Synthesis and evaluation of 3-salicyloylpyridine derivatives as cytotoxic mitochondrial apoptosis inducers

Article abstract of DOI:10.1016/j.bmcl.2014.08.010

A series of novel 3-salicyloylpyridines (4a-h) were synthesized with good yield by modified Knoevenagel-Stobbel method; o-allylation with allyl bromide lead to formation of compounds (5a-h). The synthesized compounds were characterized by spectroscopic te

Full text of DOI:10.1016/j.bmcl.2014.08.010

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4724–4728
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 3-salicyloylpyridine derivatives
as cytotoxic mitochondrial apoptosis inducers
Alisha Sood a, Vishal Sharma a, Ashun Chaudhry b, Rakesh Kumar b, Saroj Arora b, Rajnikant c,
Vivek Gupta c, Mohan Paul S. Ishar a,
⇑
a Bio-Organic and Photochemistry Laboratory, Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar 143005, Punjab, India
b Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar 143005, Punjab, India
c Post-Graduate Department of Physics, University of Jammu, Jammu Tawi, 180 006, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 3-salicyloylpyridines (4a–h) were synthesized with good yield by modified Knoevena-
gel–Stobbel method; o-allylation with allyl bromide lead to formation of compounds (5a–h). The synthe-
sized compounds were characterized by spectroscopic techniques and evaluated for cytotoxic activity
against human cancer cell lines. Compounds bearing hydroxyl group displayed high cytotoxicity (4a–h)
as compared to o-allylated molecules (5a–h). The most active compound 4b was selected for further
investigation to look for mechanism of cell death in prostate cancer (PC-3) cells. The apoptotic bodies
induced by 4b in PC-3 cells were scanned by confocal microscopy and confirmed by scanning electron
microscopy (SEM). Further results obtained from spectrofluorimetric determination of mitochondrial
Received 16 April 2014
Revised 17 July 2014
Accepted 5 August 2014
Available online 12 August 2014
Keywords:
3-Formylchromone
3-Salicyloylpyridines
Cytotoxic activity
Apoptosis
membrane potential (
DWm) and intracellular reactive oxygen species (ROS) in treated PC-3 cells revealed
that mitochondria dependent apoptosis was involved in the cell death.
Ó 2014 Elsevier Ltd. All rights reserved.
Cancer is a mortiferous disease characterized by unregulated
proliferation of cells leading to tremendous health costs with asso-
ciated high level of mortality and morbidity.1 Despite the availabil-
ity of many effective anticancer agents, still chemotherapy is
placed in the third line for the treatment of cancer due to severe
side effects. Therefore, search for novel anticancer drugs without
any side effects is the major goal for the drug designing program
of medicinal chemists. Pyridine is a six member nitrogen contain-
ing heterocyclic compound and its derivatives exhibit remarkable
biological activities such as antiviral,2 anticancer,3 antimicrobial,4
antidiabetic,5 antidote,6 antileishmanial,7 antioxidant,8 anticoagu-
lant,9 antitubercular10 etc. In the biological system, pyridine is
present as basic nucleus in important vitamins like niacin (vitamin
B3) and pyridoxine (vitamin B6), and it also exist as pharmacophore
of over 7000 existing drugs with a variety of biological activities.11
Camptothecin analogues having pyridine nucleus such as topotec-
an and irinotecan are used clinically as anticancer drugs, which act
as an effective poison of DNA topoisomerase I (TopI) by forming a
ternary complex with TopI-DNA.12 Recently, we have reported
cytotoxic pyridine based isoxazolidines as highly active against
ovarian (IGROV-1), breast (MCF-7) and human glioblastoma
(SF-295) cancer cells.13 According to literature reports, pyridine
based compounds have been found to possess anticancer activity
through varied mechanisms such as blocking the activity of
enzyme CHK1 protein kinase,14 selective inhibition of telomerase,15
inhibition of topoisomerase II,16 inhibition of tyrosine kinases,17
and inhibition of microtubule assembly.18 Taking cognizance of
promising anticancer activity of pyridine based molecules, it
was decided to synthesize some novel pyridine analogues and
evaluates their cytotoxic potential against various human cancer
cell lines. A variety of approaches have been employed for the con-
struction of pyridine nucleus.19–23 In the present study, modified
Knoevenagel–Stobbel method was utilized for the construction of
substituted pyridines.24
Substituted pyrano[4,3-b][1]benzopyrans (3a–h) were synthe-
sized by the hetero Diels–Alder reaction [4+2] of substituted 3-
formylchromones (1a–h) with excess of ethyl vinyl ether (2) in
dichloromethane at room temperature,25,26 which on reaction with
ammonium acetate in ethanol, under refluxing conditions for an
hour, afforded 3-salicyloylpyridines (4a–h, Scheme 1, Table 1). Lit-
erature reports suggested that o-allylated/prenylated compounds
have shown promising bioactivities,27 therefore, taking into con-
sideration the substituted 3-salicyloylpyridines (4a–h) were ally-
lated with allyl bromide in the presence of fused potassium
carbonate to obtain 3-(20-allyloxybenzoyl)pyridines (5a–h,
Table 1). After completion of reactions (Tlc), the residues obtained
⇑
Corresponding author. Tel.: +91 1832258802 09x3321; fax: +91 1832258820.
E-mail address: mpsishar@yahoo.com (M.P.S. Ishar).
http://dx.doi.org/10.1016/j.bmcl.2014.08.010
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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)+.

4726
A. Sood et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4724–4728
Table 2
IC50 (lM) values of various compounds (4a–h and 5a–h)
Compd No.
IC50 (lM)
PC-3 (Prostate)
MCF-7 (Breast)
IMR-32 (CNS)
Hela (Cervix)
Hep (Liver)
4a
4b
4c
4d
4e
4f
4g
4h
33.4
10.0
11.8
24.8
60.2
28.7
31.5
88.6
55
24.6
8.04
27.8
29.0
53.5
29.6
26.7
>100
>100
84
33.7
5.54
6.21
19.5
23.4
28.8
26
89
44
87
33
26
27
24
29
>100
21
37
>100
94
31
11
12
27
29
27
28
85
5a
5b
>100
72
98
5c
5d
5e
>100
>100
59
>100
54
92
91
91
88
93
75
96
>100
>100
78
5f
5g
5h
Paclitaxel
5-FU
96
85
52
94
98
—
1.3
—
34
>100
92
>100
—
0.5
—
>100
>100
—
—
1.5
>100
>100
0.2
—
—
100
>100
—
—
2.0
Mitomycin-C
membrane blebbing were observed in treated cells (Fig. 1). Cam-
ptothecin was used as positive control, which induces apoptosis
in the formation of apoptosome containing cytochrome c, Apaf-1 &
caspase-9. Therefore, to observe the pathway of apoptosis, the
in treated PC-3 cells at 2
lM concentration. Several reports have
change of mitochondrial membrane potential (DWm) in PC-3 cells
indicated that camptothecin induces apoptosis in various cancer
cells,31 however, the detailed molecular mechanisms are not fully
understood.
was determined by spectrofluorimetry using Rhodamine-123 dye.
The PC-3 cells (1 ꢂ 106) were incubated in the presence and
absence of the compound 4b at concentrations of 1, 5 and 10 lM
Scanning electron microscopy (SEM): The cell death induced by
compound 4b through apoptosis was further confirmed by scan-
ning electron microscopy (SEM). Prostate cancer cells (PC-3) with-
out treatment appeared pointed in shape with a few surface
projections under scanning electron microscope. Incubation of
the cells with compound 4b at its IC50 caused rounding or circular
cell and blebbing of plasma membrane after 12 h (Fig. 2). This
study showed the presence of apoptotic bodies and confirmed that
the cell death has occurred via the process of apoptosis.
Apoptosis is the natural death of the cells caused by either acti-
vation of extrinsic (death receptor mediated) or intrinsic (mito-
chondrion dependent) pathways.32,33 In extrinsic pathway, the
engagement of death receptors leads to the formation of death
inducing signalling complex (DISC) containing the death receptors,
adaptor proteins, caspase-8 and caspase-10. In mitochondrion
dependent apoptosis, the potential of mitochondrial membrane
decreases with release of cytochrome-c into the cytosol resulting
for 12 h at 37 °C in 5% CO2 incubator in 24 well plate and then
washed with phosphate buffered saline (PBS). After treatment
Rhodamine-123 (10 lM/ml) was added to each well for 1 h in
serum free media.34 Samples were measured directly in a multi-
purpose plate reader (Biotek synergy HT) at the excitation and
emission wavelengths of 480 nm and 530 nm, respectively. Rh
123 is a fluorescent indicator of change of mitochondrial mem-
brane potential (
when m increases and this accumulation decreases with
decreased m, which is indicated by decreased fluorescence.
DWm) and gets accumulated in the mitochondria
D
W
DW
As shown in Figure 3 the Rh123 fluorescence intensity of PC-3 cells
treated with compound 4b decreased significantly in a dose-
dependent manner as compared to the control. This indicates that
compound 4b could cause a loss of
DWm in PC-3 cells.
Literature reports35–37 suggest that decrease in the mitochon-
drial membrane potential disrupts the mitochondrial electron
transport chain that elevates the level of intracellular reactive
Figure 1. The morphological changes in treated prostate cancer (PC-3) cells examined by confocal microscopy; (A) untreated control cells, (B) cells treated with 2 lM positive
control camptothecin, (C) PC-3 cells treated with compound 4b at its IC50 showed apoptotic morphological features include spherical cell shape, plasma membrane blebbing
and small apoptotic bodies and (D) magnified treated PC-3 cells.

A. Sood et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4724–4728
4727
Figure 2. Scanning electron microscopy (SEM) images of treated PC-3 cells; (A) untreated control cells, (B) cells treated with 2
l
M camptothecin (positive control) and (C) PC-
3 cells treated with compound 4b at its IC50 value. The control cells show microvilli on cell surface (A), the treated cells after 12 h reveal smoothening of cell surface,
condensation in size and blebbing of the plasma membrane (C).
concentration of compound 4b (Fig. 4). This indicates that com-
pound 4b decreases the mitochondrial membrane potential with
consequent increase in the load of reactive oxygen species (ROS).
In conclusion, the series of 3-salicyloylpyridines (4a–h) and 3-
(20-allyloxybenzoyl)pyridines (5a–h) were synthesized and evalu-
ated for their cytotoxic potential against various human cancer cell
lines. Results of cytotoxic activity revealed that compounds bear-
ing hydroxyl group (4a–h) displayed high cytotoxicity as compared
to compounds bearing allyloxy group (5a–h). The most active com-
pound 4b was further investigated for effect on morphology of
treated PC-3 cancer cells and mechanism of cell death by using
confocal microscopy, scanning electron microscopy (SEM) and
spectrofluorimetric determination of mitochondrial membrane
potential, and intracellular ROS level. The results revealed that
compound 4b induces apoptosis, as shown by appearing of apopto-
tic bodies in confocal microscopic and SEM images, and decrease in
membrane potential of mitochondria along with increase in intra-
cellular ROS level; the latter indicating the involvement of mito-
chondrial pathway in apoptosis.
Figure 3. Spectrofluorimetric determination of mitochondrial membrane potential
m) with standard deviation (⁄SD ).
(DW
oxygen species (ROS), which can also be estimated by spectrofluo-
rimetry. For analysis of the ROS, PC-3 cells were plated at 1 ꢂ 106
cells/dish in 24 well plates, allowed to adhere for 6 h and then
grown in the presence of and in the absence of compound 4b.
The cells harvested after 12 h of treatment by collecting trypsini-
zed cells with floating cells in the medium and at 30 min before
the end of the treatment, 20,70-dichlorofluorescein diacetate
(DCFH-DA) is added. For each condition, a volume of the cell sus-
pension corresponding to 1 ꢂ 106 cells was centrifuged and the
resultant cell pellet was resuspended in ice-cold PBS (1.0 ml).
The level of intracellular peroxides in PC-3 cells treated with
Acknowledgment
We sincerely express our thanks to University Grant Commis-
sion (UGC) for providing support and funds under UPE scheme to
our university to generate necessary research facilities.
Supplementary data
compound 4b at concentrations 5, 10 and 15 lM for 12 h was
determined by using DCFH-DA. The observed results reveal that
reactive oxygen species (ROS) load increases with increase in the
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.08.
010.
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