Novel benzothiazole hybrids targeting EGFR: Design, synthesis, biological evaluation and molecular docking studies

Article abstract of DOI:10.1016/j.molstruc.2021.130595

Novel benzo[d] thiazole-based analogues were synthesized with the aim of screening their in vitro anticancer activity. All the new derivatives 4–21 were evaluated against human hepatocellular carcinoma (HepG-2) and breast cancer cells (MCF-7) using doxorubicin as a reference drug. All compounds exhibited excellent potency against MCF-7 (IC₅₀ values ranging from 0.71 ± 0.4 to 1.04 ± 0.7 μM) and variable promising potency against HepG-2 (IC₅₀ ranged from 2.53 ± 2.5 to 3.47 ± 3.4 μM) comparing with the standard (IC₅₀ = 1.03 ± 0.8 μM and 2.85 ± 1.9 μM, respectively) in addition to their safety towards the normal cell line. Compounds 5, 6, 7, 13 and 16 having the highest cytotoxic activity, were further evaluated for their EGFR inhibitory activity using Erlotinib as a reference drug. Molecular docking studies were performed for the promising compounds 5, 6 and 7 to interpret their detected enzymatic activities based upon their binding interactions with the receptor. Moreover, cell cycle analysis and detection of apoptosis induction illustrated that compounds 5 and 6 exhibited a significant pre G₁ and G₂/M cell cycle arrest, in comparison with the untreated MCF-7 cells. In addition, compounds 5 and 6 elevated the levels of the oncogenic parameters; Bax, p53 and caspase-3 with decreased level of Bcl-2. These previous encouraging results of biological evaluation of the newly synthesized benzothiazoles could recommend an excellent framework toward the detection of new potent antitumor leads.

Full text of DOI:10.1016/j.molstruc.2021.130595

Journal of Molecular Structure 1240 (2021) 130595
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Novel benzothiazole hybrids targeting EGFR: Design, synthesis,
biological evaluation and molecular docking studies
a,∗
b
c
d
Eman A. Abd El-Meguid , Gaber O. Moustafa , Hanem M. Awad , Eman R. Zaki ,
Eman S. Nossiere
a
Department of Chemistry of Natural and Microbial Products, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo
12622, Egypt
b
Peptide Chemistry Department, Chemical Industries Research Division, National Research Centre, Dokki, Cairo 12622, Egypt
Department of Tanning Materials and Leather Technology, National Research Centre, Dokki, Cairo 12622, Egypt
Department of Molecular Biology, National Research Centre, Dokki 12622, Cairo Egypt
c
d
e
Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo 11754, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel benzo[d] thiazole-based analogues were synthesized with the aim of screening their in vitro an-
ticancer activity. All the new derivatives 4–21 were evaluated against human hepatocellular carcinoma
Received 27 February 2021
Revised 7 April 2021
Accepted 28 April 2021
Available online 5 May 2021
(
HepG-2) and breast cancer cells (MCF-7) using doxorubicin as a reference drug. All compounds exhib-
ited excellent potency against MCF-7 (IC50 values ranging from 0.71 ± 0.4 to 1.04 ± 0.7 μM) and variable
promising potency against HepG-2 (IC50 ranged from 2.53 ± 2.5 to 3.47 ± 3.4 μM) comparing with the
standard (IC50 = 1.03 ± 0.8 μM and 2.85 ± 1.9 μM, respectively) in addition to their safety towards the
normal cell line. Compounds 5, 6, 7, 13 and 16 having the highest cytotoxic activity, were further evalu-
ated for their EGFR inhibitory activity using Erlotinib as a reference drug. Molecular docking studies were
performed for the promising compounds 5, 6 and 7 to interpret their detected enzymatic activities based
upon their binding interactions with the receptor. Moreover, cell cycle analysis and detection of apoptosis
induction illustrated that compounds 5 and 6 exhibited a significant pre G1 and G2/M cell cycle arrest,
in comparison with the untreated MCF-7 cells. In addition, compounds 5 and 6 elevated the levels of the
oncogenic parameters; Bax, p53 and caspase-3 with decreased level of Bcl-2. These previous encouraging
results of biological evaluation of the newly synthesized benzothiazoles could recommend an excellent
framework toward the detection of new potent antitumor leads.
Keywords:
Benzothiazole
Anticancer
EGFR
Cell cycle analysis
Bax
Bcl-2
P53
Caspase-3
Molecular docking
©
2021 Elsevier B.V. All rights reserved.
1
. Introduction
ulation of multiple bioactivities, like adhesion, regulation of the
cell cycle and motility of the cell [8]. Mutations or over-expression
of EGFR prompts cell proliferation, angiogenesis, anti-apoptosis and
metastasis leading to a variety of epidermal carcinomas, especially
breast, bladder and colon cancers [9–11]. Therefore, the design of
EGFR inhibitors is an attractive strategy for the development of
new therapeutic agents [12]. Erlotinib bearing 4-anilinoquinazoline
scaffold, is a common example of clinical EGFR inhibitors used in
treating breast and lung cancers [13–15].
Of various types of remediations applicable to treat cancer,
chemotherapy is the most common strategy [1] which includes the
use of chemotherapeutics to prevent tumor cells from growing or
kill them by various mechanisms [2]. The discovery of novel, safe
and effective chemotherapeutic entities for treatment of cancer is
considered a challenge due to several limitations like drug resis-
tance, poor bioavailability and solubility, and toxicity to normal
cells [3–6].
Benzothiazoles are versatile and unique scaffolds employed in
the design of numerous analogs of pharmacological benefits [16].
They revealed diverse pharmacological activities as anticancer [17],
analgesic, anti-inflammatory [18], anticonvulsant [19], antitubercu-
lar [20], antiviral [21], antimalarial [22], antidiabetics [23], antimi-
crobial [24] and antioxidant [25]. It was reported that several ben-
zothiazole derivatives (I-V) (Fig. 1) exhibited a unique profile of
cytotoxicity especially against MCF-7 cell lines [6,26–28]. Addition-
Kinases are overexpressed in many types of malignancies and
are considered as viable targets for development of anticancer
drugs [7]. The epidermal growth factor receptor tyrosine kinase
(EGFR-TK) has a vital role in signal transduction pathways and reg-
∗
Corresponding author
E-mail addresses: emannrc@yahoo.com, ea.mabrouk@nrc.sci.eg (E.A.A. El-
Meguid).
https://doi.org/10.1016/j.molstruc.2021.130595
0
022-2860/© 2021 Elsevier B.V. All rights reserved.

E.A.A. El-Meguid, G.O. Moustafa, H.M. Awad et al.
Journal of Molecular Structure 1240 (2021) 130595
Fig. 1. Some benzothiazole hybrid molecules as anticancer agents.
ally, the benzothiazole containing cores (VI–IX) were found to be
active as anticancer through inhibition of EGFR [27,29–31] (Fig. 2).
Guided by the afore-mentioned considerations, and upon con-
tinuance of our research plan in the area of the detection of
biologically active anticancer targets [32–39], the work depicted
herein reports the design of benzothiazole-based derivatives de-
signed via bioisosterism to the quinazoline ring system in Erlotinib
and molecular hybridization with other moieties linked to active
benzothiazoles (Figs. 1 and 2) with the proposed binding mode
within the EGFR receptor. The synthesized prototypes comprising
benzothiazole motif were subjected to in vitro antitumor screen-
ing against HepG-2 and MCF-7 cancer cell lines, in addition to
the assessment of the inhibitory activity of the most active an-
ticancer derivatives against the EGFR kinase. Molecular docking
studies were also done to describe and rationalize the binding
modes of the most potent compounds into the active site of EGFR
kinase. Moreover, the cell cycle analysis apoptosis, the levels of
Bax, Bcl2, p-53 and caspase-3 were evaluated in this study.
pounds 5-10 was established under the basis of their spectral data.
IR of compounds 5–10 showed absorption bands in the region at
3385-3400 cm 1 due to NH in addition to absorptions of C=O
−
−
1 1
amide groups in the region at 1640-1612 cm
. H-NMR revealed
signals in the region at δ 10.52-11.16 of NH in addition to the D O
2
exchangeable signals of amidic, in addition to signals in the region
at δ 3.45-3.96 of OCH3 group, as well as, multiples at 2.51 of 2CH2,
pyrrolidine-2, 5-dione. 13C-NMR revealed signals in the region at
δ 180.55-164.65 of CONH, in addition to signals in the region at
δ 54.25-61.75 of OCH3 group. The mass spectrum of compounds
5–10 showed a peak equal to its molecular weight at m/z = 497
+
+
+
+
(M , 1.09), 409 (M , 1.12), 411 (M , 0.22), 459 (M , 3.22), 775
+
+
(M , 3.03) and 437 (M , 1.12), respectively.
The novel conjugated amino acid derivatives 11–21, were syn-
thesized by a stirred compound 4 in ice bath in presence of tri-
ethylamine with different amino acids (Scheme 3). IR of Com-
pounds 11–21 showed absorption bands in the region at 3371-3400
cm 1 due to NH, as well as, absorptions of C=O acid in the re-
−
−
1
gion at 1689-1676 cm , in addition to absorptions of C=O amide
−
1 1
2. Results and discussion
groups in the region at 1632-1600 cm
. H-NMR revealed signals
in the region at δ 10.35-10.52 of NH in addition to the D O ex-
2
2
.1. Chemistry
changeable signals of amidic, signals in the region at δ 12.50-10.65
of COOH group. 13C-NMR revealed signals in the region at δ 167.51-
171.19 of (COOH) in addition to signals in the region at δ 154.02-
167.52 of CONH. The mass spectrum of compounds 11–21 showed
This study aims to synthesis of novel benzo[d]thiazole deriva-
tives (4-21), which may be expected to possess various anti-
cancer properties. Accordingly, a rational design, synthesis, purifi-
cation, and structural characterization of novel derivatives (4-21),
were optimally synthesized. The compounds 4-(benzo[d]thiazol-
+
+
a peak equal to its molecular weight at m/z = 458 (M , 0.09), 474
+
+
+
(M , 0.03), 543 (M , 0.11), 501 (M , 0.03), 515 (M , 0.08), 488
+
+
+
+
(M , 0.03), 502 (M , 0.52), 486 (M , 2.50), 504 (M , 3.05), 480
+
+
2
-yl)-2-methoxyphenol (1); ethyl 2-(4-(benzo[d]thiazol-2-yl)-2-
(M , 1.50) and 534 (M , 2.60), respectively, as well as, showed a
methoxyphenoxy) acetate (2) and 2-[4-(benzo[d]thiazol-2-yl)-2-
methoxyphenoxy]acetohydrazide (3) were composed in accor-
dance with the methods reported beforehand [40,41]. The
compound 2-(4-(benzo[d]thiazol-2-yl)-2-methoxyphenoxy)-N’-(2-
chloroacetyl)acetohydrazide (4) was synthesized by the addition
of chloroacetylchloride drop wisely to a well stirred solution of
base peak at m/z =257 (100).
2.2. Biological activity
2.2.1. Anticancer evaluation
The cytotoxic activity of all synthesized benzothiazole deriva-
tives 4–21 was evaluated by MTT assay method [42] using doxoru-
bicin as standard reference. The cytotoxic screening was done in-
vitro for all derivatives against HepG-2 and MCF-7 cancer cell lines.
By investigation of the obtained data (Table 1, Fig. 3), it was no-
ticed that all compounds displayed excellent potency against MCF-
7 with IC50 values ranging from 0.71 ± 0.4 to 1.04 ± 0.7 μM com-
parable with the reference drug (IC50 = 1.03 ± 0.8 μM). Also, all
the screened derivatives illustrated promising and variable strength
of potency (IC50 ranged from 2.53 ± 2.5 to 3.47 ± 3.4 μM) in
comparison with the standard (IC50 = 2.85 ± 1.9 μM). The de-
terioration of the normal cells by anticancer drugs is one of the
most important drawbacks in cancer treatment strategy. Thus, the
cytotoxic activity of the promising tested compounds (5 and 6)
2
-(4-(benzo[d]thiazol-2-yl)-2-methoxyphenoxy) acetohydrazide (3)
at room temperature, (Scheme 1). IR of compound 4 showed ab-
−1
sorption bands at 3396 cm
due to NH in addition to absorp-
tions of C=O amide at 1680 and 1620 cm−1, respectively. 1H-NMR
revealed two signals at δ 10.48, 10.42 of 2NH in addition to the
D O exchangeable signals of amidic, as well as, 13C-NMR revealed
2
two signals at δ166.60 and 165.50 of 2CONH. The mass spectrum
of compound 4 showed a peak equal to its molecular weight at
m/z = 406 (M+).
On
the
other
hand,
2-(4-(benzo[d]thiazol-2-yl)-2-
methoxyphenoxy)acetohydrazide (3) was refluxed with the
appropriate acid anhydride in glacial acetic acid, which produced
the novel derivatives (5–10), (Scheme 2). The structure of com-
2

E.A.A. El-Meguid, G.O. Moustafa, H.M. Awad et al.
Journal of Molecular Structure 1240 (2021) 130595
Fig. 2. Reported benzothiazoles and the design strategy for the new hybrids 4–21 targeting EGFR TK with their proposed hypothetic model.
against tested cancer cells was also examined against the human
normal fibroblast cells (WI-38) relative to Doxorubicin as a refer-
ence drug, and their IC50 values were determined by the MTT as-
say. The IC50 values of all tested compounds against WI-38 were
higher than their IC50 values against the tested cell lines (Table 1).
The most potent cytotoxic candidates 5 and 6 exhibited significant
cytotoxic activity against HepG-2 and MCF-7 cancer cell lines and
also showed safety profile against the normal cells.
garding to the gained data against HepG-2 cell line, the open
chain derivative 4 having chloroacetamide moiety revealed equipo-
tency with the reference (IC50 = 2.89 ± 3.1 μM). Replacement
with N-pivaloylpivalamide 5, pyrrole-2,5-dione 6 or pyrrolidine-
2,5-dione 7 displayed increase in the potency (IC50 = 2.53 ± 2.5,
2.56 ± 3.1 and 2.58 ± 2.3 μM, respectively). Ring fusion via re-
placement of pyrrole-2,5-dione with isoindoline-1,3-dione 8, af-
forded slight decrease in the activity but still equipotent with
the reference (IC50 = 2.86 ± 3.1 μM). Substitution with elec-
tron withdrawing groups (i.e., Br) at positions-4, 5, 6 and 7 of
isoindoline-1,3-dione moiety in compound 9, improved the anti-
Structure–activity relationship (SAR) study
It was clear that all derivatives are bearing 4-(benzo[d]thiazol-
2
-yl)-2-methoxyphenoxy)acetamide nucleus in their structures. Re-
3

E.A.A. El-Meguid, G.O. Moustafa, H.M. Awad et al.
Journal of Molecular Structure 1240 (2021) 130595
Scheme 1. Synthesis of parent benzothiazole derivatives 3 and 4.
Scheme 2. Synthesis of benzothiazole derivatives 5–10.
4

E.A.A. El-Meguid, G.O. Moustafa, H.M. Awad et al.
Journal of Molecular Structure 1240 (2021) 130595
Scheme 3. Synthesis of benzothiazole derivatives 11–21.
Fig. 3. IC50 (μM) data of benzothiazole derivatives 4–21 in comparison with doxorubicin against human HepG-2 and MCF-7 cancer cell lines according to the MTT assay.
cancer activity (IC50 = 2.66 ± 2.4 μM). Isosteric replacement of N-
pivaloylpivalamide with N-benzoylbenzamide 10, exhibited marked
decrease in the potency (IC50 = 3.03 ± 3.3 μM). As demonstrated
in Scheme 3, there was open chain elongation with different amino
acids that gave slight decrease in the potency in almost derivatives
in comparison with reference drug (IC50 ranged from 2.93 ± 3.1 to
2.2.2. In vitro inhibitory assay of EGFR
Cancer is usually associated with excessive activation of recep-
tor tyrosine kinase (RTK) signaling pathways, so blocking of these
receptors gives significant inhibitors with therapeutic potential in
treatment of cancer [7]. Regarding to the well-balanced cytotoxic
potency, the benzothiazole targets 5, 6, 7, 13 and 16 were selected
for further elucidation of their EGFR inhibitory activity using Er-
lotinib as a reference drug. As depicted in Table 2, compounds 5, 6
and 7 displayed the highest potential EGFR inhibitory activity with
IC50 values of 0.09 ± 0.42, 0.12 ± 0.22 and 0.16 ± 0.30 μM, respec-
tively but slightly less than that of Erlotinib (IC50 = 0.088 ± 0.25
3
.47 ± 3.4 μM). With exception of the derivatives bearing arginine
13, 2-amino-3-hydroxybutanoic acid 16 and L-homocysteine 19, an
excellent and a higher cytotoxic activity was obtained comparing
with the standard (IC50 = 2.57 ± 3.5, 2.59 ± 2.9 and 2.69 ± 3.6
μM, respectively).
5

E.A.A. El-Meguid, G.O. Moustafa, H.M. Awad et al.
Journal of Molecular Structure 1240 (2021) 130595
Fig. 4. Cell cycle analysis of the promising derivatives 5 and 6.
Table 1
The cytotoxic IC50 values of the newly synthesized compounds 4–21
against HepG-2, MCF-7 cancer cell lines and normal cell line WI-38 ac-
cording to the MTT assay.
IC50 (μM) ± SD
Compound
HepG-2
MCF-7
WI-38
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2.89 ± 3.1
2.53 ± 2.5
2.56 ± 3.1
2.58 ± 2.3
2.86 ± 3.1
2.66 ± 2.4
3.03 ± 3.3
2.98 ± 3.1
2.93 ± 3.1
2.57 ± 3.5
2.98 ± 3.1
3.18 ± 2.9
2.59 ± 2.9
3.43 ± 3.6
3.47 ± 3.4
2.69 ± 3.6
3.18 ± 3.1
3.29± 3.5
2.85 ± 1.9
0.78 ± 0.5
0.71 ± 0.4
0.72 ± 0.6
1.04 ± 0.7
0.96 ± 0.6
1.02 ± 0.8
0.96 ± 0.5
0.97 ± 1.1
0.93 ± 0.5
0.72 ± 0.9
0.92 ± 0.7
0.99 ± 1.1
0.77 ± 1.1
0.87 ± 0.5
0.88 ± 0.5
0.74 ± 0.5
0.91 ± 0.9
0.77 ± 0.7
1.03 ± 0.8
-
88.5 ± 1.4
72.5 ± 1.1
-
-
-
0
1
2
3
4
5
6
7
8
9
0
1
-
-
Fig. 5. Apoptotic activity of benzothiazole derivatives 5 and 6.
-
-
-
μM). However, the rest derivatives 13 and 16 revealed drastic de-
crease in the suppression effect (IC50 = 17.04 ± 0.11 and 23.80 ±
-
-
-
0
.26 μM, respectively) which might be attributed to the improper
-
fitting in the EGFR binding pocket.
-
-
-
2
.2.3. Flow cytometry analysis and apoptosis detection
Doxorubicin
78.9 ± 0.8
To investigate the molecular mechanism of the benzo[d]thiazole
IC50: Compound concentration required to inhibit the cell viability by
0%, SD = standard deviation mean; each value is the mean of three
values.
analogues against cancerous cells, the highly potent compounds
and 6 were chosen to evaluate their effect on the cell cycle
5
5
progression through flow cytometry. In current work, MCF-7 cells
were treated with 0.71 and 0.72 μM concentrations of the ana-
logues 5 and 6 for 24 h, respectively to study their consequence
on the normal profile of cell cycle and apoptosis. As illustrated
in Fig. 4, compounds 5 and 6 demonstrated cell accumulations
of 39.27 % and 34.28 % at pre G1 phase and 38.78 % and 24.3 %
Table 2
In vitro inhibitory activities of the newly synthesized
derivatives 5, 6, 7, 13 and 16 against EGFR enzyme.
IC50 (Mean ± SEM) (μM)
at G /M phase, respectively, regarding to the control values (un-
Compd.No.
EGFR
2
treated cells) 1.61 % and 4.99 % for pre G1 and G /M phases, re-
2
5
6
7
0.09± 0.42
0.12± 0.22
0.16± 0.30
17.04± 0.11
23.80± 0.26
0.088± 0.25
spectively. These obtained findings clearly revealed that derivatives
5 and 6 prompted a significant pre G1 and G2/M cell cycle arrest,
in comparison with the untreated MCF-7 cells.
1
1
3
6
Erlotinib
2
.2.4. Annexin V-FITC apoptosis study
IC50: Compound concentration required to inhibit EGFR en-
zyme activity by 50%, SEM = standard error mean; each
value is the mean of three values.
The MCF-7 cells treated with the compounds 5 and 6 were
double stained with Annexin V/PI (annexin V-FITC and propidium
iodide) and tested through flow cytometry technique comparing
6

E.A.A. El-Meguid, G.O. Moustafa, H.M. Awad et al.
Journal of Molecular Structure 1240 (2021) 130595
Fig. 6. Cell cycle analysis and the effect of the compounds 5 and 6 on the percentage of V-FITC-positive annexin staining in MCF-7 cells in comparison with the control.
Table 3
with the control cells to validate the ability of these derivatives
Apoptosis and necrosis results on MCF-7 cells induced by 5 and 6.
to cause apoptosis (Table 3, Figs. 5 and 6). It was noted that there
was an increase to 23.37 % and 20.66 % created by the investigated
compounds 5 and 6 from 0.12 % (DMSO control) in the late apop-
tosis and an increase to 6.39 % and 2.61 % in the early apopto-
sis from 0.47 % (DMSO control), respectively. Also, these analogues
produced necrosis percent of 9.51 and 11.01 versus 1.02 % caused
by DMSO control, respectively. Thus, the marked increase in the
apoptotic cells indicated the ability of the compounds 5 and 6 to
afford apoptosis induction.
Apoptosis %
Total %
Compd.
Necrosis %
Early %
Late %
5
6
/ MCF-7
/ MCF-7
39.27
34.28
1.61
6.39
2.61
0.47
23.37
20.66
0.12
9.51
11.01
1.02
Cont. / MCF-7
7

E.A.A. El-Meguid, G.O. Moustafa, H.M. Awad et al.
Journal of Molecular Structure 1240 (2021) 130595
Fig 7. A & B illustrating 2D & 3D Views of the native ligand, erlotinib re-docked in the active site of EGFR (PDB ID: 1M17) using MOE software. C illustrating 3D representation
of the superimposition of the co-crystallized (yellow) and the docking pose (red) of Erlotinib with an RMSD value of 0.85 A˚ .
2
.2.5. The impact of the derivatives 5 and 6 on Bax / Bcl-2 / p53 /
kinase complexed with 4-anilinoquinazoline inhibitor (Erlotinib,
4AQ) (PDB ID: IM17) [45] was downloaded from the protein data
bank. The docking calculations were done using MOE (Molecular
Operating Environment) software 10.2008 [46]. The original lig-
and was extracted from the X-ray co-crystallized structure and re-
docked in the ATP-active site of EGFR to validate the docking ap-
proach. This validation was confirmed through the small RMSD
caspase-3 levels
There are two different pathways that control the cell apop-
totic process: the intrinsic mitochondrial pathway and the extrinsic
death receptor pathway [43]. The anti-apoptotic Bcl-2 protein over-
expresses in the tumor cell boosting the cell survival via inhibition
of apoptosis, while the pro-apoptotic Bax performs a vital role in
cell apoptosis. So, the balance between these two proteins controls
the cell fate [44]. Moreover, the protein p53 is a tumor suppressor
acting as a transcription factor that is involved in the regulation
of the G2 to M transition preventing mitosis, thus leads to either
apoptosis or growth arrest. Also, it controls the expression of Bax
and Bcl-2 genes [45]. Caspase-3 is a member in the family of cys-
teine proteases and its induction catalyzes apoptosis process [45].
In this study, the MCF-7 cells were exposed to the derivatives 5
and 6 at their IC50 concentrations of 0.71 and 0.72 μM, respectively
for 24 h to evaluate the levels of Bax, Bcl-2, p53 and caspase-3.
The obtained results exhibited that the screened compounds 5 and
˚
value (root mean standard deviation, 0.83 A) between the experi-
mental and the docked poses of co-crystallized inhibitor, the score
energy of -12.66 kcal/mol, and the highly observed superimposi-
tion between them (Fig. 7c).
As previously described in the docking of Erlotinib [47], the
quinazoline scaffold interacted with the active site of EGFR via
hydrogen bonding between the N1 atom and the backbone of
Met769. Moreover, the methoxyethoxy group formed hydropho-
bic interaction within the hydrophobic region II (Gly772, Leu768
and Leu694 residues). While the ethynylphenyl moiety inserted in
the hydrophobic pocket I and exhibited hydrophobic interactions
with Asp831, Thr830, Leu820, Thr766, Lue764, Lys721 and Ala719.
(Fig. 7a, b)
6
boosted the levels of Bax by 6.02 and 4.90 folds, p53 by 6.03
and 5.04 folds, and caspase-3 by 9.45 and 7.41 folds, respectively
comparing with the untreated cells. Reversely, 5 and 6 reduced the
protein Bcl-2 level by 0.23 and 0.30 folds, respectively in compari-
son with the control cells (Table 4).
The docked benzo[d]thiazole targets 5, 6 and 7 displayed higher
negative energy scores of −13.55, 13.25 and -12.83 kcal/mol,
and respectively suggesting higher expected binding affinity than
the original co-crystalized ligand. It was observed that the
benzo[d]thiazole scaffold of all these analogues were fitted in the
active site of EGFR in a similar manner through two different
interactions: hydrogen bonding between nitrogen atom and the
sidechain of Lys721 and hydrophobic arene-arene interaction with
Phe699 (Figs. 8–10).
2
.3. Molecular docking study
The docking study of the promising benzo[d]thiazole analogues
, 6 and 7 was performed to rationalize the gained in vitro en-
5
zyme assessment data on EGFR enzyme. The domain of EGFR
8

E.A.A. El-Meguid, G.O. Moustafa, H.M. Awad et al.
Journal of Molecular Structure 1240 (2021) 130595
Fig. 8. A & B illustrating 2D & 3D Views of the compound 5 docked in the active site of EGFR (PDB ID: 1M17) using MOE software.
Fig. 9. A & B illustrating 2D & 3D Views of the compound 6 docked in the active site of EGFR (PDB ID: 1M17) using MOE software.
Fig. 10. A & B illustrating 2D & 3D Views of the compound 7 docked in the active site of EGFR (PDB ID: 1M17) using MOE software.
9

E.A.A. El-Meguid, G.O. Moustafa, H.M. Awad et al.
Journal of Molecular Structure 1240 (2021) 130595
Table 4
Effect of the compounds 5 and 6 on Bax, Bcl-2, p53 and caspase-3 levels.
Compd.
Bax
Bcl-2
P53
Casp-3
Conc. Pg/mL
FLD
Conc. ng/mL
FLD
Conc. Pg/mL
FLD
Conc. ng/mL
FLD
5
6
/ MCF-7
/ MCF-7
181.90± 9.60
148.10± 4.80
30.24± 1.90
6.02
4.90
1.00
1.95± 0.16
2.57± 0.11
8.43± 0.21
0.23
0.30
1.00
882.60± 20.80
737± 22
6.03
5.04
1.00
24.95± 1.14
19.56± 0.77
2.64± 0.10
9.45
7.41
1.00
Cont./MCF-7
146.30± 2.80
Fig. 11. 3D diagram of the superimposition of the original ligand Erlotinib (red), 5 (yellow), 6 (green) and 7 (blue) within the ATP-binding pocket of EGFR (PDB code: 1M17)
For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
(
By inspection of Fig. 8, the two oxygens of N-pivaloylpivalamide
moiety in compound formed two H-bond acceptors with
sidechain of the gatekeeper residue Lys766 and the backbone of
EGFR inhibitory activity using erlotinib as a standard drug. Molec-
5
ular docking studies were done to confirm the promising enzy-
matic results of the compounds 5, 6 and 7. Furthermore, mecha-
nistic studies revealed that compounds 5 and 6 arrested cell cycle
˚
the key residue Met769 (distance: 2.87 and 2.80 A, respectively).
On the other hand, the sidechain of Lys766 was bound to
at pre G1 and G /M phases and induced apoptosis through upreg-
2
the phenoxy oxygen in the compounds 6 and 7 (distance: 2.87
ulating p53, caspase-3 and Bax and downregulating Bcl-2 levels, in
comparison with the untreated MCF-7 cells.
˚
and 2.49 A, respectively). Moreover, the backbone of Met769 en-
forced fixation through hydrogen bond acceptor with the oxygen
of pyrrole-2,5-dione in 6 or pyrrolidine-2,5-dione in 7 (distance:
˚
4. Experimental
2
.55 and 2.53 A, respectively) (Figs. 9 and 10).
Furthermore, the superimposition diagram between the native
ligand Erlotinib and the new targets 5, 6 and 7 within ATP-binding
pocket of EGFR confirmed that all of them fitted in the same active
site through the same hydrophilic interactions with the key residue
Met769. The targets improved their binding via hydrophobic inter-
actions with Phe699 and hydrophilic interactions with Lys721 and
Lys766 (Fig. 11).
4.1. Chemistry
These companies: Sigma (Ronkonkoma, NY, USA), Fluka (Buchs,
Switzerland), and E. Merck (Hohenbrunn, Germany) were used to
import all chemical and related materials. All “melting points;
M.P.” uncorrected which gained from " Digital melting point appa-
ratus; model: IA9100”. The “Micro-Analytical center” in Cairo Uni-
versity of Egypt were used for measuring the following spectro-
scopic analysis; (i) Elemental Micro-analyses for C, N, and H, which
gained through a good restriction of the theoretical values (± 0.4%).
(ii) “Potassium bromide; KBr” disks were handled for prepara-
tion tested samples to implement into the “Fourier transforms in-
frared spectrophotometer; IR Shimadzu; Model: with 1S affinity”.
Finally, it could be assumed that the existence of the
benzo[d]thiazole scaffold bearing N-pivaloylpivalamide, pyrrole-
2
,5-dione or pyrrolidine-2,5-dione and separated with phenoxy
fragment allowed the chance for excellent binding profile and in-
hibitory activities against EGFR.
3. Conclusion
(
iii) Mass spectra measured by “Shimadzu gas chromatograph–
In summary, a Novel series of benzo[d]thiazole derivatives 4–
spectrometer, Model: QP2010ultra; Kyoto, Japan”. 1H- and 13C NMR
spectra were recorded at 400 (100) MHz using Jeol ECA 500 MHz
Spectrometer and performed at Micro Analytical Laboratory Center,
Faculty of Pharmacy, Cairo University, Cairo, Egypt. DMSO-d6 was
used as a solvent and chemical shifts were given in ppm relative
to TMS as internal standard.
21 bearing various heterocyclic scaffolds was designed and syn-
thesized. All newly synthesized targets were examined for their in
vitro cytotoxicity against HepG-2 and MCF-7 cancerous cell lines
in comparison with doxorubicin as a reference. The highest cyto-
toxic compounds 5, 6, 7, 13 and 16 were further screened for their
10

E.A.A. El-Meguid, G.O. Moustafa, H.M. Awad et al.
Journal of Molecular Structure 1240 (2021) 130595
H), 4.89, 4.80 (s, 2H, OCH ), 3.95-3.92 (s, 3H, OCH ). 13C-NMR (100
4.1.1. Synthesis of 4-(benzo[d]thiazol-2-yl)-2-methoxyphenol (1)
2
3
This compound was composed in accordance with the methods
MHz, δ, ppm, DMSO-d ): δ = 166.60 (CONH), 162.55, 160.00 (2C,
6
reported beforehand [40].
2 NCO, aromatic), 153.65-112.20 (15 C, aromatic), 66.25 (OCH ),
2
+
5
8.05 (OCH ). MS (EI, 70 eV): m/z (%) = 409 (M , 1.12), 258
3
4
.1.2. Synthesis of ethyl 2-(4-(benzo[d]thiazol-2-yl)-2-
(50.06), 257 (100), 256 (44.00), 109 (10.70), 69 (30.32), 57 (34.80),
55 (10.75), 51 (3.00). Molecular formula (M.wt.): C20H15N O S
methoxyphenoxy)acetate (2) and 2-[4-(benzo[d]thiazol-2-yl)-
3
5
2
-methoxyphenoxy]acetohydrazide (3)
(409.4). Calculated analysis: C, 58.67; H, 3.69; N, 10.26; found: C,
58.66; H, 3.69; N, 10.26.
These compounds were composed in accordance with the
methods reported beforehand [41].
4
.1.4.3. 2-(4-(benzo[d]thiazol-2-yl)-2-methoxyphenoxy)-N-(2,5-
4
.1.3. Synthesis of 2-(4-(benzo[d]thiazol-2-yl)-2-methoxyphenoxy)-
N’-(2-chloroacetyl) acetohydrazide (4)
To well stirred solution of 2-(4-(benzo[d]thiazol-2-yl)-2-
dioxopyrrolidin-1-yl)acetamide (7). Yield: 90 %; melting point:
180–182 ˚C, IR (cm 1): (KBr): γ = 3395 (NH stretching), 3170,
−
a
3045 (CH, aromatic), 2935 (CH, aliphatic), 1630 (C=O amide).
1
methoxyphenoxy)acetohydrazide (3) (1mmol, 0.33 g) in dry
dimethylformamide (10 ml), chloroacetylchloride (1 mmol, 0.11
ml) was drop wisely added with continuous stirring, then stir at
room temperature for 6 h. The reaction mixture was subsequently
poured over ice with continues stirring. The precipitated solid was
filtered and dried to give the crude product which was further pu-
rified by crystallization from ethanol to give analytically pure prod-
uct as a white powder.
H-NMR (400 MHz, δ, ppm, DMSO-d ): δ = 10.80 (s, 1H, 1NH,
6
D O exchangeable, amide, hydrazide), 8.10-7.22 (s, 7H, aromatic
2
H), 4.80, 4.65 (s, 2H, OCH ), 3.80-3.75 (s, 3H, OCH ), 2.51 (m, 4H,
2
3
2CH , pyrrolidine-2, 5-dione). 13C-NMR (100 MHz, δ, ppm, DMSO-
2
d ): δ = 172.05, 170.00 (2C, 2 NCO), 165.00 (CONH), 153.50-112.02
6
(15 C, aromatic), 65.00 (OCH ), 58.05 (OCH ), 33.25 (2C, CH CH ).
2
3
2
2
+
MS (EI, 70 eV): m/z (%) = 411 (M , 0.22), 355 (11.00), 300 (19.70),
257 (100), 69 (40.10), 57 (22.33), 55 (7.00). Molecular formula
(M.wt.): C20H17 N O S (411.4). Calculated analysis: C, 58.38; H,
Yield: 80%; melting point: 207–209˚C, IR (cm 1): (KBr):
−
3
5
γ = 3396 (NH stretching), 3160, 3050 (CH, aromatic), 2920 (CH,
4.16; N, 10.21; found: C, 58.38; H, 4.16; N, 10.21.
1
aliphatic), 1680, 1620 (C=O amide I and II, respectively). H-NMR
(
400 MHz, δ, ppm, DMSO-d ): δ = 10.48, 10.42 (s, 2H, 2NH,
4.1.4.4. 2-(4-(benzo[d]thiazol-2-yl)-2-methoxyphenoxy)-N-(1,3-
dioxoisoindolin-2-yl) acetamide (8). Yield: 75 %; melting point:
100–102 ˚C, IR (cm 1): (KBr): γ = 3397 (NH stretching), 3152,
6
D O exchangeable, amide, hydrazide), 8.12-7.02 (s, 7H, aromatic
2
−
H), 4.80, 4.74 (s, 2H, OCH ), 3.93, 3.92 (s, 2H, CH Cl), 3.38 (s, 3H,
2
2
OCH ). 13C-NMR (100 MHz, δ, ppm, DMSO-d ): δ = 166.60, 165.50
3
6
3033 (CH, aromatic), 2902 (CH, aliphatic), 1628 (C=O amide).
1
(
2CONH), 154.00-110.00 (13 C, aromatic), 66.75 (OCH ), 60.90
H-NMR (400 MHz, δ, ppm, DMSO-d ): δ = 10.98 (s, 1H, 1NH,
2
6
+
(
OCH ), 50.25 (COCH Cl). MS (EI, 70 eV): m/z (%) = 406 (M , 0.13),
D O exchangeable, amide, hydrazide), 8.14-7.02 (s, 11H, aromatic
3
2
2
H), 4.96, 4.80 (s, 2H, OCH ), 3.96-3.92 (s, 3H, OCH ). 13C-NMR
4
05 374 (4.10), 270 (100), 258 (31.60), 256 (77.00), 185 (18.48),
2
3
5
7 (30.30), 51 (8.00). Molecular formula (M.wt.): C18 H16 ClN O S
(100 MHz, δ, ppm, DMSO-d ): δ = 165.15 (CONH), 164.55 (2C,
3
4
6
(
405.9). Calculated analysis: C, 53.27; H, 3.97; N, 10.35; found: C,
2 NCO, aromatic), 155.02-112.00 (19 C, aromatic), 65.20 (OCH ),
2
+
5
3.26; H, 3.97; N, 10.35.
56.00 (OCH ). MS (EI, 70 eV): m/z (%) = 460 (M +1, 1.01), 459
3
+
(
M , 3.22), 317 (19.70), 258 (100), 109 (31.31), 69 (50.10), 57
4
.1.4. Synthetic routes for compounds 5–10
(34.90). Molecular formula (M.wt.): C24H17 N O S (459.5). Calcu-
3 5
2
-(4-(Benzo[d]thiazol-2-yl)-2-methoxyphenoxy)acetohydrazide
3) (1mmol, 0.33 g) and the appropriate acid anhydride (10 mmol)
were refluxed in least amount of glacial acetic acid (5 ml) for
h. The reaction mixture was subsequently poured over ice
lated analysis: C, 62.74; H, 3.73; N, 9.15; found: C, 62.74; H, 3.73;
N, 9.15.
(
5
4.1.4.5. 2-(4-(benzo[d]thiazol-2-yl)-2-methoxyphenoxy)-N-(4,5,6,7-
tetrabromo-1,3-dioxoisoindolin-2-yl)acetamide (9). Yield: 75 %;
melting point: 166–168 ˚C, IR (cm 1): (KBr): γ = 3385 (NH
with continuous stirring. The precipitated solid was filtered and
dried to give the crude product which was further purified by
crystallization from methanol to give analytically pure products.
−
stretching), 3162, 3030 (CH, aromatic), 2922 (CH, aliphatic), 1622
1
(
C=O amide). H-NMR (400 MHz, δ, ppm, DMSO-d ): δ = 10.85
6
4
.1.4.1. N’-(2-(4-(benzo[d]thiazol-2-yl)-2-methoxyphenoxy)acetyl)-N-
(s, 1H, 1NH, D O exchangeable, amide, hydrazide), 8.20-7.10 (s, 7H,
2
pivaloyl pivalohydrazide (5). Yield: 65 %; melting point: 120–122
aromatic H), 4.90, 4.70 (s, 2H, OCH ), 3.90-3.88 (s, 3H, OCH ). 13C-
2
3
˚
C, IR (cm 1): (KBr): γ = 3397 (NH stretching), 3167, 3049 (CH,
−
NMR (100 MHz, δ, ppm, DMSO-d ): δ = 164.65 (CONH), 163.05,
6
1
aromatic), 2925 (CH, aliphatic), 1625 (C=O amide). H-NMR (400
MHz, δ, ppm, DMSO-d ): δ = 10.52 (s, 1H, 1NH, D O exchangeable,
162.70 (2C, 2 NCO, aromatic), 154.22-110.02 (19 C, aromatic), 63.25
+
(OCH ), 54.25 (OCH ). MS (EI, 70 eV): m/z (%) = 776 (M +1,
6
2
2
3
+
amide, hydrazide), 8.15-7.22 (s, 7H, aromatic H), 4.90, 4.85 (s, 2H,
OCH ), 3.45 (s, 3H, OCH ), 1.40-1.20 (s, 18H, CH ). 13C-NMR (100
0.11), 775 (M , 3.03), 630 (20.90), 510 (10.70), 458 (18.25), 305
(46.80), 258 (20.60), 257 (100), 110 (60.11), 69 (20.00), 57 (44.85),
55 (10.70), 51 (5.55). Molecular formula (M.wt.): C24H13Br N O S
2
3
3
MHz, δ, ppm, DMSO-d ): δ = 180.55, 180.00 (2C, 2 NCO, aliphatic),
6
4
3
5
1
66.15 (CONH), 154.05-111.00 (13 C, aromatic), 66.60 (OCH ), 61.75
(775.1). Calculated analysis: C, 37.19; H, 1.69; N, 5.42; found: C,
37.18; H, 1.68; N, 5.42.
2
(
OCH ), 40.25 (2C, 2COCH), 27.50 (6C, 6 CHCH ). MS (EI, 70 eV):
3
3
m/z (%) = 498 (M +1, 0.19), 497 (M+ +1, 1.09), 415 (10.39), 314
+
(
09.88), 270 (17.06), 258 (22.60), 257 (100), 185 (33.55), 69 (40.75),
4.1.4.6. N’-(2-(4-(benzo[d]thiazol-2-yl)-2-methoxyphenoxy)acetyl)-N-
benzoylbenzohydrazide (10). Yield: 70 %; melting point: 248–250
˚C, IR (cm 1): (KBr): γ = 3386 (NH stretching), 3166, 3047 (CH,
5
7 (40.80), 51 (5.30). Molecular formula (M.wt.): C26H31N O S
3 5
−
(
497.6). Calculated analysis: C, 62.76; H, 6.28; N, 8.44; found: C,
6
2.75; H, 6.28; N, 8.44.
aromatic), 2920 (CH, aliphatic), 1689 (C=O, acid), 1640, 1612
1
(
C=O amide I and II, respectively). H-NMR (400 MHz, δ, ppm,
4
2
.1.4.2. 2-(4-(benzo[d]thiazol-2-yl)-2-methoxyphenoxy)-N-(2,5-dioxo-
DMSO-d ): δ = 11.16 (s, 1H, 1NH, D O exchangeable, amide,
6
2
,5-dihydro-1H-pyrrol-1-yl)acetamide (6). Yield: 80%; melting
hydrazide), 8.14-7.16 (s, 17H, aromatic H), 4.97 (s, 2H, OCH ), 3.96
2
−1
13
point: 240–242 ˚C, IR (cm ): (KBr): γ = 3400 (NH stretching),
170, 3050 (CH, aromatic), 2930 (CH, aliphatic), 1620 (C=O amide).
(s, 3H, OCH ). C-NMR (100 MHz, δ, ppm, DMSO-d ): δ = 163.70
3
6
3
(NCOPhe), 163.60 (CONH), 155.20-111.00 (25 C, aromatic), 62.20
1
+
H-NMR (400 MHz, δ, ppm, DMSO-d ): δ = 10.73 (s, 1H, 1NH,
(OCH ), 54.00 (OCH ). MS (EI, 70 eV): m/z (%) = 437 (M , 1.12),
6
2
3
D O exchangeable, amide, hydrazide), 8.13-7.02 (s, 7H, aromatic
314 (19.19), 257 (100), 256 (20.66), 109 (11.00), 57 (24.75), 55
2
11

E.A.A. El-Meguid, G.O. Moustafa, H.M. Awad et al.
Journal of Molecular Structure 1240 (2021) 130595
(
7.70), 51 (6.80). Molecular formula (M.wt.): C30H23N O S (537.6).
(100 MHz, δ, ppm, DMSO-d ): δ = 171.19 (COOH), 167.52, 166.62
3
5
6
Calculated analysis: C, 67.03; H, 4.31; N, 7.82; found: C, 67.00; H,
(2CONH), 165.32 (CNH ), 162.37-110.29 (13 C, aromatic), 67.02
2
4
.27; N, 7.81.
(OCH ), 66.91 (NHCHCOOH), 61.21 (OCH ), 56.20 (COCH NH),
2 3 2
5
4.61 (CH CH CH NH), 25.45 (2C, CH CH CH NH). MS (EI, 70
2 2 2 2 2 2
+
4
.1.5. Synthetic routes for compounds 11–21
eV): m/z (%) = 543 (M , 0.11), 424 (6.84), 328 (9.48), 314 (19.61),
282 (11.03), 258 (21.49), 257 (100), 256 (74.33), 228 (27.20), 185
(19.73), 86 (31.13), 69 (58.18), 57 (50.56), 55 (60.23). Molecular
formula (M.wt.): C24H29N O S (543.6). Calculated analysis: C,
A dichloromethane solution of compound 4 (1mmol, 50 ml) was
drop wisely added to a cold and stirred dichloromethane solution
50 ml) of free amino acid (1mmol, obtained by the addition of
(
7
6
two equivalents amount of triethylamine (2mmol) to the amino
acid to a stirred and cold dichloromethane, 50 ml) in ice bath. The
obtained mixture was additionally stirred for extra 3 h in ice bath,
then for 24 h at room temperature, washed with distilled water,
53.03; H, 5.38; N, 18.04; found: C, 52.99; H, 5.37; N, 18.04.
4
.1.5.4. 6-(2-(2-(2-(4-(benzo[d]thiazol-2-yl)-2-
methoxyphenoxy)acetyl)hydrazinyl)-2-oxoethylamino)hexanoic
acid
1
N sodium bicarbonate, 1N potassium hydrogen sulphate and dis-
−1
(
14). Yield: 80%; melting point: 202-205 ˚C, IR (cm ): (KBr):
tilled water then dried for (24 h at 0˚C) over sodium sulphate an-
hydrous. The volatile materials were evaporated till drought then
triturated with petroleum ether (B.P. = 40-60 ˚C) to get residual
material. The obtained precipitate was collected, and then recrys-
tallized from methanol to gain the compounds 11–21.
γ = 3389 (NH stretching), 3167, 3049 (CH, aromatic), 2920 (CH,
aliphatic), 1679 (C=O, acid), 1632, 1612 (C=O amide I and II,
respectively). 1H-NMR (400 MHz, δ, ppm, DMSO-d ): δ = 10.82 (s,
6
1
H, OH, acid), 10.44, 10.36 (s, 2H, 2NH, D O exchangeable, amide,
2
hydrazide), 8.12-7.10 (s, 7H, aromatic H), 4.75 (s, 2H, OCH ), 3.93,
2
3
.92 (s, 3H, OCH ), 3.46 (s, 2H, CH NH), 2.51-1.25 (10H, 5CH ,
3 2 2
4
.1.5.1. 2-(2-(2-(2-(4-(benzo[d]thiazol-2-yl)-2-
aliphatic). 13C-NMR (100 MHz, δ, ppm, DMSO-d ): δ = 167.53
6
methoxyphenoxy)acetyl)hydrazinyl)-2-oxoethylamino)propanoic acid
(
COOH), 166.64, 165.35 (2CONH), 154.00-110.31 (13 C, aromatic),
(
11). Yield: 80%; melting point: 213–215˚C, IR (cm 1): (KBr):
−
6
6.93 (OCH ), 56.21 (OCH ), 41.28 (2C, NHCH CH CH CH CH
2 3 2 2 2 2 2
γ = 3400 (NH stretching), 3167, 3047 (CH, aromatic), 2925 (CH,
aliphatic), 1679 (C=O, acid), 1632, 1612 (C=O amide I and II,
COOH), 39.00 (3C, NHCH CH CH CH CH COOH). MS (EI, 70 eV):
2
2
2
2
2
+
m/z (%) = 501 (M , 0.03), 404 (12.64), 314 (16.34), 270 (7.22), 258
21.16), 257 (100), 256 (67.70), 228 (37.12), 185 (23.19), 86 (56.74),
9 (37.46), 57 (27.97). Molecular formula (M.wt.): C24H28N O S
respectively). 1H-NMR (400 MHz, δ, ppm, DMSO-d ): δ = 10.45,
6
(
1
0.37 (s, 2H, 2NH, D O exchangeable, amide, hydrazide), 8.11-
2
6
4
6
7
.10 (s, 7H, aromatic H), 4.79, 4.73 (s, 2H, OCH ), 4.18 (s, 2H,
2
(
500.6). Calculated analysis: C, 57.59; H, 5.64; N, 11.19; found: C,
7.59; H, 5.65; N, 11.18.
CH NH), 3.93 (s, 3H, OCH ), 3.44 (m, 1H, CHCH ), 1.27-1.25 (d,
2
3
3
5
3
H, CHCH ). 13C-NMR (500 MHz, δ, ppm, DMSO-d ): δ = 167.51
3
6
(
COOH), 166.62, 165.32 (2CONH), 154.02-110.25 (13 C, aromatic),
4
.1.5.5. 5-amino-2-(2-(2-(2-(4-(benzo[d]thiazol-2-yl)-2-
6
6.99 (OCH ), 56.20 (CH NH), 7.88 (CH ). MS (EI, 70 eV): m/z
2 2 3
+
methoxyphenoxy)acetyl) hydrazinyl)-2-oxoethylamino)-5-
(
(
(
%) = 458 (M , 0.09), 405 (26.91), 314 (9.79), 258 (20.66), 257
oxopentanoic acid (15). Yield: 75 %; melting point: 207–209˚C,
IR (cm 1): (KBr): γ = 3396 (NH stretching), 3168, 3049 (CH,
100), 256 (62.00), 109 (11.71), 69 (20.12), 57 (14.80), 55 (17.75), 51
−
6.95). Molecular formula (M.wt.): C21H22N O S (458.5). Calculated
4
6
aromatic), 2920 (CH, aliphatic), 1678 (C=O, acid), 1625, 1615
analysis: C, 55.01; H, 4.84; N, 12.22; found: C, 55.00; H, 4.79; N,
2.20.
1
(
C=O amide I and II, respectively). H-NMR (400 MHz, δ, ppm,
1
DMSO-d ): δ = 10.65 (s, 1H, OH, acid), 10.45, 10.37 (s, 2H, 2NH,
6
D O exchangeable, amide, hydrazide), 8.12-7.10 (s, 7H, aromatic
2
4
.1.5.2. 2-(2-(2-(2-(4-(benzo[d]thiazol-2-yl)-2-
H), 4.75 (s, 2H, OCH ), 3.93 (s, 3H, OCH ), 3.44 (3H, CH NH and
2
3
2
methoxyphenoxy)acetyl)hydrazinyl)-2-oxoethylamino)-3-
CHCH CH ), 2.51 (q, 2H, CH , CHCH CH ), 1.27-1.25 (m, 2H, CH ,
2
2
2
2
2
2
hydroxypropanoic acid (12). Yield: 65 %; melting point: 202-205
CHCH2CH2). 13C-NMR (100 MHz, δ, ppm, DMSO-d6): δ = 167.50
C, IR (cm−1): (KBr): γ = 3395 (NH stretching), 3168, 3049 (CH,
˚
(
COOH), 166.62 (CONH ), 165.32, 154.02 (2CONH), 150.50-110.29
2
aromatic), 2920 (CH, aliphatic), 1689 (C=O, acid), 1622, 1612
1
(13 C, aromatic), 66.96 (2C, OCH2, NHCHCOOH), 56.20 (2C, OCH3,
COCH NH), 41.30, 39.56 (CH CH CONH ). MS (EI, 70 eV): m/z
(
C=O amide I and II, respectively). H-NMR (400 MHz, δ, ppm,
2
2
2
2
DMSO-d ): δ = 10.44, 10.37 (s, 2H, 2NH, D O exchangeable, amide,
6
2
+
(
(
%) = 515 (M , 0.08), 406 (12.60), 317 (26.33), 257 (100), 228
hydrazide), 8.12-7.10 (s, 7H, aromatic H), 4.75 (s, 2H, OCH ), 4.19,
2
77.10), 185 (73.10), 69 (30.06), 57 (20.90). Molecular formula
4
3
.17 (s, 2H, CH NH), 3.93 (s, 3H, OCH ), 3.48 (m, 1H, CHCH ),
2 3 2
.44, 3.42 (d, 3H, CHCH ). 13C-NMR (100 MHz, δ, ppm, DMSO-
(M.wt.): C23H25N5O7S (515.5). Calculated analysis: C, 53.58; H,
.89; N, 13.58; found: C, 53.57; H, 4.88; N, 13.60.
2
4
d ): δ = 167.54 (COOH), 166.61, 165.31 (2CONH), 154.05-110.29
6
(
13 C, aromatic), 66.83 (3C, OCH , NHCHCH ), 56.04 (2C, OCH ,
2 2 3
+
COCH ), 7.88 (CH ). MS (EI, 70 eV): m/z (%) = 474 (M , 0.03), 405
4.1.5.6. 2-(2-(2-(2-(4-(benzo[d]thiazol-2-yl)-2-
2
3
(
14.39), 374 (4.11), 314 (11.28), 270 (7.46), 258 (21.64), 257 (100),
56 (67.29), 228 (32.83), 185 (17.44), 69 (20.75), 57 (10.30), 51
8.36). Molecular formula (M.wt.): C21H22N O S (474.5). Calculated
methoxyphenoxy)acetyl)hydrazinyl)-2-oxoethylamino)-3-
2
hydroxybutanoic acid (16). Yield: 70%; melting point: 209-212
˚C, IR (cm 1): (KBr): γ = 3371 (NH stretching), 3167, 3049 (CH,
aromatic), 2921 (CH, aliphatic), 1679 (C=O, acid), 1622, 1612
−
(
4
7
analysis: C, 53.16; H, 4.67; N, 11.81; found: C, 53.12; H, 4.66; N,
1.80.
1
1
(C=O amide I and II, respectively). H-NMR (400 MHz, δ, ppm,
DMSO-d ): δ = 10.43, 10.36 (s, 2H, 2NH, D O exchangeable, amide,
6
2
4
.1.5.3. 2-(2-(2-(2-(4-(benzo[d]thiazol-2-yl)-2-
hydrazide), 8.12-7.10 (s, 7H, aromatic H), 4.74 (s, 2H, OCH ), 4.17
2
methoxyphenoxy)acetyl)hydrazinyl)-2-oxoethylamino)-5-
(s, 2H, CH NH), 3.93 (s, 3H, OCH ), 3.46 (d, 1H, NHCHCH), 3.18 (d,
2 3
guanidinopentanoic acid (13). Yield: 85%; melting point: 165-168
1H, NHCHCH). 13C-NMR (100 MHz, δ, ppm, DMSO-d ): δ = 167.53
6
˚
C, IR (cm 1): (KBr): γ = 3397 (NH stretching), 3167, 3049 (CH,
−
(COOH), 166.64, 165.35 (2CONH), 154.00-110.29 (13 C, aromatic),
66.95 (NHCHCH), 66.92 (2C, OCH2, NHCHCH), 56.21 (2C, OCH3,
aromatic), 2922 (CH, aliphatic), 1679 (C=O, acid), 1632, 1612
1
+
(
C=O amide I and II, respectively). H-NMR (400 MHz, δ, ppm,
COCH NH), 7.86 (CH ). MS (EI, 70 eV): m/z (%) = 488 (M , 0.03),
2
3
DMSO-d ): δ = 10.65 (s, 1H, OH, acid), 10.45, 10.37 (s, 2H, 2NH,
400 (22.04), 314 (10.30), 270 (77.25), 257 (100), 256 (60.60),
6
D O exchangeable, amide, hydrazide), 8.12-7.12 (s, 7H, aromatic
86 (54.78), 57 (7.90). Molecular formula (M.wt.): C22H24N O S
4 7
2
H), 4.87 (s, 2H, OCH ), 3.93 (s, 3H, OCH ), 3.55 (s, 2H, CH NH),
(488.5). Calculated analysis: C, 54.09; H, 4.95; N, 11.47; found: C,
54.08; H, 4.92; N, 11.47.
2
3
2
3
.47 (t, CHCH CH ), 1.71-1.26 (6H, 3CH2, aliphatic). 13C-NMR
2 2
12

E.A.A. El-Meguid, G.O. Moustafa, H.M. Awad et al.
Journal of Molecular Structure 1240 (2021) 130595
CH NH), 3.40 (s, 3H, OCH ), 3.30 (s, 2 H, CH -Phe). 13C-NMR (100
4
.1.5.7. 2-(2-(2-(2-(4-(benzo[d]thiazol-2-yl)-2-
2
3
2
methoxyphenoxy)acetyl)hydrazinyl)-2-oxoethylamino)succinic
acid
17). Yield: 85%; melting point: 200-203 ˚C, IR (cm 1): (KBr):
MHz, δ, ppm, DMSO-d ): δ = 167.00, 164.20 (2CONH), 163.00
6
−
(
(NHCCHCHN, pyrimidine moiety), 155.20 (CO, pyrimidine moiety),
150.00 (NHCCHCHN, pyrimidine moiety), 149.00-109.00 (13 C,
γ = 3397 (NH stretching), 3168, 3048 (CH, aromatic), 2921 (CH,
aliphatic), 1679 (C=O, acid), 1620, 1602 (C=O amide I and II,
aromatic), 91.00 (NHCCHCHN, pyrimidine moiety), 68.55 (OCH ),
2
respectively). 1H-NMR (400 MHz, δ, ppm, DMSO-d ): δ = 10.44,
6
69.50 (OCH ), 49.00 (COCH NH). MS (EI, 70 eV): m/z (%) = 480
3
2
+
1
0.36 (s, 2H, 2NH, D O exchangeable, amide, hydrazide), 8.11-7.10
(M , 1.50), 459 (11.50), 330 (42.72), 257 (100), 256 (80.20), 228
(62.80), 187 (66.00), 86 (47.18), 57 (60.11). Molecular formula
(M.wt.): C22H20N O S (480.5). Calculated analysis: C, 54.99; H,
2
(
(
(
s, 7H, aromatic H), 4.74 (s, 2H, OCH ), 4.17 (s, 2H, CH NH), 3.93
2
2
s, 3H, OCH ), 3.49 (m, 1H, CHCH ), 2.51 (d, 2H, CHCH ). 13C-NMR
3
2
2
6
5
100 MHz, δ, ppm, DMSO-d ): δ = 167.59, 166.64 (2C, 2COOH),
4.20; N, 17.49; found: C, 54.98; H, 4.21; N, 17.49.
6
1
65.35 (2CONH), 154.35-110.29 (13 C, aromatic), 66.95 (2C, OCH
2,
NHCHCH), 56.20 (2C, OCH , CH NH), 40.12 (CH COOH). MS (EI,
4.1.5.11. 2-(2-(2-(2-(4-(benzo[d]thiazol-2-yl)-2-
3
2
2
+
7
0 eV): m/z (%) = 502 (M , 0.52), 409 (1.53), 314 (22.71), 258
21.91), 257 (100), 256 (84.27), 228 (42.89), 185 (26.49), 86 (37.08),
7 (30.01), 52 (55.15). Molecular formula (M.wt.): C22H22N O S
methoxyphenoxy)acetyl)hydrazinyl)-2-oxoethylamino)-3-
(
phenylpropanoic acid (21). Yield: 75%; melting point: 256-258
˚C, IR (cm 1): (KBr): γ = 3390 (NH stretching), 3170, 3070 (CH,
aromatic), 2900 (CH, aliphatic), 1680 (C=O, acid), 1630 and 1610
−
5
4
8
(
502.5). Calculated analysis: C, 52.58; H, 4.41; N, 11.15; found: C,
1
5
2.58; H, 4.40; N, 11.14.
(C=O amide I and II, respectively). H-NMR (400 MHz, δ, ppm,
DMSO-d ): δ = 12.50 (s, H, COOH), 10.48-10.35 (s, 3H, 3NH, D O
6
2
4
.1.5.8. 2-(2-(2-(2-(4-(benzo[d]thiazol-2-yl)-2-
exchangeable), 8.12-7.02 (s, 12H, aromatic H), 4.75, 4.65 (s, 2H,
OCH2), 3.97, 3.95 (s, 2H, CH2NH), 3.87 (s, H, CHCOOH), 3.35 (s,
3H, OCH3). 13C-NMR (100 MHz, δ, ppm, DMSO-d6): δ = 170.00
(COOH), 165.75, 164.00 (2CONH), 152.00-110.50 (19 C, aromatic),
67.85 (OCH2), 62.00 (OCH3), 60.00 (CH2-Phe), 51.05 (COCH2NH).
methoxyphenoxy)acetyl)hydrazinyl)-2-oxoethylamino)pentanoic acid
18). Yield: 90%; melting point: 197-199 ˚C, IR (cm 1): (KBr):
−
(
γ = 3396 (NH stretching), 3168, 3048 (CH, aromatic), 2921 (CH,
aliphatic), 1679 (C=O, acid), 1622, 1602 (C=O amide I and II,
respectively). 1H-NMR (400 MHz, δ, ppm, DMSO-d ): δ = 10.47,
+
+
MS (EI, 70 eV): m/z (%) = 535 (M +1, 0.50), 534 (M , 2.60),
439 (11.50), 321 (20.70), 257 (100), 230 (52.80), 185 (36.00),
87 (30.28), 57 (30.21). Molecular formula (M.wt.): C27H26N4O6S
(534.6). Calculated analysis: C, 60.66; H, 4.90; N, 10.48; found: C,
60.65; H, 4.90; N, 10.50.
6
1
0.38 (s, 2H, 2NH, D O exchangeable, amide, hydrazide), 8.10-7.10
2
(
(
(
s, 7H, aromatic H), 4.75 (s, 2H, OCH ), 4.18 (s, 2H, CH NH), 3.93
2
2
s, 3H, OCH ), 3.48 (m, 1H, CHCH ), 2.51 (t, 2H, CHCH ), 1.20-1.18
m, 2H, CH CH ), 0.92-0.82 (t, 3H, CH CH ). 13C-NMR (100 MHz,
3
2
2
2
2
2
3
δ, ppm, DMSO-d ): δ = 167.52 (COOH), 166.64, 165.35 (2CONH),
6
4
.2. Biological activity
1
54.02-110.24 (13 C, aromatic), 66.96 (2C, OCH , NHCHCOOH),
2
5
7
2
8
6.19 (2C, OCH , COCH NH), 41.30-8.90 (3C, CH CH CH ). MS (EI,
3 2 2 2 3
0 eV): m/z (%) = 487 (M+ +1, 0.52), 486 (M , 2.50), 319 (20.70),
+
4.2.1. Cytotoxicity assay
The cytotoxic activities of the newly synthesized derivatives 4–
58 (20.90), 257 (100), 256 (94.27), 228 (40.80), 185 (36.40),
21 on the HepG-2, MCF-7 human cancer cell lines and normal
6 (47.18), 57 (40.04). Molecular formula (M.wt.): C23H26N O S
4
6
fibroblast cell line (WI-38) were assessed, employing the 3-[4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
assay according to the reported method [42]. More details were
indicated in the supplementary file.
(
486.5). Calculated analysis: C, 56.78; H, 5.39; N, 11.52; found: C,
5
6.78; H, 5.38; N, 11.53.
4
.1.5.9. 2-(2-(2-(2-(4-(benzo[d]thiazol-2-yl)-2-
methoxyphenoxy)acetyl)hydrazinyl)-2-oxoethylamino)-4-
4
.2.2. EGFR inhibitory assay
mercaptobutanoic acid (19). Yield: 80%; melting point: 195-197
The promising cytotoxic targets 5, 6, 7, 13 and 16 were es-
˚
C, IR (cm 1): (KBr): γ = 3371 (NH stretching), 3164, 3046 (CH,
−
timated for their in vitro inhibitory activity against epidermal
growth factor receptor (EGFR). Erlotinib was allowed as a refer-
ence following the previously mentioned process [45]. More details
were indicated in the supplementary file.
aromatic), 2919 (CH, aliphatic), 1676 (C=O, acid), 1623 and 1602
1
(
C=O amide I and II, respectively). H-NMR (400 MHz, δ, ppm,
DMSO-d ): δ = 10.37, 10.28 (s, 2H, 2NH, D O exchangeable, amide,
6
2
hydrazide), 8.12-7.02 (s, 7H, aromatic H), 4.80 (s, 2H, OCH ), 4.20
2
(
s, 2H, CH NH), 3.93 (s, 3H, OCH ), 3.48 (m, 1H, CHCH ), 2.53,
2 3 2
4
.2.3. Flow cytometry analysis and annexin V-FITC apoptosis study
2
1
.51 (t, 2H, CHCH ), 2.20-2.00 (m, 2H, CH CH ), 1.5 (s, 1H, CH SH).
2 2 2 2
Cell cycle analysis and apoptosis investigation were done by
3
C-NMR (100 MHz, δ, ppm, DMSO-d ): δ = 167.51 (COOH), 166.62,
6
flow cytometry [45]. MCF-7 cells were incubated at 37°C and
treated with the tested compounds, for 24 h. More details were
indicated in the supplementary file.
1
65.33 (2CONH), 154.01-110.24 (13 C, aromatic), 66.98, 66.95 (2C,
OCH , NHCHCOOH), 56.20 (2C, OCH , COCH NH), 41.30, 27.78 (2C,
2
3
2
+
CH CH SH). MS (EI, 70 eV): m/z (%) = 505 (M +1, 0.75), 504
2
+
2
(
(
M , 3.05), 429 (51.50), 344 (20.00), 258 (27.71), 257 (100), 256
74.77), 227 (32.89), 187 (16.40), 87 (47.00), 57 (30.00), 50 (50.10).
4
.2.4. The impact of the derivatives 5 and 6 on Bax / Bcl-2 / p53 /
caspase-3 levels
Molecular formula (M.wt.): C22H24N O S (504.6). Calculated
4
6
2
The levels of the apoptotic marker Bax, anti-apoptotic marker
Bcl-2 and the tumor suppressor gene p53 were estimated using
BIO RAD iScript TM One-Step RT-PCR kit with SYBR® Green. The
procedure of the used kit was done according to the manufac-
turer’s instructions. The activity of caspases-3 was assessed using
DRG Caspase-3 (human) ELISA (EIA-4860) kit (DRG International
Inc., USA) according to the manufacturer instructions.
analysis: C, 52.37; H, 4.79; N, 11.10; found: C, 52.36; H, 4.77; N,
1.10.
1
4
.1.5.10. 2-(4-(benzo[d]thiazol-2-yl)-2-methoxyphenoxy)-N’-(2-(2-
oxo-2,3-dihydropyrimidin-4-ylamino)acetyl)acetohydrazide (20).
Yield: 85%; melting point: 118-120 ˚C, IR (cm ): (KBr): γ = 3400
NH stretching), 3100 (CH, aromatic), 2910 (CH, aliphatic), 1630
and 1600 (C=O amide
and II, respectively). 1H-NMR (400
−1
(
I
4.3. Molecular docking study
MHz, δ, ppm, DMSO-d ): δ = 10.52, 10.45 (s, 2H, 2NH, D O
6
2
exchangeable, amide, hydrazide), 8.15 (s, H, NH, D O exchange-
The molecular docking is a powerful instrument to rationalize
the obtained biological data. The interactions of the newly synthe-
sized compounds 5, 6, and 7 having the highest EGFR inhibitory
2
able, pyrimidine moiety), 8.02-7.25 (s, 9H, 7 H aromatic H, 2 H
pyrimidine moiety), 4.80, 4.70 (s, 2H, OCH ), 3.93, 3.90 (s, 2H,
2
13

E.A.A. El-Meguid, G.O. Moustafa, H.M. Awad et al.
Journal of Molecular Structure 1240 (2021) 130595
activity were examined with the active site of the target enzyme to
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The authors declare no conflict of interest.
[
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