Novel series of benzo[d]thiazolyl substituted-2-quinolone hybrids: Design, synthesis, biological evaluation and in-silico insights

Article abstract of DOI:10.1016/j.molstruc.2020.129413

A novel series of 3-(2-(4-(substituted-benzo[d]thiazol-2-yl)phenylamino)acetyl)-4?hydroxy-1-methyl/phenyl quinolin-2(1H)-one (7a-f and 8a-f) were synthesized. Reaction of appropriately substituted-2-(4-amino phenyl)benzo[d]thiazole (4a-f) with 3-(2-bromoacetyl)-4?hydroxy-1-methyl/phenyl quinolin-2(1H)-one (5/6) in the presence of glacial acetic acid resulted in desired compounds. Structures of the synthesized compounds were characterized based on their spectral (IR, ¹H NMR, ¹³C NMR and MS) and elemental analysis. The cytotoxicity screening studies revealed that MCF-7 and WRL68 cancer cells were sensitive to all the tested compounds. Out of twelve novel hybrids, compound 8f displayed the most significant anticancer activity. Docking studies were performed in order to understand the binding mode of the title compounds at the active site of the target enzyme (EGFR tyrosine kinase, 1M17). Compounds 8f and 7f displayed prominent and conserved binding interactions against 1M17. In addition, compounds 7e, 7f, 8e, and 8f exhibited interesting in vitro antibacterial activity, especially against Gram-negative bacteria E. coli. In summary, the novel benzo[d]thiazolyl substituted-2-quinolone hybrid (8f) could be considered as promising hit and could be further exploited for developing potential anticancer/antimicrobial agents.

Full text of DOI:10.1016/j.molstruc.2020.129413

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Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Novel series of benzo[d]thiazolyl substituted-2-quinolone hybrids:
Design, synthesis, biological evaluation and in-silico insights
a,∗
b
c
b
Girish Bolakatti , Mahesh Palkar , Manjunatha Katagi , Girish Hampannavar ,
f
d
e
Rajshekhar V. Karpoormath , Shilpa Ninganagouda , Arvind Badiger
a
Department of Pharmaceutical Chemistry, GM Institute of Pharmaceutical Sciences and Research, Davangere, 577006, Karnataka, India
Department of Pharmaceutical Chemistry, K.L.E.U’s College of Pharmacy, Vidyanagar, Hubli, 580031, Karnataka, India
Department of Pharmaceutical Chemistry, Bapuji Pharmacy College, Davangere, 577004, Karnataka, India
Department of Pharmaceutical Chemistry, School of Health Sciences, University of KwaZulu-Natal, Westville Campus, Durban, 4000, South Africa
Bapuji Institute of Engineering and Technology, Davangere, 577004, Karnataka, India
b
c
d
e
f
BDR Pharmaceutical International Pvt Ltd, Baroda, Vadodara, 390020, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A
novel series of 3-(2-(4-(substituted-benzo[d]thiazol-2-yl)phenylamino)acetyl)-4–hydroxy-1-methyl/
phenyl quinolin-2(1H)-one (7a-f and 8a-f) were synthesized. Reaction of appropriately substituted-2-
4-amino phenyl)benzo[d]thiazole (4a-f) with 3-(2-bromoacetyl)-4–hydroxy-1-methyl/phenyl quinolin-
(1H)-one (5/6) in the presence of glacial acetic acid resulted in desired compounds. Structures of the
Received 7 May 2020
Revised 4 October 2020
Accepted 5 October 2020
Available online xxx
(
2
synthesized compounds were characterized based on their spectral (IR, 1H NMR, 13 C NMR and MS) and
elemental analysis. The cytotoxicity screening studies revealed that MCF-7 and WRL68 cancer cells were
sensitive to all the tested compounds. Out of twelve novel hybrids, compound 8f displayed the most sig-
nificant anticancer activity. Docking studies were performed in order to understand the binding mode of
the title compounds at the active site of the target enzyme (EGFR tyrosine kinase, 1M17). Compounds 8f
and 7f displayed prominent and conserved binding interactions against 1M17. In addition, compounds 7e,
Keywords:
Benzo[d]thiazole
2-quinolone
Antimicrobial
Anticancer
MTT assay
Docking
7f, 8e, and 8f exhibited interesting in vitro antibacterial activity, especially against Gram-negative bacteria
E. coli. In summary, the novel benzo[d]thiazolyl substituted-2-quinolone hybrid (8f) could be considered
as promising hit and could be further exploited for developing potential anticancer/antimicrobial agents.
©
2020 Elsevier B.V. All rights reserved.
1. Introduction
ing tumor represents one of the most significant challenges for
chemists and oncologists [3].
A grave health problem across the globe ‘Cancer’ is a chronic
pathological condition, where there is a rapid uncontrolled pro-
liferation of abnormal premature cells. Cancer may affect people
at all ages, even fetuses, but risk tends to increase with age [1].
The active cases of cancer are increasing at an alarming rate due
to drastic change in lifestyle of humans over the years. It is the
second most common cause of death after heart diseases. Approx-
imately 21% annual deaths worldwide occur due to cancer i.e. 7.6
million deaths annually and is expected to reach up to 13 million
in 2030 [2]. Despite increase in sophisticated approach in design-
ing cancer chemotherapy, there is still no curative treatment that
is 100% effective. Currently cancer chemotherapeutic agents lack
selectivity and specificity against cancerous cells/tissues, which is
a significant setback. Consequently, cancer chemotherapy target-
Heterocyclic skeletons serve as ideal scaffolds on which phar-
macophores can be appended to yield potent and selective drugs
[4]. In the past two decades, 2-substituted benzo[d]thiazole ana-
logues have been extensively studied for their anticancer activ-
ity and continued to magnetize considerable attention in anti-
cancer research [5–8]. Literature reports suggest that 2-(4-Amino
phenyl)benzo[d]thiazoles [9] and their corresponding N-acetylated
derivatives [10] possess remarkable in vitro anticancer activity es-
pecially against breast, colon, and ovarian cell lines. Fig. 1 illus-
trates the importance of benzo[d]thiazole nucleus and its analogs
with their potential antitumor activities [11–15]. This versatile and
potent scaffold has provided an impetus for the anticancer drug
discovery in recent years.
Another important class of heterocyclic scaffold is 2-quinolone
(
also known as carbostyril or 1-aza coumarin), which serves as
a privileged structure for the generation of drug-like libraries
in drug-discovery programs. In addition, 2-quinolones are iso-
∗
Corresponding author.
E-mail address: principal.gmips@gmit.ac.in (G. Bolakatti).
https://doi.org/10.1016/j.molstruc.2020.129413
0
022-2860/© 2020 Elsevier B.V. All rights reserved.
Please cite this article as: G. Bolakatti, M. Palkar, M. Katagi et al., Novel series of benzo[d]thiazolyl substituted-2-quinolone hybrids: De-
sign, synthesis, biological evaluation and in-silico insights, Journal of Molecular Structure, https://doi.org/10.1016/j.molstruc.2020.129413

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Fig. 1. Reported and Proposed structures of anticancer benzo[d]thiazole and 2-quinolone derivatives. Design of a novel series of 2-phenylbenzo[d]thiazolyl substituted 2-
quinolone analogs using molecular hybridization approach.
meric to 4-quinolones and isosteric to coumarins and are associ-
ated with interesting biologic activities such as antibacterial, an-
ticancer, antiviral, cardiotonic, etc. [16–21]. During the last decade,
ological evaluation for anticancer and antimicrobial activities is
communicated.
2
-quinolones have been extensively studied for their anticancer ac-
2. Results and discussion
tivity as represented in Fig. 1. Further, Ruiz et al. reported cytotoxic
mechanism of quinolone derivatives as alkylating agents [22]. Dif-
ferent substituted 2-quinolones have also show in-vitro and in vivo
growth inhibition activity against various strains of bacteria and
fungi; moreover quinolones are also known to inhibit DNA syn-
thesis by promoting cleavage of bacterial DNA gyrase and type-IV
topoisomerase, resulting in rapid cell death [23–25].
2.1. Synthetic and spectral studies
The synthesis of series of twelve novel 3-(2-(4-(6,7-
substituted-benzo[d]thiazol-2-yl)phenylamino)acetyl)−4–hydroxy-
1-methyl/phenyl quinolin-2(1H)-one (7a-f and 8a-f) derivatives
was achieved through convenient and efficient synthetic route
Over the years, molecular hybridization based drug design ap-
proach [26,27] has been exploited by many researchers in order
to develop some promising new hybrid chemical entities (NHCEs),
displaying significant therapeutic values. The combination of two
pharmacophores into a single molecular skeleton is a well estab-
lished approach for designing more potent drugs with significant
increase in activity. A hybrid molecule acting on manifold tar-
gets is considered to be a better drug candidate than drug com-
binations, since administration of single drug will have more pre-
dictable pharmacokinetic and pharmacodynamic properties with
improved patient compliance [28]. Owing to their well appreciated
anticancer and antimicrobial properties, it was envisaged to design
hybrid structures having substituted 2-phenylbenzo[d]thiazole and
substituted 2-quinolone motifs connected with a linker (Fig. 1).
Therefore, in view of the above facts and in continuation of
search on biologically active hybrid molecules [29–33], in this
report the synthesis of novel benzo[d]thiazolyl-2-quinolone hy-
brid analogs (7a-f and 8a-f) with their subsequent in vitro bi-
as outlined in Schemes
1
and 2. The cyclization of N-(3,4-
with potassium
disubstituted-phenyl)−4-nitrobenzothioamide
ferricynide in presence of aqueous sodium hydroxide afforded
6,7-substituted-2-(4-nitrophenyl)benzo[d]thiazole, which upon re-
duction with stannous chloride yielded the key intermediate com-
pounds i.e. 6,7-disubstituted-2-(4-aminophenyl)benzo[d]thiazole
(4a-f) following the Jacobson synthetic method [34] as depicted
in Scheme 1. The structures of these intermediates were charac-
terized by IR and NMR spectral studies (Table 1). The IR spectrum
of compound 4a showed two characteristic sharp absorption
bands at 3425.63 cm 1 and 3333.24 cm , which was attributed
−
−1
to the amino (-NH ) group at para position of phenyl ring of
2
2-phenylbenzo[d]thiazole moiety, while the absorption band
appeared at 1654.11 cm 1 due to C = N bond confirmed the
formation of benzo[d]thiazole ring. This is further substantiated
from 1H NMR spectrum of compound 4a, which showed presence
of characteristic singlet peak at δ 7.87 ppm attributed for amino
−
(NH ) protons, while the aromatic protons appeared as multiplets
2
2

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∗
Scheme 1. Synthetic outline of key intermediates 6,7-disubstituted-2-(4-aminophenyl)benzo[d]thiazole (4a-f). Reagents and Conditions: (a) Pyridine, reflux, 2–5 h; (b)
Lawesson’s reagent, HMPA, 100 °C, stir, 6–9 h; (c) 10% Aqueous NaOH, Potassium ferricynide, 90 °C, stir, 1–2 h; (d) Tin(II)chloride dehydrate, Absolute ethanol, nitrogen
environment, stir, 3–5 h.
Scheme 2. Synthetic route of 3-(2-bromoacetyl)−4-hydroxy-1-methyl/phenyl quinolin-2(1H)-one (5 and 6) and the corresponding benzo[d]thiazolyl disubstituted-2-
∗
quinolone hybrid analogs (7a-f and 8a-f). Reagents and Conditions: (a) Bromine, Glacial acetic acid, ꢀ, 80 °C, stir; (b) Glacial acetic acid, stir, reflux, 6–13 h.
3

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Table 1
Physicochemical and spectral characterization data of 6,7-disubstituted-2-(4-aminophenyl)benzo[d]thiazole (4a-f).
∗
−1
)
1H NMR (DMSO–d6 δ, ppm)
Comp
R1
R2
Mol formula
MP °C
Rf
FTIR (KBr disk, Cm
4
4
4
4
4
4
a
b
c
d
e
f
F
H
C13H9FN2S
171–
0.25
0.64
0.34
0.79
0.59
0.91
3425.63, 3420.24 (NH2 Str), 3125.35 (C–H Str),
6.67–7.38 (m, 6H, Ar-H), 7.87 (s, 2H, NH2), 7.97
(m, 1H, Ar-H)
1
73
1654.11 (C = N)
Cl
Br
H
H
H
H
Cl
Cl
C13H9ClN2S
C13H9BrN2S
C13H10N2S
194–
96
3498.74, 3489.29 (NH2 Str), 3077.67 (C–H Str),
6.81–7.04 (m, 5H, Ar-H), 7.20–7.29 (m, 1H,
Ar-H), 7.40 (s, 2H, NH2), 7.91 (m, 1H, Ar-H)
1
1674.80 (C = N)
161–
63
3469.06, 3450.57 (NH2 Str), 3078.69 (C–H Str),
6.71–7.30 (m, 5H, Ar-H), 7.35 (s, 2H, NH2),
7.60–7.65 (m, 1H, Ar-H)
1
1662.20 (C = N)
267–
69
3454.17, 3433.57 (NH2 Str), 3080.10 (C–H Str),
7.01–8.08 (m, 8H, Ar-H), 7.57 (s, 2H, NH2)
2
1659.11 (C = N)
F
C13H8ClFN2S
C13H8Cl2N2S
180–
82
3433.45, 3426.20 (NH Str), 3083.46 (C–H Str),
6.93–7.18 (m, 5H, Ar-H), 7.16–7.22 (m, 1H,
Ar-H), 7.91 (s, 2H, NH2)
1
1648.10 (C = N)
Cl
189–
91
3477.73, 3462.41 (NH2 Str), 3101.12 (C–H Str),
7.04–7.77 (s, 5H, Ar-H), 7.85 (s, 2H, NH2), 8.02
(m, 1H, Ar-H)
1
1653.47 (C = N)
between δ 6.67–7.97 ppm, indicating its formation by simple
and methylene (–C H –CO) resonating at δ 8.50–8.74 and 4.87–
2
cyclization process.
5.64 ppm, respectively. Aromatic protons appeared as multiplets
between δ 6.58–8.18 ppm while in the case of compounds 7a-f,
the N–C H3 proton signals were observed around δ 3.15–3.62 ppm,
respectively. Further, the formations of desired hybrid compounds
were confirmed by recording their mass spectra, which were in
full agreement with their molecular weights.
Further,
compounds
3-(2-bromoacetyl)−4–hydroxy-1-
methyl/phenyl quinolin-2(1H)-one (5 and 6) were prepared [35],
which involves the reaction of 3-acetyl-4–hydroxy-1-methyl/phenyl
quinolin-2 (1H)-one with the calculated quantities of bromine in
the presence of glacial acetic acid as shown in Scheme 2. The
spectral details of compounds 5 and 6 are summarized in ex-
perimental section. The IR spectrum of 5 showed a characteristic
2.2. Anticancer activity
broad band at 3469.02 cm 1 due to the hydroxyl (OH) group,
−
while the most prominent peaks appeared at 1737.08 cm 1 and
−
Anticancer drug development has become an extremely com-
petitive and expensive process with high rate of failures. Gener-
ally, anticancer activities of most of the chemotherapeutic agents
are due to their cytotoxicity. Anticancer activity of newly syn-
thesized compounds (7a-f and 8a-f) was assessed by MTT bioas-
say [36] and their respective cytotoxicity data (IC50 values) are
summarized in Table 4. The title compounds were evaluated for
their in vitro cytotoxic activity against four human cancer cell lines
namely Ehrlich Ascites Carcinoma cells (EAC), hormone dependant
breast cancer cells (MCF7), human colon carcinoma cells (HT29),
and hepatic cancer cells (WRL68). Doxorubicin was taken as refer-
ence drug (positive control) in the study. These synthesized com-
pounds exhibited an interesting anticancer activity profile against
tested panel of cell lines and a brief structure activity relationship
is discussed below.
1
658.22 cm 1 attributed to the carbonyl groups (C = O) of acetyl
−
(
-COCH Br) and amide (-CONH of 2-quinolone), respectively. This
2
is further evidenced from the 1H NMR spectrum of compound 5,
which showed a characteristic singlet at δ 3.23 ppm indicating
the presence of acetyl (-COC H Br) protons, while the N–CH3
2
protons appeared as singlet δ 3.64 ppm and the aromatic protons
resonated as multiplets between δ 7.22–8.68 ppm. From 13C NMR
spectrum of compound 5, it was further confirmed that the most
characteristic signals appeared at δ 33.45 and 201.03 ppm due to
N-methyl (N–CH ) carbon and acetyl (-COCH ) carbon, respectively.
3
2
Further,
the
desired
compounds
amino)acetyl)−4–hydroxy-1-
methyl/phenyl quinolin-2(1H)-one (7a-f and 8a-f) were
obtained by the reaction of 6,7-disubstituted-2-(4-
3-(2-(4-(substituted-
benzo[d]thiazol-2-yl)phenyl
aminophenyl)benzo[d]thiazole (4a-f) with 3-(2–bromo acetyl)−4–
hydroxy-1-methyl/phenylquinolin-2(1H)-one (5 or 6) in presence
of glacial acetic acid as illustrated in Scheme 2. Structures of final
hybrid derivatives (7a-f and 8a-f) were characterized based on
their physicochemical, spectral (IR, 1H NMR, 13C NMR and MS),
and elemental analysis. All the newly synthesized compounds
showed acceptable analysis of their anticipated structures, which
are summarized in Tables 2 and 3. The IR spectra of the title com-
pounds (7a-f and 8a-f) showed moderately strong bands around
EAC were found to be sensitive towards two of the twelve syn-
thesized compounds (7a-f and 8a-f). Among them, compounds 8f
(IC50 = 6.98, 4.44, 1.817 μM) and 7f (IC = 9.34, 5.40, 3.08 μM)
50
exhibited highest anticancer activity as compared to Doxorubicin
(IC50 = 4.10, 1.86, 0.843 μM) against EAC at different time points
of drug exposure. In the case of hormone dependant breast cancer
cells, four compounds were found to be active out of twelve com-
pounds tested. In particular, compound 8f (IC50 = 2.22, 0.954 and
0.398 μM) displayed the notable anticancer activity while com-
−
−1
3
426.38–3498.74 cm 1, 3335.23–3385.07 cm , 3071.11–3089.96
pounds 7e (IC50 = 5.06, 3.33 and 1.76 μM), 7f (IC = 10.51, 5.78
50
cm 1, and 1630.26–1639.87 cm , which are characteristics of
−
−1
and 2.97 μM) and 8e (IC50 = 11.09, 7.08 and 5.55 μM) displayed
moderate to good activity against MCF7 at 24 h, 48 h and 72 h, re-
spectively. Compound 8f exhibited almost equipotent activity when
compared to reference drug Doxorubicin (IC50 = 1.60, 0.625 and
0.259 μM). Further compound 8f (IC50 = 10.21, 7.20 and 3.01 μM)
also displayed most significant anticancer activity against HT29
cancer cells at different time intervals of drug exposure. This was
the only compound from the series to exhibit potent anticancer
the O–H, N–H, C–H, and C = N groups, respectively. The most
−
1
informative and sharp bands appeared at 1716.03–1728.13 cm
−
1
and 1660.22–1672.90 cm
confirmed the presence of two dis-
tinctive carbonyl groups (C = O) of acetyl (-COCH Br) and amide
2
1
(
-COCH NH of 2-quinolone), respectively. In H NMR spectra, the
2
characteristic O–H proton (singlet) of 2-quinolone ring appears at
δ 10.39–11.45 ppm and the two distinctive singlets of N–H protons
4

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Table 2
Physicochemical and elemental analysis data of a novel series of 3-(2-(4-(6,7-disubstituted-benzo[d]thiazol-2-yl)phenylamino)acetyl)−4-hydroxy-1-methyl/phenyl
quinolin-2(1H)-one derivatives.
∗
Comp
R1
R2
Molecular formula
MP ( °C)
Rf
Elemental (CHN) Analysis:% Found (Calcd)
C
H
N
7
7
7
7
7
7
8
8
8
8
8
8
a
b
c
d
e
f
F
H
H
H
H
Cl
Cl
H
H
H
H
Cl
Cl
C25H18FN3O3S
C25H18ClN3O3S
C25H18BrN3O3S
C25H19N3O3S
194–196
176–178
209–211
154–156
203–205
245–247
222–224
199–201
265–267
168–170
233–235
269–271
0.57
0.53
0.47
0.31
0.41
0.77
0.39
0.68
0.36
0.61
0.72
0.69
65.34 (65.34)
63.03 (63.09)
57.68 (57.70)
67.99 (68.01)
60.78 (60.79)
58.86 (58.83)
69.11 (69.09)
67.00 (66.97)
61.89 (61.86)
71.25 (71.55)
64.80 (64.81)
62.96 (62.94)
3.92 (3.95)
3.79 (3.81)
3.43 (3.49)
4.35 (4.34)
3.44 (3.47)
3.33 (3.36)
3.89 (3.87)
3.76 (3.75)
3.44 (3.46)
4.21 (4.20)
3.43 (3.44)
3.36 (3.35)
9.16 (9.14)
8.89 (8.83)
8.10 (8.07)
9.51 (9.52)
7.16 (7.18)
8.21 (8.23)
8.04 (8.06)
7.79 (7.81)
7.21 (7.21)
8.36 (8.34)
7.56 (7.56)
7.32 (7.34)
Cl
Br
H
F
C25H17ClFN3O3S
C25H17Cl2N3O3S
C30H20FN3O3S
C30H20ClN3O3S
C30H20BrN3O3S
C30H21N3O3S
Cl
F
a
b
c
d
e
f
Cl
Br
H
F
C30H19ClFN3O3S
C30H19Cl2N3O3S
Cl
∗
TLC Solvent system: Chloroform: Methanol: Strong ammonia-(10:5:3).
Table 3
Spectral data of a series of 3-(2-(4-(6,7-disubstituted-benzo[d]thiazol-2-yl)phenylamino)acetyl)−4-hydroxy-1-methyl/phenylquinolin-2(1H)-one (7a-f and 8a-f) derivatives.
+
Mass: M (m/z
Comp FTIR (KBr disk, Cm 1
−
)
1H NMR (DMSO–d6, δ, ppm)
value)
7
7
7
7
7
7
8
8
8
8
8
8
a
b
c
d
e
f
3456.76 (OH Str), 3385.07 (NH Str), 3078.39 (C–H Str), 1725.94
C = O Str), 1662.45(C = O Str), 1639.49 (C = N Str)
3498.74 (OH Str), 3356.44 (NH Str), 3089.96 (C–H Str), 1716.03
C = O Str), 1670.10(C = O Str), 1631.78 (C = N Str)
3469.06 (OH Str), 3350.65 (NH Str), 3078.39 (C–H Str), 1726.29
C = O Str), 1666.27(C = O Str), 1637.45 (C = N Str)
3468.30 (OH Str), 3382.50 (NH Str), 3084.39 (C–H Str), 1726.28
C = O Str), 1663.55(C = O Str), 1635.13 (C = N Str)
3476.39 (OH Str), 3338.21 (NH Str), 3083.27 (C–H Str), 1720.62
C = O Str), 1672.90(C = O Str), 1638.28 (C = N Str)
3478.29 (OH Str), 3372.42 (NH Str), 3071.11 (C–H Str), 1728.13
C = O Str), 1660.22(C = O Str), 1639.87 (C = N Str)
3426.38 (OH Str), 3384.33 (NH Str), 3079.32 (C–H Str), 1719.37
C = O Str), 1669.32(C = O Str), 1635.29 (C = N Str)
3492.74 (OH Str), 3353.21 (NH Str), 3089.96 (C–H Str), 1718.63
C = O Str), 1664.36(C = O Str), 1633.17 (C = N Str)
3461.25 (OH Str), 3363.06 (NH Str), 3078.21 (C–H Str), 1727.93
C = O Str), 1665.66(C = O Str), 1632.11 (C = N Str)
3468.20 (OH Str), 3382.50 (NH Str), 3084.39 (C–H Str), 1726.31
C = O Str), 1666.42(C = O Str), 1636.10 (C = N Str)
3472.76 (OH Str), 3335.23 (NH Str), 3082.20 (C–H Str), 1720.54
C = O Str), 1667.35(C = O Str), 1635.44 (C = N Str)
3473.48 (OH Str), 3370.38 (NH Str), 3074.88 (C–H Str), 1723.67
C = O Str), 1661.75(C = O Str), 1630.26 (C = N Str)
2.26 (s, 3H, CH3), 5.64 (s, 2H, CH2), 6.68–7.78 (m, 11H, Ar-H),
7.67 (s, 1H, NH), 9.85 (s, 1H, OH)
459.24
(
3.23 (s, 3H, CH3), 5.59 (s, 2H, CH2), 6.69–7.88 (m, 11H, Ar-H),
8.59 (s, 1H, NH), 11.26 (s, 1H, OH)
475.96
519.03
441.14
493.11
509.07
521.21
537.10
581.52
503.52
556.05
572.32
(
3.33 (s, 3H, CH3), 5.60 (s, 2H, CH2), 6.98–8.03 (m, 11H, Ar-H),
8.62 (s, 1H, NH), 11.04 (s, 1H, OH)
(
3.15 (s, 3H, CH3), 4.99 (s, 2H, CH2), 7.01–8.08 (m, 12H, Ar-H),
8.57 (s, 1H, NH), 11.30 (s, 1H, OH)
(
3.62 (s, 3H, CH3), 5.09 (s, 2H, CH2), 6.91–8.08 (m, 10H, Ar-H),
8.50 (s, 1H, NH), 11.45 (s, 1H, OH)
(
3.56 (s, 3H, CH3), 5.06 (s, 2H, CH2), 6.86–8.00 (m, 10H, Ar-H),
8.53 (s, 1H, NH), 10.64 (s, 1H, OH)
(
a
b
c
d
e
f
4.95 (s, 2H, CH2), 6.66–7.82 (m, 16H, Ar-H), 8.64 (s, 1H, NH),
11.09 (s, 1H, OH)
(
5.05 (s, 2H, CH2), 6.92–8.14 (m, 16H, Ar-H), 8.56 (s, 1H, NH),
10.63 (s, 1H, OH)
(
5.57 (s, 2H, CH2), 6.58–8.12 (m, 16H, Ar-H), 8.74 (s, 1H, NH),
10.86 (s, 1H, OH)
(
5.03 (s, 2H, CH2), 7.12–7.85 (m, 17H, Ar-H), 8.62 (s, 1H, NH),
11.06 (s, 1H, OH)
(
4.87 (s, 2H, CH2), 7.04–8.18 (m, 15H, Ar-H), 8.71 (s, 1H, NH),
10.81 (s, 1H, OH)
(
5.16 (s, 2H, CH2), 6.73–8.09 (m, 15H, Ar-H), 8.54 (s, 1H, NH),
10.39 (s, 1H, OH)
(
activity against HT29 cancer cells. However, five compounds were
found to be highly active against hepatic (liver) cancer cells, from
which compound 7e (IC50 = 7.63, 3.15 and 1.80 μM) presented
the most promising anticancer activity. Whereas compounds 7f
sults evidently indicated that the presence of halogens especially
fluoro and/or chloro at 6th and 7th position of benzo[d]thiazole
nucleus contributed to the increase in anticancer activity, which
is in agreement with previously reported studies [37,38]. Anti-
cancer drugs, which are active against MCF-7 and WRL68 cells
usually, cause apoptosis through the expression of caspase-3, re-
sulting in generation of reactive oxygen species (ROS) and lead-
ing to DNA destruction [39,40]. From literature, it is also known
that substitution with electronegative atoms (chloro/fluoro) at
6th and/or 7th position on benzo[d]thiazole ring increases the
lipophilicity of a molecule, which could be responsible for en-
hanced cytotoxicity in MTT bioassay [6,7,12,41]. Thus in the ti-
(
IC50 = 5.12, 3.73 and 2.48 μM), 8b (IC = 9.75, 6.47 and 3.76 μM),
50
8
e (IC = 10.84, 7.57 and 5.00 μM), and 8f (IC = 9.37, 4.47
5
0
50
and 2.25 μM) demonstrated moderate to good activity as compared
to standard Doxorubicin (IC50 = 2.25, 1.48 and 0.827 μM) against
WRL68 at 24 h, 48 h, and 72 h respectively.
The present study revealed that among the four human can-
cer cell lines tested, MCF-7 and WRL68 cells were more sensi-
tive to all the tested compounds. The cytotoxicity screening re-
5

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Journal of Molecular Structure xxx (xxxx) xxx
Table 4
Cytotoxic activity data of the newly synthesized compounds (7a-f and 8a-f) against various human cancer cell lines at different
time points of drug exposure by MTT assay method.
Compounds
IC50 (μM)a in EACb
IC50 (μM) in MCF-7b
IC50 (μM) in HT29b
IC50 (μM) in WRL68b
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
7
7
7
7
7
7
8
8
8
8
8
8
a
b
c
d
e
f
21.26
43.65
35.23
19.87
11.45
09.34
18.33
31.23
29.07
28.54
22.18
06.98
04.10
16.02
35.88
26.56
17.99
09.20
05.40
13.88
23.86
23.67
20.84
16.90
04.44
01.86
11.92
29.21
20.76
12.19
06.93
03.08
10.39
14.44
16.30
14.86
12.34
1.817
0.843
13.56
25.26
31.21
25.26
05.06
10.51
23.85
40.98
34.21
33.98
11.09
02.22
1.60
09.76
19.03
22.88
16.04
03.33
05.78
13.67
32.09
22.74
20.66
07.08
0.954
0.625
06.33
10.02
12.74
08.53
01.76
02.97
07.86
23.23
16.99
12.87
05.55
0.398
0.259
56.23
39.56
61.25
71.02
26.20
25.29
58.99
47.04
57.02
40.56
38.34
10.21
06.14
35.26
30.22
50.03
63.05
22.36
20.59
39.65
31.28
37.76
31.87
33.56
07.20
03.45
27.86
24.15
42.05
55.25
19.54
18.55
30.48
24.56
28.41
26.73
28.69
03.01
01.50
25.20
29.63
22.91
23.25
07.63
05.12
28.44
09.75
25.50
33.62
10.84
09.37
02.25
19.25
16.53
17.38
16.09
03.15
03.73
20.34
06.47
18.14
24.00
07.57
04.47
1.48
12.04
10.19
14.20
10.63
01.80
02.48
13.60
03.76
11.40
18.77
05.00
02.25
0.827
a
b
c
d
e
f
Standardc
a
The IC50 values of tested compounds at 48 h and 72 h were significantly reduced in comparison with 24 h values.
b
EAC (Ehrlich Ascites Carcinoma cells); MCF7 (hormone dependant breast cancer cells); HT29 (human colon carcinoma cells);
WRL68 (hepatic cancer cells).
c
Standard used was Doxorubicin.
Table 5
In vitro antimicrobial activity data of title compounds (7a-f and 8a-f).
Compound
MIC (μg/ml)
Staphylococcus aureus
Bacillus substilis
E. coli
Pseudomonas aeruginosa
Candida albicans
Asparagus niger
7
7
7
7
7
7
8
8
8
8
8
8
a
b
c
d
e
f
64
64
8
16
8
>128
64
>128
64
>128
64
>128
>128
>128
32
4
4
4
32
64
32
8
4
>128
32
>128
>128
>128
>128
>128
>128
>128
32
16
0.5
0.5
4
2
8
64
0.5
8
64
a
b
c
d
e
f
16
16
32
16
32
2
2
64
32
64
4
8
32
>128
64
>128
128
16
4
2
>128
32
0.5
0.5
0.5
0.25
ND
2
16
1
32
>128
ND
Ampicillin
Gatifloxacin
Fluconazole
0.5
0.25
ND
0.5
0.5
0.25
ND
ND
ND
0.5
0.25
ND
ND
0.5
∗ND= Not determined.
tle compounds, similar substitutions on the benzo[d]thiazole scaf-
fold exhibited enhanced anticancer activity. In addition, it was ob-
served that replacement of N-phenyl for N-methyl group on 4–
hydroxy-quinoline-2-one moiety contributed to increase in anti-
cancer activity. Overall, the anticancer activity profile indicated
that the synthesized benzo[d]thiazolyl substituted-2-quinolone hy-
brids exhibited encouraging activity against human breast cell line
showed significant antibacterial activity against E. coli. Compounds
7e, 7f, 8e, and 8f exhibited excellent antibacterial activity with
MIC 0.5 μg.mL 1against E. coli, while moderate to good activity
(MIC = 1.0 - 8.0 μg.mL 1) was observed for the remaining com-
pounds in this series. In the case of P. aeruginosa, compound 7f
(MIC = 0.5 μg.mL 1) displayed significant antibacterial activity,
whereas other compounds displayed good to moderate antibac-
−
−
−
terial activity with MIC values ranging from 1.0 to 16.0 μg.mL 1.
However, moderate activity against S. aureus was observed for
compound 7f (MIC = 8.0 μg.mL 1) while other compounds in this
series exhibited little or poor activity against Gram-positive bacte-
ria. With respect to antifungal activities (Table 5), all compounds
displayed poor or no activity against C. albicans and A. niger. The
−
(
MCF-7).
−
2
.3. Antimicrobial activity
All the newly synthesized compounds (7a-f and 8a-f) were
screened for their in-vitro antimicrobial activity against a panel
of microorganisms; Gram positive bacteria Staphylococcus aureus
MIC values were ranging from 32.0 to >128.0 μg.mL 1, while the
−
MIC value of standard drug Fluconazole was 0.5 μg.mL 1. In gen-
eral, from the antibacterial activity data, an interesting correlation
was observed between the compounds and activity against E. coli
and P. aeruginosa. The activity was considerably enhanced by elec-
tron withdrawing substituents like chloro or fluoro at 6th and/or
−
(
ATCC 11,632), Bacillus substilis (ATCC 60,511) and Gram nega-
tive bacteria E. coli (ATCC 10,536), Pseudomonas aeruginosa (ATCC
0,145). For antifungal activity, the title compounds were screened
against Candida albicans (ATCC 2501) and Aspergillus niger (ATCC
781). Ampicillin, Gatifloxacin and Fluconazole were used as ref-
1
1
7
th position of benzo[d]thiazole ring. However, in the case of com-
pounds 7a-d and 8a-d, which have no substitution (R2 = H) at
th position of benzo[d]thiazole ring, slightly decrease in activity
erence drugs for antibacterial and antifungal activities. The MIC
values of in vitro antimicrobial screening are summarized in
Table 5. A systematic analysis of the data revealed that some
of the synthesized compounds exhibited enhanced antibacterial
activity, especially against Gram negative E. coli (ATCC 10,536),
P. aeruginosa (ATCC 10,145) organisms. All the title compounds
7
was noticed. It was also perceived that replacing N-phenyl group
in place of N-methyl group on 4–hydroxy-quinoline-2-one moiety
resulted in equipotent antibacterial activity.
6

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Journal of Molecular Structure xxx (xxxx) xxx
2
.4. Molecular docking studies
interactions with the NH spine of Met-769 (3.5167 A˚ ). Normally,
this amino acid residue forms very crucial hydrogen bond with N-1
of the adenine ring in ATP and helps to fix it in the binding pocket.
This hydrogen bond can be considered as decisive for orienting the
benzo[d]thiazole ring in the pocket. A second hydrogen bond inter-
The epidermal growth factor receptor (EGFR) and transform-
ing growth factor-∝ are one of the earliest characterized mem-
bers of the growth factor/receptor tyrosine kinase (RTK) family. The
EGFR induces cell proliferation and cell differentiation upon bind-
ing and activation by one of a number of its known ligands. Mem-
bers of the EGFR family are frequently overactive in solid tumors
actions were observed with carbonyl oxygen atom of 2-quinolone
ring and the OH group in side chain of Thr-766 (3.0474 A˚ ), which
is located at the beginning of the extended coil stretch deep in
the binding cleft. Similarly compound 7f also displayed five hydro-
gen bonding interactions as depicted in Fig. 3. In the case of com-
pounds 7e and 8e, prominent hydrogen bonding interactions were
[
42]. For example, the epidermal growth factor receptor tyrosine
kinases (EGFRTK) of the erbB family (which includes erbB1-erbB4)
is frequently expressed at high levels in certain carcinomas par-
ticularly in breast, colon, and bladder cancers [43]. A number of
therapeutic approaches that interfere with aberrant EGFR family
signaling are being investigated [44]. A relatively new therapeutic
approach to kinase inhibition is the use of ATP-competitive small
molecules [45]. From literature, it is well known that anticancer
benzo[d]thiazole and quinazoline analogs act via competing with
ATP for binding at the catalytic domain of tyrosine kinase [7,46].
The in vitro cytotoxic activity results especially against MCF-7
were inspiring and in order to validate experimental results fur-
ther and to explore the possible binding interactions. Molecular
docking studies for the title compounds (7a-f and 8a-f) were car-
ried out using Molegro Virtual Docker (MVD-2013, 6.0). Fig. 2 rep-
resents the native crystal structure of Erlotinib (AQ4) bound to
the active site of EGFRTK obtained from Protein Data Bank (http:
observed, that is between nitrogen atom (N-1) of 2-quinolone ring
and OH group of the residue Thr 766 (3.0558 A˚ and 2.7788 A˚ ), ni-
trogen atom of the linker (-Ph–NH–CH -) and NH group of Lys 721
2
(3.4616 A˚ and 3.1567 A˚ ) and carbonyl oxygen atom of 2-quinolone
ring with the OH group of Thr 830 (2.7415 A˚ and 2.8145 A˚ ), respec-
tively. Nevertheless, the less effective compounds 8b (3.2038 A˚ ),
8c (3.1357 A˚ ), and 8d (3.1764 A˚ ), displayed a prominent hydrogen
bonding interactions with NH group of Cys773 residue with the
oxygen atom of a keto group on the 3rd position of 2-quinolone
nucleus. Besides hydrogen bonding interactions, the docking re-
sults of the active compounds also exhibited steric and aromatic
π-π stacking interactions with the various amino acid residues
in the active site of 1M17, which are essential for the inhibi-
tion of EGFRTK [47]. The above results demonstrate that these
hybrid compounds could be effective inhibitors of EGFRTK. Thus,
the in-silico results provide an insight into the binding mode of
benzo[d]thiazolyl substituted-2-quinolone hybrids in the active site
of 1M17.
/
/www.rscb.org/pdb) with the PDB ID: 1M17 [47]. The EGFR con-
sists of a single polypeptide chain of 1186 amino acids that is
expressed on the cell membrane of numerous cell types. An es-
sential feature of the binding site is the conservation of hydrogen
bondings, steric and aromatic π-π stacking interactions. The active
pocket consisted of amino acid residues such as Val702, Ala719,
Lys721, Met742, Thr766, Gln767, Leu768, Met769, Pro770, Gly772,
Leu820, Thr830, and Asp831. Hence to identify other residual in-
teractions of the tested compounds, a grid box (include residues
2.5. Conclusion
In this paper, the synthesis, spectral studies, and biological
evaluation of
a
series of 3-(2-(4-(substituted-benzo[d]thiazol-
quinolin-
˚
within a 10.0 A radius) large enough to accommodate the ac-
2-yl)phenylamino)acetyl)−4–hydroxy-1-methyl/phenyl
tive site was constructed. AQ4 being a known inhibitor, the cen-
ter of this site was considered as the center of search space for
docking. The 2D structures of the synthesized ligands were con-
verted to energy minimized 3D structures and were used for in
silico protein–ligand docking calculations. Docking of the synthe-
sized compounds with the EGFRTK exhibited the well-conserved
interactions like hydrogen bonding, hydrophobic bonding, and van
der Waals’s interactions with one or more amino acid residues of
the active pocket. The molecular docking results are summarized
in Table 6, from which the following assumptions can be drawn.
For the test compounds (7a-f and 8a-f), the MolDock score ranged
from –77.7254 to –129.7780 while the Moldock score of standard
drug AQ4 was –114.2710. The best poses (orientations) of AQ4 and
of the docked compounds are represented in Figs. 2 and 3. It
was clearly observed that the nitrogen atom (N-1 of quinazoline)
and oxygen atom (methoxyl side chain) of standard drug (AQ4)
2(1H)-one (7a-f and 8a-f) derivatives are reported. The in vitro
anticancer results obtained indicated that the compound 8f was
most active hybrid in the series and from the SAR studies it was
concluded that the presence of electronegative groups (Cl/F) on the
benzo[d]thiazole nucleus was favorable and the presence of quina-
zoline moiety is also equally responsible for substantial increase
in anticancer activity. Molecular binding interaction pattern of the
title compounds with the target enzyme (EGFR tyrosine kinsae)
was effectually generated with the help of docking simulations and
the results indicate that the compounds 8f and 7f displayed most
relevant conserved hydrogen bond interactions requisite for 1M17
inhibition. Moreover, in many respects the SAR profile exhibited
by these compounds is almost similar to that shown by the previ-
ously reported quinazoline series of EGFRTK inhibitors. Hence, the
molecular hybridization strategy chosen in the current study to
combine key pharmacophoric features (2-phenyl benzo[d]thiazole
and substituted 2-quinolone) connected with suitable linker was
fruitful. The in vitro antimicrobial screening of the title compounds
7e, 7f, 8e, and 8f exhibited notable antibacterial activity espe-
cially against Gram-negative bacterium (E. coli). In conclusion, the
novel series of benzo[d]thiazolyl substituted-2-quinolone hybrids
resulted in identifying a hit molecule 8f, which could be explored
further to develop potential anticancer and antibacterial agents.
˚
exhibited distinctive hydrogen bonding with Met769 (2.8387 A)
˚
and Cys773 (3.3126 A), respectively. Many researchers reported the
similar binding mode in protein kinase structures with the high
degree of sequence conservation present in the catalytic core of
protein kinases are in agreement the present work. Among the
tested series, compound 8f displayed maximum conserved hydro-
gen bond interactions with the surrounding amino acid residues of
the active site. It was observed that the oxygen atom (4–hydroxyl
group of 2-quinolone ring) exhibited three hydrogen bonding inter-
3. Experimental
˚
actions with hydroxyl (OH) group of Thr830 (2.8306 A), of Asp831
˚
˚
(
3.0922 A), and amino group (NH) of Asp831 (3.1830 A). The ni-
3.1. Synthetic protocol
trogen atom on 2-phenyl ring (linker:-Ph–NH–CH -) displayed two
2
˚
hydrogen bonding interactions with OH group of Thr830 (3.5802 A)
All research chemicals were purchased from Sigma–Aldrich (St.
Louis, Missouri, USA) or Lancaster Co. (Ward Hill, MA, USA) and
used as such for the reactions. Solvents except laboratory reagent
˚
and Thr766 (2.7066 A) respectively. Interestingly, The N-3 of the
benzo[d]thiazole moiety generates much conserved hydrogen bond
7

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Journal of Molecular Structure xxx (xxxx) xxx
Fig. 2. (A) Secondary structure (complete protein) epidermal growth factor receptor tyrosine kinase (EGFRTK, PDBID: 1M17) in complex with inhibitor (AQ4: Erlotinib); (B)
showing the binding orientation of AQ4 forming 1H bond with Met769 and 1H bond with Cys773.
grade were dried and purified according to the literature when
necessary. The reference drugs used in this work was received as a
gift sample from Fourrtis (India) Lab. Pvt. Ltd., Chennai (Tamilnadu,
India). The progress of reactions and purity of compounds were
monitored by thin-layer chromatography (TLC) on pre-coated alu-
minum sheets with GF254 silica gel plates procured from E. Merck
and Co. (Darmstadt, Germany). Methanol (12%) in dichloromethane
was used as mobile phase and short wavelength UV radiations as
well as iodine vapours were used as visualizing agent.
dia). Chemical shifts are reported in δ ppm units with respect
to TMS as internal standard. Mass spectra were recorded on Shi-
madzu LC MS-2010A at Quest Research and Training Institute Pri-
vate Limited, (Bangalore, India).
3.2. General procedure for synthesis of N-(substituted
phenyl)−4-nitrobenzamide (1a-f)
A mixture of substituted aniline (0.02 mol) and 4-nitrobenzoyl
chloride (0.02 mol, 0.371 g) and 50 mL of pyridine (The excess
of solvent is used to get pure compound) was refluxed for 2–5 h.
The reaction mixture was cooled to room temperature and poured
into ice-cold water. The precipitate formed was collected, washed
with ice-cold water, and recrystallized from either ethyl alcohol or
methyl alcohol.
Melting points of synthesized compounds were determined in
Veego (VMP-MP) melting point apparatus and were uncorrected. IR
spectra were recorded on ThermoNicolet IR200 FT-IR Spectrometer
(
Madison, WI, USA) by using KBr pellets and Brucker Alpha I FTIR
Spectrophotometer. The 1H NMR spectra were recorded on Bruker
Avance III 400 NMR spectrometer (Bruker, Rheinstetten/Karlsruhe,
Germany) using appropriate solvent at Sophisticated Analytical and
Instrumentation Facility (SAIF), Punjab University (Chandigarh, In-
N-(4-fluorophenyl)−4-nitrobenzamide (1a): It is light green color
crystalline needles (EtOH); yield 4.3 g (82.69%); mp 176–178 °C;
8

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Table 6
Molecular docking results based on Moldock score, E-inter, hydrogen bonding interaction, hydrophobic/steric interaction, Docking scores and Rerank scores of the title
compounds by MVD-2013 (6.0) against 1M17.
E-Inter
Residues
Moldock
Score
(protein–
ligand)
Residues involved in
involved in
Docking
Score
Rerank
score
hydrophobic and steric
interactions (within 5 A˚ ) bonding
hydrogen
Heavy atoms
H-Bonds count
Compds (Kcal/mol)
(Kcal/mol)
LE1 LE3
(Kcal/mol)
(Kcal/mol)
No.
(Kcal/mol)
7
7
a
−88.7340
−111.281
Leu694, Ala719, Glu738,
Thr766, Leu768, Met769,
Gly772, Leu820
—–
00
0.0
33
33
−2.6889 −0.8373
−3.0764 −1.0360
−511.185
−535.941
−27.6334
−34.1897
b
−101.5240 −125.862
Val702, Lys721, Met742,
Thr766, Gln767, Met769,
Leu820
Cys773
01
−0.5681
7
7
c
−77.7254
−89.4629
−105.273
−114.103
Leu694, Ala719, Met769
Leu694, Leu768, Met769,
Gly772, Leu820
Gly772
Thr766
01
01
−0.6800
−1.5403
33
32
−2.3553 −0.8252
−2.7957 −0.5223
−511.824
−532.235
−27.2316
−16.7163
d
7
7
e
f
−117.8740 −146.335
Ala719, Cys751, Gln767,
Met769, Leu820
Lys721,
Thr766,
Thr830
Thr766,
Thr830,
Asp831
——-
03
05
−5.6917
34
39
−3.4668 −1.2522
−584.646
−42.5758
−120.6290 −150.548
Val702, Met742, Thr766,
Gln767, Leu820
−9.2427
−3.0930 −2.2980
−584.646
−89.6235
8
8
a
−101.0260 −114.103
−105.1150 −131.158
Val702, Ala719, Met742,
Leu768, Met769, Leu820
Val702, Ala719, Met742,
Gln767, Leu768, Met769,
Leu820
00
01
0.0
38
38
−2.6585 −0.9297
−2.7661 −0.8585
−519.330
−526.940
−35.3300
−32.6260
b
Cys773
−1.9806
8
8
8
c
−108.3960 −132.463
−113.6400 −143.527
−117.8990 −148.116
Val702, Gln767, Leu768,
Met769, Leu820
Cys773
Cys773
01
01
03
−2.3213
−2.1175
−7.2163
38
37
39
−2.8525 −1.6943
−3.0713 −2.2006
−3.0230 −2.1324
−531.134
−563.963
−569.095
−64.3870
−81.4240
−83.1648
d
e
Val702, Ala719, Leu768,
Met769, Leu820
Ala719, Cys751, Thr766,
Gln767, Met769, Leu820
Lys721,
Thr766,
Thr830
Thr766,
Met769,
Thr830,
Asp831
Met769,
Cys773
∗
8
f
−129.7780 −161.837
−114.2710 −131.574
Val702, Ala719, Met742,
Thr766, Gln767, Leu768,
Met769, Leu820, Asp831
07
02
−9.5463
34
29
−3.8170 −2.7854
−3.9403 −3.2729
−592.379
−94.7051
AQ4
Pro770, Gly772
−3.93697
−579.7700 −94.9144
∗
Compound exhibited high binding energy (Moldock score); H. Bonds: Hydrogen bonding energy between protein and ligand; LE1(Ligand Efficiency): MolDock score divided
by heavy atoms count; LE3: Rerank score divided by heavy atoms count; AQ4: Erlotinib (4-Anilinoquinazoline derivative).
IR (KBr, ν, cm 1): 3325.45 (N–H str) 3078.24 (-CH Str), 1627.30 (-
−
(C = O carbon of amide), 162.30, 155.32, 137.32, 125.22, 122.65,
1
C = O str of amide); H NMR (400 MHz, DMSO–d , δ, ppm): 8.29
116.51; ESI MS: m/z 242.05; calcd. 242.07.
6
(
s, 1H, NH), 8.03–6.92 (m, 8H, Ar-H); 13C NMR (400 MHz, DMSO–
N-(3–chloro–4-fluorophenyl)−4-nitrobenzamide(1e): It is light
d , δ, ppm): 172.54 (C = O carbon of amide), 160.41, 155.39, 137.00,
green crystalline powder (EtOH); yield 1.5 g (54.42%); mp 202–
6
204 °C; IR (KBr, ν, cm 1): 3331.45 (N–H str) 3123.34 (-CH Str),
1660.05 (-C = O str of amide); 1H NMR (400 MHz, DMSO–d6,
δ, ppm): 8.23 (s, 1H, NH), 7.92–7.01 (m, 7H, Ar-H); 13C NMR
(400 MHz, DMSO–d6, δ, ppm): 169.58 (C = O carbon of amide),
161.54, 153.69, 136.11, 127.72, 125.20, 122.37, 119.65, 117.45; ESI
MS: m/z 294.02; calcd. 294.04.
−
1
35.10, 128.32, 125.96, 123.40, 116.43; ESI MS: m/z 260.31; calcd.
60.22.
N-(4-chlorophenyl)−4-nitrobenzamide(1b): It is white crystalline
needles (EtOH); yield 2.5 g (93.63%); mp 232–234 °C; IR (KBr, ν,
2
−1
cm ): 3345.39 (N–H str) 3014.02 (-CH Str), 1645.13 (-C = O str of
amide); 1H NMR (400 MHz, DMSO–d , δ, ppm): 8.12 (s, 1H, NH),
6
7
.92–7.08 (m, 8H, Ar-H); 13C NMR (400 MHz, DMSO–d , δ, ppm):
N-(3,4-dichlorophenyl)−4-nitrobenzamide(1f): It is white crys-
6
1
71.15 (C = O carbon of amide), 163.32, 155.33, 138.35, 135.14,
talline compound (EtOH); yield 2.1 g (67.74%); mp 197–199 °C;
−1
1
27.02, 125.34, 122.41, 116.44; ESI MS: m/z 276.11; calcd. 276.03.
IR (KBr, ν, cm ): 3261.76 (N–H str) 3087.34 (-CH Str), 1651.03 (-
C = O str of amide); 1H NMR (400 MHz, DMSO–d6, δ, ppm): 8.43
(s, 1H, NH), 7.95–6.96 (m, 7H, Ar-H); 13C NMR (400 MHz, DMSO–
d6, δ, ppm): 168.43 (C = O carbon of amide), 161.39, 153.69, 135.94,
126.99, 125.22, 123.18, 119.61, 117.65; ESI MS: m/z 310.01; calcd.
309.99.
N-(4-bromophenyl)−4-nitrobenzamide(1c): It is light brown crys-
talline powder (MeOH); yield 2.7 g (84.37%); mp 217–219 °C; IR
KBr, ν, cm 1): 3278.30 (N–H str) 3021.43 (-CH Str), 1643.57 (-
−
(
1
C = O str of amide); H NMR (400 MHz, DMSO–d , δ, ppm): 8.76
6
(
s, 1H, NH), 8.02–6.75 (m, 8H, Ar-H); 13C NMR (400 MHz, DMSO–
d , δ, ppm): 171.45 (C = O carbon of amide), 162.84, 155.30, 137.30,
6
1
35.34, 127.65, 125.22, 122.11, 116.91; ESI MS: m/z 320.05; calcd.
19.98.
-nitro-N-phenylbenzamide(1d): It is white crystalline powder
3
.3. General procedure for synthesis of N-(substituted
3
phenyl)−4-nitrobenzothioamide (2a-f)
4
EtOH); yield 2.0 g (82.64%); mp 181–183 °C; IR (KBr, ν, cm 1):
−
(
A
mixture of one of the N-(substituted phenyl)−4-
3
1
223.56 (N–H str) 2968.43 (-CH Str), 1639.97 (-C = O str of amide);
nitrobenzamide (1a-f, 0.01 mol) and Lawesson’s reagent (0.6 mol
H NMR (400 MHz, DMSO–d , δ, ppm): 8.14 (s, 1H, NH), 8.12–
6
Equivalent (Eq)) in 30 mL of hydroxymethylphospharamide
6
.58 (m, 9H, Ar-H); 13C NMR (400 MHz, DMSO–d , δ, ppm): 172.60
6
(
HMPA) was stirred at 100 °C for 6–9 h. The reaction mass was
9

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G. Bolakatti, M. Palkar, M. Katagi et al.
Journal of Molecular Structure xxx (xxxx) xxx
Fig. 3. Showing the compounds docked in best of its conformation into the binding site of EGFRTK (1M17); broken lines (blue color) indicate hydrogen bonding interactions.
A) Binding mode of compound 7e forming 1H bond with Lys721, 1H bond with Thr766 and 1H bond with Thr830; (B) Binding mode of compound 7f forming 2H bonds
(
with Thr766, 1H bond with Thr830 and 2H bonds with Asp831; (C) Binding mode of compound 8e forming 1H bond with Lys721, 1H bond with Thr766 and 1H bond with
Thr830; (D) Binding mode of compound 8f forming 2H bonds with Thr766, 1H bond with Met769, 2H bonds with Thr830 and 2H bonds with Asp831. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)
then poured into ice-cold water. Thus obtained desired products
a-f were collected as crystalline solids and washed with ice-cold
water, dried, and recrystallized from methanol.
final concentration of 10% aqueous NaOH. Aliquots of this mixture
(5 mL) were carefully added at 1 min intervals to a well stirred so-
lution of potassium ferricynide (4 mol Eq) in hot water maintained
at 90 °C. The reaction mixture was further heated for 1–2 h and
then cooled to the room temperature. The products (3a-f) formed
were collected, washed with ice-cold water, and recrystallized from
methanol.
2
3
6
.4. General procedure for jacobson’s synthesis of
,7-substituted-2-(4-nitrophenyl) benzo[d]thiazole (3a-f) [34]
6
-fluoro-2-(4-nitrophenyl) benzo[d]thiazole (3a): Yield 1.19 g
N-(substituted phenyl)−4-nitrobenzothioamide (2a-f, 0.05 mol)
−
(
69.20%); mp 188–190 °C; IR (KBr, ν, cm 1): 3072.58 (-CH Str),
were drenched in 5 mL of ethanol, to which 30% sodium hydrox-
ide (NaOH) solution (8 mol Eq) was slowly added with constant
stirring. The mixture was diluted with distilled water to provide a
1
1
662.18 (-C = N str); H NMR (400 MHz, DMSO–d , δ, ppm): 7.95–
6
6
.96 (m, 7H, Ar-H); 13C NMR (400 MHz, DMSO–d , δ, ppm): 168.56,
6
10

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Journal of Molecular Structure xxx (xxxx) xxx
1
53.50, 148.64, 138.41, 128.19, 126.22, 123.39, 122.25; ESI MS: m/z
73.96; calcd. 274.02.
–chloro–2-(4-nitrophenyl) benzo[d]thiazole (3b): Yield 2.10 g
4-(6-bromobenzo[d]thiazol-2-yl)benzenamine (4c): Yield 1.07 g
(35.31%); Rf 0.34; mp 161–163 °C; IR (KBr, ν, cm 1): 3469.06,
−
2
6
3450.57 (-NH2 Str), 3078.69 (-CH Str), 1662.20 (-C = N); 1H NMR
72.66%); mp 213–215 °C; IR (KBr, ν, cm 1): 3094.21 (-CH Str),
−
(400 MHz, DMSO–d , δ, ppm): 7.65–7.60 (m, 1H, Ar-H), 7.35 (s,
(
6
1
2H, NH ), 7.30–6.71 (m, 6H, Ar-H); 13C NMR (400 MHz, DMSO–
1
658.68 (-C = N str); H NMR (400 MHz, DMSO–d , δ, ppm): 8.04–
6
2
6
.94 (m, 7H, Ar-H); 13C NMR (400 MHz, DMSO–d , δ, ppm): 166.45,
d6, δ, ppm): 170.11, 156.21, 154.29, 150.18, 133.42, 128.22, 125.36,
124.65, 121.77, 121.05, 113.28; LCMS: m/z 303.93; Calcd. 303.97;
Anal. Calcd: C, 51.16; H, 2.97; N, 9.18. Found: C, 51.04; H, 3.01; N,
9.10.
6
1
54.34, 148.33, 136.35, 128.12, 125.20, 123.34, 121.21; ESI MS: m/z
90.06; calcd. 289.99.
–bromo–2-(4-nitrophenyl) benzo[d]thiazole (3c): Yield 2.60 g
2
6
77.84%); mp 234–235 °C; IR (KBr, ν, cm 1): 3156.54 (-CH Str),
−
2-(4-aminophenyl)benzo[d]thiazole (4d): Yield 1.78 g (78.76%); Rf
−1
(
1
1
652.61 (-C = N str); H NMR (400 MHz, DMSO–d , δ, ppm): 7.84–
0.79; mp 157–159 °C; IR (KBr, ν, cm ): 3454.17, 3433.57 (-NH2
6
7
.04 (m, 7H, Ar-H); 1 C NMR (400 MHz, DMSO–d , δ, ppm): 167.35,
3
Str), 3080.10, (-CH Str), 1659.11 (-C = N); 1H NMR (400 MHz,
6
1
54.31, 148.31, 136.32, 128.52, 124.54, 119.01; ESI MS: m/z 334.02;
DMSO–d , δ, ppm): 8.08–7.01 (m, 8H, Ar-H), 7.57 (s, 2H, NH );
6
2
13
calcd. 333.94.
C NMR (400 MHz, DMSO–d , δ, ppm): 168.12, 153.86, 151.91,
6
2
-(4-nitrophenyl)benzo[d]thiazole (3d): Yield 1.80 g (70.31%); mp
151.85, 133.72, 128.62, 125.92, 124.07, 121.64, 121.52, 120.31, 113.58,
113.54; LCMS: m/z 225.98; Calcd. 226.04; Anal. Calcd: C, 69.00; H,
4.45; N, 12.38. Found: C, 68.70; H, 4.39; N, 12.36.
−1
2
26–228 °C (lit35 229–231 °C); IR (KBr, ν, cm ): 3124.68 (-
1
CH Str), 1656.39 (-C = N str); H NMR (400 MHz, DMSO–d , δ,
6
13
ppm): 8.39–7.47 (m, 8H, Ar-H); C NMR (400 MHz, DMSO–d , δ,
4-(7–chloro–6-fluorobenzo[d]thiazol-2-yl)benzenamine (4e): Yield
6
1.60 g (57.55%); Rf 0.59; mp 180–182 °C; IR (KBr, ν, cm 1):
−
ppm): 148.64, 153.50, 164.56, 138.41, 135.04, 128.19, 126.80, 126.11,
1
24.30, 123.39, 122.25; ESI MS: m/z 256.06; calcd. 256.03.
3433.45, 3426.20 (-NH Str), 3083.46, (-CH Str), 1648.10 (-C = N);
1
7
–chloro–6-fluoro-2-(4-nitrophenyl)benzo[d]thiazole (3e): Yield
H NMR (400 MHz, DMSO–d , δ, ppm): 7.91 (s, 2H, NH ), 7.22–
6
2
.45 g (79.36%); mp 140–142 °C; IR (KBr, ν, cm 1): 3074.43 (-
−
7.18 (m, 1H, Ar-H), 7.16–6.93 (m, 5H, Ar-H); 13C NMR (400 MHz,
2
1
CH Str), 1639.34 (-C = N str); H NMR (400 MHz, DMSO–d , δ,
DMSO–d , δ, ppm): 167.93, 153.39, 152.01, 133.71, 128.88, 125.96,
6
6
ppm): 8.12–7.01 (m, 6H, Ar-H); 3C NMR (400 MHz, DMSO–d6,
1
124.00, 121.51, 120.38, 114.67; LCMS: m/z 277.97; Calcd. 278.01;
Anal. Calcd: C, 56.02; H, 2.89; N, 10.05. Found: C, 56.00; H, 2.37;
N, 10.04.
δ, ppm): 168.32, 152.54, 149.43, 145.78, 139.22, 135.62, 128.20,
122.50, 114.32; ESI MS: m/z 308.03; calcd. 307.98.
6
,7-dichloro-2-(4-nitrophenyl)benzo[d]thiazole (3f): Yield 2.25 g
4-(7–chloro–6-fluorobenzo[d]thiazol-2-yl)benzenamine (4f): Yield
−
−1
(
69.02%); mp 164–166 °C; IR (KBr, ν, cm 1): 3088.41 (-CH Str),
1.45 g (49.32%); R 0.91; mp 189–191 °C; IR (KBr, ν, cm ): 3477.73,
f
1
3462.41 (-NH2 Str), 3101.12, (-CH Str), 1653.47 (-C = N); 1H NMR
1
641.03 (-C = N str); H NMR (400 MHz, DMSO–d , δ, ppm): 8.11–
6
13
6
.86 (m, 6H, Ar-H); C NMR (400 MHz, DMSO–d , δ, ppm): 167.47,
(400 MHz, DMSO–d , δ, ppm): 8.02 (m, 1H, Ar-H), 7.85 (s, 2H,
6
6
1
52.78, 148.10, 144.54, 140.21, 134.27, 128.65, 124.17, 122.30; ESI
NH ), 7.77–7.04 (m, 5H, Ar-H); 13C NMR (400 MHz, DMSO–d , δ,
2
6
MS: m/z 323.92; calcd. 323.95.
ppm): 168.01, 154.30, 151.67, 133.35, 128.09, 125.50, 124.08, 121.11,
20.84, 114.39; LCMS: m/z 294.04; Calcd. 293.98; Anal. Calcd: C,
1
5
2.89; H, 2.73; N, 9.49. Found: C, 52.65; H, 2.56; N, 9.42.
3
6
.5. General procedure for synthesis of
,7-substituted-2-(4-aminophenyl)benzo[d]thiazole (4a-f) [48]
3
.6. General procedure for synthesis of
A
mixture of an appropriately substituted-2-(4-nitrophenyl)
3-(2-bromoacetyl)−4–hydroxy-1-methyl/phenyl quinolin-2(1H)-one (5
benzo[d]thiazole (one of 3a-f, 0.015 mol) and tin (II) chloride di-
hydrate (0.075 mol) in 50 mL boiling ethanol was stirred under ni-
trogen environment for 3–5 h. The excess ethanol was removed by
vacuum evaporation using rota-evaporator and the residue formed
was successively extracted with 75 ml of ethyl acetate (3 times).
The combined organic fraction was further thoroughly washed first
with 50 mL of 2 M aqueous NaOH (3 times) followed by 50 mL of
distilled water (2 times). To the resultant organic fraction, anhy-
and 6) [35]
As shown in Scheme 2, these compounds were prepared by
dissolving 3-acetyl-4–hydroxy-1-methyl/phenyl quinolin-2(1H)-one
(0.065 mol) in glacial acetic acid (50 mL) and gently heated at
80 °C with constant stirring for 30 min. The equimolar quantity
of bromine (0.065 mol) in glacial acetic acid (10 mL) was slowly
added in a drop wise manner for a period of 1 h and heating of
the solution was continued until a slight change in the color of the
solution was observed. The reaction mixture was cooled to room
temperature. The products (5 and 6) were obtained as yellow crys-
talline solid and recrystallized from ethanol.
drous Na SO4 was added, filtered, and the filtrate was evaporated
2
in-vacuum to yield the desired compounds 4a-f.
4-(6-fluorobenzo[d]thiazol-2-yl)benzenamine (4a): Yield 1.63 g
(
66.80%); Rf 0.25; mp 171–173 °C; IR (KBr, ν, cm 1): 3425.63,
−
3
420.24 (-NH2 Str), 3125.35 (-CH Str), 1654.11 (-C = N str); 1H
3-(2-bromoacetyl)−4–hydroxy-1-methylquinolin-2(1H)-one
(5):
−1
NMR (400 MHz, DMSO–d , δ, ppm): 7.97 (m, 1H, Ar-H), 7.87 (s,
Yield 12.4 g (64%), mp 172–174 °C, IR (KBr, ν, cm ): 3469.02
6
2
H, NH ), 7.38–6.67 (m, 6H, Ar-H); 13C NMR (400 MHz, DMSO–
(-OH), 2985.31(-CH str), 1737.08 (-C = O str of COCH Br), 1658.22
2
2
1
d6, δ, ppm): 168.42, 159.39, 153.64, 151.42, 133.46, 128.20, 125.36,
24.04, 121.49, 120.35, 113.27; LCMS: m/z 243.99; Calcd. 244.05;
Anal. Calcd: C, 63.92; H, 3.71; N, 11.47. Found: C, 63.84; H, 3.48; N,
1.37.
-(6-chlorobenzo[d]thiazol-2-yl)benzenamine (4b): Yield 0.96 g
(-C = O str of amide); H NMR (400 MHz, DMSO–d , δ, ppm):
6
1
15.68 (s, 1H, OH), 8.68–7.22 (m, 4H, Ar-H), 3.23 (s, 2H, CH ), 2.64
2
(s, 3H, N–CH ); 13C NMR (101 MHz, DMSO–d , δ, ppm): 201.03
3
6
1
(C = O carbon of COCH Br), 167.07 (C = O of 2-quinolone), 137.06,
2
4
128.87, 126.36, 124.45, 118.17, 115.60, 107.04 (Aromatic carbons),
36.92%); Rf 0.64; mp 194–196 °C; IR (KBr, ν, cm 1): 3498.74,
−
(
33.45 (N–CH ), 35.98 (CH Br); LCMS: m/z 294.92; calcd. 294.98.
3
2
3
489.29 (-NH2 Str), 3077.67 (-CH Str), 1674.80 (-C = N); 1H NMR
3-(2-bromoacetyl)−4–hydroxy-1-phenylquinolin-2(1H)-one
(6):
−1
(
400 MHz, DMSO–d , δ, ppm): 7.91 (m, 1H, Ar-H), 7.40 (s, 2H,
Yield 11.6 g (50%), mp 264–266 °C, IR (KBr, ν, cm ): 3478.43 (-OH
6
NH ), 7.29–7.20 (m, 1H, Ar-H), 7.04–6.81 (m, 5H, Ar-H); 13C NMR
Str), 2990.30 (-CH str), 1732.05 (-C = O str of COCH Br), 1655.33
2
2
1
(
400 MHz, DMSO–d , δ, ppm): 168.11, 158.23, 153.61, 152.49,
(-C = O str of amide); H NMR (400 MHz, DMSO–d , δ, ppm):
6
6
1
33.44, 128.60, 125.36, 124.34, 122.51, 119.88, 114.71; LCMS: m/z
59.86; Calcd. 260.02; Anal. Calcd: C, 59.88; H, 3.48; N, 10.74.
Found: C, 59.85; H, 3.29; N, 10.65.
16.89 (s, 1H, OH), 8.28–6.12 (m, 9H, Ar-H), 2.82 (s, 2H, CH ); 13C
2
2
NMR (400 MHz, DMSO–d , δ, ppm): 205.09 (C = O carbon of
6
COCH Br), 174.39 (C = O of 2-quinolone), 163.42, 141.09, 137.00,
2
11

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1
35.11, 129.77, 129.07, 128.24, 125.66, 120.47, 116.93, 114.42, 104.47,
3-(2-(4-(7–chloro–6-fluorobenzo[d]thiazol-2-
yl)phenylamino)acetyl)−4–hydroxy-1-methylquinolin-2(1H)-one
3
6.03 (CH Br); LCMS: m/z 256.73; calcd. 257.00.
2
(
7e): Yield 3.90 g (79.10%); Rf 0.41; mp 203–205 °C; IR (KBr, ν,
cm 1): 3478.06 (OH Str), 3278.06 (NH Str), 3098.39 (-CH Str),
−
3
3
.7. General procedure for synthesis of
1723.23 (C = O Str), 1678.78 (C = O Str), 16.38.57 (C = N Str);
1
-(2-(4-(6,7-substituted-benzo[d]thiazol-2-yl)phenyl
H NMR (400 MHz, DMSO–d6, δ, ppm): 11.45 (s, 1H, OH), 8.50
aminoacetyl)−4–hydroxy-1-methyl/phenyl quinolin-2(1H)-one (7a-f
(s, 1H, NH), 8.08–6.91 (m, 10H, Ar-H), 5.09 (s, 2H, CH2), 3.62 (s,
3H, CH3); 13C NMR (400 MHz, DMSO–d6, δ, ppm): 193.33, 167.22,
and 8a-f)
165.30, 159.45, 153.31, 151.67, 134.64, 128.62, 125.41, 124.72,
As illustrated in Scheme 2,
ately substituted-2-(4-amino
a
mixture of an appropri-
123.61, 122.33, 119.45, 113.56, 115.76, 109.24, 57.62, 28.31; LCMS:
m/z 492.95; Calcd. 493.07; Anal. Calcd: C, 60.79; H, 3.47; N, 8.51.
Found: C, 60.78; H, 3.44; N, 8.49.
phenyl)benzo[d]thiazole (one
of 4a-f, 0.004 mol) and 3-(2-bromoacetyl)−4–hydroxy-1-
methyl/phenylquinolin-2(1H)-one (5/6, 0.004 mol) was dissolved
in 45 mL of glacial acetic acid with constant stirring. The reaction
mixture was then heated at reflux temperature for about 6–13 h.
The progress of the reaction was monitored by the TLC. The
reaction mixture was allowed to attain the room temperature. The
solvent was removed under vacuum. The residue was dissolved
in ethyl acetate, washed twice with water, and dried (Na SO ).
3-(2-(4-(6,7-dichlorobenzo[d]thiazol-2-yl)phenylamino)acetyl)−4–
hydroxy-1-methylquinolin-2(1H)-one (7f): Yield 3.33 g (65.42%); Rf
0.77; mp 245–247 °C; IR (KBr, ν, cm 1): 3478.29 (OH Str), 3372.42
−
(NH Str), 3071.11 (-CH Str), 1728.13 (C = O Str), 1660.22 (C = O Str),
1
1639.87 (C = N Str); H NMR (400 MHz, DMSO–d6, δ, ppm): 10.64
(s, 1H, OH), 8.53 (s, 1H, NH), 8.00–6.86 (m, 10H, Ar-H), 5.06 (s, 2H,
CH2), 3.56 (s, 3H, CH3); 13C NMR (400 MHz, DMSO–d6, δ, ppm):
193.37, 167.18, 164.12, 159.30, 154.56, 151.90, 151.27, 134.34, 128.65,
125.76, 124.65, 123.33, 122.98, 119.19, 113.83, 113.33, 110.20, 56.24,
28.96; LCMS: m/z 508.93; Calcd. 509.04; Anal. Calcd: C, 58.83; H,
3.36; N, 8.23. Found: C, 58.85; H, 3.32; N, 8.20.
2
4
Further, the title compounds were purified by removal of the
solvent followed by column chromatography using 200–400 mesh
silica gel eluting with methanol (12%) in dichloromethane.
3
-(2-(4-(6-fluorobenzo[d]thiazol-2-yl)phenylamino)acetyl)−4–
hydroxy-1-methylquinolin-2(1H)-one (7a): Yield 3.25 g (70.80%); Rf
3-(2-(4-(6-fluorobenzo[d]thiazol-2-yl)phenylamino)acetyl)−4–
hydroxy-1-phenylquinolin-2(1H)-one (8a): Yield 2.90 g (55.02%); Rf
0.39; mp 222–224 °C; IR (KBr, ν, cm 1): 3444.45 (OH Str), 3346.34
(NH Str), 3139.13 (-CH Str), 1717.14 (C = O Str), 1675.77 (C = O Str),
.57; mp 194–196 °C; IR (KBr, ν, cm 1): 3456.76 (-OH Str), 3385.07
−
0
−
(
-NH Str), 3078.39 (-CH Str), 1725.94 (C = O Str), 1662.45 (C = O
1
Str), 1639.49 (C = N Str); H NMR (400 MHz, DMSO–d , δ, ppm):
6
1
9
.85 (s, 1H, OH), 7.67 (s, 1H, NH), 6.68–7.78 (m, 11H,Ar-H), 5.64
1637.17 (C = N Str); H NMR (400 MHz, DMSO–d6, δ, ppm): 11.09
13
(
s, 2H, CH ), 0.26 (s, 3H, CH ); C NMR (400 MHz, DMSO–d , δ,
(s, 1H, OH), 8.64 (s, 1H, NH), 7.82–6.66 (m, 16H, Ar-H), 4.95 (s,
2H, CH2); 13C NMR (400 MHz, DMSO–d6, δ, ppm): 192.55, 168.53,
167.06, 151.45, 149.41, 138.51, 136.28, 136.01, 128.97, 128.60, 127.37,
125.29, 124.70, 122.76, 122.16, 119.32, 113.41, 113.16, 109.38, 57.39;
LCMS: m/z 521.04; Calcd. 521.12; Anal. Calcd: C, 69.09; H, 3.87; N,
8.06. Found: C, 69.11; H, 3.87; N, 8.04.
2
3
6
ppm): 193.48, 167.27, 165.37, 158.51, 153.33, 151.98, 151.22, 134.67,
28.66, 125.44, 124.75, 123.60, 122.30, 119.32, 113.45, 113.01,
09.28, 56.64, 27.30; LCMS: m/z 459.06; Calcd. 459.11; Anal. Calcd:
C, 65.35; H, 3.95; N, 9.14. Found: C, 65.35; H, 3.92; N, 9.16.
-(2-(4-(6-chlorobenzo[d]thiazol-2-yl)phenylamino)acetyl)−4–
hydroxy-1-methylquinolin-2(1H)-one (7b): Yield 3.50 g (73.68%); Rf
1
1
3
3-(2-(4-(6-chlorobenzo[d]thiazol-2-yl)phenylamino)acetyl)−4–
hydroxy-1-phenylquinolin-2(1H)-one (8b): Yield 2.60 g (48.41%); Rf
0.68; mp 199–201 °C; IR (KBr, ν, cm 1): 3363.09 (OH Str), 3266.33
(NH Str), 3156.20 (-CH Str), 1708.55 (C = O Str), 1617.61 (C = N
Str); 1H NMR (400 MHz, DMSO–d6, δ, ppm): 10.63 (s, 1H, OH),
.53; mp 176–178 °C; IR (KBr, ν, cm 1): 3498.74 (OH Str), 3356.44
−
0
−
(
NH Str), 3089.96 (-CH Str), 1716.03 (C = O Str), 1670.10(C = O
1
Str), 1631.78 (C = N Str); H NMR (400 MHz, DMSO–d , δ, ppm):
6
1
1.26 (s, 1H, OH), 8.59 (s, 1H, NH), 7.88–6.69 (m, 11H,Ar-H), 5.59
13
8.56 (s, 1H, NH), 8.14–6.92 (m, 16H, Ar-H), 5.05 (s, 2H, CH2); 13
C
(
s, 2H, CH ), 3.23 (s, 3H, CH ); C NMR (400 MHz, DMSO–d , δ,
2
3
6
ppm): 192.05, 168.06, 166.22, 158.18, 154.20, 151.87, 151.04, 133.61,
28.26, 125.05, 124.88, 123.23, 122.11, 119.35, 114.27, 114.03,
10.23, 57.70, 28.28; LCMS: m/z 475.00; Calcd. 475.08; Anal. Calcd:
C, 63.09; H, 3.81; N, 8.83. Found: C, 63.03; H, 3.79; N, 8.89.
-(2-(4-(6-bromobenzo[d]thiazol-2-yl)phenylamino)acetyl)−4–
hydroxy-1-methylquinolin-2(1H)-one (7c): Yield 3.12 g (60.81%); Rf
NMR (400 MHz, DMSO–d6, δ, ppm): 191.79, 166.64, 166.73, 151.40,
149.52, 138.52, 136.21, 136.01, 129.04, 128.66, 127.20, 125.20,
124.74, 122.71, 122.11, 119.29, 113.63, 113.12, 111.30, 56.30; LCMS:
m/z 538.92; Calcd. 537.09; Anal. Calcd: C, 66.97; H, 3.75; N, 7.81.
Found: C, 67.00; H, 3.76; N, 7.79.
1
1
3
3-(2-(4-(6-bromoobenzo[d]thiazol-2-yl)phenylamino)acetyl)−4–
hydroxy-1-phenylquinolin-2(1H)-one (8c): Yield 3.10 g (53.35%); Rf
0.36; mp 265–267 °C; IR (KBr, ν, cm 1): 3443.04 (OH Str), 3357.88
(NH Str), 2923.04 (C–H Str), 1711.96 (C = O Str), 1654.35 (C = O
−
0
.47; mp 209–211 °C; IR (KBr, ν, cm 1): 3469.06 (-OH Str), 3350.65
−
(
NH Str), 3078.39 (-CH Str), 1726.29 (C = O Str), 1666.27(C = O
1
Str), 1637.45 (C = N Str); H NMR (400 MHz, DMSO–d , δ, ppm):
6
1
1
1.04 (s, 1H, OH), 8.62 (s, 1H, NH), 8.03–6.98 (m, 11H,Ar-H), 5.60
Str), 1605.49 (C = N Str); H NMR (400 MHz, DMSO–d6, δ, ppm):
1
(
s, 2H, CH ), 3.33 (s, 3H, CH ); 3C NMR (400 MHz, DMSO–d6,
10.86 (s, 1H, OH), 8.74 (s, 1H, NH), 8.12–6.58 (m, 16H, Ar-H), 5.57
(s, 2H, CH2); 13C NMR (400 MHz, DMSO–d6, δ, ppm): 192.55,
165.60, 164.70, 151.34, 149.02, 138.17, 136.22, 136.98, 129.20,
128.61, 126.00, 125.65, 124.29, 122.65, 122.01, 120.01, 114.34,
114.11, 110.29, 57.44; LCMS: m/z 580.97; Calcd. 581.04; Anal. Calcd:
C, 61.86; H, 3.46; N, 7.21. Found: C, 61.89; H, 3.44; N, 7.21.
2
3
δ, ppm): 192.67, 168.52, 163.19, 157.82, 153.42, 151.73, 151.38,
33.49, 128.41, 125.41, 124.05, 123.62, 122.30, 119.38, 113.62,
13.21, 110.39, 57.04, 26.37; LCMS: m/z 518.97; Calcd. 519.03; Anal.
Calcd: C, 57.70; H, 3.49; N, 8.07. Found: C, 57.68; H, 3.43; N, 8.10.
-(2-(4-(benzo[d]thiazol-2-yl)phenylamino)acetyl)−4–hydroxy-1-
methylquinolin-2(1H)-one (7d): Yield 3.60 g (81.63%); R 0.57; mp
1
1
3
3-(2-(4-(benzo[d]thiazol-2-yl)phenylamino)acetyl)−4–hydroxy-1-
phenylquinolin-2(1H)-one (8d): Yield 3.50 g (69.59%); Rf 0.61; mp
168–170 °C; IR (KBr, ν, cm 1): 3478.43 (OH Str), 3336.81 (NH Str),
2990.30 (-CH Str), 1732.05 (C = O Str), 1655.33 (C = O Str), 1614.62
f
−
1
54–156 °C; IR (KBr, ν, cm 1): 3468.30 (OH Str), 3382.50 (NH
−
Str), 3084.39 (-CH Str), 1726.28 (C = O Str), 1663.55(C = O Str),
1
1
635.13 (C = N Str); H NMR (400 MHz, DMSO–d , δ, ppm): 10.30
6
1
(
s, 1H, OH), 8.57 (s, 1H, NH), 8.08–7.01 (m, 12H, Ar-H), 4.99 (s, 2H,
(C = N Str); H NMR (400 MHz, DMSO–d6, δ, ppm): 11.06 (s, 1H,
1
3
CH ), 3.15 (s, 3H, CH ); C NMR (400 MHz, DMSO–d , δ, ppm):
OH), 8.62 (s, 1H, NH), 7.85–7.12 (m, 17H, Ar-H), 5.03 (s, 2H, CH2);
13C NMR (400 MHz, DMSO–d6, δ, ppm): 191.27, 168.76, 166.48,
153.76, 149.90, 139.29, 136.33, 135.78, 128.78, 128.30, 127.30,
125.09, 124.20, 122.37, 122.10, 119.06, 113.74, 113.25, 109.22, 57.30;
2
3
6
1
94.62, 168.62, 163.39, 158.42, 153.00, 151.53, 134.53, 128.62,
25.86 124.74, 123.62, 122.53, 119.44, 113.34, 109.29 56.31, 27.86;
LCMS: m/z 441.10; Calcd. 441.11; Anal. Calcd: C, 68.01; H, 4.34; N,
.52. Found: C, 67.99; H, 4.34; N, 9.51.
1
9
12

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Journal of Molecular Structure xxx (xxxx) xxx
LCMS: m/z 503.05; Calcd. 503.13; Anal. Calcd: C, 71.55; H, 4.20; N,
.34. Found: C, 71.25; H, 4.21; N, 8.36.
-(2-(4-(7–chloro–6-fluorobenzo[d]thiazol-2-
yl)phenylamino)acetyl)−4–hydroxy-1-phenylquinolin-2(1H)-one (8e):
4.1.2. In vitro antimicrobial activity
4.1.2.1. Medium. The solid media Mullere Hinton agar (MHA; beef
8
−
1, casein acid hydrolysate 17.5 g.L 1, starch
−
3
infusion 300 g.L
1.5 g.L 1, agar 17 g.L 1, and distilled water 1000 mL, adjusted
to pH 7.4) was used for the antibacterial activity. For antifungal
activity, solidified sterile Sabouraud’s Dextrose agar (SDA) was used
as medium.
−
−
−
1
Yield 3.90 g (70.27%); R 0.72; mp 233–235 °C; IR (KBr, ν, cm ):
f
3
472.76 (OH Str), 3335.23 (NH Str), 3082.20 (-CH Str), 1720.54
(
(
C = O Str), 1667.35(C = O Str), 1635.44 (C = N Str); 1H NMR
400 MHz, DMSO–d , δ, ppm): 10.81 (s, 1H, OH), 8.71 (s, 1H, NH),
6
8
.18–7.04 (m, 15H, Ar-H), 4.87 (s, 2H, CH ); 13C NMR (400 MHz,
4.1.2.2. Test microorganisms. In vitro antimicrobial activity was car-
ried out against a panel of microorganisms including Gram pos-
itive bacteria Staphylococcus aureus (ATCC 11,632), Bacillus subtilis
(ATCC 60,511) and Gram negative bacteria E. coli (ATCC 10,536),
Pseudomonas aeruginosa (ATCC 10,145). For the antifungal activity,
the title compounds were screened against Candida albicans (ATCC
2501) and Aspergillus niger (ATCC 1781).
2
DMSO–d , δ, ppm): 193.50, 167.88, 165.02, 152.27, 148.41, 139.31,
6
1
36.73, 135.65, 128.11, 128.65, 127.11, 125.22, 124.09, 122.45,
22.00, 118.96, 114.54, 114.05, 109.20, 56.72; LCMS: m/z 555.05;
Calcd. 555.08; Anal. Calcd: C, 64.81; H, 3.44; N, 7.56. Found: C,
4.80; H, 3.43; N, 7.56.
-(2-(4-(6,7-dichlorobenzo[d]thiazol-2-yl)phenylamino)acetyl)−4–
hydroxy-1-phenylquinolin-2(1H)-one (8f): Yield 2.75 g (48.16%); Rf
1
6
3
.69; mp 269–271 °C; IR (KBr, ν, cm 1): 3498.74 (OH Str), 3356.44
−
0
4.1.2.3. Minimum inhibitory concentration [49]. The in-vitro antimi-
crobial activity for newly synthesized compounds (7a-f and 8a-
f) was evaluated using the conventional agar-dilution method ac-
cording to the guidelines of Clinical and Laboratory Standards In-
stitute (CLSI) [50]. All bacterial strains were subcultured in MHA
plates and incubated overnight at 37 °C and fungal strains were
subcultured in SDA plates at 35 °C for 24–48 h. The microor-
ganisms were processed at least twice to ensure purity and vi-
ability. Twofold serial dilutions of the compounds and reference
drugs (Ampicilin, Gatifloxacin, and Fluconazole) were prepared in
MHA/SDA. Drugs (10.0 mg) were dissolved in DMSO (1 mL) and
the solution was diluted with water (9 mL). Further progressive
double dilution with melted MHA was performed to obtain the re-
quired concentrations of 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, 1,
(
NH Str), 3089.96 (-CH Str), 1716.03 (C = O Str), 1661.75(C = O
1
Str), 1606.00 (C = N Str); H NMR (400 MHz, DMSO–d , δ, ppm):
6
1
0.39 (s, 1H, OH), 8.54 (s, 1H, NH), 8.09–6.73 (m, 15H, Ar-H),
5
.16 (s, 2H, CH ); 13C NMR (400 MHz, DMSO–d , δ, ppm): 192.18,
2
6
1
68.58, 166.39, 154.33, 148.01, 139.44, 137.03, 136.03, 128.12,
28.62, 127.12, 125.22, 124.77, 122.39, 122.30, 118.92, 114.90,
14.25, 109.21, 56.74; LCMS: m/z 577.32; Calcd. 571.05; Anal. Calcd:
C, 62.94; H, 3.35; N, 7.34. Found: C, 62.96; H, 3.36; N, 7.32.
1
1
4
. Biological evaluation: anticancer activity
4
.1. Human cancer cell lines
0
.5, 0.25, 0.125, and 0.0625 μg.mL 1. The bacterial inoculates were
−
Compounds were evaluated for their in vitro cytotoxic activ-
prepared by suspending 24 h old bacterial colonies from MHA me-
dia in 0.85% saline. The inoculate were adjusted to 0.5 McFarland
ity against four human cancer cell lines (EAC, MCF-7, HT29, and
WRL68 cells), which were cultured in MEM medium supplemented
8
Standard (1.5 × 10 CFU/mL) [51]. The suspensions were then di-
−1
with 10% FBS, 1% l-glutamine, and 50 mg.mL gentamicin sulfate
in a CO2 incubator in a humidified atmosphere of 5% CO2 and 95%
air. The EAC cells were maintained for 12–14 days in the peritoneal
cavity of Swiss albino mice. The tumor cell cultures were started
from mouse Ehrlich Ascites with at least one passage in vitro prior
to use.
luted in 0.85% saline to give 107 CFU/mL. Test tubes were spot-
inoculated with 10 μL of each prepared bacterial suspension (104
CFU/spot) and incubated at 37 °C for 24 h. The experiments were
carried out in triplicate for all the organisms tested. At the end
of the incubation period, MIC was determined, which is the low-
est concentration of the test compound that resulted in no visible
growth on the plate. A control test was also performed with test
medium supplemented with DMSO at the same dilutions as used
in the experiment in order to ensure that the solvent had no in-
fluence on bacterial growth. The yeast suspensions used for inoc-
ulation were prepared at 104 cfu/mL by diluting fresh cultures at
MacFarland 0.5 density (106 cfu/mL). Suspensions of the yeast at
104 cfu/mL concentration were inoculated to the twofold diluted
solution of the compounds. There were 103 cfu/mL bacteria in the
wells after inoculations. A 10 μL yeast inoculum was added to each
well of the test tubes, which were incubated at 35 °C in a humid
chamber and MIC endpoints were read after 48 h of incubation.
The experiments were carried out in triplicate for all the organ-
isms tested.
4
.1.1. In vitro cytotoxicity studies (MTT assay)
In vitro cytotoxicity was determined using a standard MTT as-
say [36] with protocol appropriate for the individual test sys-
tem. In brief, exponentially growing cells were plated in 96-well
plates (104 cells/well in 100 mL of medium) and incubated for
2
4 h to enable them to adhere properly in the each well of 96-
well polystyrene microplate (Grenier, Germany). Test compounds
7a-f and 8a-f) were prepared prior to the experiment by dis-
(
solving in 0.1% dimethyl sulfoxide (DMSO, Merck, Germany) and
diluted with medium. The cells were then exposed to different
concentrations of the drugs (1–100 μM) in the volume of 100
μL per well. Cells in the control wells received the same vol-
ume of medium containing 0.1% DMSO. After 24 h incubation, 10
μL of MTT [3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium
4.1.2.4. Docking methodology. Molecular docking studies of the
synthesized compounds (7a-f and 8a-f) were performed in order to
rationalize the obtained anticancer results especially against hor-
mone dependant breast cancer cells. Molecular docking studies fur-
ther help in understanding the various interactions between the
ligand and enzyme active site in detail. Docking study was car-
ried out for the target compounds using MVD-2013 (version: 6.0).
MolDock scoring function used by MVD program is defined by:
−1
bromide; Sigma M 2128; 1 mg.mL ] was added in each well and
plates were further incubated at 37 °C for 4 h. The formazan pro-
duced by the viable cells was solubilized by addition of 100 μL
DMSO. The suspension was placed on micro-vibrator for 5 min
and absorbance was recorded by a SpectraMax 190 Microplate
ELISA reader (Molecular Devices Inc. USA) at 540 nm. The exper-
iment was performed in triplicate. The percentage cytotoxicity and
IC50 values were determined at 24, 48, and 72 h of drug incu-
bation. IC50 value is the concentration (reported in μM) required
for 50% inhibition of cell growth as compared to that of untreated
control.
Escore = Einter + Eintra
where, Escore = MolDock score.
13

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JID: MOLSTR
[m5G;October 22, 2020;11:47]
G. Bolakatti, M. Palkar, M. Katagi et al.
Journal of Molecular Structure xxx (xxxx) xxx
References
Einter = ligand − −Protein interaction
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and electrostatic terms. To further improve docking accuracy, a re-
ranking scoring function was introduced, which identifies the most
promising docking explanation from the results obtained by the
docking algorithm [52].
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Declaration of Competing Interest
The authors declare that they have no known competing finan-
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influence the work reported in this paper. The authors declare the
following financial interests/personal relationships which may be
considered as potential competing interests: The authors state no
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Girish Bolakatti: Conceptualization, Methodology. Mahesh
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Visualization, Investigation. Girish Hampannavar: Writing - re-
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This work was supported by financial assistance received from
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