Synthesis of 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives as platelet aggregation inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.11.027

Novel 2H-benzo[b][1,4]oxazin-3(4H)-ones have been synthesized by condensation, reduction, O-alkylation and Smiles rearrangement using 3-bromo-4-hydroxy benzaldehyde, anilines, and chloroacetyl chloride as starting materials. All the synthesized compounds

Full text of DOI:10.1016/j.bmcl.2011.11.027

Bioorganic & Medicinal Chemistry Letters 22 (2012) 204–206
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Bioorganic & Medicinal Chemistry Letters
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Synthesis of 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives as platelet
aggregation inhibitors
Xiao Tian a, Li-Ying Wang a, Shuai Xia a, Zhu-Bo Li a, Xing-Hui Liu a, Yuan Yuan a, Liang Fang b, , Hua Zuo a,
⇑
⇑
a College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
b Department of Oncology, The Ninth People’s Hospital of Chongqing, Chongqing 400700, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 2H-benzo[b][1,4]oxazin-3(4H)-ones have been synthesized by condensation, reduction, O-alkyl-
ation and Smiles rearrangement using 3-bromo-4-hydroxy benzaldehyde, anilines, and chloroacetyl chlo-
ride as starting materials. All the synthesized compounds have been characterized by 1H NMR, 13C NMR,
and HRMS, and tested for the inhibitory ability on platelet aggregation. The results have shown that the
ADP (adenosine 50-diphosphate)-induced platelet aggregation was inhibited by 7a–g with the IC50 value
Received 12 August 2011
Revised 20 October 2011
Accepted 9 November 2011
Available online 16 November 2011
at 10.14–18.83
among all the compounds, but less potent than the control drug ticlopidine (3.18
(6.07 mol/L). The preliminary structure–activity relationship (SAR) was initially investigated in the
study.
l
mol/L. Compound 7a exhibited the most potent inhibitory effect (IC50 = 10.14
lmol/L)
Keywords:
l
mol/L) and aspirin
2H-Benzo[b][1,4]oxazin-3(4H)-one
Smiles rearrangement
Platelet aggregation inhibition
ADP
l
Ó 2011 Elsevier Ltd. All rights reserved.
Structure–activity relationship
Arterial thromboembotic diseases, for example, ischemic heart
failure, myocardial infarction, unstable angina, and stroke, which
are caused by platelet aggregation, are the major causes of death
in developed countries.1 Platelets, or thrombocytes, show no inter-
action with the inner surface of normal blood vessel endothelium
(BVE) but adhere promptly where endothelial cells are altered or
extracellular matrix substrates are exposed. The final pathway in
platelet aggregation induced by diverse agents, such as collagen,
ADP (adenosine 50-diphosphate), and thrombin, involves binding
of the adhesive protein fibrinogen to the glycoprotein IIb/IIIa
(GPIIb/IIIa) on the surface of activated platelets.2 Since under path-
ological conditions platelet is a primary factor in thrombosis and
heart disease the inhibition of platelet function represents a
well-established approach to prevent these diseases.3
A number of current clinically used anti-platelet drugs are rep-
resented as aspirin, ticlopidine, dipiridamole, and tirofiban (Fig. 1).
In spite of the therapeutic anti-platelet agents, improvements are
needed due to the limited number of the agents and their side ef-
fects.4 Therefore, it is of great interest to explore new agents, not
only for use as drugs but also because such compounds could be
used as pharmacological tools to provide important information
regarding platelet function.
pounds with various biological activities such as anti-microbial,5
anti-inflammatory,6 anti-tumor,7 and act as Na/H exchange inhib-
itors,8 renin inhibitors,9 COX-2-inhibitors,10 and nonsteroidal pro-
gesterone receptor agonists.11
Jakobsen et al. found that benzoxazines showed inhibitory
activities on the tissue factor/factor VIIa-induced activation of fac-
tor X. The factor X is required to convert prothrombin to thrombin,
which then converts fibrinogen to fibrin as a final stage in forming
fibrin clot.12 The in vitro anti-aggregatory study on 2,7-di-substi-
tuted 2H-1,4-benzoxazine-3(4H)-ones indicated that such benzox-
azines exhibited high affinity towards aIIbb3 integrins. The binding
acts like Arg-Gly-Asp (RGD) peptides, a key recognition signal for
GPIIb/IIIa, whose importance was confirmed by the inhibition of
glycoprotein adhesion in the presence of small RGD-containing
peptides.13,14
Molecular modeling revealed that 4,7-di-substituted 2H-1,4-
benzoxazine-3(4H)-one analogs should have more ‘cup-shaped’
minimum conformations while retaining the generally accepted
distance requirement between carboxylate and amidine moie-
ties.13 Based on these findings, we describe the design, the synthe-
sis, and the evaluation of the anti-aggregatory activities of a series
of 4,7-di-substituted 2H-benzo[b][1,4]oxazin-3(4H)-ones including
the preliminary interpretation of their structure–activity relation-
ships. The set consists of 2H-benzo[b][1,4]oxazin-3(4H)-ones con-
taining a alkyl fragment at 4-position, as well as an aryl unit at
7-positions (Scheme 1).
On the other hand, benzoxazines represent a very important
heterocyclic scaffold which gives accessibility of specific com-
⇑
Corresponding authors. Tel.: +86 23 6821 3059; fax: +86 23 6886 5904.
According to our previous work,15 Smiles rearrangement, an
intramolecular nucleophilic reaction, is employed in the preparation
E-mail addresses: fangliangfranc@gmail.com (L. Fang),
zuohua@swu.edu.cn
(H. Zuo).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.027

X. Tian et al. / Bioorg. Med. Chem. Lett. 22 (2012) 204–206
205
H
N
R
Ph
O
N
HO2C
CO2H
N
N
Ph
O
S
NH
Ph
S
Cl
R = H Ticlopidine
4
S
O
O
OCOCH3
O
BuO
O
Aspirin
Tirofiban
Sulfinpyrazone
R = CO2Me Clopidogrel
Figure 1. Structures of the most prominent anti-platelet agents currently.
of this set of compounds. 2H-Benzo[b][1,4] oxazin-3(4H)-ones (7a–
g) were synthesized in satisfactory yields as shown in Scheme 1. The
Schiff’s bases 4 were synthesized by applying the condensation reac-
tion of anilines 3 and 3-bromo-4-hydroxybenzaldehyde in anhy-
drous ethanol. The reduction of compounds 4 by NaBH4 provided
compound 5, which then reacted with 2-chloro-N-alkylacetamides
2 in K2CO3/MeCN system to afford the O-alkylated compounds 6.
The following Smiles rearrangement of compound 6 in refluxing
DMF with Cs2CO3 as base gave 2H-benzo[b][1,4]oxazin-3(4H)-ones
7 as desired compounds.
As we mentioned above, benzoxazines are associated with var-
ious pharmacological functions. Based on these surveys, we stud-
ied the anti-platelet aggregation activity of compounds 7a–g. In
the platelet aggregation assays, ADP was employed as the inducer.
The ability of the tested compounds to inhibit platelet aggregation
was evaluated by IC50, calculated from the inhibition ratio linearity
regression equation which was fitted based on the inhibition ratio.
The result is presented in Table 1.
the nitrogen atom of the substituent aniline at 7-position of the
2H-benzo[b][1,4]oxazin-3(4H)-one and the nitrogen atom at the 4-
position can give the derivatives with a significantly lower pharma-
cological potency than compound 7a. This is the case for compound
7a, which does not contain substituent near the nitrogen atom in the
aniline fragment. Therefore, the platelet receptors may easily bond
to the compound, which contributed to the highest inhibitory activ-
ity of product 7a. The highest steric blockade o-CH3 around nitrogen
atom on the aniline hindered the binding of the compound 7c to the
platelet receptor, and decreased the anti-platelet effect. Thus, the
compound 7c showed the highest IC50 value among all the synthe-
sized compounds. Similarly, it was observed that compound 7b
exhibited weaker anti-platelet aggregation activity than compound
7a, due to the presence of n-butyl group attached to the nitrogen
atom of the oxazine ring.
Unfortunately, the detailed structure–activity relationship
(SAR) could not be established at present due to the limited variety
of compounds. The further SAR of the 2H-benzo[b][1,4]oxazin-
3(4H)-ones in order to determine in detail both the mechanism
of action and the structural requirements will be studied in our
laboratory.
In conclusion, set of 2H-benzo[b][1,4]oxazin-3(4H)-one deriva-
tives were synthesized with the assistance of Smiles rearrange-
ment. According to the pharmacological experiments, all the
compounds exhibited inhibitory activity against platelet aggrega-
tion, weaker than the control drugs aspirin and ticlopidine. The
preliminary study of structure–activity relationship indicated that
reducing the steric blockade at 4- and 7-position increased the
inhibitory ability of the compounds bonded to platelet receptors.
For presenting more detailed relationship between the structures
and the anti-platelet aggregation, more compounds need to be
approached.
All the compounds generally showed inhibitory effects on plate-
let aggregation induced by ADP in a concentration-dependent man-
ner, similar to the reference drugs, aspirin, and ticlopidine. The
tested compounds showed lower anti-aggregatory activity with
IC50 10.14–18.83 lmol/L, than aspirin and ticlopidine (Table 1).
The presence of propyl group at 4-position of 7-((phenyl amino)-
methyl)-2H-benzo[b][1,4]oxazin-3(4H)-one gave rise to the highest
activity of the series, in the product 7a. The introduction of methyl
group on the benzene ring at the 7-position of the substituted 2H-
benzo[b][1,4]oxazin-3(4H)-one produced a decrease of the anti-
platelet effect, with the IC50 value at 12.68–18.83 lmol/L (7c–g),
and the presence of prolonged alkyl chain at 4-position of nitrogen
atom reduced the activity as well (7b), compared with compound
7a. These results indicated that the stereospecific blockade around
R2
R2
OH
Br
NH2
Br
Br
NaBH4
EtOH
R2
EtOH
N
N
H
+
OHC
OH
OH
5
3
4
o-CH3
m-CH3
p-CH3
R2
=
O
Cl
K2CO3
R1HN
Cl
MeCN, K2CO3
R1NH2
Cl
MeCN,Reflux
O
2
1
NH2
R1
=
NH2
R1
N
O
O
O
NHR1
H
H
N
N
O
Br
R2
R2
reflux
7
6
Scheme 1. Synthesis of 2H-benzo[b][1,4]oxazin-3(4H)-ones.

206
X. Tian et al. / Bioorg. Med. Chem. Lett. 22 (2012) 204–206
Table 1
for Graduate Students (ky2010011) of Southwest University, China
to Li-Ying Wang, for financial support.
Anti-platelet aggregation activity of compounds 7a–g
Compounds
IC50 (lmol/L)
Supplementary data
N
O
O
O
O
Supplementary data (General procedures and spectral data.)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2011.11.027.
H
N
10.14
15.17
18.83
7a
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O
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N
O
O
H
N
7f
N
O
H
14.24
N
O
7g
Ticlopidine
Aspirin
3.18
6.07
Acknowledgments
We would like to thank the National Natural Science Founda-
tion of China (21002081) and Technology Innovation Foundation