Anti-proliferative potential of triphenyl substituted pyrimidines against MDA-MB-231, HCT-116 and HT-29 cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2020.127468

A series of triphenyl substituted pyrimidines as analogous of colchicine and combretastatin A-4 was synthesized and evaluated for the antiproliferative potential. The compounds were screened against MDA-MB-231, HCT-116 and HT-29 cell lines using MTT assay. Most of the compounds displayed antiproliferative activity in low to sub micro molar concentration. Amongst the synthesized derivatives, compounds HK-2, HK-10 and HK-13 were found to be effective against all the three cancer cell lines. HK-2 exhibited IC₅₀ values of 3.39 μM, 4.78 μM and 4.23 μM, HK-10 showed IC₅₀ values of 0.81 μM, 5.89 μM, 4.96 μM and HK-13 showed IC₅₀ values 3.24 μM, 4.93 μM and 4.73 μM against MDA-MB-231, HCT-116 and HT-29 cancer cell lines, respectively. HK-10 was found to be the most potent compound in the series with IC₅₀ values of 0.81 μM against MDA-MB-231. In the cell cycle analysis, HK-2 and HK-10 showed cell arrest at G2/M phase of the cell cycle while HK-13 inhibited cell growth at the G1/G0 phase. All the three compounds showed cell death induced through apoptosis. In the docking studies, HK-2, HK-10 and HK-13 were found to fit well in the colchicine binding site of the tubulin. Some of the compounds in the current series were found to be promising against all the three cancer cell lines and may act as potent leads for further development.

Full text of DOI:10.1016/j.bmcl.2020.127468

Journal Pre-proofs
Anti-proliferative Potential of Triphenyl Substituted Pyrimidines Against
MDA-MB-231, HCT-116 and HT-29 cancer cell lines
Ashish Ranjan Dwivedi, Vijay Kumar, Harmeet Kaur, Naveen Kumar, Ravi
Prakash Yadav, P Ramarao, Somesh Baranwal, Vinod Kumar
PII:
S0960-894X(20)30579-5
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127468
BMCL 127468
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
15 May 2020
26 July 2020
3 August 2020
Please cite this article as: Ranjan Dwivedi, A., Kumar, V., Kaur, H., Kumar, N., Prakash Yadav, R., Ramarao, P.,
Baranwal, S., Kumar, V., Anti-proliferative Potential of Triphenyl Substituted Pyrimidines Against MDA-
MB-231, HCT-116 and HT-29 cancer cell lines, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://
doi.org/10.1016/j.bmcl.2020.127468
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Anti-proliferative Potential of Triphenyl Substituted Pyrimidines Against MDA-MB-231,
HCT-116 and HT-29 cancer cell lines
Ashish Ranjan Dwivedia, Vijay Kumara, Harmeet Kaura, Naveen Kumara, Ravi Prakash
Yadavb, P Ramaraoa, Somesh Baranwalb* and Vinod Kumara,c*
aDepartment of Pharmaceutical Sciences and Natural Products, Central University of Punjab,
Bathinda, Punjab, India-151001.
bDepartment of Microbiology, School of Basic and Applied Sciences, Central University of
Punjab, Bathinda, Punjab, India-151001.
cLaboratory of Organic and Medicinal Chemistry, Department of Chemistry, Central
University of Punjab, Bathinda, Punjab, India-151001.
Corresponding Author:
*Dr Vinod Kumar
Laboratory of Organic and Medicinal Chemistry, Department of Chemistry, Central University
of Punjab, Bathinda, Punjab, India-151001.
E-mail: vpathania18@gmail.com; vinod.kumar@cup.edu.in
Phone No. +911642864214
Co-corresponding author:
Dr. Somesh Baranwal
Department of Microbiology, School of Basic and Applied Sciences, Central University of
Punjab, Bathinda, Punjab, India
E-mail: someshbaranwal@gmail.com; somesh.baranwal@cup.edu.in
Conflict of Interest Statement: Authors declare no potential conflict of interest.

Abstract
A series of triphenyl substituted pyrimidines as analogous of colchicine and combretastatin
A-4 was synthesized and evaluated for the antiproliferative potential. The compounds were
screened against MDA-MB-231, HCT-116 and HT-29 cell lines using MTT assay. Most of the
compounds displayed antiproliferative activity in low to sub micro molar concentration.
Amongst the synthesized derivatives, compounds HK-2, HK-10 and HK-13 were found to be
effective against all the three cancer cell lines. HK-2 exhibited IC50 values of 3.39 µM, 4.78
µM and 4.23 µM, HK-10 showed IC50 values of 0.81 µM, 5.89 µM, 4.96 µM and HK-13
showed IC50 values 3.24 µM, 4.93 µM and 4.73 µM against MDA-MB-231, HCT-116 and HT-
29 cancer cell lines, respectively. HK-10 was found to be the most potent compound in the
series with IC50 values of 0.81 µM against MDA-MB-231. In the cell cycle analysis, HK-2 and
HK-10 showed cell arrest at G2/M phase of the cell cycle while HK-13 inhibited cell growth
at the G1/G0 phase. All the three compounds showed cell death induced through apoptosis.
In the docking studies, HK-2, HK-10 and HK-13 were found to fit well in the colchicine
binding site of the tubulin. Some of the compounds in the current series were found to be
promising against all the three cancer cell lines and may act as potent leads for further
development.
Keywords: Antiproliferative agents, Pyrimidines, Colchicine binding site, Tubulin
polymerization inhibitors, Combretastatin

Cancer is one the leading cause of death all over the world. Despite of continuous
advancements in the treatment and prevention strategies, the successful treatment of cancer, in
particular metastases, still remains a big challenge.1 Discovery of new and safer anticancer
agents that are more potent and effective with improved cytotoxicity in cancer cells would
improve the management of cancer.2 In 2018, the disease led to nearly 700,000 deaths in India.3
Cancer is a multifactorial disease and a number of cellular pathways are associated with the
progression of the disease. Major problems associated with the cancer treatment are toxicity to
the normal cells and development of resistance to therapy.4 More safe and effective anticancer
ligands that target EGFR (Epidermal Growth Factor Receptors), VEGF (Vascular Endothelial
Growth Factor), PARP (poly ADP ribose polymerase), telomerase, and tubulin are being
developed.5-8
Cancer cell has the ability to evade apoptosis, a programmed cell death, and microtubule
targeting drugs may trigger apoptosis by disrupting the tubulin-microtubule equilibrium.9
Microtubules are composed of α,ß-tubulin heterodimers that play crucial role in cell division,
intracellular trafficking, cell migration and angiogenesis.10 These tubulin polymers exist in
dynamic equilibrium through polymerization and depolymerization processes. Numerous
structurally different natural as well as synthetic compounds have been identified that target
microtubule polymerization. Colchicine, a natural product, bind to the colchicine binding site
of the tubulin and inhibit tubulin polymerization. Similarly, combretastatin A-4 (CA-4) also
bind to the colchicine binding site and disturb the tubulin-microtubule equilibrium by inhibiting
tubulin polymerization. Thus, the colchicine binding site (CBS) present on the tubulin is being
explored as a promising target for developing new drugs for the treatment of cancer.11-13 All
the CBS ligands inhibit the cell division in G-2/M phase of the cell cycle and causes cell death
either through necrosis or apoptosis.14 Most of the CBS ligands are made up of three fragments
having two binding groups and a bridge.15 CA-4, a potent microtubule targeting and vascular
damaging agent, is capable of drastically inhibiting cancer cell proliferation in vitro.16 CA-4P,
a comparatively safer phosphate derivative of CA-4 is under clinical trials and it has revealed
its ability to regress tumor vasculature in variety of cancers.17 CA-4P was granted fast track
status by the FDA for the treatment of platinum-resistant ovarian cancer.18-20 Thus, a large
number of CA-4 derivatives containing different heterocyclic rings such as tetrazole, triazoles,
21, 22
imidazole,23 pyrazole,24 pyrimidine,25 have been designed, synthesized and screened for
the anticancer potential. However, clinical application of these agents is not successful till

now.26 It is a big challenge to synthesize CA-4 analogues with improved activity along with
therapeutic potency.
In the careful analysis of the approved drugs (FDA.gov2019), it has been observed that more
than 60% of the approved anticancer drugs in 2019 including entrectinib, zanubrutinib etc
contain nitrogen based heterocyclic compounds.27 Pyrimidine represent one of the important
nitrogen containing heterocycles present in many drugs such as imatinib and rosuvastatin.28
The pyrimidine ring can be optionally substituted to synthesize different types of compounds
with diverse pharmacological profile.29 Various pyrimidine ring containing scaffolds such as
imatinib, monastrol and ispinesib (Fig-1) have been explored as attractive agents for the cancer
31
treatment.30,
Lefebvre et al. screened 4-aryl-6-(piperidin-1-yl)pyrimidines with a
sulphonamide group against different cancer cell lines and reported that the presence of a
bulkier substituent on the aromatic ring has increased the inhibition potential of the
compounds.32 Czudor et al. synthesized a series of 2,4-disubstituted pyrimidine derivatives
and tested against the Cyclin-dependent kinases (CDKs) and Polo-like kinases (PLKs). Most
of these compounds displayed nanomolar activity against CDK9 and significant
antiproliferative potential against multiple myeloma cell lines (RPMI-8226).33 Zonghui et al.,
developed a series of tetrahydropyrido[4,3-d]pyrimidine-2,4-dione based compounds that
showed potent anticancer activity.34 Similarly, Jin et al. designed and synthesized compounds
based on phenylpyrimidine pharmacophore model. The lead compounds showed good activity
against HepG2 and MCF-7 cancer cell lines.35
OH
O
HN
NH
O
O
N
N
O
HN
O
O
O
N
N
S
N
O
O
N
Imatinib
H
NH
Monastrol
Colchicine
NH2
N
N
O
N
O
O
O
A
B
N
O
Cl
R
N
O
O
HO
O
O
Combretastatin A-4
Designed molecule
Ispinesib
Fig-1, Target compounds and their structural comparison with colchicine, combretastatin A-4 (CA-4) and other
pyrimidine derivatives.

Recently, we have synthesized and screened a series of CA-4 inspired biphenyl pyrimidines
for their anticancer potential.14 Most of the compounds displayed anticancer activity with IC50
valuesin low micro-molar range. Taking leads from our previous study and literature reports14,
in the current research work we decided to increase the bulk on the pyrimidine ring by
incorporating additional phenyl ring at the C-2 position. 3,4,5-trimethoxyphenyl group was
kept as ring A as it is reported to play crucial role in the binding of ligands to the colchicine
binding site.36 Pyrimidine ring act as linker while aromatic ring C was substituted with different
electron withdrawing and electron releasing groups to develop structure-activity relationship
profile. C-2 position of the pyrimidine ring was substituted with an aromatic ring to increase
the bulk.32 The synthesized compounds were screened against MDA-MB-231, HCT-116 and
HT-29 cancer cell lines for their antiproliferative activity. Most of the compounds were found
to be effective against all the three cancer cell lines and displayed IC50 values in low to sub
micro molar range. HK-2, HK-10 and HK-13 were found to be the potent lead compounds
against all the three cancer cell lines. HK-10 with IC50 values of 0.81± 0.11 µM, 5.89 ± 0.57
µM, 4.96 ± 0.39 µM against MDA-MB-231, HCT-116, HT-29 respectively, was found to be
the most potent compound in the series and displayed comparable activity as compared to the
standard colchicine. The molecular modelling studies of the potent compounds were also
performed in order to understand the binding orientation and interactions of the compounds
with the tubulin protein. The cell cycle and apoptosis analysis of the potent compounds
revealed that these compounds kill the cancer cells in G-2/M phase of the cell cycle and cell
death is caused due to apoptosis.
Target compounds were synthesized via two step procedure as described in Scheme 1. In the
first step, the intermediate 1,3-diphenyl-2-propen-1-one chalcones (3) were synthesized
through acid catalyzed Claisen-Schimdt condensation between trimethoxy substituted
aldehydes (1) and different acetophenones (2), and the intermediates were recrystallized to
obtain pure products.37-39 In the second step, the intermediate chalcones (3) were reacted with
benzamidine hydrochloride using sodium carbonate and acetonitrile to get corresponding
pyrimidine bridged final products (HK-1 to HK-13).40 All the synthesized compounds were
characterized by 1H NMR, 13C NMR, ESI-MS and HRMS.41-43
Scheme 1. Synthesis of pyrimidine bridged analogues of combretastatins

O
H
O
CH3
R1
O
N
N
O
O
a
O
O
b
R1
O
O
R1
O
1
O
O
2
3
HK-1 to HK-13
b)
Benzamidine hydrochloride (1.4 eq),
Reagent and conditions: a)
10 mol % H2SO4 in aceticacid, strring, reflux
anhyd Na2CO3 (2.5 eq), acetonitrile (10ml) , reflux,
All the synthesized compounds (HK-1 to HK-13) were investigated for their in vitro
antiproliferative activity against human MDA-MB-231 (breast), HCT-116 (colon) and HT-
29 (colon) cancer cell lines using standard MTT assays (Table 1) with colchicine as a
reference compound. Four different concentrations (1 µM, 5 µM, 10 µM and 25 µM) of
the test compounds were used and the results were analyzed after 48 h of drug treatment.
Each concentration was evaluated in triplicate and the cells incubated without the test
compounds were used as control. Cells viability was assessed and antiproliferative activity
was calculated in terms of IC50 values recorded in Table 1. Most of the compounds
displayed activity in low to sub micromolar range against all the three cancer cell lines. As
evident from Table 1, compounds HK-2, HK-3, HK-4, HK-10 and HK-13 were found
effective against all the three cancer cell lines. HK-10 was found to be the most potent
against MDA-MB-231 cancer cell line with IC50 value of 0.81 µM. Similarly, HK-6 was
found to be the most potent against HCT-116 and HT-29 cancer cell lines with IC50 values
of 4.32 µM and 3.17 µM, respectively. In all the three cases IC50 values of the test
compounds were found to be better than the standard colchicine. In this series, HK-2, HK-
10 and HK-13 showed best antiproliferative activity against all the three cancer cell lines.
Compound HK-2 exhibited IC50 values of 3.39 µM, 4.78 µM and 4.23 µM against MDA-
MB-231, HCT-116 and HT-29 cell lines respectively. Similarly, HK-10 and HK-13
showed IC50 values of 0.81 µM, 5.89 µM, 4.96 µM and 3.24 µM, 4.93 µM, 4.73 µM against
MDA-MB-231, HCT-116, HT-29 cell lines, respectively (48 h post treatment). The
antiproliferative activities of all the compounds, HK-1 to HK-13 and reference colchicine
against MDA-MB-231, HCT-116 and HT-29 are described in Table 1.

D
B
N
N
O
O
A
C
R1
O
Table 1: Evaluation of in vitro antiproliferative activity (IC50 in µM) of compounds HK-1 to HK-13 against
MDA-MB-231, HCT-116 and HT-29 cancer cell lines (Data are presented as the mean ± SDs of three independent
experiments)
R1
IC50 (µM)
Compound
C
MDAMB231
HCT116
HT29(µM)
ring
(µM)
(µM)
HK-1
HK-2
HK-3
HK-4
HK-5
HK-6
4-CH3
4-OCH3
-H
5.11 ± 0.13
3.39 ± 0.06
5.87 ± 0.09
1.60 ± 0.37
>25
15.67 ± 0.13
4.78 ± 0.33
9.95 ± 0.51
>25
4.23 ± 0.16
7.93 ± 0.25
4-OH
4-Cl
11.85 ± 0.65 11.34 ± 0.18
7.98 ± 0.41
4.32 ± 0.21
6.23 ± 0.15
3.17 ± 0.68
4-Br
>25
HK-7
HK-8
-H
5.11 ± 0.49
1.44 ± 0.08
16.97 ± 0.27
>25
>25
>25
S
2,4-
OCH3
3-OH
HK-9
HK-10
HK-11
HK-12
HK-13
1.99 ± 0.18
0.81 ± 0.11
2.44 ± 0.51
1.01 ± 0.13
3.24 ± 0.23
20.35 ± 1.58
5.89 ± 0.57
>25
>25
4.96 ± 0.19
>25
2,4-OH
3-NO2
4-NH2
-H
12.34 ± 0.57
4.93 ± 0.46
>25
4.73 ± 0.16

Colchicine
0.93 ± 0.13
5.13 ± 0.35
5.19 ± 0.42
Cell cycle and apoptosis studies of the lead compounds
HK-2, HK-10 and HK-13 have displayed promising antiproliferative activities and selected for
cell cycle analysis and apoptosis studies to understand the mechanism of action of the
compounds. This series of compounds are derived from colchicine and combretastatin A-4 and
are expected to bind to the colchicine binding site of the tubulin. Ligands binding to the
colchicine site, act as tubulin polymerization inhibitors and arrest the cell cycle at G2/M phase.
Thus, the cell cycle analysis of these three compounds was performed on HCT-116 cancer
cells. Cells were grown in 6 well-treated corning plate, and treated with 12.5 μM concentration
of the compounds. It was found that HK-2, and HK-10 showed cell cycle arrest of 88.8 %, and
79.5 %, respectively at the G2/M phase while HK-13 showed cell cycle arrest of 78.5 % at
G1/G0 phase. Colchicine was used as a positive control and as expected it showed cells arrest
of 90.8 % in G2/M phase of the cell cycle. Non-treated cells showed cells arrest of 50.1 %, at
G1/G0 phase,16.2 % at S phase, and 28.4 % at G2/M phase of the cell cycle. (Fig-2). Thus,
HK-2 and HK-10 might be acting like colchicine and CA-4 by binding to the colchicine
binding site of the tubulin.
Table 2: Cell cycle analysis of HK-2, HK-10 and HK-13 against HCT-116 cancer cells
S. No. Compound name /
The phase of the cell cycle
G1/G0
Control Colchicine HK-2 HK-10 HK-13
1
2
3
50.1%
16.2%
28.4%
5.1%
5.6%
4.6%
11.3% 78.5%
6.8% 6.8%
S
4.1%
G2/M
90.8%
88.8% 79.5% 8.1%
Colchicine
HK-2
Control
HK-10
HK-13

Figure 2: Cell cycle assay with propidium iodide at 12.5 µM concentration of the test compounds treated for
24h with colchicine as a positive control.
In order to understand the mechanism of cell death induced by HK-2, HK-10 and HK-13, a
cytofluorimetric analysis was performed on HCT-116 cancer cells using Muse™ Annexin V
and Dead Cell kit to detect the phosphatidyl serine membrane translocation. This is considered
as a major hallmark of the late stage apoptosis. HCT-116 human cancer cells were treated with
HK-2, HK-10, HK-13 as test compounds and colchicine as reference at 5 µM concentrations.
It has been found that HK-2, HK-10, and HK-13 showed apoptotic cell death of 48.3 %, 35.2
%, and 49.6 %, respectively as compared to 2.4 % apoptosis of the non-treated cells.
Colchicine as a positive control, showed 56.4 % apoptosis after 24h treatment (Fig-3). Hence,
it can be concluded that the primary mode of cell death with the tested compounds is through
apoptosis.
Colchicine
HK-2
Control
HK-10
HK-13
Figure-3: Quantitative analysis of apoptotic cells using AnnexinV FITC/PI double staining and flow-cytometry
calculations.
in silico studies
Docking studies
From the literature search it was apparent that most of the combretastatin A-4 and colchicine
derivatives bind to the colchicine binding site of the tubulin and inhibit tubulin polymerization.
44,45 HK-2, HK-10, and HK-13 displayed good antiproliferative activities and in the cell cycle
analysis it was found that HK-2 and HK-10 arrest the cell cycle at G2/M phase, typical

Fig:4, Docking poses of HK-2, HK-10 and HK-13 along with CA-4 and colchicine in colchicine binding site.
HK-2, HK-10, HK-13, CA-4 and colchicine showed dock score of -5.82 kcal/mol, -7.85 kcal/mol, -6.15 kcal/mol,
-7.95 kcal/mol and -5.5 kcal/mol, respectively.

characteristics of colchicine site binding agents. Thus, these compounds were docked into the
colchicine binding site of the tubulin (PDB entry: 4O2B) to explore their interactions with the
binding pocket and to investigate their binding pattern.46 Glide (GLIDE 11.1 module of
Schrödinger Suite) was used for scoring the interaction between ligands and receptor. The
colchicine domain consists of colchicine binding site (main site) and its neighbouring pockets.
This domain was divided into three zones i.e. zone 1, zone 2 (main site) and zone 3.47 The
docking orientations of HK-2 and HK-10 (Fig-4a & 4b) inside the active cavity revealed that
the phenyl ring with the trimethoxy substituents fitted in the zone 2 which is delimited by
Lysβ352, Asnβ350, Leuβ378, Alaβ316, Leuβ255, Lysβ254, Alaβ250 and Leuβ242, and one of
the methoxy group of A ring is involved in a hydrogen bonding interaction with the distant
amino group Lysβ254. In HK-10, the C-ring confined in zone 3 and makes hydrogen-bonding
interaction with hydrogen of p-hydroxy group and Valβ238, while D-ring orient towards zone
1 and lined with Valα181 and Alaα180. In HK-2, the C-ring is also confined in zone 3 and
makes hydrophobic interactions with the surrounding amino acids residues such as Thrβ239,
Valβ238, which may impart better binding affinity. In HK-13 (Fig 4c), in contrast to other
colchicine binding site agents such as CA-4 (Fig 4d), the phenyl ring with trimethoxy
substituents align in different direction and showed hydrogen bonding interaction with
Ashβ329(a neutral Asp) and surrounded by Ashβ329, Metβ325, Leuβ248, Glnβ247. The C-
ring (naphthyl ring) of HK-13 confined in zone 1 and showed hydrophobic interaction with
Serα178, Thrα179, Valα181 while D-ring stayed outside the colchicine domain. Both, HK-10
and HK-2 showed lower dock scores of -7.25 kcal/mol and -5.82 kcal/mol, respectively as
compared to reference compound colchicine with a dock score of -5.5 kcal/mol. This may be
due to proper fitting of the test compounds in the active site and due to presence of additional
interactions. HK-13 with naphthyl ring (unsubstituted) as ring C showed dock score of -6.15
kcal/mol and expected to display hydrophobic interactions only. 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.
ADME studies
The physicochemical properties of the synthesized compounds were investigated by
performing ADME studies on Schrodinger’s QikProp module. QikProp module utilises
different parameters of Lipinski’s rule of five for the analysis of drug like properties.
Lipophilicity is one of the property of compounds that determine whether the molecule will
cross the biological membrane or not, and for that Log P (<5) is an important physiochemical

property. HK-2 and HK-10 displayed lipophilicity within the range of 5.71 to 6.36. Although
these two compounds displayed Log P values greater than 5 but less than 6.5 which is the upper
limit for druggable compounds indicating that these molecules are slightly lipophilic in nature.
However, HK-13 showed exceptionally high log P value of 7.48 and higher log P value
indicates that it has some undesired characteristics such as poor aqueous solubility, rapid
metabolic turnover, high plasma protein binding, and tissue accumulation. As lipophilicity
plays crucial role in determining the solubility of drug candidates in biological system, and
hence log S values of these compounds were also predicted. HK-2, HK-10 and HK-13 showed
log S values of -6.98, -6.39, and -8.13, respectively as compared to standard drug CA-4 (-4.14)
and colchicine (-3.07). It indicates that all the three compounds, more particularly HK-13
would display low aqueous solubility that may compromises bioavailability during in vivo
study. Thus, there is scope in further modification of these compounds and additional polar
functionalities may be incorporated in the scaffold to increase the hydrophilicity and making
these more druggable. The low values of the docking score indicate that these molecules may
bind strongly with the active site. Molecular weights of these compounds were less than 500
and number of hydrogen bond donors and hydrogen bond acceptors also lies within the
acceptable range (Table 2). Low value of the dipole moment, one of the factors that decide the
binding of compounds with protein, of the ligands binding to the colchicine binding site is
found to be better for the activity. All the three compounds showed 100 % human oral
absorption and compound HK-2 and HK-13 undergo lesser number of metabolic reactions as
compared to the standard tubulin inhibitors colchicine and combretastatin A-4 (Table 3). Thus,
the tested compounds satisfied the Lipinski rule of five except for the lipophilic characteristics
which needs to be taken in consideration during the lead optimization.
Table 3: Drug like characteristics of compounds HK-2, HK-10 and HK-13 as determined by QikProp application
of Schrodinger
Name
Mol.
Wt.
Log P Log S
HB
HB
% human dipole #metb
donor acceptor
oral
absorption
HK-2
HK-10
HK-13
428.48
430.45
448.52
6.36
5.71
7.48
-6.98
-6.39
-8.13
0
2
0
1
1
5
5
4
7
4
100
100
3.806
2.005
3.854
6.91
4
5
3
5
5
100
Colchicine 399.44 2.704 -3.07
CA-4 316.35 4.045 -4.14
95.43
100
4.02

SAR studies
In the current study, 3,4,5-trimethoxyphenyl containing pyrimidine bridged combretastatin A-
4 and colchicine derived compounds have been synthesized and evaluated for antiproliferative
activities against MDA-MB-231, HCT-116 and HT-29 cancer cell lines. Most of the
compounds displayed antiproliferative activities against these cancer cell lines in low to sub
micromolar range. All the synthesized compounds composed of a trimethoxyphenyl fragment
as ring A, pyrimidine moiety as ring B and a phenyl substituent on the pyrimidine as ring D.
Ring C was kept as a variable fragment and selected from a group of naphthyl, thiophene, and
phenyl ring optionally substituted with different types of electron withdrawing and electron
releasing groups such as methyl, methoxy, amine, chloro, bromo, nitro etc. HK-1 with para
toluene group as ring C was found to be active against MDA-MB-231cell line however it
showed low potency against HCT-116 and HT-29 cancer cell lines. Replacement of methyl
substituent to methoxy in HK-2 improved the activity of the compounds against all the three
cell lines with IC50 values of 3.39 µM, 4.78 µM and 4.23 µM against MDA-MB-231, HCT-
116 and HT-29, respectively. HK-3, with an unsubstituted phenyl ring showed almost 2 folds
decrease in the activity against all the three cell lines as compared to HK-2. Incorporation of a
hydroxy substituent at para position (HK-4) increased the activity against MDA-MB-231cells
but decreased activity was observed against HCT-116 and HT-29 cell lines. Presence of
electron withdrawing groups like chloro (HK-5) and bromo (HK-6) on the phenyl ring found
to decrease the potency against MDA-MB-231cell line while these compounds were found to
be effective against HCT-116 and HT-29 cell lines. In the series, HK-6 was found to be the
most potent against HT-29 cell line with IC50 value of 3.17 µM. Thiophene as ring C (HK-7),
was found to be effective against MDA-MB-231cells but it showed low activity against other
two cancer cell lines. Similarly, HK-8 and HK-9 with 2,4-dimethoxy and 3-hydroxy
substituents displayed high potency against MDA-MB-231cells but showed very low activity
against HCT-116 and HT-29 cell lines. HK-10 with 2,4-dihydroxy substitution, was found
effective against all the three cancer cell lines with IC50 values of 0.81 µM, 5.89 µM and 4.96
µM against MDA-MB-231, HCT-116 and HT-29, respectively. It was found to be the most
potent compound in the current series against MDA-MB-231 cell line. HK-11 and HK-12
with 3-nitro and 4-amino substituents, respectively showed high potency against MDA-MB-
231 but these compounds were found to be less active against HCT-116 and HT-29 cell lines.
Compound HK-13 with naphthyl group as ring C, displayed significant antiproliferative
activities with IC50 values of 3.24 µM, 4.93 µM and 4.73 µM against MDA-MB-231, HCT-
116 and HT-29 cancer cell lines, respectively. In this series, many compounds were found to

show similar potency as displayed by the reference compound colchicine. Although the effect
of substitution pattern on the activity, varies on different cell lines but in general it has been
observed that the compounds with electron donating substituents showed higher
antiproliferative activities as compared to compounds with electron withdrawing groups HK-
5 and HK-6. Interestingly, in this series most of the compounds were found to be more potent
against MDA-MB-231 cell line as compared to other two cell lines. Thus, it can be concluded
that some of the promising compounds from the current series especially HK-2, HK-10 and
HK-13 can act as leads for further developments.
Pyrimidine is a versatile nucleus and indispensable constituent of many drugs and active
pharmaceutical ingredients. Current series of triphenyl pyrimidines were designed as
colchicine and combretastatin A-4 analogous. A total of thirteen compounds (HK-1 to HK-13)
were synthesized and evaluated for antiproliferative activities against MDA-MB-231, HCT-
116 and HT-29 cancer cell lines. Most of the compounds showed antiproliferative activities in
low to sub micromolar range. Compounds HK-2, HK-10 and HK-13 were found to be
effective against all the three cancer cell lines. HK-10 was found to be the most potent
compound in the series with IC50 values of 0.81 µM against MDA-MB-231 cell line. In the
series, some of the compounds displayed comparable activities to the reference colchicine.
Although these compounds showed very high binding in the docking study however, low
activities may be due to the high lipophilicity and their absorption & retention in the cell
membrane during in vitro study. The promising compounds i.e. HK-2, HK-10 and HK-13
were subjected to cell cycle analysis and apoptosis studies to understand the mode of action of
these compounds. In the cell cycle analysis, it was found that HK-2 and HK-10 arrest the cells
in G2/M phase of the cell cycle while HK-13 inhibit cell growth in G0/G1 phase. The ligands
binding to the colchicine site arrest the cell cycle at G2/M phase and hence it was proposed
that HK-2 and HK-10 might be binding to the colchicine site of the tubulin and killing the cells
by disturbing the equilibrium between tubulin polymerization-depolymerization. It has been
observed that all the three compounds inhibited cell proliferation of cancer cells by inducing
apoptosis. In the docking studies, it was found that HK-2 and HK-10 fit well in the colchicine
binding site of the tubulin while some portion of HK-13 stay outside the colchicine domain.
Tubulin binding studies of these compounds are under process. These molecules were found
to be highly lipophilic and hence further modifications in the structure can be performed to
make them more potent and druggable. In conclusion, some of the triphenyl substituted
pyrimidines were found to be effective against all the three cancer cell lines and these
compounds can act as important leads for further studies.

Acknowledgement
A.R.D. and Vinod K. are thankful to the DST-SERB (EMR/2015/002339) for providing
financial assistance. Vijay K. and NK are thankful to the UGC for the Senior and Junior
Research Fellowships, respectively. SB lab is supported by a DBT Ramalingaswami
Fellowship (BT/HRD/02/09/2013), DST-SERB (ECR/2016/000903) grant; RPY is supported
by CSIR-Junior Research Fellowship (09/1051(0016)/2018. Authors thanks Central University
of Punjab, Bathinda for providing required infrastructure. This manuscript is dedicated to Prof.
P Ramarao on his superannuation from the Central University of Punjab, Bathinda on June
30, 2020.
Supplementary Material
Supplementary information describing detailed synthetic protocols, spectral data, biological
protocols associated with this research article can be accessed in the online version.
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Graphical Abstract
IC50
=
0.81 µM (MDAMB-231)
5.89 µM (HCT-116)
4.96 µM (HT-29)
R1
N
O
O
N
O
IC50
=
Antiproliferative potential of
triphenylpyrimidines
3.24 µM (MDAMB-231)
4.93 µM (HCT-116)
4.73 µM (HT-29)
IC50
=
3.39 µM (MDAMB-231)
4.78 µM (HCT-116)
4.23 µM (HT-29)
Highlights:
A series of triphenyl substituted pyrimidines as analogous of colchicine and combretastatin
A-4 was synthesized.
Synthesized compounds were evaluated for the antiproliferative potential against MDA-MB-
231, HCT-116 and HT-29 cell lines
Compounds displayed antiproliferative activity in low to sub micro molar concentration
Some of the compounds showed comparable activity as shown by standard inhibitor
colchicine
Lead compounds showed cell arrest at G2/M phase and cell death induced through apoptosis