Benzothiazoles: Search for anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2012.05.028

Novel derivatives of 2-amino benzothiazoles 4(a-j) have been synthesized and tested for their antitumor activity using National Cancer Institute (NCI) disease oriented antitumor screen protocol against nine panel of cancer cell lines. Among the synthesized compounds, two compounds were granted NSC code and screened at National Cancer Institute (NCI)-USA for anticancer activity at a single high dose (10^-5 M) and five dose in full NCI 60 cell panel. Among the selected compounds, 7-chloro-N-(2,6-dichlorophenyl)benzo[d]thiazol-2- amine (4i) with GI₅₀ values of 7.18 × 10^-8 M against Non-Small Cell HOP-92 Lung Cancer cell line proved to be the most active members in this study. Virtual screening was carried out through docking the designed compounds into the ATP binding site of epidermal growth factor receptor (EGFR) to predict if these compounds have analogous binding mode to the EGFR inhibitors.

Full text of DOI:10.1016/j.ejmech.2012.05.028

European Journal of Medicinal Chemistry 54 (2012) 447e462
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Benzothiazoles: Search for anticancer agents
,
b
c
Malleshappa N. Noolvi a , Harun M. Patel , Manpreet Kaur
*
a Department of Pharmaceutical Chemistry, Shree Dhanvantary Pharmacy College, Kim (Surat)-3941110, Gujrat, India
b Department of Pharmaceutical Chemistry, R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur (Dhule)-425405, Maharashtra, India
c Department of Pharmaceutical Chemistry, ASBASJSM College of Pharmacy, Bela (Ropar)-140111, Punjab, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< Synthesis and in vitro anticancer
activity of
a
novel 2-amino
benzothiazoles.
< Characterized by IR, 1H NMR, 13C
NMR, and HRMS technique.
< The title compound 4e shown
promising anti-tumor activity.
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel derivatives of 2-amino benzothiazoles 4(aej) have been synthesized and tested for their antitumor
activity using National Cancer Institute (NCI) disease oriented antitumor screen protocol against nine
panel of cancer cell lines. Among the synthesized compounds, two compounds were granted NSC code
and screened at National Cancer Institute (NCI)-USA for anticancer activity at a single high dose (10ꢀ5 M)
and five dose in full NCI 60 cell panel. Among the selected compounds ,7-chloro-N-(2,6-dichlorophenyl)
benzo[d]thiazol-2-amine (4i) with GI50 values of 7.18 ꢁ 10ꢀ8 M against Non-Small Cell HOP-92 Lung
Cancer cell line proved to be the most active members in this study. Virtual screening was carried out
through docking the designed compounds into the ATP binding site of epidermal growth factor receptor
(EGFR) to predict if these compounds have analogous binding mode to the EGFR inhibitors.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 25 January 2012
Received in revised form
16 May 2012
Accepted 22 May 2012
Available online 30 May 2012
Keywords:
Synthesis 2-amino benzothiazoles
Antitumor
Pharmacophore mapping
Docking
1. Introduction
have been shown to be key regulators of normal cellular processes
and additionally play a critical role in the development and
Nowadays, health is one of the most important domains which
we human beings have focused on in our society. However, tumor is
the biggest killer of our lives, so there has been steadily increasing
research in the field of anticancer therapy over recent years. With
the current chemotherapy, lack of selectivity of chemotherapeutic
agents against cancerous cells is a significant problem. Receptor
tyrosine kinases (RTKs) are high affinity cell surface receptors that
bind polypeptide growth factors, cytokines and hormones. They
progression of many types of cancer [1]. Protein tyrosine kinases
occupy a central position in the control of cellular proliferation.
Over expression of certain RTKs show association with promotion
and maintenance of malignancies. For example, the epidermal
growth factor (EGF) receptor tyrosine kinases of the erbB family
(which includes erbB1eerbB4) is frequently expressed at high
levels in certain carcinomas and shows an inverse correlation with
survival (particularly breast, colon and bladder cancers) [2]. Thus,
inactivation of the specific tyrosine kinases those are responsible
for the malignant phenotype of certain cancers represent a poten-
tial approach for design of antiproliferative drugs [3].
* Corresponding author. Tel.: þ91 9417563874; fax: þ91 1881263655.
E-mail address: mnoolvi@yahoo.co.uk (M.N. Noolvi).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.05.028

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M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
Benzothiazole type compounds have attracted considerable
medicinal importance of benzothiazole moiety, we synthesized and
in vitro evaluated benzothiazoles at National Cancer Institute
(NCI-USA) for anti-tumor activity. We have also tried to dock the
synthesized compounds with the crystal structure of EGFR to
explore the possible anticancer mechanism of our compounds.
Prior compounds have been reported [30,31] to compare the anti-
cancer activity of such compounds.
attention to anticancer research [4e14], and several attempts were
made for modifying the benzothiazole nucleus to improve their
antitumoractivities. Modificationsonthebenzothiazolenucleushave
resulted in a large number of compounds having diverse pharmaco-
logical activities. Among them imidazo benzothiazoles as well as
polymerized benzothiazoles and other substituted benzothiazoles
such as 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (PMX 610)
(Fig. 1) has been shown to exhibit exquisitely potent (GI50 < 0.1 nM)
and selective in vitro antitumor properties in human cancer cell lines
(e.g.,colon, nonsmalle celllungandbreastsubpanels)oftheNational
Cancer Institute (NCI) 60 human cancer cell line screen [15] and also
exhibited remarkable antitumor activity against malignant cell lines
[16]. 2-(4-aminophenyl)-benzothiazole (CJM 126) and its analogs
comprise a novel mechanistic class of antitumor agents [17,18]. These
nucleuses come from the related structure polyhydroxylated
2-phenylbenzothiazoles, flavone quercetin and the isoflavone genis-
tein, which are tyrosine kinase inhibitors bearing potent antitumor
activity [19,20]. The isoflavone, genistein [21] and the flavone, quer-
cetin [22] are competitive inhibitors at the ATP-binding site of kinases
[23,24]. As the crystal structure of 5,6-dimethoxy-2-(4-methox-
yphenyl)benzothiazole was solved [20], the preliminary analysis
based on comparisons between polyhydroxylated 2-phenyl-
benzothiazoles and the adenine fragment of ATP suggested that
suitablysubstituted benzothiazoles mightmimictheATPcompetitive
binding of genistein and quercetin at tyrosine kinases.
2. Rational and design
Benzothiazoles act via competing with ATP for binding at the
catalytic domain of tyrosine kinase [20]. The ATP binding site has
the following features; Adenine region e contains two key
Hydrogen bonds formed by the interaction of N-1 and N-6 amino
group of the adenine ring. Many potent inhibitors use one of these
Hydrogen bonds. Sugar region e a hydrophilic region, except a few
e.g. EGFR. Hydrophobic pocket e though not used by ATP but
plays an important role in inhibitor selectivity. Hydrophobic
channels e it is not used by ATP and may be exploited for inhibitor
specificity. Phosphate binding region e This is used for improving
inhibitor selectivity [32].
In this study, we present a new sub-family of compounds con-
taining 2-anilino benzothiazole core as EGFR inhibitors. Our
strategy is directed toward designing a variety of ligands which are
structurally similar with basic skeleton, 4-anilino quinazoline of
tinibs (erlotinib, lapatinib, gefitinib and canaratinib) with diverse
chemical properties (Fig. 2). We replaced quinazoline ring with
benzothiazole since both are isosteric with adenine portion of ATP
Hence in continuation of our efforts on the design and synthesis
of novel anti-cancer agents [25e29] and keeping in mind the
Fig. 1. Reported and proposed antitumor benzothiazole derivatives.

M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
449
interactions with the protein mimicking the 30-chloro-40-[(3-fluo-
robenzyl) oxy] aniline group of lapatinib (Fig. 3). We put both
electron withdrawing (Cl) and electron donating (CH3) anilines at
2nd position just to check the effect of these substituents over
anticancer activity since it is well known that presence of electron
withdrawing group on anilines provides protection from metabo-
lism and provides specificity to the molecules.
3. Chemistry
In Scheme 1 the compounds were synthesized using various
isothiocyanates 2(aee), which was prepared from different
aromatic primary amines 1(aee) [33]. Prepared isothiocyanates
2(aee) yielded thioureas
2,6-dimethyl and 2,6-dichloro aniline [25]. Oxidative cyclization of
(aee) by bromine resulted in the synthesis of proposed
compounds 4 (aej). Physical data of the synthesized compound is
given in Table 1.
The derivatives were characterized by spectral studies using IR,
1H NMR, 13C NMR and HRMS. The structures of thiocynates were
confirmed through the followingspectral data 2 (aee). IR absorption
peak at w2200-2000 cmꢀ1 corresponding to e SCN and 1H NMR
3
(aee) on condensation with
3
Fig. 2. Proposed hypothetical model of the highly active 7-chloro-N-(2,6-
dichlorophenyl)benzo[d]thiazol-2-amine (4i) bound to ATP binding site of EGFR
protein tyrosine kinase.
and can mimic the ATP competitive binding regions of EGFR tyro-
sine kinase. Like 4-aniline group in tinibs (erlotinib, lapatinib,
gefitinib and canaratinib) we introduced substituted aniline ring at
2nd position of benzothiazole since secondary amino group at 2nd
position of benzothiazoles is acting as conformational lock and
extending substituted aniline portion into the hydrophobic pockets
of EGFR e tyrosine kinase, making predominantly hydrophobic
showing a wd 6.80e7.90 ppm for aromatic protons of iso-
thiocyanates 2 (aee). Thioureas 3 (aej) were confirmed by the
absence of characteristic IR absorption peak at w2200e2000 cmꢀ1
Fig. 3. Rational designing of proposed compounds based upon known EGFR-Tyrosine
kinase inhibitor.
Scheme 1.

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M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
of e SCN group. Bands at w1610e1600 cmꢀ1 confirmed formation of
thioureas eNHeCSeNHe. Further occurrence of two broad peaks
4. Pharmacology
at w
tiated the formation of thioureas. 13C NMR confirmed conversion of
e SCN toeNHeCSeNHe by a peak at w 181e179 ppm corre-
d
9.23 and 8.14 ppm corresponding two e NH groups substan-
The in vitro anticancer screening at NCI is a two-stage process,
beginning with the evaluation of all compounds against the 60 cell
lines at a single dose of 10 mM. The output from the single dose
d
sponding to >C]S. The novel 2-amino benzothiazoles 4 (aej)
showed IR absorption band at w3300 (NH-strech), w3000 (CH-
strech), w1600 (NH-bend) and w700 (CeCl) cmꢀ1. 1H NMR spectra
screen is reported as a mean graph and is available for analysis by
the COMPARE program. Compounds which exhibit significant
growth inhibition are evaluated against the 60 cell panel at five
concentration levels. The human tumor cell lines of the cancer
screening panel are grown in RPMI 1640 medium containing 5%
fetal bovine serum and 2 mM L-glutamine. For a typical screening
experiment, cells are inoculated into 96 well microtiter plates in
revealed all the corresponding peaks at w
d 2.30 ppm for eCH3,
wd
4.50 for eNHe and w C
d
6.76e8.23 ppm for aromatic protons. 13
NMR gave valuable information to confirm cyclisation of substituted
thioureas to respective substituted 2-amino benzothiazoles with
characteristic peak at w
d
172 ppm for C-2. Further HRMS gave all the
100 mL at plating densities ranging from 5000 to 40,000 cells/well
molecular ion peaks corresponding to molecular weight of
confirmed novel compounds.
depending on the doubling time of individual cell lines. After cell
inoculation, the microtiter plates are incubated at 37 ꢂC, 5% CO2,
Table 1
Physicochemical properties of the synthesized compounds 4(aej).
No.
Compounds
NSC code
Molecular formula
C16H16N2S
Melting point (ꢂC)
146-148
Percentage yield (%)
32
4a
105624/759789
4b
e
C15H13BrN2S
156-160
28
4c
e
e
C15H13N3O2S
162-166
128-134
41
23
4d
C15H13ClN2S
4e
4f
e
e
C15H13N3O2S
124-128
138-142
30
38
C14H10Cl2N2S
4g
e
C13H7BrCl2N2S
122-125
42
4h
4i
e
C13H7Cl2N3O2S
172-174
165-168
44
31
105628/759790
C13H7Cl3N2S
4j
e
C13H7Cl2N3O2S
152-156
34

M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
451
Fig. 4. 2D and 3D view of 2-anilino benzothiazole template used for pharmacophoric mapping alignment.
95% air and 100% relative humidity for 24 h prior to addition of
experimental drugs.
with 1% acetic acid and the plates are air dried. Bound stain is
subsequently solubilized with 10 mM trizma base, and the absor-
bance is read on an automated plate reader at a wavelength of
515 nm. For suspension cells, the methodology is the same except
that the assay is terminated by fixing settled cells at the bottom of
After 24 h, two plates of each cell line are fixed in situ with TCA,
to represent a measurement of the cell population for each cell line
at the time of drug addition (Tz). Experimental drugs are solubi-
lized in dimethyl sulfoxide at 400 fold the desired final maximum
test concentration and stored frozen prior to use. At the time of
drug addition, an aliquot of frozen concentrate is thawed and
diluted to twice the desired final maximum test concentration with
the wells by gently adding 50 mL of 80% TCA (final concentration,
16% TCA). Using the seven absorbance measurements [time zero,
(Tz), control growth, (C), and test growth in the presence of drug at
the five concentration levels (Ti)], the percentage growth is
calculated at each of the drug concentrations levels. Percentage
growth inhibition is calculated as:
complete medium containing 50
10-fold or ½ log serial dilutions are made to provide a total of five
drug concentrations plus control. Aliquots of 100 L of these
different drug dilutions are added to the appropriate microtiter
wells already containing 100 L of medium, resulting in the
required final drug concentrations.
mg/ml gentamicin. Additional four,
m
½ðTiꢀTzÞ=ðCꢀTzÞꢃꢁ100forconcentrationsforwhichTi>= ¼ Tz
m
½ðTi ꢀ TzÞ=Tzꢃ ꢁ 100 for concentrations for which Ti < Tz:
Following drug addition, the plates are incubated for an addi-
tional 48 h at 37 ꢂC, 5% CO2, 95% air, and 100% relative humidity.
For adherent cells, the assay is terminated by the addition of cold
Three dose response parameters are calculated for each exper-
imental agent. Growth inhibition of 50% (GI50) is calculated from
[(TiꢀTz)/(CꢀTz)] ꢁ 100 ¼ 50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as
measured by SRB staining) in control cells during the drug incu-
bation. The drug concentration resulting in total growth inhibition
(TGI) is calculated from Ti ¼ Tz. The LC50 (concentration of drug
resulting in a 50% reduction in the measured protein at the end of
the drug treatment as compared to that at the beginning) indicating
TCA. Cells are fixed in situ by the gentle addition of 50 mL of cold
50% (w/v) TCA (final concentration, 10% TCA) and incubated for
60 min at 4 ꢂC. The supernatant is discarded, and the plates are
washed five times with tap water and air dried. Sulforhodamine B
(SRB) solution (100 ml) at 0.4% (w/v) in 1% acetic acid is added to
each well, and plates are incubated for 10 min at room tempera-
ture. After staining, unbound dye is removed by washing five times
Fig. 5. Common biopharmacophore of 4(aej).

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M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
a net loss of cells following treatment is calculated from [(TiꢀTz)/
Tz] ꢁ 100 ¼ ꢀ50. Values are calculated for each of these three
parameters if the level of activity is reached; however, if the effect is
not reached or is exceeded, the value for that parameter is
expressed as greater or less than the maximum or minimum
concentration tested [34e36].
5. Pharmacophore mapping
A pharmacophore is the ensemble of steric and electronic
features that is necessary to ensure the optimal supramolecular
interactions with a specific biological targets and to trigger (or to
block) its biological response. 3D pharmacophore modeling is
Fig. 6. Binding mode of Lapatinib (A) and compound 4i (B) in the ATP binding site of EGFR-TK showing H bonds between N-1 of quinazoline (Lapatinib) and N-3 of benzothiazole
(4i) with Met 793.

M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
453
Fig. 7. Binding mode of Lapatinib (C) and compound 4i (D) in the ATP binding site of EGFR-TK showing residue within 5 Å.

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M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
Table 2
Glide docking results based on hydrophobic interaction, hydrogen bonding interaction, glide dock score, E-model and good Vdw-interaction.
No.
Compounds
Hydrophobic interaction (within 5 Å)
H-bond interaction
Glide score E-model Good Vdw
(kcal/mol) -interaction
4a
ALA-743, ARG-841, ASP-855, CYS-797, GLY-791,
GLY-796, ILE-744, LEU-718, LEU-777, LEU-788,
LEU-792, LEU-844, LEU-858, LYS-745, MET-1002,
MET-766, MET-793, PHE-856, PRO-794, THR-790,
THR-854, VAL-726, PHE-795, VAL-845
N- of benzothiazole and H atom of ꢀ7.85
ꢀ70.781 221
amino acid backbone of MET-793
4b
ALA-743, ARG-776, ARG-841, ASN-842, ASP-855,
CYS-775, CYS-797, GLN-791, GLY-857, LEU-718,
LEU-777, LEU-788, LEU-792, LEU-844, LEU-858,
LYS-745, MET-766, PHE-856, THR-790, VAL-726,
VAL-774, LEU-795, VAL-845
N- of benzothiazole and H atom of ꢀ8.23
ꢀ80.710 234
amino acid backbone of MET-793
4c
ALA-743, ARG-841, ASN-842, ASP-855, CYS-775,
CYS-797, GLY-796, GLY-857, LEU-718, LEU-777,
LEU-788, LEU-858, LYS-745, MET-766, PHE-856,
THR-790, THR-854, VAL-726, VAL-769, LEU-778
N- of benzothiazole and H atom of ꢀ8.48
ꢀ85.15
ꢀ95.56
268
198
amino acid backbone of MET-793
4d
ALA-743, ARG-841, ASN-842, ASP-855, CYS-797,
GLY-796, ILE-744, ILE-789, LEU-718, LEU-777,
LEU-788, LEU-792, LEU-844, LYS-745, MET-1002,
MET-766, MET-793, THR-790, THR-854, VAL-726,
VAL-774, GLY-721, TYR-727, VAL-843
N- of benzothiazole and H atom of ꢀ8.44
amino acid backbone of MET-793
4e
4f
ALA-743, ARG-841, ASN-842, ASP-855, CYS-797,
GLY-796, ILE-744, ILE-789, LEU-718, LEU-777,
LEU-788, LEU-792, LEU-844, LYS-745, MET-1002,
MET-766, MET-793, PHE-856, THR-790, THR-854,
VAL-720, VAL-843, GLY-721, TYR-727
N- of benzothiazole and H atom of ꢀ8.80
amino acid backbone of MET-793
N- of Nitro group and H atom of
amino acid backbone of ASP-855
ꢀ89.86
ꢀ74.92
298
231
ALA-743, ARG-841, ASN-842, ASP-855, CYS-797,
GLY-719, GLY-796, ILE-744, ILE-789, LEU-718,
LEU-777, LEU-788, LEU-792, LEU-844, LYS-745,
MET-1002, MET-766, MET-793, SER-720, THR-790,
THR-854, VAL-726, LEU-778, GLY-721
N- of benzothiazole and H atom of ꢀ8.94
amino acid backbone of MET-793
4g
4h
4i
ALA-743, ARG-776, ARG-841, ASN-842, ASP-885,
CYS-775, CYS-797, GLY-857, LEU-718, LEU-788,
LEU-792, MET-766, MET-793, PHE-856, THR-790,
THR-854, VAL-726, VAL-769, VAL-774, LYS-745
N- of benzothiazole and H atom of ꢀ8.68
ꢀ71.03
ꢀ79.45
ꢀ98.16
282
221
301
amino acid backbone of MET-793
ALA-743, ARG-841, ASN-842, ASP-855, CYS-797,
GLY-719, ILE-744, ILE-789, LEU-718, LEU-777,
LEU-788, LEU-844, LYS-745, MET-1002, SER-720,
THR-790, THR-854, VAL-720, GLY-721
N- of benzothiazole and H atom of ꢀ8.48
amino acid backbone of MET-793
ALA-743, ARG-841, ASP-855, CYS-797,
GLN-791, ILE-744, ILE-789, LEU-718,
N- of benzothiazole and H atom of ꢀ9.95
amino acid backbone of MET-793
LEU-777, LEU-788, LEU-844, LYS-745,
MET-1002, MET-766, MET-793, THR-790,
THR-854, VAL-726, LEU-758, GLY-797, TYR-727
4j
ALA-743, ARG-841, ASN-842, ASP-855,
N- of benzothiazole and H atom of ꢀ9.49
amino acid backbone of MET-793
N- of Nitro group and H atom of
amino acid backbone of ASP-855
ꢀ92.18
254
CYS-797, GLY-719, LEU-718, LEU-771, LEU-792,
LEU-842, LYS-745, MET-766, MET-793, PHE-856,
THR-854, ILE-789, GLY-721, ILE-744
a technique for designing the interaction of a small molecule ligand
with a macromolecular target. Vlife MDS MolSign module is used
for the identification, generation and analysis of pharmacophore by
aligning small organic molecules based on their pharmacophore
features using PLS method [37].
A pharmacophore can be derived in a ligand-based manner, by
flexibility overlaying a set of active molecules and determining
those conformations that are able to be overlaid in such a way that
a maximum number of important chemical features geometrically
overlap. To be a useful tool for drug design, a pharmacophore model

M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
455
has to provide valid information for medicinal chemist investi-
gating structure-activity relationship. The pharmacophore model
has to describe the nature of the functional groups involved in
ligand-target interactions, as well as the type of the non-covalent
bonding and interchange distances.
All the structures of the compounds were drawn in 2D-APPL
mode of software and exported to 3D. Three-dimensional struc-
tures of all compounds have been constructed using MDS 3.5
version of Vlife science and their geometries were subsequently
optimized to make the conformations having least potential
energy. Energy minimizations were performed using Merck
molecular force field (MMFF) and MMFF charge [38] followed by
considering distance-dependent dielectric constant of 1.0 and
convergence criterion of 0.01 kcal/mol. The compounds were then
subjected to conformational analysis using Montecarlo conforma-
tional search with RMS gradient of 0.001 kcal/mol using
a MMFF force field. Molecular alignment is a crucial step for
pharmacophoric study to obtain meaningful results. This method is
based on moving of molecules in 3D space, which is related to the
conformational flexibility of molecules. The goal is to obtain
optimal alignment between the molecular structures [39]. All
molecules in the data set were aligned by template-based method
using 2-anilino benzothiazole as template, where a template is
built by considering common substructures in the series as shown
in Fig. 4. A highly bioactive energetically stable conformation in this
class of compounds is chosen as a reference molecule on which
other molecules in the data set are aligned, considering template as
a basis for the alignment. All the synthesized molecules were taken
for pharmacophore development. Highly active molecule to set as
reference. The reference molecule is the molecule on which other
molecules of the aligned data set get aligned. For pharmacophoric
identification tolerance limit and maximum distance allowed
between two features were set up to 10 Å. This abstract model
containing chemical functionalities such as hydrogen bond donor,
Fig. 8. Single dose assay of compound 4a (NSC: 105624/759789).

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M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
hydrogen bond acceptor and aromatic carbon center, can serve as
an effective search filter.
protein preparation was carried out using ‘protein preparation
wizard’ in Maestro 9.0 in two steps, preparation and refinement.
After ensuring chemical correctness, water molecules in the crystal
structures were deleted and hydrogens were added, where they
were missing. Using the OPLS 2005 force field energy of crystal
structure was minimized [41]. Grids were defined centering them
on the ligand in the crystal structure using the default box size. The
ligands were built using maestro build panel and prepared by
Ligprep 2.2 module which produce the low energy conformer of
6. Docking study
The molecular docking tool, GLIDE (Schordinger Inc., USA)
(2006) was used for ligand docking studies into tyrosine kinase
receptor binding pocket. The crystal structures of tyrosine kinase
was obtained from protein data bank (PDB ID: 1XKK) [40]. The
Fig. 9. Five dose assay of compound 4a (NSC: 105624/759789).

M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
457
ligands using OPLS 2005 force field. The low energy conformation
of the ligands was selected and was docked into the grid generated
from protein structures using standard precision (SP) docking
mode. The final evaluation is done with glide score (docking score)
and single best pose is generated as the output for particular ligand.
pharmacophore having four larger orange color tessellated sphere
shows aromatic and aliphatic carbon. Magneta color larger tessel-
lated sphere shows the hydrogen bond donor as shown in Fig. 5. The
distance among the various chemical features are as follows;
1. AroS (Benzothiazole) to AroC (Aniline) ¼ 4.872 Å
2. AroS (Benzothiazole) to AliC (1st methyl of aniline) ¼ 4.202 Å
3. AroS (Benzothiazole) to HDr (eNHe) ¼ 5.392 Å
4. AroC (Aniline) to HDr (eNHe) ¼ 5.351 Å
7. Results and discussion
7.1. Pharmacophore modeling
5. AroC (Aniline) to AliC (1st methyl of aniline) ¼ 3.275 Å
6. HDr (eNHe) to AliC (1st methyl of aniline) ¼ 3.621 Å
7. HDr (eNHe) to AliC (6th methyl of aniline) ¼ 4.872 Å
The pharmacophore mapping was carried out to map the
chemical features or functional groups present in the synthesized
compounds which are essential for anticancer activity. We gener-
ated different pharmacophore pattern based on a set of synthesized
aligned molecules. Selected pharmacophore shows five chemical
features which were present in all the synthesized molecules
(indicated by 100%) as shown in Fig. 5. The information shows that
the five features used were two AroC feature (Aromatic), AliC feature
(Aliphatic) one HDr (Hydrogen bond donor) features. The average
RMSD of the pharmacophore alignment of each two molecule is
0.010511 Å. These five chemical features were found in all the
synthesized molecules. The larger tessellated spheres are indicative
of the common pharmacophore identified in the molecule. The
smaller solid features are of the individual molecules. The common
Hypothesis generation was performed using low energy
conformers of the molecules. All adapted models showed that the
donor atoms of the NH fragments (magenta color larger tessellated)
as hydrophilic element and the aryl moieties as hydrophobic
element were well superimposed within the set distance tolerance.
This confirms the important role of the hydrophilic and hydro-
phobic moieties for recognition and binding to receptor sites.
According to the pharmacophore generated by Molsign the
minimal structural requirements for antitumor activity consist of
an aromatic ring (hydrophobic region) attached to NH fragment
(H-bonding donor region), and a hydrophobic region represented
Fig. 10. Five dose assay graph of compound 4a (NSC: 105624/759789) against nine panel cancer cell line at NCI.

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M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
by benzothiazole core. This pharmacophoric assumption was in
consistence with biological data.
with many structurally related lead compounds such as lapatinib
and gefitinib we could observe how our compounds might bind to
the kinase binding site, based on the knowledge of the structure of
similar active sites. We redocked lapatinib into the active site of the
enzyme and then we replaced with our compounds in order to
compare the binding mode of both ligand and the test compound.
These docking studies have revealed that the benzothiazole ring
binds to a narrow hydrophobic pocket in the N-terminal domain of
EGFR TK where N- of the benzothiazole ring interacts with the
backbone NH of Met-793 via a hydrogen bond. These interactions
underscore the importance of both nitrogen atoms for binding and
the subsequent inhibitory capacity. The aniline moiety at C-2 of
benzothiazole lies in a deep and hydrophobic pocket similar to the
30-chloro-40-(3-fluorobenzyl)oxy moiety of lapatinib. The results of
this virtual screening could support the postulation that our active
compounds may act on the same enzyme target where EGFR
inhibitor acts.
7.2. Docking study
Docking study was carried out for the target compounds into
EGFR using GLIDE (Schordinger Inc., USA) (2006). The crystal
structure of the enzyme with lapatinib (1XKK) was obtained from
protein data bank PDB [40]. Since it was found that gefitinib mimic
ATP and bind to the ATP binding region of the kinase active site. Our
compounds were modeled by positioning them in the lapatinib
binding site in accordance with the published crystal structures of
quinazoline derivatives bound to kinase [42]. The entire complex
was then subjected to alternate cycles of minimization and
dynamics. The intent was to get a satisfactory structure for the
complex that was consistent with the published crystal structure
[43,44]. From the comparative docking study of our compounds
Fig. 11. Single dose assay of compound 4i (NSC: 105628/759390).

M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
459
Figs. 6 and 7 demonstrate binding mode of lapatinib and ben-
zothiazoles (4i) in the ATP binding site. Lapatinib forms hydrogen
bonding with MET-793 via N-1 of quinazoline and similar
hydrogen bonding interaction is also shown by N-3 of
benzothiazoles (4i) with MET-793 and in case of compound 4e and
4j, we noticed additional H bond between N of nitro group and H
atom of amino acid backbone of ASP-855. Residues within 5 Å
areas of lapatinib and benzothiazoles (4i) are shown in Fig. 7. Some
Fig. 12. Five dose assay of compound 4i (NSC: 105628/759390).

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M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
common residues involved in this type of interaction within 5 Å
area are ALA-743, ARG-841, ASP-855, CYS-797, LEU-718, LEU-777,
LEU-788, LYS-745, MET-766, THR-790 and THR-854 as shown in
Table 2.
7.4. In vitro 5 dose full NCI 60 cell panel assay
All the cell lines (about 60), representing nine tumor subpanels,
were incubated at five different concentrations (0.01, 0.1, 1, 10 &
100 mM). The outcomes were used to create log concentration Vs %
growth inhibition curves and three response parameters (GI50, TGI
and LC50) were calculated for each cell line. The GI50 value (growth
inhibitory activity) corresponds to the concentration of the
compound causing 50% decrease in net cell growth, the TGI value
(cytostatic activity) is the concentration of the compound resulting
in total growth inhibition and LC50 value (cytotoxic activity) is the
concentration of the compound causing net 50% loss of initial cells
at the end of the incubation period of 48 h [34e36].
Compound under investigation 4a exhibited remarkable anti-
cancer activity against most of the tested cell lines representing
nine different subpanels with GI50 value 4.12 ꢁ 10ꢀ7 M against
Leukemia SR cell line, 5.52 ꢁ 10ꢀ7 M against Non-Small Cell Lung
Cancer NCI-H5222 cell line, 9.20 ꢁ 10ꢀ7 M, 6.29 ꢁ 10ꢀ7 M,
9.47 ꢁ 10ꢀ7 M against Colon Cancer HCT-116, HCT-15and SW-620
cancer cell line respectively, 7.25 ꢁ 10ꢀ7 M against CNS cancer SF-
295 cancer cell line, 2.42 ꢁ 10ꢀ7 M against Melanoma MDA-MB-
435 cancer cell line, 6.39 ꢁ 10ꢀ7 M against Ovarian NCI/ADR-RES
cancer cell line, 5.64 ꢁ 10ꢀ7 M against Renal Cancer CAKI-1
7.3. Primary single high dose (10ꢀ5 M) full NCI 60 cell panel
in vitro assay
The tumor growth inhibition properties of the two compounds
4a and 4i with the NCI codes 105624/759789 and 105628/759390
selected among 4(aej) by the National Cancer Institute (NCI), USA,
were screened on human tumor cell lines at single high dose
(10ꢀ5 M) and five dose level at the NIH, Bethesda, Maryland, USA,
under the drug discovery program of the NCI.
With regard to sensitivity against individual cell lines at primary
single high dose (10ꢀ5 M). Compound 4a showed remarkably
lowest cell growth promotion against CNS cancer SNB-75 cell line
and Melanoma MDA-MB-435 cancer cell line with cell growth
promotion of ꢀ15.50 and ꢀ23.68 respectively as shown in Fig. 8. On
the other hand compound 4i was found to be broad spectrum
against all nine panel of cancer cell line at primary single high dose
(10ꢀ5 M) as shown in Fig. 11.
Fig. 13. Five dose assay graph of compound 4i (NSC: 105628/759390) against nine panel of cancer cell line at NCI.

M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
461
cancer cell line, 5.28 ꢁ 10ꢀ7 M and 3.30 ꢁ 10ꢀ7 M against Breast
MDA-MB-231/ATCC and MDA-MB-468 cancer cell line. A sign of
“>” is used as prefix to the concentration for those cell lines which
were found to be insensitive at the highest tested concentration i.e.
GF254 silica gel, 0.2 mm layer thickness (E. Merck). Melting points
of the synthesized compounds were recorded on the Veego
(VMP-MP) melting point apparatus. IR spectrum was acquired on
a Shimadzu Infra Red Spectrometer, (model FTIR-8400S). Both
1H-NMR (DMSO) and 13C NMR (DMSO) spectra of the synthesized
compounds were performed with Bruker Avance-II 400 NMR
Spectrometer operating at 400 MHz in SAIF, Punjab University
(Chandigarh). Chemical shifts were measured relative to internal
100 mM as shown in Figs. 9 and 10.
With regard to the sensitivity against some individual cell lines
the compound 4i showed highest activity against Non-Small Cell
HOP-92 Lung cancer cell line with GI50 value of 7.18 ꢁ 10ꢀ8 M.
Obtained data revealed an obvious broad spectrum sensitivity
profile of 4i toward all 9 subpanel of cancer cell line with GI50 value
ranging from 1.60 ꢁ 10ꢀ6 M to 7.18 ꢁ 10ꢀ8 M, least for Non-Small
Cell HOP-92 Lung cancer and maximum for OVCAR-SK-OV-3 cell
line as shown in Figs. 12 and 13.
standard TMS (d:0). Chemical shifts are reported in d scale (ppm).
Mass spectra of the synthesized compounds were recorded at MAT
120 in SAIF, Punjab University.
9.1. Synthesis of substituted isothiocyanates 2(aee) [33]
On the basis of results obtained it was found that compound 4i
(NSC: 105628/759390) was most active compound of the series.
Based on close examination on substitutions, it may be concluded
that the role of electron withdrawing groups (eCl) has great
influence on anticancer activity, it is also proved by the docking
study where compound 4a is having dock score of ꢀ7.85 kcal/mol
and 4i is having ꢀ9.95 kcal/mol. Finally it is conceivable that further
derivatization of such compounds will be of interest with the hope
to get more selective anticancer agents.
9.1.1. Synthesis of substituted thioureas 3(aej) [25]
9.1.1.1. General procedure for the synthesis of substituted 2-amino
benzothiazoles 4(aej). Substituted thioureas 3 (aej) (0.01 mol)
were dissolved in chloroform (15 ml), the reaction mixture was
cooled in an ice bath and then bromine: chloroform (1:9) mixture
was added drop wise. The reaction was monitored by TLC and
after an hour, was poured on to crushed ice. The solid that
separated was filtered, dried in each case and then purified by
column chromatography using silica gel. It was eluted with CHCl3:
EtOAc (7:3) and the eluents on evaporation and crystallization
yielded pure solid substituted 2-amino benzothiazoles.
8. Conclusion
Compounds 7-chloro-N-(2,6-dichlorophenyl)benzo[d]thiazol-
2-amine (4a) with GI50 values of 7.18 ꢁ 10ꢀ8 M against Non-Small
Cell Lung Cancer HOP-92 cancer cell line proved to be the most
active members in this study. This benzothiazole analog could be
considered as useful templates for future development to obtain
more potent antitumor agent(s). Flexible alignment was conducted
on the basis of experimental data from which five featured phar-
macophore model was developed. This pharmacophore model
could be very useful for the virtual screening in the development of
new antitumor agents. According to the pharmacophore generated
by Molsign the minimal structural requirements for antitumor
activity consist of an aromatic ring (hydrophobic region) attached
to NH fragment (H-bonding donor region), and a hydrophobic
region represented by benzothiazole core. Molecular docking
studies further supports our assumption that the synthesized
compounds have analogous binding mode to the EGFR inhibitors
and demonstrates the various interactions between the ligands and
enzyme active sites and thereby help to design novel potent
inhibitors. The overall outcome of this model revealed that: (i) the
benzothiazole ring is a satisfactory backbone for antitumor activity,
(ii) the presence of substituted aniline moiety at the C-2 amino
position is necessary for the activity as hydrophobic region, (iii) the
presence of electron withdrawing group on benzothiazole ring and
on substituted aniline at 2nd position enhances the anticancer
activity. (iv) secondary amino group at 2nd position of benzothia-
zoles is acting as conformational lock and extending substituted
aniline portion into the hydrophobic pockets of EGFR-tyrosine
kinase, making predominantly hydrophobic interactions with the
protein mimicking the 30-chloro-40-[(3-fluorobenzyl)oxy] aniline
group of lapatinib. These preliminary encouraging results of bio-
logical screening of the tested compounds could offer an excellent
framework in this field that may lead to discovery of potent anti-
tumor agent.
9.1.1.1.1. N-(2,6-dimethylphenyl)-6-methylbenzo[d]thiazol-2-amine
(4a). This compound was prepared and purified as per the above
mentioned procedure; IR (KBr) ymax 3301 (NH-strech), 3039 (Arom.
CH strech), 2937 (Aliph. CH strech), 1602 (NH-bend) cmꢀ1; 1H NMR
(DMSO-d6)
d
ppm: 7.46e8.33 (m, 6H, AreH), 4.54 (s,1H, NH), 2.32 (s,
ppm:172.3, 154.3,
6H, CH3), 2.46 (s, 3H, CH3); 13C NMR (DMSO-d6)
d
138.7, 134.2, 130.7, 124.2, 120.6, 21.6 (benzothiazole), 140.2, 138.2,
131.3, 124.6, 20.2 (2,6-dimethyl aniline); HRMS (EI) m/z calcd for
C16H16N2S: 268.1034; found: 268.1038.
9.1.1.1.2. 6-bromo-N-(2,6-dimethylphenyl)benzo[d]thiazol-2-amine
(4b). This compound was prepared and purified as per the above
mentioned procedure; IR (KBr) ymax 3256 (NH-strech), 3049 (Arom.
CH strech), 2919 (Aliph. CH strech), 1563 (NH-bend), 623 (CeBr)
cmꢀ1
;
1H NMR (DMSO-d6)
d
ppm: 6.76e7.35 (m, 6H, AreH), 4.26
(s, 1H, NH), 1.99 (s, 6H, CH3); 13C NMR (DMSO-d6)
d
ppm: 173.1,
156.2, 134.2, 130.1, 126.3, 120.8, 118.6 (benzothiazole), 141.8, 138.6,
130.3, 125.5, 21.3 (2,6-dimethyl aniline); HRMS (EI) m/z calcd for
C15H13BrN2S: 331.9983; found: 331.9981.
9.1.1.1.3. N-(2,6-dimethylphenyl)-6-nitrobenzo[d]thiazol-2-amine
(4c). This compound was prepared and purified as per the above
mentioned procedure; IR (KBr) ymax 3379 (NH-strech), 3118 (Arom.
CH strech), 2952 (Aliph. CH strech), 1583 (NH-bend),1552,1349
(NO2) cmꢀ1; 1H NMR (DMSO-d6)
d
ppm: 6.98e7.85 (m, 6H, AreH),
4.54 (s, 1H, NH), 2.23 (s, 6H, CH3); 13C NMR (DMSO-d6)
d
ppm:
174.8, 162.3, 149.2, 134.4, 126.4, 122.2, 119.2 (benzothiazole),
139.6, 136.2, 130.2, 126.2, 20.4 (2,6-dimethyl aniline); HRMS (EI)
m/z calcd for: C15H13N3O2S: 299.0728; found: 299.0732.
9.1.1.1.4. 7-chloro-N-(2,6-dimethylphenyl)benzo[d]thiazol-2-amine
(4d). This compound was prepared and purified as per the above
mentioned procedure; IR (KBr) ymax 3391 (NH-strech), 3134 (Arom.
CH strech), 2817 (Aliph. CH strech), 1584 (NH-bend), 727 (CeCl)
cmꢀ1
;
1H NMR (DMSO-d6)
d
ppm: 6.85e8.17 (m, 6H, AreH), 5.47
(s, 1H, NH), 2.20 (s, 6H, CH3); 13C NMR (DMSO-d6)
d
ppm: 171.3,
154.2, 136.3, 134.2, 132.1, 130.6, 118.2 (benzothiazole), 141.2, 139.4,
131.3, 125.1, 20.3 (2,6-dimethyl aniline); HRMS (EI) m/z calcd for
C15H13ClN2S: 288.0488; found: 288.0492.
9. Experimental
All chemicals and solvents were supplied by Merck, S.D. Fine
Chemical Limited, Mumbai. All the solvents were distilled and dried
before use. The reactions were monitored with the help of thin-
layer chromatography using pre-coated aluminum sheets with
9.1.1.1.5. N-(2,6-dimethylphenyl)-7-nitrobenzo[d]thiazol-2-amine
(4e) [45]. This compound was prepared and purified as per the
above mentioned procedure; IR (KBr) ymax 3291 (NH-strech), 3051
(Arom. CH strech), 2962 (Aliph. CH strech), 1574 (NH-bend),

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M.N. Noolvi et al. / European Journal of Medicinal Chemistry 54 (2012) 447e462
1548,1354 (NO2) cmꢀ1 1H NMR (DMSO-d6)
; d ppm: 6.76e8.14
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Med. Chem. 13 (2005) 2509e2522.
(m, 6H, AreH), 4.23 (s, 1H, NH), 2.28 (s, 6H, CH3); 13C NMR
(DMSO-d6)
d ppm: 171.3, 152.3, 146.2, 132.8, 131.4, 128.4, 122.3
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1323e1331.
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D.W. Andrew, J. Med. Chem. 49 (2006) 179e185.
(benzothiazole), 140.2, 136.4, 132.6, 124.9, 20.8 (2,6-dimethyl
aniline); HRMS (EI) m/z calcd for C15H13N3O2S: 299.0728; found:
299.0724.
9.1.1.1.6. N-(2,6-dichlorophenyl)-6-methylbenzo[d]thiazol-2-amine
(4f). This compound was prepared and purified as per the above
mentioned procedure; IR (KBr) ymax 3312 (NH-strech), 3061 (Arom.
CH strech), 2923 (Aliph. CH strech), 1589 (NH-bend), 684 (CeCl)
cmꢀ1
;
1H NMR (DMSO-d6)
d
ppm: 6.81e8.10 (m, 6H, AreH), 4.21
(s, 1H, NH), 2.21 (s, 3H, CH3); 13C NMR (DMSO-d6)
d
ppm: 173.4,
152.4, 136.5, 132.6, 128.4, 124.4, 118.4, 21.3 (benzothiazole), 140.2,
137.2, 130.3, 122.6 (2,6-dichloro aniline); HRMS (EI) m/z calcd
forC14H10Cl2N2S: 307.9942; found: 307.9945.
9.1.1.1.7. 6-bromo-N-(2,6-dichlorophenyl)benzo[d]thiazol-2-amine
(4g). This compound was prepared and purified as per the above
mentioned procedure; IR (KBr) ymax 3331 (NH-strech), 3012 (CH
strech), 1591 (NH-bend), 711 (CeCl), 623 (CeBr) cmꢀ1 1H NMR
;
(DMSO-d6)
NMR (DMSO-d6)
d
ppm: 6.76e7.33 (m, 6H, AreH), 4.36 (s, 1H, NH); 13C
ppm: 171.3, 156.2, 134.8, 130.2, 125.1, 122.6, 118.3
[16] Y.A. Al-Soud, Arkivoc 15 (2008) 225e238.
d
[17] E. Kashiyama, I. Hutchinson, M.S. Chua, S.F. Stinson, L.R. Phillips, G. Kaur,
E.A. Sausville, T.D. Bradshaw, A.D. Westwell, M.F.G. Stevens, J. Med. Chem. 42
(1999) 4172e4184.
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Cancer 77 (1998) 745e752.
(benzothiazole), 139.2, 138.6, 129.8, 124.6 (2,6-dichloro aniline);
HRMS (EI) m/z calcd for C13H7BrCl2N2S: 371.8890; found: 371.8895.
9.1.1.1.8. N-(2,6-dichlorophenyl)-6-nitrobenzo[d]thiazol-2-amine
(4h). This compound was prepared and purified as per the above
mentioned procedure; IR (KBr) ymax 3308 (NH-strech), 3008
(CH strech), 1594 (NH-bend), 1552,1346 (NO2), 725 (CeCl) cmꢀ1; 1H
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J. Med. Chem. 37 (1994) 1689e1695.
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M. Shibuya, Y. Fukami, J. Biol. Chem. 262 (1987) 5592e5595.
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798e806.
NMR (DMSO-d6)
d
ppm: 6.88e8.23 (m, 6H, AreH), 4.01 (s, 1H, NH);
ppm: 170.3, 162.9, 146.4, 134.8, 124.6, 120.2,
13C NMR (DMSO-d6)
d
118.5 (benzothiazole), 141.3, 138.5, 129.8, 121.3 (2,6-dichloro
aniline); HRMS (EI) m/z calcd for C13H7Cl2N3O2S: 338.9636;
found: 338.9641.
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J.A. Hickman, Anti-Cancer Drug Des 7 (1992) 365e384.
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A.K. Gadad, G. Singh, N.G. Kutty, M. Rao, Eur. J. Med. Chem. 44 (2009)
2923e2929.
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Pharm. Bull. 40 (1992) 2387e2390.
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M.F.G. Stevens, J. Med. Chem. 44 (2001) 1446e1455.
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J. Liebetanz, T. O’Reilley, P. Traxler, B. Chaudhari, H. Fretz, J. Zimmermann,
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9.1.1.1.9. 7-chloro-N-(2,6-dichlorophenyl)benzo[d]thiazol-2-amine
(4i). This compound was prepared and purified as per the above
mentioned procedure; IR (KBr) ymax 3256 (NH-strech), 3112 (CH
strech), 1576 (NH-bend), 761 (CeCl), cmꢀ1 1H NMR (DMSO-d6)
;
d
d
ppm:6.72e8.10(m, 6H,AreH), 4.51(s,1H,NH);13CNMR(DMSO-d6)
ppm: 172.4, 152.4, 132.4, 130.2, 128.4, 125.4, 121.3 (benzothiazole),
140.7, 138.4, 128.2, 120.6 (2,6-dichloro aniline); HRMS (EI) m/z calcd
forC13H7Cl3N2S: 327.9396; found: 327.9391.
9.1.1.1.10. N-(2,6-dichlorophenyl)-7-nitrobenzo[d]thiazol-2-amine
(4j). This compound was prepared and purified as per the above
mentioned procedure; IR (KBr) ymax 3323 (NH-strech), 3075
(CH strech), 1584 (NH-bend),1555, 1351 (NO2) 703 (CeCl) cmꢀ1; 1H
NMR (DMSO-d6)
d
ppm: 7.12e7.94 (m, 6H, AreH), 4.52 (s, 1H, NH);
ppm: 173.5, 152.3, 146.2, 136.2, 135.8, 125.6,
13C NMR (DMSO-d6)
d
120.3 (benzothiazole), 142.3, 139.5, 131.6, 121.7 (2,6-dichloro
aniline); HRMS (EI) m/z calcd for C13H7Cl2N3O2S: 338.9636;
found: 338.9632.
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Acknowledgment
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The authors would like to thank Director General, Department
of Science and Technology, Government of India, New Delhi, for
funding the project (Grant No. SR/FT/LS-0024/2008).
References
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