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A. Gupta et al. / Biochimie 92 (2010) 1180e1185
2.3.2. Benzofuran-3,6-diyl diacetate (2b)
(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,
It was obtained as a yellow solid (85%); mp: 80 ꢀC (Literature
mp ¼ 81 ꢀC) [14]; 1H NMR (CDCl3, 300 MHz):
2.33, 2.37 (2 ꢂ s, 6H,
d
2 ꢂ 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):
1020.78, 972.71, 899.24, 850.96, 801.00, 702.96 and 671.11 cmꢁ1
;
d
20.83, 21.14 (2 ꢂ -OCOCH3), 105.74 (C-7), 117.23, 118.65 (C-4 and
UV (MeOH) lmax: 252 and 318 nm; HRMS: Calculated for C10H7O4Cl
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 ꢂ 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 cmꢁ1; UV (MeOH) lmax: 249 nm; HRMS: Calculated for
C12H10O5 [M]þ 234.0528, found 234.0248.
[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
2.3.3. 5-Chlorobenzofuran-3,6-diyl diacetate (2c)
sucrose and 1.4 mM
b-mercaptoethanol, pH ¼ 7.0 using Potter
It was obtained as a red solid (80%); mp: 72 ꢀC; 1H NMR (CDCl3,
Elvehjem homogenizer. The homogenate was centrifuged using
Sorvall Centrifuge at 10,000 ꢂ 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 ꢂ 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.
300 MHz):
d
2.36, 2.38 (2 ꢂ s, 6H, 2 ꢂ -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 ꢂ 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 ꢂ 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 cmꢁ1; UV (MeOH) lmax: 254 and 294 nm; HRMS:
Calculated for C12H9O5Cl [M þ H]þ 269.0139, found 268.8932.
2.6. Glutathione S-transferase assay
2.4. General procedure for the synthesis of acetoxy derivatives
of 3-oxobenzofurans (3aec)
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
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).
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.
2.4.1. 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 ꢂ s, 6H, 2 ꢂ 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
2.6.1. Assay of CRTAase
NMR (CDCl3, 75 MHz):
d
20.19, 20.65 (2 ꢂ eOCOCH3), 75.99 (C-2),
The assay of CRTAase was performed by preincubation of rat
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 ꢂ 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 cmꢁ1; UV (MeOH)
lmax: 258 and 319 nm; HRMS: Calculated for C12H10O6 [M]þ
250.2042, found 250.5470.
liver microsome (12
toxy benzofurans (50
the CRTAase activity was expressed in terms of inhibition of GST
under the conditions of the assay [1].
m
m
g) with cytosol (20
M), followed by the assay of GST. The unit of
mg protein) and the ace-
2.7. Preparation of platelet rich plasma (PRP) for platelet
aggregation studies
The method of O0Brein [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,
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
SYSMEX Model No. FA20 and was adjusted to 250,000 per
platelet poor plasma (PPP).
mL with
(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
PPP was prepared by centrifugation of remainder of blood at
2500 ꢂ g for 10 min.
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 cmꢁ1; UV (MeOH) lmax: 271 and 318 nm; HRMS:
Calculated for C10H8O4 [M]þ 192.0423, found 192.0276.
Platelet counts (PC) were adjusted according to the formula:
PCðPRPÞ ꢂ mL PRP=250; 000 ¼ mL PRPð250; 000Þ
2.4.3. 5-Chloro-3-oxo-2,3-dihydrobenzofuran-6-yl acetate (3c)
2.8. Aggregometry
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,
20.66
Platelet count was adjusted to 250,000/
mL with homologous
H-7), 7.75 (s, 1H, H-4); 13C NMR (CDCl3, 75 MHz):
d
PPP. Various PAs and polyphenols were preincubated at 37 ꢀC with