light
CDCl ) δ 167.7 (C O), 163.0 (C O), 145.0 (C), 137.0
3
ꢀ
1
yellow solid; mp 133.3–134.0 C; H NMR (400 MHz,
CDCl ) δ 9.09 (s, 1H), 7.31–7.23 (m, 4H; ArH),
6
(C), 132.9 (C), 128.4 (CH), 127.5 (CH), 126.5 (CH),
124.0 (C), 114.3 (CH), 112.9 (CH), 108.0 (CH), 66.9
(CH ), 62.1 (CH ), 61.1 (CH ), 52.7 (CH ), 52.4 (CH ),
3
.96–6.88 (m, 4H; ArH), 4.58 (s, 2H; CH ), 3.91 (t,
2
2
2
2
2
2
J = 7.6 Hz, 2H; CH ), 3.27 (t, J = 4.8 Hz, 4H; CH ),
40.1 (CH ), 28.3 (CH ), 19.3 (CH ), 13.0 (CH ) ppm.
2 2 2 3
HRMS (ESI): Calcd for [M + H] (C H N O ) requires
25 33 4 3
m/z 437.2547, found m/z 437.2540.
N-(4-Butyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
2
2
+
3
1
.20 (s, 2H; CH ), 2.79 (t, J = 5.0 Hz, 4H; CH ),
2
2
.67–1.59 (m, 2H; CH ), 1.39 (m, 2H; CH ), 0.95 (t,
2
1
2
3
J = 7.4 Hz, 3H; CH3); C NMR (100 MHz, CDCl ) δ
3
1
(
1
68.1 (C=O), 163.7 (C=O), 150.9 (C), 145.8 (C), 133.6
C), 129.2 (CH), 124.9 (C), 120.2 (CH), 116.3 (CH),
15.1 (CH), 113.7 (CH), 108.8 (CH), 67.7 (CH ), 61.9
(morpholin-4-yl)acetamide (BOAP-AM23). Yellow oil;
1
H NMR (400 MHz, CDCl ) δ 8.97 (s, 1H), 7.24–7.15
3
(m, 2H; ArH), 6.85 (d, J = 8.8 Hz, 1H; ArH), 4.49 (s,
2H; CH ), 3.82 (t, J = 7.6 Hz, 2H; CH ), 3.69 (t,
2
(
CH ), 53.6 (CH ), 49.5 (CH ), 40.8 (CH ), 29.1 (CH ),
2 2 2 2 2
2
2
2
0.1 (CH ), 13.8 (CH ) ppm. HRMS (ESI): Calcd for
J = 4.6 Hz, 4H; CH ), 3.06 (s, 2H; CH ), 2.54 (t,
2 2
2
3
+
[
M + H] (C H N O ) requires m/z 423.2391, found
J = 4.6 Hz, 4H; CH ), 1.58–1.51 (m, 2H; CH ), 1.31 (m,
2 2
2
4
31
4
3
1
3
m/z 423.2399.
2H; CH ), 0.87 (t, J = 7.4 Hz, 3H; CH ); C NMR
2 3
N-(4-Butyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
(100 MHz, CDCl ) δ 167.0 (C O), 162.7 (C O), 144.7
3
(
4-(2-fluorophenyl)piperazin-1-yl)acetamide (BOAP-AM20).
(C), 132.5 (C), 123.8 (C), 114.1 (CH), 112.7 (CH), 107.8
(CH), 66.6 (CH ), 66.0 (CH ), 61.4 (CH ), 52.8 (CH ),
ꢀ
1
Light yellow powder; mp 161.5–162.3 C; H NMR
400 MHz, CDCl ) δ 9.02 (s, 1H), 7.21–7.19 (m, 2H; ArH),
2
2
2
2
(
7
39.8 (CH ), 28.1 (CH ), 19.0 (CH ), 12.8 (CH ) ppm.
2 2 2 3
3
+
.03–6.85 (m, 5H; ArH), 4.51 (s, 2H; CH ), 3.84 (t,
HRMS (ESI): Calcd for [M + H] (C H N O ) requires
18 26 3 4
2
J = 7.6 Hz, 2H; CH ), 3.14 (s, 2H; CH ), 3.11 (t,
m/z 348.1918, found m/z 348.1916.
N-(4-Butyl-2H-benzo[b][1,4]oxazin-3(4H)-one-7-yl)-2-
(4-methylpiperazin-1-yl)acetamide (BOAP-AM24).
2
2
J = 4.6 Hz, 4H; CH ), 2.74 (t, J = 4.6 Hz, 4H; CH ),
2
2
1
.60–1.52 (m, 2H; CH ), 1.37–1.28 (m, 2H; CH ), 0.88 (t,
2 2
Bull. Korean Chem. Soc. 2018
© 2018 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
5

BULLETIN OF THE
Article
N-(1,4-Benzoxazinone)Acetamides with Antiplatelet Aggregation Activity
KOREAN CHEMICAL SOCIETY
1
Yellow oil; H NMR (400 MHz, CDCl ) δ 9.03 (s, 1H),
n-Octanol/Water Partition Coefficient
3
7
.24–7.17 (m, 2H; ArH), 6.88–6.84 (m, 1H; ArH), 4.52–4.50
Materials. The n-octanol was purchased from Ruijinte
Chemical. Co. Ltd. (Tianjin, China). Water used in experi-
ments was double distilled water and the n-octanol and
water were mutually presaturated before performing the
experiments. All the chemicals were of analytical grade.
Partition Coefficient Determinations. The n-octanol/water
partition coefficient of all compounds was measured by a
(
m, 2H; CH ), 3.84 (m, 2H; CH ), 3.07–3.05 (m, 2H; CH ),
2
2
2
2
.59 (s, 4H; CH ), 2.44 (s, 4H; CH ), 2.27–2.25 (m, 3H; CH ),
2 2 3
.57–1.54 (m, 2H; CH ), 1.36–1.27 (m, 2H; CH ), 0.91–0.86
m, 3H; CH3); C NMR (100 MHz, CDCl ) δ 167.4 (C O),
62.8 (C O), 144.8 (C), 132.7 (C), 123.8 (C), 114.2 (CH),
12.7 (CH), 107.8 (CH), 66.7 (CH ), 60.8 (CH ), 54.3 (CH ),
2.5 (CH ), 45.0 (CH ), 39.9 (CH ), 28.2 (CH ), 19.1 (CH ),
2.8 (CH ) ppm. HRMS (ESI): Calcd for [M + H]
1
(
2
2
13
3
1
1
5
1
2
2
2
24
shake flask-ultraviolet spectrophotometry method. The
solution of each compound in n-octanol was prepared at a
concentration of 10–15 μg/mL and the concentration of
each compound was obtained through their respective stan-
dard curve. And then 10.0 mL of aqueous solution was
added to 10.0 mL n-octanol solution in glass flask. The
mixtures obtained were then placed into an ultrasonic
cleaner (SB-3200, Xinzhi Biotechnology, Ningbo, China)
for 30 min at room temperature and then maintained at
2
2
3
2
2
+
3
(
C H N O ) requires m/z 361.2234, found m/z 361.2239.
19 29 4 3
Biological Assay
In vitro Assay
Materials and Methods. Materials. ADP was purchased
from Sigma-Aldrich St. Louis, MO, USA. Ticlopidine was
purchased from Wuhan Yuancheng Tech. Development Co.,
Ltd Wuhan, China. All the chemicals were of analytical
grade.
Inhibition Against Platelet Aggregation. Each chemical
compound was dissolved in 20% dimethyl sulfoxide
DMSO) in water. The artery blood sample collected from
Male New Zealand rabbit (Tengxin Experimental Animal
Ltd. Co., Chongqing, China) was mixed with just enough
sodium citrate with a concentration 3.8% to prevent clotting,
and centrifuged at room temperature for 5 min at 800 rpm to
give platelet-rich plasma (PRP). The remaining blood was
further centrifuged for 15 min at 3000 rpm to give platelet-
poor plasma (PPP). In PRP, the number of platelets was
ꢀ
37 ꢁ 0.01 C in a shaking thermostatic oscillator (SHA-C,
Changzhou Aohua Instrument Co., Ltd., Jiangsu, China)
for at least 72 h. This led to the formation of two clear and
transparent phases, with a well-defined interface. The con-
centration of each compound in the n-octanol phase col-
lected was determined through their respective maximum
wavelength (λmax) by measuring the ultraviolet absorbance
using a UV–visible spectrophotometer (UV-2550, Shi-
madzu, Kyoto, Japan). The partition coefficients were
obtained by calculating the ratio of the concentration (cal-
culated based on the absorbance) in organic layer to the
concentration in water layer and the experimental measure-
ments were repeated at least three times. The reproducibil-
ity of the measured concentration was smaller than 0.1%,
and the maximum deviation from the average value was
always <0.15%.
(
6
adjusted to 250 ꢁ 25 × 10 /mL by dilution with PPP. A
2
0 μL of blank solvent or test compounds at various concen-
trations (200, 100, 50, 25 μmol/L) was added to 170 μL of
PRP and after 5 min the aggregation was initiated by adding
The n-octanol/water partition coefficient was defined as:
10 μL of ADP (4 μmol/L), in each well of 96-well plates,
respectively. Optical density (OD) at 630 nm was measured
with a Microplate Reader (BioTek ELX800) after 1, 2,
Pow = c =c−c :
o
o
4
, 6 min of the addition. All the experiments were performed
Here, Pow is n-octanol/water partition coefficient of the
23
in triplicate. The platelet aggregation rate was expressed as
PAR (%) and was calculated as follows:
compounds and c represents the concentration of the com-
o
pounds in n-octanol phase at equilibrium (Note that the
concentration c was obtained from the standard curve previ-
ously). Pow is actually the equilibrium constant for com-
pounds partitioned in n-octanol phase and aqueous phase
saturated with each other at certain temperature.
PARð%Þ = ð1 – OD =OD Þ × 100%:
1
0
Here, OD and OD indicated the optical density before the
0
1
ADP was added and after the ADP was added, respectively.
The following equation was used to calculate the inhibition
rate of platelet aggregation (IR).
Molecular Docking. Docking study was performed using
2
4
FlexX program in SYBYL1.1 software package in the
catalytic binding site of the X-ray structure of GPIIb/IIIa
23
receptor, with a resolution of 2.9 Å (PDB: 2VDM). Mole-
cules BOAP-AM6, BOAP-AM21, 7a, and 7b were built
using SYBYL1.1 molecular modeling package and mini-
mized using Powell method. Acceptor protein was prepared
by removing water molecules and adding hydrogens using
Prepare Protein Structure tool in SYBYL Biopolymer mod-
ule. Docking calculations were carried out using FlexX pro-
gram and 20 top ranked docking poses were saved for each
docking run.
IRð%Þ = ð1 – PARtest=PARblankÞ × 100%:
Here, PARtest and PARblank represented the PAR of the
tested compounds and the blank solvent, respectively. The
ability of the tested compounds to inhibit platelet aggrega-
tion was evaluated by IC , calculated from the inhibition
5
0
ratio linearity regression equation, which was fitted based
on the inhibition ratio.
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N-(1,4-Benzoxazinone)Acetamides with Antiplatelet Aggregation Activity
KOREAN CHEMICAL SOCIETY
Results and Discussion
Table 1. In vitro antiplatelet aggregation evaluation of the N-(1,4-
benzoxazinone)acetamides derivatives in platelet-rich plasma and
their logPow values.
Design and Structure–Activity Relationship. In order to
explore the binding mode of BOAP-L9u to GPIIb/IIIa, we
have performed molecular docking for its interaction with
GPIIb/IIIa using the crystal structure of GPIIb/IIIa in com-
a
b
Comp.
Structure
IC50 (μM)
LogPow
BOAP-AM1
9.24
2.027
2
1
plex with BOAP-L9u (PDB CODE: 2VDM) and com-
2
5
BOAP-AM2
BOAP-AM3
BOAP-AM4
BOAP-AM5
BOAP-AM6
BOAP-AM7
BOAP-AM8
BOAP-AM9
10.31
11.52
9.77
1.594
1.419
1.350
1.194
1.139
2.149
1.840
2.024
pared the QSAR study on benzoxazinone. Our results
showed that the hydrophobic pocket of the active site of
GPIIb/IIIa mediates the interactions with BOAP-L9u. In
addition, the carboxyl group forms hydrogen bond with the
amino acid residue ARG214.
To further explore the hydrogen bond and hydrophobic
interaction, using BOAP-L9u as the lead compound, we
designed BOAP-AM, in which the new acetyl bridged the
10.29
8.93
1
(
(
,4-benzoxazinone and piperazine ring. We expect that:
1) the 1,4-benzoxazinone plays a pharmacophore role;
2) the C O at 2-position forms the hydrogen bond with
amino acid ARG214; (3) 7-substituted C O of the new
acetyl forms hydrogen bond with Arg-Gly-Asp (RGD)
sequence of fibrinogen GPIIb/IIa, to further enhance the
antiaggregation activity (Figure 2). The set of compounds
vary in the substitutions on the 4-N of 1,4-benzoxazinone
ring and the new acetamide.
Chemistry. The synthesis of the compounds in Table 1
proceeded by linear stepwise processes as shown in
Scheme 1. Of the routes the alkylation of 2-chloro-4-
nitrophenol by 2-chloro-N-substituted acetamide 2 in DMF
was the most favored approach. The alkylation product
underwent intramolecular cyclization, namely Smiles rear-
13.12
12.19
12.81
BOAP-AM10
BOAP-AM11
BOAP-AM12
BOAP-AM13
BOAP-AM14
12.85
10.96
9.84
0.732
0.710
0.545
1.403
1.117
23,26
rangement
3
and directly afforded the benzoxazinone
via one-step reaction, which was then reduced with
hydrogen and Pd/C in methanol and provided the corre-
sponding 7-amino-1,4-benzoxazinone 4. Conversion of the
intermediate 4 to BOAP-AM was readily achieved by first
generating the acylation product 5 in ice bath and then N-
alkylation with piperazine/morpholine derivative 6. This
route demonstrated the Smiles rearrangement and further
derivatization is applicable to the preparation for the series
of desired compound BOAP-AM in reasonably good yield.
While it is apparent that benzoxazinone 3 may be pre-
pared by 2-chloro-N-substituted acetamide 2 and 2-chloro-
13.88
12.35
BOAP-AM15
11.97
1.556
BOAP-AM16
BOAP-AM17
BOAP-AM18
BOAP-AM19
BOAP-AM20
BOAP-AM21
12.99
12.29
11.01
13.01
11.88
8.67
0.959
0.754
0.347
1.744
1.957
2.147
4
-nitrophenol in K CO /CH CN system followed by the
2 3 3
Smiles rearrangement, the generation of intermediate
occurred very slowly and resulted in incomplete reac-
tions, and afforded extremely low yield of the products.
Alternatively, the nucleophilic substitution reaction and the
3
(
continued overleaf )
Figure 2. The identification of novel compound BOAP-AM.
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N-(1,4-Benzoxazinone)Acetamides with Antiplatelet Aggregation Activity
KOREAN CHEMICAL SOCIETY
Table 1 (continued)
acetamides improved the platelet aggregation inhibitory
activity with IC50 values ranged from 8.67 to 13.88 μM.
This trend suggested that the acetylamino moiety or the het-
erocycles formed additional interactions with the protein
surface. Among all the synthesized compounds, the pro-
longed alkyl group (ethyl, propyl and i-propyl) at the 4-
position of benzoxazinone moiety decreased the inhibition
activity (BOAP-AM1, BOAP-AM7, and BOAP-AM13, or
other serials) due to the steric hindrance for which the lon-
ger alkyl group might exceed the binding site of the GPIIb/
IIIa receptor. For the compounds containing methylpipera-
zinyl or morpholinyl group they exhibited higher inhibition
activity. It is noteworthy that the in vitro inhibitory activity
positively correlated with logPow (n-octanol/water partition
coefficient). For the compounds containing a methylpipera-
zine group (BOAP-AM6, BOAP-AM12, BOAP-AM18,
and BOAP-AM24), compound BOAP-AM6 had a lowest
IC50 value at 8.93 μM, with the highest logPow value at
a
b
Comp.
Structure
IC50 (μM)
LogPow
BOAP-AM22
BOAP-AM23
BOAP-AM24
12.92
11.57
11.06
1.587
0.959
0.849
Aspirin
Ticlopidine
6.17
3.65
—
—
1
.139. Morpholinyl-containing compound BOAP-AM5,
with logPow at 1.194, showed stronger antiaggregatory
activity than compound BOAP-AM11, BOAP-AM17, and
BOAP-AM23. The best platelet aggregation inhibitor, to
our surprise, was compound BOAP-AM21 with its IC50
value at 8.67 μM with an optimum n-octanol/water parti-
tion coefficient at 2.147.
Scheme 1. Synthetic route for the target molecule N-(1,4-benzoxa-
zinone)acetamides BOAP-AM.
Molecular Docking. Then we tried to find the interactions
of compound BOAP-AM6 and BOAP-AM21 with GPIIb/
IIIa receptor through molecular docking studies, to propose
their plausible binding mode against the active site of GPIIb/
IIIa receptor. The representative molecules BOAP-AM6
and BOAP-AM21 were docked into the binding pocket of
GPIIb/IIIa receptor using the surflex-dock module of Sybyl-
Smiles rearrangement were then conducted under more
alkaline conditions through a one-pot procedure. Screened
from the conventional bases such as NaOH, Cs CO ,
2
3
K CO and NaH, 2.5–3.5 equiv of NaH was proven to be
2
3
the most effective for the reactions at the temperature of
ꢀ
28
1
40 C in DMF. Moreover, the yield of the one-step cycli-
X 1.1 software. The X-ray structure of GPIIb/IIIa receptor
zation was moderate to good (61–78%).
(PDB: 2VDM) was selected for the docking study since it
has been proved that GPIIb/IIIa is an important reason for
Biological Activity. According to Born’s research that the
OD at the wavelength of 600 nm decreased quantitatively
29
the development of antiaggregation process. The binding
model of compound BOAP-AM6 with the active site of
GPIIb/IIIa receptor is shown in Figure 3(a). The hydropho-
bic pocket of the active site of GPIIb/IIIa mediates the inter-
actions with BOAP-AM6. The oxygen atom in the carbonyl
group of the benzoxazine ring formed a total of three
2
7
when the platelet aggregated, the platelet aggregation rate
and inhibition rate of platelet aggregation could be obtained
by measuring the changes in OD values before and after
the platelet aggregation. The IC50 values (concentration
required to inhibit platelet aggregation by 50%) for the syn-
thesized compounds BOAP-AM in ADP-induced rich
platelet plasma aggregation of New Zealand rabbit were
detected and the results were presented in Table 1 and the
antiplatelet aggregation drugs aspirin and ticlopidine were
used as positive controls.
ꢂ ꢂ ꢂ
hydrogen bonds represented by (–C O HO–TYR166),
ꢂ ꢂ ꢂ
ꢂ ꢂ ꢂ
(–C O HN–ARG214) and another (–C O HN–
ARG214). Figure 3(b) illustrates the binding way of com-
pound BOAP-AM21 with the active site of GPIIb/IIIa recep-
tor. Besides the two hydrogen bonds between the oxygen
atom in the carbonyl group of the benzoxazine ring and the
2
0–22
In the previous letters,
SARs in potential antiaggregatory compounds based on
H-benzo[b][1,4]oxazin-3(4H)-one core. Herein, we contin-
we demonstrated the basic
ꢂ ꢂ ꢂ
ꢂ ꢂ ꢂ
GPIIb/IIIa receptor revealed as two different (–C O HN–
ASN215), another hydrogen bond shown as (–C O HN–
2
ued our work on optimizing the structures by introducing
piperazinyl/morpholinyl to the benzoxazinone core and an
acetylamino group to the C-7 position of the benzoxazinone
skeleton and then assayed logPow (n-octanol/water partition
coefficient) to explore the relationship between the antipla-
telet aggregation activity and solubility of the compounds.
As depicted Table 1, these novel N-(1,4-benzoxazinone)
ALA218) was bridged from the oxygen atoms of the car-
bonyl at the 7-position to the nitrogen atom attaching to the
receptor, which could partly explain the excellent perfor-
mance of BOAP-AM21 in antiaggregation experiment.
To further explore the specific binding position of the
synthesized compounds, we then performed the docking
process which combined the reported α β antagonists 7a
IIb 3
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N-(1,4-Benzoxazinone)Acetamides with Antiplatelet Aggregation Activity
KOREAN CHEMICAL SOCIETY
synthesis, and evaluation of activities of a set of 1,4-
benzoxazinones capable of acting as an inhibitor against
ADP-induced platelet aggregation and analyzed the SAR.
To improve the inhibition activity, we optimized the scaf-
fold by developing a new group of N-(1,4-benzoxazinone)
acetamides BOAP-AM. The bioassay results verified that
all the synthesized compounds performed good antiplatelet
aggregation activity, with IC50 values ranging from 8.67 to
1
3.88 μM. The most potent antiaggregatory compounds
BOAP-AM6 and BOAP-AM21, which had even equally
aggregation inhibition activity as the control drug aspirin,
can serve as lead compounds for the next generation of
platelet inhibition agents, making use of established binding
modes and SARs.
Acknowledgments. We would like to thank the Science and
technology Support Program Project from the Science and
Technology Department of Sichuan Province, China (2015F
Z0070 and 2014sz0004-5), the Fundamental Research Funds
for the central Universities, P. R. China (XDJK2016A015,
XDJK2014C175), and the National Research Foundation of
Korea (NRF-2016R1D1A1B1011019) for financial support.
Figure 3. Molecular docking of compound BOAP-AM6 and
BOAP-AM21 into the binding site of GPIIb /IIIa receptor (PDB:
2
VDM). (a) Compound BOAP-AM6 docked into the active site
of GPIIb/IIIa receptor. (b) Compound BOAP-AM21 docked into
the active site of GPIIb/IIIa receptor.
Supporting Information. Additional supporting informa-
tion is available in the online version of this article.
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