Synthesis, cytotoxic evaluation, and in silico studies of substituted N-alkylbromo-benzothiazoles

Article abstract of DOI:10.1007/s00044-012-0424-0

In efforts to develop a new class of anticancer agents with improved efficacy and selective action, a series of N-alkylbromo-benzothiazoles were synthesized and evaluated for in vitro cytotoxic activity against various human cancer cell lines such as lung (A-549), prostate (PC-3), leukemia (THP-1), and colon (Caco-2). They were found to be highly active against prostate (PC-3) and leukemia (THP-1) cancer cells, moderately active against colon (Caco-2) cancer cells and less active against lung (A-549) cancer cells. Of the 12 compounds, two (11d, 11j) exhibit IC₅₀ values of ≤ 1 μM against leukemia (THP-1) cancer cell lines. Compound 11l showed significant cytotoxic activity against the PC-3 (IC₅₀ = 0.6 μM), THP-1 (IC₅₀ = 3 μM) and Caco-2 cell lines (IC₅₀ = 9.9 μM), respectively. Docking study of the synthesized ligand was done on epidermal growth factor receptor using ArgusLab flexible docking, to determine their observed activity. Further QSAR investigations with stepwise multiple linear regression analysis were applied to find correlation between various physicochemical parameters and anticancer activity. The QSAR results showed that anticancer activity could be modeled with descriptors. The predictive ability of models was cross-validated by observation of the low residual activity values and adjusted coefficient of variation (r_adj²) obtained by leave-one-out technique.

Full text of DOI:10.1007/s00044-012-0424-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0424-0
ORIGINAL RESEARCH
Synthesis, cytotoxic evaluation, and in silico studies of substituted
N-alkylbromo-benzothiazoles
•
Rupinder Kaur Gill Gagandeep Singh
•
•
Anuradha Sharma P. M. S. Bedi
•
A. K. Saxena
Received: 12 October 2012 / Accepted: 14 December 2012
Ó Springer Science+Business Media New York 2012
Abstract In efforts to develop a new class of anticancer
agents with improved efficacy and selective action, a series of
N-alkylbromo-benzothiazoles were synthesized and evalu-
ated for in vitro cytotoxic activity against various human
cancer cell lines such as lung (A-549), prostate (PC-3), leu-
kemia (THP-1), and colon (Caco-2). They were found to be
highly active against prostate (PC-3) and leukemia (THP-1)
cancer cells, moderately active against colon (Caco-2) cancer
cells and less active against lung (A-549) cancer cells. Of the
12 compounds, two (11d, 11j) exhibit IC50 values of B 1 lM
against leukemia (THP-1) cancer cell lines. Compound 11l
showed significant cytotoxic activity against the PC-3
(IC50 = 0.6 lM), THP-1 (IC50 = 3 lM) and Caco-2 cell
lines (IC50 = 9.9 lM), respectively. Docking study of the
synthesized ligand was done on epidermal growth factor
receptor using ArgusLab flexible docking, to determine their
observed activity. Further QSAR investigations with stepwise
multiple linear regression analysis were applied to find cor-
relation between various physicochemical parameters and
anticancer activity. The QSAR results showed that anticancer
activity could be modeled with descriptors. The predictive
ability of models was cross-validated by observation of the
low residual activity values and adjusted coefficient of vari-
ation (ra2dj) obtained by leave-one-out technique.
Keywords 2-Aminobenzothiazole ꢀ Dibromoalkane ꢀ
N-Alkylation ꢀ Cytotoxic activity ꢀ Sulforhodamine assay
Introduction
Cancer is a renegade system of growth that originates within
a patient’s biosystem, which are of different types but all
share one hallmark characteristic, unchecked growth that
progresses toward limitless expansion (www.cancer.gov.,
2012). Currently, one in four deaths in the United States is
due to cancer (Siegel et al., 2011). Despite the treatment
efficacy, cytotoxicity of anticancer drugs to the normal cells
is the major problem in cancer therapy that engenders the risk
of inducing secondary malignancy (Saeed et al., 2010). To
improve this, in the recent years, there has been a concerned
research for the discovery and development of novel selec-
tive anticancer agents that impede the progression of
malignant tumors or prevent their recurrence.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-012-0424-0) contains supplementary
material, which is available to authorized users.
A number of nitrogen and sulfur-containing heterocyclic
compounds such as indole (Singh et al., 2009), isatin (Sol-
omon et al., 2009), benzothiazole, oxadiazole (Zhang et al.,
2011), etc., show anticancer activities. Among them, ben-
zothiazole moiety shows interesting anticancer activity by
inhibiting tyrosine kinase (Bhuva and Kini, 2010), aurora
kinase (Tasler et al., 2009), topoisomerase II (Pinar et al.,
2004), and ubiquitin proteasome system (Guedat and Col-
land, 2007). In addition, benzothiazole derivatives also
exhibit a variety of biological functions such as anticonvul-
sant (Siddiqui et al., 2007), antitubercular (Palmer et al.,
R. K. Gill ꢀ G. Singh ꢀ P. M. S. Bedi (&)
Department of Pharmaceutical Sciences, Guru Nanak Dev
University, Amritsar 143 005, Punjab, India
e-mail: bedi_preet@yahoo.com; research.medchem@gmail.com
A. Sharma
University Institute of Pharmaceutical Sciences, Panjab
University, Chandigarh 160014, India
A. K. Saxena
Pharmacology Division, Indian Institute of Integrative Medicine,
Jammu 180 001, J&K, India
123

Med Chem Res
1971), antimalarial (Burger and Sawhney, 1968), antidia-
betic (Moreno-Diaz et al., 2008), and antimicrobial activities
(Bondock et al., 2010). N-Alkylated indoles (Liu et al., 2003;
Bacher et al., 2001) and isatins (Singh et al., 2011) are also
well known to exhibit anticancer activity.
Taking cognizance and our interest toward the syn-
thesis of novel heterocyclic compounds with diverse
medicinal potential, we report herein the synthesis and
cytotoxic evaluation of novel N-alkylbromo-benzothia-
zoles against four different human cancer cell lines
namely lung (A-549), prostate (PC-3), leukemia (THP-1),
and colon (Caco-2). Then several attempts have been
made in generating the predictive QSAR models on dif-
ferent cancer cell lines using various types of descriptors.
The alkyl chain length and substitution at 4, 5, and 6
positions have been varied on benzothiazole moiety to
ascertain the quantitative structure–activity relationship of
the resulting scaffolds.
Some of the benzothiazole derivatives as anticancer agents
(Fig. 1) include 2-acetyl-3-(6-methoxy-benzothiazol)-2-yl-
amino-acrylonitrile (AMBAN, compound 1) induced apop-
tosis in HL60 and U937 cells through mitochondrial/caspase
3-dependent pathway (Repicky et al., 2009). Research on
benzothiazole moiety has been further extended through
introduction of amide (compound 2) (Song et al., 2008), urea
substituents (compound 3) (Song et al., 2008), isopropylam-
idino (compound 4) (Caleta et al., 2009), and imidazolinyl
groups(compound 5) (Caleta et al., 2009) which increased the
antiproliferative effect on the wide range of tumor cell lines.
Benzothiazole linked with pyrrolobenzodiazepine conjugates
(compound 6) reported to have significant cytotoxicity due to
promising DNA-binding ability and apoptosis caused by G0/
G1 phase arrest at sub-micromolar concentrations (Kamal
et al., 2010).
Results and discussion
Chemistry
The compounds 11a–11l described in this study were pre-
pared as shown in Scheme 1. The substituted 2-amin-
obenzothiazoles (compounds 10a–10l) were synthesized by
treating substituted aniline with ammonium thiocyanate and
bromine in chloroform (Saeed et al., 2010). This reaction
involves the nucleophilic attack at the electrophilic carbon of
the thiocyanate ion by the amine. The arylthiourea synthe-
sized, was then cyclized in the presence of bromine and
chloroform to form 2-aminobenzothiazole (compounds
10a–10l). Finally, synthesis of N-alkylbromo-benzothiazole
derivatives (compounds 11a–11l) was achieved by N-alkyl-
ation of substituted 2-aminobenzothiazole (compound 10)
with appropriate dibromoalkane in the presence of potassium
carbonate in DMF. The completion of reaction was monitored
In silico studies play an important role in the drug design
and discovery, as well as in the mechanistic study by placing
a molecule into the binding site of the target macromolecule
in a non-covalent fashion. Probable mode of action could be
analyzed by prediction of correct binding geometry of each
ligand at the active site, which reveals the docking score with
surrounding amino acids. The docking score of a protein/
ligand complex is estimated in the term of binding free
energy using ArgusLab (ArgusLab 4.0.1, 2011). It consists
of a user interface that displays the graphical structure of the
molecules and runs quantum mechanics calculation using
Argus Computing Server.
Z
X
N
X
N
N
NH
NH
CN
NH
S
Z
S
S
H3CO
NH
Y
Y
O
COCH3
O
3 a-c
2 a-c
1
a: X = 6-F, Y = H, Z = m-NO2
b: X = 6-Cl, Y = H, Z = m-NO2
c: X = 5-Me, Y = 6-Me, Z = o-Me
a: X = 4-OMe, Y = H, Z = Me
b: X = 5-Me, Y = 7-Me, Z = NO2
c: X = 5-Me, Y = 7-Me, Z = CN
H
H
N
N
S
N
OCH3
N
S
NH
HN
R1
R1
N
N
O
O
N
H
N
N
5
N
H
N
H3CO
S
O
4 a-c
5 a-c
6
a: R1= 4-CH3
b: R1= 5-CH3
c: R1= 6-CH3
a: R1= 5-CH3
b: R1= 6-CH3
Fig. 1 Some benzothiazole analogs as anticancer agents
123

Med Chem Res
by thin layer chromatography. All the compounds were sep-
arated and purified by column chromatography.
final results expressed in terms of IC50 (lM, concentration
that required to inhibit cancer cell proliferation by 50 %
after exposure of cells to test compounds) values as shown
in Table 2.
All the structures of novel N-alkylbromo-benzothiazoles
were characterized by rigorous spectroscopic analysis i.e.,
1H & 13C NMR, IR, and Mass. The IR (KBr) spectra of
compound 11a compound has strong N–H absorptions at
about 3465.88 cm-1, and displayed absorptions at about
1585.38 cm-1 and 1230.50 cm-1which indicates the for-
mation of imine bond (C=NH) and CH2Br, respectively. In
1H-NMR-spectra, two triplets were observed confirmed by
their coupling constants. A triplet at d 4.49 (J = 7.5 Hz) is
observed for CH2Br and at d 4.66 (J = 7.5 Hz) for CH2–
NH. Peak of NH protons are extremely variable and weak,
therefore, confirmed by exchange of NH proton with D2O.
Reaction time and yield (%) of various compounds are
summarized in Table 1.
Present study reveals that among the human cancer cell
lines tested, prostate (PC-3) and leukemia (THP-1) cancer
cell lines are more sensitive to all the tested compounds than
colon (Caco-2) and lung (A-549) cancer cell lines. As shown
in Table 2, most of the tested compounds exhibit significant
cytotoxic activities against all the tested cell lines. Com-
pound 11a bearing chlorine group at 4th position of benzo-
thiazole ring was active only against colon (Caco-2) cancer
cell line with IC50 value 26.9 lM. However, by increasing
chain length as in compound 11g having the same substitu-
tion, shows significant cytotoxic activity against prostate
Table 1 Reaction time and yield (%) of various products (com-
pounds 11a–11l) are:
In vitro cytotoxicity
S. no. Compounds Substituted
benzothiazole
Reaction Yield (%)
time (h) of various
products
In vitro cytotoxicity of all the synthesized compounds
(11a–11l) was assessed by Sulforhodamine-B (SRB) assay
against various human cancer cell lines. The SRB assay
was performed to assess growth inhibition. This is a col-
orimetric assay which estimates cell number indirectly by
staining total cellular protein with the SRB dye. Initially
cancer cells were treated with each compound for 48 h,
followed by measuring cell growth rates by SRB-based
spectrophotometry as described (Skehan et al., 1990).
Percentage growth inhibition of all the synthesized com-
pounds were evaluated at 10, 50, and 100 lM concentra-
tion against lung (A-549), prostate (PC-3), leukemia (THP-
1), and colon (Caco 2) cancer cell lines using paclitaxel,
mitomycin-C, and 5-fluorouracil as positive controls. The
concentration dependent cytotoxicity of these compounds
against human cancer cell lines are shown in Fig. 2 and the
Br
Br
n
R
R1 R2
n = 2, 3
1
11a
11b
11c
11d
11e
11f
11g
11h
11i
Cl
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
2
2
2
2
2
2
3
3
3
3
3
3
18
18
24
18
40
34
18
18
24
18
40
34
68
70
72
67
66
61
70
65
74
65
71
65
2
Cl
3
Cl Cl
4
H
H
H
H
H
F
5
CH3
OCH3
H
6
7
8
Cl
9
Cl Cl
10
11
12
11j
11k
11l
H
H
H
F
CH3
OCH3
NH2
R
R
R
NH2
S
R
R1
R2
NH2.HCl
R1
NH
C
R1
R2
NH4SCN
H2O
N
S
HCl / H2O
Br2/CHCl3
liq NH3
NH2
R1
R2
R2
8a-l
9a-l
10a-l
7a-l
g) R=Cl, R1=H, R2=H, n=3
h) R=H, R1=H, R2=Cl, n=3
i) R=H, R1=Cl, R2=Cl, n=3
j) R=H, R1=H, R2=F, n=3
k) R=H, R1=H, R2=CH3, n=3
l) R=H, R1=H, R2=OCH3, n=3
a) R=Cl, R1=H, R2=H, n=2
b) R=H, R1=H, R2=Cl, n=2
c) R=H, R1=Cl, R2=Cl, n=2
d) R=H, R1=H, R2=F, n=2
e) R=H, R1=H, R2=CH3, n=2
f) R=H, R1=H, R2=OCH3, n=2
K2CO3,
DMF
Br
Br
n
R
4
3
R1
3a
N
5
H
N
Br
2
6
S
7a
n
R2
7
1
11a-l
Scheme 1 Synthesis of substituted N-alkylbromo-benzothiazoles
123

Med Chem Res
Fig. 2 Cytotoxicity of a N-ethylbromo-benzothiazoles (compounds 11a–11f); b N-propylbromo-benzothiazoles (compounds 11g–11l)
(IC50 = 1 and 0.9 lM) were higher than that of Mitomycin-
C (IC50 = 1.5 lM). Compound 11j also exhibit significant
cytotoxic activity against prostate (PC-3) cancer cell line
with IC50 = 2.5 lM. The difference in cytotoxic activity of
compounds 11g and 11h against prostate (PC-3) cancer cell
line (IC50 = 3.1 and 62.8 lM, respectively) and leukemia
(THP-1) cancer cell line (IC50 = 1.72 and 60 lM, respec-
tively) is attributed to the change in position of chlorine
group at 4th and 6th position of benzothiazole derivatives.
When comparing compounds 11k and 11l (6-methyl vs 6-
methoxy), 11l bearing methoxy group showed good cyto-
toxic activity against all the tested cell lines with IC50 value
of 0.6, 3, and 9.9 lM on prostate (PC-3), leukemia (THP-1),
and colon (Caco-2) cancer cell lines, respectively. Most
interestingly their activity against colon (Caco-2) cell line
was higher than 5-fluorouracil. Present study showed that on
elongation of the alkyl chain, cytotoxic activity of the
compound increases. However, the differences in the IC50
values may be attributable to such factors as the nature of the
N-alkylbromides and hydrophilic or hydrophobic substitu-
tion on 4, 5, or 6th position of benzothiazole ring.
Table 2 IC50 determination of cytotoxicity of compounds (11a–11l)
against human cancer cell lines
Compounds
IC50 value (lM)
Lung
(A549)
Prostate
(PC-3)
Leukemia
(THP-1)
Colon
(Caco-2)
11a
[100
[100
85.5
110
99
110
97.8
8.46
1
26.9
[100
8.4
11b
11c
24
11d
67.6
9.9
40.9
100
3.1
62.8
28
16.8
67.7
47.9
[100
33
11e
[100
[100
[100
[100
[100
[100
[100
89.9
39.9
110
1.72
60
11f
11g
11h
11i
7
63
11j
2.5
53.3
0.6
NT
NT
1.5
0.9
30.3
3
93.7
81.6
9.9
11k
11l
Paclitaxel
5-Fluorouracil
Mitomycin-C
2.7
NT
NT
1.5
NT
NT
21
0.4
NT
Although in most of the cases, the exact mechanism of
cytotoxic activity is not known for benzothiazole deriva-
tives; however, the varied modes of action of benzothiazole
derivatives have been reported. Previous studies have
shown that the purposed mechanism of cytotoxicity of
benzothiazoles is mediated via activation of arylhydrocar-
bon receptor (AhR) signaling pathway by translocation
from the cytosol to the nucleus. Therein the induced
cytochrome P450 CYP1A1 enzyme activity leads to the
generation of a reactive electrophilic species that selec-
tively generates DNA adducts, cause DNA damage, ulti-
mately resulting in cell death by activation of apoptic
machinery (Loaiza-Perez et al., 2002; Trapani et al., 2003;
NT Not tested
(PC-3) and leukemia (THP-1) cancer cell line (IC50 = 3.1
and 1.72 lM, respectively); whereas it was found to be not
active against colon cancer cell line. Dichloro substituted
compound 11c displayed IC50 = 8.4 lM against colon
(Caco-2) cancer cell line, which is comparable to 5-Fluoro-
uracil (IC50 = 21 lM). Compound 11c and 11i also possess
moderate activity against leukemia (THP-1) cancer cell line
with IC50 = 8.46 and 7 lM, respectively. Furthermore, the
activity of compound 11d and 11j against THP-1 cell line
123

Med Chem Res
Leong et al., 2004). Docking studies confirmed that ben-
zothiazole containing hydroxamic acid act as inhibitior of
histone deacetylases (HDAC) that could be a prominent
action mechanism for the cytotoxicity (Oanh et al., 2011).
Various benzothiazole derivatives were proposed as
inhibitors of fatty acid amide hydrolase (FAAH) (Wang
et al., 2009), Raf kinase (Raf-1) (Song et al., 2008), and
B-cell lymphoma protein BCL-2 (Zheng et al., 2007).
(Fig. 3c). Comparison of most active and least active
compound shows the importance of hydrogen bond inter-
actions with Cys797 amino acid present on binding site
contributing the inhibitory activity toward the target
(Fig. 3d).
Quantitative structure activity relationship
QSAR analysis applies statistical methods to describe the
relationship between chemical structure and biological
activities of a series of N-alkylbromo-benzothiazoles
quantitatively. In the present study, we report the synthesis
and QSAR studies of substituted benzothiazoles. The
groups of calculated thermodynamic descriptors included
bend energy, heat of formation, torsion energy, boiling
point, melting point, Gibbs free energy, Henry’s Law
constant, ideal gas thermal capacity, exact mass, and
molecular weight. Steric descriptors derived were steric
energy, connolly accessible area, connolly molecular area,
connolly solvent excluded volume, molar refractivity, and
ovality apart from this partition coefficient calculated as
Log P. Electronic descriptors included kinetic energy,
potential energy, and total energy were calculated.
Molecular topology descriptors included balaban index,
cluster count, molecular topological index, num rotatable
bonds, polar surface area, radius, shape attribute, sum of
degrees, sum of valence degrees, topological diameter,
total connectivity, total valence connectivity, and wiener
index.
Docking study
Tyrosine kinases of the epidermal growth factor receptor
(EGFR) family are involved in cancer proliferation suggest
that an inhibitor which blocks the tyrosine kinase activity
of the entire EGFR family, could play significant role in
cancer (Mendelsohn and Baselga, 2000).
So EGFR was selected as a biological target for carrying
out the docking study of our synthesized compounds to
know probable mode of action. The crystal structure of
EGFR kinase domain in complex with an irreversible
inhibitor (PDB ID: 2J5F) was obtained from the protein
data bank. The protein structure with co-crystal was given
in the Fig. 3a.
The co-crystallized ligand lying within the receptor was
isolated. The receptor was optimized and saved as pdb file,
which was used for docking simulation. Structures of ligands
were sketched by means of Chem-Draw Ultra 8.0 (http://
www.cambridgesoft.com, 2012) and were exported to
Weblab Pro (http://weblab-viewer-pro.fyxm.net/, 2012).
Three-dimensional structures of all molecules were gener-
ated. The 3-D structures were then energetically minimized
up to the rms gradient of 0.01 using Universal Force Field
(UFF). Binding site was determined with the help of Ligplot
(Fig. 3e). The parameter fixed for docking simulation were
50 population size, 1000 maximum generation, 0.8 crossover
rate, 0.2 mutation rate, 5 elitism, 0.0015936 kcal/mol con-
vergence, 0.06 local search rate, 0.00015936 kcal/mol local
search convergence, 20 local search max steps, 0.4 grid
resolution, and 15 9 15 9 15 angstroms binding site box
size. The docking simulation was done using ArgusDock
including flexible docking, lamarckian genetic algorithm
and AScore scoring function. Docking was carried out to
determine the probable mode of action of the synthesized
ligands.
It is important to note that all these models were
developed using the entire set (n = 12), since no outliers
were identified. The quality of the models is indicated by
the following parameters: r—correlation coefficient; F—
Fisher’s statistics; and s—standard error of estimation,
ra2dj—adjusted coefficient of variation r2 obtained by
‘leave-one-out’ (LOO) method. Stepwise multiple linear
regression analysis method was used to perform QSAR
analysis employing Minitab software (Darlington, 1990).
The best model was selected on the basis of various sta-
tistical parameters such as correlation coefficient (r),
standard error of estimation (SEE) and sequential Fischer
test (F).
The main objective of this work is to generate quanti-
tative models using different physicochemical descriptors
which can be useful in identifying potent anticancer agents.
The reference drugs were not included in model generation
as they belong to different structural series. The co-crystal
natural substrate was taken out of the active site. The data
presented in Table S2 demonstrates the feeble correlation
of molecular descriptors of different substituted benzo-
thiazoles with their anticancer activity. It will prompt us to
develop QSAR model using consensus method by statis-
tical approach.
Docking score clears the importance of electron-
releasing methoxy group and carbon linkers (Table S1,
supplementary). The proposed interaction modes of the
ligand with the 2J5F binding site were determined as the
highest scored conformation among the thousand confor-
mations. Hypothetical binding motif of all ligands in the
enzyme pocket of 2J5F was given in the Fig. 3b. Signifi-
cant hydrogen bond interaction of methoxy group of most
active compound was observed in the binding pocket
123

Med Chem Res
Fig. 3 a The 3-D structure of
Tyrosine kinase in the ribbon
form with co-crystal (PDB ID:
2J5F); b hypothetical binding
motif of all ligands in the
enzyme pocket of 2J5F;
c binding orientation of
compound 11l (most active)
showing hydrogen bond
interaction with Cys797 at
˚
distance of 2.9 A; d comparison
of most active (11l, pink color)
and least active (11a, yellow
color) compound in the enzyme
pocket of 2J5F; e ligplot of 2J5F
showing the active amino acid
present at the binding site
(Color figure online)
s ¼ 0:0425; r2 ¼ 99:87; ra2dj ¼ 99:72;
PRESS ¼ 0:062391; rp2red ¼ 99:13
pIC50 ¼ ð3:542 ꢁ ð0:3166 ꢂ Torsion energyÞ
ꢁ ð1:296 ꢂ Topological diameterÞ
þ ð0:2111 ꢂ Sum of valence degreesÞ
þ ð0:8112 ꢂ Num rotatable bondsÞ
The consensus of six types of descriptors torsion energy,
topological diameter, sum of valence degrees, radius, num
rotatable bonds, and ideal gas thermal capacity was found
to be very significant in generating the predictive equation.
þ ð0:0076 ꢂ Ideal gas thermal capacityÞÞ ðM1Þ
123

Med Chem Res
As shown in the Model 1 (M1), the inhibitory activity is
proportional to the sum of valence degrees, it means higher
the sum of valence degrees more the inhibitory activity as in
the case of ligand number 11a, 11b, 11c, and 11d. Torsion
energy has not favorable contribution toward the binding
affinity i.e., high torsion energy leads to repress the activity.
Topological diameter has an unfavorable contribution as
evidenced by negative regression coefficient. Num rotatable
bonds have a favorable contribution toward the binding
affinity as in the case of ligand number 11i, 11j, and 11k.
The above model explains 99.72 % of variance (adjus-
ted coefficient of variation-ra2dj and 99.87 % of r2 which
indicates the robustness of fit (Fig. 4a) and the predicted
potential was further validated by predicted r2 (rp2red) (Table
S3).
QSAR models could not be developed for A549 cancer
cell line, while for the Caco-2 cancer cell line there is low
quality of predictive ability. In this case, the influences of
the synthesized compounds are quite different, making the
unsatisfactory QSAR models.
The statistical quantities of different models are given in
Table-S5, explain the model quality, internal and external
validation parameters.
On comparing the compounds 11b (R2 = Cl), 11d
(R2 = F), 11h (R2 = Cl), and 11j (R2 = F), QSAR models
suggest that cytotoxic activity increases in the order of
11b \ 11d and 11h \ 11j, which is the same order as the
substituent’s electron withdrawing ability as shown in the
Fig. 5. However, two chlorine atoms substituted compound
11c and 11i exhibited increased activity in PC-3, THP-1,
and Caco-2 cancer cell lines than compounds 11b and 11h.
Among the electron-releasing group-substituted com-
pounds such as 11k with a methyl group at the 6-position
did not show good cytotoxic activity; however, compound
11l having a stronger electron-releasing methoxy group
showed highest activity. Whereas, when compounds 11l
and 11j (6-methoxy vs 6-fluorine) were compared, 11l
displayed higher cytotoxic activity against prostate and
colon cancer cell lines and 11j displayed highest cytotoxic
activity against leukemia cancer cell line. These results
suggest that compounds bearing stronger electron-releasing
(methoxy) or withdrawing (fluorine) substituents show
improved cytotoxic activity against their particular cancer
cell lines. Furthermore, the cytotoxic activity increases by
increasing carbon linkers between nitrogen and bromo
group as evidenced by compounds 11g, 11h, 11i, 11j, 11k,
and 11l which showed maximum growth inhibition in case
of prostate (PC-3) and leukemia (THP-1) cancer cell lines
while the compounds 11a, 11b, 11c, 11d, 11e, and 11f
presented moderate growth inhibition.
pIC50 ¼ ð1:819 ꢁ ð0:179 ꢂ Torsion energyÞ
þð0:236 ꢂ Sum of valence degreesÞ
ꢁ ð1:68 ꢂ Topological diameterÞ
þ ð1:86 ꢂ RadiusÞ þ ð1:27 ꢂ Num rotatable bondsÞ
þ ð0:007 ꢂ Ideal gas thermal capacityÞ
ꢁð0:00121 ꢂ Molecular topological indexÞÞ
ðM2Þ
s ¼ 0:205; r2 ¼ 97:18; ra2dj ¼ 92:25;
PRESS ¼ 0:823715; rp2red ¼ 86:19
As shown in the Model 2 (M2), torsion energy and
topological diameter has adverse contribution toward
binding affinity. Sum of valence degree has a positive
contribution, can be understood as in the case of ligand
number 11j, 11k, and 11l. Num rotatable bond and radius
has played a positive role toward binding affinity as evi-
denced by positive correlation. Molecular topological
index has decreased the activity as in the case of ligand
number 11f and 11g. The low residual activity values
observed (Table S4), justify the selection of the linear
regression model expressed by Eq. M2. Further the plot of
linear regression predicted values against the observed
values also favors the model expressed by Fig. 4b.
Validation methods
QSAR modeling is done to develop model, which should
be strong enough to be capable for reliable predictions of
biological activities of new ligands. Robustness of QSAR
The r2, ra2dj andrp2red wasfoundtobe97.18,92.25,and86.19
respectively, shows the reliability of above explained model.
Fig. 4 Correlation between
(b)
(a)
Model 1
Model 2
experimental and predicted
activity using a model 1 and b
model 2
7
6
5
4
3
2
1
0
7
6
5
4
3
2
1
0
y = 0.9985x + 0.0067
R² = 0.9987
y = 0.9709x + 0.1013
R² = 0.973
0
2
4
6
8
0
2
4
6
8
Experimental pIC50
Experimental pIC50
123

Med Chem Res
Fig. 5 Structure activity
relationship of N-alkylbromo-
benzothiazoles (11a–11l)
Substitution of chlorine at bothC5 and C6
position show better cytotoxic activity
With increase in alkyl chain cytotoxic
activity increases against prostate and
leukemia cancer cell lines and
than compounds having mono substituted
chlorine group
decreases against colon cancer cell line
X1
X2
N
S
H
N
Br
n
n=2,3
Substitution of methoxy group atC6 increases
cytotoxic activity against prostate and colon cancer
cell line when compared to fluorine group whereas,
fluorine shows increase in cytotoxic activity against
leukemia cancer cell line.
models is generally verified by means of different types of
validation criteria such as (i) internal validation or cross-
validation, (ii) validation by dividing the dataset into
training and test compounds, (iii) data randomization or
Y-scrambling, and (iv) true external validation by appli-
cation of model on new external data (Roy, 2007). Pre-
diction error sum of squares (PRESS) is a standard index to
measure the accuracy of a modeling method based on the
cross-validation technique (Roy, 2007; Roy and Roy, 2008)
Experimental
General
Melting points (mp) were taken in open capillaries on the
1
Veego VMP-PM melting point apparatus. The H and 13C
NMR-spectra were recorded on a JEOL-JNM—300-spec-
trometer at 300 and 75 MHz respectively, in CDCl3 as
solvent. Chemical shifts were expressed as d values relative
to TMS as internal standard. IR spectra were recorded on
Shimadzu 8400 S FT-IR spectrophotometer with KBr
pellets. Mass spectra were reported on Shimadzu GCMS-
QP-2000A mass spectrograph. The progress of the reaction
was monitored on silica-gel plates using hexane: ethyl
acetate as solvent. Iodine was used as a developing agent.
Chromatographic purification was carried out using silica
gel (60–120 mesh).
Conclusion
In present report, we describe synthesis and in vitro cyto-
toxic evaluation of a series of N-alkyl-bromo benzothiaz-
oles. Some of these derivatives exhibit potent cytotoxic
activity. The benchmark active compound was 11l (3-
bromo-propyl-6-methoxy-benzothiazol-2-yl-amine) shows
significant cytotoxic activity on PC-3, THP-1, and Caco-2
cancer cell lines confirmed by their IC50 values as 0.6, 3,
and 9.9 lM, respectively. Other compounds exhibited IC50
value of B 1 lM were 11d and 11j against leukemia
(THP-1) cancer cell line. These molecules shall serve as
useful ‘‘lead’’ for further anticancer drug development.
On the comparison of docking score of synthesized
ligand with inhibitory activity, it may be concluded that
ligand may be acted by inhibiting tyrosine kinase of EGFR
family to have anticancer activity.
Chemistry
General procedure for the synthesis of N-c-bromo-alkyl-
(benzothiazol-2-yl)-amines (compound 11a–11l)
All the synthesized compounds were prepared according to
the following two-step procedures:
Step 01: The intermediates 10a–10l was prepared
according to the previous reported procedure with certain
modifications (Saeed et al., 2010). Equimolar quantities of
substituted aniline (25 ml) and concentrated hydrochloric
acid (25 ml) were warmed for 30 min. A saturated solution
of ammonium thiocyanate in water (30 g in 60 ml) was
added slowly in above solution, and then the mixture was
boiled until the solution got turbid. The turbid solution was
poured in cold water. The resulting precipitate was filtered
and re-crystallized from aqueous ethanol to get pure phen-
ylthiourea. The solution of substituted phenylthiourea
(26 mmol) in chloroform (50 ml) was brominated using
bromine solution in chloroform (5 %) till the orange-yellow
color appeared. After completion of total addition of
Further the analysis of multiple stepwise linear regres-
sion model give an insight into characteristic feature and
the result suggests that the inhibitory potency of benzo-
thiazole analogs is increased by augmenting the carbon
chain length. The models were validated using Leave-one-
out procedure. The QSAR methodology can greatly help
this effort being firmly founded on physicochemical and
statistical basis. Therefore, developed QSAR models could
be used to predict the range of activities for new anticancer
agents and play a significant role to facilitate the process of
design of new potent anticancer agents.
123

Med Chem Res
bromine stirring was continued for a period of 4 h. The
slurry was kept overnight in freezer. The precipitate
obtained was filtered and washed with sodium bicarbonate
and extracted with ethyl acetate. The organic layer was
dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The precipitate, as hydrobromide, was
dissolved in rectified spirit (150 ml) and basified with
ammonia solution. The precipitate was filtered, washed
with water, dried and re-crystallized using rectified spirit to
afford 2-aminobenzothiazole (compound 10a–10l).
Br), 1101.28 cm-1 (Aryl-Cl). MS (m/z): 292 (M?). Anal.
Calcd for C9H8BrClN2S: C, 37.07; H, 2.77; N, 9.61.
Found: C, 37.09; H, 2.73; N, 9.57.
(2-Bromo-ethyl)-(5,6-dichloro-benzothiazol-2-yl)-amine
(compound 11c)
Yield: 72 %, mp: 162–166 °C. 1H NMR (300 MHz,
CDCl3) d: 7.48–7.21 (m, 2H, ArH), 4.29 (t, 2H,
J = 7.4 Hz, CH2–NH), 3.52 (t, 2H, J = 7.5 Hz, CH2Br).
13C NMR (75 MHz, CDCl3) d: 163.21 (C2), 142.16 (C3a),
131.02 (C6), 126.65 (C5), 123.23 (C7), 121.46 (C4), 106.95
(C7a), 48.75 (CH2–NH), 29.80 (CH2Br). IR (KBr, cm-1) t:
3343, 1321.44 (C–NH), 1641.31 (C=N), 1248.01 (CH2Br),
1113.03 cm-1 (Aryl-Cl). MS (m/z): 292 (M?). Anal. Calcd
for C9H7BrCl2N2S: C, 33.15; H, 2.16; N, 8.59. Found: C,
33.12; H, 2.19; N, 8.63.
Step 02: To a solution of 2-aminobenzothiazoles (com-
pound 10a–10l) in DMF, K2CO3 (1.6 equiv.) was added
slowly and the mixture was stirred vigorously for 15 min at
room temperature. To the resulting solution, terminally
substituted dibromoalkane (1 equiv.) in 2 ml of DMF was
added drop wise. The reaction mixture was heated at 60 °C,
and the progress of the reaction was monitored by TLC. The
reaction mixture was quenched by addition of water and
extracted with ethyl acetate. The organic layer was washed
with brine two times and dried over anhydrous Na2SO4.
After filtration, organic layer is concentrated under reduced
pressure. Then, the crude product was purified by column
chromatography using ethyl acetate/hexane (20:80) to afford
the desired product compound 11.
(2-Bromo-ethyl)-(6-fluoro-benzothiazol-2-yl)-amine
(compound 11d)
Yield: 67 %, mp: 142–145 °C. 1H NMR (300 MHz,
CDCl3) d: 7.31–7.06 (m, 3H, ArH), 4.78 (t, 2H,
J = 6.8 Hz, CH2–NH), 4.53 (t, 2H, J = 6.9 Hz, CH2Br).
13C NMR (75 MHz, CDCl3) d: 160.91 (C2), 155.92 (C6),
141.56 (C3a), 115.76 (C7a), 112.62 (C4), 109.93 (C5),
100.01 (C7), 57.21 (CH2–NH), 35.02 (CH2Br). IR (KBr,
cm-1) t: 3360.11, 1312.46 (C–NH), 1625.88 (C=N),
1230.50 (Aryl-F), 1211.22 cm-1 (CH2Br). MS (m/z): 276
(M?). Anal. Calcd for C9H8BrFN2S: C, 39.29; H, 2.93; N,
10.18. Found: C, 39.32; H, 2.95; N, 10.15.
Characterization data for all synthesized compounds
(2-Bromo-ethyl)-(4-chloro-benzothiazol-2-yl)-amine
(compound 11a)
Yield: 68 %, mp: 149–153 °C. 1H NMR (300 MHz, CDCl3)
d: 7.79 (d, 1H, J = 4.7 Hz, ArH), 7.66 (dd, 1H, J = 8.4 Hz,
4.7 Hz, ArH), 7.42 (d, 1H, J = 8.2 Hz, ArH), 4.66 (t, 2H,
J = 7.5 Hz, CH2–NH), 4.49 (t, 2H, J = 7.5 Hz, CH2Br).
13C NMR (75 MHz, CDCl3) d: 165.91 (C2), 147.48 (C3a),
129.49 (C4), 126.54 (C6), 124.56 (C5), 122.44 (C7a), 119.84
(C7), 44.97 (CH2–NH), 35.67 (CH2Br). IR (KBr, cm-1) t:
3465.84, 1332.72 (C–NH), 1517.87, 1585.38 (C=N),
1230.50 (CH2Br), 1033.77 cm-1 (Aryl-Cl). MS (m/z): 290.8
(M?). Anal. Calcd for C9H8BrClN2S: C, 37.07; H, 2.77; N,
9.61. Found: C, 37.11; H, 2.74; N, 9.64.
(2-Bromo-ethyl)-(6-methyl-benzothiazol-2-yl)-amine
(compound 11e)
Yield: 66 %, mp: 132–135 °C. 1H NMR (300 MHz, CDCl3)
d: 7.63 (d, 1H, J = 3.6 Hz, ArH), 7.57 (s, 1H, ArH), 7.51 (d,
1H, J = 3.4 Hz, ArH), 4.65 (t, 2H, J = 6.9 Hz, CH2–NH),
4.44 (t, 2H, J = 6.8 Hz, CH2Br), 2.46 (s, 3H, CH3). 13C
NMR (75 MHz, CDCl3) d: 169.72 (C2), 138.13 (C3a), 133.85
(C6), 127.56 (C5), 121.13 (C7), 120.88 (C7a), 120.40 (C4),
49.94 (CH2–NH), 35.37 (CH2Br), 21.13 (CH3). IR (KBr,
cm-1) t: 3310.05, 1323.81 (C–NH), 2923.45 (sp3-C),
1618.17 (C=N), 1235.11 cm-1 (CH2Br). MS (m/z): 270
(M?). Anal. Calcd for C10H11BrN2S: C, 44.29; H, 4.09; N,
10.33. Found: C, 44.32; H, 4.11; N, 10.35.
(2-Bromo-ethyl)-(6-chloro-benzothiazol-2-yl)-amine
(compound 11b)
Yield: 70 %, mp: 154–158 °C. 1H NMR (300 MHz,
CDCl3) d: 7.66 (s, 1H, ArH), 7.38 (d, 1H, J = 2.4 Hz,
ArH), 7.35 (d, 1H, J = 2.3 Hz, ArH), 4.65 (t, 2H,
J = 8.2 Hz, CH2–NH), 4.40 (t, 2H, J = 8.4 Hz, CH2Br).
13C NMR (75 MHz, CDCl3) d: 164.28 (C2), 138.56 (C3a),
122.98 (C4), 128.27 (C6), 125.21 (C5), 123.45 (C7a), 121.76
(C7), 49.28 (CH2–NH), 32.26 (CH2Br). IR (KBr, cm-1) t:
3396.41, 1332.72 (C–NH), 1590.19 (C=N), 1230.50 (CH2–
(2-Bromo-ethyl)-(6-methoxy-benzothiazol-2-yl)-amine
(compound 11f)
Yield: 60 %, mp: 158–160 °C. 1H NMR (300 MHz,
CDCl3) d: 7.89–7.62 (m, 3H, ArH), 4.65 (t, 2H,
J = 7.8 Hz, CH2–NH), 4.34 (t, 2H, J = 7.8 Hz, CH2Br),
123

Med Chem Res
3.74 (s, 3H, OCH3). 13C NMR (75 MHz, CDCl3) d: 168.32
(C2), 154.27 (C6), 143.04 (C3a), 121.88 (C7a), 120.31 (C4),
111.17 (C5), 96.21 (C7), 56.95 (O–CH3), 56.63 (CH2–NH),
38.69 (CH2Br). IR (KBr, cm-1) t: 3431.13, 1328.86
(C–NH), 2937.38 (sp3-C), 1598.88 (C=N), 1267.14 cm-1
(CH2Br). MS (m/z): 286.9 (M?). Anal. Calcd for
C10H11BrN2OS: C, 41.82; H, 3.86; N, 9.75. Found: C,
41.86; H, 3.80; N, 9.81.
(3-Bromo-propyl)-(6-fluoro-benzothiazol-2-yl)-amine
(compound 11j)
Yield: 65 %. 1H NMR (300 MHz, CDCl3) d: 7.46–7.09 (m,
3H, ArH), 4.46 (t, 2H, J = 6.1 Hz, CH2–NH), 4.26 (t, 2H,
J = 6.3 Hz, CH2Br), 2.38–2.29 (m, 2H, CH2). 13C NMR
(75 MHz, CDCl3) d: 161.82 (C2), 156.03 (C6), 139.71
(C3a), 120.45 (C7a), 117.42 (C4), 112.85 (C5), 107.26 (C7),
45.87 (CH2–NH), 34.24 (CH2), 30.90 (CH2Br). IR (KBr,
cm-1) t: 330.42, 1294.91 (C–NH), 1598.95 (C=N),
1273.61 (CH2Br), 1145.23 cm-1 (Aryl-F). MS (m/z): 288
(M?). Anal. Calcd for C10H10BrFN2S: C, 41.54; H, 3.49;
N, 9.69. Found: C, 41.57; H, 3.43; N, 9.72.
(3-Bromo-propyl)-(4-chloro-benzothiazol-2-yl)-amine
(compound 11g)
1
Yield: 70 %. H NMR (300 MHz, CDCl3) d: 7.61 (d, 1H,
J = 4.5 Hz, ArH), 7.50 (dd, 1H, J = 7.8 Hz, 4.8 Hz, ArH),
7.41 (d, 1H, J = 7.6 Hz, ArH), 4.45 (t, 2H, J = 5.4 Hz,
CH2–NH), 4.26 (t, 2H, J = 6.0 Hz, CH2Br), 2.35–2.28 (m,
2H, CH2). 13C NMR (75 MHz, CDCl3) d: 168.24 (C2),
142.58 (C3a), 125.56 (C4), 125.11 (C6), 122.24 (C5), 120.63
(C7a), 119.24 (C7), 49.93 (CH2–NH), 37.24 (CH2), 29.98
(CH2Br). IR (KBr, cm-1) t: 3350.08, 1282.14 (C–NH),
1607.67 (C=N), 1242.43 (CH2Br), 1042.28 cm-1 (Aryl-Cl).
MS (m/z): 305 (M?). Anal. Calcd for C10H10BrClN2S: C,
39.30; H, 3.30; N, 9.17. Found: C, 39.34; H, 3.27; N, 9.15.
(3-Bromo-propyl)-(6-methyl-benzothiazol-2-yl)-amine
(compound 11k)
Yield: 71 %. 1H NMR (300 MHz, CDCl3) d: 7.37–7.07 (m,
3H, ArH), 3.72 (t, 2H, J = 6.7 Hz, CH2–NH), 3.47 (t, 2H,
J = 6.7 Hz, CH2Br), 2.92–2.89 (m, 2H, CH2), 2.39 (s, 3H,
CH3). 13C NMR (75 MHz, CDCl3) d: 170.21 (C2), 142.46
(C3a), 135.49 (C6), 122.47 (C5), 121.67 (C7), 121.22 (C7a),
120.98 (C4), 44.94 (CH2–NH), 29.93 (CH2), 21.47
(CH2Br), 20.76 (CH3). IR (KBr, cm-1) t: 3362.45, 1341.12
(C–NH), 2800.08 (sp3–C), 1605.28 (C=N), 1219.32 cm-1
(CH2Br). MS (m/z): 286 (M?). Anal. Calcd for
C11H13BrN2S: C, 46.32; H, 4.59; N, 9.82. Found: C, 46.28;
H, 4.54; N, 9.93.
(3-Bromo-propyl)-(6-chloro-benzothiazol-2-yl)-amine
(compound 11h)
1
Yield: 65 %. H NMR (300 MHz, CDCl3) d: 7.25 (s, 1H,
ArH), 7.22 (d, 1H, J = 2.3 Hz, ArH), 7.20 (d, 1H,
J = 2.4 Hz, ArH), 4.48 (t, 2H, J = 5.4 Hz, CH2–NH),
4.25 (t, 2H, J = 6.0 Hz, CH2Br), 2.33–2.24 (m, 2H, CH2).
13C NMR (75 MHz, CDCl3) d: 168.51 (C2), 144.24 (C3a),
131.42 (C6), 123.45 (C5), 123.13 (C7a), 122.91 (C4), 122.05
(C7), 49.10 (CH2–NH), 35.24 (CH2), 29.26 (CH2Br). IR
(KBr, cm-1) t: 3361, 1300.26 (C–NH, 1627.81 (C=N),
1220.18 (CH2Br), 1120.06 cm-1 (Aryl-Cl). MS (m/z): 306
(M?). Anal. Calcd for C10H10BrClN2S: C, 39.30; H, 3.30;
N, 9.17. Found: C, 39.33; H, 3.32; N, 9.20.
(3-Bromo-propyl)-(6-methoxy-benzothiazol-2-yl)-amine
(compound 11l)
Yield: 65 %. 1H NMR (300 MHz, CDCl3) d: 7.69–6.98 (m,
3H, ArH), 3.59 (t, 2H, J = 7.0 Hz, CH2–NH), 3.33 (t, 2H,
J = 7.2 Hz, CH2Br), 2.70–2.66 (m, 2H, CH2), 2.35 (s, 3H,
OCH3). 13C NMR (75 MHz, CDCl3) d: 159.93 (C2),
149.93 (C6), 132.21 (C3a), 120.64 (C7a), 120.49 (C4),
108.24 (C5), 96.53 (C7), 56.80 (O–CH3), 46.18 (CH2–NH),
34.73 (CH2), 29.63 (CH2Br). IR (KBr, cm-1) t: 3421.48,
1379.01 (C–NH), 2848.67 (sp3–C), 1618.17 (C=N),
1267.14 (CH2Br), 1215.07 (C–O). MS (m/z): 300.8 (M?).
Anal. Calcd for C11H13BrN2OS: C, 43.86; H, 4.35; N, 9.30.
Found: C, 43.82; H, 4.32; N, 9.34.
(3-Bromo-propyl)-(5,6-dichloro-benzothiazol-2-yl)-amine
(compound 11i)
1
Yield: 74 %. H NMR (300 MHz, CDCl3) d: 7.51 (s, 1H,
ArH), 7.35 (s, 1H, ArH), 4.16 (t, 2H, J = 6.3 Hz, CH2–
NH), 4.40 (t, 2H, J = 7.5 Hz, CH2Br), 2.26–2.18 (m, 2H,
CH2). 13C NMR (75 MHz, CDCl3) d: 156.58 (C2), 141.28
(C3a), 129.90 (C6), 127.73 (C5), 121.98 (C7), 121.52 (C4),
106.20 (C7a), 46.26 (CH2–NH), 35.23 (CH2), 29.75
(CH2Br). IR (KBr, cm-1) t: 3348.19, 1319.22 (C–NH),
1618.17 (C=N), 1213.14 (CH2Br), 1100.28 cm-1 (Aryl-
Cl). MS (m/z): 341 (M?). Anal. Calcd for C10H9BrCl2N2S:
C, 35.32; H, 2.67; N, 8.24. Found: C, 35.29; H, 2.64; N,
8.20.
Biological assay
For evaluating cytotoxicity, the compounds were dissolved
in DMSO and stock solution of 2 9 104 lM was prepared.
Stock solutions were further diluted with complete growth
medium supplemented with 50 lg/ml gentamicin to obtain
test concentrations of 10, 50, and 100 lM. 5-fluorouracil
and paclitaxel were dissolved in DMSO and stock solution
of 2 9 103 lM was prepared. Mitomycin-C was dissolved
123

Med Chem Res
in double-distilled water and stock solution of 2 9 103 lM
was prepared. Stock solutions were further diluted with
complete growth medium supplemented with 50 lg/ml
gentamicin to obtain desired concentrations. All the cells
were maintained in RPMI-1640 medium, supplemented
with fetal bovine serum (10 %), 100 U/ml penicillin and
100 lg/ml streptomycin (complete medium). The cells
were seeded into 96 well cell culture plates (1 9 104 cells/
100 ll/well) and incubated in CO2 incubator (37 °C, 5 %
CO2, 95 % relative humidity) for 24 h. After 24 h, com-
pounds 11a–11l and positive controls (100 ll/well) were
added in quadruplets, and the plates were further incubated
in CO2 incubator for 48 h. Suitable controls were also
included in each experiment. After 48 h chilled trichloro-
acetic acid (50 % w/v, 50 ll) was laid gently on top of the
medium in all the wells. The plates were incubated at 4 °C
for 1 h to fix the cells. All the contents of the wells were
gently pipette out and discarded. The plates were washed
five times with distilled water to remove trichloroacetic
acid, growth medium, low molecular weight metabolites,
and serum proteins, etc. The plates were air-dried. Sulfo-
rhodamine-B (0.4 % SRB in 1 % acetic acid, 100 ll/well)
was added to each well of the 96 well plates for 30 min.
Excess of the dye was washed off using 1 % acetic acid,
and the plates were air-dried. Tris buffer (10 mM, pH 10.5,
100 ll/well) was added to each well and plates were sha-
ken on a mechanical stirrer for 10 min and O. D. was
recorded on ELISA reader at 540 nm. Viability of cells was
evaluated by trypan blue exclusion method immediately
before setting up the experiment for cytotoxicity determi-
nation. Cells with [98 % viability were used in the assay.
based on forward selection and backward elimination
techniques for inclusion and rejection of descriptors. The
selection of the significant descriptors for developing the
model was done using ‘‘stepping criteria’’ (F) with F = 4
for inclusion and F = 3.9 for exclusion (Darlington, 1990;
Mitra et.al., 2009).
Acknowledgments Authors thanks to the Department of Chemistry,
Guru Nanak Dev University, Amritsar, for 300 MHz NMR facility
generated by Department of Science and Technology, Government of
India.
Conflict of interest The authors have declared no conflict of
interest.
References
ArgusLab (2011) http://www.chem.ac.ru/Chemistry/Soft/ARGUS.en.
html. Accessed 02 Sept 2012
Bacher G, Nickel B, Emig P, Vanhoefer U, Seeber S, Shandra A,
Klenner T, Beckers T (2001) D-24851, a novel synthetic
microtubule inhibitor, exerts curative antitumoral activity
in vivo, shows efficacy toward multidrug-resistant tumor cells,
and lacks neurotoxicity. Cancer Res 61:392–399
Bhuva HA, Kini SG (2010) Synthesis, anticancer activity and docking
of some substituted benzothiazoles as tyrosine kinase inhibitors.
J Mol Graph Model 29:32–37
Bondock S, Fadaly W, Metwally MA (2010) Synthesis and antimi-
crobial activity of some new thiazole, thiophene and pyrazole
derivatives containing benzothiazole moiety. Eur J Med Chem
45:3692–3701
Burger A, Sawhney SN (1968) Antimalarials. 3. Benzothiazole amino
alcohols. J Med Chem 11:270–273
Caleta I, Kralj M, Marjanovic M, Bertosa B, Tomic S, Pavlovic G,
Pavelic K, Karminski-Zamola G (2009) Novel cyano- and
amidinobenzothiazole derivatives: synthesis, antitumor evalua-
tion, and X-ray and quantitative structure-activity relationship
(QSAR) analysis. J Med Chem 52:1744–1756
Binding energy calculation using ArgusLab
Cancer (2012) www.cancer.gov/cancertopics/understandingcancer/
cancer/page1. Accessed 10 Sept 2012
Chem-Draw Ultra 8.0 (2012) http://www.cambridgesoft.com. Accessed
02 Sept 2012
Darlington RB (1990) Regression and linear models. McGraw-Hill,
New York
Guedat P, Colland F (2007) Patented small molecule inhibitors in the
ubiquitin proteasome system. BMC Biochem 8:S14
Kamal A, Reddy KS, Khan MN, Shetti RV, Ramaiah MJ, Pushpavalli
SN, Srinivas C, Pal-Bhadra M, Chourasia M, Sastry GN, Juvekar
A, Zingde S, Barkume M (2010) Synthesis, DNA-binding ability
and anticancer activity of benzothiazole/benzoxazole-pyrrolo[2,1-c]
[1,4]benzodiazepine conjugates. Bioorg Med Chem 18:4747–4761
Leong CO, Suggitt M, Swaine DJ, Bibby MC, Stevens MFG,
Bradshaw TD (2004) In vitro, in vivo, and in silico analyses of
the antitumor activity of 2-(4-amino-3-methylphenyl)-5-fluor-
obenzothiazoles. Mol Cancer Ther 3:1565–1575
It is the software used for calculating the binding energies of
the ligands with receptors i.e., proteins. Dock score is
obtained in the form of binding energy (kcal/mol). Docking
is done in two steps: Optimization of geometry and then
docking with Argusdock. After adding hydrogen atoms,
ligand molecules were minimized using the Universal Force
Field (UFF) implemented in Arguslab. For docking tests,
ArgusDock was evaluated using Ascore scoring function
˚
with grid resolution 0.4 A. Furthermore, size of the binding
site bounding box was determined automatically using Ar-
˚
guslab (22.351 9 15.765 9 13.957 A). Docked poses with
energy were displayed in the molecule tree view window.
Liu Y, Lashuel HA, Choi S, Xing X, Case A, Ni J, Yeh LA, Cuny GD,
Stein RL, Lansbury PT Jr (2003) Discovery of inhibitors that
elucidate the role of UCH-L1 activity in the H1299 lung cancer
cell line. Chem Biol 10:837–846
Minitab software
The prediction models were developed using the stepwise
multiple linear regression method (MINITAB software)
Loaiza-Perez AI, Trapani V, Hose C, Singh SS, Trepel JB, Stevens
MFG, Bradshaw TD, Sausville EA (2002) Aryl hydrocarbon
123

Med Chem Res
receptor mediates sensitivity of MCF-7 breast cancer cells to
antitumor agent 2-(4-amino-3-methylphenyl) benzothiazole. Mol
Pharmacol 61:13–19
Identification of highly promising leads. Bioorg Med Chem Lett
19:3054–3058
Singh P, Kaur S, Kumar V, Bedi PM, Mahajan MP, Sehar I, Pal HC,
Saxena AK (2011) Synthesis and in vitro cytotoxic evaluation of
N-alkylbromo and N-alkylphthalimido-isatins. Bioorg Med
Chem Lett 21:3017–3020
Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D,
Warren JT, Bokesch H, Kenney S, Boyd MR (1990) New
colorimetric cytotoxicity assay for anticancer-drug screening.
J Natl Cancer Inst 82:1107–1112
Mendelsohn J, Baselga J (2000) The EGF receptor family as targets
for cancer therapy. Oncogene 19:6550–6565
Mitra I, Saa A, Roy K (2009) Quantitative structure–activity
relationship modeling of antioxidant activities of hydroxybenz-
alacetones using quantum chemical, physicochemical and spatial
descriptors. Chem Biol Drug Des 73:526–536. doi:10.1111/j.1747-
0285.2009.00801.x
Moreno-Diaz H, Villalobos-Molina R, Ortiz-Andrade R, Diaz-Coutino
D, Medina-Franco JL, Webster SP, Binnie M, Estrada-Soto S,
Ibarra-Barajas M, Leon-Rivera I, Navarrete-Vazquez G (2008)
Antidiabetic activity of N-(6-substituted-1,3-benzothiazol-2-yl)ben-
zenesulfonamides. Bioorg Med Chem Lett 18:2871–2877
Oanh DT, Hai HV, Park SH, Kim HJ, Han BW, Kim HS, Hong JT,
Han SB, Hue VT, Nam NH (2011) Benzothiazole-containing
hydroxamic acids as histone deacetylase inhibitors and antitumor
agents. Bioorg Med Chem Lett 21:7509–7512
Palmer PJ, Trigg RB, Warrington JV (1971) Benzothiazolines as
antituberculous agents. J Med Chem 14:248–251
Pinar A, Yurdakul P, Yildiz I, Temiz-Arpaci O, Acan NL, Aki-Sener
E, Yalcin I (2004) Some fused heterocyclic compounds as
eukaryotic topoisomerase II inhibitors. Biochem Biophys Res
Commun 317:670–674
Repicky A, Jantova S, Cipak L (2009) Apoptosis induced by 2-acetyl-
3-(6-methoxybenzothiazo)-2-yl-amino-acrylonitrile in human
leukemia cells involves ROS-mitochondrial mediated death
signaling and activation of p38 MAPK. Cancer Lett 277:55–63
Roy K (2007) On some aspects of validation of predictive quantitative
structure-activity relationship models. Expert Opin Drug Discov
2:1567–1577. doi:10.1186/1752-153X-2-S1-P9
Roy PP, Roy K (2008) On some aspects of variable selection for
partial least squares regression models. QSAR Comb Sci 27:
302–313. doi:10.1002/qsar.200710043
Solomon VR, Hu C, Lee H (2009) Hybrid pharmacophore design and
synthesis of isatin-benzothiazole analogs for their anti-breast
cancer activity. Bioorg Med Chem 17:7585–7592
Song EY, Kaur N, Park MY, Jin Y, Lee K, Kim G, Lee KY, Yang JS,
Shin JH, Nam KY, No KT, Han G (2008) Synthesis of amide and
urea derivatives of benzothiazole as Raf-1 inhibitor. Eur J Med
Chem 43:1519–1524
Tasler S, Muller O, Wieber T, Herz T, Pegoraro S, Saeb W, Lang M,
Krauss R, Totzke F, Zirrgiebel U, Ehlert JE, Kubbutat MH,
Schachtele C (2009) Substituted 2-arylbenzothiazoles as kinase
inhibitors: hit-to-lead optimization. Bioorg Med Chem 17:6728–
6737
Trapani V, Patel V, Leong CO, Ciolino HP, Yeh GC, Hose C, Trepel
JB, Stevens MFG, Sausville EA, Loaiza-Perez AI (2003) DNA
damage and cell cycle arrest induced by 2-(4-amino-3-methyl-
phenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is atten-
uated in aryl hydrocarbon receptor deficient MCF-7 cells. Br J
Cancer 88:599–605
Wang X, Sarris K, Kage K, Zhang D, Brown SP, Kolasa T, Surowy C,
El Kouhen OF, Muchmore SW, Brioni JD, Stewart AO (2009)
Synthesis and evaluation of benzothiazole-based analogues as
novel, potent, and selective fatty acid amide hydrolase inhibitors.
J Med Chem 52:170–180
weblab Pro (2012) http://weblab-viewer-pro.fyxm.net/. Accessed 04
Sept 2012
Saeed S, Rashid N, Jones PG, Ali M, Hussain R (2010) Synthesis,
characterization and biological evaluation of some thiourea
derivatives bearing benzothiazole moiety as potential antimicro-
bial and anticancer agents. Eur J Med Chem 45:1323–1331
Siddiqui N, Pandeya SN, Khan SA, Stables J, Rana A, Alam M,
Arshad MF, Bhat MA (2007) Synthesis and anticonvulsant
activity of sulfonamide derivatives-hydrophobic domain. Bioorg
Med Chem Lett 17:255–259
Zhang XM, Qiu M, Sun J, Zhang YB, Yang YS, Wang XL, Tang JF,
Zhu HL (2011) Synthesis, biological evaluation, and molecular
docking studies of 1,3,4-oxadiazole derivatives possessing 1,4-
benzodioxan moiety as potential anticancer agents. Bioorg Med
Chem 19:6518–6524
Zheng CH, Zhou YJ, Zhu J, Ji HT, Chen J, Li YW, Sheng CQ, Lu JG,
Jiang JH, Tang H, Song YL (2007) Construction of a three-
dimensional pharmacophore for Bcl-2 inhibitors by flexible
docking and the multiple copy simultaneous search method.
Bioorg Med Chem 15:6407–6417
Siegel R, Ward E, Brawley O, Jemal A (2011) Cancer statistics, 2011.
CA Cancer J Clin 61:212–236
Singh P, Kaur M, Verma P (2009) Design, synthesis and anticancer
activities of hybrids of indole and barbituric acids—
123