Rational design and synthesis of 2-anilinopyridinyl-benzothiazole Schiff bases as antimitotic agents

Article abstract of DOI:10.1016/j.bmcl.2017.03.089

Based on our previous results and literature precedence, a series of 2-anilinopyridinyl-benzothiazole Schiff bases were rationally designed by performing molecular modeling experiments on some selected molecules. The binding energies of the docked molecules were better than the E7010, and the Schiff base with trimethoxy group on benzothiazole moiety, 4y was the best. This was followed by the synthesis of a series of the designed molecules by a convenient synthetic route and evaluation of their anticancer potential. Most of the compounds have shown significant growth inhibition against the tested cell lines and the compound 4y exhibited good antiproliferative activity with a GI₅₀ value of 3.8?μM specifically against the cell line DU145. In agreement with the docking results, 4y exerted cytotoxicity by the disruption of the microtubule dynamics by inhibiting tubulin polymerization via effective binding into colchicine domain, comparable to E7010. Detailed binding modes of 4y with colchicine binding site of tubulin were studied by molecular docking. Furthermore, 4y induced apoptosis as evidenced by biological studies like mitochondrial membrane potential, caspase-3, and Annexin V-FITC assays.

Full text of DOI:10.1016/j.bmcl.2017.03.089

Accepted Manuscript
Rational design and synthesis of 2-anilinopyridinyl-benzothiazole Schiff bases
as antimitotic agents
Thokhir B. Shaik, S.M. Ali Hussaini, V. Lakshma Nayak, M. Lakshm i
Sucharitha, M. Shaheer Malik, Ahmed Kamal
PII:
S0960-894X(17)30337-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.089
BMCL 24841
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 October 2016
27 March 2017
29 March 2017
Please cite this article as: Shaik, T.B., Hussaini, S.M.A., Nayak, V.L., i Sucharitha, M.L., Malik, M.S., Kamal, A.,
Rational design and synthesis of 2-anilinopyridinyl-benzothiazole Schiff bases as antimitotic agents, Bioorganic &
Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.03.089
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Rational design and synthesis of 2-anilinopyridinyl-benzothiazole Schiff bases
as antimitotic agents
Thokhir B. Shaik, a,c,§ S. M. Ali Hussaini, a,d,§ V. Lakshma Nayak,a M. Lakshmi Sucharitha,b M.
Shaheer Malik,a,e Ahmed Kamal *a,b
aMedicinal Chemistry and Pharmacology, CSIR ‒ Indian Institute of Chemical Technology,
Hyderabad 500 007, India; bDepartment of Medicinal Chemistry, National Institute of
Pharmaceutical Education & Research (NIPER), Hyderabad 500 037, India.
cDepartment of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur
522510, India
dPresent address: Department of Chemistry, University of Saskatchewan, 110 Science Place,
Saskatoon, SK S7N 5C9, Canada
ePresent address: Science and Technology Unit, Umm Al-Qura University, Makkah, Saudi
Arabia.
§Contributed equally.
Abstract: Based on our previous results and literature precedence, a series of 2-anilinopyridinyl-
benzothiazole Schiff bases were rationally designed by performing molecular modelling
experiments on some selected molecules. The binding energies of the docked molecules were
better than the E7010, and the Schiff base with trimethoxy group on benzothiazole moiety, 4y
was the best. This was followed by the synthesis of a series of the designed molecules by a
convenient synthetic route and evaluation of their anticancer potential. Most of the compounds
have shown significant growth inhibition against the tested cell lines and the compound 4y
exhibited good antiproliferative activity with a GI50 value of 3.8 µM specifically against the cell
line DU145. In agreement with the docking results, 4y exerted cytotoxicity by the disruption of
the microtubule dynamics by inhibiting tubulin polymerization via effective binding into
colchicine domain, comparable to E7010. Detailed binding modes of 4y with colchicine binding
site of tubulin were studied by molecular docking. Furthermore, 4y induced apoptosis as
evidenced by biological studies like mitochondrial membrane potential, caspase-3, and Annexin
V-FITC assays.
Keywords: E7010, Benzothiazole, Cytotoxicity, Cell cycle, Tubulin polymerization.
∗,a,bCorresponding authors. Tel.: +91-40-27193157; fax: +91-40-27193189 (A.K.); e-mail:
ahmedkamal@iict.res.in (A. Kamal).

Mitosis is the last stage of cell cycle characterized by splitting of a parent cell into two
daughter cells. This process is aided by microtubules which are key components of the
cytoskeleton in eukaryotic cells.1 Microtubules play a crucial role in several cellular processes,
one of which include playing out from opposite ends of the cell thereby pulling the sister
chromatids of each chromosome apart leading to cell division.2 These are long, filamentous,
tube-shaped structures formed by the polymerization of α, β-tubulin heterodimers. Microtubules
are highly dynamic polymers that dissociate to tubulin heterodimers and associate back to form
microtubules.3 Any perturbation in these dynamics results in a programmed cell death and this
provides a scope for the development of anticancer agents. Moreover, microtubules are also
reported to play a significant role in angiogenesis, a process by which new blood vessels are
formed, providing nutrients (blood) to the tumor. Interestingly, microtubule-targeting agents
demonstrated antiangiogenic effects cutting off the blood supply by disrupting tumor vasculature
at substantially lower concentrations than those at which antimitotic effect is observed.4
Therefore, targeting microtubules is a validated approach for cancer drug development. As
tubulin is the main component of microtubules, compounds inhibiting tubulin intervene with
microtubule dynamics and arrest the cell cycle in the G2/M phase leading to apoptosis. Among
the four well known binding sites on tubulin, colchicine binding site named after naturally
occurring colchicine (1) is one of the extensively studied targets for developing tubulin
polymerization inhibitors.5 However, in recent past, quite a few molecules structurally distinct
from colchicine have been crystallized in the colchicine binding site. The sulphonamide E7010
(2), is an orally bioavailable tubulin binding agent that exerts cytotoxic effects by binding to the
colchicine binding site.6 It is reported to possess a wide spectrum of antitumor activity and is
even effective against certain multidrug resistant cells. Comparative structural analysis of E7010
binding to colchicine binding site of tubulin indicates that its sulphonamide bridge overlaps with
the B ring while the pyridine and methoxy groups superimpose with A and C rings of the
colchicine respectively.
On the other hand, benzothiazole is a privileged heterocyclic scaffold with diverse biological
properties including anticancer activity.7 Several structurally simple benzothiazoles are reported
to possess excellent in vitro and in vivo cytotoxicity at low nanomolar concentrations. Phortress
(3), a prodrug is one of the best examples of such drug candidates presently undergoing phase I
clinical trials. 8 Moreover, it is evident from the literature that a large number of molecules

possessing potential anticancer activity are the outcome of modifications done on the
benzothiazole scaffold. Therefore, based on our previous work on the development of newer
anticancer agents based on E7010 10 as well as benzothiazole structures,11 a series of 2-
anilinopyridinyl-benzothiazole Schiff bases were rationally designed by performing molecular
docking experiments taking into account the pharmaceutical background of Schiff bases.12 We
herein report the synthesis and biological evaluation of these 2-anilinopyridinyl-benzothiazole
Schiff bases (4a-ad), their ability to inhibit tubulin polymerization apart from inducing
apoptosis.
Figure 1: Anticancer agents.
In recent years, our research endeavors on the development of newer anticancer agents
based on E7010 has produced fruitful results.10 From the previous reports and experiences, we
have observed that 2-anilinopyridine moiety of E7010 is crucial for the anticancer activity and
attempts to cyclize the secondary amine was deleterious for the cytotoxic activity.13 In this
context, one of the 2-anilinopyridinyl-oxadiazole conjugates (compound 5) developed in our
laboratory was found to be an effective inhibitor of tubulin polymerization.14 Moreover in
another effort, improved cytotoxicity was observed on conjugating benzothiazole and 2-
anilinopyridine moieties via an amide functionality.15 In continuation of our research in this area,

we have rationally designed a new molecular structure retaining 2-anilinopyridine moiety and
replacing the amino oxadiazole core of 5 with imino thiazole fused to a benzene ring (Figure 2).
The trimethoxy anilinopyridine was also retained as the trimethoxy group is known to show
crucial interactive potential with microtubules.16 The imine functionality as Schiff base, was
selected taking into account the conformational flexibility it imparts, which is presumed to be
helpful in efficiently interacting at the binding pocket of colchicine domain.12
2.1 Design:
Figure 2: Strategy of the design.
In this regard, in silico molecular modeling studies have been carried out on certain
structures obtained by tethering 2-trimethoxyanilinopyrdine and benzothiazole moieties via an
imine linkage. These structures with variable substituents on benzothiazole nucleus were docked
into the colchicine binding site of tubulin. The protein structure of tubulin was downloaded from
the protein data bank (PDB code: 3E22) and the docking studies were performed using the
software Autodock 4.2.17 Figures 3A, 3B, 3C, and 3D represent the binding modes of
compounds possessing fluoro, chloro, methoxy and trimethoxy substituents on the benzothiazole
with colchicine binding site of tubulin. As evident from these figures (3A, 3B, and 3C),
compounds with fluoro, chloro and methoxy substituents on the benzothiazole interact with the
protein by establishing two and one hydrogen bonding interactions each, respectively. Whereas
the compound bearing trimethoxy substituent on benzothiazole establishes three hydrogen
bonding interactions as shown in Figure 3D. It is noteworthy that in the case of monosubstitution
on benzothiazole, either benzothiazole or 2-anilinopyridine moiety is involved in the hydrogen
bonding interaction. However, in the case of trimethoxy substituent, two out of the three

hydrogen bonding interactions are established by 2-anilinopyridine moiety while benzothiazole
is involved in only one interaction (discussed in detail in molecular modeling section). This
presupposition is supported by the binding energies of these molecules with the tubulin.
Compounds with fluoro, chloro, methoxy and trimethoxy substituents have binding energies in
the order -9.02, -9.20, -8.80, -9.26 kcal/mol, respectively. Interestingly, the standard molecule
E7010, a known inhibitor of colchicine binding site shows a binding energy of -8.64 kcal/mol.
Therefore, it was considered of interest to synthesize these conjugates along with related ones
and study their biological profile experimentally.
Figure 3: Binding modes of compounds 4g (A), 4h (B), 4x (C) and 4y (D) with colchicine
binding site.
The aniline intermediates required for the synthesis of the final Schiff bases were
obtained by following a convenient synthetic route outlined in Scheme 1. Commercially
available 2-chloro-3-nitropyridine (6) was heated with substituted anilines (7a-f) in ethylene
glycol at 140 ˚C for 8 hours. This aromatic nucleophilic substitution reaction yielded substituted
2-anilino-3-nitropyridines (8a-f). The substituents on anilines used for this reaction include
fluoro, chloro, methoxy, dimethoxy and trimethoxy. Reduction of 2-anilino-3-nitropyridine
derivatives with the help of reducing agent stannous chloride (SnCl2.2H2O) in methanol gave the
amine precursors in good yields (9a-f).

Scheme 1. Reagents and conditions: a) Ethylene glycol, 140 ˚C, 8 h; b) SnCl2.2H2O, methanol,
80 ˚C, 3 h.
The aldehyde precursors were synthesized by following the synthetic route described in
Scheme 2. Commercially available substituted anilines (7a-e) were acetylated using acetic
anhydride (10) in dichloromethane (CH2Cl2). Substituents of different nature such as fluoro,
chloro, methoxy and trimethoxy along with neutral hydrogen were used on aniline to study their
effect on anticancer activity. The acetanilides formed (11a-e) were converted to their
thioacetylated product (12a-e) by heating with Lawesson’s reagent in dioxane. The
thioacetanilides were cyclized to substituted 2-methylbenzothiazole derivatives (13a-e) via
Jacobsen cyclization procedure i.e., by charging sodium hydroxide solution of thioacetanilides
into preheated aqueous potassium ferricyanide K3Fe(CN)6 solution. Subsequently, the 2-
methylbenzothiazole derivatives (13a-e) were refluxed with the oxidizing agent selenium dioxide
(SeO2) in dioxane to yield the aldehyde precursors (14a-e) in significant yields.

H
H
N
X
Y
N
X
Y
X
Y
NH2
O
O
a
b
+
S
O
O
Z
Z
Z
12a-e
7a-e
11a-e
10
a: X=Y=Z=H
b: X=Z=H, Y=F
c
c
: X=Z=H, Y=Cl
d: X=Z=H, Y=OCH3
e: X=Y=Z=OCH3
X
Y
X
Y
H
O
N
S
N
d
S
Z
Z
14a-e
13a-e
Scheme 2. Reagents and conditions: a) CH2Cl2, rt, 15 mins; b) Lawesson’s Reagent, dioxane,
110 ˚C, 2 h; c) K3Fe(CN)6, 8N NaOH, 80 ˚C, 0.5 h; d) SeO2, dioxane, 110 ˚C, 12 h.
With both the amine and aldehyde fragments in hand, the synthesis of target Schiff bases
was achieved in a convenient manner as described in Scheme 3. The amine precursors (8a-f)
obtained from Scheme 1 and aldehyde precursors (13a-e) prepared according to the Scheme 2
were condensed in ethanol at room temperature to afford the diversely substituted desired
compounds (Schiff bases) in good yields (4a-ad).
Scheme 3. Reagents and conditions: a) Ethanol, rt, 1-2 h.

These 2-anilinopyridinyl-benzothiazole Schiff bases were evaluated for their anticancer
potential against three different human cancer cell lines, namely A549, DU145, and MCF7.
Cancer cells were seeded in 96 well plate at a density of 2.5 X 104 cells per well. All the
compounds were tested against each cell line at various concentrations namely 0.1, 1, 5 and 10
µM and their growth inhibitory effects were determined by Sulforhodamine B assay after 48 h.
Sulphonamide E7010 along with the standard drug 5-fluorouracil (5-FU) were taken as positive
controls. From the growth inhibition data, GI50 values have been calculated which are tabulated
in Table 1.
As seen from the data, 2-anilinopyridinyl-benzothiazole conjugates showed considerable
cytotoxicity. The inhibitory effects due to these conjugates were observed to be of varied
magnitude against these test cell lines. Some of these conjugates were found to be active below
20 µM concentration,however some were not active even at 100 µM. Interestingly, these
conjugates have displayed specificity toward the prostate cancer cell line (DU145) and most of
them have demonstrated significant cytotoxicities against it. Compound 4y was the most potent
conjugate of the series exhibiting significant to good antiproliferative activities against the cell
lines A549, MCF 7 and DU145 with GI50 values of 17, 12 and 3.8 µM, respectively. Similarly,
compound 4t was found to be an effective conjugate from the series displaying a GI50 of 4.3 µM
against the cell line A549. Moreover, other conjugates such as 4e, 4h, 4l, 4s, 4t and 4w have also
exhibited significant antiproliferative activities against the corresponding cell lines. Further,
these conjugates were evaluated for their cytotoxic effects on the normal human lung cells
(MRC5). All the conjugates along with the standard were tested at various concentrations
ranging from 1 to 100 µM and the results demonstrated that all the conjugates showed no
significant activity up to 100 µM concentration except 4j which showed the GI50 value of 91.1
µM.
During the discussion in chemistry part, the various substituents used in the synthesis of
2-anilino pyridine and benzothiazole moities of the desired Schiff bases have been described.
The main objective of diversifying the final target structure is to study the effect of substituents
on the anticancer activity. From the anticancer activity data shown in Table 1 it was analyzed
that trimethoxy group on the benzothiazole nucleus is fruitful for cytotoxicity. In the presence of
trimethoxy group on the benzothiazole moiety, both ring activating as well as deactivating

substituents at the 4th position of the aryl ring of 2-anilinopyridinyl moiety bestow significant
cytotoxicity. This effect was observed in the absence of substituent also, but not for a 3,5-
dimethoxy substituent. Effect of fluoro and chloro substituents on benzothiazole nucleus was
also observed to be significant. These groups confer good cytotoxicity in the presence of ring
deactivating substituents on 2-anilinopyridinyl moiety.
Compounds containing ring deactivating groups on 4th position of the aryl ring of 2-
anilinopyridinyl moiety have demonstrated better antiproliferative effects than the compounds
with ring activating substituents. Thus, fluoro and chloro groups on 4th position of the aryl ring
of 2-anilinopyridinyl moiety are desirable while methoxy group at this position is not tolerable.
However 3,5-dimethoxy substituent on 2-anilinopyridinyl confers significant activity.
Interestingly the trimethoxy group, on both anilinopyridine and benzothiazole moiety, imparts
most potent activity in comparison to others.
Table 1: Cytotoxic activity (aGI50 in µM) data of compounds, 4a-4ad by SRB method.
Compd
aGI50 values in µM
bA549
cMCF 7
85.1 0.42
N.A
dDU145
N.A
N.A
eMRC5
f
N.A
42.7 0.36
N.A
76.3 0.28
N.A
N.A
N.A
14.2 0.16
> 100
12.3 0.22
16.7 0.18
N.A
8.6 0.19
31.6 0.26
12.4 0.22
11.1 0.19
N.A
-
-
-
-
-
-
-
-
4a
4b
4c
4d
4e
4f
f
f
56.5 0.48
32.9 0.23
12.4 0.18
10.7 0.26
16.3 0.15
11.6 0.22
40.5 0.31
20.0 0.29
17.7 0.20
31.4 0.14
41.6 0.43
N.A
N.A
N.A
N.A
N.A
N.A
N.A
44.4 0.22
N.A
f
8.8 0.11
10.7 0.14
N.A
N.A
16.6 0.16
N.A
25.2 0.30
15.7 0.35
4.8 0.24
12.7 0.21
N.A
8.6 0.36
32.6 0.48
18.9 0.19
47.4 0.24
N.A
f
f
f
4g
4h
4i
f
f
-
91.1 0.5
4j
f
-
-
-
-
-
-
-
-
4k
4l
f
f
4m
4n
4o
4p
4q
4r
4s
f
f
f
f
f
N.A
f
5.8 0.26
4.3 0.17
N.A
-
-
-
f
12.5 0.16
N.A
4t
f
4u

f
f
f
f
f
f
f
f
f
f
f
N.A
N.A
N.A
17.0 0.18
N.A
18.9 0.22
30.7 0.28
14.0 0.26
58.6 0.49
1.31
N.A
10.8 0.13
7.9 0.26
20.7 0.16
3.8 0.15
N.A
-
-
-
-
-
-
-
-
-
-
-
4v
18.6 0.16
30.0 0.52
12.0 0.24
15.1 0.16
11.3 0.22
21.8 0.25
19.9 0.34
19.8 0.29
1.53
4w
4x
4y
4z
N.A
4aa
4ab
4ac
4ad
E7010
5-FU
16.2 0.18
15.9 0.24
N.A
1.81
5.82
2.09
1.67
Note: a Concentration of drug causing 50% inhibition of cell growth, bA549- lung carcinoma cell
line, cMCF 7- breast cancer cell line, dDU145- prostate cancer cell line. eMRC5 normal lung
cells, -f GI50 values >100 µM.
In order to understand the mechanism of action of Schiff base with two trimethoxy
groups 4y, the microtubule inhibitory ability was investigated and correlated with the docking
results. The progression of tubulin polymerization18 was thus examined by monitoring the
absorbance at 340 nm in 384 well plate for 1 h at 37 °C with and without the conjugate at 3 µM
concentration in comparison with reference compound E7010. Thus compound (4y) significantly
inhibited tubulin polymerization by 59.57 %, whereas the reference compound (E7010) exhibited
60.64 % inhibition (Figure 4). This was followed by evaluation of IC50 values for this compound
and the results are shown in Table 2. The test compound 4y showed tubulin-assembly inhibition
with an IC50 value of 4.22 µM, whereas the standard E7010 exhibited IC50 of 2.51 µM. The
effect of these compounds on the inhibition of tubulin assembly correlated well with the in silico
molecular modeling studies.
Table 2: Inhibition of tubulin polymerization (IC50) of compounds 4y and E7010.
Compound
4y
aIC50 SD (in µM)
4.22 0.13
E7010
2.51 0.15
a
Note: Concentration of drug to inhibit 50% of tubulin assembly.

3.2. Effect of compounds on tubulin polymerization
Figure 4: Effect of compound on tubulin polymerization: Tubulin polymerization was monitored
by increase in absorbance at 340 nm for 1 h at 37 ˚C. E7010 was used as a positive control.
Values indicated are the mean SD of two different experiments performed in triplicates.
As conjugate 4y, showed good inhibitory effects on tubulin polymerization similar to that
of E7010 which is known to bind into colchicine binding site of the tubulin, it was considered of
interest to investigate the binding of 4y on this site of the tubulin. Hence, a fluorescence-based
assay19 was carried out taking E7010 as the positive control and vinblastine as the negative
control. Thus 4y, E7010 and vinblastine were separately coincubated with colchicine at 37 ˚C for
60 minutes and subsequently their fluorescence were measured. Figures 5A, 5B and 5C represent
the scan of fluorescence wavelength for tubulin-colchicine complex from 600 to 390 nm fixing
the excitation wavelength at 350 nm when coincubated with 4y, E7010 and vinblastine
respectively at various concentrations. From the Figures it is clear that the tubulin-colchicine
complex gives fluorescence at 440 nm when excited at 350 nm, therefore the fluorescence was
measured at the same wavelength. It is also noteworthy from the figures that 4y and E7010
caused a significant decrease in the fluorescence whereas no such effect was observed in the case

of vinblastine. Similarly, Figure 5D represents the change in fluorescence intensity of tubulin-
colchicine complex with the concentration of 4y, E7010 and vinblastine. As evident from the
figure, a significant decrease in the fluorescence was observed in the case of 4y and E7010.
However, vinblastine exerts no effect on the complex fluorescence as it binds at a different site
on the tubulin. These observations indicate that 4y and E7010 compete to bind to the tubulin at
the colchicine binding site. Interestingly, 4y caused greater lowering of the complex fluorescence
than E7010 indicating that it is an effective inhibitor of colchicine binding site.
3.3. Competitive colchicine binding assay
Figure 5: Scan of fluorescence wavelength of tubulin colchicine complex coincubated at
different concentrations with A) 4y B) E7010 C) Vinblastine D) Variation of fluorescence of
tubulin-colchicine complex observed from Fluorescence based colchicine competitive binding
assay of 4y were carried out at various concentrations containing 3 µM of tubulin and colchicine
for 60 min at 37 ˚C. E7010 was used as a positive control whereas vinblastine was used as
negative control which binds at vinca site. Fluorescence values are normalized to DMSO
(control).

As discussed previously, molecular modeling studies have been carried out to get an
insight into the binding modes of 4y with tubulin. The protein structure of tubulin was
downloaded from protein data bank (PDB code: 3E22) and the docking studies were performed
in the colchicine binding domain of tubulin using the software Autodock 4.2. The results suggest
that 4y binds at the interface of α and β chains of tubulin heterodimer which is depicted in Figure
6A. As evident from the figure several amino acid residues such as Asn 101, Thr 179, Ala 180,
Val 181, Asn 249, Ala 250, Leu 252, Arg 253, Lys 254, Leu 255, Lys 352, Thr 353 etc surround
4y in the binding pocket. A series of hydrophobic interactions have been observed between most
of these amino acid residues with aryl and pyridine rings of 4y. Additionally, two methoxy group
of 2-trimethoxyanilinopyridine moiety establishes a hydrogen bonding interaction each with the
carboxylate group of the amino acid residue αAsn 101 (distance 2.9 and 3.3 Å respectively). In
the same way, one of the methoxy groups of benzothiazole interacts with the hydroxyl group of
βTyr202. As shown in Figure 6B, the benzothiazole moiety of 4y is deeply buried in the β
subunit of the tubulin while the 2-anilinopyridine moiety is exposed outside towards the α chain,
wherein it establishes several polar and hydrophobic interactions including hydrogen bonding
interactions with the surrounding amino acid residues. All these observations illustrate that 4y
exerts a cytotoxic effect by effectively interacting with colchicine binding domain of the tubulin
which is evident from the competitive colchicine binding assay.
3.4. Molecular modeling studies
Figure 6: A) Binding pose of 4y (orange) with tubulin. α and β chains of the protein are colored
as green and cyan for better understanding. B) Binding pose of 4y in β subunit of tubulin.

Drug likeliness properties of the test compound 4y was carried out using computational
approach as described in the experimental section along with E7010. The predicted values of 4y
was compared with the standard E7010 and the values are presented in the Table 3. It was
observed that 4y and E7010 showed resemblance in CYP2C19, CYP2C9, CYP2D6 inhibition
and bioavailability score. Both the compounds inhibited the CYP2C9 enzyme with no inhibition
of CYP2C19 and CYP2D6 enzymes. Moreover, compound 4y acted as a weak substrate to
CYP3A4 enzyme compared to E7010, which does not act as substrate to this enzyme. Plasma
protein binding values of 4y and E7010 suggested that lower dose of 4y is required compared to
E7010 as blood plasma protein has less bound 4y compared to E7010. According to the
molinspiration calculation both the compounds have comparable bioactive properties with the
acceptable limits, such as TPSA values are less than 140 Å, whereas greater than this value may
show poor intestinal absorption.20,21 HIA prediction was important to understand human
intestinal absorption of drug, preADMET predictor can be used to identify the percent human
intestinal absorption of drug.22 HIA percentage values of 4y and E7010 suggested that 4y has
more human intestinal permeability compared to E7010. Further, identification of drug molecule
interaction with P-gp protein is an important property for lead optimization. Multi drug
resistance (MDR) is also one the major challenge for the new molecules and P-gp, that was
identified as one of the main ABC transporter protein responsible for MDR development in
cancer cells. To improve the ADMET profile of drug molecules FDA has suggested the
importance of finding whether a new molecule is a P-gp substrate or inhibitor. Most of the drug
candidates that are identified as P-gp substrate can be readily effluxed, leading to decreased
bioavailability in blood plasma.23-25 In silico preADMET prediction showed that 4y was a P-gp
inhibitor, whereas E7010 was neither inhibitor nor substrate. The above ADMET prediction data
suggested that the compound 4y showed moderate to good ADMET properties compared to
E7010 which provides further opportunities for the development of similar therapeutic potential
derivatives (Table 3).

Table 3: ADME properties of the compounds 4y and E7010 studied by in silico preADMET
prector and molinspiration programme.
Properties
E7010
0.352325
Non
Inhibitor
Non
Non
Inhibitor
Non
91.716935
Non
100
0.55
6
4y
0.477624
Non
Inhibitor
Non
Non
Non
Weakly
98.583151
Inhibitor
89.509825
0.55
BBB
CYP_2C19_inhibition
CYP_2C9_inhibition
CYP_2D6_inhibition
CYP_2D6_substrate
CYP_3A4_inhibition
CYP_3A4_substrate
HIA
Pgp_inhibition
Plasma_Protein_Binding
Bioavailability Score
Rotatable bonds
H-bond acceptors
H-bond donors
miLogP
10
9
1
4.12
5
3
3.33
100.55
26
105.58
36
TPSA
natoms
371.42
510.57
MW
7
10
nON
3
0
1
1
nOHNH
nviolations
nrotb
6
312.5
10
442.19
volume
Many anticancer compounds exert their growth inhibitory effect either by arresting the
cell cycle at a particular checkpoint or by induction of apoptosis or a combined effect of both cell
cycle block and apoptosis.26 Furthermore regulation of the cell cycle and apoptosis are
considered to be effective cancer therapeutic methods.27 In general, the inhibition of tubulin
polymerization is associated with the cell cycle arrest in G2/M phase.28 Therefore, it was
considered of interest to study the cell cycle arrest and the apoptosis inducing ability of
compound 4y. In this investigation, DU-145 cells were treated with compound 4y at 2.5 and 5
µM concentrations along with the reference compound E7010 (5 µM) for 48 h. The data

obtained clearly indicated that this compound arrested cell cycle at G2/M phase as compared to
the untreated control (Figure 7). The test compound, 4y showed 37.85 and 66.09 % of cell
accumulation in G2/M phase at 2.5 and 5 µM concentrations, whereas the reference compound
E7010 showed 50.21 % cell accumulation in G2/M phase at 5 µM concentration.
3.5. Cell cycle analysis
Figure 7 : Cell cycle distribution in DU-145 cells treated with compound 4y at 2.5 and 5 µM
concentrations for 48h. After 48 h of drug treatment with 4y samples were analyzed by flow
cytometry. A: Control cells (DU-145), B: E7010 (5 µM), C: 4y (2.5 µM) and D: 4y (5 µM).

Table 4: Distribution of DU145 cells in various phases of cell cycle
Sample
Sub G1 % G0/G1 % S %
G2/M %
11.85
A: Control
0.68
82.09
34.94
55.96
30.47
3.04
0.97
2.52
0.71
B: E7010 (5µM) 0.42
62.59
C: 4y (2.5 µM) 1.00
38.51
D: 4y (5 µM)
0.46
67.56
The maintenance of mitochondrial membrane potential (∆Ψm) is significant for
mitochondrial integrity and bioenergetic function.29 Mitochondrial changes, including loss of
mitochondrial membrane potential (∆Ψm), are key events that take place during drug-induced
apoptosis. Mitochondrial injury by 4y was evaluated by detecting drops in mitochondrial
membrane potential (∆Ψm). In this study, we investigated the involvement of mitochondria in
the induction of apoptosis by compound 4y. After 48 h of drug treatment with 4y at 2.5 and 5
µM, reduction in mitochondrial membrane potential (∆Ψm) of DU-145 cells was observed which
was assessed by JC-1 staining (Figure 8).
3.6. Measurement of Mitochondrial Membrane Potential (∆Ψm)

Figure 8: Compound 4y triggers mitochondrial injury. Drops in membrane potential (∆Ψm) was
assessed by JC-1 staining of DU-145 cells treated with test compound and samples were then
subjected to flow cytometry analysis on a FACScan (Becton Dickinson) in the FL1, FL2 channel
to detect mitochondrial potential. Shown are representative dot plots (left panels) and
quantification of membrane potential (right panel). A: Untreated control cells (DU-145), B:
E7010 (2.5 µM), C: 4y (2.5 µM) and D: 4y (5 µM).
The apoptotic effect of compound 4y was further evaluated by Annexin V FITC/PI (AV/PI) dual
staining assay30 to examine the occurrence of phosphatidylserine externalization and also to
understand whether it is due to physiological apoptosis or nonspecific necrosis. In this study,
DU- 145 cells were treated with compound 4y for 48 h at 2.5 and 5 µM concentrations. It was
observed that this compound showed significant apoptosis against DU- 145 cells as shown in
Figure 9. Results indicated that compound 4y, showed 16.74 and 25.37 % of apoptosis at 2.5 and
5 µM concentrations, respectively whereas the standard E7010 showed 18.26 % of apoptosis at
2.5 µM concentration. This experiment suggested that 4y induced cell death by apoptosis in
human prostate cancer cells.
3.7. Annexin V-FITC for apoptosis

Figure 9 Annexin V-FITC assay; quadrants represent, lower left (LL) represents live cells, lower
right (LR) represents early apoptotic, upper right (UR) represents late apoptotic and upper left
(UL) represents necrotic cells. A: Control cells (DU-145), B: E7010 (2.5 µM), C: 4y (2.5 µM)
and D: 4y (5 µM).
Table 5: Distribution of apoptotic cells in Annexin-V FITC experiment
Sample
Upper left
%
Upper right
%
Lower left
%
Lower right
%
0.14
0.93
0.57
0.75
9.88
86.77
80.80
82.69
73.87
3.21
1.35
0.86
1.16
A: Control (DU-145)
B: E7010 (2.5 µM)
C: 4y (2.5 µM)
16.91
15.88
24.21
D: 4y (5 µM)
From previous reports, it is well established that molecules affecting microtubule
polymerization cause mitotic arrest and ultimately lead to apoptosis.31 Caspases are a family of
cysteine-aspartic proteases that are crucial mediators of apoptosis. Among them, caspase-3 is the
best understood in the mammalian caspases in terms of its specificity and role in apoptosis.
Furthermore, there are some reports26 that indicate that the cell cycle arrest at G2/M phase takes
place by the induction of cellular apoptosis. Hence, it was considered of interest to understand
the correlation of cytotoxicity with apoptosis by 4y. DU-145 cells were treated with 4y (2.5 and
5 µM) and examined for the activation of caspase-3 activity. Results indicated that there was
nearly 1.5 to 2 fold-induction in caspase-3 levels compared to the control (Figure 10).
3.8. Effect on activation of caspase 3 activity

Figure 10: Effect of compound 4y on caspase-3 activity: DU-145 cells were treated with
compound 4y at 2.5 and 5 µM concentrations for 48 h. Values indicated are the mean SD of
two different experiments performed in triplicates; *P < 0.0003, **P < 0.0007, and ***P <
0.0002 compared to control.
In conclusion, a new molecular structure of Schiff bases resulting from tethering 2-
anilinopyridinyl and benzothiazole moieties has been designed by in silico molecular modeling
technique. The Schiff base with trimethoxy groups on both the moieties 4y was found to be
better than E7010 in binding to the colchicine domain of tubulin, in silico. Executing the design,
a series of thirty related Schiff bases have been synthesized by diversifying various substituents,
both on 2-anilinopyridinyl as well as benzothiazole nucleus. These conjugates were tested for
their cytotoxicity against three different human cancer cell lines. Some compounds have shown
specificity towards prostate cancer cell line (DU145). Compound 4y was found to be the potent
conjugate from this series exhibiting cytotoxic effects on all the tested cell lines more
specifically against DU145 with a GI50 value of 3.8 µM. Moreover, further studies were carried
out on 4y to determine the mechanistic aspects and correlate with the docking results, which
indicated that it caused cell cycle arrest in G2/M phase by inhibiting tubulin polymerization.
Competitive colchicine binding assay revealed that 4y competes with colchicine in binding to
tubulin. Other detailed biological investigations such as mitochondrial membrane potential,
Annexin V-FITC, and caspase-3 assays suggested that this compound significantly triggers the
apoptotic cell death.
Acknowledgements
T.B.S thanks to UKIERI and British Council and S.M.A.H thanks to CSIR, New Delhi for the
award of a research fellowship. This work was also supported under the 12th Five Year plan
project “Affordable Cancer Therapeutics (ACT)” (CSC0301).

References
1. Perez, E. A. Mol. Cancer Ther. 2009, 8, 2086.
2. Zhou, J.; Yao, J.; Joshi, H. J. Cell Science. 2002, 115, 3547.
3. a) Jordan, M. A.; Wilson, L. Nat. Rev. Cancer. 2004, 4, 253; b) Kingston, D. G. I. J.
Nat. Prod. 2009, 72, 507.
4. a) Schwartz, E. L. Clin Cancer Res. 2009, 15, 2594.; b) Pasquier, E.; Andrè, N.; Braguer,
D. Curr. Cancer Drug Targets. 2007, 7, 566.
5. Lu, Y.; Chen, J.; Xiao, M.; Li, W.; Miller, D. D. Pharm. Res. 2012, 29, 2943.
6. Yokoi, A.; Kuromitsu, J.; Kawai, T.; Nagasu, T.; Sugi, N. H.; Yoshimatsu, K.; Yoshino,
H.; Owa, T. Mol. Cancer Ther. 2002, 1, 275.
7. a) Solomon, V. R.; Hu, C.; Lee, H. Bioorg. Med. Chem. 2009, 17, 7585; b) Hutchinson,
I.; Jennings, S. A.; Vishnuvajjala, B. R.; Westwell, A. D.; Stevens, M. F. J. Med. Chem.
2002, 45, 744.
8. Prota, A.E.; Bargsten, K.; Diaz, J.F.; Marsh, M.; Cuevas, C.; Liniger, M.; Neuhaus, C.;
Andreu, J.M.; Altmann, K.H.; Steinmetz, M.O. Proc Natl Acad Sci U S A. 2014, 111,
13817.
9. a) Kamal, A.; Hussaini, S. M. A.; Nayak, V. L.; Malik, M. S.; Sucharitha, M. L.; Shaik,
T. B.; Ashraf, M.; Bagul, C. Bioorg. Med. Chem. 2014, 22, 6755; b) Kamal, A.; Reddy,
N. V. S.; Nayak, V. L.; Bolla, N. R.; Rao, A. V. S.; Prasad, B. Bioorg. Med. Chem. 2014,
22, 3465; c) Kamal, A.; Khan, M. N. A.; Reddy, K. S.; Rohini, K. Bioorg. Med. Chem.
2007, 15, 1004.
10. a) Kamal, A.; Sultana, F.; Ramaiah, M. J.; Srikanth, Y. V. V.; Viswanath, A.; Kishor, C.;
Sharma, P.; Pushpavalli, S. N. C. V. L.; Addlagatta, A.; Pal-Bhadra, M.
Chem.Med.Chem. 2012, 7, 292; b) Kamal, A.; Mallareddy, A.; Suresh, P.; Shaik, T. B.;
Nayak, V. L.; Kishore, C.; Shetti, R. V. C. R. N. C.; Rao, N. S.; Tamboli, J. R.;
Ramakrishna, S.; Addlagatta, A. Bioorg. Med. Chem. 2012, 20, 3480.
11. Sztanke, K.; Maziarka, A.; Osinka, A.; Sztanke, M. Bioorg. Med. Chem. 2013, 21, 3648.
12. Kamal, A.; Ashraf, M.; Vishnu Vardhan, M. V. P. S.; Faazil, S.; Nayak, V. L. Bioorg.
Med. Chem. Lett. 2014, 24, 147.

13. Kamal, A.; Srikanth, Y. V. V.; Shaik, T. B.; Khan, M. N. A.; Ashraf, M.; Reddy, M. K.;
Kumar, K. A.; Kalivendi, S. V. Med. Chem. Commun. 2011, 2, 819.
14. Kamal, A.; Srikanth, Y. V. V.; Khan, M. N. A.; Ashraf, M.; Reddy, M. K.; Sultana, F.;
Kaur, T.; Chashoo, G.; Suri, N.; Sehar, I.; Wani, Z. A.; Saxena, A.; Sharma, P. R.;
Bhushan, S.; Mondhe, D. M.; Saxena, A. K. Bioorg. Med. Chem. 2011, 19, 7136.
15. Negi, A. S.; Gautam, Y.; Alam, S.; Chanda, D.; Luqman, S.; Sarkar, J.; Khan, F.;
Konwar, R. Bioorg. Med. Chem. 2015, 23, 373.
16.
Cormier, A.; Marchand, M.; Ravelli, R. B. G.; Knossow, M.; Gigant, B. EMBO Rep.
2008, 9, 1101; b) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.;
Goodsell, D. S.; Olson, A. J. J. Comput. Chem. 2009, 16, 2785.
http://autodock.scripps.edu/.
17. Huber, K.; Patel, P.; Zhang, L.; Evans, H.; Westwell, A. D.; Fischer, P. M.; Chan, S.;
Martin, S. Mol. Cancer Ther. 2008, 7, 143.
18. a)Kamal, A.; Balakishan, G.; Ramakrishna, G.; Shaik, T. B.; Sreekanth, K.; Balakrishna,
M.; Rajender, Dastagiri, D.; Kalivendi, S.V. Eur J Med Chem. 2010, 45, 3870.
b)Larocque, K,; Ovadje, P,; Djurdjevic, S,; Mehdi, M,; Green, J,; Pandey, S. PloS one.
2014, 9, e87064.
19. Bhattacharyya, B,; Wolff, J. PNAS-USA. 1974, 71, 2627.
20. Tanwara O.P,; Sahaa, R,; Marellaa, A,; Alama, M.M,; Akhtera, M,; Kumar, D.V. Journal
of Advanced Bioinformatics Applications and Research. 2014, 5, 172-182.
21.
Kovačević, SZ,; Jevrić, LR,; Kuzmanović, SO,; Lončar, ES. Iranian journal of
pharmaceutical research: IJPR. 2014, 13, 899.
22. Nerkar, AG,; Kudale, SA,; Joshi, PP,; Chikhale, HU. Int J Pharm Pharm Sci. 2012, 4,
449-53.
23. Poongavanam, V,; Haider, N,; Ecker, GF. Bioorganic & medicinal chemistry. 2012, 20,
5388-95.
24. Li, D,; Chen, L,; Li, Y,; Tian, S,; Sun, H,; Hou, T. Molecular pharmaceutics. 2014, 11,
716-26.
25. Srivalli, KM,; Lakshmi, PK. Brazilian Journal of Pharmaceutical Sciences. 2012, 48,
353-67.

26. Liu, Z.; Zhou, Z.; Tian, W.; Fan, X.; Xue, D.; Yu, L.; Yu, Q.; Long, Y.-Q.
Chem.Med.Chem. 2012, 7, 680.
27. Chan, K. T.; Meng, F. Y.; Li, Q.; Ho, C. Y.; Lam, T. S.; To, Y.; Lee, W. H.; Li, M.; Chu,
K. H.; Toh, M. Cancer Lett, 2010, 294, 24-118; b) J. K. Shen, H. P. Du, M. Yang, Y. G.
Wang, J. Jin, Ann. Hematol. 2009, 88, 743-752.
28. Blank, M.; Shiloh, Y.; Cell Cycle. 2007, 6, 686.
29. Budihardjo, I.; Oliver, H.; Lutter, M.; Luo, X.; Wang, X. Annu. Rev. Cell Dev. Biol.
1999, 15, 269.
30. (a) Hengartner, M. O. Nature 2000, 407, 770. (b) Sun, S.-Y.; Hail, N., Jr.; Lotan, R. J.
Natl. Cancer Inst. 2004, 96, 662
31. Weir, N. M.; Selvendiran, K.; Kutala, V. K.; Tong, L.; Vishwanath, S.; Rajaram, M.;
Tridandapani, S.; Anant, S.; Kuppusamy, P. Cancer Biol. Ther. 2007, 6, 178.
32. Zhang, L. H.; Wu, L.; Raymon, H. K.; Chen, R. S.; Corral, L.; Shirley, M. A.; Narla, R.
K.; Gamez, J.; Muller, G. W.; Stirling, D. I.; Bartlett, B. J.; Schafer, P. H.; Payvandi, F.
Cancer Res. 2006, 66, 951.

Graphical Abstract