Biochimie 92 (2010) 1180e1185

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Biochimie
journal homepage: www.elsevier.com/locate/biochi

Research paper

Specificity of Calreticulin Transacetylase to acetoxy derivatives of benzofurans:

Effect on the activation of platelet Nitric Oxide Synthase
Anjali Gupta a, Nivedita Priya b, Sarah Jalal a, Prabhjot Singh b, Karam Chand a, Hanumantharao G. Raj b,
Virinder S. Parmar a, Anthony L. DePass c, Sunil K. Sharma a, *
a
Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
b
Department of Biochemistry, V.P. Chest Institute, University of Delhi, Delhi 110 007, India
c
Department of Biology, Long Island University-Brooklyn, 1 University Plaza, Brooklyn, NY 11201, USA

a r t i c l e i n f o a b s t r a c t

Article history: Calreticulin Transacetylase (CRTAase) catalyzes the transfer of acetyl group(s) from polyphenolic acetates
Received 15 March 2010 (PAs) to functional proteins, such as Glutathione S-transferase (GST), NADPH Cytochrome c reductase and
Accepted 11 June 2010 Nitric Oxide Synthase (NOS) resulting in the modulation of biological activities. A comparison of the
Available online 19 June 2010
specificities of the acetoxy derivatives of coumarins, biscoumarins, chromones, flavones, isoflavones and
xanthones has been carried out earlier by us with an aim to study the effect of nature and position of the
Keywords:
acetoxy groups on the benzenoid ring and the position of the carbonyl group with respect to oxygen/
Acetoxy benzofurans
nitrogen heteroatom for the catalytic activity of CRTAase. In this communication for the first time, we
CRTAase
GST inhibition
have studied the influence of differently substituted benzofurans on the CRTAase activity to study the
Platelet NOS effect of the replacement of pyran ring of coumarin with furan ring, presence of carbonyl at C-3,
substitution of C-3 carbonyl group with acetoxy group and presence of various substituents (OAc/OH/Cl)
on the benzenoid ring. It was observed that acetoxy derivatives of benzofurans lead to inhibition of ADP
induced platelet aggregation by the activation of platelet Nitric Oxide Synthase catalyzed by CRTAase.
Accordingly, the formation of NO in platelets by 3-oxo-2,3-dihydrobenzofuran-6,7-diyl diacetate (3a) was
found to be comparable with that of model polyphenolic acetate (PA), 7,8-diacetoxy-4-methylcoumarin
(DAMC).
Ó 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction CRTAase catalyzes the transfer of acetyl group from 7,8-diac-

The acetylation of proteins in biological systems is largely
catalyzed by specific acetyl transferases utilizing acetyl CoA as the
acetyl donor. The enzymatic acetylation of proteins independent of
acetyl CoA was unknown until a unique membrane bound enzyme
Acetoxy Drug: protein Transacetylase (TAase) was discovered in our
laboratory. TAase was found to catalyze the transfer of acetyl
groups from PA to certain enzyme proteins such as GST, NADPH
Cytochrome c reductase and NOS, resulting in the modulation of
their catalytic activities [1e8]. Our earlier studies confirmed TAase
as the Calreticulin (CR), a calcium binding protein and hence
designated as CRTAase [9]. Several PAs being the substrate of
CRTAase were found to be effective in eliciting several biological
effects by virtue of acetylating the concerned functional proteins
[10,11].
* Corresponding author. Tel.: þ91 11 27666646x191.
E-mail address: sk.sharma90@gmail.com (S.K. Sharma).
etoxy-4-methylcoumarin (DAMC), a model acetoxy drug, to GST
and results in the acetylation of several lysine residues in its active
site and subsequently to inhibition of catalytic activity of GST [2,3].
Acetoxycoumarins (DAMC and 7-acetoxy-4-methylcoumarin,
MAMC) were found to modulate the activities of liver microsomal
cytochrome P-450 catalyzed mixed function oxidases (MFO) and
NADPH Cytochrome c reductase catalyzed by CRTAase, by way of
acetylation of these enzyme proteins [4,5]. A number of acetoxy
derivatives of coumarins, biscoumarins, xanthones, flavones and
quinolones were examined for their specificity to liver microsomal
CRTAase. These investigations highlighted the structural features of
PAs such as the proximity of acetoxy groups to the oxygen/nitrogen
heteroatom, fundamental role of carbonyl group, and the influence
of various substituents in controlling their specificities for the
CRTAase [6,7].
In the present investigation, efforts have been made to compare
the specificities of diacetoxy/monoacetoxy as well as 3-oxo/3-ace-
toxy derivatives of benzofurans on CRTAase activity and also to
delineate the structureeactivity relationship (SAR) with special

0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2010.06.011
 A. Gupta et al. / Biochimie 92 (2010) 1180e1185
reference to the effect of furan ring, number and position of acetoxy
groups on the benzenoid ring and the presence of carbonyl group at
the C-3 position of the furan moiety and its replacement with
acetoxy group.
The acetylation of NOS by acetoxy benzofurans is an effective
approach to activate NOS, thereby enhancing NO levels in human
platelets. Platelets are involved in the cellular mechanisms of
primary homeostasis leading to the formation of blood clots. The
damage to the blood vessel walls exposes the sub endothelium
proteins, most notably collagen. The circulating platelets bind
collagen with collagen-specific glycoprotein Ia/IIa receptors. The
adhesion is strengthened further by the large, multimeric circu-
lating 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-trans-
membrane domain receptor family, expressed on platelets, exten-
sively bind to ADP resulting in the release of dense granules
containing PAF (Platelet Activating Factors) vWF, serotonin,
thromboxane A2 (TxA2) which further activate the other circu-
lating platelets [12]. 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.
Derivatives of benzofurans have not been fully explored in terms of
their biological activity, we have in this report demonstrated for the
first time the effect of acetoxy benzofuran derivatives on the acti-
vation of NOS and inhibition of platelet aggregation.
2. Materials and methods
2.1. Chemicals
The organic solvents (acetone, acetic anhydride, chloroform,
tetrahydrofuran, petroleum ether and ethyl acetate) were dried and
distilled prior to their use. Reactions were monitored 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 (100e200 mesh) was used for column chromatography.
Melting points were recorded in capillaries in sulphuric acid bath
and are uncorrected. Infrared spectra were recorded on Per-
kineElmer FT-IR model Spectrum 2000. The 1H NMR and 13C NMR
spectra were recorded 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 values (J) are in hertz. The EI/HR mass spectra were
recorded on Agilent-6210 ES-TOF. NADPH, Cytochrome c, reduced
glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB), dichloro
fluorescein-diacetate (DCFH-DA), N-nitro L-arginine methyl ester
(L-NAME), adenosine diphosphate (ADP) and L-arginine were
purchased from Sigma Chemical Co., St. Louis, MO (USA). Clopi-
dogrel was a generous gift from BIODATA CORPORATION. All other
chemicals used were of high purity and were obtained from local
suppliers.
2.2. General procedure for the synthesis of substituted
hydroxybenzofuran-3(2H)-ones (1aec)
To a stirred solution of the appropriate phenol (10 g, 80 mmol)
and aluminium chloride (22 g, 160 mmol) in carbon disulphide
(100 mL) was added chloroacetyl chloride (6.4 mL, 70 mmol)
dropwise at 0  C in about 15 min.
The reaction mixture was refluxed for 12 h and then poured on
a mixture of crushed ice (500 g) and conc. HCl (15 mL). The
resulting precipitate was filtered, washed with water and dried. The
1181
crude solid so obtained was then refluxed with sodium acetate
(16 g) in absolute ethanol (100 mL) for 6 h. Ethanol was distilled off
and remaining mixture was poured on ice. The precipitate was
filtered and recrystallized from absolute ethanol to obtain pure
benzofuran-3-ones (1aec).
2.2.1. 6,7-Dihydroxybenzofuran-3(2H)-one (1a)
It was obtained as a yellow solid (90%); mp: 232  C (literature
mp ¼ 230e232  C) [13]; 1H NMR (DMSO-d6, 300 MHz): d 4.71
(s, 2H, OCH2CO), 6.58 (d, J ¼ 8.1 Hz, 1H, H-5), 6.95 (d, J ¼ 8.4 Hz, 1H,
H-4); 13C NMR (DMSO-d6, 75 MHz): d 75.70 (C-2), 112.24, 114.78
(C-4 and C-5), 114.66 (C-9), 130.81 (C-7), 154.55 (C-8), 163.98 (C-6)
and 198.22 (C-3); IR (KBr) nmax: 3438.22 (OH), 3130.49, 2986.36,
2934.77, 1634.95 (CO), 1599.10, 1540.44, 1461.88, 1402.13, 1335.12,
1288.21, 1206.07, 1173.87, 1094.50, 1036.53, 1002.62, 975.30, 792.19,
756.91, 718.98, 659.53 and 634.47 cm1; UV (MeOH) lmax: 295 nm;
HRMS: Calculated for C8H6O4 [M]þ 166.0266, found 165.9986.
2.2.2. 6-Hydroxybenzofuran-3(2H)-one (1b)
It was obtained as a yellow solid (85%); mp: 242  C (Literature
mp ¼ 245  C) [14]; 1H NMR (Acetone-d6, 300 MHz): d 4.62 (s, 2H, H-
2), 6.54 (s, 1H, H-7), 6.65 (d, J ¼ 8.4 Hz, 1H, H-5), 7.47 (d, J ¼ 8.4 Hz,
1H, H-4); 13C NMR (DMSO-d6, 75 MHz): d 75.18 (C-2), 98.25 (C-7),
111.72 (C-5), 112.91 (C-9), 125.00 (C-4), 166.67 (C-8), 175.56 (C-6)
and 196.76 (C-3); IR (KBr) nmax: 3384.73 (OH), 3084.28, 2974.18,
2840.47, 2714.50, 1663.28 (CO), 1595.07, 1523.09, 1461.00, 1396.47,
1331.69, 1273.04, 1204.99, 1158.70, 1114.93, 1051.69, 1000.32,
846.58, 810.60, 768.43, 654.50 and 612.97 cm1; UV (MeOH) lmax:
272 and 319 nm; HRMS: Calculated for C8H6O3 [M þ H]þ 151.0317,
found 151.1180.
2.2.3. 5-Chloro-6-hydroxybenzofuran-3(2H)-one (1c)
It was obtained as a yellow solid (80%); mp: 199  C; 1H NMR
(Acetone-d6, 300 MHz): d 4.68 (s, 2H, H-2), 6.76 (s, 1H, H-7), 7.58
(s, 1H, H-4); 13C NMR (DMSO-d6, 75 MHz): d 75.55 (C-2), 99.25
(C-7), 113.29 (C-9), 116.20 (C-5), 124.15 (C-4), 161.43 (C-8), 173.40
(C-6) and 196.16 (C-3); IR (KBr) nmax: 3433.23 (OH), 3146.74,
2944.68, 2499.29, 1668.74 (CO), 1602.41, 1490.53, 1467.54, 1416.51,
1376.48, 1298.44, 1260.43, 1195.22, 1155.48, 1023.32, 976.12, 886.33,
848.66, 765.75, 720.62, 686.08 and 639.70 cm1; UV (MeOH) lmax:
260 and 338 nm; HRMS: Calculated for C8H5O3Cl [M]þ 183.9927,
found 184.1263.
2.3. General procedure for the synthesis of 3-acetoxy
derivatives of benzofurans (2aec)
To a mixture of hydroxy benzofuran-3-one (1aec, 500 mg) and
dimethylaminopyridine (10 mg) in THF was added acetic anhydride
(1.4 mL, 15 mmol) and the reaction mixture was stirred at room
temperature for 24 h. On completion of the reaction, 50 mL
ice/water was added. The crude solid was then filtered and washed
with water to obtain the desired products.
2.3.1. Benzofuran-3,6,7-triyl triacetate (2a)
It was obtained as a yellow solid (85%); mp: 106  C (Literature
mp ¼ 104e105  C) [15]; 1H NMR (CDCl3, 300 MHz): d 2.33, 2.37,
2.39 (3  s, 9H, 3  eOCOCH3), 7.09 (d, J ¼ 8.5 Hz, 1H, H-5), 7.42 (d,
J ¼ 8.5 Hz, 1H, H-4), 8.06 (s, 1H, H-2); 13C NMR (CDCl3, 75 MHz):
d 20.34, 20.64, 20.79 (3  eOCOCH3), 115.68, 118.48 (C-4 and C-5),
121.37 (C-9), 128.04 (C-7), 134.38 (C-3), 134.84 (C-2), 140.19 (C-8),
144.32 (C-6), 167.14, 167.22, 168.56 (3  eOCOCH3); IR (KBr) nmax:
3195.15, 2924.36, 1781.12, 1762.48, 1633.56, 1580.92, 1505.90,
1433.81, 1363.47, 1213.95, 1181.08, 1087.64, 1024.63, 971.95, 893.05,
818.85, 772.41, 750.62 and 617.00 cm1; UV (MeOH) lmax: 250 nm;
HRMS: Calculated for C14H12O7 [M]þ 292.0583, found 291.9289.
1182 A. Gupta et al. / Biochimie 92 (2010) 1180e1185
2.3.2. Benzofuran-3,6-diyl diacetate (2b)
It was obtained as a yellow solid (85%); mp: 80  C (Literature
mp ¼ 81  C) [14]; 1H NMR (CDCl3, 300 MHz): d 2.33, 2.37 (2  s, 6H,
2  eOCOCH3), 7.03 (d, J ¼ 8.3 Hz, 1H, H-5), 7.25 (s, 1H, H-7), 7.54 (d,
J ¼ 8.4 Hz, 1H, H-4), 8.04 (s, 1H, H-2); 13C NMR (CDCl3, 75 MHz):
d 20.83, 21.14 (2  -OCOCH3), 105.74 (C-7), 117.23, 118.65 (C-4 and
C-5), 119.28 (C-9), 134.27 (C-3), 134.31 (C-2), 148.60 (C-8), 152.21
(C-6), 167.34, 169.63 (2  eOCOCH3); IR (KBr) nmax: 3187.50,
2924.36, 1757.06, 1618.85, 1489.59, 1433.51, 1367.87, 1209.24,
1123.16, 1084.07, 1017.14, 951.33, 894.77, 826.47, 798.58 and
577.26 cm1; UV (MeOH) lmax: 249 nm; HRMS: Calculated for
C12H10O5 [M]þ 234.0528, found 234.0248.
2.3.3. 5-Chlorobenzofuran-3,6-diyl diacetate (2c)
It was obtained as a red solid (80%); mp: 72  C; 1H NMR (CDCl3,
300 MHz): d 2.36, 2.38 (2  s, 6H, 2  -OCOCH3), 7.29 (s, 1H, H-7), 7.64
(s, 1H, H-4), 8.05 (s, 1H, H-2); 13C NMR (CDCl3, 75 MHz): d 20.62, 20.72
(2  eOCOCH3), 107.57 (C-7), 119.09 (C-4), 120.28, 122.40 (C-5 and
C-9), 133.66 (C-3), 135.23 (C-2), 144.47 (C-8), 150.47 (C-6), 167.13,
168.73 (2  eOCOCH3); IR (KBr) nmax: 3188.73, 3074.60, 2943.52,
1784.61, 1759.66, 1599.31, 1577.52, 1469.96, 1432.87, 1370.24, 1339.23,
1205.43, 1137.32, 1105.99, 1086.29, 1012.48, 901.16, 889.49, 848.58,
774.48 and 670.13 cm1; UV (MeOH) lmax: 254 and 294 nm; HRMS:
Calculated for C12H9O5Cl [M þ H]þ 269.0139, found 268.8932.
2.4. General procedure for the synthesis of acetoxy derivatives
of 3-oxobenzofurans (3aec)
A mixture of acetic anhydride and acetic acid (1:4) was added to
hydroxy benzofuran-3-ones (1aec) in THF. The mixture was heated
at 70  C for 24 h and then poured on crushed ice. The resulting
precipitate was filtered and the product was purified through
column chromatography in ethyl acetate/petroleum ether (1:5).
2.4.1. 3-Oxo-2,3-dihydrobenzofuran-6,7-diyl diacetate (3a)
It was obtained as brown crystals (30%); mp: 137  C; 1H NMR
(CDCl3, 300 MHz): d 2.34, 2.36 (2  s, 6H, 2  eOCOCH3), 4.71 (s, 2H,
H-2), 6.93 (d, J ¼ 8.4 Hz, 1H, H-5), 7.58 (d, J ¼ 8.4 Hz, 1H, H-4); 13C
NMR (CDCl3, 75 MHz): d 20.19, 20.65 (2  eOCOCH3), 75.99 (C-2),
117.59, 121.51 (C-4 and C-5), 120.82 (C-9), 129.21 (C-7), 149.21 (C-8),
166.41 (C-6), 166.92, 167.50 (2  eOCOCH3) and 197.64 (C-3); IR
(KBr) nmax: 3082.89, 2934.52, 1782.89, 1722.97, 1617.87, 1497.97,
1452.77, 1368.60, 1337.15, 1313.79, 1247.41, 1201.39, 1178.50,
1154.86, 1071.95, 1038.58, 1017.86, 982.18, 891.81, 857.33, 824.39,
784.29, 768.47, 722.08, 692.21, 632.13 and 612.97 cm1; UV (MeOH)
lmax: 258 and 319 nm; HRMS: Calculated for C12H10O6 [M]þ
250.2042, found 250.5470.
2.4.2. 3-Oxo-2,3-dihydrobenzofuran-6-yl acetate (3b)
It was obtained as a yellow solid (25%); mp: 76  C (Literature
value ¼ 77e78  C) [16]; 1H NMR (CDCl3, 300 MHz): d 2.34
(s, 3H, eOCOCH3), 4.67 (s, 2H, H-2), 6.84 (d, J ¼ 8.4 Hz, 1H, H-5), 6.94
(s, 1H, H-7), 7.69 (d, J ¼ 8.4 Hz, 1H, H-4); 13C NMR (CDCl3, 75 MHz):
d 21.15 (eOCOCH3), 75.48 (C-2), 107.10 (C-7), 116.44, 124.97 (C-4 and
C-5), 118.88 (C-9), 158.34 (C-8), 168.38 (C-6), 174.81 (eOCOCH3) and
198.23 (C-3); IR (KBr) nmax: 3084.28, 2974.18, 2840.47, 2714.50,
1663.28, 1595.07, 1523.09, 1461.00, 1396.47, 1331.69, 1273.04,
1204.99, 1158.70, 1114.93, 1051.69, 1000.32, 846.58, 810.60, 768.43,
654.50 and 612.97 cm1; UV (MeOH) lmax: 271 and 318 nm; HRMS:
Calculated for C10H8O4 [M]þ 192.0423, found 192.0276.
2.4.3. 5-Chloro-3-oxo-2,3-dihydrobenzofuran-6-yl acetate (3c)
It was obtained as a red solid (15%); mp: 60  C 1H NMR (CDCl3,
300 MHz): d 2.39 (s, 3H, eOCOCH3), 4.69 (s, 2H, H-2), 7.00 (s, 1H,
H-7), 7.75 (s, 1H, H-4); 13C NMR (CDCl3, 75 MHz): d 20.66
(eOCOCH3), 75.69 (C-2), 109.34 (C-7), 115.24, 124.94 (C-4 and C-5),
120.08 (C-9), 153.48 (C-8), 168.07 (C-6), 173.00 (eOCOCH3) and
197.27 (C-3); IR (KBr) nmax: 2926.00, 2855.67, 1776.35, 1615.89,
1480.81, 1441.68, 1370.92, 1300.00, 1243.17, 1188.35, 1136.45,
1020.78, 972.71, 899.24, 850.96, 801.00, 702.96 and 671.11 cm1;
UV (MeOH) lmax: 252 and 318 nm; HRMS: Calculated for C10H7O4Cl
[M þ H]þ 227.0033, found 226.9749.
2.5. Preparation of microsomes and cytosol
Rats were sacrificed by decapitation. The liver from rats was
pooled and washed in chilled saline and the tissue was minced and
homogenized in 30% 10 mM phosphate buffer containing 0.25 M
sucrose and 1.4 mM b-mercaptoethanol, pH ¼ 7.0 using Potter
Elvehjem homogenizer. The homogenate was centrifuged using
Sorvall Centrifuge at 10,000  g for 30 min at 4  C. The pellet was
discarded and supernatant was again centrifuged in Beckmann
Ultracentrifuge Model L7 at 1,00,000  g for 1 h at 4  C. The cytosolic
fraction was set-aside at 20  C. The pellet obtained was washed with
0.15 M KCl and then resuspended in storage buffer (20% glycerol in
10 mM K3PO4, 2 mM EDTA (ethylenediamine tetraacetic acid), 2 mM
DTT (dithiothreitol), 2 mM PMSF (phenylmethanesulfonylfluoride),
pH ¼ 7.2) and stored at 20  C in aliquots.
2.6. Glutathione S-transferase assay
The method of Habig et al. [17] was followed for GST assay using
GSH (Glutathione reduced form) and CDNB (1-chloro-2,4-dinitro-
benzene) as substrates. The assay was carried out in 1 mL spec-
trophotometric cuvette. The reaction mixture consisted of 0.25 M
phosphate buffer (pH ¼ 6.5), cytosol (20 mg protein) and 1 mM
CDNB and 1 mM GSH in a total volume of 1.0 mL. The contents were
mixed and the progress of reaction was followed at 340 nm using
Cary Spectrophotometer Model Cary Bio 100 with kinetic software.
It was ensured that reaction should be linear with respect to time of
incubation and enzyme concentration.
2.6.1. Assay of CRTAase
The assay of CRTAase was performed by preincubation of rat
liver microsome (12 mg) with cytosol (20 mg protein) and the ace-
toxy benzofurans (50 mM), followed by the assay of GST. The unit of
the CRTAase activity was expressed in terms of inhibition of GST
under the conditions of the assay [1].
2.7. Preparation of platelet rich plasma (PRP) for platelet
aggregation studies
The method of O0 Brein [18] was used for carrying platelet
aggregation studies. The citrated blood was used for the prepara-
tion of PRP by the method of Vickers and Thompson [19]. The
platelet count was determined in PRP using electronic counter,
SYSMEX Model No. FA20 and was adjusted to 250,000 per mL with
platelet poor plasma (PPP).
PPP was prepared by centrifugation of remainder of blood at
2500  g for 10 min.
Platelet counts (PC) were adjusted according to the formula:
PCðPRPÞ  mL PRP=250; 000 ¼ mL PRPð250; 000Þ
2.8. Aggregometry
Platelet count was adjusted to 250,000/mL with homologous
PPP. Various PAs and polyphenols were preincubated at 37  C with
 A. Gupta et al. / Biochimie 92 (2010) 1180e1185
Fig. 1. Keto-enol tautomerism.
PRP to make the final volume of 0.5 mL. Platelet aggregation was
induced by the addition of 15 mM ADP. Platelet aggregation studies
were performed using BIODATA CORPORATION, Platelet aggrega-
tion profiler and Model No. PAP4. Platelet aggregation was
expressed as the maximum percentage of light transmittance
change from the base line at the end of the recording time using
PPP as the reference. Platelet aggregation curves were recorded for
6 min and analyzed according to internationally established stan-
dards [20].
2.9. Assay of NOS by flow cytometry
The method outlined by Imrich and Kobzik was followed for the
assay of NOS by flow cytometry [21]. Measurements were made
with a 488 nm laser based flow cytometer (FACS calibur, Becton and
Dickenson, USA) and data (light scatter and green fluorescence)
was acquired using the Cell Quest software (Becton and Dickinson,
USA). Analysis was performed by applying appropriate gates with
reference to the auto fluorescence measured under similar condi-
tions. Platelets isolated as described earlier were suspended in
phosphate buffered saline and platelet count was adjusted to
106/mL using electronic particle counter (SYSMEX, Model no. FA
20). DCFH-DA (Dichloro fluoresein diacetate) (2 mM) and poly-
phenolic acetates (180 mM) were then added to the cell suspension
and separately preincubated at 37  C for 30 min. In order to confirm
the enhancement of NO level in platelets, L-NAME (50 mM),
a specific nitric oxide inhibitor was also added. To stop the assay,
samples were placed on ice for 10 min in the dark. Relative green
DCF fluorescence was measured.
3. Results and discussion
In our earlier work, we elucidated the role of acetoxy groups on the
benzenoid ring of various PAs like coumarins, biscoumarins, chro-
mones, xanthones and quinolones in facilitating the acetylation of the
receptor protein catalyzed by CRTAase. As a part of the quantitative
HO OH HO
ClCH2COCl
R R
AlCl3, CS2
Scheme 1. Synthesis of acetates of hydroxybenzofuran-3(2H)-ones.
1183
structureeactivity relationship (QSAR) studies on CRTAase, we have
demonstrated earlier (a) positional specificity of acetoxy groups on
the benzenoid ring of certain polyphenols, (b) absolute requirement
of carbonyl group and (c) role of various substituents on benzopyrans
in controlling the protein acetylation [5e8,22].
However, the effect of benzofurans as substrates for CRTAase
activity has not been studied so far. In this report, we have for the first
time compared the specificities of acetoxy derivatives of a number of
benzofurans as substrate to CRTAase to study the effect of the
replacement of pyran ring of coumarin with furan ring, presence of
carbonyl at C-3, substitution of C-3 carbonyl group with acetoxy
group and presence of various substituents (OAc/OH/Cl) on the
benzenoid ring. Such a study would allow us to study the effect
thereof on the rate of catalytic activity of CRTAase and the efficacy of
these acetoxy benzofurans to activate platelet NOS. Hydroxy benzo-
furan-3(2H)-ones (1aec) were synthesized via Friedel Crafts acyla-
tion reaction, followed by ring closure with sodium acetate as base.
For transacetylase activity, phenolic acetates were prepared by
acetylation of corresponding hydroxy benzofuran-3(2H)-ones
(2aec). There exist two possibilities of acetylation of benzofuran-3-
ones due to the presence of acidic hydrogen at C-2 position and that
can exhibit keto-enol tautomerism and thus isomeric forms are
possible (Fig. 1). In acidic medium, keto form is predominant, thus
under acidic conditions, i.e. acetic acid/acetic anhydride, acetylation
occurs only on hydroxyl groups of benzenoid nucleus which leads to
the formation of 3-oxo acetoxy products (3aec). However, under
basic conditions, benzofuranones exist preferably in enol form and
acetylation under these conditions (Ac2O/DMAP) results in the
formation of 3-acetoxy derivatives (2aec) bearing acetoxy groups on
benzenoid ring as well as the C-3 position (Scheme 1). All the
compounds were fully characterized on the basis of their physical and
spectral data, and of total nine benzofuran derivatives, two i.e. 2c and
3c are novel. Though the remaining seven compounds are known in
literature but their complete spectral data is not given, we have
reported the data for all the compounds in the experimental section.
The rat liver microsomal CRTAase activities utilizing acetoxy
benzofurans as the substrates were compared with DAMC. The
results clearly indicated that among the acetoxy benzofurans, the
diacetoxy derivative 3-oxo-2,3-dihydrobenzofuran-6,7-diyl diac-
etate 3a showed highest affinity for the CRTAase. It is clearly
evident from Fig. 2 that activities of diacetoxy benzofurans 3a and
2a having two acetoxy groups on the benzenoid ring are twice than
that of the corresponding monoacetoxy benzofurans 3bec and
2bec having one acetoxy group on the benzenoid ring.
7 1
OH HO 6 8 O
CH3COONa 2
o R5
Cl EtOH, 78 C 4
9 3
O O
1a R = 7-OH
1b R = H
1c R = 5-Cl
Ac2O, Ac2O,
DMAP, THF AcOH, THF
AcO O AcO O
R R
OAc O
2a R = 7-OAc 3a R = 7-OAc
2b R = H 3b R = H
2c R = 5-Cl 3c R = 5-Cl
1184 A. Gupta et al. / Biochimie 92 (2010) 1180e1185
Fig. 2. CRTAase catalyzed inhibition of GST by acetoxybenzofurans. Polyphenols were
separately preincubated with rat liver microsomes (20 mg) and rat liver cytosol (12 mg)
followed by assay of GST, the activity was expressed in terms of % inhibition of GST.
Values are mean of three observations with variation less than 5%.
Fig. 3. Inhibition of ADP induced platelet aggregation: effect of acetoxy benzofurans.
PRP was incubated with test compound (180 mM) for 10 min at 37  C followed by the
addition of ADP (15 mM) and platelet aggregation was monitored by aggregometry.
Values are mean  SEM of 5 observations.
Fig. 4. Enhancement of intracellular NO levels by acetoxy benzofurans. Platelets were
incubated with benzofurans (180 mM), L-Arginine (100 mM) and DCFH-DA (2 mM) at
37  C for 30 min. L-NAME (50 mM) where used was preincubated with the platelets and
NO levels were measured as described earlier. Values are mean of three observations
with variation less than 5%.
Fig. 5. 7,8-Diacetoxy-4-methylcoumarin (DAMC) and 7-acetoxy-4-methylcoumarin
(MAMC).
From Fig. 2, it is clear that inhibition of GST by acetoxy deriva-
tives of benzofuran-3-ones 3a, 3b and 3c is almost similar to that of
the corresponding 3-acetoxy benzofuran derivatives 2a, 2b and 2c.
Compounds 2aec are the analogues of 3aec having an additional
acetoxy group at C-3 position in place of carbonyl group. As it was
proved from the earlier studies that carbonyl group leads to the
increased activity, whereas in the present study on benzofurans no
considerable loss of CRTAase activity was observed by substituting
carbonyl by acetoxy group. So, it could be suggested that decline in
activity due to removal of carbonyl group at the C-3 position is
compensated by its replacement with acetoxy group. Moreover, the
introduction of chloro group at the C-5 position, i.e. 5-chloro-3-
oxo-2,3-dihydrobenzofuran-6-yl acetate (3c) has no significant
influence on CRTAase activity.
In recent times the antiplatelet drugs are commonly used for the
treatment of heart diseases [23]. The influence of acetoxy benzo-
furans on the inhibition of platelet aggregation has been clearly
brought out in Fig. 3. It is clear from the result that 3-oxo-2,3-
dihydrobenzofuran-6,7-diyl diacetate (3a) was found superior in
exhibiting the inhibition of ADP induced platelet aggregation in
comparison with the other acetoxy derivatives. Thus, the structural
modifications of acetoxy benzofurans were found to influence their
effect on inhibition of platelet aggregation in tune with the speci-
ficity of platelet CRTAase to these compounds.
We have in this communication elucidated for the first time the
action of CRTAase on acetoxy derivatives of benzofurans and the
consequent enhancement of NO levels in platelets leading to inhi-
bition of ADP induced platelet aggregation. PAs by virtue of
enhancing NO levels in platelets were considered to merit as
antiplatelet agents. A significant elevation of NO levels in platelets
by PAs was considered due to the acetylation of NOS [24]. NO level
in platelets was assayed by measuring the DCF fluorescence caused
by oxidation of DCFH by NO radical. Addition of L-NAME was found
to reduce the NO production to the basal level confirming the
formation of NO. The influence of benzofurans on the NO level in
platelets has been clearly brought out in Fig. 4. The ability of various
acetoxy benzofurans to activate platelet NOS was in the order
3aw2a > 3bw2b > 3cw2c (Fig. 5).
4. Conclusion
In conclusion, benzofurans are well documented as substrates
for CRTAase. The CRTAase activity of diacetoxy compound, 3-oxo-
2,3-dihydrobenzofuran-6,7-diyl diacetate (3a) is almost compa-
rable to that of the model drug, DAMC. The carbonyl group in
compounds 1aec undergoes enolization under basic conditions
and this newly generated hydroxyl group can then be acetylated
and leads to compounds 2aec. Thus the compounds 2aec are
analogues of 3aec and have an additional acetoxy group at C-3
position in place of carbonyl group. Since no significant loss of
CRTAase activity was observed by substituting carbonyl by acetoxy
group, it may be suggested that the loss in CRTAase activity due to
absence of carbonyl group is compensated by the presence of
acetoxy group at the same position. Furthermore, the presence of
two acetoxy groups on the benzenoid ring of benzofurans enhances
 A. Gupta et al. / Biochimie 92 (2010) 1180e1185
the CRTAase activity as has been observed in the case of DAMC.
These observations have amply revealed for the first time that 3-
oxo-2,3-dihydrobenzofuran-6,7-diyl diacetate (3a) is an effective
antiplatelet agent.
Acknowledgements
The financial assistance from the Department of Scientific and
Industrial Research, Council of Scientific and Industrial Research,
Government of India, and the University of Delhi is gratefully
acknowledged.
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