Synthesis and biological evaluation of pyrimidine bridged combretastatin derivatives as potential anticancer agents and mechanistic studies

Article abstract of DOI:10.1016/j.bioorg.2018.02.027

A number of pyrimidine bridged combretastatin derivatives were designed, synthesized and evaluated for anticancer activities against breast cancer (MCF-7) and lung cancer (A549) cell lines using MTT assays. Most of the synthesized compounds displayed good

Full text of DOI:10.1016/j.bioorg.2018.02.027

Accepted Manuscript
Synthesis and Biological Evaluation of Pyrimidine Bridged Combretastatin De-
rivatives as Potential Anticancer Agents and Mechanistic Studies
Bhupinder Kumar, Praveen Sharma, Vivek Prakash Gupta, Madhu Khullar,
Sandeep Singh, Nilambra Dogra, Vinod Kumar
PII:
S0045-2068(18)30063-4
DOI:
https://doi.org/10.1016/j.bioorg.2018.02.027
YBIOO 2287
Reference:
Bioorganic Chemistry
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24 February 2018
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Please cite this article as: B. Kumar, P. Sharma, V. Prakash Gupta, M. Khullar, S. Singh, N. Dogra, V. Kumar,
Synthesis and Biological Evaluation of Pyrimidine Bridged Combretastatin Derivatives as Potential Anticancer
Agents and Mechanistic Studies, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.02.027
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Synthesis and Biological Evaluation of Pyrimidine Bridged
Combretastatin Derivatives as Potential Anticancer Agents and
Mechanistic Studies
Bhupinder Kumar1, Praveen Sharma2, Vivek Prakash Gupta1, Madhu Khullar3,
Sandeep Singh2, Nilambra Dogra3# and Vinod Kumar1*
1Laboratory of Organic and Medicinal Chemistry, Department of Pharmaceutical Sciences and
Natural Products, Central University of Punjab, Bathinda, Punjab, India-151001
2Laboratory of Molecular Medicine, Department of Human Genetics and Molecular Medicines,
Central University of Punjab, Bathinda, Punjab, India-151001
3Department of Experimental Medicine and Biotechnology, PGIMER, Chandigarh, India
160012.
*Corresponding author:
Vinod Kumar, Laboratory of Organic and Medicinal Chemistry, Department of Pharmaceutical
Sciences and Natural Products, Central University of Punjab, Bathinda, Punjab, India-151001
E-mail: vinod.kumar@cup.edu.in; vpathania18@gmail.com; Tel: +917696255588
#Co-corresponding author:
Nilambra Dogra, DST-SERB Young Scientist, Department of Experimental Medicine and
Biotechnology, PGIMER, Chandigarh, India 160012.
E-mail: nilambra@gmail.com; Tel: +91 9876481757
1

Abstract: A number of pyrimidine bridged combretastatin derivatives were designed,
synthesized and evaluated for anticancer activities against breast cancer (MCF-7) and lung
cancer (A549) cell lines using MTT assays. Most of the synthesized compounds displayed good
anticancer activity with IC50 values in low micro-molar range. Compounds 4a and 4p were found
most potent in the series with IC50 values of 4.67 µM & 3.38 µM and 4.63 µM & 3.71 µM
against MCF7 and A549 cancer cell lines, respectively. Biological evaluation of these
compounds showed that selective cancer cell toxicity (in vitro using human lung and breast
cancer cell lines) might be due to the inhibition of antioxidant enzymes instigating elevated ROS
levels which triggers intrinsic apoptotic pathways. These compounds were found nontoxic to the
normal human primary cells. Compound 4a, was found to be competitive inhibitor of colchicine
and in the tubulin binding assay it showed tubulin polymerization inhibition potential
comparable to colchicine. The molecular modeling studies also showed that the synthesized
compounds fit well in the colchicine-binding pocket.
Key words: Pyrimidine, combretastatin derivatives, Apoptosis, Tubulin inhibitors, Colchicine
binding site, Anticancer
2

1.
Introduction
Cancer is a multifactorial disease and considered as the most serious health problem all over the
world. Despite recent advances in our understanding of the biological processes leading to the
development of cancer, there is still need for the development of more potent and effective
anticancer agents for the complete eradication of the disease [1]. Multi-drug resistance and acute
toxicity are the two major issues with most of the currently available chemotherapeutic agents [2,
3]. Therefore, novel anticancer agents need to be developed that are more potent, safe and
selective. In a quest for the discovery of more effective anticancer drugs, a large number of
structurally diverse synthetic and natural products have been screened for their anticancer
potential [4, 5]. Microtubules have been explored as an important target for the anticancer drug
development. Microtubules play crucial role in the spindle formation during cell-division. A
highly dynamic equilibrium exists between the microtubules and the free tubulin dimers [4]. Any
disruption in the dynamic equilibrium of tubulin-microtubule blocks cell-division at the
metaphase–anaphase transition that leads to the induction of the mitochondrial apoptosis [6]
Numerous structurally different natural as well as synthetic compounds have been identified that
target microtubule polymerization [7]. Since last few years combretastatins have received special
attention due to their simple structure and easy synthesis [8, 9]. Combretastatin A-4 (CA-4) binds
with the colchicine binding site and disrupts microtubule polymerization and induces rapid
vascular disruption which leads to tumor cell death [10, 11]. Fosbretabulin, a water-soluble
phosphate prodrug of CA-4, is under phase II/III clinical trials alone and/or in combination with
other chemotherapeutic agents. Although, numerous CA-4 analogs have been developed with
improved antitubulin activities, but clinical application of these agents is not successful till now
[12]. Thus, it is a challenge to synthesize CA-4 analogs with improved activity along with
therapeutic potency.
Most of the combretastatin derivatives have been synthesized by carrying out modifications at
the ring A and ring B. In addition, olefinic bond of combretastatins have been replaced with
number of heterocyclic rings such as, thiazole [13], tetrazole, triazoles [14, 15], imidazole [16],
pyrazole [17], Oxazole [18], pyrrole [19], indole, furan [20], benzene, pyridine [21], pyrimidine
[22], quinoline, isoquinoline, isoxazole, thiazoline, pyrazoline, thiophene [23], benzofuran [24]
3

etc. Many of these compounds displayed cytotoxic and antitubulin activities. In combretastatins,
a two carbon olefinic bridge and cis conformation was considered crucial for the cytotoxicity and
antitubulin activity of the compounds. However, a number of other compounds with varying
length of linker chain between the two phenyl rings have been reported with antitubulin activity.
For example, phenstatin, with a carbonyl group in place of double bond displayed antitubulin
activity [25]. Likewise, chalcone with a three carbon chain, strongly inhibit tubulin
polymerization with improved cytotoxicity [26]. In the present study, we have designed,
synthesized and evaluated a new series of pyrimidine bridged combretastatin derivatives for their
anticancer activities.
Different compounds with a core pyrimidine ring have been reported as inhibitors of
cyclin-dependent kinases (CDK) [27], tumor necrosis factor R (TNF-R) [28], ableson protein
tyrosine kinase (Abl) [29], 3-phosphatidylinositol kinases (PI-3K) [30], protein kinase B
(Aktkinase), and cytokines [31]. Gangjee et al. synthesized two series of the compounds based
on the 6-CH3 cyclopenta[d]pyrimidine and pyrrolo[2,3-d]pyrimidine scaffolds. These
compounds displayed potent antiproliferative activity at nanomolar concentration and target the
colchicine binding site of tubulin [32]. Xie et al. synthesized 2,4,5-substituted pyrimidine
derivatives and evaluated for antiproliferative and anti-tubulin activities. These compounds
arrested cell cycle at G2/M phases of the cell cycle (EC50 = 20nM) and were found as
competitive inhibitors of the colchicine binding site [31]. Zhang et al. synthesized a series of
4-substituted 2,6-dimethylfuro[2,3-d]pyrimidines as dual inhibitors of the microtubules and
tyrosine kinases (RTKs). These compounds exhibited microtubules de-polymerization activity
with EC50 values in nano molar range [22]. Zheng et al. evaluated anticancer activities of
3-carbon and 4-carbon linker analogous of combretastatin-A4. 3-Carbon linkers with bridged
pyridine ring showed better activities as compared to 4-carbon linkers and a non-heterocyclic
bridged system [33].
In the current studies, we have introduced a pyrimidine ring as a 3-carbon linker between the two
phenyl rings (Fig. 1) to maintain the cis locked conformation of combretastatin-A4. Ring A and
ring B were optionally substituted with different substituents having electron releasing and
electron withdrawing effects. Molecular modeling studies were also performed in order to
understand the binding interactions of the synthesized compounds with tubulin proteins. The
mechanistic studies revealed that this series of compounds displayed anticancer activity through
4

inhibition of antioxidant enzymes which may destabilizes the mitochondrial membrane and
trigger intrinsic apoptotic pathway. Further, colchicine site binding agents arrest the cell cycle at
G2/M phase that lead to mitochondrial apoptosis [6].
Fig. 1. Target compounds and their structural comparison with colchicine and combretastatin (CA-4)
2.
Results and Discussion
Chemistry
2.1
Target compounds were synthesized as described in Scheme 1. The intermediate chalcones were
synthesized through Aldol condensation. Aldehydes (1) and acetophenones (2) were condensed
to give 1,3-Diphenyl-2-propen-1-one (chalcones, 3) in varying yields, and recrystallized to
obtain pure products. These intermediate chalcones (3) were further reacted with
acetamidine/formamidine/guanidine to give corresponding pyrimidine bridged analogous of
1
combretastatins (4a-4t). All the synthesized compounds were characterized by H NMR, 13C
NMR and ESI-MS.
Scheme 1. Synthesis of pyrimidine bridged analogues of combretastatins.
2.2
Biological evaluation of compounds and discussion
5

Synthesized compounds exhibited significant cytotoxicity and selectively against cancer cell
lines
All the synthesized compounds (4a-4t) were investigated for their in vitro cytotoxicity against
human MCF-7 (breast cancer) and A549 (lung cancer) cancer cell lines using standard MTT
assays (Table 1 and supplementary figures 1a & 1b) while colchicine was used as a reference
compound. Three different concentrations (1 µM, 5 µM, and 25 µM in triplicate) of the
compounds were used and the results were analyzed after 48 h of drug treatment. It has been
found that compound 4a-4c, 4e, 4f, 4j, 4m-4p showed better antiproliferative activities as
compared to the reference colchicine against both the cell lines. In this series of compounds, 4a
and 4p showed best antiproliferative activity against both the cancer cell lines. Compound 4a
displayed IC50 values of 4.67 µM and 3.38 µM while 4p showed IC50 values of 4.63 µM and
3.71 µM against MCF-7 and A549 cell lines, respectively (48h post treatment). These
compounds were also tested on human peripheral blood cells (hPBMCs) which represented the
normal cell types. The results indicated that this series of compounds did not display cytotoxicity
against the tested cells upto 5µM concentration of the test drugs. Thus, this series of compounds
can selectively target cancer cells and can be used as lead for the development of effective
anticancer drug (supplementary information Fig. S1a and S1b).
Table 1: In vitro antiproliferative activity (IC50 in µM) of compounds 4a to 4t against MCF-7 and A549 cancer cell
lines
MCF-7 (µM)
4.67 ± 0.16
4.78 ± 0.33
7.95 ± 0.51
16.49 ± 0.65
5.98 ± 0.41
4.88 ± 0.21
A549 (µM)
3.38 ± 0.29
3.41 ± 0.54
4.8 ± 0.14
>25
Compound
R1
-H
-H
R2
-H
-H
R3
-H
-H
-H
R4
R5
H
-CH3
4a
4b
4c
4d
4e
4f
p- OCH3 -CH3
-CH3
-OCH3 -OCH3
H
-OCH3 -OCH3 -OCH3 p- OCH3 -CH3
-H
-H
-OCH3
-H
-H
-H
H
H
-H
-H
3.16 ± 0.33
3.17 ± 0.68
6

-OCH3 -OCH3 -OCH3
-OCH3 -OCH3 -OCH3
p-Cl
-CH3
-CH3
-CH3
-CH3
11.01 ± 0.27
>25
>25
>25
4g
H
4h
-OCH3 -OCH3 -OCH3 p- CH3
20.58 ± 1.58
4.89 ± 0.57
>25
>25
4i
-OCH3 -OCH3
-OCH3 -OCH3
-H
-H
-H
-H
p-CH3
3.37 ± 0.39
>25
4j
p-OCH3 -CH3
4k
-H
-H
-H
H
H
-NH2
-NH2
10.84 ± 0.57
4.93 ± 0.46
4.85 ± 0.33
5.10 ± 0.93
4.63 ± 0.87
19.37 ± 0.85
>25
>25
4l
-OCH3
4.85 ± 0.41
5.91 ± 0.52
4.23 ± 0.43
3.71 ± 0.21
>25
4m
-OCH3 -OCH3 -OCH3 p- OCH3 -NH2
4n
-OCH3 -OCH3
-H
p-Cl
-NH2
-NH2
-NH2
-NH2
-NH2
-NH2
4o
-OCH3 -OCH3 -OCH3
2,4-Cl
4p
-OCH3 -OCH3 -OCH3 p-CH3
4q
-OCH3 -OCH3 -OCH3
-OCH3 -OCH3 -OCH3
-OCH3 -OCH3 -OCH3
-Ph
p-Cl
H
>25
4r
>25
>25
4s
4t
>25
>25
5.13 ± 0.35
5.19 ± 0.42
Colchicine
2.3
The anticancer activity of synthesized compounds is via induction of apoptosis
Compounds 4a and 4p showed significant activity against the tested human cancer cell lines and
hence, these were selected for further mechanistic investigations. Compounds 4a, 4b, and 4f are
least substituted compounds while 4p is highly substituted compound. Hence, out of the least
substituted compound, 4a was selected and 4p was picked from highly substituted derivatives as
most potent compounds for cell line studies. In order to understand the mechanism of cell death
induced by compounds 4a and 4p, a cytofluorimetric analysis was performed at A549 cancer cell
7

Fig. 2. Apoptosis effect on human A549 cell line induced by compound A) control, B) colchicine (5 µM), C) 4a (5
µM) and D) 4p (5 µM). The lower left quadrants represent live cells, the lower right quadrants are for early/primary
apoptotic cells, upper right quadrants are for late/secondary apoptotic cells, while the upper left quadrants represent
cells damaged during the procedure.
lines using Muse™ Annexin V and Dead Cell kit to detect the phosphatidyl serine membrane
translocation. This is considered as a major hallmark of late stage apoptosis. A549 human cancer
cell lines were treated (24h) with 4a, 4p and colchicine as reference compound at 5 µM
concentrations. It has been found that compounds 4a and 4p caused apoptotic cell death upto
64.10% and 44.34% while colchicine showed cell death upto 49.72% as compared to the control
experiments (73.58% live cells). Hence, from these results it can be concluded that the primary
mode of cell death with the tested compounds is through apoptosis.
2.4
Compounds induce apoptosis via deregulation of antioxidant defense system
Mitochondrial changes including variations in the mitochondrial membrane potential ( m), are
the key events during drug-induced apoptosis [34]. If mitochondrial membrane potential is
altered, it leads to leakage of cytochrome-c into the cytoplasm which triggers the intrinsic
apoptotic pathway. Thus, to understand the mode of apoptosis, we analyzed mitochondrial
membrane
potential
(
m),
using
a
fluorescence
probe,
JC-1
(5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine) which has been shown to be
more specific for mitochondria than for other fluorescent dyes such as DiOC6(3) or rhodamine
123. In normal state, mitochondria with normal
JC-1 is aggregated (red/orange
m,
fluorescence), while in mitochondria with depolarization, JC-1 forms monomers (green
fluorescence). Thus, A549 cancer cells were treated with the synthesized compounds at the 5µM
8

and 25µM concentrations. Compounds 4a, 4c, 4e, 4f, 4m, 4o and colchicine caused
destabilization of the mitochondrial membrane at 5μM concentration (Fig. 3) whereas compound
4b, 4c, 4e, 4n and 4p were more active at 25 μM concentration. It has been observed that there
were significant alterations in the mitochondrial membrane potential upon drug treatment which
indicated that the intrinsic pathway of apoptosis (mitochondrial mediated) might be responsible
for the selective cancer cell apoptosis.
Major modulators of intrinsic pathway are mostly internal signaling cascades and free radicals
are known to be one of the most important modulator. Thus, we further hypothesized that the
alterations of antioxidant defense system of the cells might be responsible for the mitochondrial
membrane potential alterations. In order to test the hypothesis, we measured the impact of
selected compounds (4a and 4p) in vitro, in three enzymes namely super oxide dismutase (SOD),
catalase and glutathione reductase which are involved in the free radical detoxification.
Superoxide
Fig. 3. Treatment of A549 cancer cells with selected compounds and their JC1 analysis showing variations in the
mitochondrial membrane potential (
)
m
dismutase enzyme catalyzes the dismutation of superoxide radical (O2)2- into oxygen and
hydrogen peroxide. Thereafter, catalase converts the hydrogen peroxide into oxygen and water.
Thus, these enzymes are responsible for the antioxidant defense in almost all the cells. It has
been found that there was no significant change in SOD activity (Fig. 4a) while catalase and
9

glutathione reductase activities were reduced significantly upon treatment with compounds 4a
and 4p (Fig. 4b and 4c).
A
B
C
Fig. 4. Impact of 4a and 4p on a) super oxide dismutase (SOD) activity, b) catalase activity and c)
glutathione reductase activity
Glutathione plays crucial role in the DNA synthesis by maintaining reduced levels of
glutaredoxin or thioredoxin. In addition, a direct correlation has been observed between
glutathione levels and cell proliferation. Moreover, we performed in vitro DPPH assay and found
that the compounds have no antioxidant property of their own (supplementary information Fig.
S2, results not shown). Collectively these results indicated that the compounds are able to disturb
the cellular antioxidant defense system, which in-turn, lead to elevated intracellular ROS, and
triggers intrinsic apoptotic pathway.
2.5 Tubulin polymerization inhibition
10

This series of compounds are expected to mimic combretastatins and chalcones for their
antitubulin activity. Hence, tubulin binding efficiency of these compounds was evaluated by an
in vitro tubulin polymerization assay. Purified bovine tubulin was incubated with 10 μM 4a, 4j,
4o or 4p, and tubulin polymerization was recorded over time. Although all the four compounds
showed inhibition of tubulin polymerization as compared to control, compound 4a showed an
inhibition comparable to colchicine which was used as a positive control (Fig. 5).
1.12
1.1
1.08
1.06
1.04
1.02
1
0.98
0.96
0.94
Con
4a
4j
4o
4p
Colchicine
0.92
0
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
Time (min)
Fig. 5. In vitro studies of compounds 4a, 4j, 4o and 4p for their tubulin polymerization inhibition.
2.6 Competitive colchicine binding site assay
An in vitro competitive colchicine binding assay was performed in order to verify whether
the compounds bind to the colchicine binding site of the tubulin. It has been found that
addition of compound 4a showed reduced fluorescence of the tubulin-colchicine complex.
From these results it can be concluded that compound 4a displaces colchicine from the
tubulin-colchicine complex (Fig. 6). Thus, compound 4a was found to be competitive
inhibitor of colchicine.
11

*
Fig. 6: Binding of 4a, 4j, 4o and 4p on colchicine binding site of tubulin determined by competitive colchicine
binding assay.
2.7
Docking studies
From the literature search it was evident that most of the combretastatins and their derivatives
bind to the colchicine binding site of the tubulin and inhibit tubulin polymerization [35, 36]. The
compounds 4a and 4p displayed good anticancer activities. These compounds were docked into
the active site of the tubulin (PDB entry: 4O2B) to explore their interactions with the colchicine
binding site and to investigate their binding pattern (Fig. 7). The interactions between the ligands
and receptor site were scored using Glide (GLIDE 11.1 module of Schrödinger Suite). Validation
of the docking protocol was done by re-docking of colchicine into the crystal structure of tubulin.
In the validation results, it has been found that compounds 4a and 4p occupy the same binding
pocket as done by colchicine. The root-mean square deviation (RMSD) of docked colchicine to
the co-crystallized ligand was found to be 0.224 Å, displaying good reproducibility of the ligand
binding mode. Both the compounds fit well in the colchicine binding site and presence of the
pyrimidine ring showed extended interaction pattern.
Interestingly, docking poses of the 4p showed that trimethoxy substituents in ring A were
oriented towards α-tubulin while trimethoxy groups in colchicine were directed toward β-tubulin
(Fig. 7). Colchicine forms hydrogen bond through its carbonyl oxygen present in the ring with
Val-181 residue of α-tubulin while most of its structural part fit inside the hydrophobic pocket of
β-tubulin formed by Leu-242, Leu-248, Ala-250, Leu-252 and Leu-255 residues. On the other
12

hand, trimethoxy substituents of compound 4p were exposed to α-tubulin residues Ser-178,
Thr-179 and Ala-180 and –NH2 group present on the pyrimidine ring form hydrogen bonds with
Ala-317 and Lys-352 residues of β-tubulin. Phenyl ring with dichloro substitutions and
pyrimidine ring fit well in the hydrophobic pocket of colchicine binding site of tubulin formed
by Ile-318, Leu-242, Leu-248, Ala-250, Leu-252, Leu-255 and Ile-378 of β-tubulin. These
findings suggest that the synthesized pyrimidine bridged derivatives of combretastatins might act
as dual site inhibitors of α- and β-tubulin complexes and their cytotoxicity could be due to the
dual inhibition of α- and β-tubulin heterodimer.
Further these compounds were docked into the active site of the tubulin (PDB entry: 5LYJ) to
explore their interactions with the combretastatin binding site and to investigate their binding
pattern (Fig. 8). In the validation results, it has been found that compounds 4a and 4p bind to the
same binding pocket as done by combretastatin. Combretastatin forms hydrogen bond through its
hydroxy group present in the ring with Thr-179 residue of α-tubulin while trimethoxy ring of its
structural part fit inside the hydrophobic pocket of β-tubulin formed by Leu-242, Leu-248, Ala-
A
B
13

C
Fig. 7: A) overlapping of compound 4p (cyan) with colchicine (yellow) at colchicine binding site between α- and
β–tubulin, B) close view of the overlapped compound 4p (cyan) with colchicine (yellow) at the colchicine binding
site and C) interactions of 4p at the hydrophobic pocket of colchicine binding site (Yellow line shows hydrogen
bonding) with various amino acid residues of the α- and β–tubulin.
250, Leu-255, Ala-316, Ala-317, Ile-318 and Ile-378 residues. Similarly, in case of compound
4p, -NH2 group present at pyrimidine ring forms the hydrogen bond with Thr-179 residue of
α-tubulin while trimethoxy ring of its structural part fit inside the hydrophobic pocket of
β-tubulin formed by Leu-242, Leu-248, Ala-250, Leu-255, Ala-316, Ala-317, Ile-318 and
Ile-378 residues. It showed additional π-π interactions with Lys-352 residue of β-tubulin due to
presence of pyrimidine ring. Thus, it can be concluded that the introduction of pyrimidine ring as
C3 linker between two phenyl rings improve the interaction at the combretastatin binding site.
14

A
B
C
Fig. 8: A) overlapping of compound 4p (cyan) with combretastatin (yellow) at combretastatin binding site between
α- and β–tubulin, B) close view of the overlapped compound 4p (cyan) with combretastatin (yellow) at the
colchicine binding site and C) interactions of 4p at the hydrophobic pocket of combretastatin binding site (Yellow
line shows hydrogen bonding) with various amino acid residues of the α- and β–tubulin
2.8
ADME studies
ADME studies were performed to investigate the physicochemical properties of the synthesized
compounds. In order to analyze the drug like characteristics of the compounds, these were
evaluated for different parameters as described by Lipinski’s rule of five using qikprop
application of Schrodinger. The optimum Log P (<5) value is an important physiochemical
property indicating the lipophilicity and the ability of the molecule to cross various biological
membranes. The compounds 4a and 4p displayed lipophilicity within the range of 4.46 to 4.62.
Number of hydrogen bond donors and hydrogen bond acceptors also lies within the range and
molecular weight of the synthesized compounds were also less than 500 (Table 2). The
compound with low dipole moment was found more efficiently binding to the colchicine binding
site and have higher cytotoxic potential. Compound 4p also showed less number of metabolic
reactions (3) as compared to the standard tubulin inhibitors colchicine (5) and combretastatin (5).
Most importantly, 4a and 4p showed 100 % human oral absorption. Thus, it can be concluded
15

that the compounds with high lipophilicity and low dipole moment exhibit good drug like
characteristics.
Table 2: Drug like characteristics of compounds 4a and 4p as determined by qikprop application of Schrodinger
Name
Mol.
Log P HB
HB
% human oral dipole
#metb
Wt.
donor acceptor absorption
246.311 4.465
406.268 4.629
399.443 2.704
0
2
1
1
2
5
8
4
100
1.95
1.48
6.91
4.02
1
3
5
5
4a
100
4p
Colchicine
95.43
100
Combretastatin 316.353 4.045
2.9 SAR studies
In the current study, different optionally substituted pyrimidine bridged combretastatin
derivatives have been synthesized and evaluated. Acetamidine, formadine and guanidine were
used for the synthesis of pyrimidine ring and to keep the phenyl rings in cis locked confirmation.
Most of the compounds displayed anti-cancer activities against MCF7 and A549 cancer cell lines
in low micromolar range. In general, it has been observed that unsubstituted or monosubstituted
compounds (4a, 4b, 4e, 4f and 4m) were more active as compared to the di and tri substituted
derivatives (Table 1). Most of the trimethoxy substituted derivatives (4d, 4h, 4i, 4r, 4s and 4t)
were found 3 to 5 fold less active except compound 4n and 4p. Compound 4n with trimethoxy
substitution on ring A and 4-methoxy substitution on ring B showed IC50 values of 4.85 µM and
5.91 µM against MCF7 and A549 cancer cell lines, respectively. However, compound 4p with a
2,4-dichloro substitution on ring B was found most potent in the series with IC50 values of 4.63
µM and 3.71 µM against MCF7 and A549 cancer cell lines, respectively. Similarly, compound
4a was also found effective against both the cancer cell lines with IC50 values of 4.67 µM
(MCF7) and 3.38 µM (A549). There was no significant effect on the activity when CH3, H and
NH2 groups were interchanged on the pyrimidine ring (4b, 4e and 4m). Replacement of electron
releasing methyl group in ring B (4i) with electron withdrawing chloro group (4g) increased the
activity by almost two-fold. In contrast, compound with a para methyl substitution on ring B (4j)
was 5-fold more active than corresponding para methoxy derivative (4k). Replacement of ring B
by a naphthyl ring (4r) decreases the activity, might be due to increase in the lipophilicity
16

(higher Log P) of the compound. Unsubstituted derivative with no substituent on any of the three
rings also showed good anticancer activity with IC50 values of 4.88 µM and 3.17 µM against
MCF7 and A549 cancer cell lines, respectively.
3.
Conclusion
Twenty pyrimidine bridged combretastatin derivatives (4a-4t) have been synthesized and
evaluated for anticancer activities against MCF7 and A549 cancer cell lines. Most of the
compounds displayed anticancer activities in low micromolar range and some of the compounds
in this series were found even better as compared to the reference colchicine. Compounds 4a and
4p were found most active in this series and hence various mechanistic studies were performed
on these compounds. It has been observed that compounds 4a and 4p inhibit the cell proliferation
of cancer cells by inducing apoptosis. JC-1 analysis showed this series of compounds alter the
mitochondrial membrane potential which leads to leakage of cytochrome-c into the cytoplasm
triggering intrinsic apoptotic pathway. Further investigations on the antioxidant defense system
of the cells revealed that treatment of compounds 4a and 4p did not produce any change in SOD
activity however, catalase and glutathione reductase activities were reduced significantly. In the
competitive tubulin binding assay, it has been found that compound 4a binds to the tubulin at the
colchicine binding site. The docking studies of compounds 4a and 4p showed that these
compounds fit well in the colchicine binding site of the tubulin. Further, the tested compounds
inhibited tubulin polymerization and compound 4a showed significant inhibition potential
comparable to colchicine. Collectively, it can be concluded that the synthesized compounds
might be able to deregulate multiple pathways to induce cancer cell specific apoptosis
(colchicine like action as well as modulation of intrinsic apoptotic pathway).
4.
Experimental
4.1 General
All the reagents were of AR/GR quality and purchased from Sigma-Aldrich, Loba-Chemie Pvt.
Ltd., S.D. Fine Chemicals, Spectrochem, Sisco Research Laboratory and Avra Synthesis Ltd.
and were used without further purification. The progress of the reaction was monitored by TLC
using petroleum ether/ethyl acetate and chloroform/methanol as the mobile phase on pre-coated
Merck TLC plates & glass plates made of F254 UV grade silica in JSGW UV/fluorescent
17

analysis cabinet and/or iodine chamber. Melting points were recorded on the Stuart melting point
apparatus (SMP-30) with open glass capillary tubed. Infrared (IR) spectra of compounds were
1
recorded with KBr on a Bruker FT-IR spectrophotometer. H and 13C Nuclear magnetic
resonance (NMR) spectra was obtained in CDCl3/d6-DMSO on a Bruker Avance II (400 MHz)
NMR spectrometer using TMS (δ = 0) as internal standard at Panjab University, Chandigarh.
Mass spectra were recorded in Shimazdu GC-MS (ESI), Central University of Punjab, Bathinda,
Punjab, India.
4.2 General Procedure for synthesis of 3
The mixture of benzaldehyde 1 (2g), acetophenone 2 (1eq), sodium hydroxide (10% in water,
5eq) in methanol (15 ml) was stirred at room temperature until completion of the reaction. The
product formation was monitored by TLC. After complete product formation, the reaction
mixture was evaporated at rota-evaporator. Water was added to the residue and the precipitated
product was filtered. dried and recrystallized in the methanol.
4.3 General Procedure for synthesis of 4a-4t
The mixture of the chalcone 3 (0.2g), amidine (1.2eq), and sodium carbonate (2.4eq) in
acetonitrile (10 ml) was refluxed for overnight at 80 °C. The time required for the product
formation varies from different products (24-48hrs). The progress of reaction was monitored by
the TLC in 30% ethylacetate/petroleum ether. After the completion of the reaction, the reaction
mixture was dried on the rotavapor. To the residue water was added and crude product was
extracted with with ethyl acetate. The organic portion was separated and washed with water,
brine, dried over anhydrous sodium sulfate and rota-evaporated. Compounds were purified
through column chromatography using chloroform and methanol as solvent. Final product
formation was confirmed by EI-MS, CHN and HRMS.
1
2-methyl-4,6-diphenylpyrimidine (4a): yield 85%, light yellow solid, H NMR (400MHz,
13
CDCl3, TMS = 0) 2.87 (3H, s), 7.51-7.53 (6H, m), 7.89 (1H, s), 8.11-8.14 (4H, m), C NMR:
(100 MHz, CDCl3, TMS= 0) δ: 168.68, 164.99, 137.60, 130.76, 129.066, 127.36, 110.24, 26.64.
HRMS: m/z [M+H]+ for C17H14N2, calculated 247.1235; observed: 247.1215.
1
4-(4-methoxyphenyl)-2-methyl-6-phenylpyrimidine (4b):[37] yield 78%, white solid, H
NMR (400MHz, CDCl3, TMS = 0) 2.84 (3H, s), 3.88 (3H, s), 7.02 (2H, d, J=8 Hz ), 7.50-7.52
18

13
(3H, m), 7.82 (1H, s), 8.10-8.12 (4H, m), C NMR: (100 MHz, CDCl3, TMS = 0) δ: 168.51,
164.74, 164.39, 161.90, 137.79, 130.62, 129.94, 129.02, 128.85, 128.68, 127.33, 114.37, 109.34,
55.54, 26.64. MS-EI: m/z [M]+ for C18H16N2O, calculated 276.13; observed: 276, CHN:
calculated C- 78.24, H-5.84, N-10.14, observed C- 78.98, H-5.68, N-10.49.
1
4-(3,4-dimethoxyphenyl)-2-methyl-6-phenylpyrimidine (4c): yield 73%, cream solid, H
NMR (400MHz, CDCl3, TMS = 0) 2.85 (3H, s), 3.95 (3H, s), 4.02 (3H, s), 6.97 (1H, d, J= 8Hz
), 7.49-7.53 (3H, m), 7.67-7.69 (1H, m), 7.77 (1H, d, J= 1.96 Hz ), 7.82 (1H, s), 8.09-8.12 (2H,
13
m), C NMR: (100 MHz, CDCl3, TMS = 0) δ: 168.42, 164.67, 164.31, 151.36, 149.37, 137.61,
130.55, 130.19, 128.93, 127.25, 120.28, 111.02, 109.98, 109.40, 56.08, 56.03, 26.53. HRMS:
m/z [M+H]+ for C19H18N2O2, calculated 307.1447; observed: 307.1438.
4-(4-methoxyphenyl)-2-methyl-6-(3,4,5-trimethoxyphenyl)pyrimidine (4d): yield 72%, light
brown solid, 1H NMR (400MHz, CDCl3, TMS = 0) 2.83 (3H, s), 3.88 (3H, s), 3.92 (3H, s), 3.99
(6H, s), 7.04 (2H, d, J= 7 Hz ), 7.33 (2H, s), 7.73 (1H, s), 8.11 (2H, d, J= 7 Hz) 13C NMR: (100
MHz, CDCl3, TMS = 0) δ: 168.33, 164.32, 164.31, 161.84, 153.61, 140.31, 133.23, 129.85,
128.77, 114.29, 109.02, 104.50, 61.01, 56.36, 55.45, 26.51. HRMS: m/z [M+H]+ for
C21H22N2O2, calculated 367.1658; observed: 366.1644.
4-(4-methoxyphenyl)-6-phenylpyrimidine (4e): yield 74%, off white solid, 1H NMR
(400MHz, CDCl3, TMS = 0) 3.88 (3H, s), 7.02 (2H, dd), 7.51-7.54 (3H, m), 8.03 (1H, s),
8.10-8.14 (4H, m), 9.26 (1H, s) 13C NMR: (100 MHz, CDCl3, TMS = 0) δ: 164.45, 164.18,
162.06, 159.11, 137.21, 130.84, 129.36, 129.02, 128.74, 127.18, 114.39, 111.98, 55.47. MS-EI:
m/z [M]+ for C17H14N2O, calculated 262.11; observed: 262, CHN: calculated C- 77.84, H-5.38,
N-10.68, observed C- 77.37, H-5.29, N-10.87.
1
4,6-diphenylpyrimidine (4f):[38, 39] yield 75%, off white solid, H NMR (400MHz, CDCl3,
TMS = 0) 7.50-7.56 (6H, m), 8.09 (1H, s), 8.11-8.16 (4H, m), 9.31 (1H, s), 13C NMR: (100
MHz, CDCl3, TMS = 0) δ: 164.73, 159.22, 137.03, 130.99, 129.07, 127.22, 112.89. HRMS: m/z
[M+H]+ for C16H12N2, calculated 233.1079; observed: 233.1067.
4-(4-chlorophenyl)-2-methyl-6-(3,4,5-trimethoxyphenyl)pyrimidine (4g): yield 82%, off
yellow solid, 1H NMR (400MHz, CDCl3, TMS = 0) 2.77 (3H, s), 3.85 (3H, s), 3.92 (6H, s), 7.27
(2H, s), 7.43 (2H, d, J= 8 Hz ), 7.69 (1H, s), 8.01 (2H, d, J= 8 Hz ) 13C NMR: (100 MHz,
19

CDCl3, TMS = 0) δ: 167.57, 163.70, 162.57, 152.64, 139.60, 135.88, 134.89, 131.79, 128.15,
127.54, 108.49, 103.60, 59.98, 55.37, 28.68, 25.43. MS-EI: m/z [M]+ for C20H19ClN2O3,
calculated 370.11; observed: 370, CHN: calculated C- 64.78, H-5.16, N-7.55, observed C-
64.03, H-5.05, N-7.47.
2-methyl-4-phenyl-6-(3,4,5-trimethoxyphenyl)pyrimidine (4h): yield 84%, light brown
1
solid, H NMR (400MHz, CDCl3, TMS = 0) 2.86 (3H, s), 3.92 (3H, s), 3.99 (6H, s), 7.36 (2H,
13
s), 7.51-7.53 (3H, m), 7.80 (1H, s), 8.10-8.13 (2H, m) C NMR: (100 MHz, CDCl3, TMS = 0)
δ: 168.50, 164.92, 164.51, 153.65, 140.50, 137.55, 133.00, 130.66, 128.96, 127.28, 109.88,
104.59, 61.00, 56.38, 26.50. MS-EI: m/z [M]+ for C20H20N2O3, calculated 336.15; observed: 336,
CHN: calculated C- 71.41, H-5.99, N-8.33, observed C- 72.00, H-6.27, N-8.33.
2-methyl-4-(p-tolyl)-6-(3,4,5-trimethoxyphenyl)pyrimidine (4i): yield 81%, light yellow
1
solid, H NMR (400MHz, CDCl3, TMS = 0) 2.43 (3H, s), 2.84 (3H, s), 3.92 (3H, s), 3.99 (6H,
s), 7.26-7.34 (4H, m), 7.77 (1H, s), 8.01 (2H, d, J= 8 Hz ) 13C NMR: (100 MHz, CDCl3, TMS =
0) δ: 168.41, 164.82, 164.40, 153.63, 141.03, 140.42, 134.68, 133.14, 129.67, 127.17, 109.53,
104.58, 60.99, 56.38, 26.50, 21.46. MS-EI: m/z [M]+ for C21H22N2O3, calculated 350.16;
observed: 350, CHN: calculated C- 71.98, H-6.33, N-7.99, observed C- 71.41, H-6.52, N-7.29.
4-(3,4-dimethoxyphenyl)-2-methyl-6-(p-tolyl)pyrimidine (4j): yield 79%, light yellow solid,
1H NMR (400MHz, CDCl3, TMS = 0) 2.42 (3H, s), 2.83 (3H, s), 3.95 (3H, s), 4.02 (3H, s), 6.98
(1H, d, J= 8 Hz ), 7.32 (2H, d, J= 8 Hz ), 7.66- 7.68 (1H, m), 7.76 (1H, s), 7.79 (1H, s), 8.02 (2H,
d, J = 8 Hz ) 13C NMR: (100 MHz, CDCl3, TMS = 0) δ: 168.33, 164.58, 164.19, 151.31, 149.37,
140.87, 134.81, 130.34, 129.64, 127.13, 120.24, 111.04, 110.04, 109.02, 56.08, 56.02, 26.53,
21.45. MS-EI: m/z [M]+ for C20H20N2O2, calculated 320.15; observed: 320, CHN: calculated C-
74.98, H-6.29, N- 8.74, observed C- 75.30, H-6.57, N-8.31.
4-(3,4-dimethoxyphenyl)-6-(4-methoxyphenyl)-2-methylpyrimidine (4k):[37] yield 78%, off
yellow solid, 1H NMR (400MHz, CDCl3, TMS = 0) 2.82 (3H, s), 3.86 (3H, s), 3.96 (3H, s), 4.02
(3H, s), 6.99 (1H, d, J= 8 Hz ), 7.03 (2H, d, J= 8 Hz ), 7.65-7.68 (1H, m), 7.76 (2H, s), 8.09-8.11
(2H, m) 13C NMR: (100 MHz, CDCl3, TMS = 0) δ: 168.27, 164.10, 161.75, 151.27, 149.36,
130.74, 130.42, 128.72, 120.21, 114.27, 113.80, 111.03, 110.03, 108.52, 56.08, 56.03, 55.44,
26.52. HRMS: m/z [M+H]+ for C20H20N2O3, calculated 337.1552; observed: 337.1534.
20

1
4, 6-diphenylpyrimidin-2-amine (4l):[40] yield 53%, white solid, H NMR (400MHz, CDCl3,
TMS = 0) 5.47 (2H, s), 7.45 (1H, s), 7.457.51 (6H, m), 8.04-8.06 (4H, m), 13C NMR: (100 MHz,
CDCl3, TMS = 0) δ: 166.36, 163.79, 137.85, 130.55, 128.88, 127.23, 104.42. MS-EI: m/z [M]+
for C16H13N3, calculated 247.11; observed: 247.
1
4-(4-methoxyphenyl)-6-phenylpyrimidin-2-amine (4m):[40] yield 55%, off white solid, H
NMR (400MHz, CDCl3, TMS = 0) 3.87 (3H, s), 5.23 (2H, s), 7.01 (2H, d, J = 9.2 Hz), 7.41 (1H,
s), 7.487.49 (3H, m), 8.02-8.05 (4H, m), 13C NMR: (100 MHz, CDCl3, TMS = 0) δ: 166.11,
165.77, 163.60, 161.76, 138.03, 130.40, 128.88, 128.74, 127.21, 114.22, 103.69, 55.54. MS-EI:
m/z [M]+ for C17H15N3O, calculated 277.12; observed: 277.
4-(4-methoxyphenyl)-6-(3, 4, 5-trimethoxyphenyl) pyrimidin-2-amine (4n):[41] yield 77%,
light yellow solid, 1H NMR (400MHz, CDCl3, TMS = 0) 3.87 (3H, s), 3.91 (3H, s), 3.97 (6H, s),
5.27(2H, s), 7.02 (2H, d, J = 6.96 Hz), 7.28 (2H, s), 7.33 (1H, s), 8.04 (2H, d, J = 6.84 Hz), 13C
NMR: (100 MHz, CDCl3, TMS = 0) δ: 165.70, 165.61, 163.47, 161.69, 153.45, 140.13, 133.42,
130.07, 128.64, 114.12, 104.36, 103.29, 60.99, 56.32, 55.4. MS-EI: m/z [M]+ for C20H21N3O4,
calculated 367.15; observed: 367.
4-(4-chlorophenyl)-6-(3, 4-dimethoxyphenyl) pyrimidin-2-amine (4o): yield 64%, light
brown solid, 1H NMR (400MHz, CDCl3, TMS = 0) 3.95 (3H, s), 4.00 (3H, s), 5.22 (2H, s), 6.96
(1H, d, J = 8.44 Hz), 7.37 (2H, s), 7.47 (2H, d, J = 6.72Hz), 7.63 (1H, dd, J1 = 8.4Hz, J2 =
13
2.04Hz), 7.70 (1H, s), 8.00 (2H, d, J = 6.76 Hz), C NMR: (100 MHz, CDCl3, TMS = 0) δ:
165.88, 164.67, 163.51, 151.33, 149.25, 136.51, 136.27, 130.23, 128.9, 128.41, 120.18, 110.90,
109.93, 103.29, 56.05, 56.0. MS-EI: m/z [M]+ for C18H16ClN3O2, calculated 341.09; observed:
341.
4-(2, 4-dichlorophenyl)-6-(3, 4, 5-trimethoxyphenyl) pyrimidin-2-amine (4p): yield 70%, off
1
yellow solid, H NMR (400MHz, CDCl3, TMS = 0) 3.91 (3H, s), 3.95 (6H, s), 5.32 (2H, s),
7.26-7.28 (1H, m), 7.39 (1H, dd, J1 = 8.24Hz, J2 =1.96 Hz), 7.52 (1H, d, J = 2.04 Hz), 7.58 (1H,
d, J = 8.32 Hz), 13C NMR: (100 MHz, CDCl3, TMS = 0) δ: 165.29, 164.84, 163.29, 153.49,
140.46, 136.23, 135.76, 132.94, 132.63, 131.80, 130.14, 127.50, 108.20, 104.42, 61.00, 56.28
CHN: calculated C- 56.17, H-4.22, N-10.34, observed C- 56.18, H-4.57, N-9.34. MS-EI: m/z
[M]+ for C19H17Cl2N3O3, calculated 405.06; observed: 405.
21

4-(p-tolyl)-6-(3, 4, 5-trimethoxyphenyl) pyrimidin-2-amine (4q): yield 68%, off white solid,
1H NMR (400MHz, CDCl3, TMS = 0) 2.42 (3H, s), 3.91 (3H, s), 3.97 (6H, s), 5.30 (2H, s),
7.26-7.31 (4H, m), 7.35 (1H, s), 7.96 (2H, d, J = 8.16 Hz), 13C NMR: (100 MHz, CDCl3, TMS =
0) δ: 166.22, 165.73, 163.52, 153.46, 140.85, 140.16, 134.89, 133.36, 129.53, 127.05, 104.37,
103.80, 61.0, 56.32, 21.4. MS-EI: m/z [M]+ for C20H21N3O3, calculated 351.16; observed: 351.
4-(naphthalen-2-yl)-6-(3, 4, 5-trimethoxyphenyl) pyrimidin-2-amine (4r): yield 72%, white
1
solid, H NMR (400MHz, CDCl3, TMS = 0) 3.91 (3H, s), 3.94 (6H, s), 5.43 (2H, s), 7.25 (1H,
s), 7.30 (2H, s), 7.49-7.53 (2H, m), 7.53-7.59 (1H, m), 7.65-7.67 (1H, m), 7.91-7.96 (2H, m),
8.15-8-8.18 (1H, m), 13C NMR: (100 MHz, CDCl3, TMS = 0) δ: 168.48, 165.37, 163.27, 153.50,
140.36, 136.95, 133.88, 132.85, 130.61, 129.77, 128.50, 126.90, 126.80, 126.21, 125.39, 125.27,
108.61, 104.39, 61.00, 56.29, 50.73. MS-EI: m/z [M]+ for C23H21N3O3, calculated 387.16;
observed: 387, CHN: calculated C- 71.30, H-5.46, N-10.85, observed C- 71.39, H-5.27,
N-10.56.
4-(4-chlorophenyl)-6-(3, 4, 5-trimethoxyphenyl) pyrimidin-2-amine (4s):[41] yield 67%, off
yellow solid, 1H NMR (400MHz, CDCl3, TMS = 0) 3.80 (3H, s), 3.94 (6H, s), 6.43 (2H, s), 7.46
(2H, s), 7.51 (2H, d, J = 8.56 Hz), 7.60 (1H, s), 8.22 (1H, d, J = 8.6 Hz), 13C NMR: (100 MHz,
CDCl3, TMS = 0) δ: 164.77, 163.59, 163.54, 152.84, 139.51, 136.00, 135.33, 132.67, 128.50,
128.31, 104.24, 101.86, 60.06, 55.90. MS-EI: m/z [M]+ for C19H18ClN3O3, calculated 371.10;
observed: 371.
4-phenyl-6-(3, 4, 5-trimethoxyphenyl) pyrimidin-2-amine (4t): yield 58%, yellowish brown
1
solid, H NMR (400MHz, CDCl3, TMS = 0) δ 3.91 (3H, s), 3.97 (6H, s), 5.31(2H, s), 7.30 (2H,
13
s), 7.38 (1H, s), 7.49-7.51 (3H, m), 8.03-8.05 (2H, m), C NMR: (100 MHz, CDCl3, TMS = 0)
δ: 166.33, 165.86, 163.54, 153.48, 140.26, 137.77, 133.23, 130.51, 128.81, 127.51, 104.40,
104.10, 61.00, 56.33. MS-EI: m/z [M]+ for C19H19N3O3, calculated 337.14; observed: 337, CHN:
calculated C- 67.64, H-5.68, N-12.46, observed C- 67.13, H-5.41, N-12.66.
4.4 Biological Studies
4.4.1 Cytotoxic studies
22

MTT is a colorimetric assay used for the measurement of cell proliferation. The tetrazolium
yellow compound MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyl tetrazolium bromide) is
reduced to an insoluble purple coloured formazan product by mitochondrial reductase or
succinate dehydrogenase in metabolically active cells only. When formazan passes to the
mitochondria it gets solubilized with DMSO and measured spectrophotometrically.
To carry out the MTT assay, cell lines (MCF7) were grown in DMEM medium with 5ml serum
containing medium. Upon the 70-80% growth, the sub-culturing of cells was done. Cells were
counted using automated cell counter. Nearly 8,000-10,000 cells were seeded in each well of the
96 well plates. The plate was incubated at 37 ˚C with 5% CO2 for 24 h followed by serum
starvation for 8hrs for synchronization and replenishing with complete media. The treatment was
given to the cells in triplicate concentrations of 1 μM, 5 μM and 25 μM and incubated for 48 h.
MTT solution (5 mg/10mL) was added after removing the media from each well and incubated
in the dark for 4 h. At the end of 4 h, the MTT solution was removed from each well and the
intracellular precipitate was dissolved in DMSO solution and the absorbance of the violet color
formed as consequence of DMSO addition, is read spectrometrically at 570 nm and expressed as
% inhibition (Mean ± S.D).
4.4.2 Free radical scavenging assay (DPPH assay)
Tubes were filled with 3ml methanol containing extracts inside it and then DPPH was added to
test tubes (1 ml) in dark and kept for incubation for 30 minutes. The absorbance was measured at
517nm on UV-Spectrophotometer.
4.4.3 Evaluation of antioxidant enzyme inhibition
4.4.3.1 Superoxide Dismutase (SOD) inhibition assay
1.5 ml of 0.1 M Tris HCl buffer was added to cuvette followed by 0.5 ml of 6 mM EDTA
followed by 1 ml of 6 mM Pyrogallol solution and 0.1ml of sample extract was added and then
mixed properly and then absorbance was measured at 420nm after intervals of 30 sec on
UV-spectrophotometer.
4.4.3.2 Catalase inhibition assay
23

In 900 µL of 50 mM sodium phosphate buffer, 0.1 ml of test compound and hydrogen peroxide
was added. Decrease in absorbance was measured after intervals of 30 sec for 3 min at 240 nm.
4.4.3.3 Glutathione reductase inhibition assay
Using UV-spectrophotometer, in a cuvette 0.945 ml PB200 followed by addition of 5 µl DTNB
and 10 µL sample then NADPH was added. Rapidly GR enzyme was added and absorbance at
412 nm was recorded after intervals of 30 sec. and then GSSG was added and again recorded in
intervals of 30 sec till 3 min.
4.4.4 JC1 analysis
In 96-well plate cancer cells were seeded with appropriate treatment. Then, after 24 h cells were
washed with PBS and incubated with JC 1 for 30 min. Then, wash the cells to remove extra dye
with 1x PBS, and then fluorescence was measured using Synergy H1 microplate reader (green
monomers with Ex 485 nm/Em 535 nm and red J aggregate with Ex 560 nm/Em 595 nm).
4.4.5 Apoptosis analysis
Apoptosis utilising Muse™ Annexin V and Dead Cell kit (Catalogue No. 15-0180) was
performed using Muse™ cell analyser. Cellular samples (A549 previously treated with
compound Cochicine, 4a and 4p for 24 h) were prepared by the predefined protocol of Muse™
Annexin V and Dead Cell kit. The total cells and media were centrifuged at 1200 rpm for 5 min
and washed with 1x PBS. 50 µL of kit reagent was added and incubated for 30 min at room
temperature in dark before the analysis.
4.4.6 Tubulin polymerization assay
Polymerization of the bovine tubulin was measured according to Beyer et al. [42]. The indicated
drugs were added in triplicate into the wells of a 96 well plate. Bovine tubulin (1.8 mg/mL;
Sigma) was added to ice-cold polymerization buffer (PEM: 80 mM PIPES, 0.5 mM EGTA, 2
mM MgCl2, 10% glycerol, and 1mM GTP) and centrifuged at maximum speed in a
microcentrifuge for 5 minutes at 4 °C. Supernatant (100 uL/well) was immediately added to
wells of the 96-well plate containing the drugs or dimethyl sulfoxide control in PEM buffer.
After addition of tubulin, the plate was immediately placed in a spectrophotometer, which was
maintained at 37 °C, and the absorbance was measured at 340 nm every 5 min for 2.5 hours.
24

4.4.7 Competitive tubulin binding assay
4a, 4j, 4o and 4p (10 μM each) were co-incubated with 3 μM colchicine in tubulin
polymerization buffer containing 3 μM tubulin at 37 °C for 60 min. After incubation, the
fluorescence of tubulin- colchicine complex was measured by using Tecan multimode reader at
excitation wavelength of 380 nm and emission wavelength of 435 nm. The raw fluorescence
values were normalized by setting the fluorescence of 3 μM tubulin with 3 μM colchicine to
100%.
4.5 Docking Studies
To determine the mode of interaction between synthesized ligands and tubulin docking studies
were performed. 2D structure of chemicals was prepared in ChemBioDraw ultra 8.0 software
and converted to 3D structures in ligand preparation application of Maestro 11.1 (Schrödinger)
software. The X-ray crystal structure of tubulin (PDB ID: 4O2B) co-crystallized with colchicine
[43] and co-crystallized with Combretastatin A4 (PDB ID: 5LYJ) [40] was downloaded from the
Protein Data Bank (www.rcsb.org). Further modification for protein preparations such as polar
hydrogen addition, addition of side chains, and removal of water molecules was performed by
Maestro 11.1 software. All Synthesized ligands along with co-crystallized colchicine and
combretastatin were docked into the binding site of tubulin using Maestro 11.1 software. All
atoms within a 5 Å around the co-crystallized ligand in crystal coordinates of tubulin was chosen
as binding site. For each compound, the top-score docking poses were chosen for final
ligand-target interaction analysis employing XP interaction visualizer of Maestro 11.1 software.
Validation of docking procedure was first evaluated by re-docking of co-crystalized ligands into
the tubulin binding sites. Qikprop application of Schrodinger suit was used to determine the drug
like and ADME properties of the compounds.
5. Acknowledgement
VK and SS are thankful to the SERB-Department of Science and Technology, New Delhi for the
extramural financial support grant number EMR/2015/002339. The authors declare no potential
conflicts of interest.
References:
25

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