Novel scaffold hopping of potent benzothiazole and isatin analogues linked to 1,2,3-triazole fragment that mimic quinazoline epidermal growth factor receptor inhibitors: Synthesis, antitumor and mechanistic analyses

Article abstract of DOI:10.1016/j.bioorg.2020.104133

A series of benzothiazole/isatin linked to 1,2,3-triazole moiety and terminal sulpha drugs 5a-e and 6a-e were synthesized and evaluated for cytotoxic activity against a panel of cancer cell lines. The novel compounds showed variable IC₅₀ range

Full text of DOI:10.1016/j.bioorg.2020.104133

Journal Pre-proofs
Novel Scaffold Hopping of Potent Benzothiazole and Isatin Analogues linked
to 1,2,3-Triazole Fragment that Mimics Quinazoline Epidermal Growth Fac-
tor Receptor Inhibitors: Synthesis, Antitumor and Mechanistic Analyses
Nadjet Rezki, Meshal A. Almehmadi, Saleh Ihmaid, Ahmed M. Shehata,
Abdelsattar M. Omar, Hany E. A. Ahmed, Mohamed Reda Aouad
PII:
S0045-2068(20)31430-9
DOI:
https://doi.org/10.1016/j.bioorg.2020.104133
YBIOO 104133
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Bioorganic Chemistry
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11 June 2020
11 July 2020
18 July 2020
Please cite this article as: N. Rezki, M.A. Almehmadi, S. Ihmaid, A.M. Shehata, A.M. Omar, H. E. A. Ahmed,
M. Reda Aouad, Novel Scaffold Hopping of Potent Benzothiazole and Isatin Analogues linked to 1,2,3-Triazole
Fragment that Mimics Quinazoline Epidermal Growth Factor Receptor Inhibitors: Synthesis, Antitumor and
Mechanistic Analyses, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104133
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Novel Scaffold Hopping of Potent Benzothiazole and Isatin Analogues linked to 1,2,3-Triazole Fragment that Mimics
Quinazoline Epidermal Growth Factor Receptor Inhibitors: Synthesis, Antitumor and Mechanistic Analyses
Nadjet Rezki,1,* Meshal A. Almehmadi,1 Saleh Ihmaid,2 Ahmed M. Shehata ,3,4 Abdelsattar M. Omar,5,6 Hany E. A. Ahmed, 2,7* Mohamed Reda
Aouad1
1Department of Chemistry, College of Science, Taibah University, Al-Madinah Al-Munawarah 30002, Saudi Arabia
2Pharmacognosy and Pharmaceutical Chemistry Department, College of Pharmacy, Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia
3Pharmacology and Toxicology Department, College of Pharmacy, Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia.
4Department of Pharmacology and Toxicology, Faculty of Pharmacy, Beni-Suef University, Egypt.
5Pharmaceutical Chemistry Department, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
6Pharmaceutical Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Nasr City 11884, Cairo, Egypt
7Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar University, 11884 Nasr City, Cairo, Egypt
*Corresponding author
E-mail address: nadjetrezki@yahoo.fr , heahmad@taibahu.edu.sa
Abstract
A series of benzothiazole/isatin linked to 1,2,3-triazole moiety and terminal sulpha drugs 5a-e and 6a-e were synthesized and evaluated for cytotoxic
activity against a panel of cancer cell lines. The novel compounds showed variable IC50 range of activity and some of them were potent compared to
reference drug. The promising compounds were subjected as postulated the mimicry proposal for quinazoline-based EGFR inhibitors for their inhibitory
profile against EGFR TK enzyme. That data obtained revealed that most of these compounds were potent EGFR TK inhibitors at nanomolar
concentrations. Among these, compounds 5a and 5b showed more potent activity on EGFR compared to erlotinib (IC50 103 and 104 versus 67.6 nM).
Based upon the results, molecular docking analysis was performed on EGFR receptor and proved the strong contribution of fragments; benzothiazole,
isatin, and triazole to the binding ATP pocket. When these selected compounds 5a and 5b were tested in an HepG2 model, they could effectively
inhibited tumor growth, strongly induced cancer cell apoptosis, and suppressed cell cycle progression leading to DNA fragmentation. Well-DMET
profile of the most active derivatives was presented and compared to the reference drugs. Taken together, we introduced novel triazole-sulpha drug
hybrid for the first time as EGFR inhibitors and the results of our studies indicate that the newly discovered inhibitors have significant potential for
anticancer treatment.
Keywords: Triazole; benzothiazole; isatin; EGFR TK; apoptosis; cell cycle analysis; anticancer activity.
1

Introduction:
Cancer is considered as one of the most threatening disease affecting many of populations. WHO reported in 2020, that the incidence of population is
18.1 million cancer cases and mortality about 9.6 million deaths occurred [1]. Over the past decade, different strategies were offered to prevent cancer
from developing and stop patients from deteriorating, and lastly, a novel series of selective chemotherapeutic drugs such as enzyme-mediated drugs like
imatinib, gefitinib, erlotinib, and afatinib have been launched worldwide for the treatment of cancer [2]. The action targets of these innovative drugs are
mainly directed to impact the signal transduction pathway of the epidermal growth factor receptor (EGFR) [3]. The over-expression and/or mutation of
EGFR plays an important role in the growth of cancer including cell proliferation, anti-apoptosis, metastasis, and angiogenesis [4-7]. The EGFR has
been reported as an attractive target for cancer therapy [8, 9]. Over recent years, many scientists have discovered the mechanism of the EGFR that the
binding of specific ligands such as epidermal growth factor (EGF) and transforming growth factor α (TGFα) to the extracellular domain of EGFR
induces the dimerization, activation of intrinsic kinase activity, and subsequent autophosphorylation of EGFR at multiple residues in the cytoplasmic
region, while the downstream signaling proteins including phosphoinositide 3-kinase (PI3-K), Akt, mammalian target of rapamycin (mTOR), Ras, Raf,
MEK, and extracellular signal-regulated kinase (ERK) stimulate several signal transduction cascades, leading to DNA synthesis and cell proliferation
[10, 11]. As it can obviously influence the proliferation of human cancer cells, and even result in the apoptosis of human cancer cells, the EGFR has
become a very luminous target for various antitumor candidates [12, 13].
There are different heteroaromatic cyclic structures representing a core structures for most of marketed drugs [14]. Among these, benzothiazole and
isatin, which are considered as promising chemical scaffolds for designing of numerous biologically and pharmacologically active drugs [15-20]. 1,2,3-
Triazoles, one of the most fascinating classes of nitrogen containing heterocycles, have the ability to form various non-covalent interactions such as
hydrophobic interactions, hydrogen bonds, van der Waals forces and dipole-dipole bonds with different biological targets that possess diverse
pharmaceutical properties [21-27]. In addition, triazole-linked drugs were introduced as novel panel in medicinal chemistry for discovery of potent
anticancer agents [28]. The triazole-based drugs exhibited ideal features as metabolically stable bioisosteres of trans-amide bonds and the 1,2,3-triazoles
mimic amide bonds in terms of planarity, size, dipolar moment, and hydrogen bonding features [29-31]. Kumbhare et al. introduced novel compounds
formed of triazoles and isoxazoles linked 2-phenyl benzothiazole as potential anticancer agents [32]. In the previous work, we introduced a novel
scaffold consisting of benzothiazole-linked to triazole derivatives that offering anticancer activity [25, 33-35]. These trials have motivated us to optimize
these rationales and changed scaffold based upon the hybridization of hetroaromatic cyclic scaffold linked to the triazole moiety and terminal side chain
polar sulphonamides to improve physical properties and confer a favorable pharmacokinetic profile in animals and humans, Fig. 1. According to
scaffold-hopping strategy [36, 37], the quinazoline fragment of gefitinib was replaced by triazole core structure with methylene linker to
benzothiazole/isatin ring as pharmacodynamic group as aniline fragment and ending with polar sulphonamide part. This molecular replacement strategy
affords more metabolic stability and hydrogen bonding features for the novel analogs. Moreover, to gain an understanding of the interactions between
these novel derivatives and EGFR, hereinafter docking studies are also presented.
2

Query scaffold definition
O
O
N
O
O
O
O
N
N
N
N
O
HN
HN
Cl
F
Erlotinib
Gefitinib
O
O
N
N
O
N
N
N
H
F
Afatinib
Cl
Fragment mapping
N
O
HN
N
Cl
F
O
O
N
O
N
O
F
N
Cl
NH
O
O
HN
O
O
New ligand generation
O
N
O
N
N
N
S
S
N
N
N
N
O
O
S
R
R
N
S
O
N
H
O
H
5a-e
6a-e
Target compounds
Fig.1. Scaffold hopping exercise on EGFR quinazoline-based inhibitors mapped to the designed ligands. Fragmentation patterns for reference ligands
and mapped with target scaffolds.
2. Results and discussion
2.1. Target scaffold design
The marketed EGFR inhibitors are described in Fig.1. and structurally analyzed into three main components; aromatic core scaffold substituted with
hydrophobic aromatic part and terminal hydrophilic part. The structural fragments were mapped into the proposed fragments of the target compounds.
The 1,2,3 triazole scaffold is identified as a candidate for replacement of 6-membered quinazoline as a core structure for the novel compounds connected
to benzothiazole or isatin fragment as lipophilic moiety and substituted with sulphonamides as hydrophilic part. These new proposed scaffolds; isatin-
or benzothiazole-triazole-sulphonamide were analyzed using the molecular surface maps and binding behavior towards EGFR pocket and compared to
reference drugs.
3

The electrostatic potential of all compounds; reference drugs and proposed ligands are displayed with different energy levels (in kcal/mol) of the
hydrophobic (green zones), H-bond acceptor (red zones), and H-bond donor (blue zones) maps. Actually, it appears that the comparison of the patterns
from Fig. 3 reveals the molecular features controlling the inhibitory activity are trapped in the properties of the active fragments contributing in the
interaction patterns. These fragments vary from the core scaffold to terminal hydrophilic parts as electron-acceptor rich parts and this quite clear in the
analysis of the electrostatic potential maps of reference drugs (Fig. A-B); red zones. This pattern looks like the electrostatic maps of the target scaffolds
as they are rich in electron-acceptors properties and consistent with the other hydrophilic H-donors and neutral properties (Fig. C-D).
A)
B)
C)
D)
Fig.2. Electrostatic map distribution of selected quinazoline-based EGFR TK inhibitors mapped with proposed benzothiazole and isatin-triazole
scaffolds.
2.1.2. EGFR pocket mapping
X-ray structure of EGFR is shown in (Fig. 2A) indicated that 4-anilinoquinazoline erlotinib causes inhibition through binding to the site occupied by
ATP during phosphotransfer. The N1 of the quinazoline accepts an H-bond from the Met769 amide nitrogen. We find Thr766, Lys721, and Leu764 are
4 Å from the acetylene moiety on the anilino ring (Thr766 and Leu764 are >3.4 Å). Both Met742 and Cys751 have been suggested to contact inhibitors
very similar to erlotinib, but contact distances seen here for both are greater than 4.5 Å. This map of interactions are derived from the analyses of both
crystal structures was consistent with previous findings [38, 39].
4

A
B
Fig. 3. X-ray structures of A) EGFR TK with bound erlotinib inhibitor, and B) EGFR TK with bound inhibitor erlotinib aligned with proposed
benzothiazole-triazole scaffold.
On the basis of the observed interaction patterns and bound inhibitors, we carried out an alignment step of proposed scaffolds to the reference erlotinib
ligand in the ATP-pocket and feature mapping were analyzed using the Molecular Operating Environment (MOE) [40] and further refined the alignment
though intra- and inter-molecular energy minimization. Based on these alignments; triazole mimics quinazoline, sulphonamide mimics hydrophilic part,
and benzothiazole or isatin mimics the flurophenyl moiety (Fig. 3A). The model represented substructures of inhibitors that formed characteristic
interactions or occupied a hydrophobic pocket within the active site and were considered important for inhibitory activity.
Guided by our structure-based EGFR analysis and comparison of the new proposed scaffold to quinazoline-based inhibitors, we then designed different
triazole-based compounds and examined their detailed fit to the EGFR PDB model, which represented the central part of our structure-based design
exercise. The novel compounds derived on the basis of a newly proposed triazole-sulphonamide scaffold yielded an excellent fit to binding pocket, as
illustrated in (Fig. 3B). In exemplary compound, the triazole moiety mapped to quinazoline binding site represented by the donor/acceptor feature. The
excellent fit of the newly designed 2-aminothiazole-based compounds to the ATP active site motivated us to synthesize these compounds and test them
for anticancer activity targeting the EGFR TK. In this study, a series of 1,2,3 triazoles with sulphonamide side chain were designed and synthesized.
Next, their inhibitory potencies of EGFR kinase activity were compared to erlotinib in vitro.
2.2. Organic synthesis
The synthetic route to the target 1,2,3-triazoles 5a-e and 6a-e was performed by the click 1,3-dipolar cycloaddition reaction of the appropriate
benzothiazole and/or isatin based alkyne with several sulfa drug azides as depicted in Scheme 1. Thus, we began our investigation by the conversion of
sulfadrug amines 1a-e namely sulfamethazine, sulfadiazine, sulfapyridine, sufathiazole and sulfaguanidine into their corresponding azides 2a-e,
respectively; via diazotization in situ catalyzed by sodium nitrite in HCl followed by treatment with sodium azide according to reported procedure [41].
On the other hand, 2-mercaptobenzothiazole and/or isatin on being alkylated with propargyl bromide [34], resulted in the formation of the propargylated
benzothiazole 3 and the propargylated isatin 4 in 91 % and 95 % yields, respectively.
Click 1,3-dipolar cycloaddition reaction of the freshly prepared sulfa drug azides 2a-e to a stirring solution of 2-(prop-2-yn-1-ylthio)benzo[d]thiazole
(3), copper sulfate and sodium ascorbate; in a mixture of DMSO:H2O (1:1) at room temperature for 8-12 h; led to the formation of new 1,2,3-triazole
derivatives tethering benzothiazole-sulfa drug molecular conjugates in 84-90 % yields.
The structures of the newly synthesized 1,2,3-triazole-benzothiazole conjugates 5a-e have been fully characterized by IR, NMR and elemental analysis.
The IR spectra showed clearly the disappearance of the alkyne groups of the precursor propargylated 3, and revealed absorption bands in obvious of
5

their proposed structures (See experimental section). The formation of the resulted triazoles 5a-e was also evidences by their 1H NMR spectra, which
revealed the absence of the terminal acetylenic proton (≡C-H) confirming its involvement in the cycloaddition reaction. The spectra also exhibited
diagnostic singlets around δH 8.91-8.93 ppm attributed to the aromatic CH-triazolyl ring. In addition, the SCH2 and NH protons were resonated as two
singlets at δH 4.79-4.81 ppm and δH 12.19-12.92 ppm, respectively. The 13C NMR spectra also confirmed the success of the click reactions. The absence
of the carbons attributed to the alkyne chain and the presence of new signals assigned to SCH2 between δC 27.68-27.70 ppm agreed with the proposed
triazole structures. Extra aromatic protons and carbons were recorded in the aromatic area and were listed in the experimental section.
Application of our optimized Cu(I) catalyzed click reaction conditions to the propargylated isatin 4 afforded the desired isatin-1,2,3-triazole-sulfa drug
hybrids 6a-e in good to excellent yields 86-91 %. The structures of the resulted 1,2,3-triazole based isatin nucleus 6a-e were elucidated based on their
spectroscopic data. Their IR spectra confirmed the involvement of the terminal alkyne group in the cycloaddition reaction due to the disappearance of
1
the corresponding absorption bands (C≡C and ≡C-H). The examination of the H NMR spectra also revealed the absence of the Sp proton and the
presence of characteristic singlets at δH 5.08-5.10 and 8.93-8.96 ppm, attributed to the NCH2 and 1,2,3-triazolyl ring protons, respectively. The
sulfonamide NH protons were observed in the downfield around δH 12.02-12.83 ppm. The remaining protons resonated at their usual chemical shift
(See experimental section). In addition, the 13C NMR spectra recorded new aromatic carbons of the triazole and phenyl rings. The NCH2 were observed
between δC 34.93-35.48 ppm.
N
S
S
N
N
N
N
S
3
S
O
S
R
H2N
N3
N
O
H
O
O
i
ii
5a-e
S
O
R
S
R
N
N
O
H
H
O
1a-e
2a-e
O
O
N
N
N
N
O
N
i: 1. HCl, NaNO2, 2. NaN3
ii: CuSO4, Na ascorbate, DMSO/H2O (1:1)
O
4
S
R
O
N
H
6a-e
NH
NH2
N
N
N
S
R =
H3C
N
N
N
CH3
a
b
c
d
e
Scheme 1. Click synthesis of benzothiazole/Isatin-1,2,3-triazole-sulfa drug molecular hybrids 5a-e and 6a-e
2.3. Biological evaluation
2.3.2. EGFR TK inhibitory activity
Based upon the anticancer data, the following mechanism of action can be proposed for benzothiazole/isation-1,2,3-triazole-sulfa drug hybrids
derivatives since their chemical structure, in some respect, resembles benzothiazole/isation-1,2,3-triazole-sulfa drug hybrids -based EGFR inhibitors
which inhibits EGFR tyrosine kinase by binding to the ATP binding site. The epidermal growth factor receptor (EGFR) is considered a cellular trans-
membrane tyrosine kinases that is over-expressed in a different types of human tumors (e.g., breast, ovarian, colon, and prostate), their expression levels
often correlate with vascularity and is associated with poor prognosis in patients [61]. Thus, the activation process of the EGFR tyrosine kinase and
hence activating the anti-apoptotic rap signal transduction cascade is inhibited and the growth of malignant cancer cells is inhibited. The synthesized
6

benzothiazole/isation-1,2,3-triazole-sulfa drug hybrids derivatives may be a selective inhibitor of the epidermal growth factor receptor (EGFR) tyrosine
kinase over expression through the inhibition of EGFR autophosphorylation and EGF-stimulated signal transduction [62]. However, a detailed receptor
study is necessary to reach a concrete conclusion at the close future. Based upon the anticancer screening data, the EGFR enzyme assay was done for
all compounds. All results are presented in Table 1 and assays were done for starting materials and final products. Benzothiazoles showed more
inhibitory effect on EGFR target better than isatin ones. The data revealed that derivatives with sulpha substituents of large aromatic rings size are more
activity than lower ones and aliphatic side chains. This might be due to the hydrophobic aromatic interactions involved in binding data. The data overall
showed that most of the derivatives have potent EGFR inhibition compared to inactive precursors and comparable activity to erlotinib. Among these
active derivatives, the 5a and 5b analogs with pyrimidine part showed potent nanomolar inhibitory activity, 104-103 nM compared to the reference
drug, erlotinib. As already outlined, lipophilicity has been shown in numerous studies to be a general predictor for high P-gp inhibitory potency. This
most probably is due to the proposed access path of the compounds, which seems to be directly from the membrane bilayer. On the other hand, high
lipophilicity is very often associated with poor oral drug-like properties. This led to the assumption that LogP values between 2 and 3.5 are considered
optimal in an oral drug program and prompted Leeson et al., to introduce the concept of lipophilic efficiency (LPE) [42]. LPE is a parameter that
combines both potency and lipophilicity and is defined as a measure of how efficiently a ligand exploits its lipophilicity to bind to a given target. Briefly,
in a lead optimization series, there is a greater likelihood of achieving good in vivo performance when potency can be increased without increasing logP
or logD values. To explore this concept also for EGFR inhibitors, we calculated LPE values for the whole set of target compounds and reference erlotinib
drug Table 1, Fig. 4. The LogP values vary from -0.4 to 4.4, leading to a lipophilic efficiency range between 6.73 and 1.71. This was done for underlying
both relatively good oral absorption and potential for penetration. It was shown that erlotinib has 3.7 LPE and the novel potent derivatives 5a and 5b
showed less LPE values than it due to somehow the higher LogP values.
Table 1. In vitro inhibitory EGFR wild type assay (IC50, nM) of new candidate compounds.
O
O
N
N
N
O
N
S
N
S
N
O
S
S
N
N
N
N
O
S
O
R
N
O
R
S
H
N
O
5a-e
3
4
6a-e
H
Cpd. ID
IC50
LogP LPE
3.02
3.86
3
>50000
5a
5b
5c
5d
5e
104.29±0.20 4.37
103.79±1.20 3.76
254.53±0.82 4.36
712.29±1.20 4.42
413.3±0.55 2.13
2.61
3.11
2.23
1.72
4.25
4
>25000
0.849
5.691
6a
6b
6c
6d
6e
175.62±0.88 2.20
290.30±0.82 1.58
424.20±1.20 2.19
668.97±1.25 2.25
468.21±0.82 -0.41
4.55
4.95
4.18
3.91
6.73
Erlotinib 76.6±1.20
3.40
3.71
-
-
Data are presented as average IC50 ± SD (nM) values for at least three experiments. P-value is < 0.05.
Lipophilic Efficiency was calculated by (LPE = PIC50 – LogP) equation that is critical for drug likeness.
7

8
7
6
5
4
3
PIC50
LogP
LPE
2
1
0
-1
Target Compounds
Fig. 4. Bar chart shows the distribution analysis of inhibitory activity (PIC50) of target compounds compared to LogP and LPE values. SE data were
presented on red bars of activity.
2.3.1. Cell proliferation inhibitory activity
In-vitro anti-cancer activities of the compounds of interest were evaluated against three different cancer cell lines- MCF7 (Michigan cancer foundation-7,
a breast cancer cell line), HCT116 (a human colorectal carcinoma cell lines) and HepG2 (a human liver carcinoma cell lines) following 3,(4,5-
dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) assay method [43, 44]. Staurosporine was employed as positive protein
kinase inhibitor. The half maximal inhibitory (IC50) of the compounds of interest and that of positive control are presented in Table 2 and in Figure 1.
IC50 results indicate variable response to different cell lines.
Table 2. In Vitro Cell Growth Inhibitory Effects of compounds benzothiazole/isation-1,2,3-triazole-sulfa drug hybrids derivatives, and staurosporine
against MCF-7, HCT116 and HepG2 cancer cell lines.
IC50 (µM) a
HCT-116
Cpd.
MCF-7
HepG2
3
4
556±0.77
1002±0.1
3166±0.24
4482±0.42 1797±1.4 646.2±0.33
2.45±0.04 39±0.59 1.491±0.01
23.8±0.38 58.6±1.3 1.786±0.01
36.4±0.81 30.7±0.55 24.25±0.75
6.01±0.07 36.4±0.95 6.305±0.04
34.3±0.46 7.52±0.07 17.57±0.21
5a
5b
5c
5d
5e
6a
6b
6c
36.6±0.93 37.4±0.74 2.594±0.07
34.4±0.72 21.6±0.28 40.71±0.76
12.3±0.12 46.2±0.93 9.817±0.10
6d
6e
65.3±1.51
33.5±0.33 17.4±0.29 91.04±0.33
8.8±0.1 13.1±0.25
Staurosporine 25.8±0.88 6.75±0.07 16.98±0.25
a IC50 values expressed in µM as the mean values of triplicate wells from at least three experiments and are reported as the mean ± standard error. P-
value is < 0.05.
Some of the newly designed click products displayed excellent to moderate growth inhibitory activity against the tested cancer cell lines. Compounds
5a and 5b were the most potent derivatives against cancer cell lines such asHepG2 with IC50 1.49±0.01 and 1.78±0.01 µM, respectively, as compared
to the standard Staurosporine with 16.98±0.25 µM. While compounds 5a, 5d, 6c and 3 arranged in the order of potency exhibited inhibitory activities
on MCF7 cells with IC50 2.45±0.04, 6.01±0.07, 12.3±0.12 and 3166±0.24 µM, respectively, compared to Staurosporine 25.8±0.88 µM. Compounds 5e
and 6d exhibited inhibition against HCT116 cells IC50 7.52±0.07 and 8.8±0.1comparable to Staurosporine IC50 6.75±0.07 µM. In particular, 5c, 6b, 6e
were inactive against these three control cell lines. Additionally, compounds 6a, 6c, 6d and 5d with IC50 2.59±0.07, 9.81±0.10, 13.1±0.25 and 6.30±0.04
8

µM, respectively, possessed excellent antiproliferative activities against cancer cell line HepG2, compare to Staurosporine with IC50 16.98±0.25. Also,
5b and 6a showed selectivity pattern against cancer cell lines HepG2. As a precaution test for normotoxic effect, 5a the most active compound does not
show any cytotoxicity against WI38 cells (520 µM).
2.3.3. Cell apoptosis
Apoptosis induction was studied in HepG2 cells for three selected compounds based on the results from the EGFR assay results, Table 1 and IC50 of
compounds on different cell line, Table 2. Representative potent compounds 5a, 5b and 6a were tested for apoptosis and their results were compared
with untreated HepG2 cells which served as control for the experiment. Determined apoptosis with Annexin/PI double staining flow cytometric assay
was presented in Fig. 5A-C. The percentage of total apoptotic cells (early and late apoptotic cells) increased from 2.72% for the control to 25 % in the
case of compound 5a, Fig. 5A-B. While the percentage of total apoptotic cells (early and late apoptotic cells) increased from 2.13% for the control to
30% in the case of compounds 5b and 6a induced apoptosis at a higher rate in HepG2 cells. Necrosis cell death leads to the release of the intracellular
contents that affects neighboring cells and triggers an inflammatory reaction.[45] However, we did not observe cells undergoing necrosis upon treatment
with compound 5a, 5b and 6a (Fig. 5B-C) indicating that cells death occurred primarily through apoptosis. In cancer therapy, the most efficient and
safe anticancer agent typically interfere with the balance between cell proliferation and apoptosis and shift cells toward the induction of apoptosis.[46]
These results are consistent with other studies showing that erlotinib drug inhibitsHepG2 tumor proliferation by inducing apoptosis, [47-49] as clearly
detected for compounds.
A)
B)
9

C)
40
35
30
25
20
15
10
5
5a
5b
6a
HepG2
0
Total
Early
Late
Necrosis
Apoptosis
Fig.5. A) and B) Dot plot of Annexin V/PI double staining of HepG2 control and treated with analogues; 5a, 5b, and 6a. C) Statistical analysis of the
apoptosis percentage of HepG2 cells after incubation with compounds 5a, 5b, and 6ain conc. (1.87, 1.41, 0.7μg/mL respectively) for 24 h. The data are
reported as the mean ± SD of three independent experiments in triplicate.
2.3.4. Cell cycle analysis
For the reduction of possible nonspecific effects of drugs in cancer treatment, the design and synthesis of novel chemotherapeutic agents that can
regulate cell cycle progression and apoptosis is an attractive approach.[50] Therefore, further investigation of the effects of compound 5a, 5b, and 6a
on regulating cell cycle progression was explored using the Propidium Iodide Flow Cytometry Kit assay (Fig. 6A-C). The cell cycle histograms of the
target HepG cancer cell treated with potent selected compounds (at IC50 cytotoxicity) and compared to that of the untreated cells are illustrated in Fig.
4. The percentages of compound 5a treated HepG cells in the G2/M phase were significantly higher than those in the control group. The distribution of
cells in G1 and S phases was notably decreased compared to the control cells (Fig. 6A). The percentages of compound 5b and 6a treated HepG cells in
the G2/M phase were significantly higher than those in the control group. The distribution of cells in G1 and S phases was notably decreased compared
to the control cells (Fig. 6B-C). Previous studies showed that triazole-based EGFR drugs aligned along the microtubular network within the cells during
the mitotic phase [51]. In addition, intracellular EGFR inhibitors have been associated with antimitotic action.[52, 53] This might provide a possible
explanation of cell cycle arrest observed at G2/M phase in cells treated with compounds 5a,5b and 6a.
A)
10

B)
C)
70
60
50
40
30
20
10
0
%G0-G1
%S
%G2/M
%Pre-G1
5a
5b
6a
HepG2
Target Compounds
Fig. 6. A) and B) DNA content distribution histograms of control and treated cells. C) Statistical analysis of cell cycle phases percentage of HepG2
cells after incubation with compounds 5a, 5b, 6a (1.87, 1.41, 0.7μg/mL) for 24 h. The data are reported as the mean ± SD of three independent
experiments in triplicate. P-value is <0.05.
11

2.4. EGFR target docking simulations
The integration process of experimental and computational methods is an extremely attractive strategy for the design and optimization of drug candidates
[7, 54, 55]. Based upon the information obtained from the biology, molecular docking studies were performed as a crucial step toward understanding
the mode of interaction of these novel compounds. The experiments were done on the basis of docking calculations using MOE [40] and Autodock.[56]
From the Protein Data Bank [57] the X-ray structures of EGFR TK in complex with the erlotinib inhibitor (PDB entry 1M17) and used as template for
docking. Compounds 5a and 5b were built using MOE the geometry was optimized by energy minimization with the CHARMm force field and was
then prepared for docking calculations with Python scripts available in the Autodock package. Prior to docking, bound crystallographic inhibitors were
removed from the templates. For re-docking of crystallographic inhibitors as validation step and docking of compounds 5a and 5b, 50 flexible docking
runs were performed using Autodock, and the resulting poses were clustered with 1.8 Å tolerance. Lamarckian genetic algorithm was used for the
conformational space search with the initial population set to 150. The most populated clusters of low energy conformations were selected for analysis.
For EGFR target, the applied docking protocol closely reproduced the experimental binding modes of re-docked inhibitors. Furthermore, the predicted
binding modes of compounds 5a and 5b corresponded to the proposed fit of 5a to the bound orientation of the crystallographic inhibitors. Previous
studies [39, 58] have indicated that 4-anilinoquinazolines such as erlotinib cause inhibition through binding to the ATP site. The inhibitor was located
well in the ATP pocket and anchored by Met769 through a hydrogen bond of 3.4 A0 length to N quinazoline part, and affording more stability by
hydrophobic interactions with Leu694, Leu820, Lys721, and Thr766 (hinge region; Fig. 7A and D) with binding energy = -21.5 Kcal/mol. We find our
active compounds 5a (binding energy = -20.5 Kcal/mol) and 5b (binding energy = -19.75 Kcal/mol) bind as the same manner to reference erlotinib drug
and showed strong H bonding with conserved Met769 residue (3.4 , 3.5 A0 lengths for 5a and 5b respectively) through the conserved novel triazole N3
with consistent double aromatic stabilization with Leu694 and Gly772 residues in hinge region, Fig. 7B-C. This proves that the importance of triazole
fragment as isostere for quinazoline ring. In addition, the terminal benzothiazole part exhibited quite similar interaction with Leu820 as hydrophobic
conserved residue as did aniline aromatic part. The Asp831 acidic residues contributed for stabilization of benzothiazole in hydrophobic pocket more
than aniline fragment. All behaviors of novel derivatives 5a and 5b in binding pocket were mapped to reference erlotinib drug as aligned structures in
Fig.7 D-E. Encouragingly, the results of binding mode predictions for 5a and 5b were fully consistent with our 2D fitting as the underlying structure-
based inhibitor design strategy. The novel scaffold was introduced for the first time as EGFR inhibitor and mapped with reference drugs could used as
lead compound for design of low resistance anticancer agents.
12

A)
B)
C)
D)
E)
Fig. 7. Docking modes of active compound 5a and 5b. Shown are predicted complexes of A) Erlotinib drug with EGFR pocket; B) Compound 5a with
EGFR ATP pocket; C) Compound 5b with EGFR ATP pocket; D) Aligned 5a with erlotinib in pocket; E) Aligned 5b with erlotinib in pocket. For
comparison, the bound crystallographic inhibitors are shown. Carbon atoms of 5a and 5b and crystallographic reference inhibitors are colored red and
blue, respectively. Possible hydrogen bonding interactions between compounds and EGFR are indicated by dashed lines.
3.2.5. Computational analysis of drug-likeness and ADME properties
13

PK properties such as absorption, distribution, metabolism, excretion and toxicity (ADMET) profiling of compounds were determined using the pkCSM
ADMET descriptors algorithm protocol [59-61]. Two important chemical descriptors correlate well with PK properties, the 2D polar surface area
(PSA_2D, a primary determinant of fractional absorption) and the lipophilicity levels in the form of atom-based LogP. The absorption of drugs depends
on factors including membrane permeability [indicated by colon cancer cell line (Caco-2)], intestinal absorption, skin permeability levels, P-glycoprotein
substrate or inhibitor. The distribution of drugs depends on factors that include the blood–brain barrier (logBB), CNS permeability, and the volume of
distribution (VDss). Metabolism is predicted based on the CYP models for substrate or inhibition (CYP2D6, CYP3A4, CYP1A2, CYP2C19, CYP2C9,
CYP2D6, and CYP3A4). Excretion is predicted based on the total clearance model and renal OCT2 substrate. The toxicity of drugs is predicted based
on AMES toxicity, hERG inhibition, hepatotoxicity, and skin sensitization. These parameters were calculated for potent 5a, 5b and less potent 6b
analogs compared to marketed drugs erlotinib and checked for compliance with their standard ranges. After calculating the ADMET properties (Table
3), we can verify that the ligands 5a, 5b and the reference erlotinib have almost similar Log P value (3.76, 4.37 and 3.40 respectively), while the ligand
6b has law LogP value 1.58. On the other hand, we can verify that the ligands 5a, 5b and the reference erlotinib have a high percentage of human
intestinal absorption (98.58%, 97.66% and 94.58% respectively), as they have very hydrophobic groups in their chemical structures, which would make
it easy to pass through the biological membranes [61]. Conversely, 6b has human intestinal absorption values less 90%, demonstrating that this
compound may have poor oral bioavailability when tested experimentally [61]. Analyzing the central nervous system (CNS) permeability, 5a, 5b and
erlotinib have good penetration in the CNS (-3.55 and -3.45 and -3.21 respectively), while 6b is penetrate the CNS (Log PS <-3.50) [61]. These data
may help that 5a and 5b present lower side effects in the (CNS) compare 6b, contributing to a better tolerability among patients. It was also possible to
observe that 5a, 5b and 6b are able to inhibit the cytochrome P450, CYP3A4 isoform, the main enzyme responsible for drug metabolism. This possibly
occurs because these ligands exhibit high molecular size, thus favoring the inhibition of this enzyme and may lead to interactions with other drugs
metabolized by this CYP and may cause serious adverse effects [61]. The excretion was evaluated as Total Clearance. This parameter is related to
bioavailability and is important in determining dosage rates to achieve steady state concentrations. Thus, these compounds, due to their high
hydrophobicity, could not be excreted rapidly by the kidneys, requiring longer administration intervals to maintain desired therapeutic concentrations.
The last parameter analyzed in our studies was hepatotoxicity. In (Table 3), we can verify that ligand showed hepatotoxic. Therefore, these ligands
exhibit interaction with TcTR may not have a physiological or pathological event that could be associated with hepatic injury, suggesting that these
compounds may be well tolerated by the liver when they are tested in a biological environment.
Table 3. Predicted ADMET properties of selective target compounds.
Parameter
5b
5a
6b
Erlotinib
Molecular properties
461.46
1.58
Molecular Weight
LogP
Rotatable Bonds
Acceptors
Donors
Surface Area
481.58 509.64
393.44
3.40
10
3.76
7
4.37
7
6
9
1
10
1
10
1
7
1
187.32
191.17 203.90
169.53
Absorption
-3.45
0.19
86.67
-2.73
No
Water solubility
Caco2 permeability
-3.09
0.56
97.66 98.58
-2.73
Yes
Yes
Yes
-3.28
0.60
-4.73
1.43
94.58
-2.74
No
Intestinal absorption (human)
Skin Permeability
P-glycoprotein substrate
P-glycoprotein I inhibitor
P-glycoprotein II inhibitor
-2.73
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Distribution
-0.40
0.144
-1.807
-3.97
VDss (human)
0.114 0.136
0.091 0.084
-1.818 -1.807
0.199
0.059
-0.745
-3.21
Fraction unbound (human)
BBB permeability
CNS permeability
-3.55
-3.45
Metabolism
No
CYP2D6 substrate
No
No
No
14

Yes
Yes
No
No
No
CYP3A4 substrate
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
Yes
No
Yes
Yes
No
Yes
No
No
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Excretion
0.165
No
Total Clearance
Renal OCT2 substrate
0.178 0.066
0.702
No
Yes
No
Toxicity
No
0.672
No
AMES toxicity
Max. tolerated dose (human)
hERG I inhibitor
hERG II inhibitor
Oral Rat Acute Toxicity (LD50)
Oral Rat Chronic Toxicity (LOAEL) 0.31
Hepatotoxicity
Skin Sensitization
T. Pyriformis toxicity
Minnow toxicity
Yes
0.573 0.545
No
Yes
2.69
No
No
0.839
No
No
Yes
2.79
0.31
Yes
No
Yes
Yes
2.81
0.47
Yes
No
0.285
-1.105
2.39
1.37
Yes
No
0.309
-0.102
Yes
No
0.285 0.285
-1.739 -1.503
3.2.6. SAR analysis of target compounds
The above analyses suggested different preliminary SARs for the triazole anticancer agent scaffold that mimics the quinazoline-based inhibitors targeting
EGFR TK, that can be summarized in Fig. 8. Different factors affect activity; the terminal aromatic part weather benzothiazole or isatin, linker to
aromatic part, and terminal sulpha fragments. The installation of benzothiazole affording more active anticancer and EGFR inhibitors than isatin moiety
that behaves as more aromatic system and able to accommodate by aromatic hydrophobic interactions. In addition, the S linker to triazole scaffold
combined with benzothiazole part increases the activity than no linker in case of isatin. As well, the sulpha parts used in synthesis of final analogs also
showed prominent increase in activity according to their structures of terminal part. Full 6-membered aromatic systems with small methyl substituents
are of great importance in anticancer and EGFR activity and are favorable for interactions.
S linker increases
activity
- Benzothiazole is better
N
in activity than isatin
N
A
Aromatic part
N
O
Terminal part
S
N
O
H
Aromatic structures increase cytotoxic and EGFR
-
activity than non-armoatic and aliphatic.
- Enlarging of ring size increases activity.
- Aliphatic substituents on aromatic systems increase
activity
Fig. 8. Summary of the SARs of triazole-based EGFR anticancer derivatives.
4. Concluding remarks
In this study, a series of cytotoxic agents 1,2,3 triazole-based compounds containing different aromatic substructures and different sulpha termini have
been designed, synthesized, mimics quinazoline-based EGFR inhibitors and thus evaluated for EGFR inhibitory activity. Most of the synthesized
derivatives had promising activity compared to reference inhibitor. Compounds of benzothiazole offered more potency than isatin ones and especially
5a and 5b were the most potent EGFR inhibitors with IC50 values of 103 nM and 104 nM, respectively. We also evaluated anti-proliferative activities
of the novel compounds against MCF-7, HCT-116, and HepG2 cells and found them with remarkable promising activity compared to the reference
drug. However, the pyridine and methylpyridine derivatives of benzothiazole series 5a and 5b were the most effective cytotoxic agents. in addition,
upon treatment of HepG2 cells with the most active derivatives 5a and 5b, they cause cell cycle arrest observed at G2/M phase. Finally, we discovered
15

a novel triazole-based analogue with remarkable cytotoxicity and EGFR inhibitory activity that might be further subjected for extensive optimization
to offer a new anticancer compounds.
3. Experimental Section
3.1. General Information
All reagents and solvents used were of the highest quality of analytical reagent grade and were used without further purification. Fine chemicals
including 2-mercaptobenzothiazole, Isatin and potassium carbonate were purchased from BDH Chemicals Ltd., UK. The other fine chemicals used were
sulfamethazine, sulfadiazine, sulfapyridine, sufathiazole and sulfaguanidine sodium azide, sodium nitrate, triethylamine, propargyl bromide (80 wt %
in toluene), CuSO4 and Na-ascorbate (Sigma-Aldrich, USA). The solvents used ethyl acetate, hexane, ethanol and DMF were purchased from Sigma-
Aldrich, USA. Melting points were measured on a Stuart Scientific SMP1 and are uncorrected. TLC was performed on UV fluorescent Silical gel Merck
60 F254 plates, and the spots were visualized using a UV lamp (254 nm). SHIMADZU FTIR-8400S spectrometer was used for identification of
functional groups in the range of 4000-400 cm-1. The NMR spectra were run with Bruker spectrotometer (400 MHz) with TMS as internal reference.
Synthesis and characterization of 2-(prop-2-yn-1-ylthio)benzo[d]thiazole (3)
To a mixture of 2-mercaptobenzothiazole (10 mmol), triethylamine (11 mmol) and ethanol (20 ml) was added with stirring propargyl bromide (15
mmol). The mixture was stirred at reflux for 2 h. Ethanol was removed under pressure and the resulting solid was collected and recrystallized from
ethanol to afford the targeted alkyne 2 as white pellets, 91 %, mp: 80-81 °C (lit. mp: 82-83 °C [[34]). IR (υ, cm−1): 1590 (C=C), 1610 (C=N), 2150
(C≡C), 2940 (C-H al), 3060 (C-H ar), 3310 (≡CH). 1H NMR (400 MHz, CDCl3): δH 2.33 (s, 1H, ≡CH), 4.16 (s, 2H, SCH2), 7.33 (t, 1H, J = 8.0 Hz,
Ar-H), 7.45 (t, 1H, J = 8.0 Hz, Ph-H), 7.79 (d, 1H, J = 8.0 Hz, Ph-H), 7.94 (d, 1H, J = 8.0 Hz, Ar-H). 13C NMR (100 MHz, CDCl3): δC 21.62 (SCH2);
72.39, 78.35 (C≡C); 121.11, 121.85, 124.54, 126.28, 135.49, 153.03, 164.60 (Ar-C, C=N) ppm.
Synthesis and characterization of 1-(prop-2-yn-1-yl)indoline-2,3-dione (4)
A mixture of isatin (10 mmol), potassium carbonate (11 mmol) in DMF (25 mL) was stirred at room temperature for 2 h. Then, propargyl bromide (15
mmol) was added and the reaction mixture was stirred at room temperature overnight. The mixture was poured onto crushed ice water and the precipitate
thus formed was collected by filtration and recrystallized from ethanol to afford the desired alkyne 4 as orange crystals, 95 %, mp: 155-156 °C (lit. mp:
157-158 [[34]]). IR (υ, cm−1): 1570 (C=C), 1610 (C=N), 1720 (C=O), 2150 (C≡C), 2930 (C-H al), 3050 (C-H ar), 3310 (≡CH). 1H NMR (400 MHz,
CDCl3): δ = 2.33 (s, 1H, ≡CH), 4.55 (s, 2H, NCH2), 7.14-7.21 (m, 2H, Ar-H), 7.64-7.68 (m, 2H, Ar-H). 13C NMR (100 MHz, CDCl3): δ = 29.46 (SCH2);
73.37, 75.68 (C≡C); 111.11, 117.68, 124.23, 125.49, 138.46, 149.61, 151.22, 182.56 (Ar-C, C=N, C=O) ppm.
General procedure for the synthesis of 1,4-disubstituted-1,2,3-triazoles 5a-e and 6a-e
A solution of copper sulfate (0.10 g) and sodium ascorbate (0.15 g) in water (10 mL) was added with stirring to a solution of the appropriate heterocyclic
alkyne 3 and/or 4 (1 mmol) in DMSO (10 mL). Thereafter, the appropriate sulfa-drug azide 2a-e (1 mmol) was added, and the reaction mixture was
stirred at room temperature for 8-12 h, until the consumption of the starting material as indicated by TLC (hexane-ethyl acetate). The iced-water was
added in the mixture and the precipitate thus formed was collected by filtration, washed with saturated solution of ammonium chloride and recrystallized
from ethanol/DMF to give the targeted 1,2,3-triazoles 5a-e and 6a-e.
Characterization of the click products 5a-e and 6a-e
4-(4-((Benzo[d]thiazol-2-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide (5a)
Green solid; Yield: 85 %; mp: 195-196 C; IR (υ, cm−1): 1560 (C=C), 1610 (C=N), 2960 (C-H al), 3060 (C-H Ar), 3370 (N-H). 1H NMR (400 MHz,
DMSO-d6) δH = 2.24 (s, 6H, 2 x CH3), 4.79 (s, 2H, SCH2), 6.67 (bs, 1H, Ar-H), 7.37 (t, 1H, J = 8.0 Hz, Ar-H), 7.47 (t, 1H, J = 8.0 Hz, Ar-H), 7.93 (d,
13
1H, J = 8.0 Hz, Ar-H), 8.00-8.11 (m, 5H, Ar-H), 8.91 (s, 1H, CH-1,2,3-triazole), 12.56 (bs, 1H, NH). C NMR (100 MHz, DMSO-d6) δC = 27.68
(SCH2); 29.48 (CH3); 121.77, 122.33, 122.79, 125.06, 126.88, 130.22, 135.19, 144.90, 153.02, 166.03 (Ar-C, C=N); Found: C, 47.66; H, 3.27; N,
17.59; C16H13N5O2S3 requires C, 47.63; H, 3.25; N, 17.36.
4-(4-((Benzo[d]thiazol-2-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(pyrimidin-2-yl)benzenesulfonamide (5b)
Green solid; Yield: 86 %; mp: 204-205 C; IR (υ, cm−1): 1575 (C=C), 1615 (C=N), 2945 (C-H al), 3045 (C-H Ar), 3370 (N-H). 1H NMR (400 MHz,
DMSO-d6) δH = 4.80 (s, 2H, SCH2), 7.30-7.35 (m, 3H, Ar-H), 7.41-7.46 (m, 2H, Ar-H), 7.91-7.98 (m, 5H, Ar-H), 8.29 (bs, 1H, Ar-H), 8.93 (s, 1H,
CH-1,2,3-triazole), 12.58 (bs, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δC = 27.72 (SCH2); 121.77, 122.32, 125.03, 126.87, 135.21, 153.02, 166.05
(Ar-C, C=N); Found: C, 49.80; H, 3.16; N, 20.54; C20H15N7O2S3 requires C, 49.88; H, 3.14; N, 20.36.
4-(4-((Benzo[d]thiazol-2-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(pyridin-2-yl)benzenesulfonamide (5c)
16

Green solid; Yield: 85 %; mp: 231-232 C; IR (υ, cm−1): 1570 (C=C), 1615 (C=N), 2960 (C-H al), 3070 (C-H Ar), 3335 (N-H). 1H NMR (400 MHz,
DMSO-d6) δH = 4.80 (s, 2H, SCH2), 6.86 (bs, 1H, Ar-H), 7.22 (bs, 1H, Ar-H), 7.34-7.38 (m, 1H, Ar-H), 7.45-7.48 (m, 1H, Ar-H), 7.76 (bs, 1H, Ar-H),
13
7.91-8.05 (m, 7H, Ar-H), 8.92 (s, 1H, CH-1,2,3-triazole), 12.60 (bs, 1H, NH). C NMR (100 MHz, DMSO-d6) δC = 27.72 (SCH2); 120.80, 121.78,
122.33, 122.83, 125.05, 126.87, 128.72, 135.20, 144.58, 153.02, 166.05 (Ar-C, C=N); Found: C, 52.45; H, 3.34; N, 17.35; C21H16N6O2S3 requires C,
52.48; H, 3.36; N, 17.49.
4-(4-((Benzo[d]thiazol-2-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(thiazol-2-yl)benzenesulfonamide (5d)
Green solid; Yield: 84 %; mp: 180-181 C; IR (υ, cm−1): 1570 (C=C), 1625 (C=N), 2945 (C-H al), 3070 (C-H Ar), 3385 (N-H). 1H NMR (400 MHz,
DMSO-d6) δH = 4.79 (s, 2H, SCH2), 7.35-7.46 (m, 3H, Ar-H), 7.91-8.09 (m, 7H, Ar-H), 8.91 (s, 1H, CH-1,2,3-triazole), 12.92 (bs, 1H, NH). 13C NMR
(100 MHz, DMSO-d6) δC = 27.68 (SCH2); 120.88, 121.78, 122.32, 125.04, 126.88, 127.95, 135.19, 138.87, 153.01, 166.01 (Ar-C, C=N); Found: C,
46.87; H, 3.01; N, 17.23; C19H14N6O2S4 requires C, 46.90; H, 2.90; N, 17.27.
4-(4-((Benzo[d]thiazol-2-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(diaminomethylene)-benzenesulfonamide (5e)
Green solid; Yield: 90 %; mp: 264-265 C; IR (υ, cm−1): 1580 (C=C), 1610 (C=N), 2965 (C-H al), 3040 (C-H Ar), 3250-3370 (NH2). 1H NMR (400
MHz, DMSO-d6) δH = 4.81 (s, 2H, SCH2), 6.79 (bs, 4H, 2 x NH2), 7.37 (t, 1H, J = 8.0 Hz, Ar-H), 7.48 (t, 1H, J = 8.0 Hz, Ar-H), 7.91-7.94 (m, 3H,
Ar-H), 8.01-8.04 (m, 3H, Ar-H), 8.92 (s, 1H, CH-1,2,3-triazole). 13C NMR (150 MHz, DMSO-d6) δC = 27.70 (SCH2), 120.66, 121.78, 122.34, 122.81,
125.06, 126.89, 127.84, 135.21, 138.49, 144.48, 144.79, 153.03, 158.63, 166.03 (Ar-C, C=N); Found: C, 45.81; H, 3.36; N, 21.97; C17H15N7O2S3
requires C, 45.83; H, 3.39; N, 22.01.
N-(4,6-Dimethylpyrimidin-2-yl)-4-(4-((2,3-dioxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (6a)
Orange crystals; Yield: 90 %; mp: 211-212 C; IR (υ, cm−1): 1560 (C=C), 1610 (C=N), 1730 (C=O), 2950 (C-H al), 3070 (C-H Ar), 3340 (N-H). 1H
NMR (400 MHz, DMSO-d6) δH = 2.26 (s, 6H, 2 x CH3), 5.10 (s, 2H, NCH2), 6.70-6.74 (m, 1H, Ar-H), 7.10-7.19 (m, 2H, Ar-H), 7.61-7.66 (m, 2H,
Ar-H), 7.97-8.14 (m, 4H, Ar-H), 8.96 (bs, 1H, CH-1,2,3-triazole), 12.31 (bs, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δC = 27.75 (CH3), 34.93 (NCH2),
110.98, 117.81, 119.45, 122.31, 124.65, 129.35, 132.34, 133.07, 138.24, 139.15, 140.45, 143.25, 149.86, 155.95, 158.05, 158.56 (Ar-C, C=N), 182.74
(C=O); Found: C, 56.40; H, 3.88; N, 20.01; C23H19N7O4S requires C, 56.43; H, 3.91; N, 20.03.
4-(4-((2,3-Dioxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(pyrimidin-2-yl)benzenesulfonamide (6b)
Orange crystals; Yield: 88 %; mp: 234-235 C; IR (υ, cm−1): 1575 (C=C), 1620 (C=N), 1725 (C=O), 2970 (C-H al), 3045 (C-H Ar), 3370 (N-H). 1H
NMR (400 MHz, DMSO-d6) δH = 5.08 (s, 2H, NCH2), 7.03-7.07 (m, 1H, Ar-H), 7.12-7.20 (m, 2H, Ar-H), 7.58-7.65 (m, 2H, Ar-H), 8.05-8.08 (m, 2H,
Ar-H), 8.13-8.17 (m, 2H, Ar-H), 8.51 (d, 2H, J = 4.0 Hz, Ar-H), 8.95 (s, 1H, CH-1,2,3-triazole), 12.02 (bs, 1H, NH). 13C NMR (100 MHz, DMSO-d6)
δC = 34.99 (NCH2), 111.06, 117.68, 119.96, 122.07, 123.40, 124.45, 129.46, 129.58, 138.10, 139.08, 140.22, 143.18, 149.98, 156.64, 157.83, 158.33
(Ar-C, C=N), 182.96 (C=O); Found: C, 54.56; H, 3.25; N, 21.27; C21H15N7O4S requires C, 54.66; H, 3.28; N, 21.25.
4-(4-((2,3-Dioxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(pyridin-2-yl)benzenesulfonamide (6c)
Orange crystals; Yield: 89 %; mp: 248-249 C; IR (υ, cm−1): 1565 (C=C), 1610 (C=N), 1730 (C=O), 2960 (C-H al), 3075 (C-H Ar), 3350 cm-1 (N-H).
1H NMR (400 MHz, DMSO-d6) δH = 5.08 (s, 2H, NCH2), 6.85 (s, 1H, Ar-H), 7.12-7.20 (m, 3H, Ar-H), 7.58-7.65 (m, 2H, Ar-H), 7.73-7.77 (m, 1H,
Ar-H), 7.97-8.03 (m, 5H, Ar-H), 8.93 (s, 1H, CH-1,2,3-triazole), 12.45 (bs, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δC = 34.99 (NCH2), 110.08,
117.67, 120.12, 122.02, 123.40, 124.45, 128.25, 138.10, 138.48, 143.10, 150.00, 157.83 (Ar-C, C=N), 182.96 (C=O), Found: C, 57.56; H, 3.23; N,
18.23; C22H16N6O4S requires C, 57.38; H, 3.50; N, 18.25.
4-(4-((2,3-Dioxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(thiazol-2-yl)benzenesulfonamide (6d)
Orange crystals; Yield: 86 %; mp: 260-263 C; IR (υ, cm−1): 1580 (C=C), 1615 (C=N), 1720 (C=O), 2940 (C-H al), 3055 (C-H Ar), 3380 cm-1 (N-H).
1H NMR (400 MHz, DMSO-d6) δH = 5.08 (s, 2H, NCH2), 6.86 (bs, 1H, Ar-H), 7.12-7.21 (m, 3H, Ar-H), 7.58-7.66 (m, 2H, Ar-H), 7.97-8.03 (m, 4H,
13
Ar-H), 8.93 (s, 1H, CH-1,2,3-triazole), 12.83 (bs, 1H, NH). C NMR (100 MHz, DMSO-d6) δC = 34.99 (NCH2), 111.07, 117.70, 120.18, 121.92,
123.40, 124.46, 127.60, 138.10, 138.61, 143.17, 150.03, 157.85 (Ar-C, C=N), 182.89 (C=O), Found: C, 51.50; H, 3.06; N, 17.94; C20H14N6O4S2 requires
C, 51.49; H, 3.02; N, 18.02.
N-(Diaminomethylene)-4-(4-((2,3-dioxoindolin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (6e)
Orange crystals; Yield: 91 %; mp: 270-271 C; IR (υ, cm−1): 1560 (C=C), 1625 (C=N), 1715 (C=O), 2930 (C-H al), 3065 (C-H Ar), 3330-3445 cm-1
(N-H). 1H NMR (400 MHz, DMSO-d6) δH = 5.09 (s, 2H, NCH2), 6.78 (bs, 4H, 2 x NH2), 7.15 (t, 1H, J = 8.0 Hz, Ar-H), 7.22 (d, 1H, J = 8.0 Hz, Ar-
13
H), 7.61 (d, 1H, J = 8.0 Hz, Ar-H), 7.65 (t, 1H, J = 8.0 Hz, Ar-H), 7.92-8.01 (m, 4H, Ar-H), 8.95 (s, 1H, CH-1,2,3-triazole). C NMR (100 MHz,
DMSO-d6) δC = 35.48 (NCH2), 111.58, 118.19, 120.44, 122.48, 123.90, 124.98, 127.87, 138.47, 138.60, 143.60, 144.80, 150.49, 158.36, 158.63 (Ar-
C, C=N), 183.49 (C=O), Found: C, 50.79; H, 3.49; N, 23.01; C18H15N7O4S requires C, 50.82; H, 3.55; N, 23.05.
4.2. Cytotoxicity evaluation using a viability assay
17

The cytotoxic activity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay as reported
previously [43, 44, 62]. In brief, the tumor cell lines were suspended in medium at concentration 5 x 104 cell/well in Corning® 96-well tissue culture
plates and then incubated for 24 h. The tested compounds with concentrations ranging from 0 to 50 μg/ml were then added into 96-well plates (six
replicates) to achieve different conc. for each compound. Six vehicle controls with media or 0.5 % DMSO were run for each 96 well plate as a control.
After incubating for 24 h, the numbers of viable cells were determined by the MTT test. Briefly, the media was removed from the 96 well plates and
replaced with 100 μl of fresh culture RPMI 1640 medium without phenol red then 10 µl of the 12 mM MTT stock solution (5 mg of MTT in 1 mL of
PBS) to each well including the untreated controls. The 96-well plates were then incubated at 37 οC and 5 % CO2 for 4 h. An 85-μl aliquot of the media
was removed from the wells, and 50 µl of DMSO was added to each well and mixed thoroughly with the pipette and incubated at 37 οC for 10 min.
Then, the optical density was measured at 590 nm with the microplate reader (Sunrise, TECAN, Inc, USA) to determine the number of viable cells and
the percentage of viability was calculated as [1 - (ODt/ODc)] x 100 % where ODt is the mean optical density of wells treated with the tested sample
and ODc is the mean optical density of untreated cells. The relation between surviving cells and drug concentration is plotted to get the survival curve
of each tumor cell line after treatment with the specified compound. The 50 % inhibitory concentration (IC50), the concentration required to cause toxic
effects in 50 % of intact cells, was estimated from graphic plots of the dose response curve for each conc. using Graph pad Prism software (San Diego,
CA. USA).
4.3. EGFR wild type assay
EGFR kinase kit was purchased from BPS Bioscience. Briefly, EGFR-ErbB1 (catalog number PV3872), 0.200ꢀmg/mL were used. An ATP solution and
a kinase/peptide mixture were prepared right before use. The solutions on the plate were mixed thoroughly, and the plate was incubated for 1ꢀh at room
temperature. After that, 5ꢀml of the developing solution was added to each well. The plate was incubated for 1ꢀh at room temperature and then read by
ELISA Reader (PerkinElmer, USA). Curve fitting was performed using Graph Pad Prism. Every experiment was repeated three times. Data represented
as meansꢀ±ꢀSD from three independent experiments.
4.4. Quantification of apoptosis by flow cytometry
Annexin V-FITC apoptosis assay was performed by using Annexin V-FITC/PI double staining detection kit (BD Pharmingen, USA).[63, 64] Flow
cytometric analysis was performed on FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Annexin V-FITC was detected
through (FL1) channel while PI was detected through the (FL2) channel. Finally, a minimum of 10,000 cells per sample were acquired and analyzed
using Cell Quest Pro software (BD Biosciences).
4.6. Cell cycle analysis
Cell cycle arrest and distribution was assessed, using the Propidium Iodide Flow Cytometry Kit (ab139418, Abcam) followed by flow cytometry
analysis. The DNA content in each cell nucleus was determined by a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Finally,
cell cycle phase distribution was analyzed using Cell Quest Pro software (BD Biosciences) showing collected propidium iodide fluorescence intensity
on FL2 [65].
Molecular docking
Molecular docking of target compounds into the EGFR protein enzyme compromising the crystal structure of enzyme (PDB ID: 1M17) were retrieved
from PDB server (http://www.pdb.org) with acceptable resolution using the most active compounds 5a and 5b derivatives. The AutoDock 3.0 [56] and
the MOE software [40] were used for all the docking calculations. The detailed protocol was adopted in [39]. The binding energy file generated from
docking experiments was used for calculation of the inhibition constants. The protein-ligand interaction plots were generated, using MOE 2012.10. The
standard parameter settings were applied. High-scoring binding poses were selected on the basis of visual inspection.
18

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20

Highlights
1. Introducing of 1,2,3-Triazole-based scaffold for design of EGFR inhibitors.
2. Anticancer screening of novel analogues against a panel of cancer cell lines.
3. Molecular docking analyses was done for SAR interpretation.
4. ADMET studies were done for most active derivatives.
21