Design and synthesis of cis-restricted benzimidazole and benzothiazole mimics of combretastatin A-4 as antimitotic agents with apoptosis inducing ability

Article abstract of DOI:10.1016/j.bmcl.2016.06.044

A series of colchicine site binding tubulin inhibitors were designed and synthesized by the modification of the combretastatin A-4 (CA4) pharmacophore. The ring B was replaced by the pharmacologically relevant benzimidazole or benzothiazole scaffolds, and

Full text of DOI:10.1016/j.bmcl.2016.06.044

Accepted Manuscript
Design and synthesis of cis-restricted benzimidazole and benzothiazole mimics
of combretastatin A-4 as antimitotic agents with apoptosis inducing ability
Md. Ashraf, Thokhir B. Shaik, M. Shaheer Malik, Riyaz Syed, Prema L.
Mallipeddi, M.V.P.S. Vishnu Vardhan, Ahmed Kamal
PII:
S0960-894X(16)30655-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.06.044
BMCL 23997
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 April 2016
14 June 2016
16 June 2016
Please cite this article as: Ashraf, Md., Shaik, T.B., Shaheer Malik, M., Syed, R., Mallipeddi, P.L., Vishnu Vardhan,
M.V.P.S., Kamal, A., Design and synthesis of cis-restricted benzimidazole and benzothiazole mimics of
combretastatin A-4 as antimitotic agents with apoptosis inducing ability, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.06.044
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Design and synthesis of cis-restricted benzimidazole and benzothiazole mimics
of combretastatin A-4 as antimitotic agents with apoptosis inducing ability
Md. Ashraf,a Thokhir B Shaik,ac M Shaheer Malik,a Riyaz Syed, a Prema L.Mallipeddib M V P S
Vishnu Vardhan,a Ahmed Kamal,*a
aMedicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology,
Tarnaka, Hyderabad 500 007, India
bGVK Biosciences Pvt. Ltd., Plot No 79, IDA, Mallapur, Hyderabad 500076, Andhra Pradesh,
India.
cAcharya Nagarjuna University, Nagarjuna Nagar, Guntur 522510, India.
Key words: Tubulin inhibitor, combretastatin A-4, benzimidazole, benzothiazole, apoptosis.
Abstract: A series of colchicine site binding tubulin inhibitors were designed and synthesized by
the modification of the combretastatin A-4 (CA4) pharmacophore. The ring B was replaced by
the pharmacologically relevant benzimidazole or benzothiazole scaffolds, and the cis-
configuration of the olefinic bond was restricted by the incorporation of a pyridine ring which is
envisaged by the structural resemblance to a tubulin inhibitor like E7010. These compounds
were evaluated for their antiproliferative activity on selected cancer cell lines and an insight in
the structure activity relationship was developed. The most potent compounds (6c and 6l)
demonstrated an antiproliferative effect comparable and superior to that of CA4 (GI50 up to 40
nM). Mitotic cell cycle arrest in G2/M phase revealed the disruption of microtubule dynamics
that was confirmed by tubulin polymerization assays and immunocytochemistry studies at the
cellular level. The molecular docking studies suggested that the binding of these mimics at the
colchichine site of the tubulin is similar to that of combretastatin A-4.
Introduction:
Microtubules are one of the key structural components of the cytoskeleton in eukaryotic cells
comprising of α and β-tubulin heterodimers. They play a crucial role in various cellular processes
1

and have emerged as an attractive and viable target in the development of anticancer drugs
mainly due to their indispensability in mitotic cell division.1 Generally, drugs that target
microtubules bind to one of three main sites of tubulin,2 which includes the paclitaxel site for the
microtubule stabilizing agents,3,4 the vinca domain5 and the colchicine domain6,7 for the
destabilizing agents. Interfering with the dynamic stability of microtubules, these agents acts as
spindle poisons arresting the dividing cells in G2/M phase of the cell cycle, causing mitotic
catastrophy and finally leading to apoptotic cell death. Some of the well-known naturally
occurring tubulin binding ligands that affect the microtubule dynamics by binding to distinct
colchicine domain of tubulin are colchicines and combretastatin A-4 (Figure 1).8,9
Figure 1. Colchicine site binding microtubule inhibitors
2

Combrestatin, the cis stilbene derivative, isolated from the bark of the South African tree
Combretum caffrum is one of the simplest and promising molecules among the structurally
diverse microtubule perturbing agents. Combretastatin A-4 exhibits remarkably strong inhibition
of tubulin polymerization by binding to the colchicine site as well as potent cytotoxicity against
murine lymphocytic leukemia, human ovarian and colon cancer cell lines resulting in vascular
shutdown in solid tumor.10-14 In recent years CA4 emerged as a preferred lead compound in the
development of new inhibitors of tubulin polymerization because of its high potency and ease of
synthesis.15,16 Similar to other colchicine site binders, CA4 has three important pharmacophoric
substructures, the two hydrophobic rings (A and B) and the linking olefinic bridge with
appropriate dihedral angle resulting from the cis configuration. It was previously postulated that
the presence of trimethoxy substitution on ring A is crucial for efficient binding at colchicine
site.8 However, of late this substitution pattern on ring A is considered to be not as indispensable
as previously assumed.10 On the other hand, the ring B was found to be tolerant to structural
modification and a number of reports defined this ranging from substituted phenyl ring to
heterocyclic and nonsubstituted aromatic rings.10,17 Importantly, the olefinic bond with cis
configuration plays a fundamental role in binding at the colchicine site by positioning the rings at
appropriate distance to maximize interactions.18 Several attempts have been reported to modify
this cis-olefinic bond to prevent its isomerisation under amenable conditions. This mostly
included either modification of the olefinic bond by the introduction of saturation, substituents
and other functionalities or its replacement with a three to six membered ring system, which
resulted in cis restricted analogues of CA4. 16,19-22
Similar to CA4, a number of colchicine site binding ligands are known to inhibit tubulin
polymerization and are considered as potential lead like molecules. One of them, E7010 is the
first orally active antimitotic sulphonamide derivative that has received much interest in recent
years and currently it is in clinical trials.23 It binds reversibly to colchicine site of β-tubulin and
inhibits tubulin polymerization resulting in cell cycle arrest in M phase. Structure-activity
relationship studies of E7010 suggested that the replacement of pyridine moiety and the methoxy
as well as hydroxy groups resulted in lowered in vitro and in vivo activity.24-26 Research efforts
have been directed towards the development analogues of E7010 as inhibitors of tubulin
polymerization in cancer drug discovery programs.27-28
3

Figure 2. Designing of combretastatin conjugates
Our recent research studies have been mainly focused on the synthesis, evaluation and
mechanistic aspects of newer molecules based on different heterocyclic scaffolds as potential
anticancer agents.29,30 In particular, the targeting of tubulin polymerization by new diversified
ligands based on CA4 and E7010 have led to promising and interesting results.31 Herein, we
describe different modifications on CA4 scaffold, specifically the incorporation of
benzimidazole and benzothiazole moieties of pharmacological relevance by replacement of ring
B. The rationale behind this is the significance of these moieties as privileged structures and the
opportunity offered by them as anchors that could be harnessed for further diversification,
besides they are present in number of antimitotic agents such as nocodazole and DF-203.32,33
Moreover, the structural resemblance to E7010, a colchicine site binder (with crucial pyridine
ring) was envisaged by the replacement of the olefinic bond by a pyridine ring to maintain the cis
configuration. Such replacements of the olefinic bond with a ring system have been reported to
provide potential advantage of promoting tubulin binding activity and tumor selectivity besides
minimizing toxicity.10 In addition, since the constitutive role of the trimethoxybenzene moiety
(ring A) is ambiguous in tubulin binding, we modified it with different substitution patterns. The
resulting cis-restricted benzimidazole and benzothiazole mimics of CA4 were evaluated for
antiproliferative activity and examined for their structure activity relationship (SAR) followed by
studies to elucidate the mechanism of action which included cell cycle progression, tubulin
polymerization assay and molecular docking studies. Further to confirm the induction of
4

apoptotic cell death by the potent mimics like 6c and 6l, studies such as caspase activation,
mitochondrial membrane potential.
The synthesis of benzimidazole and benzothiazole mimics of CA4 (5a-l and 6a-n) was
accomplished by the synthetic route illustrated in Schemes 1 and 2. In the synthetic route 2-
chloronicotinic acid (7) was employed as the starting material, which was converted to its ester
derivative, ethyl 2-chloronicotinate (8) by using sulphuric acid and ethanol in quantitatively
yield31 (Scheme 1).
Scheme 1. Synthesis of benzimidazole mimics of CA4 (5a-l). Reagents and conditions: (a)
H2SO4, ethanol, 80 °C, 2 h. (b) ArB(OH)2, Pd(PPh3)4, Na2CO3, toluene-EtOH-water, 120 °C, 8 h.
(c) 2N NaOH, 1N HCl, ethanol, 80 °C, 4 h. (d) polyphosphoric acid, 180 °C, 2 h.
The ester 8 was subjected to Suzuki coupling by condensation with a variety of substituted
boronic acids in the presence of tetrakis(triphenylphosphine)palladium and potassium carbonate
in toluene and ethanol-water to afford the intermediates 9a-e, which on subsequent hydrolysis
with 2N NaOH solution in ethanol provided 2-phenyl substituted nicotinic acids 10a-e.34 These
key intermediates were utilized in the synthesis of the benzimidazole and benzothiazole mimics
5

of CA4. These intermediates 10a-e were condensed with various substituted o-diaminobenzenes
(11) in the presence of polyphosphoric acid under refluxing condition to afford the desired cis-
restricted benzimidazole mimics of CA4 (5a-l).
Table 1. Percentage of yields of compounds 5a-l
Comp R1
R2
R3
R4
X
Y
Yield (%)
OMe OMe OMe H
OMe OMe OMe H
OMe OMe OMe H
OMe
H
OMe 51
5a
5b
5c
5d
5e
5f
H
H
58
44
OMe
OMe
H
H
OMe OMe H
OMe OMe H
OMe OMe H
OMe 60
H
H
H
56
67
H
OMe
OMe
OMe
Cl
H
OMe H
OMe H
OMe H
H
H
H
H
H
OMe 48
5g
5h
5i
H
H
H
H
H
H
50
58
46
63
60
H
H
CF3
CF3
H
H
H
H
OMe
Cl
5j
H
5k
5l
CF3
Cl OMe
To access the benzothiazole mimics of CA4, the same intermediates 10a-e were employed as
depicted in Scheme 2. An amide linkage was established by condensation of substituted nicotinic
acids (10a-e) with a variety of substituted anilines 12 to yield nicotinamide deriviatives (13a-n)
by employing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1H-benzo[d][1,2,3]triazol-1-
ol in N,N-dimethylformamide.34 This was followed by the conversion of carbonyl functionality
to its analogous of thiocarbonyl group employing Lawessons reagent under refluxing in toluene
to provide thioamides 14a-n. Finally, the intramolecular cyclization was performed by using a
standard method of potassium ferricyanide in presence of 10% NaOH solution furnishing the
desired cis-restricted benzothiazole mimics of CA4 (6a-n).35
6

Scheme 2. Synthesis of benzothiazole mimics of CA4 (6a-n). Reagents and conditions: (a)
°
EDCI.HCl, HOBT, DMF, rt. (b) Lawessons reagent, toluene, 100 C, 3 h. (c) K3[Fe(CN)6], 10%
NaOH, 100 °C, 2 h.
Table 2. Percentage of yields of compounds 6a-n
Comp R1
R2
R3
R4
H
H
H
H
H
H
H
H
H
H
X
Y
Yield (%)
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
CF3
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe OMe 41
6a
6b
6c
6d
6e
6f
OMe
OMe
H
H
H
H
48
45
OMe
OMe OMe 50
H
H
H
H
38
46
H
OMe
H
OMe OMe 55
6g
6h
6i
H
H
OMe
Cl
H
H
H
56
51
55
H
H
H
H
OMe
6j
7

H
CF3
H
H
H
H
H
H
Cl
H
H
H
H
40
46
30
42
6k
6l
CF3
CF3
CF3
Cl OMe
Cl Cl
H
6m
6n
H
Cl
H
Initially we evaluated the antiproliferative activity of the compounds 5a-h against selected
human cancer cell lines viz., cervix (HeLa), liver (HepG2), lung adenocarcinoma (A549) and
prostate (DU-145), using SRB assay.36 The results of growth inhibitory activities (GI50 values)
are shown in micromolar concentrations and CA4 was used as the reference compound (Table
3). Most of the compounds from these series displayed potent broad spectrum growth inhibitory
activities against all the tested cancer cell lines. In the SAR studies it was considered to
investigate whether the presence of 3,4,5-trimethoxyphenyl substitution influences the activity
along with other substituents on the benzimidazole ring. It was observed that the compounds (5a-
c) containing trimethoxy substitution on ring-A showed relatively strong inhibitory effect than
the 3,4-dimethoxy and 4-methoxy substitution with GI50 values in the range of 0.29-14.6 µM
against some representative cancer cell lines. Among them, compound 5c with a methoxy group
on C-6 position of the benzimidazole moiety exhibited promising activity with a GI50 value of
0.29 µM against prostate cancer cell line. On the replacement of trimethoxy substituted ring A
with di and monomethoxy substituted phenyl ring resulted in moderate activity and compound
5d was the most potent among them exhibiting GI50 value of 1.9 µM in the liver cancer cell line.
The substitution effect on ring A by these studies reaffirmed the importance of 3,4,5-trimethoxy
substitution on the cytotoxic effect by these CA4 mimics. We then proceeded to explore the
influence of electron withdrawing group on ring A and benzimidazole moiety with respect to the
antiproliferative activity. Compounds having electron withdrawing substituents like
trifluoromethyl on ring A (5j-k) with a methoxy and chloro group on C-6 position of
benzimidazole moiety exhibited significant decrease in activity compared to 5c. Surprisingly
when two electron withdrawing substituents were incorporated on the ring A, a substantial
increase in the cytotoxic potency was observed. Compound 5l with trifluoromethyl on C-5 and
chloro on C-2 position of ring A and methoxy group on C-6 position of benzimidazole moiety
was the most active benzimidazole mimic of CA4 exhibiting an GI50 range of 0.15-0.76 µM
against certain cancer cell lines.
8

In the benzothiazole counterparts (6a-h), in which one of the nitrogen of the benzimidazole
moiety was replaced by sulphur, a substantial enhancement in the antiproliferative activity was
observed with GI50 values in sub-micromolar concentrations against some of the cancer cell lines
tested. Similar to benzimidazole mimics, we initially examined the effect of 3,4,5-trimethoxy
substitution on ring A with methoxy substituent on C-5 and C-6 position of benzothiazolyl ring.
An identical pattern of activity was observed with trimethoxy substitution on ring A exerting
relatively stronger influence on activity than di and monomethoxy substitutions. Compound 6c
with trimethoxy groups on ring A and a methoxy substitution on C-6 position of benzothiazole
moiety was one of the most potent mimic exhibiting GI50 of 0.06 µM against HeLa liver cancer
cell line. On replacement of 3,4,5-trimethoxyphenyl ring with 3,4-dimethoxy and 4-methoxy
phenyl ring, the compounds (6d-i) exhibited lower growth inhibition against all the tested cell
lines, however 6e displayed promising activity with GI 50<1M against HepG2 and A549 cancer
cell lines. In the mimics (6j-k) with one electron withdrawing substituent, decreased
antiproliferative potency was exhibited similar to benzimidazole counterparts. Nanomolar
activity was observed in case of 6l with two electron withdrawing groups on C-2 and C-5
positions of ring A and methoxy group on benzothiazole moiety against all the tested cancer cell
lines with GI50 ranging from 0.04-0.09 µM. Interestingly, mimic 6l exhibited remarkable and
superior antiproliferative activity compared to the standard CA4 particularly against HeLa and
DU-145 cancer cell lines and were significantly more potent than sulphonamide E7010.
In order to understand that the effect exerted by the two electron withdrawing groups, that is CF3
at C-5 and Cl on C-2 on ring A towards the antiproliferative activity. We synthesized and
examined some mimics (6m-n) in this series. Compound 6m with chloro group on C-6 position
of the benzothiazole ring displayed good activity (GI50 <1.5 µM in two cancer cell lines) whereas
the absence of substitution in 6n lowered the antiproliferative activity. Overall, SAR studies of
these cis-restricted mimics of CA4 suggested the superiority of 3,4,5-trimethoxy substitution
compared to the 3,4-dimethoxy and 4-methoxy substitution on ring A which are in agreement
with some similar studies on CA4 analogues described in earlier reports.8 The introduction of
single electron withdrawing group on ring A decreased the antiproliferative activity, however
when two electron withdrawing groups were incorporated at C-2 and C-5 position, a remarkable
enhancement in the activity was observed. This is when a methoxy substituent is present on C-6
9

position of the benzimidazole or benzothiazole moiety, which is considered crucial for imparting
potent antiproliferative properties.
Table 3. Growth Inhibition values of compounds 5a-l and 6a-n on HeLa, HepG2, A549 and DU-
145 cancer cell lines.
GI50 µM
Compound
5a
5b
5c
HeLaa
HepG2b
A549c
DU-145d
1.7± 0.22
2.6± 0.24
5± 0.54
1.5 ± 0.14
3.5 ± 0.15
0.84± 0.01
1.9± 0.26
12.4± 0.32
44.8± 0.17
42.5± 0.41
2.5 ± 0.19
4.5 ± 0.19
1.5± 0.21
7.9± 0.17
0.6± 0.14
14.6± 0.24
0.29± 0.12
4.19± 0.41
5.8± 0.33
12.0± 0.16
7.7± 0.11
5d
5e
e
e
-
-
16.6± 0.41
21.4± 0.09
31.1± 0.09
27.6± 0.54
19.5± 0.19
21.5± 0.25
5f
e
-
5g
5h
5i
e
-
19.6± 0.09
34.7± 0.17
32.1± 0.09
e
e
-
24.5± 0.15
-
e
e
-
-
38.2± 0.12
18.4± 0.33
0.75±0.02
0.5 ± 0.01
3.2± 0.26
0.13± 0.01
7.58 ±0.32
0.2 ± 0.19
1.7± 0.11
59.1
5j
e
e
15.8± 0.16
0.15±0.01
1.6± 0.02
4.8± 0.19
0.06± 0.001
13.5± 0.11
1.8± 0.22
6.2± 0.14
11.3± 0.11
8.1± 0.17
7.7± 0.14
15.7± 0.12
20.4± 0.09
-
-
5k
5l
0.76±0.01
0.5 ± 0.01
3.5± 0.21
0.26± 0.02
1.2 ± 0.62
0.7 ± 0.14
2.2± 0.21
30.9± 0.14
0.21±0.01
0.6± 0.04
4.1 ± 0.11
0.18± 0.03
5.91± 0.16
3.6± 0.24
6.8± 0.32
6a
6b
6c
6d
6e
6f
e
-
6g
6h
6i
e
e
-
13.3± 0.09
7.9± 0.17
17.0± 0.16
19.5± 0.19
-
3.9± 0.26
0.19± 0.41
e
e
-
-
6j
e
32.5± 0.41
-
6k
10

0.04 ± 0.001
5.8± 0.12
4.89
0.079 ± 0.001
1.2± 0.16
15.0
0.091± 0.002
1.5± 0.16
7.41
0.052± 0.001
6l
e
-
6m
5.49
6n
CA4
E7010
0.06 ± 0.002
4.8 ± 0.22
b
0.08 ± 0.002
2.8 ± 0.22
0.06 ± 0.003
1.8 ± 0.22
d
0.07 ± 0.003
1.2 ± 0.3
aHuman Cervical cancer; Liver cancer; cNon-Small Cell Lung Cancer, Prostrate cancer, GI50 value not
e
attained at the concentration used in the assay.
Mechanistic studies were performed to understand the mode of action of these CA4 based
mimics and the most potent compounds (6c and 6l) were selected for further biological
investigations. Initially, cell cycle analysis was performed using the cell permeable DNA binding
dye propidium iodide to determine whether the antiproliferative effect resulted from cell cycle
arrest. For this purpose, HeLa cells were treated with 6c and 6l at concentrations of 40 nM as
well as 60 nM for 48 h to study the distribution of cells at different phases of cell cycle. The
results demonstrated that compared to untreated cells, the cells treated with 6c and 6l displayed
significant arrest in G2/M phase, the arrest is not only comparable but superior to CA4,
particularly at 60 nM concentration (Figure 3). Compounds 6c and 6l arrested 40.8% and 54.05%
cells, respectively, in G2/M phase compared to 41.48% blocked by CA4. These results
demonstrate that the growth inhibitory effects of the CA4 mimics (6c and 6l) are induced
because of the arrest in G2/M phase during cell cycle progression in a concentration dependent
manner.
Table 4. The percentage of cells in G2/M phase of cell cycle was quantified by flow cytometry.
Compound
control
G0
G1
S
G2/M
17.56
23.65
40.80
24.26
54.05
38.87
41.48
5.64
73.28
51.22
19.54
43.00
15.19
17.92
17.29
3.52
6c-40nm
6c-60nm
6l-40nm
13.27
25.35
8.13
11.86
14.30
24.62
10.30
17.34
16.44
6l-60nm
20.47
25.87
24.78
CA4-40nm
CA4-60nm
11

Figure 3. FACS analysis of cell cycle distribution of HeLa cells after treatment with 6c and 6l at 40 nM
and 60 nM concentrations and CA4 for 48 h. Cell cycle analysis was performed employing propidium
iodide as indicated under materials and methods. The percentage of cells in each phase of cell cycle was
quantified by flowcytometry.
The G2/M phase arrest of cells takes place due to the perturbation in the mitosis cell division
machinery and the tubulin is one of the important structural proteins in the mitosis process.
Therefore, as 6c and 6l showed significant antiproliferative activity as well as arrest in the G2/M
phase in the cell cycle analysis, it is likely that they would inhibit the polymerization of tubulin.
The progression in tubulin polymerization was investigated by monitoring the increase in
fluorescence emission at 420 nm at 3 μM concentration employing CA4 as the positive control.
These conjugates showed significant activity to inhibit the tubulin polymerization which is
comparable to CA4, whereas 6c inhibited the tubulin polymerization by 60.1% while 6l inhibited
by 68.3% (Figure. 4). The IC50 values of 6c, 6l and CA4 are 2.01±0.2 μM, 1.91±0.23 μM and
1.87±0.01 μM respectively (Table 5).
12

Figure 4. Effect of compounds on tubulin polymerization: Tubulin polymerization was monitored by the

increase in fluorescence at 360 nm (excitation) and 420 nm (emission) for 1 h at 37 C. Synthesized
compounds 6c and 6l were included at a final concentration of 1 µM. CA4 was used as a positive control.
A) Real time kinetic graph of tubulin polymerization: compounds 6c and 6l and CA4 at 1 µM
concentration. B) Percentage of tubulin polymerization inhibition compared to control.
Table 5. IC50 concentration of tubulin polymerization inhibition by mimics 6c, 6l and CA4.
Compounds
Tubulin assembly
IC50 ± SD (μM)
2.01±0.2
6c
1.91±0.23
6l
CA4
1.87±0.01
To validate the effect of these compounds on the disruption of microtubule dynamics in living
cells, studies were carried out to examine the in situ effects of 6c and 6l on cellular microtubules.
HeLa cells seeded on sterile cover slips were treated with these compounds and CA4 was
employed as a standard at a concentration of 60 nM for 48 h. The confocal images depicted in
Figure 5 showed that the untreated human cervical cancer cell line displayed the normal
distribution of microtubules. However, cells treated with compounds 6c and 6l showed disrupted
microtubule organization as seen in Figure 5, thus demonstrating the inhibition of tubulin
polymerization. The density of microtubules was pronounced at the cell periphery with
disorganized central networks and the standard CA4 also showed disrupted microtubule
organization. This immunofluorescence study showed that the level of tubulin polymerization
inhibition was comparable to that of CA4 for both 6c and 6l.
13

Figure 5. HeLa cells were treated with compounds 6c and 6l at 60 nM concentration for 48 h followed by
staining with α-tubulin antibody. Microtubule organization was clearly observed by green colour tubulin
network like structures in control cells and was found to be disrupted in cells treated with compounds 6c
and 6l. CA4 was used as positive control. All pictures were taken in confocal microscopy equipped with
60X objective at scale bar 50 µM.
Docking Studies
The above studies confirmed that both the potent mimics (6c and 6l) disrupt the microtubule
assembly by inhibition of tubulin polymerization as in case of CA4. We then proceeded to
understand the possible mode of binding of by 6c and 6l at the colchicine site by performing a
series of molecular docking analysis. These compounds were designed on the basis of the CA4
skeleton with modification of ring A and B in addition to ring replacement of the olefinic bridge.
The lowest energy docking model resembles cochicine binding pose in the crystal structure
(Figure 6A).37 Pyridine ring mimics role of olefinic bond and CA4 mimics in cis-configuration.38
The trimethoxyphenyl group in the benzothiazole mimic 6c binds in hydrophobic region lined by
residues Cys241β, Leu242β, Leu252β, Leu255β, Ala316β, Val318β, and Ile378β. Specifically,
R1 methoxy fits in a hydrophobic pocket formed by Cys241β, Leu242β, Leu252β and Leu255β.
Occupancy of this pocket seems to provide anchor point for the benzothiazole/benzimidazole
14

CA4 mimics. Activity of 5l/6c further establishes that steric factor at R1 is crucial for activity and
trimethoxy group is not essential. Moderate activity of 5d-k and 6g-k (where R1 is ‘H’ and R2 is
‘OMe/CF3’), further strengthens this argument. This assumption agrees well with prior
observations where modifications of trimethoxyphenyl moiety were well tolerated.39 Further, the
backbone carbonyl of Val238β is close enough to R2 position. It might be interesting to
substitute a hydrogen-bond donor at R2 position. 6-Methoxy group of benzimidazole moiety fits
into a hydrophobic pocket formed by residues Val181α, Thr314β, Val315β, Ala316β and
Met259β. It is notable that benzimidazole rings fits in between Asn258β and Lys352β. Lys350β
(equivalent to Lys352β) has been identified as a key residue by mutational studies.40 Hence,
addition of polar groups at position 4/5 of benzimidazole ring might improve potency by
hydrogen-bonding with Lys352 side chain. The docked pose (Figure 6B) predicted along with
the key interacting residues, also agrees well with the pharmacophore model developed based on
diverse colchicine binding site inhibitors.41
Fig 5A
Fig 5B
Figure. 6. A) 6c docked pose at the αβ interface of tubulin. Residues within 3.5 A of 6c are shown.
Backbone of residues is shown in cartoon representation. Backbone of α chain is shown in green and β
chain in purple. B) Overlay of DAMA-Colchicine and 6c docked pose is shown in orange and DAMA-
Colchicine is colored by element. Backbone of α chain is shown in green and β chain in purple
Apoptosis Induction
Apoptosis is a tightly regulated process of destruction of undesirable cells during development or
homeostasis in multicellular organisms. The induction of apoptosis is a well established strategy
in the cancer drug discovery programs. In general, the microtubule perturbing agents are known
15

to arrest cell cycle in G2/M phase, which is followed by the loss of the mitochondrial
transmembrane potential. This result in mitochondrial release of apoptogenic proteins leading to
DNA fragmentation.42 In the present work, the apoptosis inducing ability of these cis restricted
mimics of CA4 was studied which includes perturbation of mitochondrial membrane potential,
activation of caspase cascade and subsequent DNA fragmentation.
Effect on Mitochondrial Membrane Depolarization
In order to quantify the loss of mitochondrial membrane potential using JC-1 stain, we also
investigated the effects of 6c, 6l and CA4 on the JC-1 red/green ratio signals from isolated
mitochondria. This was studied because the interference from the cytoplasm sometimes renders
the interpretations of JC-1 signals in intact cells to be difficult.
Figure 7. The emission ratio of the red and green fluorescence is shown. The first significant increase in
ratio can be seen at 40 nM and 60 nM concentration of 6c and 6l respectively.
The use of isolated mitochondria largely removed the contributions from the cytoplasm redox-
dependent signal transduction pathways. In this study, the mitochondria were challenged with 6c,
6l and CA4 in the presence of JC-1 stain. Results indicated that the JC-1 red/green ratio signal
did not change in the control condition, indicating that the inner mitochondrial membrane
potential was stable. Compounds 6c, 6l and CA4 were treated at 40 and 60 nM concentrations for
48 h. After incubation for 40 min, compounds 6c and 6l caused a slight decrease in the JC-1
16

red/green ratio signal when compared to control, whereas at 60 nM the compounds rapidly
decreased the JC-1 red/green signal. The standard CA4 showed similar effect at both the
concentration and 6l exhibited comparable result to CA4 at higher concentration. The results
demonstrated that 6c, 6l and CA4 rapidly decreased the JC-1 red/green ratio signal,
corresponding to depolarization of the inner mitochondrial membrane potential (Figure 7).
In the present investigation we performed experiments to study the effect of 6c and 6l in
triggering apoptosis in cancer cells.43 It is well known that caspases play a crucial role in the
induction of apoptosis and amongst the caspase cascade. Caspase-3 is one of the effector caspase
which in turn is activated by caspase-9. Therefore to determine apoptosis inducing activity,
conjugated caspase-3 and -9 substrates were employed and HeLa cells were treated with 40 nM
and 60 nM concentrations of 6c and 6l along with CA4 as the positive control. The activation of
caspase-9 and -3 were examined at two different time intervals as described in the procedure.
Results indicate that 6l exhibits superior caspase-3 activation than the standard CA4 with nearly
2 fold induction in both the caspases at 48 h (Figure 8).
30000
Caspase 9
25000
Caspase 3
20000
15000
10000
5000
0
24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h
Control
6c 40 nM 6c 60 nM 6l 40 nM 6l 60 nM CA-4 40 nM CA-4 60 nM
Figure 8. Effect of 6c and 6l at 40 nM and 60 nM concentrations on caspase-3 and caspase-9 in HeLa
cells determined by fluorimetry at different time intervals, and CA4 is employed as the positive control.
In the present investigation, a series of cis-restricted benzimidazole (5a-l) and benzothiazole (6a-
n) mimics of CA4 were designed and synthesized. On evaluation of cytotoxic activities against
17

selected human cancer cell lines, these mimics exhibited potent activity and the lead compounds
like 6c and 6l displayed potent GI50 values in the range of 0.06±0.001µM and 0.04 ± 0.001 µM,
respectively against HeLa cells (human cervical cancer cell line). SAR studies suggested that
importance of 3,4,5-trimethoxy substitution on ring A compared to 3,4-dimethoxy and 4-
methoxy substitution pattern. The methoxy group on C-6 position of benzimidazole and
benzothiazole moiety was essential for imparting potent antiproliferative activity and was crucial
in these mimic with two electron withdrawing groups on ring A. In the case of mimic 6l with
trifluoromethyl on C-5 and chloro on C-2 position of ring A and methoxy group on C-6 position
of benzothiazole moiety not only exhibited remarkable activity but was superior to CA4. FACS
analysis demonstrated that the growth inhibitory effects of these CA4 mimics (6c and 6l) were
induced because of the arrest of cell cycle progression in G2/M phase comparable to CA4 in a
concentration dependent manner. Tubulin polymerization assay showed that these mimics (6c
and 6l) are also potent inhibitors of tubulin polymerization and moreover the
immunofluorescence studies demonstrated that they disrupt the microtubule organization. To
understand the mode of binding, molecular docking studies were undertaken which demonstrated
these mimics bind at the colchicine site of the tubulin. Further, 6c and 6l demonstrated loss of
mitochondrial dysfunction which leads to two fold increase in induction of caspase-3 as well as
caspase-9 compared to a standard at 40 nM and 60 nM concentrations. The SAR provided a
useful insight that could be utilized in developing the lead compound 6l as a potential
antiproliferative agent. The mechanistic studies discussed are useful for the development of
molecules based on such mimics as potential anticancer agents targeting tubulin.
Associated content
Supplementary data
Experimental procedures and spectra of 1H NMR, 13C NMR, MS and HR-MS of compounds (5a-
l) and (6a-n).
Author Information
Corresponding Author
* Phone: +91 40 27193157; Fax: +91-40-27193189. E-mail: ahmedkamal@iict.res.in.
Acknowledgments
18

M.A acknowledge CSIR-UGC, New Delhi and for the award of senior research fellowship. We
also acknowledge CSIR for financial support under the 12th Five Year plan project “Affordable
Cancer Therapeutics (ACT)” (CSC0301).
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Graphical Abstract
Design and synthesis of cis-restricted benzimidazole and benzothiazole mimics of
combretastatin A-4 as antimitotic agents with apoptosis inducing ability
Md. Ashraf, Thokhir B Shaik, M Shaheer Malik, Riyaz Syed, Prema L Mallipeddi, M V P S
Vishnu Vardhan, Ahmed Kamal*
Design and synthesis of cis-restricted benzimidazole and benzothiazole mimics of combretastatin
A-4 as antimitotic agents with apoptosis inducing ability
23