4246
Shakeel-u-Rehman et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4243–4246
(PC-3) cell line, while as, triazoles with ortho substituted R0 groups
are selectively cytotoxic against lung (A-549) cancer cell line. How-
ever further studies especially the in vivo studies need to be carried
out for revealing the exact mechanism of action.
(prop-2-ynyloxy)-2H-chromen-2-one (2) (Scheme 1): To
a solution of 6-
hydroxycoumarin (1) (2 g, 12.3 mmol) in acetone (10 ml), K2CO3 (1.87 g,
13.1 mmol) was added portionwise and stirred for 15 min at 25 °C. Solution of
propargyl bromide (1.1 ml, 14.8 mmol) in acetone was then added and the
suspension was stirred for 1 h. Progress of reaction was monitored using TLC at
regular intervals. After the completion of reaction, the product was extracted
which on subsequent purification over silica gel column resulted in the
isolation of pure product 2 in 95% yield. Colourless solid, mp: 155–158 °C. 1H
NMR (400 MHz, CDCl3) d: 2.56 (s, 1H), 4.74 (s, 2H), 6.45 (d, J = 9.6 Hz, 1H), 7.04
(d, J = 2.8 Hz, 1H), 7.19 (dd, J = 8.4, 2.8 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.67 (d,
J = 9.6 Hz, 1H). 13C NMR (126 MHz, CDCl3) d: 56.52, 76.15, 77.97, 111.88,
117.26, 117.98, 119.19, 120.24, 143.03, 149.04, 153.93, 160.77. HR-ESIMS:
201.0544 (Calculated for C12H8O3, 200.0473). General procedure for synthesis
isoxazolyl derivatives 3–14 (Scheme 1): To a suspension of 10 mmol of aldehyde
in a 1:1:2 mixture of H2O/EtOH/ice (10 ml) was added 10 mmol of hydroxyl-
amine hydrochloride, followed by 25 mmol of NaOH (as a 50% solution in
water), while keeping the temperature below 30 °C. After being stirred at room
temperature for 1 h, the solution was extracted with diethyl ether. The
aqueous phase was acidified to pH 6 by adding concentrated HCl while keeping
the temperature below 30 °C and extracted with Et2O. The organic phase was
dried over MgSO4, and the solvent was evaporated to give the oxime products
in 85–95% yield, which were used directly in the next reaction. To a solution of
10 mmol of oxime in tetrahydrofuran (10 ml) containing 6-(prop-2-ynyloxy)-
2H-chromen-2-one (2), 10 mmol of N-chlorosuccinimide (NCS) was added
using K2CO3 (40 mmol) as base and the suspension was stirred for 1–2 h at
room temperature till its completion. The crude reaction mixture was
extracted with ethyl acetate (3 ꢂ 30 ml) and the combined organic layer was
dried over sodium sulphate and purified through column chromatography to
afford pure 3–14 in 85–89 % yield. Spectral data of 3: Colourless solid, yield:
87%, mp: 140–142 °C, 1H NMR (400 MHz, CDCl3) d: 5.27 (s, 2H), 6.46 (d,
J = 9.6 Hz, 1H), 7.05 (d, J = 2.9 Hz, 1H), 6.84 (s, 1H), 7.22 (dd, J = 8.4, 2.9 Hz, 1H),
7.32 (d, J = 8.4 Hz, 1H), 7.42-7.36 (m, 2H), 7.50 (d, J = 7.7 Hz, 1H), 7.67 (d,
J = 9.6 Hz, 1H), 7.74 (dd, J = 7.7, 1.9 Hz, 1H). 13C NMR (126 MHz, CDCl3) d: 62.00,
105.14, 111.76, 117.47, 118.24, 119.32, 120.14, 127.22, 127.84, 130.49, 130.99,
131.14, 132.90, 142.97, 149.19, 154.21, 160.74, 161.24, 166.89. HR-ESIMS:
354.0525 (calculated for C19H12ClNO4, 353.0455). Similarly other compounds
were isolated and characterised using spectral analysis. General procedure for
synthesis triazolyl derivatives 15–26 (Scheme 1): 6-(Prop-2-ynyloxy)-2H-chro-
men-2-one 2 (3 mmol) and different freshly prepared organic azides (3 mmol)
were suspended in 10 ml of a 1:1 water/t-butanol mixture. Sodium ascorbate
Supplementary data
Supplementary data associated with this article can be found, in
References and notes
(0.3 mmol, 300
lL of freshly prepared 1 M solution in water) was added,
followed by copper(II) sulfate pentahydrate (0.03 mmol, in 100
lL of water).
The heterogeneous mixture was stirred vigorously till the completion of
reaction. The reaction mixture was extracted with ethyl acetate (30 ꢂ 3 ml)
and the combined organic layer was dried over sodium sulfate and purified
through column chromatography to give pure 15–26 in excellent yields of 90–
95 %. Spectral data of 15: Colourless solid, yield: 92%, mp: 160–166 °C, 1H NMR
(400 MHz, CDCl3) d: 5.34 (s, 2H), 6.44 (d, J = 9.6 Hz, 1H), 7.12 (d, J=2.9 Hz, 1H),
7.22 (dd, J = 9.1, 2.9 Hz, 1H), 7.29 (d, J = 9.1 Hz, 1H), 7.46 (t, J = 7.4 Hz, 1H), 7.55
(m, 2H), 7.68 (d, J = 9.6 Hz, 1H), 7.75 (m, 2H), 8.07 (s, 1H). 13C NMR (126 MHz,
CDCl3) d: 62.55, 111.34, 117.24, 118.10, 119.27, 119.27, 120.26, 120.63, 121.13,
129.08, 129.86, 129.86, 136.87, 143.20, 144.31, 148.86, 154.58, 160.91. HR-
ESIMS: 320.1029 (calculated for
C18H13N3O3, 319.0957). Similarly other
compounds were isolated and characterised using spectral data analysis.
39. Evaluation of cytotoxicity using MTT assay: All the compounds were evaluated
against a panel of five different human cancer cell lines viz. prostate (PC-3),
colon (HCT-116 and Colo-205), leukemia (HL-60) and lung (A-549) using MTT
assay in a 96 well plate. Cells were routinely maintained in RPMI 1640 (Sigma
Aldrich) supplemented with 10% FBS (Merck) and 1% penicillin
G and
streptomycin (Sigma Aldrich) at 37 °C in a humidified incubator with 5% CO2
and were subcultured at 1:5 ratio once a week. For antiproliferative activity,
compounds were dissolved in cell culture grade DMSO. Briefly, cells (104 cells/
well) were cultured in 96 well tissue culture plates and treated with different
concentrations of compounds for 48 h. At the end of incubation, 20 lL of MTT
(2.5 mg/mL) was added to the wells and incubated for 4 h. Absorbance was
recorded at 570 nm using Eliza Plate Reader. Inhibition of formation of
coloured MTT formazan was taken as an index of cytotoxicity activity. The IC50
values on the cancer cells of different tissue origin used for screening were
determined by non-linear regression analysis using graph pad software.40
33. Isolation of 6-hydroxycoumarin: 6-Hydroxycoumarin was isolated in bulk
quantity from the root parts of Prangos pabularia (duly authenticated by the
taxonomist of our institute). 6-Hydroxycoumarin used in present study was of
98% purity (HPLC analysis) achieved through repeated column
chromatography over silica gel 60–120 mesh and the natural product was
characterized by spectral data analysis in light of literature.34 Synthesis of 6-