A. Sood et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4724–4728
4725
O
H
O
OH
R3
O
R3
R2
R3
R1
R2
O
O
DCM, rt
N
reflux, EtOH
CH3COO- NH4
+
R2
O
OC2H5
O
R1
R1
OC2H5
2
1a-h
3a-h
4a-h
allyl bromide, K2CO3
dry acetone, reflux
a) R1 = H, R2 = H, R3 = H
b) R1 = H, R2= H, R3 = CH3
c) R1 = H, R2 = H, R3 = F
d) R1 = H, R2 = H, R3 = Cl
e) R1 = H, R2 = H, R3 = Br
f) R1 = Cl, R2 = H, R3 = Cl
g) R1 = Cl, R2 = H, R3 = H
h) R1 = H, R2= Br, R3 = H
O
O
R1
N
R2
R3
5a-h
5c
Scheme 1. Synthesis of compounds 4a–h and 5a–h.
Table 1
The cytotoxic activity of all the synthesized compounds (4a–h
and 5a–h) was determined against various human cancer cell lines
such as Prostate (PC-3), Breast (MCF-7), CNS (IMR-32), Cervix
(Hela) and Liver (Hep) according to procedure of Skehan et al.29
The growth inhibition (%) was determined at concentrations of
Reaction time (min) and yield (%) of various purified products 4a–h and 5a–h
Compd No
R1
R2
R3
Time (min)
Yield (%)
4a
4b
4c
4d
4e
4f
4g
4h
5a
5b
5c
5d
5e
5f
H
H
H
H
H
Cl
Cl
H
H
H
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
Br
H
H
H
H
H
H
H
Br
H
CH3
F
Cl
Br
Cl
H
60
60
60
60
60
60
60
60
45
45
45
45
45
45
45
45
72
78
77
75
72
70
67
74
67
72
74
68
73
70
65
68
10, 30 and 100
cin-C as positive controls. IC50
l
M by using paclitaxel, 5-fluorouracil and mitomy-
M) values, which is the concentra-
(l
tion required to inhibit cancer cell proliferation by 50% after
exposure of cells to test compounds, have also been determined
(Table 2). In the case of prostate cancer cell line (PC-3), compounds
4b and 4c showed maximum cytotoxicity with IC50 = 10.8 and 11.8,
respectively, whereas compound 4d, 4f, 4g and 4a exhibited mod-
erate cytotoxic potential with IC50 <33.4. Against breast cancer cell
line (MCF-7) compound 4b displayed high cytotoxic potential with
IC50 = 8.0, followed by 4a with IC50 = 24.6 and 4g with IC50 = 26.7.
In the case of CNS cancer cell line (IMR-32) compound 4b showed
significant inhibitory activity with IC50 = 5.5, followed by com-
pound 4c with IC50 = 6.2 and compound 4d with IC50 = 19.5. In cer-
vix cancer cell line, almost all compounds showed inhibitory
potential with IC50 <30. Compounds 4b and 4c were found to be
active against liver cancer cell line (Hep) with IC50 = 11 and 12,
respectively.
From the results of cytotoxic activity, it was observed that com-
pounds with hydroxyl group (4a–h) displayed more cytotoxic effect
as compared to o-allylated compounds (5a–h). Compound 4b dis-
played significant inhibitory activity against all the cancer cell
lines, therefore it was used to investigate the mechanism of cell
death by using microscopic techniques such as confocal micros-
copy and scanning electron microscopy (SEM):
Confocal microscopy: The fluorescent dye DAPI were used to
visualize the changes of cell morphological features (nuclei and
mitochondria).15,30 DAPI is a popular cell-permeable nuclear stain
that emits blue fluorescence when bound to dsDNA. For imaging,
PC-3 cells (1 ꢂ 106) in 1 ml of culture medium were transferred
into 24 well plates and grown to confluence at 37 °C under 5%
CO2 for 24 h. The cultures were then exposed to compound 4b at
its IC50 value and incubated for another 12 h. After incubation,
the medium was aspirated with 1 ml culture medium without
serum and then stained with DAPI (10 mM) for 1 h at 37 °C. The
confocal fluorescence images of the cells treated with compound
4b were scanned on a Nikon eclipse TiE inverted fluorescence
microscope equipped with a Nikon AiR laser scanning confocal
microscope system (Nikon Corp., Japan) equipped with NIS ele-
ment viewer software. The confocal scanning micrographs showed
the difference in distribution pattern of mitochondria and DNA in
the treated cells compared to control. Moreover, the typical fea-
tures of apoptosis, that is, cell shrinkage, cell rounding and plasma
H
H
CH3
F
Cl
Br
Cl
H
5g
5h
H
on removal of solvent under vacuum were purified by column
chromatography, using neutral silica gel (pHꢀ7, 60–120 mesh)
and eluted with 1:10 chloroform in hexane. All the purified prod-
ucts (4a–h and 5a–h) were characterized by rigorous spectroscopic
techniques (IR, 1H and 13C NMR, mass). Finally, the structure of
compound 5c was confirmed by X-ray28crystallography
(Scheme 1).
The assigned structures (4a–h and 5a–h) are based on rigorous
spectroscopic techniques such as 1H & 13C NMR, IR and mass. 1H
NMR spectrum of compound (4a) showed the appearance of two
doublets at d 8.79 and 8.00 ppm due to the pyridine C6-H and
C4-H protons with J = 4.8 and 7.5 Hz, respectively. The presence
of a hydroxyl proton involved in a hydrogen bond with the carbonyl
group was identified by its characteristic singlet at d 11.6 ppm. 13
C
NMR spectrum (4a) showed resonance at d 198.50 attributed to the
(C@O) group, which was further confirmed by IR spectrum through
a strong band at 1650.2 cmꢁ1. IR spectrum also showed broad band
of O–H at 3416 cmꢁ1 and C–N stretching at 1471.5 cmꢁ1 of pyri-
dine ring. The structure of compound 4a is further corroborated
by mass spectrum, which showed a molecular ion peak at m/z
200 (M+H)+ which corresponds to compound 4a. For allylated com-
pound 5a 1H NMR spectrum showed allylic proton resonances at
regions of d 5.71–5.59 (m, 1H, C200-H), d 5.06–4.92 (m, 2H, C300-H)
and d 4.44 (d, 2H, J = 4.8 Hz, C100-H). 13C NMR spectrum of 5a
showed resonance at d 193.50 due to C@O group, which was
further corroborated by IR spectrum showing a strong band at
1685 cmꢁ1
. IR spectrum also showed the alkene stretching
at 3013 cmꢁ1 and mass spectrum revealed a molecular ion peak
at m/z 240 (M+H)+.