Characterization of 4-methyl-2-oxo-1,2-dihydroquinolin-6-yl acetate as an effective antiplatelet agent

Article abstract of DOI:10.1016/j.bmc.2010.04.011

We have studied earlier a membrane bound novel enzyme Acetoxy Drug: protein transacetylase identified as Calreticulin Transacetylase (CRTAase) that catalyzes the transfer of acetyl groups from polyphenolic acetates (PAs) to the receptor proteins and thus modulating their biological activities. In this communication, we have reported for the first time that acetoxy quinolones are endowed with antiplatelet action by virtue of causing CRTAase catalyzed activation of platelet Nitric Oxide Synthase (NOS) by way of acetylation leading to the inhibition of ADP/Arachidonic acid (AA)-dependent platelet aggregation. The correlation of specificity of platelet CRTAase to various analogues of acetoxy quinolones with intracellular NO and consequent effect on inhibition of platelet aggregation was considered crucial. Among acetoxy quinolones screened, 6-AQ (4-methyl-2-oxo-1,2-dihydroquinolin-6-yl acetate/6-acetoxyquinolin-2-one, 22) was found to be the superior substrate to platelet CRTAase and emerged as the most active entity to produce antiplatelet action both in vitro and in vivo. 6-AQ caused the inhibition of cyclooxygenase-1 (Cox-1) resulting in the down regulation of thromboxane A2 (TxA2) and the inhibition of platelet aggregation. Structural modification of acetoxy quinolones positively correlated with enhancement of intracellular NO and antiplatelet action.

Full text of DOI:10.1016/j.bmc.2010.04.011

Bioorganic & Medicinal Chemistry 18 (2010) 4085–4094
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Characterization of 4-methyl-2-oxo-1,2-dihydroquinolin-6-yl acetate as an
effective antiplatelet agent
Nivedita Priya a, Anjali Gupta b, Karam Chand b, Prabhjot Singh a, Abha Kathuria b, Hanumantharao G. Raj a,
,
*
Virinder S. Parmar b, Sunil K. Sharma b,
*
a Department of Biochemistry, V P Chest Institute, University of Delhi, Delhi 110 007, India
b Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We have studied earlier a membrane bound novel enzyme Acetoxy Drug: protein transacetylase
identified as Calreticulin Transacetylase (CRTAase) that catalyzes the transfer of acetyl groups from poly-
phenolic acetates (PAs) to the receptor proteins and thus modulating their biological activities. In this
communication, we have reported for the first time that acetoxy quinolones are endowed with antiplate-
let action by virtue of causing CRTAase catalyzed activation of platelet Nitric Oxide Synthase (NOS) by
way of acetylation leading to the inhibition of ADP/Arachidonic acid (AA)-dependent platelet aggregation.
The correlation of specificity of platelet CRTAase to various analogues of acetoxy quinolones with intra-
cellular NO and consequent effect on inhibition of platelet aggregation was considered crucial. Among
acetoxy quinolones screened, 6-AQ (4-methyl-2-oxo-1,2-dihydroquinolin-6-yl acetate/6-acetoxyquino-
lin-2-one, 22) was found to be the superior substrate to platelet CRTAase and emerged as the most active
entity to produce antiplatelet action both in vitro and in vivo. 6-AQ caused the inhibition of cyclooxygen-
ase-1 (Cox-1) resulting in the down regulation of thromboxane A2 (TxA2) and the inhibition of platelet
aggregation. Structural modification of acetoxy quinolones positively correlated with enhancement of
intracellular NO and antiplatelet action.
Received 27 January 2010
Revised 1 April 2010
Accepted 2 April 2010
Available online 8 April 2010
Keywords:
Quinolin-2-ones
Platelet CRTAase
Antiplatelet agents
Cox-1
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
expressed on platelets, extensively bind to ADP resulting in the re-
lease of dense granules containing PAF (Platelet Activating Factors)
Platelets are involved in the cellular mechanisms of primary
homeostasis leading to the formation of blood clots. Platelets are
activated when brought into contact with agents such as collagen,
thrombin, and ADP. The damage to the blood vessel walls exposes
the sub endothelium proteins, most notably collagen. The
circulating platelets bind collagen with collagen-specific glycopro-
tein Ia/IIa receptors. The adhesion is strengthened further by the
large, multimeric circulating protein like von Willebrand factor
(vWF), which forms links between the platelets glycoprotein Ib/
IX/V and the collagen fibrils. This adhesion activates the platelets.
ADP receptors P2Y1 and P2Y12, both belonging to the
G-protein-coupled seven-transmembrane domain receptor family,
vWF, serotonin, thromboxane A2 (TxA2) which further activate the
other circulating platelets.1 Numerous antiplatelet agents were
developed based on their ability to block the receptors responsible
for platelet activation. Further, the agents causing the inhibition of
cyclooxygenase catalyzed TxA2 synthesis would also lead to the
inhibition of platelet aggregation.2 Previous investigations carried
out in our laboratory documented for the first time, the remarkable
activation of endothelial NOS by a certain class of PAs by way of
acetylation of NOS mediated by CRTAase.3 Accordingly, PAs were
found to be effective in the inhibition of ADP induced platelet
aggregation.
In the present investigation, efforts have been made to compare
the specificities of acetoxy quinolones on CRTAase mediated acti-
vation of NOS and also to delineate the structure activity relation-
ship (SAR) with reference to the effect of position of substitution of
acetoxy group on benzenoid ring and pyridone ring, alkyl group at
C-3 position of the quinolone moiety, and substitution at N- and
O- of the pyridone ring of PAs. The results clearly demonstrated
6-acetoxy-4-methylquinolin-2-one (6-AQ, 22) to be the best
substrate to platelet CRTAase compared to the other acetoxy quin-
olones resulting in inhibition of ADP induced platelet aggregation.
Abbreviations: AA, arachidonic acid; AQ, acetoxy quinolone; CDNB, 1-cloro 2,4-
dinitro benzene; Cox-1, cyclooxygenase-1; Cox-2, cyclooxygenase-2; CRTAase,
calreticulin transacetylase; DAMC, 7,8-diacetoxy-4-methyl coumarin; DCFH-DA,
dichlorofluorescin diacetate; NOS, nitric oxide Synthase; GSH, reduced glutathione;
GST, glutathione-S-transferase; NO, nitric oxide; PAs, polyphenolic acetates; PPP,
platelet poor plasma; PRP, platelet rich plasma; TxA2, thromboxane A2; TxB2,
thromboxane B2.
* Corresponding authors. Tel.: +91 11 27666646x191 (S.K.S.).
E-mail addresses: rajhg@yahoo.com (H.G. Raj), sk.sharma90@gmail.com
(S.K. Sharma).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.011

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N. Priya et al. / Bioorg. Med. Chem. 18 (2010) 4085–4094
formation of two isomers is due to the existence of tautomerism
2. Results
in the amidic bond. 4-Hydroxy quinolone (32) was prepared by
reaction of aniline with malonic acid in the presence of zinc chlo-
ride and phosphorus oxychloride and further its acetylation gives
4-acetoxy quinolone (4-AQ, 33), as shown in Scheme 3. All the
compounds were fully characterized on the basis of their physical
and spectral data, and of total 33 quinolone derivatives, 21, that is,
2, 3, 5, 6, 8, 9, 12, 14, 15, 17, 18, 21, and 23–31 are novel.
In our earlier work we elucidated the role of acetoxy groups on
the benzenoid ring of chromones, coumarins, xanthones, and flav-
ones in facilitating the acetylation of receptor proteins catalyzed by
CRTAase. In this regard we also studied the factors, such as the
proximity of the acetoxy group to the oxygen heteroatom, the role
of carbonyl group on the benzopyran nucleus, and the effect of
substituents on the coumarin molecule in controlling the protein
acetylation.4,5
2.1. CRTAase activity
However, the action of CRTAase on acetoxy quinolones has not
been studied so far. Herein, we have elucidated the action of CRTA-
ase on a series of acetoxy quinolones and consequent effect on the
enhancement of NO levels in platelets and inhibitory effect on
ADP/AA induced platelet aggregation. We have compared the spec-
ificities of acetoxy quinolones by varying the position of acetoxy
group on benzenoid ring, replacing 4-methyl group by acetoxy
group, and by incorporating alkyl groups of varying size at C-3
position. Also, we have synthesized N- and O-substituted alkyl es-
ters of acetoxy quinolin-2-ones to study the CRTAase substrate
specificity. Such a study would allow us to observe the effect there-
of on the rate of catalytic activity of CRTAase and the efficacy of
these acetoxy quinolones to activate platelet NOS. The methoxy
derivatives of C-3 alkyl quinolones (1–9) were synthesized via
Knorr reaction of 2-alkyl ethyl acetoacetate with anisidines.
Demethylation was carried out with a mixture of hydrobromic acid
and acetic acid to yield corresponding hydroxy quinolones (10–18)
which were then acetylated with acetic anhydride in acetic acid to
yield acetoxy derivatives 19–27 (Scheme 1). The 6- and 7-acetoxy
derivatives of quinolones were further derivatized into N- (28 and
29) and O-alkyl esters (30 and 31) by reaction with ethyl bromo-
acetate in the presence of potassium carbonate (Scheme 2). The
The results documented in Table 1 revealed the deferential
specificity of platelet CRTAase to a number of acetoxy quinolones
and the position of acetoxy group played a key role in deciding
specificity of platelet CRTAase and was found to be in the order:
6-AQ > 7-AQꢀ8-AQ o 4-AQ. It is evident from the results (Table 1)
that substitution of alkyl group at C-3 position resulted in drastic
reduction of CRTAase activity and longer the alkyl chain greater
was the inhibition. The substitution of ester group at N-, that is,
in compounds 28 and 29 resulted in the marginal decline of CRTA-
ase activity of platelets, while O-substitution in compounds 30 and
31 hardly affected the activity. Moreover, substitution of 4-methyl
group with acetoxy group (compound 33) showed poor inhibition
of platelet aggregation.
H
N
H
N
NH
O
2 CH2(COOH)2
O
(CH3CO)2O
CH3COOH
ZnCl2, POCl3
OH
OAc
32
33
Scheme 3. Synthesis of 4-acetoxy derivative of quinolin-2-one.
R
H
NH2
O
O
H2SO4
100ºC
135-150º C
N
+
H3CO
O
H3CO
O
O
R
R = H / C2H5 / C6H13
R = H / C2H5 / C6H13
1
H
H
H
8
5
2
9
N
O
R
N
O
R
N
O
R
7
Ac2O / CH3COOH
HBr / CH3COOH
reflux
R1
R1
R1
reflux
6
10
3
4
R = H; R1 = 7-OAc
1 R = H; R1 = 7-OCH3
10
R = H; R1 = 7-OH
19
20 R = C2H5; R1 = 7-OAc
21 R = C6H13; R1 = 7-OAc
2 R = C2H5; R1 = 7-OCH3
11 R = C2H5; R1 = 7-OH
12 R = C6H13; R1 = 7-OH
R = C6H13; R1 = 7-OCH3
3
R = H; R1 = 6-OAc
4 R = H; R1 = 6-OCH3
13
R = H; R1 = 6-OH
22
23 R = C2H5; R1 = 6-OAc
24 R = C6H13; R1 = 6-OAc
5 R = C2H5; R1 = 6-OCH3
14 R = C2H5; R1 = 6-OH
15 R = C6H13; R1 = 6-OH
R = C6H13; R1 = 6-OCH3
6
R = H; R1 = 8-OAc
7 R = H; R1 = 8-OCH3
16
R = H; R1 = 8-OH
25
26 R = C2H5; R1 = 8-OAc
27 R = C6H13; R1 = 8-OAc
8 R = C2H5; R1 = 8-OCH3
17 R = C2H5; R1 = 8-OH
18 R = C6H13; R1 = 8-OH
R = C6H13; R1 = 8-OCH3
9
Scheme 1. Synthesis of 3-alkyl derivatives of quinolin-2-ones.
CH2COOEt
H
N
N
OCH2COOEt
N
O
O
BrCH2COOEt
K2CO3 / DMF
+
R
R
R
R = 7-OAc
R = 6-OAc
28
30
R = 7-OAc
29 R = 6-OAc
R = 7-OAc
31 R = 6-OAc
Scheme 2. Synthesis of N- and O-substituted alkyl esters of quinolin-2-ones.

N. Priya et al. / Bioorg. Med. Chem. 18 (2010) 4085–4094
4087
Table 1
modification of acetoxy quinolones was found to influence their ef-
fect on NO production in platelets in tune with the specificity of
platelet CRTAase to these compounds (Table 1).
Assay of CRTAase activity of platelets using acetoxy quinolones as the acetyl group
donor
Acetoxy quinolones
CRTAase activity (units)
2.2.2. Effect of acetoxy quinolones on platelet aggregation
2.2.2.1. Platelet aggregation in vitro. The effect of acetoxy quino-
lones on the ADP and AA induced platelet aggregation in vitro is
shown in Figure 2. PRP was incubated with acetoxy quinolones fol-
lowed by the measurement of platelet aggregation by the addition
of ADP/AA. 6-AQ (22) inhibited the ADP induced platelet aggrega-
tion to a greater extent as compared to 7-AQ (19) and 8-AQ (25)
derivatives. The modification of carbonyl group of the quinolone
moiety (30 and 31) hardly affected the platelet aggregation while
the presence of alkyl ester at N- (28 and 29) resulted in appreciable
reduction of platelet aggregation. 6-AQ has profound effect on AA
induced platelet aggregation as compared to that induced by
ADP. The trend of activities followed the same manner, higher in
case of 6-acetoxy as compared to 7- and 8-acetoxy derivatives.
These observations have amply revealed that the structural modi-
fications of acetoxy quinolones alter their effectiveness in the man-
ner dependent on their specificities to CRTAase as the substrate.
The 6-hydroxyquinolone (13) was devoid of antiplatelet activity.
19
20
21
28
30
22
23
24
13
29
31
25
26
27
33
13.25 0.085
13.0 0.073
2.0 0.052
10.0 0.044
13.2 0.081
31.0 0.095
26.0 0.071
7.0 0.067
Nil
23.0 0.077
28.6 0.268
12 0.052
11.5 0.045
Nil
Nil
CRTAase activity was assayed in platelet lysate as described in Section 4.1. The unit
of CRTAase was expressed in terms of % inhibition of GST under the experimental
conditions. Values are mean SEM of five observations (p <0.01).
2.2. Enhancement of intracellular nitric oxide (NO) levels
2.2.2.2. Platelet aggregation in vivo. The effect of 6-AQ (22) on
the ADP/AA induced platelet aggregation in vivo is shown in Fig-
ure 3. Rats were treated with 6-AQ p.o., sacrificed after 24 h and
the platelet aggregation phenomenon was assessed by aggregom-
etry. 6-AQ was also found to effectively inhibit both ADP as well
as AA induced platelet aggregation in vivo. Like aspirin, 6-AQ has
also effectively inhibited AA induced platelet aggregation much
more than induced by ADP. Accordingly, 6-AQ exhibited greater
antiplatelet activity in rats while the 6-hydroxy derivative (13)
had no ability to inhibit platelet aggregation.
2.2.1. Measurement of NO level by flow cytometry
The influence of acetoxy quinolones on the NO level in platelets
has been clearly brought out in Figure 1. Platelets were incubated
with acetoxy quinolones followed by the measurement of NO lev-
els by flow cytometry. It is clear from the results that 6-AQ (22)
profoundly enhanced NO level in platelets as compared to 7-acet-
oxy- (19) and 8-acetoxyquinolone (25) derivatives. The structural
2.3. Cox-1 activity assay
The effect of 6-AQ (22) on the Cox-1 activity in vivo is shown in
Figure 4. Rats were treated with 6-AQ p.o., sacrificed after 24 h and
the Cox-1 activity was measured by ELISA. 6-AQ was found to
effectively inhibit Cox-1 activity like aspirin by approximately
3.5-fold. Accordingly, 6-AQ exhibited greater inhibition of Cox-1
activity in rats while the 6-hydroxy derivative (13) could not.
3. Discussion
Cardiovascular diseases such as myocardial infarction, unstable
angina, and deep vein thrombosis greatly contribute to the mortal-
ity in the developed world. For the treatment of such heart condi-
Figure 1. Influence of acetoxy quinolones on NO levels in washed platelets.
Figure 2. Screening of acetoxy quinolones for antiplatelet activity in vitro.

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N. Priya et al. / Bioorg. Med. Chem. 18 (2010) 4085–4094
Table 2
100
90
80
70
60
50
40
30
20
10
0
Kinetic analysis of PAs to CRTAase catalyzed transacetylase activity
Compound
Km
980
1528
1050
1558
mmax
DAMC
352
258
327
240
7-AQ (19)
6-AQ (22)
8-AQ (25)
Substrates were separately preincubated (37 °C, 10 min) with CRTAase and GST in
potassium phosphate buffer (pH 6.5) followed by addition of GSH and CDNB. The
absorbance was measured at 340 nm. Initial reaction velocities of CRTAase were
determined at varying substrates concentrations.
Aspirin
22
13
Treatment
platelet CRTAase to a number of acetoxy quinolones and shows
that the position of acetoxy group on the phenyl ring of quinolones
plays an important role in deciding specificity of platelet CRTAase.
It is evident from the results (Table 1) that the addition of alkyl
group at C-3 results in drastic reduction of CRTAase activity, that
is, longer the alkyl chain greater is the inhibition. The addition of
ester group at N- results in the marginal decline of CRTAase activity
of platelets, while the substitution of ethyl ester group at O- hardly
affects the activity of platelet CRTAase when used as substrate.
It is hypothesized that the enzyme catalyzed acetylation led to
the generation of phenoxide ion, the partial charge calculated
using ChemBio3D Ultra 11.0 (2008) indicates the amidic nitrogen
to have substantial positive charge (+0.6824) and to partially offset
this charge lone pair of electrons on oxygen attached to the benze-
noid ring may undergo partial conjugation with amidic nitrogen
(Fig. 5). This conjugation seems to be more effective in case of 6-
acetoxy quinolones as compared to its 7-/8-acetoxy analogues.
It is pertinent to point out that the ability of 6-AQ to inhibit
platelet aggregation by the enhancement of NO levels in platelets.
The enhanced NO formation in blood vessels is known to regulate
the vascular functions.13 6-AQ effectively inhibited the Cox-1 activ-
ity like aspirin through the blunting of Cox-1 activity and eventu-
ally TxA2, an AA metabolite acting as an endogenous platelet
activator, intensifies the extent of platelet aggregation. It is note-
worthy that 6-hydroxyquinolone (13), the deacetylated product
of 6-AQ was totally ineffective for the inhibition of platelet aggre-
gation and the similar pattern was observed for Cox-1 activity also.
This observation highlighted the acetyl group of quinolone as the
active moiety responsible for activation of platelet NOS through
acetylation leading to the antiplatelet action of acetoxy quinolones.
Fluoroquinolones are important and effective antibacterial agents.
They are recommended for a number of serious bacterial infections
and in some cases of life threatening infections. Quinolones in gen-
eral are well tolerated drugs. However, the toxicity of 6-AQ if any,
has to be ascertained before they are chosen for antiplatelet ther-
apy.14 The results documented in this paper have projected for
the first time the antiplatelet action of a PA, 6-AQ implicating the
cardinal role of CRTAase in the mechanism of action.
Figure 3. Antiplatelet activity of 6-AQ in vivo.
Figure 4. Assay for Cox-1 activity.
tions there is a greater need for the application of antiplatelet
drugs.6 Intensive search for the newer antiplatelet drugs is going
on worldwide. We have in this report characterized for the first
time acetoxy quinolones as the effective antiplatelet agents. The
rationale for viewing these compounds as the possible antiplatelet
targets had its origin with our novel finding that PAs such as acet-
oxy coumarins were the enhancers of intracellular NO by virtue of
causing acetylation of NOS.7 Recently, it was also observed that
aspirin acetylates NOS-3 in platelets that increases the activity of
this enzyme which would give rise to an increase in platelet NO
biosynthesis and thus contribute to the antiplatelet effect of aspi-
rin. Phosphorylation of NOS-3 has been shown to occur on serine
residues 114, 615, 633, and 1177 as well as on threonine-495
and can have important modulatory effects on their activity inde-
pendent of any changes in Ca2+ concentration.8 However, our ear-
lier work convincingly established that Calreticulin, an important
Ca2+ binding protein of lumen of endoplasmic reticulum, mediate
the transfer of acetyl group from PAs to target protein such as
NOS.7,9,10 The protein acetyl transferase function of Calreticulin
utilizing PAs as the acetyl group donors was termed CRTAase.11
PAs by virtue of enhancing NO levels in platelets were considered
to merit as antiplatelet agents. Accordingly, 7,8-diacetoxy 4-meth-
ylcoumarin (DAMC), a model PA, was found to significantly inhibit
ADP induced platelet aggregation.12 We have in this paper sought
to correlate the ability of acetoxy quinolones to inhibit platelet
aggregation with special reference to the specificity of platelet
CRTAase to acetoxy quinolones and NOS activation. The results
presented in the Table 1 revealed that various acetoxy quinolones
are the substrates for platelet CRTAase to varying degrees. Accord-
ingly, among the acetoxy quinolones screened, 6-AQ (22) was
found to be the most suitable substrate to platelet CRTAase that
activated platelet NOS to a greater extent compared to the other
acetoxy quinolones resulting in inhibition of ADP/AA induced
platelet aggregation. The kinetic analysis depicts a clear picture
of structural activity relation where the affinity for various quino-
lones is in the order: 6 AQ > 7AQꢀ8 AQ (Table 2). The results doc-
umented in Tables 1 and 2 reveal the deferential specificity of
4. Experimental
4.1. Materials and methods
4.1.1. Chemicals
The organic solvents (acetone, acetic anhydride, chloroform,
tetrahydrofuran, DMF, petroleum ether, and ethyl acetate) were
dried and distilled prior to their use. Reactions were monitored
Figure 5. Conjugation in case of 6-substituted quinolone.

N. Priya et al. / Bioorg. Med. Chem. 18 (2010) 4085–4094
4089
by precoated TLC plates (Merck Silica Gel 60F254); the spots were
visualized either by UV light, or by spraying with 5% alcoholic FeCl3
solution. Silica gel (100–200 mesh) was used for column chroma-
tography. Sodium hydride (60% dispersed in mineral oil) was sup-
plied from Spectrochem. Pvt. Ltd, India. Melting points were
recorded in capillaries in sulfuric acid bath and are uncorrected.
Infrared spectra were recorded on Perkin–Elmer FT-IR model
BXspectrophotometer. The 1H NMR and 13C NMR spectra were re-
corded on Bruker AC-400 (400 MHz, 100 MHz) NMR spectrometer
and Avance-300 spectrometer using TMS as internal standard. The
chemical shift values are on d scale and the coupling constant val-
ues (J) are in Hertz. The EI/HR mass spectra were recorded on Agi-
lent-6210 ES-TOF. Ethyl 2-ethyl-3-oxobutanoate, reduced
glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB), dichloro-
7.60 (d, 1H, J = 8.9 Hz, H-5), 12.29 (br s, 1H, NH); 13C NMR (CDCl3,
75 MHz): d 11.80 (CH2CH3), 14.48 (C-4 CH3), 23.09, 27.27, 29.57,
30.01, 32.21 ((CH2)5CH3), 55.79 (OCH3), 98.72, 111.94 (C-6 and C-
8), 115.71 (C-10), 125.96 (C-3), 129.05 (C-5), 138.92 (C-9), 143.51
(C-4), 160.97 (C-7), 164.76 (C-2); IR (KBr) mmax: 1660.22, 1612.15,
1562.22, 1514.39, 1462.82, 1398.69, 1260.73, 1225.71, 1173.39,
1028.91, 940.95, 927.99, 798.50, 607.85 cmÀ1; UV (methanol) kmax
:
325 and 339 nm; HRMS: C17H23O2N [M]+: 273.1893.
4.1.2.4. 6-Methoxy-4-methylquinolin-2(1H)-one (4). It was ob-
tained as white solid (75%); mp: 260–262 °C, (Literature va-
lue = 260–262 °C);18 1H NMR (DMSO-d6, 300 MHz): d 2.42 (s, 3H,
C-4 CH3), 3.82 (s, 3H, OCH3), 6.39 (s, 1H, H-3), 7.13–7.18 (m, 2H,
H-5 and H-7), 7.26 (dd, 1H, J = 1.8 and 5.3 Hz, H-8), 11.46 (br s,
1H, NH); 13C NMR (DMSO-d6, 75 MHz): d 19.45 (C-4 CH3), 56.37
(OCH3), 107.69, 117.52 (C-5 and C-7), 119.87 (C-10), 121.06 (C-
8), 122.11 (C-9), 131.38 (C-3), 148.27 (C-4), 154.96 (C-6), 162.03
fluorescin diacetate (DCFH-DA), L-arginine, adenosine diphosphate
(ADP), were obtained from M/S Sigma Chemical Co. St. Louis. Mo.
USA. All other chemicals used were of high purity and were ob-
tained from local suppliers.
(C-2); IR (KBr) mmax: 3433.06, 2821.76, 1653.70, 1621.98,
1503.71, 1421.31, 1275.63, 1240.22, 1202.18, 1179.30, 1044.05,
835.98, 628.40 cmÀ1; UV (methanol) kmax: 269 and 350 nm; HRMS:
C11H11O2N [M]+: 189.8188.
4.1.2. General procedure for the synthesis of 3-alkyl-methoxy-4-
methylquinolin-2-ones (1–9)
Anisidine (20 g, 164 mmol) was added drop wise to alkylated
ethyl acetoacetate15 (277 mmol) and the reaction mixture was
refluxed for 20 h. After the completion of reaction, the contents
were cooled and then poured on sodium carbonate solution. The
compound was extracted with chloroform and the solvent was
evaporated in vacuo. 70% Sulfuric acid (40 mL) was added and
the reaction mixture was stirred at 95 °C. The progress of reaction
was monitored on TLC (5% methanol–chloroform). On completion
of the reaction, the solution was cooled and poured on crushed
ice (500 g). The resulting precipitate was filtered and washed with
water and petroleum ether. The crude product was recrystallized
from ethanol to give methoxy quinolin-2-ones 1–9.16
4.1.2.5. 3-Ethyl-6-methoxy-4-methylquinolin-2(1H)-one (5). It
was obtained as white solid (65%); mp: 204–206 °C; 1H NMR
(DMSO-d6, 300 MHz): d 1.01 (t, 3H, J = 7.2 Hz, CH2CH3), 2.39 (s,
3H, C-4 CH3), 2.63 (q, 2H, J = 7.2 Hz, 2H, CH2CH3), 3.79 (s, 3H,
OCH3), 7.06–7.14 (m, 2H, H-5 and H-7), 7.21 (d, 1H, J = 8.7 Hz, H-
8), 11.49 (br s, 1H, NH); 13C NMR (DMSO-d6, 75 MHz): d 13.21
(CH2CH3), 14.52 (C-4 CH3), 19.71, (CH2CH3), 55.40 (OCH3), 106.83,
116.19 (C-5 and C-7), 117.65 (C-8), 120.64 (C-10), 131.45 (C-3),
132.90 (C-9), 140.99 (C-4), 154.02 (C-6), 160.85 (C-2); IR (KBr)
mmax
:
3434.17, 2965.75, 1642.07, 1502.12, 1462.63, 1414.19,
1371.42, 1271.48, 1218.23, 1131.27, 1038.23, 927.21, 836.80,
633.80 cmÀ1
UV (methanol) kmax 272 and 349 nm; HRMS:
C13H15O2N [M]+: 217.7774.
4.1.2.1. 7-Methoxy-4-methylquinolin-2(1H)-one (1). It was ob-
tained as white solid (83%); mp: 195 °C, (Literature va-
lue = 196 °C);17 1H NMR (acetone-d6, 300 MHz): d 2.45 (s, 3H, C-4
CH3), 3.88 (s, 3H, OCH3), 6.30 (s, 1H, H-3), 6.84 (d, 1H, J = 8.7 Hz,
H-6), 6.94 (s, 1H, H-8), 7.66 (d, 1H, J = 8.9 Hz, H-5), 11.00 (br s,
1H, NH); 13C NMR (DMSO-d6, 75 MHz): d 19.36 (C-4 CH3), 56.14
(OCH3), 99.12, 111.12 (C-6 and C-8), 114.68 (C-3), 118.56 (C-10),
127.02 (C-5), 141.26 (C-9), 148.85 (C-4), 161.80 (C-7), 162.89 (C-
;
:
4.1.2.6. 3-Hexyl-6-methoxy-4-methylquinolin-2(1H)-one (6). It
was obtained as white solid (70%); mp: 146–148 °C; 1H NMR
(CDCl3, 300 MHz): d 0.91 (br s, 3H, CH2CH3), 1.26–1.57 (m, 8H,
CH2(CH2)4CH3), 2.47 (s, 3H, C-4 CH3), 2.82 (t, 2H, J = 7.1 Hz,
CH2CH2), 3.87 (s, 3H, OCH3), 7.08–7.11 (m, 2H, H-5 and H-7),
7.35 (d, 1H, J = 8.4 Hz, H-8), 12.35 (br s, 1H, NH); 13C NMR (CDCl3,
75 MHz): d 14.18 (CH2CH3), 15.21 (C-4 CH3), 22.67, 27.13, 29.08,
29.56, 31.81 ((CH2)5CH3), 55.71 (OCH3), 106.57, 117.34 (C-5 and
C-7), 117.73 (C-8), 121.72 (C-10), 131.51 (C-3), 132.18 (C-9),
2); IR (KBr)
1417.53, 1261.70, 1217.14, 1177.11, 1023.59, 856.63, 808.71,
710.74 cmÀ1
UV (methanol) kmax 323 and 337 nm; HRMS:
C11H11O2N [M]+: 189.9665.
mmax: 2957.41, 1658.97, 1629.05, 1549.71, 1474.57,
;
:
4.1.2.2. 3-Ethyl-7-methoxy-4-methylquinolin-2(1H)-one (2). It
was obtained as yellow solid (65%); mp: 126 °C; 1H NMR (CDCl3,
300 MHz): d 1.17 (t, 3H, J = 7.4 Hz, CH2CH3), 2.46 (s, 3H, C-4 CH3),
2.81 (q, 2H, J = 7.5 Hz, CH2CH3), 3.89 (s, 3H, –OCH3), 6.78 (s, 1H,
H-8), 6.81 (d, 1H, J = 9.2 Hz, H-6), 7.59 (d, 1H, J = 8.7 Hz, H-5),
11.95 (br s, 1H, NH); 13C NMR (CDCl3, 75 MHz): d 13.50 (CH2CH3),
14.84 (C-4 CH3), 23.74 (CH2CH3), 55.51 (OCH3), 107.09, 119.39 (C-6
and C-8), 122.56 (C-10), 124.94 (C-3), 131.05 (C-5), 143.97 (C-9),
142.28 (C-4), 154.83 (C-6), 163.52 (C-2); IR (KBr) mmax: 3148.91,
2928.00, 2850.90, 1656.22, 1624.10, 1560.71, 1506.61, 1462.53,
1418.38, 1382.73, 1279.60, 1209.01, 1173.66, 1039.55, 855.83.
804.15, 725.86, 643.24 cmÀ1
;
UV (methanol) kmax
: 274 and
348 nm; HRMS: C17H23O2N [M]+: 273.4577.
4.1.2.7. 8-Methoxy-4-methylquinolin-2(1H)-one (7). It was ob-
tained as white solid (30%); mp: 192–194 °C, (Literature va-
lue = 188–190 °C);19 1H NMR (DMSO-d6, 300 MHz): d 2.41 (s, 3H,
C-4 CH3), 3.89 (s, 3H, OCH3), 6.42 (s, 1H, H-3), 7.14–7.15 (m, 2H,
H-5 and H-7), 7.27–7.30 (m, 1H, H-6), 10.58 (br s, 1H, NH); 13C
147.56 (C-4), 152.31 (C-7), 160.38 (C-2); IR (KBr)
mmax: 2932.78,
1660.95, 1621.90, 1560.80, 1512.79, 1461.85, 1396.94, 1255.88,
1223.07, 1179.61, 1141.37, 1029.98, 925.23, 842.09, 763.03 cmÀ1
;
UV (methanol) kmax: 324 and 339 nm; HRMS: C13H15O2N [M+H]+:
NMR (DMSO-d6, 75 MHz):
d 18.74 (C-4 CH3), 56.07 (OCH3),
218.3962.
110.89, 116.36 (C-5 and C-7), 120.08 (C-9), 121.36, 121.57 (C-3
and C-6), 128.57 (C-10), 145.80 (C-4), 148.15 (C-8), 161.14 (C-2);
4.1.2.3. 3-Hexyl-7-methoxy-4-methylquinolin-2(1H)-one (3). It
was obtained as yellow solid (75%); mp: 140 °C; 1H NMR (CDCl3,
IR (KBr) mmax: 3163.95, 2933.15, 1648.26, 1605.82, 1462.84,
1389.90, 1265.97, 1154.88, 1049.33, 860.83, 791.04, 739.93,
726.00, 636.77 cmÀ1; UV (methanol) kmax: 278 and 335 nm; HRMS:
C11H11O2N [M]+: 189.7739.
300 MHz):
d 0.91 (br s, 3H, CH2CH3), 1.26–1.55 (m, 8H,
CH2(CH2)4CH3), 2.47 (s, 3H, C-4 CH3), 2.76 (t, 2H, J = 7.3 Hz,
CH2CH2), 3.89 (s, 3H, OCH3), 6.81–6.86 (m, 2H, H-6 and H-8),

4090
N. Priya et al. / Bioorg. Med. Chem. 18 (2010) 4085–4094
4.1.2.8. 3-Ethyl-8-methoxy-4-methylquinolin-2(1H)-one (8). It
was obtained as white solid (20%); mp: 186–188 °C; 1H NMR
(DMSO-d6, 300 MHz): d 1.03 (t, 3H, J = 7.5 Hz, CH2CH3), 2.41 (s,
3H, C-4 CH3), 2.65 (q, 2H, J = 7.5 Hz, CH2CH3), 3.89 (s, 3H, OCH3),
7.07–7.16 (m, 2H, H-5 and H-6), 7.32 (dd, 1H, J = 2.1 and 7.5 Hz,
H-7), 10.48 (br s, 1H, NH); 13C NMR (DMSO-d6, 75 MHz): d 13.12
(CH2CH3), 14.69 (C-4 CH3), 19.69 (CH2CH3), 56.02 (OCH3), 109.83,
116.28 (C-5 and C-7), 120.47 (C-3), 121.33 (C-6), 126.93 (C-9),
132.98 (C-10), 141.66 (C-4), 145.47 (C-8), 160.75 (C-2); IR (KBr)
CH2(CH2)4CH3), 2.34 (s, 3H, C-4 CH3), 2.50–2.56 (m, 2H, CH2CH2),
6.60–6.67 (m, 2H, H-6 and H-8), 7.51–7.54 (m, 1H, H-5), 9.86 (br
s, 1H, OH), 11.35 (br s, 1H, NH); 13C NMR (CDCl3, 125 MHz): d
14.82 (CH2CH3), 15.54 (C-4 CH3), 22.98, 27.02, 29.53, 29.78, 32.09
((CH2)5CH3), 100.61, 111.93 (C-6 and C-8), 114.10 (C-10), 126.78
(C-3), 128.13 (C-5), 139.64 (C-9), 142.67 (C-4), 159.22 (C-7),
162.86 (C-2); IR (KBr)
1564.06, 1510.64, 1405.93, 1325.26, 1257.31, 1191.41, 1109.40,
807.53, 721.67, 692.46 cmÀ1
UV (methanol) kmax 326 and
340 nm; HRMS: C16H21O2N [M]+: 260.0510.
mmax: 3218.05, 2955.52, 2923.83, 1620.25,
;
:
mmax
:
3148.45, 2927.25, 1638.11, 1485.25, 1390.38, 1259.63,
1214.22, 1051.94, 866.49, 766.44, 727.12, 677.02 cmÀ1; UV (meth-
anol) kmax
:
256, 281 and 333 nm; HRMS: C13H15O2N [M]+:
4.1.3.4. 6-Hydroxy-4-methylquinolin-2(1H)-one (13). It was ob-
tained as white solid (80%); mp: 324–326 °C, (Literature va-
lue = 326–330 °C);22 1H NMR (DMSO-d6, 300 MHz): d 2.34 (s, 3H,
C-4 CH3), 6.36 (s, 1H, H-3), 7.00 (br s, 2H, H-5 and H-7), 7.17 (br
s, 1H, H-8), 9.40 (br s, 1H, OH), 11.42 (br s, 1H, NH); 13C NMR
(DMSO-d6, 75 MHz): d 19.36 (C-4 CH3), 109.41, 117.43 (C-5 and
C-7), 120.39 (C-10), 121.09 (C-8), 121.80 (C-9), 132.74 (C-3),
217.1143.
4.1.2.9. 3-Hexyl-8-methoxy-4-methylquinolin-2(1H)-one (9). It
was obtained as white solid (25%); mp: 112–114 °C; 1H NMR
(CDCl3, 300 MHz): d 0.89 (br s, 3H, CH2CH3), 1.31–1.52 (m, 8H,
CH2(CH2)4CH3), 2.46 (s, 3H, C-4 CH3), 2.74 (t, 2H, J = 7.2 Hz,
CH2CH2), 3.96 (s, 3H, OCH3), 6.93 (d, 1H, J = 7.8 Hz, H-5), 7.12 (t,
1H, J = 8.1 Hz, H-6), 7.29 (d, 1H, J = 8.4 Hz, H-7), 9.10 (br s, 1H,
NH); 13C NMR (DMSO, 75 MHz): d 14.14 (CH2CH3), 15.36 (C-4
CH3), 22.70, 27.22, 29.00, 29.60, 31.84 ((CH2)5CH3), 55.98 (OCH3),
108.75, 116.36 (C-5 and C-7), 121.37 (C-3), 121.46 (C-6), 126.84
(C-9), 132.73 (C-10), 142.63 (C-4), 145.39 (C-8), 161.64 (C-2); IR
148.01 (C-4), 152.93 (C-6), 161.99 (C-2); IR (KBr) mmax: 3489.59,
3187.46, 1661.73, 1634.87, 1608.22, 1513.42, 1433.01, 1294.34,
1191.88, 866.12, 853.10, 810.02, 642.28 cmÀ1; UV (methanol) kmax
:
269 and 352 nm; HR MS: C10H9O2N [M]+: 175.7805.
4.1.3.5. 3-Ethyl-6-hydroxy-4-methylquinolin-2(1H)-one (14). It
was obtained as white solid (75%); mp: 262–264 °C; 1H NMR
(DMSO-d6, 300 MHz): d 0.98 (t, 3H, J = 7.2 Hz, CH2CH3), d 2.31 (s,
3H, C-4 CH3), 2.59 (q, 2H, J = 7.05 Hz, CH2CH3), 6.91 (d, 1H,
J = 8.4 Hz, H-7), 7.01 (d, 1H, J = 1.8 Hz, H-5), 7.10 (d, 1H,
J = 8.7 Hz, H-8), 9.32 (br s, 1H, OH), 11.39 (br s, 1H, NH); 13C
NMR (DMSO-d6, 75 MHz): d 13.24 (CH2CH3), 14.42 (C-4 CH3),
19.68 (CH2CH3), 108.49, 116.05 (C-5 and C-7), 118.24 (C-8),
120.91 (C-10), 130.33 (C-9), 132.60 (C-3), 140.63 (C-4), 151.92
(KBr) mmax
:
2924.14, 1654.17, 1647.83, 1459.82, 1271.95,
1217.55, 1051.29, 862.42, 765.87, 727.19, 618.33 cmÀ1; UV (meth-
anol) kmax: 281 and 334 nm; HRMS: C17H23O2N [M]+: 273.2189.
4.1.3. General procedure for the synthesis of 3-alkyl-hydroxy-4-
methylquinolin-2-ones (10–18)
A mixture of 10 mL hydrobromic acid and acetic acid (7:3) was
added to 1 g of 3-alkyl-methoxy-4-methyl-1H-quinolin-2-ones (1–
9). The reaction mixture was refluxed for 75 h and then poured on
crushed ice. The resulting precipitate was filtered and washed with
water to yield 3-alkyl-7-hydroxyquinolin-2-ones (10–18).20
(C-6), 160.78 (C-2); IR (KBr)
mmax: 3231.81, 2973.40, 1641.50,
1620.48, 1505.72, 1480.33, 1431.71, 1392.90, 1319.33, 1279.99,
1207.53, 1170.02, 939.73, 880.82, 859.94, 700.63, 642.18 cmÀ1
;
UV (methanol) kmax: 274 and 350 nm; HRMS: C12H13O2N [M]+:
4.1.3.1. 7-Hydroxy-4-methylquinolin-2(1H)-one (10). It was ob-
tained as white solid (85%); mp: >300 °C, (Literature value = 306–
307 °C);21 1H NMR (DMSO-d6, 500 MHz): d 2.28 (s, 3H, C-4 CH3),
6.08 (s, 1H, H-3), 6.59 (d, 1H, J = 8.8 Hz, H-6), 6.65 (d, 1H,
J = 2.4 Hz, H-8), 7.45 (d, 1H, J = 8.8 Hz, H-5), 10.02 (br s, 1H, OH),
11.33 (br s, 1H, NH); 13C NMR (DMSO-d6, 125 MHz): d 19.03 (C-4
CH3), 100.61, 111.80 (C-6 and C-8), 113.32 (C-3), 117.50 (C-10),
126.68 (C-5), 141.04 (C-9), 148.44 (C-4), 159.95 (C-7), 162.69 (C-
203.7640.
4.1.3.6. 3-Hexyl-6-hydroxy-4-methylquinolin-2(1H)-one (15). It
was obtained as white solid (80%); mp: 221–223 °C; 1H NMR
(DMSO-d6, 300 MHz): d 0.80 (br s, 3H, CH2CH3), 1.23 (br s, 8H,
CH2(CH2)4CH3), 2.27 (s, 3H, C-4 CH3), 2.55 (br s, 2H, CH2CH2),
6.88 (d, 1H, J = 8.1 Hz, H-7), 6.97 (s, 1H, H-5), 7.07 (d, 1H,
J = 8.7 Hz, H-8), 9.24 (br s, 1H, OH), 11.34 (br s, 1H, NH); 13C
NMR (DMSO-d6, 75 MHz): d 13.89 (CH2CH3), 14.70 (C-4 CH3),
22.06, 26.45, 28.51, 28.89, 31.17 ((CH2)5CH3), 108.48, 116.04 (C-5
and C-7), 118.21 (C-8), 120.91 (C-10), 130.32 (C-9), 131.38 (C-3),
2); IR (KBr)
mmax: 3425.24, 2927.52, 2364.39, 1652.89, 1541.56
(amide-II), 1474.84, 1406.35, 1255.85, 1220.96, 1074.07, 905.44,
817.60, 689.94 cmÀ1; UV (methanol) kmax: 324 and 337 nm; HRMS:
C10H9O2N [M+H]+: 176.2008.
140.86 (C-4), 151.92 (C-6), 160.95 (C-2); IR (KBr)
3143.50, 2952.85, 1647.12, 1624.99, 1504.90, 1422.73, 1317.46,
1288.39, 1208.76, 1165.02, 937.98, 826.24, 645.45 cmÀ1
UV
274 and 350 nm; HRMS: C16H21O2N [M]+:
mmax: 3565.89,
4.1.3.2. 3-Ethyl-7-hydroxy-4-methylquinolin-2(1H)-one (11). It
was obtained as yellow solid (70%); mp: 250–252 °C; 1H NMR
(CDCl3, 300 MHz): d 1.01 (t, 3H, J = 7.3 Hz, CH2CH3), 2.35 (s, 3H,
C-4 CH3), 2.59 (q, 2H, J = 7.2 Hz, CH2CH3), 6.56 (d, 1H, J = 7.6 Hz,
H-6), 6.63 (s, 1H, H-8), 7.53 (d, 1H, J = 8.7 Hz, H-5), 9.87 (br s, 1H,
OH), 11.34 (br s, 1H, NH); 13C NMR (CDCl3, 75 MHz): d 13.89
(CH2CH3), 14.82 (C-4 CH3), 19.82 (CH2CH3), 100.21, 111.56 (C-6
and C-8), 113.67 (C-10), 126.39 (C-3), 128.92 (C-5), 139.23 (C-9),
;
(methanol) kmax
:
259.7355.
4.1.3.7. 8-Hydroxy-4-methylquinolin-2(1H)-one (16). It was ob-
tained as white solid (70%); mp: 250–254 °C, (Literature
value = 248 °C);23 1H NMR (DMSO-d6, 300 MHz): d 2.37 (s, 3H, C-
4 CH3), 6.34 (s, 1H, H-3), 6.89–6.98 (m, 2H, H-5 and H-6), 7.10
(d, 1H, J = 7.8 Hz, H-7), 10.25 (br s, 2H, NH and OH); 13C NMR
(DMSO-d6, 75 MHz): d 18.70 (C-4 CH3), 114.36, 114.96 (C-5 and
C-7), 120.48 (C-9), 121.04, 121.60 (C-3 and C-6), 127.84 (C-10),
142.07 (C-4), 158.85 (C-7), 162.30 (C-2); IR (KBr) mmax: 3291.86,
3229.49, 2966.43, 1629.42, 1573.05, 1514.05, 1417.29, 1319.53,
1256.84, 1191.89, 1066.27, 972.79, 862.40, 791.89, 690.01,
657.09 cmÀ1
;
UV (methanol) kmax
: 325 and 339 nm; HRMS:
C12H13O2N [M+H]+: 204.3963.
143.67 (C-4), 148.19 (C-8), 160.91 (C-2); IR (KBr)
mmax: 3401.25,
1639.37, 1601.19, 1553.68, 1478.15, 1398.60, 1288.58, 1208.23,
1145.95, 924.47, 861.73, 792.88, 735.68, 600.31 cmÀ1
; UV
4.1.3.3. 3-Hexyl-7-hydroxy-4-methylquinolin-2(1H)-one (12). It
was obtained as yellow solid (75%); mp: 202 °C; 1H NMR (DMSO-
d6, 500 MHz): d 0.85–0.87 (m, 3H, CH2CH3), 1.23–1.38 (m, 8H,
(methanol) kmax: 254, 279 and 336 nm; HRMS: C10H9O2N [M]+:
175.9968.

N. Priya et al. / Bioorg. Med. Chem. 18 (2010) 4085–4094
4091
4.1.3.8. 3-Ethyl-8-hydroxy-4-methylquinolin-2(1H)-one (17). It
was obtained as white solid (65%); mp: 212–214 °C; 1H NMR
(DMSO-d6, 300 MHz): d 0.94 (t, 3H, J = 7.2 Hz, CH2CH3), 2.31 (s,
3H, C-4 CH3), 2.56 (q, 2H, J = 7.2 Hz, CH2CH3), 6.83 (d, 1H,
J = 7.5 Hz, H-5), 6.89–6.94 (m, 1H, H-6), 7.11 (d, 1H, J = 8.1 Hz, H-
7), 10.11 (br s, 2H, NH and OH); 13C NMR (DMSO-d6, 75 MHz): d
13.19 (CH2CH3), 14.68 (C-4 CH3), 19.71 (CH2CH3), 113.31, 114.90
(C-5 and C-7), 120.94 (C-3), 121.47 (C-6), 126.24 (C-9), 132.63
1H NMR (DMSO-d6, 500 MHz): d 0.93 (br s, 3H, CH2CH3), 1.38–
1.58 (m, 8H, CH2(CH2)4CH3), 2.35 (s, 3H, C-4 CH3), 2.49 (s, 3H,
OCOCH3), 2.81 (br s, 2H, CH2CH2), 6.98 (d, 1H, J = 8.2 Hz, H-5),
7.14 (s, 1H, H-8), 7.70 (d, 1H, J = 8.4 Hz, H-6), 12.25 (br s, 1H,
NH); 13C NMR (CDCl3, 125 MHz): d 14.14 (CH2CH3), 15.23 (C-4
CH3), 21.14 (OCOCH3), 22.65, 26.92, 28.99, 29.48, 31.73
((CH2)5CH3), 108.42, 116.25 (C-6 and C-8), 119.09 (C-10), 125.53
(C-3), 131.45 (C-5), 137.59 (C-9), 142.68 (C-4), 151.11 (C-7),
(C-10), 141.84 (C-4), 143.31 (C-8), 160.62 (C-2); IR (KBr) mmax
:
164.01 (C-2), 169.24 (OCOCH3); IR (KBr) mmax: 3449.06, 2927.86
3178.10, 2970.45, 1924.13, 1654.16, 1606.43, 1560.17, 1606.43,
1462.64, 1397.96, 1290.56, 1205.28, 1011.15, 831.96, 768.59,
732.46, 680.38, 639.97 cmÀ1; UV (methanol) kmax: 257, 282 and
334 nm; HRMS: C12H13O2N [M]+: 203.6310.
(C-H str), 2855.18, 2367.37, 1765.40, 1656.22, 1564.92, 1511.10,
1459.84, 1370.74, 1223.68, 1014.71, 924.77 cmÀ1; UV (methanol)
kmax: 323 and 368 nm; HRMS: C18H23O3N [M]+: 301.9141.
4.1.4.4. 4-Methyl-2-oxo-1,2-dihydroquinolin-6-yl acetate25 (22). It
was obtained as white solid (85%); mp: 270 °C; 1H NMR (DMSO-
d6, 300 MHz): d 2.30 (s, 3H, C-4 CH3), 2.40 (s, 3H, OCOCH3), 6.46
(s, 1H, H-3), 7.29–7.35 (m, 2H, H-5 and H-7), 7.47 (dd, 1H, J = 1.5
and 7.5 Hz, H-8), 11.71 (br s, 1H, NH); 13C NMR (DMSO-d6,
75 MHz): d 18.39 (C-4 CH3), 20.76 (OCOCH3), 116.27, 117.06 (C-5
and C-7), 120.01 (C-10), 121.47 (C-8), 124.63 (C-3), 136.38 (C-9),
144.72 (C-4), 147.43 (C-6), 161.45 (C-2), 169.53 (OCOCH3); IR
4.1.3.9. 3-Hexyl-8-hydroxy-4-methylquinolin-2(1H)-one (18). It
was obtained as white solid (65%); mp: 174–176 °C; 1H NMR
(CDCl3, 300 MHz): d 0.88–0.90 (m, 3H, CH2CH3), 1.31–1.57 (m,
8H, CH2(CH2)4CH3), 2.51 (s, 3H, C-4 CH3), 2.80 (t, 2H, J = 6.9 Hz,
CH2CH2), 7.09–7.26 (m, 3H, H-5, H-6 and H-7), 10.52 (br s, 2H,
OH and NH); 13C NMR (CDCl3, 75 MHz): d 14.14 (CH2CH3), 15.50
(C-4 CH3), 22.68, 27.14, 29.06, 29.58, 31.78 ((CH2)5CH3), 114.68,
115.18 (C-5 and C-7), 122.20 (C-3), 122.98 (C-6), 125.76 (C-9),
130.98 (C-10), 144.03 (C-4), 145.18 (C-8), 162.80 (C-2); IR (KBr)
(KBr) mmax
:
3433.37, 2835.40, 1751.37, 1656.34, 1559.57,
1502.72, 1425.27, 1373.09, 1212.40, 1166.23, 1135.37, 1012.90,
905.94, 871.27, 842.91, 683.29 cmÀ1; UV (methanol) kmax: 264
and 334 nm; HRMS: C12H11O3N [M+H]+: 218.4582.
mmax
: 3377.54, 1638.80, 1624.56, 1600.44, 1552.36, 1394.62,
1278.70, 1229.22, 1200.55, 773.31, 710.18, 624.22 cmÀ1
; UV
(methanol) kmax: 255, 283 and 333 nm; HRMS: C16H21O2N [M]+:
259.4653.
4.1.4.5. 3-Ethyl-4-methyl-2-oxo-1,2-dihydroquinolin-6-yl ace-
tate (23). It was obtained as white crystals (80%); mp: 258–
260 °C; 1H NMR (DMSO-d6, 300 MHz): d 0.99 (t, 3H, J = 7.2 Hz,
CH2CH3), 2.26 (s, 3H, C-4 CH3), 2.35 (s, 3H, OCOCH3), 2.62 (q, 2H,
J = 7.2 Hz, CH2CH3), 7.19–7.29 (m, 2H, H-5 and H-7), 7.44 (s, 1H,
H-8), 11.67 (br s, 1H, NH); 13C NMR (DMSO-d6, 75 MHz): d 13.10
(CH2CH3), 14.45 (C-4 CH3), 19.68 (CH2CH3), 20.78 (OCOCH3),
115.76, 116.95 (C-5 and C-7), 120.50 (C-10), 123.36 (C-8), 133.32
(C-9), 134.89 (C-3), 141.59 (C-4), 144.60 (C-6), 161.15 (C-2),
4.1.4. General procedure for the synthesis of 3-alkyl-4-methyl-
2-oxo-1,2-dihydroquinolin-yl acetate (19–27)
A solution of 12 mL acetic anhydride and acetic acid (1:4) was
added to 1 g of 3-alkyl-hydroxy-4-methylquinolin-2(1H)-ones
10–18. The reaction mixture was refluxed for 6 h and then poured
on crushed ice.24 The resulting precipitate was filtered and washed
with water to yield acetoxy-3-alkylquinolin-2-ones 19–27.
170.04 (OCOCH3); IR (KBr) mmax
: 2934.63, 1752.29, 1644.31,
4.1.4.1. 4-Methyl-2-oxo-1,2-dihydroquinolin-7-yl acetate
(19). It was obtained as white solid (90%); mp: 258 °C, (Literature
value = 257–258 °C);21 1H NMR (DMSO-d6, 500 MHz): d 2.30 (s, 3H,
C-4 CH3), 2.42 (s, 3H, OCOCH3), 6.39 (s, 1H, H-3), 6.98 (dd, 1H,
J = 2.4 and J = 6.2 Hz, H-6), 7.05 (s, 1H, H-8), 7.73 (d, 1H,
J = 6.1 Hz, H-5), 11.67 (br s, 1H, NH); 13C NMR (DMSO-d6,
125 MHz): d 19.38 (C-4 CH3), 21.76 (OCOCH3), 108.77, 116.71 (C-
6 and C-8), 118.36 (C-3), 121.21 (C-10), 126.98 (C-5), 140.43 (C-
9), 148.52 (C-4), 152.59 (C-7), 162.63 (C-2), 169.82 (–OCOCH3);
1500.24, 1458.60, 1420.02, 1386.26, 1371.75, 1258.49, 1221.17,
1182.79, 1046.76, 1020.62, 953.84, 897.95, 705.93, 681.39 cmÀ1
;
UV (methanol) kmax: 269 and 332 nm; HRMS: C14H15O3N [M+H]+:
246.5055.
4.1.4.6. 3-Hexyl-4-methyl-2-oxo-1,2-dihydroquinolin-6-yl ace-
tate (24). It was obtained as white crystals (80%); mp: 158–
160 °C; 1H NMR (CDCl3, 300 MHz): d 0.90 (t, 3H, J = 6.8 Hz,
CH2CH3),1.34–1.57 (m, 8H, CH2(CH2)4CH3), 2.34 (s, 3H, C-4 CH3),
2.45 (s, 3H, OCOCH3), 2.80 (t, 2H, J = 7.5 Hz, CH2CH2), 7.18 (dd,
1H, J = 2.4 and 8.7 Hz, H-7), 7.39–7.43 (m, 2H, H-5 and H-8)
12.46 (br s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz): d 14.15
(CH2CH3), 15.15 (C-4 CH3), 21.11 (OCOCH3), 22.65, 27.05, 28.95,
29.51, 31.76 ((CH2)5CH3), 116.53, 117.04 (C-5 and C-7), 121.63
(C-10), 123.06 (C-8), 132.62 (C-3), 134.70 (C-9), 142.45 (C-4),
IR (KBr) mmax: 2927.12, 2855.27, 1751.47, 1678.55, 1561.24,
1510.88, 1458.53, 1362.63, 1232.32, 116 6.29, 1025.06, 906.26,
856.71, 645.56 cmÀ1; UV (methanol) kmax: 323 and 335 nm; HRMS:
C12H11O3N [M]+: 217.2915.
4.1.4.2. 3-Ethyl-4-methyl-2-oxo-1,2-dihydroquinolin-7-yl ace-
tate (20). It was obtained as yellow solid (75%); mp: 176–178 °C;
1H NMR (CDCl3, 300 MHz): d 1.20 (t, 3H, J = 7.2 Hz, CH2CH3), 2.36
(s, 3H, C-4 CH3), 2.50 (s, 3H, OCOCH3), 2.84 (q, 2H, J = 7.3 Hz,
CH2CH3), 6.84 (d, 1H, J = 7.8 Hz, H-6), 7.14 (s, 1H, H-8), 7.70 (d,
1H, J = 8.7 Hz, H-5), 12.12 (br s, 1H, NH); 13C NMR (CDCl3,
75 MHz): d 13.32 (CH2CH3), 14.83 (C-4 CH3), 20.09 (OCOCH3),
21.09 (CH2CH3), 108.49, 116.18 (C-6 and C-8), 118.98 (C-10),
125.37 (C-3), 132.41 (C-5), 137.59 (C-9), 142.44 (C-4), 151.08 (C-
145.38 (C-6), 163.83 (C-2), 169.90 (OCOCH3); IR (KBr) mmax
:
3432.83, 2926.24, 1761.26, 1656.07, 1500.98, 1369.83, 1219.35,
1176.07, 1014.16, 940.47, 628.69 cmÀ1; UV (methanol) kmax: 269
and 333 nm; HRMS: C18H23O3N [M]+: 301.6057.
4.1.4.7. 4-Methyl-2-oxo-1,2-dihydroquinolin-8-yl acetate (25). It
was obtained as white solid (85%); mp: 242–244 °C; 1H NMR
(DMSO-d6, 300 MHz): d 2.19 (s, 3H, C-4 CH3), 2.25 (s, 3H, OCOCH3),
6.27 (s, 1H, H-3), 7.01 (t, 1H, J = 7.9 Hz, H-6), 7.12 (d, 1H, J = 7.7 Hz,
H-5), 7.43 (d, 1H, J = 7.9 Hz, H-7), 11.27 (br s, 1H, NH); 13C NMR
(DMSO-d6, 75 MHz): d 18.68 (C-4 CH3), 21.35 (OCOCH3), 121.19
(C-9), 121.23, 121.48 (C-7 and C-3), 122.36, 123.84 (C-5 and C-6),
131.54 (C-10), 136.74 (C-4), 148.00 (C-8), 161.63 (C-2), 169.64
7), 163.89 (C-2), 169.21 (–OCOCH3); IR (KBr)
mmax: 3448.86,
2931.73, 2851.67, 1770.34, 1658.08, 1562.63, 1510.84, 1370.03,
1207.92, 1157.16, 1012.24, 911.90 cmÀ1; UV (methanol) kmax
:
323 and 336 nm; HRMS: C14H15O3N [M+H]+: 246.7908.
4.1.4.3. 3-Hexyl-4-methyl-2-oxo-1,2-dihydroquinolin-7-yl ace-
(OCOCH3); IR (KBr)
mmax: 3431.86, 2996.30, 1763.12, 1671.14,
tate (21). It was obtained as yellow solid (80%); mp: 134–136 °C;
1647.83, 1605.69, 1475.41, 1422.01, 1366.81, 1195.13, 1168.09,

4092
N. Priya et al. / Bioorg. Med. Chem. 18 (2010) 4085–4094
1138.04, 1016.39, 932.32, 869.41, 744.18, 689.19 cmÀ1; UV (meth-
123.72 (C-10), 124.79 (C-5), 146.89 (C-9), 147.29 (C-4), 151.31
(C-7), 161.07 (C-2), 169.24 (–OCOCH3), 169.35 (COOCH2CH3); IR
anol) kmax: 268 and 328 nm; HRMS: C12H11O3N [M+H]+: 218.1072.
(KBr) mmax
:
2989.04, 2928.22, 1761.41, 1641.32, 1580.26,
4.1.4.8. 3-Ethyl-4-methyl-2-oxo-1,2-dihydroquinolin-8-yl ace-
tate (26). It was obtained as white solid (80%); mp: 210–212 °C;
1H NMR (DMSO-d6, 300 MHz): d 1.04 (t, 3H, J = 7.5 Hz, CH2CH3),
2.37 (s, 3H, C-4 CH3), 2.44 (s, 3H, OCOCH3), 2.66 (q, 2H, J = 7.5 Hz,
CH2CH3), 7.15–7.25 (m, 2H, H-5 and H-6), 7.64 (dd, 1H, J = 1.5
and 7.8 Hz, H-7), 11.42 (br s, 1H, NH); 13C NMR (DMSO-d6,
75 MHz): d 13.11 (CH2CH3), 14.72 (C-4 CH3), 19.67 (CH2CH3),
21.35 (OCOCH3), 120.99 (C-7), 121.60 (C-3), 122.11, 122.58 (C-5
and C-6), 129.99 (C-9), 133.23 (C-10), 136.46 (C-4), 141.50 (C-8),
1516.42, 1463.00, 1426.20, 1371.03, 1210.92, 1175.98, 1136.43,
1081.82, 1014.69, 943.65, 912.83, 862.57, 797.55, 692.03,
599.54 cmÀ1; UV (methanol) kmax: 324 nm; HRMS: C16H17O5N
[M]+: 303.5980.
4.1.5.3. Ethyl 2-(6-acetoxy-4-methyl-2-oxoquinolin-1(2H)-yl)ace-
tate (29). It was obtained as white crystals (60%); mp: 166 °C; 1H
NMR (CDCl3, 300 MHz): d 1.26 (t, 3H, J = 7.2 Hz, CH2CH3), 2.34 (s,
3H, C-4 CH3), 2.45 (s, 3H, OCOCH3), 4.23 (q, 2H, J = 6.9 Hz, CH2CH3),
5.08 (s, 2H, NCH2), 6.65 (s,1H, H-3), 7.10 (d, 1H, J = 9.0 Hz, H-8),
7.28 (dd, 1H, J = 2.4 and 8.7 Hz, H-7), 7.44 (d, 1H, J = 2.1 Hz, H-5);
13C NMR (CDCl3, 75 MHz): d 14.07 (CH2CH3), 19.07 (C-4 CH3),
21.03 (OCOCH3), 43.68 (NCH2), 61.74 (CH2CH3), 114.77, 117.86
(C-5 and C-7), 121.37 (C-8), 122.17 (C-10), 124.24 (C-3), 136.81
(C-9), 145.36 (C-4), 146.74 (C-6), 161.35 (C-2), 168.04 (OCOCH3),
161.34 (C-2), 169.61 (OCOCH3); IR (KBr) mmax: 3162.22, 1770.93,
1632.36, 1421.68, 1361.50, 1188.50, 1021.12, 955.93, 867.96,
735.74, 675.53 cmÀ1; UV (methanol) kmax: 272 and 328 nm; HRMS:
C14H15O3N [M]+: 245.4751.
4.1.4.9. 3-Hexyl-4-methyl-2-oxo-1,2-dihydroquinolin-8-yl ace-
tate (27). It was obtained as white solid (80%); mp: 162–164 °C;
1H NMR (CDCl3, 300 MHz): d 0.89 (t, 3H, J = 6.6 Hz, CH2CH3),
1.31–1.54 (m, 8H, CH2(CH2)4CH3), 2.47 (s, 3H, C-4 CH3), 2.48 (s,
3H, OCOCH3), 2.75 (t, 2H, J = 6.9 Hz, CH2CH2), 7.16–7.31 (m, 2H,
H-5 and H-6), 7.56 (d, 1H, J = 8.4 Hz, H-7), 9.65–9.74 (br m, 1H,
NH); 13C NMR (CDCl3, 75 MHz): d 14.11 (CH2CH3), 15.37 (C-4
CH3), 21.20 (OCOCH3), 22.68, 27.20, 29.00, 29.57, 31.82
((CH2)5CH3), 121.34, 121.74 (C-5 and C-7), 121.80 (C-6), 122.53
(C-3), 128.95 (C-9), 132.84 (C-10), 136.37 (C-4), 142.65 (C-8),
169.54 (COO); IR (KBr) mmax: 3474.88, 2988.51, 1763.26, 1745.76,
1664.42, 1600.02, 1570.50, 1441.61, 1424.06, 1373.46, 1313.63,
1207.62, 1170.43, 1122.79, 1024.86, 953.86, 899.96, 813.98,
681.48, 615.15 cmÀ1; UV (methanol) kmax: 276 and 336 nm; HRMS:
C16H17O5N [M]+: 303.8917.
4.1.5.4. Ethyl 2-(6-acetoxy-4-methylquinolin-2-yloxy)acetate
(31). It was obtained as white crystals (30%); mp: 62 °C; 1H NMR
(CDCl3, 300 MHz): d 1.25–1.34 (m, 3H, CH2CH3), 2.36 (s, 3H, C-4
CH3), 2.60 (s, 3H, OCOCH3), 4.21–4.30 (m, 2H, CH2CH3), 5.00 (s,
2H, OCH2CO), 6.91 (s, 1H, H-3), 7.34 (dd, 1H, J = 2.7 and 9.0 Hz,
H-7), 7.57 (d, 1H, J = 2.4 Hz, H-5), 7.79 (d, 1H, J = 9.0 Hz, H-8); 13C
NMR (CDCl3, 75 MHz): d 14.21 (CH2CH3), 18.73 (C-4 CH3), 21.16
(OCOCH3), 61.04 (CH2CH3), 62.49 (OCH2CO), 113.13, 115.17 (C-5
and C-7), 124.04 (C-8), 126.00 (C-10), 129.15 (C-3), 144.02 (C-9),
146.85 (C-4), 147.10 (C-6), 160.47 (C-2), 169.34 (OCOCH3),
162.16 (C-2), 168.80 (OCOCH3); IR (KBr) mmax: 3428.77, 2930.79,
2372.19, 1754.72, 1650.93, 1565.36, 1461.77, 1367.69, 1209.86,
1017.50, 907.94, 781.27, 738.35, 698.94 cmÀ1; UV (methanol) kmax
:
273 and 327 nm; HRMS: C18H23O3N [M]+: 301.4821.
4.1.5. General procedure for thesynthesis of N- and O-substituted
alkyl esters of quinolin-2-ones (28–31)
To a mixture of acetoxy-4-methylquinolin-2-ones (7.3 mmol),
K2CO3 (1 g, 7.3 mmol) in anhydrous DMF (10 mL), was added ethyl
bromoacetate (1.2 g, 7.3 mmol) and the reaction mixture was re-
fluxed for 12 h. The reaction was then cooled to room temperature
and the mixture was poured in ice cold water and extracted with
ethyl acetate. The crude product was purified through column
chromatography to give two isomeric products.
169.78 (COO); IR (KBr) mmax: 3478.18, 2990.84, 1762.23, 1750.29,
1609.84, 1580.73, 1522.33, 1467.00, 1439.28, 1421.11, 1384.17,
1364.61, 1341.67, 1219.46, 1168.78, 1088.15, 1036.15, 1017.43,
942.95, 908.61, 887.67, 847.01, 824.95, 736.28, 620.01 cmÀ1; UV
(methanol) kmax: 324 nm; HRMS: C16H17O5N [M]+: 304.0379.
4.1.5.5. General procedure for the synthesis of 2-oxo-1,2-dihy-
droquinolin-4-yl acetate (33). Aniline (5 g, 54 mmol) was added
to a mixture of fused zinc chloride (21.5 g, 161 mmol), malonic
acid (5.6 g, 54 mmol), and phosphorus oxychloride (108 mmol,
16.5 g). The reaction mixture was heated at 65 °C for 36 h. It was
then cooled and poured on ice.26 The precipitate was filtered and
purified through column chromatography using silica gel (100–
200 mesh) in methanol/chloroform (1:100). A solution of acetic
anhydride and acetic acid (1:4) was added to 4-hydroxyquinolin-
2-one (32) and the reaction mixture was refluxed for 6 h and then
poured on crushed ice (Scheme 3). The resulting precipitate was
filtered and washed with water to yield 2-acetoxyquinolin-2-one
(33).
4.1.5.1. Ethyl 2-(7-acetoxy-4-methyl-2-oxoquinolin-1(2H)-yl)ace-
tate (28). It was obtained as white crystals (60%); mp: 110 °C; 1H
NMR (CDCl3, 300 MHz): d 1.25 (t, 3H, J = 7.1 Hz, CH2CH3), 2.34 (s,
3H, C-4 CH3), 2.48 (s, 3H, OCOCH3), 4.23 (q, 2H, J = 7.1 Hz, CH2CH3),
5.04 (s, 2H, NCH2), 6.59 (s,1H, H-3), 6.87 (s, 1H, H-8), 7.03 (d, 1H,
J = 8.7 Hz, H-6), 7.73 (d, 1H, J = 8.7 Hz, H-5); 13C NMR (CDCl3,
75 MHz): d 14.09 (CH2CH3), 19.23 (C-4 CH3), 21.20 (OCOCH3),
43.76 (NCH2), 61.82 (CH2CH3), 107.08, 116.10 (C-6 and C-8),
119.32 (C-3), 120.18 (C-10), 126.70 (C-5), 140.14 (C-9), 147.10
(C-4), 152.43 (C-7), 161.73 (C-2), 168.04 (OCOCH3), 168.97
(COO); IR (KBr) mmax
: 2999.98, 2925.16, 1766.13, 1745.40,
1658.88, 1594.89, 1437.53, 1386.80, 1371.56, 1324.86, 1243.22,
1204.97, 1177.62, 1111.11, 1019.91, 958.23, 909.97, 864.10,
816.25, 745.32, 714.35, 649.75, 604.10, 561.14 cmÀ1; UV (metha-
nol) kmax: 324 nm; HRMS: C16H17O5N [M]+: 304.0316.
4.1.5.6. 4-Hydroxyquinolin-2(1H)-one (32). It was obtained as
yellow solid (70%); mp: 320 °C, (Literature value = 318–320 °C);27
1H NMR (DMSO-d6, 300 MHz): d 5.76 (s, 1H, H-3), 7.11–7.78 (m,
4H, H-5, H-6, H-7 and H-8), 11.18 (br s, 1H, OH); 13C NMR
(DMSO-d6, 75 MHz): d 98.18 (C-3), 114.95, 115.10 (C-6 and C-8),
121.04 (C-5), 122.62 (C-10), 130.82 (C-7), 139.13 (C-9), 162.43
4.1.5.2. Ethyl 2-(7-acetoxy-4-methylquinolin-2-yloxy)acetate
(30). It was obtained as white crystals (30%); mp: 98 °C; 1H NMR
(CDCl3, 300 MHz): d 1.28 (br s, 3H, CH2CH3), 2.35 (s, 3H, C-4 CH3),
2.62 (s, 3H, OCOCH3), 4.26 (br s, 2H, CH2CH3), 4.99 (s, 2H, OCH2CO),
6.87 (s, 1H, H-3), 7.17 (d, 1H, J = 7.8 Hz, H-6), 7.51 (s, 1H, H-8), 7.88
(C-4), 163.57 (C-2); IR (KBr)
mmax: 3430.12, 3094.44, 2953.09,
2861.12, 2639.30, 2364.36, 1669.52, 1633.94, 1594.78, 1560.44,
1506.18, 1471.81, 1420.44, 1378.93, 1332.01, 1259.95, 1235.09,
1160.89, 1145.18, 1102.39, 1035.00, 909.53, 867.70, 773.96,
762.96, 755.55, 671.97, 630.01 cmÀ1; UV (methanol) kmax: 269
and 314 nm; HRMS: C9H7O2N [M]+: 161.7715.
(d, 1H, J = 6.3 Hz, H-5); 13C NMR (CDCl3, 75 MHz):
d 14.16
(CH2CH3), 18.77 (C-4 CH3), 21.15 (OCOCH3), 61.01 (CH2CH3),
62.41 (OCH2CO), 112.33, 118.89 (C-6 and C-8), 119.20 (C-3),

N. Priya et al. / Bioorg. Med. Chem. 18 (2010) 4085–4094
4093
4.1.5.7. 2-Oxo-1,2-dihydroquinolin-4-yl acetate (33). It was ob-
tained as yellow solid (85%); mp: 220 °C, (Literature value = 217–
219 °C);28 1H NMR (DMSO-d6, 300 MHz): d 2.45 (s, 3H, –OCOCH3),
6.65 (s, 1H, H-3), 7.22–7.66 (m, 4H, H-5, H-6, H-7 and H-8), 11.89
(br s, 1H, NH); 13C NMR (DMSO-d6, 75 MHz): d 20.75 (–OCOCH3),
98.19 (C-3), 112.28, 115.48 (C-6 and C-8), 122.03 (C-5), 122.44
(C-10), 131.47 (C-7), 138.87 (C-9), 156.04 (C-4), 162.39 (C-2),
(saturating condition) in assay buffer. Inhibitors were added to the
incubation reaction at different time intervals before the addition
of TMPD and arachidonic acid. The enzyme activity was measured
calorimetrically by monitoring the appearance of oxidized TMPD at
590 nm.
4.6. Assay of platelet CRTAase
172.04 (–OCOCH3); IR (KBr)
mmax: 3396.31, 3009.90, 2965.78,
2858.41, 1768.58, 1668.77, 1615.91, 1561.20, 1506.34, 1475.03,
1434.55, 1398.53, 1370.26, 1268.81, 1189.23, 1160.47, 1143.99,
1078.60, 984.19, 953.88, 887.47, 770.84, 758.32, 660.04 cmÀ1; UV
(methanol) kmax: 264 and 326 nm; HRMS: C11H9O3N [M+H]+:
204.1366.
Platelet CRTAase was assayed as mentioned in the earlier pub-
lications.7 The unit of CRTAase activity was expressed in terms of
% inhibition of GST activity under the conditions of the assay.
4.7. Platelet measurement of NO level by flow cytometry
4.2. Isolation of platelet rich plasma (PRP)
The method outlined by Imrich and Kobzik as described earlier
was followed for the assay of NOS in platelets by flow cytometry.32
The citrated blood was used for the preparation of PRP by the
method of Vickers and Thompson.29 Venous blood (9 mL) was
collected from healthy human volunteer and mixed with 1.0 mL
of 3.8% trisodium citrate and centrifuged at 180g for 10 min. The
upper two-third fraction of plasma (PRP) was transferred to an-
other centrifuge tube leaving behind lower one-third layer to avoid
contamination with WBC’s and RBC’s. Platelet poor plasma (PPP)
was obtained by centrifugation of the remaining sample at 2500g
for 10 min. Platelet count was determined in PRP using electronic
counter, SYSMEX Model No. FA 20 and were adjusted to
250 Â 106/mL with PPP.
4.8. Calculation and statistics
Calculations and statistics were performed using the graph pad
prism 3.02 software. The one-way analysis of variance (ANOVA)
tests followed by the Turkey multiple comparisons test were used.
Data were expressed as mean standard error. Statistical signifi-
cance was calculated using the Student’s t-test. p values less than
0.05 (p <0.05) were considered to be statistically significant.
Acknowledgments
4.3. Aggregometry
The financial assistance from the Department of Scientific and
Industrial Research, University Grant Commission, Government of
India, and University of Delhi is gratefully acknowledged. A.G.
and P.S. are the recipients of Senior Research Fellowship from
Council of Scientific and Industrial Research, Government of India.
The test compounds (in appropriate concentrations) were sepa-
rately preincubated with PRP in a final volume of 0.5 mL at 37 °C.
Platelet aggregation was induced by the addition of ADP (15 lM)
and assessed by using a platelet aggregation profiler (BIODATA
CORPORATION, Model No. PAP-4) and the results were expressed
as the maximum percentage of light transmittance change (%
max) from the baseline at the end of the recording time, using
PPP as a reference. Platelet aggregation curves were recorded for
6 min and analyzed according to internationally established
standards.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.011.
References and notes
4.4. Platelet aggregation in vivo
1. Roberts, D. E.; Mc Nicol, A.; Bose, R. J. Biol. Chem. 2004, 297, 19421.
2. Vane, J. R. Nature (New Biol.) 1971, 231, 232.
3. Bansal, S.; Gaspari, M.; Raj, H. G.; Kumar, A.; Cuda, G.; Verheji, E.; Tyagi, Y. K.;
Ponan, P.; Rastogi, R. C.; Parmar, V. S. Appl. Biochem. Biotechnol. 2008, 144, 37.
4. Singh, I.; Kohli, E.; Raj, H. G.; Gyanda, K.; Jain, S. K.; Tyagi, Y. K.; Gupta, G.;
Kumari, R.; Kumar, A.; Pal, G.; Prasad, A. K.; Rastogi, R. C.; Olsen, C. E.; Jain, S. C.;
Parmar, V. S. Bioorg. Med. Chem. 2002, 10, 4103.
Male sprague dawely rats (weight 200–250 g) housed in mesh
cages maintained at 25 °C and illuminated at 12:12 h light dark cy-
cles. The known amount of test compound was suspended in
appropriate volume of saline, sonicated for 30 s and the prepara-
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of platelet aggregation.30
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Briefly, 50
enzymatic reaction was initiated by adding 100
methyl-p-phenylenediamine (TMPD) and 100 M arachidonic acid
lL of each lysed samples were added to the wells, the
lM N,N,N,N-tetra-
l

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