Novel benzo[4,5]thiazolo[2,3-C][1,2,4]triazoles: Design, synthesis, anticancer evaluation, kinase profiling and molecular docking study

Article abstract of DOI:10.1016/j.molstruc.2021.131138

In the current study, we report the synthesis and cytotoxic evaluation of a new series of S-benzo[4,5]thiazolo[2,3-c][1,2,4]triazole-based derivatives 8-12. Cytotoxicity of the new compounds was investigated in A549, MCF-7, and Hep3B cancer cell lines. Among these derivatives, compound 12 bearing an isatin moiety was the most active derivative (IC₅₀ = 2.40-3.53 μM). A mechanistic study of compound 12 was performed using the kinase profiling test to explore its inhibitory activity against 10 types of the oncogenic kinases and the potential activation of caspase 3/7 enzymes. The results revealed that compound 12 showed moderate inhibition of the EGFR and LCK kinases. Moreover, compound 12 also activated caspase-3/7 in A549 cells. The docking study of compound 12 into EGFR ATP-active site revealed that it fits nicely with good binding affinity. Together, the results indicated that compound 12 could serve as a good lead for developing new potential anticancer agents.

Full text of DOI:10.1016/j.molstruc.2021.131138

Journal of Molecular Structure 1246 (2021) 131138
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Novel benzo[4,5]thiazolo[2,3-C][1,2,4]triazoles: Design, synthesis,
anticancer evaluation, kinase profiling and molecular docking study
Ahmed H. Abdelazeema,b,∗, Alaa M. Alqahtanic, Hany H. Arabd, Ahmed M. Goudaa,
Asmaa G. Safi El-Dina
a Department of Medicinal Chemistry, College of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt
b Department of Pharmaceutical Sciences, College of Pharmacy, Riyadh Elm University, Riyadh 11681, Saudi Arabia
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Umm Al-Qura University, Makkah 21955, Saudi Arabia
d Department of Pharmacology and Toxicology, College of Pharmacy, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
In the current study, we report the synthesis and cytotoxic evaluation of
a new series of S-
Received 8 April 2021
Revised 5 June 2021
Accepted 16 July 2021
Available online 20 July 2021
benzo[4,5]thiazolo[2,3-c][1,2,4]triazole-based derivatives 8-12. Cytotoxicity of the new compounds was
investigated in A549, MCF-7, and Hep3B cancer cell lines. Among these derivatives, compound 12 bearing
an isatin moiety was the most active derivative (IC50 = 2.40-3.53 μM). A mechanistic study of compound
12 was performed using the kinase profiling test to explore its inhibitory activity against 10 types of the
oncogenic kinases and the potential activation of caspase 3/7 enzymes. The results revealed that com-
pound 12 showed moderate inhibition of the EGFR and LCK kinases. Moreover, compound 12 also acti-
vated caspase-3/7 in A549 cells. The docking study of compound 12 into EGFR ATP-active site revealed
that it fits nicely with good binding affinity. Together, the results indicated that compound 12 could serve
as a good lead for developing new potential anticancer agents.
Keywords:
S-benzo[4,5]thiazolo[2,3-c][1,2,4]triazole
anticancer
Kinase
caspase3/7
Apoptosis
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
small cell lung cancer [7,8]. However, resistance has been also re-
ported to some MKIs [9].
Cancer has been regarded as a major health problem worldwide
and comes after the cardiovascular diseases as the most common
causes of death. It accounts for one in each seven deaths in the
world [1]. Despite the remarkable improvement and advances in
cancer treatment, the high rate of incidence and mortality associ-
ated with cancer leads the world for continuous research for new
anticancer agents [2]. Moreover, the multi-drug resistance (MDR)
in cancer chemotherapy represents a big challenge for researchers
in this field. Several reports have addressed this problem for more
than twenty years ago [3-5]. Accordingly, many approaches have
been developed to improve patient outcomes and decrease the
probability for emerging MDR. Combination chemotherapy is one
of these approaches which hits more than one target in cancer
cells [6]. The emerging of multikinase inhibitors (MKIs) as power-
ful agents in treatment of cancer represents also a new strategy to
overcome MDR. Several derivatives of these classes displayed high
efficacy in treatment of several types of cancers including non-
The above-mentioned drawbacks enhanced the extensive re-
search to discover and develop new and more potent anticancer
agents. In this regard, we have reviewed the literature searching
for promising scaffolds with potential anticancer activity. Of these
scaffolds, the 1,2,4-triazole ring was found in many biologically ac-
tive agents with diverse biological activity such as antimicrobial
[10], anti-inflammatory [11], analgesic [12] and anticancer activities
[13-15]. Moreover, several derivative bearing benzothiazole or thi-
azole scaffolds were reported with potent and selective anticancer
activity [16,17].
Previously, we have incorporated the 1,2,4-triazole and ben-
zothiazole scaffolds into a single scaffold, benzo[4,5]thiazolo[2,3-
c][1,2,4]triazole [18,19]. In these studies, we have inves-
tigated the anticancer activity of the two series bearing
benzo[4,5]thiazolo[2,3-c][1,2,4]triazole scaffold. Of the first se-
ries, compounds 1a,b displayed potent anticancer activity with
IC50 values in the range of 11.1-21.5 μM [18]. Mechanistic studies
of compound 1a revealed weak inhibitory activity against CDK-2.
The cytotoxic activity of compound 1a was medicated by induction
of caspase 3/7.
∗
Corresponding author at: Department of Medicinal Chemistry, Faculty of Phar-
The cytotoxic activity of compounds 2a-c (Fig. 1) was exam-
ined in A549, MCF-7, and Hep3B cancer cell lines and the ob-
macy, Beni-Suef University, Beni-Suef 62514, Egypt.
E-mail address: ahmed.abdelazeem@pharm.bsu.edu.eg (A.H. Abdelazeem).
https://doi.org/10.1016/j.molstruc.2021.131138
0022-2860/© 2021 Elsevier B.V. All rights reserved.

A.H. Abdelazeem, A.M. Alqahtani, H.H. Arab et al.
Journal of Molecular Structure 1246 (2021) 131138
Fig. 1. Anticancer agents based on benzo[4,5]thiazolo[2,3-c][1,2,4]triazole.
Fig. 2. Rational design and structural modifications of scaffold A
served IC50 ranged between 3.17 and14.18 μM [18,19]. Notably,
compound 2c was the most active among the three compounds for
combating cancer cell line growth. Mechanistically, the three com-
pounds 2a-c triggered marked apoptosis as proven with the activa-
tion of caspase-3/7 pro-apoptotic markers. Regarding CDK-2 signal,
the three compounds demonstrated a weak to moderate inhibition
against the aforementioned target.
recorded using Shimadzu Qp-2010 plus apparatus at the microana-
lytical center, Faculty of Science, Cairo University. The starting ma-
terials 3-7 were prepared according to the reported synthetic path-
ways [20, 21].
2-(2-(benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-ylthio)acetyl)hydrazi
necarbothioamide (8). A mixture of KSCN (1.5 g, 15 mmol) and
conc. HCl (2 mL) was added to a solution of acid hydrazide 7 (2.79
g, 10 mmol) in CH3OH (40 mL) with stirring at room temperature
for 30 min. The solvent was immediately evaporated in vacuo
to dryness and refluxed for more 1h with another 30 mL of
CH3OH. The resulting solid was treated with water and ethanol
to afford thiosemicarbazide derivative 8 as off-white solid (75%).
m.p. 179-180°C. 1H NMR (400 MHz, DMSO-d6): δ 11.51 (s, 2H,
NH2), 8.18 (d, J = 7.9 Hz, 1H), 8.14 (s, 1H, NH), 8.10 (d, J = 7.7
Hz, 1H), 7.64 (t, J = 7.4 Hz, 1H), 7.53 (t, J = 7.4 Hz, 1H), 4.12 (s,
2H, SCH2), 3.16 (s, 1H, NH). 13C NMR (101 MHz, DMSO): δ 169.72
(C=S), 166.80 (C=O), 131.91, 129.98, 127.63, 127.58, 126.99, 126.04,
115.00, 114.82, 35.53 (SCH2). MS (EI) m/z 338 (M+). Anal. calcd.
for: C18 H11 N5O3S2: C, 52.80; H, 2.71; N, 17.10. Found: C, 52.61; H,
2.93; N, 16.97.
Encouraged by the above mentioned findings, we designed a
new series of benzo[4,5]thiazolo[2,3-c][1,2,4]triazole analogs, aim-
ing to optimize their cytotoxic potential. The novel compounds
were obtained through modification of compound 1b structure.
The study of SAR was achieved using two different modifications.
The first, was extending the length of the side chain by two atoms.
The second was chain cyclization of the acetohydrazide side chain
into various heterocyclic rings including pyrrole, pyrazole, phthalyl
and isatin rings, Fig. 2. The newly synthesized compounds were in-
vestigated for their anticancer activity and the possible mechanism
of action.
2. Materials and methods
1-(2-(benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-ylthio)acetyl)pyrazoli
dine-3,5-dione (9). Diethyl malonate (0.29 g, 1.8 mmol) was added
to a solution of acid hydrazide 7 (0.5 g, 1.8 mmol) in glacial acetic
acid (15 mL) and the mixture was refluxed overnight. The reaction
mixture was allowed to cool and the formed precipitate was
filtered, washed with diethyl ether and dried to furnish a white
solid (45%) of compound 9, m.p. 255-257°C. 1H NMR (400 MHz,
DMSO-d6): δ 10.43 (s, 1H, NH), 8.19 (d, J = 15.8 Hz, 1H), 8.08 (d,
J = 7.5 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.52 (t, J = 7.1 Hz, 1H),
4.01 (s, 2H, SCH2), 3.97 (s, 2H, CH2). 13C NMR (101 MHz, DMSO): δ
172.51 (C=O), 168.06 (C=O), 165.44 (C=O), 156.80, 142.61, 131.89,
130.03, 127.52, 126.89, 125.94, 115.04, 36.20 (SCH2), 36.08 (CH2).
2.1. Chemistry
The solvents and chemical reagents were purchased from com-
mercial vendors. Melting points (m.p.) were uncorrected and were
recorded using IA 9100MK-Digital Melting Point Apparatus. Micro-
analyses were done at the microanalytical center, Faculty of Sci-
ence, Cairo University. The proton magnetic spectra were recorded
on BRUKER AVANCE III at 400 MHz (Faculty of Pharmacy, Beni-
Suef University, Egypt) in DMSO-d6. The J constant is given in
Hz. The 13C-NMR spectra of the newly synthesized compounds in
CDCl3/DMSO-d6 were carried out at 101 MHz. Mass spectra was
2

A.H. Abdelazeem, A.M. Alqahtani, H.H. Arab et al.
Journal of Molecular Structure 1246 (2021) 131138
MS (EI) m/z 347 (M+). Anal. calcd. for: C13H9N5O3S2: C, 44.95; H,
2.61; N, 20.16. Found: C, 44.74; H, 2.39; N, 19.89.
cells for a time span of 72 h in a culture medium containing 5%
FBS. Finally, the MTT solution (0.5 mg/mL) was added for 2 h at
37°C in a humidified incubator containing 5% CO2. Finally, DMSO
(200 μL/well) was used to solubilize the resultant reduced form of
MTT, and the final absorbance was read at 570 nm with the aid of
a microplate reader, as depicted in Table 1.
2-(benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-ylthio)-N-(2,5-dioxo-
2,5-dihydro-1H-pyrrol-1-yl) acetamide (10). Maleic anhydride (0.2 g,
2 mmol) was added to a mixture of acid hydrazide 7 (0.5 g, 1.8
mmol) and anhyd. CH3COONa (0.2 g, 2.5 mmol) in gl. acetic acid
(20 mL). The reaction mixture was heated under reflux overnight.
The reaction mixture was concentrated in vacuo and poured onto
cold ice-water. The formed precipitate was filtered off, dried to
furnish a light brown solid (43%) of compound 10, m.p. 159-160°C.
1H NMR (400 MHz, DMSO-d6): δ 10.42 (s, 1H, NH), 8.17 (d, J = 8.1
Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.52 (t,
J = 7.7 Hz, 1H), 6.97 (s, 2H), 4.11 (s, 2H, CH2). 13C NMR (101 MHz,
DMSO): δ 172.45 (C=O), 166.68 (2C=O), 152.87, 145.86, 142.59,
131.88, 129.80, 127.51, 126.90, 122.71, 114.73, 138.24 (SCH2). MS
(EI) m/z 359 (M+). Anal. calcd. for: C14 H9N5O3S2: C, 46.79; H, 2.52;
N, 19.49. Found: C, 46.97; H, 2.63; N, 19.24.
2.2.2. Kinase profiling assay protocol
The assay of the kinase profiling was performed at the Reaction
Biology Corp., PA, USA, following the previous report [24]. The re-
quired cofactors were added individually to each kinase reaction.
Compound 12 was dissolved in 100% DMSO to the desired con-
centration. For the assay, the substrate solution (in fresh reaction
buffer) was added to the corresponding cofactor and then, the de-
sired kinase was supplemented. After mixing the kinase reaction
mixture, compound 12 (dissolved in DMSO) was added by means
of the Acoustic technology (Echo550; nanoliter range) for an incu-
bation time of 20 min at ambient temperature. The kinase reaction
was initiated via adding 33P-ATP (10 μCi/μl specific activity) to the
reaction mixture for 2 h at ambient temperature and the radioac-
tivity was measured by the filter-binding method. The kinase activ-
ity of the test compound was presented as the % remaining kinase
activity, versus the vehicle (DMSO), as outlined in Fig. 4.
2-(benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-ylthio)-N-(1,3-
dioxoisoindolin-2-yl)acetamide (11). A mixture of acid hydrazide 7
(0.56 g, 2 mmol), phthalic anhydride (0.3 g, 2 mmol) and anhyd.
CH3COONa (5 mmol, 295 mg) was heated under reflux in gl. acetic
acid (15 mL) overnight. The mixture was left aside to cool, the
formed precipitate was collected, washed with water, and dried
affording a white solid (81%) of compound 11, m.p. 166-167°C. 1H
NMR (400 MHz, DMSO-d6): δ 11.06 (s, 1H, NH), 8.21 (d, J = 8.1
Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.97-7.89 (m, 4H), 7.60 (t, J = 7.8
Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 4.26 (s, 2H). 13C NMR (101 MHz,
DMSO): δ 167.19 (C=O), 165.15 (2 C=O), 156.85, 142.45, 135.74,
131.89, 130.02, 129.85, 127.45, 126.90, 125.90, 124.21, 115.19, 36.00
(SCH2). MS (EI) m/z 409 (M+). Anal. calcd. for: C18 H11 N5O3S2: C,
52.80; H, 2.71; N, 17.10. Found: C, 52.61; H, 2.93; N, 16.97.
2.2.3. Caspase 3/7 activation assay
The activity of caspase 3/7 was carried out using Promega
caspase-Glo 3/7 luminescence assay kit (Promega, Madison, WI) in
A549 cells treated with the tested compound 12. The directions of
the manufacturer were strictly followed as formerly described [25].
In clear bottom/opaque wall 96-well tissue culture plates, the cells
were seeded at 1×104 (100 mL/well) for 24 h to allow adherence
to the plate bottom. Then, the test compound 12 was added for an-
other 24 h. Finally, the caspase 3/7 activity was measured in terms
of luminescence by means of a luminometer, as instructed by the
vendor, and the data were depicted in Fig. 5.
2-(benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-ylthio)-N’-(2-
oxoindolin-3-ylidene)acetohydrazide (12). Isatin (0.3 g,
2 mmol)
was added gradually to a solution of acid hydrazide 7 (0.56 g, 2
mmol) and in gl. acetic acid (15 mL) and the mixture was refluxed
overnight. During the reaction, a yellow solid was partially crys-
tallized out. The formed precipitate was filtered off after cooling,
washed with cold ethanol to give a deep yellow solid (86%) of
compound 12 m.p. 236-237°C. 1H NMR (400 MHz, DMSO-d6): δ
10.15 (s, 1H, NH), 8.14 (d, J = 8.1 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H),
7.87-7.79 (m, 4H), 7.63 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.8 Hz, 1H),
4.12 (s, 2H, CH2). 13C NMR (101 MHz, DMSO): δ 171.62 (C=O),
169.80 (C=O), 156.57, 143.01, 141.94, 141.20, 134.15, 131.91, 129.87,
127.57, 126.93, 126.06, 123.04, 121.34, 119.65, 117.34, 114.82, 36.34
(SCH2). MS (EI) m/z 408 (M+). Anal. calcd. for: C18 H12N6O2S2: C,
52.93; H, 2.96; N, 20.58. Found: C, 52.71; H, 2.70; N, 20.37.
2.2.4. Statistical analysis
Results were analyzed for the statistical significance using one-
way analysis of variance and the multi-comparison tests were car-
ried out using Neuman-Keuls test, using SPSS version 17 software
(SPSS, Chicago, IL, USA). The statistical significance was determined
at P < 0.05.
2.2.5. Molecular docking study
The crystal structure of EGFR (pdb code: 1M17) and LCK
(pdb code: 3KMM) were retrieved from the protein data bank
(http://www.rcsb.org/pdb). AutoDock 4.2 [25] was used to per-
form the docking study. The ligand, protein files were pre-
pared according to the previous report [26, 27]. The 2/3D bind-
ing modes were created using Discovery Studio Visualizer (DSV,
v16.1.0.15350)(Dassault systems BIOVIA, Discovery Studio Visual-
izer, v16.1.0.15350.2016) [28]. The results were represented in
Table 2, and Fig. 6 and 7.
2.2. Biological screening
2.2.1. Anticancer activity
2.2.1.1. Cell culture. In the current study, three cancer cell lines
(MCF-7, A549 and Hep3B; American Type Culture Collection, ATCC,
Manassas, VA) were used for the evaluation of the growth in-
hibitory activity of the synthesized compounds. As instructed by
the ATCC, the three cell lines were cultured in Dulbecco’s modi-
fied Eagle’s medium (DMEM), DMEM/ F12 medium (DMEM/F-12),
or RPMI-1640 media (Gibco, Grand Island, NY) with the provision
of 10% fetal bovine serum (FBS; Gibco). The culture conditions were
maintained at a humidified incubator containing 5% CO2 at 37°C.
3. Results and discussion
3.1. Chemistry
Synthesis of the starting materials 4 and 5 was performed
as previously described [19] (Bowe and Doyle 1957; Aboelmagd
et al. 2013) and described in Scheme 1. The reaction of ethyl
2.2.1.2. Cell viability assay. The ability of the synthesized S-
Benzo[4,5]thiazolo[2,3-c][1,2,4]triazole compounds 8-12 to inhibit
the cell viability of the three cancer cell lines (MCF-7, A549, and
Hep3B) was examined with the aid of the MTT assay, as previously
described [22, 23]. In 96-well plates, the cells were seeded first
for 24 h and the synthesized compounds were incubated with the
bromoacetate with compound
5 afforded ethyl S-triazolo[3,4-
b]benzothiazol-3-ylthioacetate 6. Subsequently, the treatment of
the produced ester 6 with hydrazine hydrate afforded the hy-
drazide 7 in a good yield.
3

A.H. Abdelazeem, A.M. Alqahtani, H.H. Arab et al.
Journal of Molecular Structure 1246 (2021) 131138
Table 1. The anticancer evaluation results of the newly synthesized S-benzothiazolotriazole
derivatives (8-12), in terms of IC50 values, against MCF-7, Hep3B, and A549 cancer cell lines.
IC50 (μM)
Comp. No
R
MCF-7
Hep3B
A549
8
6.12 0.43
5.70 0.40
9.17 0.57
9
11.07 0.67
13.23 0.90
5.07 0.47
11.70 0.70
12.80 0.87
4.93 0.70
12.20 0.80
11.40 0.73
7.03 0.63
10
11
12
2.90 0.27
2.3 0.15
2.40 0.32
2.4 0.17
3.53 0.43
1.7 0.31
Doxorubicin
The vehicle or the newly synthesized compounds were incubated for 72 h in the three can-
cer cell lines and the obtained data were expressed as the mean
S.D. (n = 6).
Table 2
Binding affinities/interactions of compounds 8-12 AQ4and LHL into EGFR and LCK.
Atoms in H-bonding
a
b
c
Pdb
Ligand
ꢀGb
Ki
HBs
In ligand
In protein
1M17
8
-7.59
-9.02
-8.55
-9.55
-9.58
-7.39
-6.58
-8.79
-8.33
-9.30
-8.96
-9.08
2.72 μM
4
2
1
2
1
1
4
2
2
2
1
2
Triazole N, S, NH2
S, CO
Cys751, Arg817, Asp831
9
242.67 nM
544.51 nM
100.12 nM
95.46 nM
3.84 μM
Lys721, Met769
10
11
12
Triazole N
Triazole N, CO
Triazole N
Quinazoline N
S, SO, CO, NH
S, CO
Thr830
Lys721, Met769
Thr830
d
AQ4
Met769
3KMM
8
15.05 μM
362.73 nM
784.16 nM
153.12 nM
270.3 nM
219.9 nM
Lys273, Glu288, Met292, Thr316, Met319,
9
Lys273, Met319
Lys273, Met319
Met319
10
11
12
LHL
S, CO
Two CO
NH
Glu288
N1, NH
Met319
a
Binding free energy (kcal/mol)
Inhibition constant
b
c
Number of hydrogen bonds
AQ4, erlotinib
d
Moreover, a singlet signal at 10.42 ppm was observed in the 1H-
NMR spectrum of compound 10, indicating the NH proton.
The acid hydrazide 7 was cyclized into various heterocyclic
rings using several reagents such as diethyl malonate, maleic anhy-
dride, isatin or phthalic anhydride (Scheme 2) to give compounds
8-12.
The spectrum of compound 11 revealed the singlet signal of the
methylene group at 4.26 ppm, while 1H-NMR spectrum of com-
pound 12 showed one singlet signal at 4.12 assigned to the methy-
lene group. On the other hand, 13C-NMR of compound 9 revealed
three signals at 172.51, 168.06, 165.44 assigned for the three car-
bonyl groups. The spectrum of compound 10 revealed two signals
at 172.45 and 166.68 ppm for the three carbonyl groups where the
two C=O groups of pyrrole-2,5-dione ring are equivalent. Further-
Characterization of compound 10-12 was done using spectral
and elemental analyses. As described by the 1H-NMR, compounds
8 showed two singlet signals at 4.12 and 11.51 ppm indicating the
methylene group (SCH2) and amino (NH2) groups, respectively. For
compound 9, the spectrum also showed two singlet signals at 4.01
and 3.97 ppm indicated the methylene groups (SCH2 and CH2).
4

A.H. Abdelazeem, A.M. Alqahtani, H.H. Arab et al.
Journal of Molecular Structure 1246 (2021) 131138
Scheme 1. The used reagents and conditions of the reaction: a) NH2NH2.H2O, reflux, EtOH, 20 h; b) i. CS2, KOH, EtOH, reflux; ii. Conc. HCl; c) Ethyl bromoacetate, K2CO3,
reflux, acetone; d) N2H4.H2O, reflux, EtOH, 1 h.
Scheme 2. The used reagents and conditions of the reaction: a) KSCN, HCl, CH3OH, rt, 30 min, reflux, 1 h; b) Diethyl malonate, AcOH, 10 h; c) Maleic anhydride, sod. acetate,
AcOH, reflux, 10 h; d) Phthalic anhydride, xylene, reflux, 12 h; e) Isatin, AcOH, reflux, 2 h.
more, two signals at δ 167.19 and 165.15 ppm indicating three two
carbonyl carbons of compound 11; two of them in the phthalyl
moiety are equivalent. Moreover, the spectrum of compound 12 re-
vealed two signals at 171.62 and 169.80 ppm for the two carbonyl
groups. Meanwhile, the molecular ion peaks were observed in the
mass spectra at m/z 338 (M+) for compound 8, 347 (M+) for com-
pound 9, 359 (M+) for compound 10, 409 (M+) for compound 11
and 408 (M+) for compound 12.
carcinoma) cell lines. The results were presented as IC50 values
in Table 1.and we used doxorubicin as the positive reference an-
ticancer agent. In the three cell lines, the new compounds demon-
strated IC50 values ranging from 2.40 to 13.23 μM against the three
cell lines. Notably, compound 12 bearing an isatin moiety showed
the highest anticancer activity in the three cell lines where the ob-
tained IC50 values ranged from 2.40 to 3.53 μM. The highest ac-
tivity was against Hep3B followed by MCF-7 cells. Compound 8
also showed comparable cytotoxic activity, as seen in Hep3B and
MCF-7 cells (IC50 = 6.12 and 5.70 μ M, respectively). Both com-
pounds 9 and 10 with the five-membered cycles were the least
active, where all the IC50 values were above 10 μM against the
three cell lines. Regarding compound 11 that harbors the phtha-
lyl moiety, it displayed better activity than compounds 9 and 10
with IC50 values between 4.93-7.03 μM. It could be concluded
that the bulkiness effect of the various substitutions has a signif-
icant impact on the anticancer activity, and this was observed in
compounds 11 and 12 with the bulky groups, isatin and phthalyl
moieties.
3.2. Biological screening
3.2.1. In vitro anticancer activity
We examined the anticancer activity of the synthesized set
of compounds by evaluating the cancer cell growth inhibi-
tion with the aid of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) assay in six replicates according to
the previously reported methodology [22,23]. The anticancer ac-
tivity was examined in MCF-7 (human breast carcinoma), A549
(human lung adenocarcinoma), and Hep3B (human hepatocellular
5

A.H. Abdelazeem, A.M. Alqahtani, H.H. Arab et al.
Journal of Molecular Structure 1246 (2021) 131138
Fig. 3. Kinase inhibitory activity % of compound 12 against 10 kinases.
3.2.2. Kinase profiling assay
(12) result in a significant increase in the cytotoxic activity against
all the three cancer cell lines. Secondly, decyclization and expan-
sion the length of the side chain of hydrazide derivatives by two
atoms as shown in compound (8) result in variable cytotoxic ac-
tivity against the three cancer cell lines. Its cytotoxic activity was
improved comparing to compound (1b), however it was still lower
than compound (12). From all aforementioned results, it was con-
cluded that replacement of pyrazole in compound (1b) by isatin in
compound (12) had the highest impact on the effectiveness in this
study promising further improvement.
It was previously reported that 1,2,4-triazole/benzothiazole-
bearing agents mediate the anticancer potential through inhibition
of several kinases [18,19]. To delineate the potential mechanism
of action of compound 12 (the most active compound in the se-
ries), the kinase-profiling assay was performed against 10 kinases
selected of various types and families. Using the radiolabeled ATP
determination method [24], the kinase profiling was carried out at
the Reaction Biology Corp., PA, USA. The results revealed that com-
pound 12 has moderate inhibitory activity against EGFR, and LCK,
while weak inhibition was observed against eight of the tested ki-
nases, Fig. 3. The highest inhibition caused by compound 12 was
against both EGFR and LCK kinases.
3.4. Docking study
The oncogenic kinases play an important role as regulators of
intracellular communication. Perturbation of the tight control of
their activity lead to tumor growth [29, 30]. Accordingly, these ki-
nases represent important targets in the discovery of new anti-
cancer agents [31]. Compound 12 displayed appreciable inhibition
against two kinase including EGFR and LCK where the highest in-
hibitory activity was against EGFR. Although this inhibitory activity
is weak, but compound 12 with its pharmacophoric groups can hit
several kinases at the same time. Therefore, this scaffold can un-
dergo future optimization into multi-kinase inhibitors. To under-
stand the biological results (Fig. 2), the new compounds 8-12 were
docked into the active sites of EGFR (pdb code: 1M17) [32] and LCK
(pdb code: 3KMM) using AutoDock 4.2 (Dassault systems BIOVIA,
Discovery Studio Visualizer, v16.1.0.15350.2016) [28]. The aim of
this study was to evaluate the binding mode, affinity, and inter-
actions of compound 12 versus those of compounds 8-11 and the
co-crystallized ligands (AQ4 and LHL).
3.2.3. Caspase 3/7 assay
According to the MTT assay, compound 12 demonstrated the
highest anticancer activity. Hence, we used the caspase 3/7 assay
to further characterize its ability to trigger apoptosis in the A549
cells. With the aid of Promega caspase-Glo 3/7 assay kit (Promega,
Madison, WI), the luminescence was used as an index of the cas-
pase 3/7 activity under strict adherence to the vendor instructions
[25], as depicted in Fig. 4. The results demonstrated that com-
pound 12 activated the caspase 3/7 enzymes in A549 cells at 5 μM,
10 μM, and 20 μM with nearly 3- to 4-fold, respectively, com-
pared to the control. Activation of caspase 3/7 indicates the ability
of compound 12 to act as an apoptosis inducer.
3.3. Structure activity relationship (SAR)
The detailed SAR study of the cytotoxic activity of compounds
8-12 was investigated and presented in Fig. 5. Firstly, the study was
carried out through the replacement of the central part in the re-
ported compound (1b) with various heterocyclic rings in attempt
to improve its cytotoxic activity. Noticeable increase in the cyto-
toxic activity against the three (MCF-7, A2780 and HT29) cancer
cell lines was observed during pyrazole replacement in compound
(1b) by diethyl malonate (9), phthalic anhydride (11). By focus-
ing, the replacement of diethyl malonate in (9) by maleic anhy-
dride in (10) lead to decrease in the cytotoxic activity. On the con-
trary, upon replacement of phthalic anhydride in (11) with isatin in
Validation of the used docking protocols into EGFR was
achieved by redocking erlotinib into the active site of the used ki-
nase. The results proved superposition of the redocked erlotinib
˚
with the co-crystallized one with RMSD of 1.44 A. In addition,
the redocked ligand showed similar binding interactions with the
key amino acids in the active site of EGFR kinase. The nitrogen
atom (N2) of the quinazoline ring in the redocked erlotinib showed
one conventional H-bond with Met769 like the co-crystallized er-
lotinib. In a similar manner for the co-crystallized ligand, the re-
docked erlotinib revealed two carbon H-bonds with Gln767 and
6

A.H. Abdelazeem, A.M. Alqahtani, H.H. Arab et al.
Journal of Molecular Structure 1246 (2021) 131138
Fig. 4. The caspases 3/7 activity for compound 12 in A549 cancer cell line. After a 48-h incubation period with the compound, the activity of caspase-3/7 was measured
with the aid of Promega Caspase-Glo Assay Kit. The obtained data were expressed as the mean
S.D. (n = 4).
Fig. 5. SAR of cytotoxicity of compounds 8-12 against the three cell lines (MCF-7, A2780 and HT29).
Thr830. The two ligands also showed similar hydrophobic inter-
which is higher than the binding free energy for the native ligand,
erlotinib (-7.39 kcal/mol), Table 2. The binding mode of compound
12 into EGFR was also investigated, Fig. 6. The thiazolo-triazole
moiety in compound 12 overlaid with the ethynyl phenyl moiety
in erlotinib. The indolin-2-one in compound 12 superposed the po-
sition of the quinazoline nucleus, while the linker in compound 12
superposed over the position of the linker in erlotinib. Moreover,
one conventional hydrogen was observed between compound 12
actions with Leu694, Ala719, Lys721, Leu820. On the other hand,
the validation of the docking study into LCK kinase was also per-
formed where the redocked ligand, LHL superposed the position of
˚
the co-crystallized ligand with RMSD of 1.45 A with similar bind-
ing interactions.
The results of the docking simulation of the new compounds 8-
12 into the active site of the two kinases are presented in Table 2.
Analysis of the docking results into EGFR revealed the high-
est binding free energy (ꢀGb) of -9.58 kcal/mol for compound 12,
˚
and MET769 (bond length = 2.62 A). Compound 12 also formed
two pi-donor hydrogen bonds with Thr766. In addition, compound
7

A.H. Abdelazeem, A.M. Alqahtani, H.H. Arab et al.
Journal of Molecular Structure 1246 (2021) 131138
Fig. 6. 2/3D Docking mode of compound 12 (shown as sticks colored by element) into EGFR: A) 3D binding mode of compound 12 into EGFR overlaid with the co-crystallized
erlotinib (shown as cyan stick), amino acids shown as lines; B) 2D Docking mode of compound 12 showing different types of interactions, hydrogen was omitted for clarity.
Fig. 7. 2/3D Docking mode of compound 12 (shown as sticks colored by element) into LCK (pdb: 3KMM): A) 3D binding mode of compound 12 into EGFR overlaid with the
co-crystallized LHL (shown as cyan stick); B) 2D Docking mode of compound 12 showing different types of interactions, hydrogen was omitted for clarity.
12 formed several hydrophobic interactions with EGFR including,
Fig. 6.
vealed excellent lipophilicity which is consistent with Lipinski rule
(logP values<5) [34]. Moreover, absorption and oral bioavailability
of the compounds were predicted through Caco2 permeability and
intestinal absorption (HIA) indicators. Among all the compounds,
11 and 12 showed excellent human intestinal absorption outcomes
(96% and 88% absorption, respectively) parallel with good Caco2
permeability potential results (1.60 and 1.58 cm/s, respectively),
so considerable absorption are predicted for the respective com-
pounds upon oral administration [35]. One the other hand, com-
pounds 8, 9 and 10 showed low theoretical dose required for uni-
form distribution in the plasma during the prediction of the dis-
tribution volume by means of a steady-state volume of distribu-
tion (VDss), while they are arranged according to diffusion through
plasma membrane in the following order 9 > 10 > 8 by virtue
of Fraction unbound-human parameter. Concerning the distribu-
tion and penetration of the compounds through the central ner-
vous system, all the screened compounds was considered as safe
promising candidates (log BB< 0.3).
Compound 12 showed a binding energy of -8.96 kcal/mol to-
ward LCK compared to LHL (ꢀGb = -9.08 kcal/mol), Table 2. One
conventional hydrogen bond was formed between the triazole ni-
trogen in compound 12 and Glu317 in LCK, Fig. 7. Moreover, it
showed multiple hydrophobic interactions with Leu251, Val259,
Ala271, Tyr318, Leu371
In conclusion, the results of the docking study into the active
sites of EGFR kinase revealed higher affinities for compound 12
compared to the remaining new compounds (8-11) and the co-
crystallized ligand (erlotinib). Compound 12 also showed compara-
ble affinity to that of LHL, the co-crystallized ligand of LCK kinase.
These findings could account for the higher activity of compound
12 compared to the remaining derivatives 8-11.
3.5. In Silico ADME profile
Furthermore, the metabolism of the screened compounds was
predicted using ones of the most significant metabolizing en-
zymes; CYP isozymes. Additionally, In-Silico toxicity prediction was
carried out through AMES toxicity test where the obtained results
revealed that only the compounds 11 and 12 are considered as
non-mutagenic agents. All the tested compounds were predicted
to be effective as hERG II inhibitors except compound 9, but not
Prediction of the pharmacokinetic properties of all the new
synthesized compounds has been performed using the computa-
tional study obtained by pkCSM tool (http://biosig.unimelb.edu.
au/pkcsm/prediction) [33]. Various parameters have been virtu-
ally screened for pharmacokinetic properties/toxicity prediction as
shown in Table 3. Among the molecular properties parameters,
partition coefficient (log P), where all the screened compounds re-
8

A.H. Abdelazeem, A.M. Alqahtani, H.H. Arab et al.
Journal of Molecular Structure 1246 (2021) 131138
Table 3
Predicted ADME study of the target compounds 8-12.
Predicted Value
8
Model Name (Unit)
9
10
11
12
Lipophilicity (LogP)
HIA (%)
0.90
0.82
0.99
2.36
2.50
88.04
1.58
74.72
0.016
87.05
0.247
88.80
0.253
96.67
1.606
Caco2 permeability
(log Papp in 10–6
cm/s)
VDss- human (log
L/kg)
−0.684
0.154
-1.568
No
−0.374
0.168
-1.302
No
−0.452
0.156
−1.231
No
−0.283
0.029
−0.956
No
−0.157
0
Fraction unbound-
human
BBB permeability
(log BB)
-1.007
No
CYP2D6 substrate/
inhibitor
CYP3A4 substrate/
inhibitor
Yes
Yes
Yes
Yes
Yes
AMES toxicity
hERG I inhibitor
hERG II inhibitor
Skin Sensitisation
Yes
No
Yes
No
Yes
No
No
No
Yes
No
Yes
No
No
No
Yes
No
No
No
Yes
No
HIA: Human Intestinal absorption Caco2: Human colon adenocarcinoma-2, VDss: Steady-state volume of distribution, BBB: Blood-brain barrier,
AMES: Salmonella typhimurium reverse mutation assay, hERG: Human ether-a-go-go-related gene.
hERG I. All the aforementioned results revealed that compound 12
possess a considerable pharmacokinetic/toxicity profile supporting
its pharmacological studies to become a promising safe anticancer
agent.
Acknowledgment
The current work was supported by Taif University Researchers
Supporting Project number (TURSP-2020/29), Taif University, Taif,
Saudi Arabia.
4. Conclusion
Supplementary materials
In the current study, we report the synthesis and cytotoxic
evaluation of five S-benzo[4,5]thiazolo[2,3-c][1,2,4]triazole-based
derivatives. The results of the MTT assay revealed growth in-
hibitory activity against the A549, MCF-7, and Hep3B cell lines
(IC50 = 2.40-13.23 μM). Compound 12 bearing an isatin moiety
was the most active (IC50 = 2.40-3.53 μM), where the highest ac-
tivity was against Hep3B. Furthermore, compounds 12 was sub-
jected to a kinase profiling test. The results revealed moderate in-
hibitory activity against EGFR and LCK kinases. In addition, com-
pound 12 activated caspase-3/7 in A549 cells. The binding modes
and interactions of compound 12 into EGFR and LCK were in-
vestigated in a molecular docking study. Among the new deriva-
tives, compound 12 exhibited the highest binding affinity toward
EGFR. In silico ADME study results revealed that compound 12
possess a good pharmacokinetic/toxicity profile. Taken together, S-
benzo[4,5]thiazolo[2,3-c][1,2,4]triazole is a novel promising anti-
cancer scaffold that needs further structural optimization and de-
velopment to share effectively in cancer treatment.
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.131138.
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