Calreticulin transacetylase: A novel enzyme-mediated protein acetylation by acetoxy derivatives of 3-alkyl-4-methylcoumarins

Article abstract of DOI:10.1016/j.bioorg.2011.10.005

Our earlier investigations culminated in the discovery of a unique membrane-bound enzyme Calreticulin transacetylase (CRTAase) in mammalian cells catalyzing the transfer of acetyl group from polyphenolic acetates (PAs) to certain functional proteins viz. Glutathione S-transferase (GST), NADPH Cytochrome c reductase and Nitric oxide synthase (NOS) resulting in the modulation of their biological activities. In order to develop SAR study, herein, we studied the influence of alkyl group at C-3 position of acetoxy coumarins on the CRTAase activity. The alkylated acetoxy coumarins lead to inhibition of catalytic activity of GST, and ADP induced platelet aggregation by the way of activation of platelet Nitric oxide synthase (NOS). Furthermore, the increase in size of the coumarin C-3 alkyl group was found to decrease the CRTAase activity.

Full text of DOI:10.1016/j.bioorg.2011.10.005

Bioorganic Chemistry 40 (2012) 131–136
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Calreticulin transacetylase: A novel enzyme-mediated protein acetylation
by acetoxy derivatives of 3-alkyl-4-methylcoumarins
a
a
a
b
b
c
Sarah Jalal , Karam Chand , Abha Kathuria , Prabhjot Singh , Nivedita Priya , Bhavna Gupta ,
b
a,
⇑
Hanumantharao G. Raj , Sunil K. Sharma
a
Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
Department of Biochemistry, VP Chest Institute, University of Delhi, Delhi 110 007, India
Department of Chemistry, University of Massachusetts Lowell, MA 01854, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 September 2011
Available online 9 November 2011
Our earlier investigations culminated in the discovery of a unique membrane-bound enzyme Calreticulin
transacetylase (CRTAase) in mammalian cells catalyzing the transfer of acetyl group from polyphenolic
acetates (PAs) to certain functional proteins viz. Glutathione S-transferase (GST), NADPH Cytochrome c
reductase and Nitric oxide synthase (NOS) resulting in the modulation of their biological activities. In
order to develop SAR study, herein, we studied the influence of alkyl group at C-3 position of acetoxy cou-
marins on the CRTAase activity. The alkylated acetoxy coumarins lead to inhibition of catalytic activity of
GST, and ADP induced platelet aggregation by the way of activation of platelet Nitric oxide synthase
Keywords:
Coumarins
Protein acetylation
Nitric oxide synthase
Glutathione transferase
Anti-platelet aggregation
(NOS). Furthermore, the increase in size of the coumarin C-3 alkyl group was found to decrease the CRTA-
ase activity.
Ó 2011 Elsevier Inc. All rights reserved.
1
. Introduction
Proteins are versatile molecules for catalyst design that play a
potentially harmful compounds. This leads to water soluble conju-
gates that can be further metabolized and eventually excreted [2].
Nitric oxide (NO) is an important regulator of a variety of
physiological and pathological conditions [3]. NOS catalyzes NO
major role to sustain life processes. Modifications of proteins (en-
zymes) result in perturbation of their natural catalytic activity.
There are two broad categories of protein modifications: (i) en-
zyme catalyzed covalent modifications whereby covalent electro-
philic groups viz. acetate, phosphate, etc. are added to the side
chain residue in protein, and (ii) cleavage of peptide backbone in
proteins by action of proteases or less commonly by auto catalytic
cleavage. Of the two methods available, covalent modification of
enzymes and proteins has been used extensively for the creation
of enzymes with new catalytic activities. Most of the time, the tar-
geted modification is accomplished by acylation of amino acid res-
idues in proteins [1]. Herein our focus is on acetyl modification on
some enzyme specific proteins such as GST and Nitric oxide syn-
thase (NOS), whereby influencing their enzymatic behavior and
properties.
biosynthesis via a reaction involving the conversion of
L-arginine
to -citrulline [4]. The acetylation of NOS is an effective approach
L
to activate it, thereby enhancing NO levels in human platelets.
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. Numerous antiplatelet agents were developed
based on their ability to block the receptors responsible for platelet
activation [5].
Protein modification by way of acetylation of the above
mentioned enzyme proteins would further extend the range of
functions performed by these enzymes. The acetylation of proteins
in biological systems was known to be acetyl CoA dependent until
a unique membrane bound enzyme Acetoxy Drug: protein Transa-
cetylase (TAase) was discovered in our laboratory [6–8]. TAase was
found to be involved in protein modifications as it catalyzes the
transfer of acetyl groups from polyphenolic acetates (PA) to the
enzyme proteins such as GST, NADPH Cytochrome c reductase,
and NOS thereby modulating their activities [9]. Several PAs, e.g.
the acetoxy derivatives of coumarins, biscoumarins, xanthones,
flavones, and quinolones have been examined for their substrate
specificity towards TAase mediated protein acetylation. 7,8-Diacet-
oxy-4-methylcoumarin (DAMC), was found to be the best substrate
GST (glutathione S-transferases) are multifunctional proteins
with representatives in all oxygen-utilizing organisms, and among
all kingdoms [2]. Mammalian GSTs are the best characterized
enzymes and their major function is to act as detoxifying agents.
GSTs catalyze the conjugation of the tripeptide glutathione (GSH)
(c
Glu-Cys-Gly) to electrophilic centers in a wide variety of
⇑
Corresponding author. Fax: +91 11 2766 7206.
E-mail address: sk.sharma90@gmail.com (S.K. Sharma).
0
045-2068/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2011.10.005

1
32
S. Jalal et al. / Bioorganic Chemistry 40 (2012) 131–136
for TAase. It catalyzes the transfer of acetyl group from DAMC to
GST and results in the acetylation of several lysine residues in its
active site and subsequently to inhibition of catalytic activity of
GST [10]. Later TAase was identified as Calreticulin (CR), a multi-
2. Results
In our efforts to target protein modifications via acetylation, a
number of PAs, e.g. coumarins, biscoumarins, flavones, quinolones,
xanthones, etc. were studied. Among these, the acetoxy coumarins
were found to be most potent substrates for CRTAase mediated acet-
ylation. The superior substrate specificity of coumarins further
prompted us to synthesize newer analogs of this class. As an exten-
sion of our previous work on 3-alkyl-4-methylcoumarins, herein we
have synthesized newer acetoxy analogs and compared their sub-
strate specificity for CRTAase [20]. All the compounds were fully
characterized on the basis of their physical and spectral data, and
of the total 15 compounds synthesized, nine, i.e. 2a, 2c, 3b, 3c, 4b,
4c, 5a, 5b, and 5c are novel. Though the remaining six compounds
(1a–1c and 2b, 3a and 4a) are known in literature, however, their
complete spectral data is not given. Herein, we have reported the
spectral data for all the compounds in the experimental section.
Due to their hydrophobic nature; the alkyl group may enhance
hydrophobic interactions between the substrate (acetoxycouma-
rins) and the target protein, thus their effect on the rate of catalytic
activity of CRTAase and the efficacy of these coumarins to activate
platelet NOS was examined. We compared the specificities of both
diacetoxy and monoacetoxy coumarins on CRTAase activity. It has
been observed that the CRTase mediated transfer of acetyl group
from diacetoxycoumarins 5a, 5b and 5c when compared with
DAMC showed a gradual decline with the increase in chain length
of the alkyl substituent at the C-3 position in the coumarin moiety.
2
+
functional Ca binding protein in Endoplasmic Reticulum (ER)
lumen and this function of CR was termed Calreticulin transacety-
lase (CRTAase) [11]. A possible mechanism for the protein acetyl
transferase function based on studies on CRTAase catalyzed acety-
lation of the receptor protein and autoacetylation of CR has already
been proposed by our group [12]. The activities of other enzymes
such as liver microsomal cytochrome P-450 catalyzed mixed func-
tion oxidase (MFO), NADPH Cytochrome c reductase, and nitric
oxide synthase (NOS) were also modulated by CRTAase mediated
acetylation of these enzyme proteins using acetoxycoumarins
DAMC and 7-acetoxy-4-methylcoumarin (MAMC), as the acetyl
donating substrates [13].
In order to develop SAR, we have 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 nucleus in modulating the substrate
specificity towards CRTAse for protein acetylation [14–19].
Herein, we have compared the specificities of C-3 alkyl diacet-
oxy and monoacetoxy coumarins on the activity of CRTAase and
CRTAase catalyzed activation of platelet NOS and also compared
the structure activity relationship (SAR) with special reference to
the effect of alkyl group at the C-3 position of the pyran moiety
of the polyphenolic acetates. To the best of our knowledge this is
the first report wherein the effect of C-3 alkyl group of coumarin
on platelet aggregation inhibition has been studied.
A
similar trend was observed with 7-acetoxy-4-methyl-3-
alkylcoumarins 4a, 4b and 4c. From the results we concluded that
the specificity of polyphenolic acetates for TAase of 3-alkyl diacet-
oxy coumarins are almost twice as that of 3-alkyl monoacetoxy
coumarins (Fig. 1). However, the TAase activities of both the
mono/diacetoxy 3-alkyl coumarins are lower than the correspond-
ing non alkylated analogs, i.e. MAMC and DAMC.
Besides the CRTase catalyzed acetylation of GST, we also studied
the influence of acetoxy coumarins (4a–c, 5a–c) towards activation
of Nitric oxide synthase (NOS) by transferring their acetyl group to
NOS. The platelets preincubated with acetoxy coumarins (4a–5c)
1
00
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
1
2
3
0
0
0
and L-arginine exhibited enhancement in the level of NO only with
compounds 4a, 5a and 5b. The diacetoxy coumarins 5a and 5b pro-
duced more enhancements of NO levels in platelets than mono-
acetoxy coumarin 4a (Fig. 2).
It has also been noticed that the acetoxy coumarins inhibit the
platelet aggregation, diacetoxy coumarin 5a having propyl group at
C-3 position is much superior in exhibiting the inhibition of ADP
induced platelet aggregation as compared to the corresponding
monoacetoxy analog 4a. The compounds 4b, 4c, 5b, and 5c having
longer alkyl group, i.e. n-heptyl and n-nonyl at C-3 position do not
inhibit platelet aggregation (Fig. 3). Thus we may conclude that
MAMC 4a
4b
4c
DAMC 5a
5b
5c
Acetoxy coumarin
Fig. 1. CRTAase catalyzed inhibition of GST by acetoxycoumarins. Acetoxy couma-
rins were separately preincubated with rat liver microsomes (25 g) and rat liver
cytosol (12.5 g) followed by assay of GST, the activity was expressed in terms of
inhibition of GST. Each histogram indicates % inhibition of GST at 10 min intervals
l
l
(
10–30 min) in succession. Values are mean ± SD of three observations.
6
5
4
3
2
1
0
Acetoxy coumarins
Fig. 2. The effect of C-3 substituted acetoxy coumarins on activation of platelet NOS. Acetoxy coumarins were incubated with platelets,
-NAME was also added along with coumarins to confirm the appearance of NO. Values are mean ± SD of three observations.
L-arginine and DCF at 37 °C for 30 min.
L

S. Jalal et al. / Bioorganic Chemistry 40 (2012) 131–136
133
modulation of enzyme activity of proteins, such as inhibition of
cystolic Glutathione-S-transferase (GST), enhancements of NO lev-
els in platelets, and inhibition of ADP dependent platelet aggrega-
tion. It was observed that the potency of 3-alkyl 7,8-diacetoxy
coumarins is almost twice as that of monoacetoxy counterparts.
It can also be inferred that with the increase in chain length at
C-3 position, the CRTAase activity decreases. However the C-3
unsubstituted coumarins, viz. MAMC and DAMC have the maxi-
mum substrate specificity for CRTAase.
6
5
4
3
2
1
0
0
0
0
0
0
0
Acetoxy coumarin
5
. Experimental
Fig. 3. The effect of C-3 substituted coumarins on platelet aggregation. PRP was
incubated with acetoxy coumarin (100 M) for 10 min at 37 °C followed by the
addition of ADP (15 M) and platelet aggregation was monitored by aggregometry.
5
5
.1. Chemistry
l
l
Values are mean ± SEM of three observations.
.1.1. Materials
The solvents were dried and distilled prior to their use. Reac-
compounds with shorter alkyl group are better substrates for the
inhibition of platelet aggregation. Among C-3 unsubstituted acet-
oxy coumarins, we found that DAMC causes highest inhibition in
platelet aggregation followed by MAMC.
tions were monitored by precoated TLC plates (Merck Silica Gel
60F254); the spots were visualized either by UV light, or by spraying
with 5% alcoholic FeCl solution. Silica gel (100–200 mesh) was
3
used for column chromatography. Sodium hydride (60% dispersed
in mineral oil) was supplied from Spectrochem. Pvt. Ltd., India, and
washed each time with petroleum ether. Melting points were re-
corded in capillaries in sulfuric acid bath and are uncorrected. UV
data was recorded on Shimadzu UV-250 IPL UV–VIS spectropho-
tometer. Infrared spectra were recorded on Perkin–Elmer FT-IR
3
. Discussion
As a part of our ongoing research program to carry out protein
modification by virtue of acetylation of its amino acid residues, a
wide variety of PAs as substrates for CRTAase were studied. A SAR
study suggests that the presence of acetoxy groups on the benzenoid
ring and the carbonyl group on the pyran nucleus are absolute
requirement for CRTAase activity [13]. Herein we have studied the
influence of replacing the C-3 hydrogen of coumarin with an alkyl
group on the catalytic activity of CRTAase. For this we have synthe-
sized a number of acetoxy coumarins and diacetoxy coumarins
bearing different alkyl (propyl, heptyl, nonyl) substituents and then
their specificities to CRTAase were determined. It was observed that
the presence of bulky alkyl groups at C-3 position in the pyran ring of
coumarins reduces its ability for the transfer of acetyl group to the
functional proteins, suggesting that bulkier alkyl groups might be
causing steric hindrance for the acetoxy substrates from accessing
the active site of CRTAase. This observation was made on the basis
that the compounds with propyl group at the C-3 position yielded
higher CRTAase activities (Fig. 1) in comparison to their heptyl or
nonyl analogs. However, the highest catalytic activity was observed
with C-3 unsubstituted acetoxy coumarins MAMC and DAMC. From
these results we can conclude that substitution at C-3 position by
1
13
model 9 spectrophotometer. The H and C NMR spectra were re-
corded on Bruker AC-400 (400 MHz, 100.6 MHz), Avance-300
(
300 MHz, 75.5 MHz), and Delta Jeol-400 (400 MHz, 100.6 MHz)
spectrometer using TMS as internal standard. The chemical shift
values are on d scale and the coupling constant values (J) are in
hertz. The EI/HR mass spectra were recorded on Waters LCT Micro-
mass-KC455. NADPH, Cytochrome c, reduced glutathione (GSH),
1
-chloro-2,4-dinitrobenzene (CDNB), dichloro fluorescein–diace-
tate (DCFH–DA), N-nitro -arginine methyl ester ( -NAME) and
-arginine were purchased from Aldrich Chemical Co., St. Louis,
L
L
L
MO (USA). Sodium nitrite was purchased from Thomas Baker
Chemicals Ltd. Mumbai, India.
5.1.2. Preparation of ethyl 2-acetylpentanoate (1a), ethyl
2-acetylnonanoate (1b) and ethyl 2-acetylundecanoate (1c)
To
a cold solution (0 °C) of ethyl acetoacetate (10 g,
76.68 mmol) in THF (30 mL) was added sodium hydride (3.13 g,
130.36 mmol). Once the addition was complete, the reaction mix-
ture was heated at 40 °C for 2 h. The contents of the flask were then
allowed to attain room temperature, further, addition of 1-bro-
(
3
C /C
7 9
/C ) alkyl groups led to gradual decrease in CRTAase activity.
The effect of alkyl substituents on CRTAase catalyzed acetyla-
tion of NOS was also examined. This proposition was examined
using human platelets as the experimental system. The platelets
3 7 7 9
moalkane (1.2 eq) i.e. C H Br/C H15Br/C H19Br dissolved in THF
was carried out under ice cold conditions. The resultant mixture
thus obtained was heated at 80 °C for 10–12 h. The reaction was
monitored by TLC (10% ethyl acetate–petroleum ether). Unreacted
sodium hydride was deactivated by addition of ethylacetate, the
solution was then filtered and the solution washed with water,
the organic layer was dried over anhydrous sodium sulfate and
the solvent evaporated. The crude product so obtained was sub-
jected to column chromatography using silica gel (100–200 mesh)
preincubated with 3-substituted acetoxy coumarins and
exhibited enhancement in the level of NO, however the inclusion of
-NAME, a well-known inhibitor of NOS successfully reduce DCF
L-arginine
L
fluorescence (a measure of NO level) to the control level. Thus
inferring that the prior increase in DCF fluorescence without the
inclusion of L-NAME was due to the increase in production of NO
radicals alone (Fig. 2).
We studied the inhibition of platelet aggregation among the
monoacetoxy and diacetoxy coumarins (4a–c, 5a–c) and found
that the results were not very promising as only two compounds,
i.e. 4a and 5a inhibit platelet aggregation while the rest of them
failed to produce any observable result (Fig. 3).
4
. Conclusion
The potency of monoacetoxy and diacetoxy coumarins (4a, 4b,
c, 5a, 5b and 5c) were evaluated for the CRTAase catalyzed
4
Scheme 1. Alkylation of ethyl acetoacetate.

1
34
S. Jalal et al. / Bioorganic Chemistry 40 (2012) 131–136
and the desired product eluted with ethyl acetate–petroleum ether
as viscous oil (Scheme 1).
on TLC (methanol–chloroform 1:19). On completion of the reac-
tion, ice cold water (100 mL) was added. The crude product so ob-
tained was then filtered, washed with water, dried and crystallized
from ethanol to give the monohydroxy (2a–c) /dihydroxy couma-
rins (3a–c) (Scheme 2).
5
.1.2.1. Ethyl 2-acetylpentanoate (1a). It was obtained as yellow oil
1
(
(
50 %); bp: 224–225 °C (literature bp: 224–225 °C) [21]; H NMR
CDCl , 300 MHz): d 0.93 (t, 3H, J = 7.2 Hz, H-5), 1.25–1.27 (m,
3
0
00
5
1
7
2
2
H, H-4 and H-2 ), 1.81 (m, 2H, H-3), 2.22 (s, 3H, H-2 ), 3.43 (t,
H, J = 7.3 Hz, H-2), 4.19 (q, 2H, J = 6.9 Hz, H-1 ); C NMR (CDCl
5.5 MHz): d 13.40 (C-5), 13.70 (C-2 ), 22.79 (C-4), 28.34 and
9.84 (C-3 and C-2 ), 59.24 (C-2), 60.81 (C-1 ), 169.53 (C-1),
5.1.3.1.
7-Hydroxy-4-methyl-3-propyl-coumarin
(2a). It
was
0
13
3
,
obtained as pale yellow solid (76%); mp: 169–171 °C (literature
0
1
mp: 169–171 °C) [24]; H NMR (DMSO-d
6
, 400 MHz): d 0.84 (t,
00
0
0
0
3H, J = 6.7 Hz, H-3 ), 1.36–1.41 (m, 2H, H-2 ), 2.28 (s, 3H, C-4
), 2.43 (t, 2H, J = 7.4 Hz, H-1 ), 6.63 (s, 1H, H-8), 6.72 (d, 1H,
J = 9.2 Hz, H-6), 7.52 (d, 1H, J = 6.8 Hz, H-5); C NMR (DMSO-d
100.6 MHz): d 14.26 (C-3 ), 15.03 (C-4 CH
(C-1 ), 102.26 (C-8), 112.97 and 113.24 (C-6 and C-10), 121.72
00
0
02.80 (C-1 ); IR (KBr)
mmax: 2963.57, 2937.60, 2875.95, 1741.74
CH
3
1
3
(
7
1
COO), 1717.86 (CO), 1466.11, 1359.67, 1188.81, 1061.86, 855.23,
6
,
ꢀ1
+
0
0
53.61 cm ; HRMS: Calcd for C
73.1120.
H
9 16
O
3
[M+H] = 173.1099. Found:
3
), 22.03 (C-2 ), 29.11
0
(
C-3), 126.88 (C-5), 147.59 (C-4), 153.61 and 160.49 (C-7 and
5
.1.2.2. Ethyl 2-acetylnonanoate (1b). It was obtained as a yellow oil
C-9), 161.60 (C-2); IR (KBr) max: 3442.52 (OH), 1736.68 (CO),
1713.63, 1677.15, 1606.44, 1555.38, 1475.22, 1356.83, 1303.11,
m
1
(
(
53 %); bp: 266–267 °C (literature bp: 266–267 °C) [22]; H NMR
CDCl , 300 MHz): d 0.87 (t, 3H, J = 7.0 Hz, H-9), 1.09–1.27 (m,
3
1266.92, 1243.80, 1207.49, 1146.54, 1063.11, 1008.00 880.11,
0
00
13
ꢀ1
1
3
3H, H-4–H-8 and H-2 ), 1.83 (brm, 2H, H-3), 2.21(s, 3H, H-2 ),
.39 (t, 1H, J = 6.7 Hz, H-2), 4.19 (q, 2H, J = 7.01 Hz, H-1 );
820.74, 802.57, 741.98, 665.60, 603.97, 435.26 cm
(methanol) kmax 323 nm; HRMS: Calcd for
218.0943. Found: 218.0099.
;
UV
0
+
C
:
13 14 3
C H O [M] =
0
NMR (CDCl
2
3
, 75.5 MHz): d 13.62 (C-9), 18.06 (C-2 ), 22.20 (C-8),
00
3.44, 25.56, 27.76, 28.62, 29.65, 32.47 (C-3–C-7 and C-2 ), 59.42
0
00
(
C-2), 60.68 (C-1 ), 171.47 (C-1), 202.46 (C-1 ); IR (KBr)
m
max
:
5.1.3.2. 3-Heptyl-7-hydroxy-4-methylcoumarin (2b). It was obtained
1
2
1
950.90, 2928.06, 2856.98, 1742.23 (COO), 1718.60 (CO),
as pale yellow solid (78%); mp: 63–65 °C ; H NMR (DMSO-d
6
ꢀ1
0
459.08, 1241.97, 1152.29, 1025.54, 858.30, 721.40 cm ; HRMS:
300 MHz): d 0.87 (t, 3H, J = 6.4 Hz, H-7 ), 1.28–1.50 (m, 10H,
+
0
0
0
Calcd for C13
H
24
O
3
[M+H] = 229.1725. Found: 229.1689.
H-2 –H-6 ), 2.40 (s, 3H, C-4 CH
3
), 2.59 (t, 2H, J = 7.5 Hz, H-1 ),
6
.70 (d, 1H, J = 2.1 Hz, H-8), 6.82 (dd, 1H, J = 2.4 and 8.7 Hz, H-6),
1
3
5
.1.2.3. Ethyl 2-acetylundecanoate (1c). It was obtained as a yellow
7.59 (d, 1H, J = 9.0 Hz, H-5); C NMR (DMSO-d
14.42 (C-7 ), 15.02 (C-4 CH
31.72 (C-1 –C-6 ), 102.29 (C-8), 113.00 and 113.20 (C-6 and
C-10), 121.97 (C-3), 126.89 (C-5), 147.34 (C-4), 153.65 and
6
, 75.5 MHz): d
), 22.56, 27.20, 28.76, 29.03, 29.42,
1
0
oil (55%); bp: 292–293 °C (literature bp: 292–293 °C) [23]; H NMR
CDCl , 300 MHz): d 0.96 (t, 3H, J = 6.9 Hz, H-11), 1.36–1.51 (m,
3
0
0
(
1
3
3
0
00
7H, H-4–H-10 and H-2 ), 1.95 (m, 2H, H-3), 2.30 (s, 3H, H-2 ),
.48 (t, 1H, J = 6.7 Hz, H-2), 4.27 (q, 2H, J = 7.2 Hz, H-1 ); C NMR
0
13
160.51 (C-7 and C-9), 161.52 (C-2); IR (KBr)
mmax: 3307.14 (OH),
0
(
CDCl
3
, 75.5 MHz): d 13.62 (C-11), 19.59 (C-2 ), 22.56 (C-10),
1713.07 (CO), 1681.12, 1576.78, 1451.86, 1379.87, 1229.95,
ꢀ
1
2
3.65, 27.29, 28.09, 28.69, 29.30, 29.61, 31.76, 32.76 (C-3–C-9
1095.63, 862.94, 777.94, 532.00 cm ; UV (methanol) kmax: 274
and 324 nm; HRMS: Calcd for C17
275.6815.
0
0
0
00
+
and C-2 ), 59.80 (C-2), 61.04 (C-1 ), 169.75 (C-1), 202.98 (C-1 );
IR (KBr) max: 2926.33, 2956.80, 2855.82, 1742.53 (COO), 1719.21
CO) 1465.13, 1244.13, 1151.81, 1025.93, 860.17, 722.07,
22 3
H O [M+H] = 275.3548. Found:
m
(
ꢀ1
+
6
46.00 cm
;
HRMS: Calcd for
C
H
15 28
O
3
[M+H] = 257.3810.
5.1.3.3. 7-Hydroxy-4-methyl-3-nonylcoumarin (2c). It was obtained
as pale yellow solid (79%); mp: 46–48 °C; H NMR (DMSO-d ,
6
00 MHz): d 0.86 (t, 3H, J = 6.6, H-9 ), 1.27–1.50 (m, 14H, H-2 –H-
3
), 2.59 (t, 2H, J = 7.6 Hz, H-1 ), 6.70 (d, 1H,
J = 2.4 Hz, H-8), 6.82 (dd, 1H, J = 2.4 and 8.7 Hz, H-6), 7.59 (d, 1H,
6
J = 8.7 Hz, H-5); C NMR (DMSO-d , 75.5 MHz): d 14.40 (C-9 ),
1
Found: 257.2038.
0
0
3
0
0
5
.1.3. General procedure for the synthesis of hydroxy coumarins (2a–c
and 3a–c)
To a cold (0 °C) mixture of resorcinol/pyragallol (1 g) in alkyl-
8 ), 2.40 (s, 3H C-4 CH
1
3
0
ated ethyl acetoacetate (1.2 eq) was slowly added concentrated
sulfuric acid (5 mL, dropwise). The mixture was stirred at room
temperature for 3–4 h. The progress of reaction was monitored
15.00 (C-4 CH
3
), 22.55, 27.19, 28.43, 29.78, 29.16, 29.41, 29.43,
0
0
31.74 (C-1 –C-8 ), 102.29 (C-8), 113.00 and 113.21 (C-6 and
C-10), 121.99 (C-3), 126.89 (C-5), 147.34 (C-4), 153.65, 160.52
Scheme 2. Synthesis of acetoxy coumarins.

S. Jalal et al. / Bioorganic Chemistry 40 (2012) 131–136
135
0
and 161.52 (C-2, C-7 and C-9); IR (KBr)
m
max: 3244.73 (OH),
1.50–1.59 (m, 2H, H-2 ), 2.31 (s, 3H, C-4 CH
3
), 2.39 (s, 3H, –
0
1
1
3
3
678.55 (CO), 1615.35, 1567.51, 1466.84, 1355.63, 1235.59,
OCOCH
3
), 2.62 (t, 2H, J = 7.8 Hz, H-1 ), 7.01–7.05 (m, 2H, H-6 and
H-8), 7.58 (d, 1H, J = 8.8 Hz, H-5); C NMR (CDCl
14.09 (C-3 ), 15.01 (C-4 CH
25.45 (C-1 ), 110.03 (C-8), 117.87 and 118.63 (C-6 and C-10),
ꢀ
1
13
091.37, 851.53, 781.53, 531.91 cm
;
UV (methanol)
k
max
:
3
, 100.6 MHz): d
+
0
0
24 nm; HRMS: Calcd for C19
03.3478.
H
26
O
3
[M+H] = 303.4079. Found:
3
), 21.14 (–OCOCH
3
), 22.00 (C-2 ),
0
1
25.28 (C-3), 126.09 (C-5), 145.52 (C-4), 151.95 and 152.63 (C-7
5.1.3.4. 7,8-Dihydroxy-4-methyl-3-propylcoumarin (3a). It was
and C-9), 161.49 and 168.95 (C-2 and –OCOCH ); IR (KBr) m :
3
max
obtained as white solid (78%); mp: 189–190 °C (literature mp:
1765.03 (OCO), 1716.32 (CO), 1611.68, 1572.19, 1421.64,
1
1
89–190 °C) [25]; H NMR (DMSO-d
6
, 400 MHz): d 0.87 (t, 3H,
1364.82, 1275.29, 1136.25, 1015.99, 909.45, 869.71, 776.87,
0
0
ꢀ1
J = 7.2 Hz, H-3 ), 1.38–1.43 (m, 2H, H-2 ), 2.29 (s, 3H C-4 CH
2
3
),
647.39, 585.88, 455.48 cm
;
UV (methanol)
H O [M+H] = 261.3016. Found:
16 4
k
max
:
274 and
0
+
.47 (t, 2H, J = 7.2 Hz, H-1 ), 6.75 (d, 1H, J = 8.4 Hz, H-6), 7.04
312 nm; HRMS: Calcd for C15
261.3524.
1
3
(
d, 1H, J = 8.8 Hz, H-5), 9.19 (s, 1H, OH) and 9.87 (s, 1H, OH);
C
),
0
NMR (DMSO-d
6
, 100.6 MHz): d 14.32 (C-3 ), 15.19 (C-4 CH
2.08 (C-2 ), 29.20 (C-1 ), 112.50 and 115.74 (C-6 and C-10),
13.75 (C-5), 121.59 (C-3), 132.30 (C-4), 142.37, 147.92 and
3
0
0
2
1
1
5.1.4.2. 7-Acetoxy-3-heptyl-4-methylcoumarin (4b). It was obtained
1
as white solid (73 %); mp: 58–60 °C; H NMR (CDCl
3
, 400 MHz):
0
0
0
48.71 (C-7, C-8 and C-9), 161.47 (C-2); IR (KBr)
mmax: 3422.80
d 0.87 (t, 3H, J = 6.45 Hz, H-7 ), 1.26–1.52 (m, 10H, H-2 –H-6 ),
(
1
8
OH), 1674.69 (CO), 1614.30, 1582.81, 1509.59, 1465.08, 1380.59,
2.33 (s, 3H, C-4 CH
3
), 2.40 (s, 3H, –OCOCH
3
), 2.65 (t, 2H,
0
357.63, 1308.54, 1247.74, 1122.09, 1086.84, 1054.53, 955.99,
J = 7.6 Hz, H-1 ), 7.02–7.07 (m, 2H, H-6 and H-8), 7.59 (d, 1H,
ꢀ1
13
0
15.40, 774.44, 671.53, 526.46 cm ; UV (methanol) kmax: 262
J = 8.4 Hz, H-5); C NMR (CDCl
3
, 100.6 MHz): d d 14.14 (C-7 ),
), 22.68, 25.62, 27.73, 28.76,
+
and 323 nm; HRMS: Calcd for C13
2
H
14
O
4
[M+H] = 235.2479. Found:
14.97 (C-4 CH ), 21.15 (–OCOCH
3
3
0
0
35.2624.
29.72 and 31.89 (C-1 –C-6 ), 110.04 (C-8), 117.86 and 118.66 (C-6
and C-10), 125.23 (C-3), 126.57 (C-5), 145.28 (C-4), 151.34 and
5
.1.3.5. 7,8-Dihydroxy-3-heptyl-4-methylcoumarin (3b). It was ob-
151.91 (C-7 and C-9), 162.74 and 168.96 (C-2 and –OCOCH
3
); IR
1
tained as white solid (76%); mp: 140–142 °C; H NMR (DMSO-d
3
H-2 –H-6 ), 2.26 (s, 3H C-4 CH
6
,
(KBr) 1770.06 (OCO), 1712.01 (CO), 1614.72, 1572.12,
max
m :
0
00 MHz): d 0.87 (t, 3H, J = 6.5 Hz, H-7 ), 1.11–1.50 (m, 10H,
1425.88, 1367.57, 1202.95, 1137.49, 1013.38, 904.82, 824.80,
0
0
0
ꢀ1
3
), 2.59 (t, 2H, J = 7.6 Hz, H-1 ), 6.84
777.78, 641.92 cm ; UV (methanol) kmax: 275 and 360 nm; HRMS:
1
3
+
(
(
2
d, 1H, J = 8.7 Hz, H-6), 7.12 (d, 1H, J = 8.7 Hz, H-5); C NMR
Calcd for C19
H
24
O
4
[M+H] = 317.1675. Found: 317.1719.
0
DMSO-d
7.25, 28.80, 29.05, 29.48, 31.74 (C-1 –C-6 ), 112.50 and 115.72
C-6 and C-10), 113.76 (C-5), 121.81 (C-3), 132.30 (C-4), 142.36,
6 3
, 75.5 MHz): d 14.44 (C-7 ), 15.15 (C-4, CH ), 22.56,
0
0
5.1.4.3. 7-Acetoxy-4-methyl-3-nonyl coumarin (4c). It was obtained
1
(
as white solid (72 %); mp: 40–42 °C; H NMR (CDCl
3
0
, 300 MHz):
0
0
1
3
1
47.75 and 148.70 (C-7, C-8 and C-9), 161.45 (C-2); IR (KBr)
m
max
:
0.87 (t, 3H, J = 6.6 Hz, H-9 ), 1.28–1.52 (m, 14H, H-2 –H-8 ), 2.33
431.39 (OH), 1673.17 (CO), 1582.51, 1510.00, 1464.89, 1380.15,
(s, 3H, C-4 CH
3
), 2.40 (s, 3H, –OCOCH
3
), 2.65 (t, 2H, J = 7.5 Hz, H-
ꢀ1
0
217.35, 1124.10, 1090.72, 812.20, 777.46, 529.83, 487.46 cm
;
1 ), 7.03–7.07 (m, 2H, H-6 and H-8), 7.59 (d, 1H, J = 8.4 Hz, H-5);
1
3
0
UV (methanol) kmax: 262 and 360 nm; HRMS: Calcd for C17
H
22
O
4
C NMR (CDCl
(–OCOCH ) 22.17, 27.17, 27.73, 28.77, 29.19, 29.68, 31.85 and
6.27 (C-1 –C-8 ), 110.04 (C-8), 117.86 and 118.66 (C-6 and
C-10), 125.23 (C-3), 126.57 (C-5), 145.28 (C-4), 151.91, 152.63
(C-7 and C-9), 161.48 and 168.96 (C-2 and –OCOCH ). IR (KBr)
mmax: 1761.78 (OCO), 1710.66 (CO), 1616.39, 1571.48, 1427.79,
3 3
, 75.5 MHz): d 14.33 (C-9 ), 14.97 (C-4 CH ), 21.15
+
[
M+H] = 291.3542. Found: 291.7042.
3
0
0
3
5
.1.3.6. 7,8-Dihydroxy-4-methyl-3-nonylcoumarin (3c). It was
1
obtained as white solid (75%); mp: 100–102 °C;
H
NMR
3
0
(
DMSO-d
6
, 300 MHz): d 0.85 (t, 3H, J = 6.6 Hz, H-9 ), 1.27–1.50
0
0
ꢀ1
(
m, 14H, H-2 –H-8 ), 2.39 (s, 3H C-4 CH
3
), 2.59 (t, 2H, J = 7.6 Hz,
1365.89, 1208.70, 1135.54, 1014.95, 903.62, 777.04, 642.03 cm ;
0
H-1 ), 6.84 (d, 1H, J = 8.7 Hz, H-6), 7.12 (d, 1H, J = 8.7 Hz, H-5);
UV (methanol) kmax: 273 and 314 nm; HRMS: Calcd for C21
[M] = 344.4446. Found: 344.4958.
H
28
O
4
1
3
0
+
C NMR (DMSO-d
6
, 75.5 MHz): d 14.40 (C-9 ), 15.12 (C-4 CH
3
0
),
0
2
1
1
1
1
7
2.55, 27.24, 28.34, 28.76, 29.17, 29.38, 29.43, 31.75 (C-1 –C-8 ),
12.50 and 115.67 (C-6 and C-10), 113.76 (C-5), 121.81 (C-3),
32.31 (C-4), 142.36, 147.69 and 148.70 (C-7, C-8 and C-9),
5.1.4.4. 7,8-Diacetoxy-4-methyl-3-propyl coumarin (5a). It was
1
3
obtained as white solid (78%); mp: 108–110 °C; H NMR (CDCl ,
0
0
61.44 (C-2); IR (KBr)
m
max: 3421.75 (OH), 1673.01 (CO), 1582.06,
400 MHz): d 0.97 (t, 3H, J = 7.4 Hz, H-3 ), 1.50–1.58 (m, 2H, H-2 ),
465.42, 1309.81, 1217.12, 1122.33, 1092.80, 954.27, 811.77,
2.31 (s, 3H, C-4 CH
3
), 2.39 (s, 6H, 2ꢁ –OCOCH
3
), 2.61 (t, 2H,
ꢀ1
0
76.02, 525.55 cm ; UV (methanol) kmax: 262 and 323 nm; HRMS:
J = 8.0 Hz, H-1 ), 7.10 (d, 1H, J = 9.2 Hz, H-6), 7.46 (d, 1H,
+
13
0
Calcd for C19
H
26
O
4
[M+H] = 319.4073. Found: 319.4775.
J = 8.8 Hz, H-5); C NMR (CDCl
3
, 100.6 MHz): d 14.08 (C-3 ),
0
1
5.13 (C-4 CH
3
), 20.38, 20.69 and 21.94 (2ꢁ –OCOCH
3
and C-2 ),
0
5
.1.4. General procedure for synthesis of 3-alkyl-7-acetoxy-4-
29.65 (C-1 ), 118.25 and 121.62 (C-6 and C-10), 119.71 (C-5),
124.91 (C-3), 130.02 (C-8), 144.06, 145.27 and 145.44 (C-4, C-7
methylcoumarin (4a–c)/7,8-diacetoxy-3-alkyl-4-methylcoumarin
(
5a–c)
To
and C-9), 160.27 (C-2), 167.61 and 167.99 (2ꢁ –OCOCH
(KBr) max: 1744.14 (OCO), 1680.29 (CO), 1610.91, 1567.15,
1421.51, 1372.85, 1207.22, 1164.61, 1040.58, 988.91, 801.73,
3
); IR
a
mixture of 3-alkyl-7-hydroxy-4-methylcoumarin
m
(2a–c)/7,8-dihydroxy-3-alkyl-4-methylcoumarin (3a–c) (1 g) and
ꢀ1
dimethylaminopyridine (20 mg) in THF (20 mL) was added acetic
anhydride (1.2/2.4 eq) and the reaction mixture was stirred at
room temperature for 24 h. On completion of the reaction, ice cold
water (100 mL) was added. The crude solid was then filtered,
washed with water, dried and crystallized from ethanol to give col-
orless crystals of 7-acetoxycoumarins (4a–c)/7,8-diacetoxycouma-
rins (5a–c) (Scheme 2).
769.75, 645.65 cm ; UV (methanol) kmax: 277 and 375 nm; HRMS:
+
Calcd for C17
18 6
H O [M] = 318.3377. Found: 318.2705.
5.1.4.5. 7,8-Diacetoxy-3-heptyl-4-methylcoumarin (5b). It was
1
obtained as white solid (74%); mp: 68–70 °C; H NMR (CDCl
3
,
0
300 MHz): d 0.88 (t, 3H, J = 6.6 Hz, H-7 ), 1.28–1.57 (m, 10H,
0
0
H-2 –H-6 ), 2.33 (s, 3H, C-4 CH
3
), 2.40 (s, 6H, 2ꢁ –OCOCH
3
), 2.64
0
(
t, 2H, J = 7.6 Hz, H-1 ), 7.12 (d, 1H, J = 9.0 Hz, H-6), 7.48 (d, 1H,
1
3
0
5
.1.4.1. 7-Acetoxy-4-methyl-3-propylcoumarin (4a). It was obtained
J = 9.0 Hz, H-5); C NMR (CDCl
(C-4 CH
), 20.37 and 20.69 (2ꢁ –OCOCH
29.15, 29.62 and 31.81 (C-1 –C-6 ), 118.21 and 121.52 (C-6 and
3
, 75.5 MHz): d 14.12 (C-7 ), 15.05
as white solid (72%); mp: 119–120 °C (literature mp: 119–120 °C)
3
3
), 22.65, 27.78, 28.66,
1
0
0
0
[
3
24]; H NMR (CDCl , 400 MHz): d 0.97 (t, 3H, J = 7.4 Hz, H-3 ),

1
36
S. Jalal et al. / Bioorganic Chemistry 40 (2012) 131–136
C-10), 119.84 (C-5), 127.05 (C-3), 130.04 (C-8), 144.02 and 145.36
fluorescence) was acquired using the Cell Quest software (Becton
and Dickinson, USA). Analysis was performed by applying appro-
priate gates with reference to the auto fluorescence measured un-
der similar conditions. PRP was incubated with polyphenolic
(
(
1
1
C-7 and C-9), 141.05 (C-4), 160.34 (C-2), 167.99 and 167.99
); IR (Nujol) max: 1777.60 (OCO), 1706.12 (CO),
612.52, 1461.21, 1377.37, 1274.79, 1221.62, 1114.75, 1164.43,
2ꢁ –OCOCH
3
m
ꢀ1
078.80, 1016.08, 952.35, 880.19, 823.50, 773.93, 723.11 cm
;
acetates (100
the addition of ADP (15
down and were washed twice with PBS and further preincubated
in buffer containing 1 L of DCF-DA (dissolved in CH OH) in a total
volume of 1 mL to make the final concentration 2 M and kept at
l
M) for 10 min and the activity was triggered by
UV (acetonitrile)
k
max
:
277 and 381 nm; HRMS: Calcd for
lM) for 5 min. Platelets were palleted
+
C
21
H
26
O
6
[M+H] = 375.4275. Found: 375.5471.
l
3
5
.1.4.6.
7,8-Dicetoxy-4-methyl-3-nonylcoumarin
(5c). It
was
l
1
obtained as white solid (71%); mp: 113–115 °C; H NMR (CDCl
3
H-2 –H-8 ), 2.32 (s, 3H, C-4 CH
3
,
37 °C for 30 min and then the reaction was stopped by placing
the tubes containing the reaction mixture over ice. One set of
PRP aliquots was preincubated with L-NAME (50 lM) for 30 min
before the addition of test compounds and subjected to NOS assay.
0
00 MHz): d 0.86 (t, 3H, J = 8.0 Hz, H-9 ), 1.25–1.57 (m, 14H,
0
0
3
), 2.39 (s, 6H, 2ꢁ –OCOCH
3
), 2.62
0
(
t, 2H, J = 7.4 Hz, H-1 ), 7.11 (d, 1H, J = 8.0 Hz, H-6), 7.47 (d, 1H,
1
3
0
J = 10.4 Hz, H-5); C NMR (CDCl
3
, 75.5 MHz): d 14.10 (C-9 ),
1
2
5.05 (C-4 CH
8.64, 29.29, 29.50, 29.52, 29.65 and 31.86 (C-1 –C-8 ), 118.19
3
), 20.34 and 20.65 (2ꢁ –OCOCH ), 22.65, 27.76,
3
Acknowledgments
0
0
and 121.52 (C-6 and C-10), 119.61 (C-5), 127.03 (C-3), 129.86 (C-
The financial assistance from the DU-DST Purse Grant, Defense
Research and Development Organization (DRDO), Government of
India, Delhi is gratefully acknowledged. We also acknowledge
University Grant Commission (Government of India, Delhi) for pro-
viding fellowship to SJ.
8
1
1
1
), 141.08, 145.10 and 144.21 (C-4, C-7 and C-9), 160.20 (C-2),
67.35 and 167.35 (2ꢁ –OCOCH ); IR (KBr) max: 1776.75 (OCO),
705.76 (CO), 1613.63, 1456.42, 1370.75, 1222.05, 1115.30,
3
m
ꢀ1
079.70, 1019.32, 881.38, 776.21, 595.79 cm ; UV (methanol)
+
30 6
kmax: 279 and 303 nm; HRMS: Calcd for C23H O [M+H] =
4
03.2042. Found: 403.2109.
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[
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5.2.2. Isolation of platelet rich plasma (PRP)
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them about the details of the experiment and taking their consent.
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3
.8% trisodium citrate (anticoagulant). The citrated blood was
centrifuged at 1200 rpm for 10 min at room temperature. 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. PRP was then further centri-
fuged at 4000 rpm for 5 min to produce a platelet button. The
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[
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[
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4.
1
[
9
5.2.3. Assay of NOS by flow cytometry
[
[
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The method outlined by Imrich and Kobzik was followed for the
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with a 488 nm laser based flow cytometer (FACS calibur, Becton
and Dickenson, USA) and data (light scatter and green
[25] M.S. Khaikin, Zhur. Obsh. Khim. 1 (1965) 356–358.
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[