Targeting EGFR tyrosine kinase: Synthesis, in vitro antitumor evaluation, and molecular modeling studies of benzothiazole-based derivatives

Article abstract of DOI:10.1016/j.bioorg.2020.104259

New benzothiazole-based derivatives were synthesized in the present work with the aim of evaluating their antitumor activity. They were in vitro tested against hepatocellular carcinoma (HepG2), colorectal carcinoma (HCT-116), mammary gland cancer (MCF-7), prostate cancer (PC-3), and epithelioid carcinoma (HeLa). The results of the in vitro antitumor evaluation revealed that the most active compounds were 39, 40, 51, 56, and 61 exhibiting IC₅₀ values comparable to the reference drug lapatinib. The most active compounds were further subjected to EGFR inhibitory activity assay to rationalize their potency mode. Notably, the most active antitumor compounds 39 and 40 represented the most potent inhibitors to EGFR with IC₅₀ values of 24.58 and 30.42 nM respectively in comparison with 17.38 nM for lapatinib as a standard drug. Molecular modeling studies were also conducted for the synthesized compounds, including docking into EGFR active site and surface mapping. Results proved the superior binding of the hydrazone derivatives 39 and 40 with EGFR suggesting them as good candidates for targeted antitumor therapy through EGFR kinase inhibition.

Full text of DOI:10.1016/j.bioorg.2020.104259

BioorganicChemistry104(2020)104259
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Targeting EGFR tyrosine kinase: Synthesis, in vitro antitumor evaluation, and
molecular modeling studies of benzothiazole-based derivatives
⁎
Amal M. Mokhtara, Shahenda M. El-Messeryb, Mariam A. Ghalya, , Ghada S. Hassana
a Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
b Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
New benzothiazole-based derivatives were synthesized in the present work with the aim of evaluating their
antitumor activity. They were in vitro tested against hepatocellular carcinoma (HepG2), colorectal carcinoma
(HCT-116), mammary gland cancer (MCF-7), prostate cancer (PC-3), and epithelioid carcinoma (HeLa). The
results of the in vitro antitumor evaluation revealed that the most active compounds were 39, 40, 51, 56, and 61
exhibiting IC50 values comparable to the reference drug lapatinib. The most active compounds were further
subjected to EGFR inhibitory activity assay to rationalize their potency mode. Notably, the most active antitumor
compounds 39 and 40 represented the most potent inhibitors to EGFR with IC50 values of 24.58 and 30.42 nM
respectively in comparison with 17.38 nM for lapatinib as a standard drug. Molecular modeling studies were also
conducted for the synthesized compounds, including docking into EGFR active site and surface mapping. Results
proved the superior binding of the hydrazone derivatives 39 and 40 with EGFR suggesting them as good can-
didates for targeted antitumor therapy through EGFR kinase inhibition.
Benzothiazole
Synthesis
Antitumor
EGFR
Molecular modeling
1. Introduction
inhibitors to treat lung and breast cancers [4–6]. They all belong to the
class of 4-anilinoquinazolines. Their quinazoline nitrogens mimic the
Cancer is an outbreak disease which causes mortality around the
world. It is characterized by uncontrolled division and abnormal pro-
liferation of body cells [1]. For years, scientists made many valuable
efforts in order to limit the spread of this disease through many stra-
tegies. One of the non-traditional strategies is concerned with epi-
dermal growth factor receptor (EGFR).
adenine of ATP in binding into the kinase active site, providing hy-
drogen bonding interactions. In addition, the aniline part is housed in
the hydrophobic pocket (Fig. 1).
Benzothiazole is a hetero bicyclic system that has a distinct phar-
macological profile including antitumor activity. Many researchers re-
ported the superior EGFR inhibitory activity of substituted benzothia-
zoles [7–9], some of them displaying more potent antitumor activity
than erlotinib as a reference drug [8].
EGFR is a transmembrane protein that belongs to tyrosine kinase
family [2]. It plays a significant role in cell transduction or commu-
nication signaling within the cell through phosphorylation (transfer of
phosphate group from ATP to the substrate) of tyrosine residues in
protein domain. Upregulation or mutation of EGFR results in abnormal
cell growth which may lead to appearance of various types of solid
malignancies e.g., colon, lung, and cervical cancers, so interruption of
EGFR communicating signals can be a suitable choice for decreasing
tumors expressed by its misregulation [3]. In contrast to conventional
cancer therapies that attack both normal cells and cancer cells resulting
in a wide spectrum of side effects which increase patients' suffering,
targeted drugs like EGFR tyrosine kinase inhibitors selectively attack
cancerous cells rather than normal ones providing a good safety profile,
less body damage and more patients' comfortability [4]. Canertinib,
erlotinib, and lapatinib are examples of clinically useful EGFR
Guided by the above considerations, the work presented herein re-
ports the synthesis of benzothiazole-based derivatives equipped with
substituted piperazinoacetamides (9–14), aminothiazolidin-4-ones
(23–29), acid hydrazones (35–41), substituted acid hydrazides (44, 45,
50–53, 60 and 61), and triazole-3-thiols (54–57) in which the ben-
zothiazole scaffold was designed as a bioistere to the quinazoline ring
system, capable of providing similar competition at the ATP binding
site of EGFR. The designed target compounds were subjected to in vitro
antitumor testing against five cell lines; hepatocellular carcinoma
(HepG2), colorectal carcinoma (HCT-116), mammary gland cancer
(MCF-7), prostate cancer (PC-3), and epithelioid carcinoma (HeLa), in
addition to assessing the EGFR kinase inhibitory activity of the most
active antitumor compounds. Molecular modeling studies were also
⁎ Corresponding author.
E-mail address: mariamaghaly@mans.edu.eg (M.A. Ghaly).
https://doi.org/10.1016/j.bioorg.2020.104259
Received 3 April 2020; Received in revised form 29 August 2020; Accepted 31 August 2020
Availableonline02September2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

A.M. Mokhtar, et al.
BioorganicChemistry104(2020)104259
Fig. 1. Clinically approved EFGR inhibitors and the designed target compounds.
performed to rationalize and describe the binding mode of the most
active compounds into EFGR kinase active site.
obtained hydrazone derivatives agreed with their suggested structures,
showing the N = CH proton as singlet signal at δ 8.54–8.99 ppm.
Scheme 3 describes the synthesis of groups of derivatives utilizing
the acid hydrazide 33 as a key compound. Compounds 44 and 45 were
obtained through the interaction of 33 with either 4-chloro or 4-
fluorobenzoyl chloride (42 and 43 respectively) in glacial acetic acid.
Appearance of additional aromatic signals due to the phenyl ring of acyl
chloride in their 1H NMR spectra proved their assigned structures.
Under the same reaction conditions, compound 33 was reacted with a
variety of isothiocyanate derivatives (46–49) to yield the corresponding
thiosemicarbazides 50–53. The structures of the latter compounds were
confirmed by the spectral data that showed the appearance of three
2. Results and discussion
2.1. Chemistry
Syntheses of the designed target compounds were carried out as
depicted in Schemes 1–3. In Scheme 1, 2-aminobenzothiazole (1 or 2)
was allowed to react with chloroacetyl chloride in chloroform in the
presence of triethylamine to afford the corresponding chloroacetamide
derivatives 4 and 5. Nucleophilic displacement of the chlorine atom of
the latter compounds with different substituted piperazine derivatives
(6–8) was achieved in DMF containing anhydrous potassium carbonate
yielding the corresponding amino compounds (9–14). The appearance
of the piperazine protons as two sets of signals, each integrating for four
protons at δ 2.53–2.80 and δ 2.66–3.26 ppm at the expected regions in
their 1HNMR spectra proved the success of the substitution reaction. In
addition, reacting 2-chloro-N-(6-methylbenzo[d]thiazol-2-yl)acetamide
(5) with ammonium thiocyanate in boiling ethanol afforded the cor-
responding 2-aminothiazol-4(5H)-one (15). The latter constructed
thiazol-4-one was condensed with a variety of aromatic and hetero-
aromatic aldehydes (16–22) in alcoholic NaOH through Knoevenagel
condensation reaction to furnish the corresponding 5-arylidenethiazol-
4(5H)-one derivatives (23–29). The 1HNMR spectra showed the ar-
ylidene protons as singlet signals appearing at δ 7.20–8.64 ppm con-
firming their proposed structures. Scheme 2 illustrates the synthesis of
ethyl benzothiazole-2-carboxylate (32) through the interaction of o-
aminothiophenol (30) with diethyl oxalate (31). Compound 32 was
reacted with hydrazine hydrate to obtain the corresponding acid hy-
drazide (33) which was utilized as a synthetic precursor for the pre-
paration of different hydrazone derivatives (35–41) through the reac-
tion with a collection of aromatic and heteroaromatic aldehydes (16,
1
singlet peaks due to NHNHCSNH at δ > 9.41 ppm in their H NMR
spectra. Additionally, the singlet peaks of tolyl CH3 and the OCH3
protons at δ 2.26 and 3.73 ppm confirmed the structure of the produced
compounds 50 and 52 respectively. The thiosemicarbazides 50–53
underwent cyclization into their corresponding 1,2,4-triazole-3-thiols
54–57 through refluxing in 2 N NaOH. The disappearance of the three
NH protons in the 1H NMR spectra confirmed the success of the cycli-
zation process. Finally, the sulfonamide derivatives 60 and 61 were
obtained through the interaction of compound 33 with 4-bromo-
benzenesulfonyl chloride 58 and tosyl chloride 59 in glacial acetic acid.
Their 1H NMR spectrum showed the appearance of doublet signals
corresponding to phenyl ring of p-substituted benzenesulfonyl group, in
addition to the mass spectrum showing the molecular ion peak
matching its molecular weight. Meanwhile, compound 61 was ad-
ditionally proved by the appearance of the tolyl CH3 protons as a singlet
peak at δ 2.37 ppm.
2.2. Biological evaluation
2.2.1. In vitro antitumor activity
The antitumor activity of the synthesized compounds was assessed
by determining their inhibitory effects on the cell growth of five cell
1
18–22, 34) in refluxing glacial acetic acid. The HNMR spectra of the
2

A.M. Mokhtar, et al.
BioorganicChemistry104(2020)104259
Scheme 1. Synthesis of compounds 9–14 and 23–29.
lines; namely hepatocellular carcinoma (HepG2), colorectal carcinoma
(HCT-116), mammary gland cancer (MCF-7), prostate cancer (PC-3),
and epithelioid carcinoma (HeLa) using the MTT assay. This colori-
metric assay is based on the conversion of the yellow tetrazolium
bromide (MTT) to a purple formazan derivative by mitochondrial suc-
cinate dehydrogenase in viable cells [10,11]. Different concentrations
of the test compounds were used (100, 50, 25, 12.5, 6.25, 3.125,
1.56 µM). The percentage inhibition of the tested compounds at a
concentration of 12.5 µM was used as the closest concentration to the
NCI assay protocol. The mentioned values and IC50 values in addition to
those of lapatinib as a reference drug, are illustrated in Table 1. The
results (Table 1) revealed that the most active antitumor compounds
were 39, 40, 51, 56, and 61 with % inhibition values ranging from 50.7
to 73.1%. Furthermore, the IC50 values of these active compounds are
comparable to the reference drug lapatinib with values ranging from
3.26 to 12.3 (µM). By taking a closer look to the obtained results, it was
found that compound 39 represents the most active compound among
the active ones with the highest inhibition range values of 66.2–73.1%
and lowest IC50 value of 3.26–7.08 µM.
to IC50 values of the tested compounds against lapatinib as a reference
drug are shown in Table 2. Results obtained revealed that all the tested
compounds exhibited high % inhibition values against EGFR ranging
from 70.6 to 86.2% in concentration of 1 µg/ml, and range of
86.7–94.9% in concentration of 10 µg/ml which is very close to the
standard drug used. Additionally, results also confirmed that the most
active antitumor compounds 39 and 40 exerted the highest potency
through EGFR kinase inhibition, with IC50 values of 24.58 and
30.42 nM, in comparison with 17.38 nM for lapatinib.
2.2.3. Cytotoxic activity against WI38 and A549 cell lines
Five of the most active compounds were selected to be further
studied for their cytotoxic activity against human lung fibroblast cell
line, WI38, as a normal cell line in order to test their safety. In addition,
they were tested for their inhibitory activity on the human EGFR-po-
sitive non-small cell lung cancer cell line; A549. Lapatinib was used as a
reference drug. The IC50 values of the most active compounds against
both cell lines are shown in Table 3. All these compounds exerted very
high IC50 values (42.8–73.2 µM) in comparison with lapatinib as a
standard drug (13.6 µM) against the normal cell line WI38 demon-
strating their safety. As for their cytotoxicity against the classic cell line
A549, which is the human EGFR-positive NSCLC cell line, three of these
most active compounds; 51, 39, and 40, exhibited inhibitory activity
comparable to lapatinib, with IC50 values of 5.41, 6.14, and 9.96 µM
respectively vs. 5.48 µM for lapatinib. These cellular assay results are in
2.2.2. EGFR kinase activity assay
In order to investigate the mode of antitumor activity of the syn-
thesized compounds, the most active ten compounds were further
subjected to EGFR kinase activity assay [12,13]. % Inhibition at four
different concentrations; 10, 100, 1000, and 10000 ng/ml, in addition
3

A.M. Mokhtar, et al.
BioorganicChemistry104(2020)104259
Scheme 2. Synthesis of compounds 35–41.
accordance with those of the enzyme assay proving the extreme po-
tency of these most active compounds.
and interactions was obtained by docking of lapatinib [14] that was
used as a reference ligand (95% inhibitory activity). It was cocrys-
tallized in X-ray crystal structure in protein data bank using pdb 1xkk
where it shows binding to Met793, Asp800 via hydrogen bonding in-
teractions in addition to Phe856 residue, as well as the key amino acid
residues of the active site, namely Lys745, Leu844, Met766 and Leu792
(Fig. 3).
2.3. Molecular modeling studies
2.3.1. Docking into EGFR kinase
Molecular docking study was carried out to obtain a better under-
standing of EGFR higher inhibitory activity results of compounds 39,
40, 56 and 60, in addition to rationalizing and describing their binding
mode into EFGR active site. The most potent inhibitors were first sub-
jected to conformational search study to get the best and least energy
conformer of each compound that will be used for docking study (Fig. 2,
supplementary file). Informative data about the binding orientations
Being docked in the active site of EGFR, compound 39 as the most
active inhibitor (94.68% inhibition) fulfilled the key interactions,
where the hydrogen bonding with Lys745 was established, as well as
the Pi-cation interaction with Val726 residue at the active site of EFGR
enzyme (Fig. 4). It was found that compound 40, which showed 94.14%
inhibition to EFGR compared to lapitinib, has shown the same pattern
Scheme 3. Synthesis of compounds 44, 45, 50–57, 60 and 61.
4

A.M. Mokhtar, et al.
BioorganicChemistry104(2020)104259
Table 1
% Inhibition and in vitro cytotoxicity (IC50, µM) of the target compounds.
of binding type as the reference ligand as follows; hydrogen bonding via
Lys745 and Pi-cation by Leu844 and Cys797 interactions which may
interpret its higher activity, (Fig. 4). Compound 56 (86.75%) showed
hydrogen bonding with Met793 and Leu792 via thiocarbonyl sulfur
atom. Moreover it was engaged in a second hydrogen bond with the
side chain Arg841 by sulfur heteroatom of thiazole ring, and an addi-
tional cationic arene interaction had been noticed with triazole ring
with Val726 (Fig. 5). The 2D binding model of compound 60 (87%) into
EGFR was depicted in Fig. 5, where hydrogen bonding interactions
were mediated via carbonyl oxygen atom to Lys745 and Met 766, in
addition to arene-cationic interaction to Val726 through thiazole ring
counterpart which may cause higher activity of compound 60 into
EGFR.
The core structures under concern adopted the same orientation as
the reference compound, that was clarified by taking compound 39 as
an example where both compound 39 and lapatinib have been
5

A.M. Mokhtar, et al.
BioorganicChemistry104(2020)104259
Table 2
% Inhibition and IC50 (nM) of EGFR kinase assay kit for the
target compounds.
Table 3
IC50 (µM) of the most active compounds against WI38 and A549 cell lines.
Comp.
WI38
A549
10
54.2
42.8
65.0
61.8
73.2
13.6
3.2
18.4
47.4
6.14
9.96
5.41
5.48
1.03
2.66
0.34
0.56
0.3
35
2.9
39
3.8
40
3.47
4.1
51
Lapatinib
0.76
0.31
completely superimposed in the active site center (Fig. 6) which may
contribute to the higher potency of the tested compound to be as potent
as lapatinib reference.
Fig. 4. 2D Binding mode and residues involved in the recognition of active
compounds 39 and 40 at active site of EFGR.
A respected point of view was to compare the shape of the docked
compounds into the active site of ATP site of EFGR. Fig. 7 clearly shows
all active compounds of interest, namely 39, 40, 56, and 60 were
deeply buried into the active site and fulfilling its cavity completely
which in turn has its impact on their higher inhibitory activity on EFGR
enzyme.
character of the molecule which is responsible for interaction with the
enzyme. A deep interaction with the hydrophobic pocket is suggested at
ATP binding back site that could be in charge for perfect fitting at active
site. In conclusion, our modeling studies were in good accordance with
the experimental results and supported them with visual evidences and
explanations too.
2.3.2. Surface mapping
In a further step for rationalizing the activity of tested compounds,
surface mapping calculations were conducted for compounds 39, 40,
56, and 60 which showed the best EFGR inhibitory profile in this study.
Fig. 8 pointed out a uniform distribution of hydrophobic regions in a
similar manner between all tested derivatives giving more lipophilic
2.4. Structure-activity correlation
Based on the obtained results, it was revealed that in general the
unsubstituted benzothiazole derivatives are more active than the
Fig. 3. Close-up view of binding of ligand (lapatinib) at binding pocket of EFGR X-ray crystal structure (pdb 1xkk).
6

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BioorganicChemistry104(2020)104259
Fig. 5. 2D Binding mode and residues involved in the recognition of active compounds 56 and 60 at active site of EFGR.
electron withdrawing group is also characteristic for activity more than
the electron donating groups as in case of compounds 56 and 61.
3. Conclusion
In the present study, different series of benzothiazole-based deri-
vatives were synthesized. The most active antitumor compounds were
the acid hydrazones 39 and 40 which also represented the most potent
inhibitors to EGFR with % inhibition values ranging from 60.2 to 73.1%
and with IC50 values of 24.58 and 30.42 nM respectively in comparison
with 17.38 nM for lapatinib as a standard drug. Docking studies into
EGFR active site revealed that compounds 39 and 40 showed a pattern
of binding similar to lapitinib as the reference ligand as they fulfilled
the key interactions, where the hydrogen bonding with Lys745 was
established. Compounds of interest, namely 39 and 40, were deeply
buried into the active site and completely housed in its cavity which in
turn has its impact on their higher inhibitory activity on EFGR enzyme.
Surface mapping displayed a deep interaction with the hydrophobic
pocket at ATP binding back site that could be in charge for perfect
fitting at active site.
Fig. 6. 3D Overview of compound 39 (pink) overlay with lapatinib reference
ligand at binding site of EFGR. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
methyl ones among the synthesized compounds. This was obvious
through the % inhibition assay against the selected tumor cell lines and
the % inhibition against EGFR kinase enzyme. The consistency of the
results revealed that such compounds are active as antitumor agents
and they act through the inhibition of EGFR kinase enzyme. Taking a
closer look for the active unsubstituted derivatives, it was found that
the presence of the acetamido structure and the thiazole-4(5H)-one
moiety doesn’t favor the activity, while the presence of the acid hy-
drazide group is very essential for activity. Specifically, the acid hy-
drazone group appeared in the most active compounds, viz. 39 and 40.
Furthermore, the presence of polycyclic ring system such as anthracene
and biphenyl group increases the activity of the compounds as in case of
compounds 39 and 40. It seems that bulkiness of substituents in vicinity
of the 2-position of the benzothiazole ring accounts for the activity of
these compounds. Moreover, compounds 23 and 35, each of which
contains a dimethoxyphenyl group, revealed activity which emphasizes
the role played by the steric factor. On the other hand, the presence of
4. Experimental protocols
The synthesis of the designed compounds was performed in the
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura
University, Mansoura, Egypt. The antitumor activity assay and cyto-
toxicity against WI38 normal cell line were conducted in the
Department of Pharmacognosy, Faculty of Pharmacy, Mansoura
University, Mansoura, Egypt. EGFR inhibitory assay and cytotoxicity
against A549 cell line were conducted at the confirmatory diagnostic
unit, VACSERA, Egypt. Melting points (°C) were determined on Stuart
melting point apparatus and are uncorrected. 1H, 13C NMR were re-
corded on a Joel 500 MHz FT spectrometer and Brucker 400 MHz
spectrometer; chemical shifts are expressed in δppm with reference to
TMS. Mass spectral (MS) data were obtained on direct inlet part to mass
analyzer in thermo scientific GCMS model ISQ at the Regional Center
7

A.M. Mokhtar, et al.
BioorganicChemistry104(2020)104259
Fig. 7. 3D View of active candidates 40, 56 and 60 deep buried into the pocket of EFGR (surface map).
Fig. 8. Surface map for active compounds 39, 40, 56, and 60. Pink: hydrogen bond, white: mild polar, green: hydrophobic. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
8

A.M. Mokhtar, et al.
BioorganicChemistry104(2020)104259
for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo,
Egypt. Thin layer chromatography was performed on precoated
(0.25 mm) silica gel GF254 plates (E. Merck, Germany), compounds
were detected with 254 nm UV lamp. All the fine chemicals and re-
agents used were purchased from Aldrich Chemicals Co, USA.
Compounds 4 [15], 5 [16], 9 [17], 10, 11 [18], 15 [19], 32, 33 [20],
44 [21], 51, 52, 55, 56 [22] were previously reported.
reflux for 24 h. The obtained precipitate was filtered, washed with
water and recrystallized from ethanol. Reddish white crystals, yield %:
87, m.p.: 250–252 °C (reported m.p. 243–245 °C) [19].
4.1.4. Synthesis of 5-arylidene-2-((6-methylbenzo[d]thiazol-2-yl)amino)
thiazol-4(5H)-ones (23–29)
To a solution of compound 15 (0.24 g, 0.001 mol) in 10% ethanolic
sodium hydroxide (1 g sodium hydroxide in 10 ml 96% ethanol), the
appropriate aldehyde (16–22) (0.001 mol) was added and the reaction
mixture was stirred at room temperature for about 10 min. The pre-
cipitate formed was filtered, washed with water and recrystallized from
the appropriate solvent (ethanol or chloroform).
4.1. Chemistry
4.1.1. Synthesis of N-(benzo[d]thiazol-2-yl)-2-chloroacetamides (4 and 5)
To a solution of the appropriate aminobenzothiazole (1 or 2,
0.01 mol) and triethylamine (5.6 ml, 0.04 mol) in chloroform (50 ml),
chloroacetyl chloride (3, 3.18 ml, 0.04 mol) was added slowly. The
reaction mixture was stirred at room temperature for 2 h. The pre-
cipitate formed was filtered, washed with water, and recrystallized
from ethanol to afford the chloroacetamide derivatives 4 and 5.
4: Yellowish white crystals, yield %: 88, m.p.: 200 °C (char.) (re-
ported m.p. 140–143 °C) [15].
23: Reddish brown crystals, yield %: 92, m.p.: 283–285 °C. 1H NMR
(DMSO‑d6): δ 2.36 (s, 3H, CH3), 3.76 (s, 3H, OCH3), 3.81 (s, 3H, OCH3),
6.93 (d, 1H, J = 7.5 Hz, Ar-H), 7.01 (d, 1H, J = 8.5 Hz , Ar-H),
7.07–7.10 (m, 2H, Ar-H), 7.47 (d, 1H, J = 8.0 Hz, Ar-H), 7.56 (s, 1H,
Ar-H), 7.70 (s, 1H, =CH). 13C NMR (DMSO‑d6): δ 21.0, 55.4, 56.0,
112.4, 112.6, 112.8, 114.7, 118.8, 119.8, 120.8, 124.7, 126.3, 131.4,
133.1, 134.6, 148.5, 151.9, 153.1, 180.3. MS (m/z, %): (M+ 411.31,
6.60), (M++1 412.33, 3.01), 394.24 (100.00). Analysis calculated for
5: Greenish brown crystals, yield %: 91, m.p: 180–182 °C (reported
m.p. 174–176 °C) [16].
C
20H17N3O3S2: C, 58.38; H, 4.16; N, 10.21.
24: Reddish brown crystals, yield %: 94, m.p.: 338–340 °C. 1H NMR
4.1.2. Synthesis of N-(benzo[d]thiazol-2-yl)-2-((4-substituted)piperazin-1-
yl)acetamides (9–14)
(DMSO‑d6): δ 2.37 (s, 3H, CH3), 3.37 (s, 1H, NH), 7.10 (dd, 1H,
J = 1.0 Hz, J = 9.0 Hz, Ar-H), 7.40 (s, 1H, =CH), 7.49 (d, 1H,
J = 8.5 Hz, Ar-H), 7.55–7.57 (m, 2H, Ar-H), 7.74 (d, 1H, J = 8.0 Hz,
Ar-H), 7.81 (d, 1H, J = 2.5 Hz, Ar-H). 13C NMR: δ 21.0, 69.7, 119.9,
120.8, 121.9, 126.3, 128.4, 130.2, 130.8, 131.1, 131.6, 135.5, 136.4.
MS (m/z, %): (M+ 420.40, 13.65), (M++1 421.64, 10.81), (M++2
422.86, 5.19), 57.19 (100.00). Analysis calculated for C18H11Cl2N3OS2:
C, 51.44; H, 2.64; N, 10.00.
A mixture of the chloroacetamide derivative (4 or 5, 0.002 mol),
potassium carbonate (2.21 g, 0.016 mol), and the appropriate piper-
azine (6–8, 0.002 mol) in DMF (25 ml) was stirred at 50 °C for 6 h. The
precipitate formed was filtered, washed with water, and recrystallized
from ethanol to obtain the piperazine derivatives 9–14.
9: White crystals, yield %: 38, m.p.: 280–282 °C (reported m.p.
257 °C) [17].
25: Reddish brown crystals, yield %: 93, m.p.: 298–300 °C. 1H NMR
(DMSO‑d6): δ 2.36 (s, 3H, CH3), 3.40 (s, 1H, NH), 7.09 (d, 1H,
J = 8.0 Hz, Ar-H), 7.18 (t, 1H, J = 4.0 Hz, Ar-H), 7.42 (d, 1H,
J = 3.0 Hz, Ar-H), 7.48 (d, 1H, J = 8.0 Hz, Ar-H), 7.56 (s, 1H, Ar-H),
7.62 (s, 1H, =CH), 7.75 (d, 1H, J = 5.5 Hz, Ar-H). MS (m/z, %): (M+
357.19, 74.63), (M++1 358.11, 18.57), 311.21 (100.00). Analysis
calculated for C16H11N3OS3: C, 53.76; H, 3.10; N, 11.76.
10: White crystals, yield %: 48, m.p.: 180–182 °C (reported m.p.
184–186 °C) [18].
11: White crystals, yield %: 46, m.p.: 140–142 °C (reported m.p.
118–119 °C) [18].
12: White crystals, yield %: 47, m.p.: 208–210 °C. 1H NMR (CDCl3):
δ 2.33 (s, 3H, piperazine-CH3), 2.47 (s, 3H, benzothiazole-CH3), 2.53
(br s, 4H, piperazine-H), 2.68 (br s, 4H, piperazine-H), 3.28 (s, 2H,
COCH2), 7.25 (d, 1H, J = 7.5 Hz, Ar-H), 7.61 (s, 1H, Ar-H), 7.68 (d, 1H,
J = 8.0 Hz, Ar-H), 10.38 (s, 1H, NH). 13C NMR (CDCl3): δ 21.4, 45.9,
53.6, 54.9, 61.0, 120.5, 121.2, 127.7, 132.3, 134.0, 146.4, 156.3,
169.0. MS (m/z, %): (M+ 304.28, 14.69), (M++1 305.30, 3.13), 70.08
(100.00). Analysis calculated for C15H20N4OS: C, 59.18; H, 6.62; N,
18.41.
26: Reddish brown crystals, yield %: 94, m.p.: 335–337 °C. 1H NMR
(DMSO‑d6): δ 2.36 (s, 3H, CH3), 3.37 (s, 1H, NH), 7.09 (d, 1H,
J = 8.5 Hz, Ar-H), 7.27 (d, 1H, J = 4.0 Hz, Ar-H), 7.31 (d, 1H,
J = 4.0 Hz, Ar-H), 7.49 (d, 1H, J = 8.0 Hz, Ar-H), 7.55 (s, 1H, =CH),
7.57 (s, 1H, Ar-H). 13C NMR: δ 21.0, 114.3, 117.0, 119.9, 120.8, 126.3,
130.8, 131.5, 131.7, 132.7, 142.5. MS (m/z, %): (M+ 436.55, 12.22),
(M++1 437.59, 4.30), 90.93 (100.00). Analysis calculated for
13: White crystals, yield %: 79, m.p.: 198–200 °C. 1H NMR (CDCl3):
δ 2.43 (s, 3H, CH3), 2.80 (s, 4H, piperazine-H), 3.26 (s, 4H, piperazine-
H), 3.35 (s, 2H, COCH2), 6.86–6.92 (m, 4H, Ar-H), 7.21–7.27 (m, 2H,
Ar-H), 7.57 (s, 1H, Ar-H), 7.63 (d, 1H, J = 8.5 Hz, Ar-H), 9.95 (s, 1H,
NH). 13C NMR (CDCl3): δ 21.4, 49.1, 53.5, 61.0, 116.4, 120.3, 120.5,
121.1, 127.7, 129.1, 132.2, 134.0, 146.2, 150.7, 156.2, 168.6. MS (m/z,
%): (M+ 366.30, 100.00), (M++1 367.34, 24.31). Analysis calculated
for C20H22N4OS: C, 65.55; H, 6.05; N, 15.29.
C
16H10BrN3OS3: C, 44.04; H, 2.31; N, 9.63.
27: Reddish brown crystals, yield %: 93, m.p.: 308–310 °C. 1H NMR
(DMSO‑d6): δ 2.36 (s, 3H, CH3), 3.37 (s, 1H, NH), 6.64 (dd, 1H,
J = 2.0 Hz, J = 5.5 Hz, Ar-H), 6.74 (t, 1H, J = 3.5 Hz, Ar-H), 7.08 (dd,
1H, J = 1.5 Hz, J = 9.5 Hz, Ar-H), 7.20 (s, 1H, =CH), 7.46 (d, 1H,
J = 8.5 Hz, Ar-H), 7.55 (s, 1H, Ar-H), 7.78 (d, 1H, J = 1.0 Hz, Ar-
H).13C NMR: δ 21.0, 111.9, 112.8, 113.2, 119.7, 120.7, 126.2, 131.1,
131.3, 144.7, 148.5, 151.2 . MS (m/z, %): (M+ 341.20, 74.85), (M++1
342.16, 38.31), 44.14 (100.00). Analysis calculated for C16H11N3O2S2:
C, 56.29; H, 3.25; N, 12.31.
14: White crystals, yield %: 63, m.p.: 234–236 °C. 1H NMR (CDCl3):
δ 2.47 (s, 3H, CH3), 2.56 (br s, 4H, piperazine-H), 2.66 (s, 4H, piper-
azine-H), 3.27 (s, 2H, COCH2), 3.55 (s, 2H, CH2-Ph), 7.25–7.28 (m, 2H,
Ar-H), 7.32–7.33 (m, 4H, Ar-H), 7.61 (s, 1H, Ar-H), 7.68 (d, 1H,
J = 8.0 Hz, Ar-H), 10.41 (s, 1H, NH). 13C NMR (CDCl3): δ 21.4, 43.5,
49.4, 60.8, 62.6, 120.5, 121.2, 127.7, 128.4, 128.5, 129.0, 146.2,
156.3, 168.8. MS (m/z, %): (M+ 380.32, 100.00), (M++1 381.37,
26.22). Analysis calculated for C21H24N4OS: C, 66.29; H, 6.36; N,
14.72.
28: Reddish brown crystals, yield %: 94, m.p.: 328–330 °C. 1H NMR
(DMSO‑d6): δ 2.35 (s, 3H, CH3), 3.40 (s, 1H, NH), 7.05 (d, 1H,
J = 7.5 Hz, Ar-H), 7.40 (d, 1H, J = 6.0 Hz, Ar-H), 7.54–7.58 (m, 5H,
Ar-H), 8.01 (d, 2H, J = 10.0 Hz, Ar-H), 8.11–8.15 (m, 3H, Ar-H), 8.64
(s, 1H, =CH). 13C NMR: δ 21.0, 119.8, 120.7, 121.9, 125.5, 125.6,
126.2, 127.2, 127.9, 128.8, 130.9, 131.0, 131.4, 141.9. MS (m/z, %):
(M+ 451.47, 12.02), 45.22 (100.00). Analysis calculated for
C26H17N3OS2: C, 69.16; H, 3.79; N, 9.31.
4.1.3. Synthesis
4(5H)-one (15)
of
2-((6-methyl-benzo[d]thiazol-2-yl)amino)thiazol-
29: Reddish brown crystals, yield %: 92, m.p.: 338–340 °C. 1H NMR
(DMSO‑d6): δ 2.37 (s, 3H, CH3), 3.37 (s, 1H, NH), 7.10 (dd, 1H,
J = 1.5 Hz, J = 9.5 Hz, Ar-H), 7.36–7.39 (m, 2H, Ar-H), 7.46–7.49 (m,
4H, Ar-H), 7.57 (s, 1H, =CH), 7.67 (d, 2H, J = 8.0 Hz, Ar-H),
A mixture of compound 5 (1.20 g, 0.005 mol) and ammonium
thiocyanate (1.52 g, 0.02 mol) in ethanol (50 ml) was heated under
9

A.M. Mokhtar, et al.
BioorganicChemistry104(2020)104259
7.71–7.74 (m, 3H, Ar-H). 13C NMR: δ 21.0, 119.8, 120.7, 124.1, 126.2,
126.6, 126.8, 126.9, 127.1, 127.7, 129.0, 129.7, 131.4, 133.0, 134.5,
139.3, 139.7, 157.8. MS (m/z, %): (M+ 427.67, 8.82), 43.31 (100.00).
Analysis calculated for C24H17N3OS2: C, 67.42; H, 4.01; N, 9.83.
40: Reddish brown crystals, yield %: 89, m.p.: 343–345 °C. 1H NMR
(DMSO‑d6): δ 7.61–7.66 (m, 2H, Ar-H), 7.72–7.84 (m, 8H, Ar-H), 7.97
(d, 1H, J = 9.0 Hz, Ar-H), 8.20 (d, 1H, J = 8.0 Hz, Ar-H), 8.26 (d, 1H,
J = 7.5 Hz, Ar-H), 8.72 (s, 1H, N = CH), 12.58 (s, 1H, NH).13C- NMR:
122.8, 123.9, 125.5, 126.2, 126.5, 125.6, 126.9, 126.9, 127.1, 127.1,
127.7, 127.8, 128.7, 128.8, 139.1, 150.0, 157.7. MS (m/z, %): (M+
357.19, 14.32), (M++1 358.26, 5.77), 360.32 (100.00). Analysis cal-
culated for C21H15N3OS: C, 70.57; H, 4.23; N, 11.76.
4.1.5. Synthesis of ethyl benzothiazole-2-carboxylate (32)
A mixture of o-aminothiophenol (30, 1.06 ml, 0.01 mol) and diethyl
oxalate (31, 4.38 ml, 0.03 mol) was heated at 110 °C for 4 h. The
precipitate formed was filtered after cooling, then washed and re-
crystallized from petroleum ether. Greenish yellow needles, yield %:
84, m.p.: 68–70 0C as reported [20].
41: Greenish white crystals, yield %: 90.3, m.p.: 333–335 °C. 1H
NMR (DMSO‑d6): δ 7.29–7.38 (m, 4H, Ar-H), 7.77–7.80 (m, 2H, Ar-H),
7.93–7.97 (m, 2H, Ar-H), 8.72 (s, 1H, N = CH), 12.39 (s, 1H, NH). 13
C
NMR: 115.9, 116.1, 129.7, 130.4, 130.7, 143.8, 150.0, 156.2, 160.5,
162.4, 162.9, 164.4, 164.9. MS (m/z, %): (M+ 299.32, 0.80), 165.10
(100.00). Analysis calculated for C15H10FN3OS: C, 60.19; H, 3.37; N,
14.04.
4.1.6. Synthesis of benzothiazole-2-carbohydrazide (33)
A mixture of compound 32 (1.9 gm, 0.01 mol) and hydrazine hy-
drate (5 ml, 0.1 mol) was stirred at room temperature for 10 min. The
carbohydrazide formed was washed with water and recrystallized from
ethanol [20]. Yellowish crystals, yield %: 91, m.p.: 200–202 °C (re-
ported m.p. 172–174 °C) [20].
4.1.8. Synthesis
of
N'-(4-substituted
benzoyl)benzo[d]thiazole-2-
carbohydrazides (44 and 45)
To a solution of benzothiazole-2-carbohydrazide (33, 0.193 g,
0.001 mol) in glacial acetic acid (10 ml), the appropriate benzoyl
chloride (42 or 43, 0.001 mol) was added dropwise and the reaction
mixture was stirred at room temperature for about 10 min. The pre-
cipitate formed was filtered, washed with water and recrystallized from
ethanol.
4.1.7. Synthesis of (E)-N'-(substituted arylidene)benzo[d]thiazole-2-
carbohydrazides (35–41)
To a solution of compound 33 (0.193 g, 0.001 mol) in glacial acetic
acid (10 ml), the appropriate aldehyde (16, 18–22, 34) (0.001 mol)
was added and the reaction mixture was heated under reflux for 1 h.
The precipitate formed was filtered on hot, washed with water and
recrystallized from the appropriate solvent (ethanol or chloroform).
35: Greenish white crystals, yield %: 91, m.p.: 283–285 °C. 1H NMR
(DMSO‑d6): δ 3.75 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 7.02–7.07 (m,
2H, Ar-H), 7.37 (d, 1H, J = 2.5 Hz, Ar-H), 7.59–7.67 (m, 2H, Ar-H),
8.18 (d, 1H, J = 8.0 Hz, Ar-H), 8.26 (d, 1H, J = 8.0 Hz, Ar-H), 8.99 (s,
1H, N = CH). 13C NMR: δ 55.4, 56.4, 109.3, 113.6, 118.1, 122.7, 123.0,
124.1, 127.1, 127.3, 136.0, 146.1, 152.6, 152.7, 153.2, 156.1, 163.7.
MS (m/z, %): (M+ 341.22, 100.00), (M++1 342.28, 30.06). Analysis
calculated for C17H15N3O3S: C, 59.81; H, 4.43; N, 12.31.
44: White crystals, yield %: 87, m.p.: 203–205 °C (reported m.p.
191 °C) [21]. 1H NMR (DMSO‑d6): δ 7.46–7.58 (m, 6H, Ar-H), 7.99 (d,
1H, J = 7.5 Hz, Ar-H), 8.17 (d, 1H, J = 8.5 Hz, Ar-H), 8.74 (s, 1H, NH),
9.95 (s, 1H, NH).
45: White crystals, yield %: 92, m.p.: 290–292 °C. 1H NMR
(DMSO‑d6): δ 7.34–7.39 (m, 2H, Ar-H), 7.60–7.68 (m, 2H, Ar-H),
7.93–8.00 (m, 2H, Ar-H), 8.19 (d, 1H, J = 7.5 Hz, Ar-H), 8.27 (d, 1H,
J = 7.5 Hz, Ar-H), 10.56 (s, 1H, NH), 10.72 (s, 1H, NH). 13C NMR: δ
115.6, 123.1, 124.2, 127.3, 128.7, 130.2, 135.9, 152.7, 158.6, 159.1,
162.6, 163.3, 164.1. MS (m/z, %): (M+ 315.46, 28.99), 155.42
(100.00). Analysis calculated for C15H10FN3O2S: C, 57.14; H, 3.20; N,
13.33.
36: Brownish white crystals, yield % 89, m.p.: 198–200 °C. 1H NMR
(DMSO‑d6): δ 7.15 (d, 1H, J = 4.0 Hz, Ar-H), 7.46–7.70 (m, 4H, Ar-H),
8.18 (d, 1H, J = 8.5 Hz, Ar-H), 8.25 (d, 1H, J = 7.5 Hz, Ar-H), 8.85 (s,
1H, N = CH), 12.54 (s, 1H, NH). MS (m/z, %): (M+ 287.10, 14.71),
(M++1 288.14, 2.29), 135.09 (100.00). Analysis calculated for
4.1.9. Synthesis
of
2-(benzo[d]thiazole-2-carbonyl)-N-(p-substituted
phenyl)hydrazine-1-carbothioamides (50–53)
C
13H9N3OS2: C, 54.34; H, 3.16; N, 14.62.
To a solution of benzothiazole-2-carbohydrazide (33, 0.193 g,
0.001 mol) in glacial acetic acid (10 ml), the appropriate phenyl iso-
thiocyanate (46–49, 0.001 mol) was added dropwise and the reaction
mixture was stirred at room temperature for 1–3 h. The precipitate
formed was filtered, washed with water and recrystallized from
ethanol.
37: Brownish white crystals, yield %: 93, m.p.: 248–250 °C. 1H NMR
(DMSO‑d6): δ 7.28 (d, 1H, J = 3.5 Hz, Ar-H), 7.32 (d, 1H, J = 4.0 Hz,
Ar-H), 7.60–7.67 (m, 2H, Ar-H), 8.18 (d, 1H, J = 7.5 Hz, Ar-H), 8.26 (d,
1H, J = 7.5 Hz, Ar-H), 8.67 (s, 1H, N = CH), 12.40 (s, 1H, NH). 13C
NMR: 115.7, 123.1, 124.1, 127.2, 131.5, 132.5, 134.7, 136.0, 140.4,
145.0, 152.6, 155.9, 163.5. MS (m/z, %): (M+ 366.00, 22.85), 213.09
(100.00). Analysis calculated for C13H8BrN3OS2: C, 42.63; H, 2.20; N,
11.47.
50: White crystals, yield %: 84, m.p.: 170–171 °C. 1H NMR
(DMSO‑d6): δ 2.26 (s, 3H, CH3), 7.11 (d, 2H, J = 8.0 Hz, Ar-H), 7.31 (d,
2H, J = 8.0 Hz, Ar-H), 7.58–7.66 (m, 2H, Ar-H), 8.16 (d, 1H,
J = 8.5 Hz, Ar-H), 8.25 (d, 1H, J = 7.5 Hz, Ar-H), 9.77 (s, 1H, NH),
9.82 (s, 1H, NH), 11.16 (s, 1H, NH). 13C NMR: δ 20.6, 116.8, 123.0,
124.1, 125.8, 127.1, 127.2, 128.5, 130.3, 135.9, 136.8, 137.9, 152.7,
163.1. MS (m/z, %): (M+ 342.27, 40.83), (M++1 343.27, 26.14),
206.44 (100.00). Analysis calculated for C16H12N4S2: C, 59.24; H, 3.73;
N, 17.27.
38: Brownish white crystals, yield %: 91, m.p.: 178–180 °C. 1H NMR
(DMSO‑d6): δ 6.66 (dd, 1H, J = 2.0 Hz, J = 5.5 Hz, Ar-H), 6.98 (d, 1H,
J = 3.5 Hz, Ar-H), 7.59–7.67 (m, 2H, Ar-H), 7.88 (d, 1H, J = 1.0 Hz,
Ar-H), 8.18 (d, 1H, J = 7.5 Hz, Ar-H), 8.26 (d, 1H, J = 7.0 Hz, Ar-H),
8.54 (s, 1H, N = CH), 12.69 (s, 1H, NH). 13C NMR: δ 112.4, 114.5,
123.1, 124.0, 127.1, 127.3, 136.0, 139.8, 145.7, 149.2, 152.7, 156.0,
163.7. MS (m/z, %): (M+ 271.20, 8.26), (M++1 272.20, 1.87), 52.19
(100.00). Analysis calculated for C13H9N3O2S: C, 57.55; H, 3.34; N,
15.49.
51: White crystals, yield %: 90, m.p.: 190–192 °C (reported m.p.
196 °C) [22]. 1H NMR (DMSO‑d6): δ 7.36–7.39 (m, 4H, Ar-H),
7.59–7.66 (m, 2H, Ar-H), 8.16 (d, 1H, J = 8.5 Hz, Ar-H), 8.26 (d, 1H,
J = 7.5 Hz, Ar-H), 9.92 (s, 1H, NH), 9.99 (s, 1H, NH), 11.21 (s, 1H,
NH), 10.86 (s, 1H, NH).
39: Reddish brown crystals, yield %: 91, m.p.: 228–230 °C. 1H NMR
(DMSO‑d6): δ 7.58–7.70 (m, 7H, Ar-H), 8.17 (d, 2H, J = 8.0 Hz, Ar-H),
8.25 (d, 1H, J = 7.5 Hz, Ar-H), 8.30 (d, 1H, J = 7.5 Hz, Ar-H), 8.77 (s,
52: White crystals, yield %: 86, m.p.: 170–172 °C (reported m.p.
168 °C) [22]. 1H NMR (DMSO‑d6): δ 3.73 (s, 3H, OCH3), 6.87–6.90 (m,
2H, Ar-H), 7.27 (d, 2H, J = 7.5 Hz, Ar-H), 7.58–7.65 (m, 2H, Ar-H),
8.15 (d, 1H, J = 7.5 Hz, Ar-H), 8.24 (d, 1H, J = 7.5 Hz, Ar-H), 9.41 (br
s, 1H, NH), 9.72 (s, 1H, NH), 10.81 (s, 1H, NH).
1H, N = CH), 8.82 (d, 2H, J = 9.0 Hz, Ar-H), 12.93 (s, 1H, NH). 13
C
NMR: δ 123.2, 124.1, 124.6, 124.9, 125.6, 127.2, 127.4, 129.1, 129.1,
129.8, 130.2, 130.9, 150.1, 152.8, 156.1, 157.8, 163.7. MS (m/z, %):
(M+ 381.25, 100.00), (M++1 382.29, 27.02). Analysis calculated for
C
21H15N3OS: C, 70.57; H, 4.23; N, 11.76.
53: White crystals, yield %: 88, m.p.: 178–180 °C. 1H NMR
10

A.M. Mokhtar, et al.
BioorganicChemistry104(2020)104259
(DMSO‑d6): δ 6.95–6.99 (m, 4H, Ar-H), 7.10–7.14 (m, 1H, Ar-H),
7.36–7.39 (m, 4H, Ar-H), 7.58–7.66 (m, 2H, Ar-H), 8.16 (d, 1H,
J = 8.0 Hz, Ar-H), 8.25 (d, 1H, J = 7.5 Hz, Ar-H), 9.84 (s, 1H, NH),
9.89 (s, 1H, NH), 11.19 (s, 1H, NH). 13C NMR: δ 118.1, 118.4, 118.7,
123.0, 123.3, 124.1, 127.1, 127.3, 130.0, 134.4, 134.6, 135.9, 152.7,
153.7, 156.8, 162.9, 180.6. MS (m/z, %): (M+ 420.65, 31.21), 114.17
(100.00). Analysis calculated for C21H16N4O2S2: C, 59.98; H, 3.84; N,
13.32.
4.2. Biological evaluation
4.2.1. Cytotoxicity assay
Cell lines were cultured in RPMI-1640 medium with 10% fetal bo-
vine serum. Antibiotics added were 100 units/ml penicillin and 100 µg/
ml streptomycin at 37 °C in a 5% CO2 incubator. The cell lines were
seeded in a 96-well plate at a density of 1.0 X 104 cells/well at 37 °C for
48 h under 5% CO2. After incubation, the cells were treated with dif-
ferent concentrations of compounds and incubated for 24 h. After 24 h
of drug treatment, 20 µl of MTT solution at 5 mg/ml were added and
incubated for 4 h. Dimethyl sulfoxide (DMSO) in volume of 100 µl was
added into each well to dissolve the purple formazan formed. The
colorimetric assay was measured and recorded at absorbance of 570 nm
using a plate reader (EXL 800, USA). The relative cell viability in per-
centage was calculated as (A570 of treated samples/A570 of untreated
sample) X 100 [10,11].
4.1.10. Synthesis of 5-(benzo[d]thiazol-2-yl)-4-(p-substituted phenyl)-4H-
1,2,4-triazole-3-thiols (54–57)
A mixture of the appropriate thiourea derivative (50–53, 0.001 mol)
in 40 ml 2 N sodium hydroxide was heated under reflux for 4–24 h, then
the reaction mixture was poured into acidic water. The separated pro-
duct was filtered, dried and recrystallized from ethanol.
54: White crystals, yield %: 63, m.p.: 188–190 °C. 1H NMR (CDCl3):
δ 2.49 (s, 3H, CH3), 7.31 (d, 2H, J = 8.0 Hz, Ar-H), 7.37 (d, 2H,
J = 8.5 Hz, Ar-H), 7.42–7.48 (m, 2H, Ar-H), 7.86 (d, 1H, J = 8.0 Hz,
Ar-H), 7.91 (d, 1H, J = 8.0 Hz, Ar-H), 11.72 (s, 1H, SH). 13C NMR: δ
21.4, 121.5, 124.5, 126.7, 126.9, 128.2, 130.2, 130.9, 134.8, 140.5,
146.4, 151.8, 152.8. MS (m/z, %): (M+ 324.28, 100.00), (M++1
325.17, 45.34). Analysis calculated for C16H12N4S2: C, 59.24; H, 3.73;
N, 17.27.
4.2.2. EGFR inhibitory activity
Lapatinib as an EGFR inhibitor was used as a positive control, in
addition to DMSO was used as a negative control, and the test com-
pounds were pre-incubated with EGFR kinase for 10 to 30 min prior to
adding ATP/substrate in order to initiate kinase reaction in 96-well
plate. 5 µl of ATP and kinase substrate were then added to each well
and left to be incubated for 30 min. Fluorimetric assay method was used
to quantify the ADP produced as a result of kinase reaction. Intensity of
fluorescence decreased as the strength of the test compound inhibitory
activity increased [12,13].
55: White crystals, yield %: 31, m.p.: 273 °C (reported m.p. 268 °C)
[22]. 1H NMR (CDCl3): δ 7.41 (d, 2H, J = 9.0 Hz, Ar-H), 7.49–7.52 (m,
2H, Ar-H), 7.56–7.57 (m, 2H, Ar-H), 7.99 (d, 1H, J = 8.0 Hz, Ar-H),
8.18 (d, 1H, J = 8.0 Hz, Ar-H).
56: White crystals, yield %: 53, m.p.: 218–220 °C (reported m.p.
242 °C) [22]. 1H NMR (CDCl3): δ 3.91 (s, 3H, OCH3), 7.07 (d, 2H,
J = 9.0 Hz, Ar-H), 7.34 (d, 2H, J = 9.0 Hz, Ar-H), 7.42–7.49 (m, 2H,
Ar-H), 7.86 (d, 1H, J = 7.5 Hz, Ar-H), 7.92 (d, 1H, J = 7.5 Hz, Ar-H),
11.68 (s, 1H, SH).
4.3. Molecular modeling studies
Three-dimensional structures of selected synthesized active EFGR
inhibitors in their neutral forms were drawn using the MOE of Chemical
Computing Group Inc software. Conformational analysis study was
performed where the lowest energy conformer ‘global-minimum’ was
identified for each derivative. The following step was docking of the
best conformer into the EFGR enzyme-binding domain. The 1xkk pdb
crystal structure of EFGR enzyme was chosen for docking, it was co-
crystallized with lapatinib ligand, a potent EFGR inhibitor [14] which
was used as a reference in our docking study. For the preparation of
docking process, the co-crystallized ligand was removed and the en-
zyme was 3D protonated. Hydrogen atoms were added at their standard
geometry, the partial charges were conducted then energy optimization
was computed. Docking was run using triangle matcher as placement
method and affinity dG as a scoring function. 30 conformers of the li-
gand were retained with the highest and best score. To validate the
docking procedure and results, lapatinib was docked into the active site
ligand receptor interaction via docking which was demonstrated by 2D
and 3D ligand views. The active site has been defined as the collection
of residues with the bound inhibitor and comprised the union of all
ligands of the ensemble. All atoms located < 10.0 A° from any ligand
atom were considered. The investigated compounds were subjected to
surface mapping and alignment experiments [23–27] using ‘Molecular
Operating Environment’ software (MOE of Chemical Computing Group
Inc., on a Core i7 workstation).
57: White crystals, yield %: 41, m.p.:128–130 °C. 1H NMR (CDCl3):
δ 7.01–7.12 (m, 6H, Ar-H), 7.33–7.36 (m, 2H, Ar-H), 7.52–7.62 (m, 2H,
Ar-H), 7.74 (d, 1H, J = 8.5 Hz, Ar-H), 8.01 (d, 1H, J = 7.5 Hz, Ar-H),
8.13 (d, 1H, J = 8.5 Hz, Ar-H), 9.31 (s, 1H, SH). 13C NMR: δ 118.4,
118.6, 119.6, 120.0, 121.4, 121.9, 122.5, 123.2, 123.2, 124.2, 127.0,
129.7, 132.3, 134.9, 137.3, 150.8, 160.5. MS (m/z, %): (M+ 402.06,
34.93), 76.24 (100.00). Analysis calculated for C21H14N4OS2: C, 62.67;
H, 3.51; N, 13.92.
4.1.11. Synthesis of N'-(benzo[d]thiazole-2-carbonyl)-p-substituted ben-
zenesulfonohydrazides (60 and 61)
To a solution of benzothiazole-2-carbohydrazide (33, 0.193 g,
0.001 mol) in glacial acetic acid (10 ml), the appropriate benzene-
sulfonyl chloride (58 or 59, 0.001 mol) was added and the reaction
mixture was stirred at room temperature for 1–3 h. The precipitate
formed was filtered, washed with water and recrystallized from
ethanol.
60: White crystals, yield %: 84, m.p.: 283–285 °C. 1H NMR (CDCl3):
δ 7.50–7.57 (m, 2H, Ar-H), 7.62 (d, 2H, J = 7.0 Hz, Ar-H), 7.75 (s, 1H,
NH), 7.81 (d, 2H, J = 7.0 Hz, Ar-H), 7.97 (d, 1H, J = 8.5 Hz, Ar-H),
8.12 (d, 1H, J = 8.0 Hz, Ar-H), 9.23 (s, 1H, NH). 13C NMR: δ 122.3,
124.8, 127.4, 127.6, 129.5, 129.9, 132.4, 135.3, 136.8, 152.5, 158.3,
158.9. MS (m/z, %): (M+ 412.14, 25.86), 178.25 (100.00). Analysis
calculated for C14H10BrN3O3S2: C, 40.79; H, 2.44; N, 10.19.
61: White crystals, yield %: 78, m.p.: 200–203 °C. 1H NMR
(DMSO‑d6): δ 2.37 (s, 3H, CH3), 7.35 (d, 2H, J = 7.5 Hz, Ar-H),
7.57–7.65 (m, 2H, Ar-H), 7.71 (d, 2H, J = 8.5 Hz, Ar-H), 8.13 (d, 1H,
J = 8.0 Hz, Ar-H), 8.21 (d, 1H, J = 7.5 Hz, Ar-H), 10.18 (s, 1H, NH),
11.30 (s, 1H, NH). 13C NMR: δ 21.0, 123.0, 124.2, 127.2, 127.3, 127.6,
129.4, 135.8, 136.3, 143.3, 152.6, 159.0, 162.0. MS (m/z, %): (M+
347.97, 6.89), (M++1 348.88, 1.54), 91.08 (100.00). Analysis calcu-
lated for C15H13N3O3S2: C, 51.86; H, 3.77; N, 12.10.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104259.
11

A.M. Mokhtar, et al.
BioorganicChemistry104(2020)104259
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