S. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 1605–1613
1611
system of ethyl acetate and petroleum ether to afford
7,8-dibenzyloxy-4-methylcoumarin (5) as a white solid
(13.0 g) in 84% yield, mp 152 ꢁC; Rf: 0.46 (petroleum
ether/ethyl acetate, 4:1); IR (KBr): 2925, 2361, 1712
fied by column chromatography using gradient system
of ethyl acetate–petroleum ether to afford 7,8-dihy-
droxy-4-methylthiocoumarin (8) as a yellow solid
(0.30 g) in 56% yield, mp 230–235 ꢁC; Rf: 0.48 (petro-
leum ether/ethyl acetate, 3:2); IR (KBr): 3312 (OH),
1
(C@O), 1607, 1293, 1088, 981 and 697 cmÀ1; H NMR
1
(CDCl3, 300 MHz): d 2.35 (3H, s, CH3), 5.17 and 5.19
(4H, 2s, 2H each, 2 · OCH2C6H5), 6.13 (1H, s, C-3H),
6.90 (1H, d, J = 8.9 Hz, C-6H), 7.21–7.51 (11H, m, C-
5H and 2 · OCH2C6H5); 13C NMR (CDCl3,
75.5 MHz): 20.01 (CH3), 72.63 and 76.94 (2 · O–CH2–
C6H5), 111.38 (C-6), 113.95 (C-3), 116.43 (C-10),
120.79 (C-5), 128.69, 129.40, 129.52, 129.55, 129.97
( 2 · OCH2C6H5), 137.56 and 138.42 (C-7 and C-8),
153.7 (C-4), 156.06 (C-9) and 161.78 (C-2). HRMS calcd
for C24H20O4 [M+Na]+ 395.1254, found 395.1243.
2925, 2855, 1561, 1434, 1366, 1254 and 1095 cmÀ1; H
NMR (DMSO-d6 + CDCl3, 300 MHz): d 2.34 (3H, s,
CH3), 6.95 (1H, d, J = 8.6Hz, C-6H), 7.02 (1H, s, C-
3H), 7.09 (1H, d, J = 8.6 Hz, C-5H), 8.83 and 8.94
(2H, 2s, 1H each, 2 · OH); 13C NMR (DMSO-
d6 + CDCl3, 75.5 MHz): d 20.68 (CH3), 116.42 (C-6),
117.72 (C-10), 118.07 (C-5), 118.31 (C-3), 134.23 (C-4),
148.55 and 148.93 (C-7 and C-8), 151.96 (C-9) and
199.31 (C-2); HRMS calcd for C10H9O3S [M+H]+
209.0272, found 209.0260.
4.3. General procedure for the thionation of coumarins 1
and 5
4.6. General procedure for the acetylation of thiocouma-
rins 6 and 8
A mixture of coumarin 1 or 5 (5 g, 28.4 or 13.4 mmol)
and LawessonÕs reagent25,26 (5.7 or 2.71 g, 14.2–
6.7 mmol) was refluxed in toluene (40 mL) for 24 h.
The progress of the reaction was monitored by TLC.
On completion, the reaction mixture was allowed to cool
to room temperature and the solvent evaporated to dry-
ness in vacuum, the residue was purified by column
chromatography on silica gel using gradient solvent
system of ethyl acetate–petroleum ether to afford 7-
hydroxy-4-methylthiocoumarin (6) and 7,8-dibenzyl-
oxy-4-methylthiocoumarin (7) as yellow solids in 44%
and 29% yields, respectively. 7-Hydroxy-4-methylthio-
coumarin (6) was identified on the basis of the spectro-
scopic data, which was found to be identical with the
spectroscopic data reported in the literature.36
To a solution of the thiocoumarin 6 or 8 (0.2 g, 1.04 or
0.96 mmol) in acetic anhydride (1.1 equiv) and pyridine
(2 equiv) was added a catalytic amount of DMAP and
the reaction mixture was stirred at 25–28 ꢁC for 6–8 h.
The progress of the reaction was monitored by TLC.
After completion, the reaction mixture was poured into
ice-cold water, the solid that precipitated was filtered
and washed with petroleum ether, dried and recrystal-
lized from CHCl3 to afford the corresponding acetates
9 and 10 in 54 or 83% yields, respectively. The structure
of monoacetoxycoumarin 9 was established on the basis
of physical and spectral data analysis and by comparing
it with the data reported in the literature.36
4.7. 7,8-Diacetoxy-4-methylthiocoumarin (10)
4.4. 7,8-Dibenzyloxy-4-methylthiocoumarin (7)
It was obtained as a yellow solid (0.15 g) in 83% yield,
mp 190–195 ꢁC; Rf: 0.52 (petroleum ether/ethyl acetate,
2:1); IR (KBr): 2925, 2854, 2361, 1774 (C@O), 1562,
It was obtained as a yellow solid (1.5 g) in 29% yield, mp
130–135 ꢁC; Rf: (petroleum ether/ethyl acetate, 4:1); IR
1
1431, 1376, 1292, 1163, 1096, 1015 and 873 cmÀ1; H
(KBr): 2924, 1596, 1378, 1103, 965 cmÀ1
;
1H NMR
NMR (CDCl3, 300 MHz): d 2.34 and 2.36 (6H, 2s, 3H
each, 2 · OCOCH3), d 2.43 (3H, s, CH3), 7.12 (1H, s,
C-3H), 7.20 (1H, d, J = 8.5Hz, C-5H) and 7.50 (1H, d,
J = 8.5 Hz, C-6H); 13C NMR (CDCl3, 75.5 MHz): d
18.36 (CH3), 20.65 and 21.00 (2 · OCOCH3), 120.04
(C-6), 120.82 (C-10), 121.79 (C-5), 129.47 (C-3), 143.16
(C-7 and C-8), 145.77 (C-4 and C-9), 167.75 and
167.93 (2 · C@O) and 196.15 (C@S); HRMS calcd for
C14H13O5S [M+H]+ 293.0484, found 293.0514.
(CDCl3, 300 MHz): d 2.27 (3H, s, CH3), 5.19 and 5.25
(4H, 2s, 2H each, 2 · OCH2C6H5) and 6.92–7.54 (13H,
m, C-3H, C-5H, C-6H and 2 · OCH2C6H5); 13C NMR
(CDCl3, 75.5 MHz): 18.39 (CH3), 71.77 and 76.20
(2 · O–CH2–C6H5), 111.74 (C-6), 116.99 (C-10), 119.77
(C-5), 127.40, 127.73, 128.55, 128.64, 129.07, 129.27
(C-3, 2 · O–CH2–C6H5), 136.45, 137.26 (C-7, C-8),
144.61 (C-4), 155.29 (C-9), 197.16 (C-2); HRMS calcd
for C24H21O3S [M+H]+ 389.1211, found 389.1221.
4.8. Cells and cell culture
4.5. Synthesis of 7,8-Dihydroxy-4-methylthiocoumarin (8)
The primary endothelial cells were isolated from the
umbilical cord by mild trypsinisation.8 Cells were main-
tained in gelatin coated tissue culture flasks in M 199
medium supplemented with 20% heat inactivated foetal
calf serum, 2 mM L-glutamine, 100 units/mL penicillin,
100 lg/mL streptomycin, 0.25 lg/mL amphotericin,
endothelial cell growth factor (50 lg/mL) and heparin
(5 U/mL). The cells were sub-cultured by dislodging
with 0.125% trypsin–0.01 M EDTA solution in Pucks
saline and HEPES buffer. For the present analysis, cells
were used between passages three to four and the viabil-
ity of cells was determined by trypan blue exclusion test.
To a solution of the thiocoumarin 7 (1.0 g, 2.58 mmol)
and N,N-dimethylaniline (1.25 g, 10.32 mmol) in
CH2Cl2 (20 mL), powdered AlCl3 (1.03 g, 7.74 mmol)
was added in portions. The reaction mixture was stirred
at room temperature for 45 min and on completion, the
reaction was quenched by the addition of 1 N HCl
(4.5 mL). The aqueous reaction mixture was extracted
with ethyl acetate (3 · 30 mL), combined organic layer
was washed with 5% NaHCO3 solution, dried over
anhydrous Na2SO4 and the solvent concentrated under
reduced pressure. The residue, thus obtained was puri-