Synthesis, biological evaluation and kinase profiling of novel S-benzo[4,5]thiazolo[2,3-c][1,2,4]triazole derivatives as cytotoxic agents with apoptosis-inducing activity

Article abstract of DOI:10.1016/j.molstruc.2020.128567

A novel set of S-benzo[4,5]thiazolo[2,3-c][1,2,4]triazoles was synthesized. Cytotoxicity of these compounds was evaluated against three cancer cell lines of different origins, Hep3B, A549, and MCF-7. Three of these compounds were screened by NCI for growth inhibitory activities against 60 cancer cell lines. The results revealed significant cytotoxic activities for compounds 13a-i. Among these derivatives, compounds 13c and 13f-13i exhibited the highest cytotoxicity against the selected cancer cell lines with IC₅₀ values between 3.17 and 14.18 μM. The structure-activity relationship of compounds 13a-i indicated favorable cytotoxic results on the expansion of the cyclic amine and the substitution with aminothiazole moiety. A mechanistic study revealed the activation of caspase-3/7 in A549 cells on treatment with compounds 13f-i at 5–20 μM. Moreover, the results of flow cytometric analysis suggested that compound 13i efficiently induced apoptosis in a dose-dependent manner. Compounds 13f,g,i also exhibited a weak to moderate inhibition of multiple kinases where compound 13i was the most active in inhibiting the activity of CDK2/Cyclin A1 (IC₅₀ = 4.65 μM). The current work provided a novel set of compounds with cytotoxic, kinase inhibition, and apoptosis-inducing activities, which can serve as a lead for further optimization.

Full text of DOI:10.1016/j.molstruc.2020.128567

Journal Pre-proof
Synthesis, biological evaluation and kinase profiling of novel S-benzo
[4,5]thiazolo[2,3-c] [1,2,4]triazole derivatives as cytotoxic agents with apoptosis-
inducing activity
Ahmed H. Abdelazeem, Alaa Alqahtani, Hany A. Omar, Syed Nasir Abbas Bukhari,
Ahmed M. Gouda
PII:
S0022-2860(20)30892-9
DOI:
https://doi.org/10.1016/j.molstruc.2020.128567
MOLSTR 128567
Reference:
To appear in: Journal of Molecular Structure
Received Date: 16 March 2020
Revised Date: 28 May 2020
Accepted Date: 28 May 2020
Please cite this article as: A.H. Abdelazeem, A. Alqahtani, H.A. Omar, S.N. Abbas Bukhari, A.M. Gouda,
Synthesis, biological evaluation and kinase profiling of novel S-benzo [4,5]thiazolo[2,3-c] [1,2,4]triazole
derivatives as cytotoxic agents with apoptosis-inducing activity, Journal of Molecular Structure (2020),
doi: https://doi.org/10.1016/j.molstruc.2020.128567.
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Credit Author Statement
Ahmed H. Abdelazeem: Conceptualization, Conducting a research (chemical
synthesis and analysis part) and Writing-Original draft preparation and Funding
acquisition.
Alaa Alqahtani: Project administration, Funding acquisition, Supervision and
Contributed to the design and implementation of the research.
Hany A. Omar: Methodology and Conducting a research (Biological part)
Syed Nasir Abbas: Writing - Review & Editing and Investigation
Ahmed M. Gouda: Writing - Review & Editing, Software, Investigation and
Contributed to the design and implementation of the research.

Synthesis, biological evaluation and kinase profiling of novel S-
benzo[4,5]thiazolo[2,3-c][1,2,4]triazole derivatives as cytotoxic
agents with apoptosis-inducing activity
Ahmed H. Abdelazeem1,*, Alaa Alqahtani2,*, Hany A. Omar3,4, Syed Nasir Abbas
Bukhari5, Ahmed M. Gouda1
1Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514,
Egypt.
2Pharmaceutical Chemistry Department, College of Pharmacy, Umm Al-Qura University, Makkah 21955,
Saudi Arabia.
3Department of Pharmacology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt.
4Sharjah Institute for Medical Research, College of Pharmacy, University of Sharjah, Sharjah 27272,
United Arab Emirates.
5Department of Pharmaceutical Chemistry, College of Pharmacy, Aljouf University, Aljouf, Sakaka 2014,
Saudi Arabia.
*To whom correspondence should be addressed:
Ahmed H. Abdelazeem, Ph.D. Department of Medicinal Chemistry, Faculty of Pharmacy,
Beni-Suef University, Beni-Suef 62514, Egypt.
Tel.: (002)-0100-2877405
E-mail address: ahmed.abdelazeem@pharm.bsu.edu.eg, ahmed.pharm@yahoo.com
Alaa Alqahtani, Ph.D. Pharmaceutical Chemistry Department, College of Pharmacy, Umm Al-
Qura University, Makkah 21955, Saudi Arabia.
E-mail address: amqahtani@uqu.edu.sa
1

Graphical Abstract:
Highlights
1- A novel set of S-benzo[4,5]thiazolo[2,3-c][1,2,4]triazoles was synthesized.
2- Compounds 13f, 13g, and 13i showed the highest cytotoxic activity.
3- Compound 13i activated caspase-3/7 and induced apoptosis.
4- Compound 13i exhibited the highest inhibitory activity against CDK2/Cyclin A1.
2

Abstract
A novel set of S-benzo[4,5]thiazolo[2,3-c][1,2,4]triazoles was synthesized. Cytotoxicity of these
compounds was evaluated against three cancer cell lines of different origins, Hep3B, A549, and
MCF-7. Three of these compounds were screened by NCI for growth inhibitory activities against
60 cancer cell lines. The results revealed significant cytotoxic activities for compounds 13a-i.
Among these derivatives, compounds 13c and 13f-13i exhibited the highest cytotoxicity against
the selected cancer cell lines with IC50 values between 3.17-14.18 μM. The structure-activity
relationship of compounds 13a-i indicated favorable cytotoxic results on the expansion of the
cyclic amine and the substitution with aminothiazole moiety. A mechanistic study revealed the
activation of caspase-3/7 in A549 cells on treatment with compounds 13f-i at 5-20 μM.
Moreover, the results of flow cytometric analysis suggested that compound 13i efficiently
induced apoptosis in a dose-dependent manner. Compounds 13f,g,i also exhibited a weak to
moderate inhibition of multiple kinases where compound 13i was the most active in inhibiting
the activity of CDK2/Cyclin A1 (IC50 = 4.65 µM). The current work provided a novel set of
compounds with cytotoxic, kinase inhibition, and apoptosis-inducing activities, which can serve
as a lead for further optimization.
Keywords: S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazole; anticancer; Apoptosis; caspase-3/7, flow
cytometry; kinase profiling; CDK2/Cyclin A1.
3

1. Introduction
Cancer is one of the leading causes of death in the world [1]. In high-income countries, the
number of deaths caused by cancers surpasses deaths caused by cardiovascular diseases [2].
Currently, combination therapy and multi-targeted anticancer agents have proved high efficacy
in the treatment of cancers [3,4]. However, their high cost, side effects, drug interaction, and
resistance problems encouraged the research to find cheap and effective alternatives [5].
Identification of molecular targets involved in cancer biology became an essential process for the
rational design of potent anticancer agents [6-8]. Of these promising targets, protein kinases
(PKs) attracted much attention due to their pivotal role in the regulation of cell survival and
proliferation [9]. In the last two decades, oncogenic kinases was emerged as potential targets in
cancer treatment [10,11]. Obviously, several kinase inhibitors succeeded to reach the drug
market exhibiting high response rate in treatment of lung cancer [12]. Among these kinases,
cyclin-dependent kinases (CDKs) which have a crucial role in the control of the cell proliferation
and transcription [13]. However, acquired resistance was reported to several kinase inhibitors
[14].
Recently, several triazoles derivatives were reported with high anticancer activities [15-17]. Lin
et al. have reported a series of 1,2,4-triazole derivatives with potent anticancer activity against a
panel of six cancer cell lines [18]. Among these derivatives, compound 1 displayed IC50 values
in the range of 0.12-0.75 µM against several cancer cell lines. This activity was mediated by
potent inhibition of multiple kinases including CDK1/cyclin B, GSK-3, and VEGF-R2, Fig. 1.
Moreover, compound 2 displayed inhibitory activity against Src kinase with IC50 value of 33.9
µM [19].
4

Fig. 1. Triazole-based multi-kinase inhibitors
In addition, several 2-aminobenzothiazole derivatives (Fig. 2) displayed broad spectra of
anticancer potential against different types of cancer cell lines [18,20]. These activities were
mediated by the induction of apoptosis [21,22]. Remarkably, compound 3 was developed as an
anti-leukemic agent with potent inhibitory activity against both wild-type BCR-ABL (BaF3/WT)
and T315I-mutated BCR-ABL (BaF3/T315I) cells. The activity of compound 3 was mediated by
cell cycle arrest at the G0/G1 phase and induction of apoptosis [23-25]. Moreover,
aminobenzothiazole 4 displayed potent antiproliferative activity against the M-CSF-dependent
murine leukemia cell line MNFS60 (EC50 = 67 nM) [26]. The mechanism of action of compound
4 was mediated by inhibiting colony-stimulating factor 1 receptor (CSF-1R) [27].
Fig. 2. Benzothiazole derivatives with anticancer/kinase inhibitory activities
More recently, our research group has reported the anticancer activity, for the first time, of a
novel series incorporating S-benzo[4,5]thiazolo[2,3-c][1,2,4]triazole scaffold that is composed of
5

two fused triazole and aminobenzothiazole heterocyclic rings [28]. Of these derivatives,
compound 5 exhibited anticancer activity against three (MCF-7, Hep3B, and A549) cancer cell
lines, Fig. 2. Kinase profiling results of compound 5 revealed weak to moderate inhibitory
activities against ten of the known oncogenic kinases. However, CDK2/Cyclin A1 was the most
sensitive kinase to the action of compound 5.
Rational Design
Based on these findings, we have designed the benzothiazolotriazole scaffold (A) incorporating
1,2,4-triazole with benzothiazole moieties. The structure activity relationship (SAR) of the
novel scaffold was investigated through structural modifications in two points. Firstly, several
derivatives were designed through variation of the substitution groups (R) using different
aliphatic, heterocyclic, and aromatic groups Fig. 3. Secondly, the impact of the spacer group
(X) on the anticancer activity of the new compounds was studied by alteration of the spacer
length/type, Fig. 3.
Fig. 3. Rational design and structural modifications of scaffold A.
6

2. Materials & Methods
2.1. Chemistry
The chemical reagents were purchased from commercial sources and used without
purification. Solvents were purchased from commercial sources and dried by standard
1
methods when necessary. H-NMR and 13C NMR spectra were measured at 400 and 100
MHz, respectively. these spectra were obtained on a Bruker APX400 at 100 MHz at
Faculty of Pharmacy, Beni-Suef University. Both mass spectra (record on Finnigan MAT,
SSQ 7000) and quantitative CHN analysis were performed in the microanalytical center,
Faculty of Science, Cairo University. Melting points (uncorrected) were determined by the
digital melting point apparatus (IA 9100MK series). The preparation of compound 8 was
achieved according to the previous reports [28,29].
2.1.1. General procedure (A) for the preparation of compounds 9, 10, and 11a-d
A mixture of benzo[4,5]thiazolo[2,3-c][1,2,4]triazole-3-thiol (0.5 g, 2.41 mmol), the appropriate
isocyanate derivative (2.41 mmol) and triethylamine (0.3 g, 3.1 mmol) was refluxed in 30 ml
ethanol for 6 hr. the reaction was monitored by TLC until completion. The reaction mixture was
left to cool, and the precipitate was filtered and washed with cold ethanol. Recrystallization was
carried out using ethanol to furnish the final targeted compounds.
2.1.1.1. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-tosylcarbamothioate (9).
The title compound was prepared using the general procedure A. Compound 9 was obtained as
1
off-white solid, m.p. 182-185 °C, yield 58%. H NMR (400 MHz, DMSO-d6): δ (ppm) 8.88 (s,
1H, NH), 8.09 (d, J = 7.9 Hz, 1H), 7.94 (d, J = 8.1 Hz, 1H), 7.65 (d, J = 7.7 Hz, 2H), 7.23-7.54
(m, 4H), 2.34 (s, 3H, CH3).13C NMR (101 MHz, DMSO-d6): δ (ppm) 174.4 (C=O), 155.3 (C),
148.8 (C), 133.2 (C), 131.6 (C), 130.6 (C), 128.9 (C), 127.3 (CH), 127.1 (CH), 125.7 (CH),
7

125.4 (CH), 122.5 (CH), 114.8 (CH), 21.4 (CH3). MS (EI) m/z 405.00 (M++1). Anal. calcd. for:
C16H12N4O3S3: C, 47.51; H, 2.99; N, 13.85. Found: C, 47.33; H, 3.24; N, 14.01.
2.1.1.1.
S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-(1R,3S,5R,7R)-adamantan-2-yl-
carbamothioate (10).
The title compound was prepared using the general procedure A. Compound 10 was obtained as
1
orange solid, m.p. 176-179 °C, yield 62%. H NMR (400 MHz, DMSO-d6): δ (ppm) 8.89 (s,
1H, NH), 8.09 (d, J = 8.4 Hz, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.54 (t, J = 7.3 Hz, 1H), 7.45 (t, J =
7.9 Hz, 1H), 1.99 (s, 2H), 1.84 (s, 4H), 1.65-1.52 (m, 4H), 1.42-1.14 (m, 5H). 13C NMR (101
MHz, DMSO-d6): δ (ppm) 171.5 (C=O), 154.7 (C), 138.7 (C), 133.4 (C), 130.5 (C), 127.0 (CH),
125.7 (CH), 122.3 (CH), 115.0 (CH), 50.1 (C), 41.6 (3CH2), 36.5 (3CH2), 29.3 (3CH). MS (EI)
m/z 385.00 (M++1). Anal. calcd. for: C19H20N4OS2: C, 59.35; H, 5.24; N, 14.57. Found: C, 59.68;
H, 5.14; N, 14.71.
2.1.1.3. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-p-tolylcarbamothioate (11a).
The title compound was prepared using the general procedure A. Compound 11a was obtained
1
as yellow solid, m.p. 200-203 °C, yield 68%. H NMR (400 MHz, DMSO-d6): δ (ppm) 9.50 (s,
1H, NH), 8.67 (d, J = 7.8 Hz, 1H), 8.12 (d, J = 7.9 Hz, 1H), 7.78 (d, J = 7.8 Hz, 2H), 7.06-7.45
(m, 4H), 2.22 (s, 3H, CH3).13C NMR (101 MHz, DMSO-d6): δ (ppm) 163.9 (C=O), 161.3 (C),
154.0 (C), 152.3 (C), 137.2 (C), 131.5 (C), 129.5 (C), 127.1 (CH), 127.0 (CH), 125.1 (CH),
118.7 (CH), 115.6 (CH), 114.8 (CH), 20.7 (CH3). MS (EI) m/z 340.00 (M+). Anal. calcd. for:
C16H12N4OS2: C, 56.45; H, 3.55; N, 16.46. Found: C, 56.12; H, 367; N, 16.94.
2.1.1.4. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-(4-chlorophenyl)carbamothioate
(11b).
8

The title compound was prepared using the general procedure A. Compound 11b was obtained
1
as yellow solid, m.p. 205-208 °C, yield 66%. H NMR (400 MHz, DMSO-d6): δ (ppm) 9.65 (s,
1H, NH), 8.18 (d, J = 8.0 Hz, 1H), 8.09 (d, J = 8.3 Hz, 1H), 7.76 (d, J = 7.7 Hz, 2H), 7.36-7.63
(m, 4H). 13C NMR (101 MHz, DMSO-d6): δ (ppm) 165.3 (C=O), 160.7 (C), 153.3 (C), 152.5
(C), 135.5 (C), 131.7 (C), 131.0 (C), 127.1 (CH), 126.9 (CH), 125.7 (CH), 115.8 (CH), 115.4
(CH), 113.7 (CH). MS (EI) m/z 360.00 (M+). Anal. calcd. for: C15H9ClN4OS2: C, 49.93; H, 2.51;
N, 15.53. Found: C, 49.51; H, 2.73; N, 15.09.
2.1.1.5. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-(4-fluoroophenyl)carbamothioate (11c).
The title compound was prepared using the general procedure A. Compound 11c was obtained
1
as yellow solid, m.p. 220-223 °C, yield 56%. H NMR (400 MHz, DMSO-d6): δ (ppm) 9.64 (s,
1H, NH), 8.86 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 7.8 Hz, 2H), 7.11-7.55
(m, 4H). 13C NMR (101 MHz, DMSO-d6): δ (ppm) 164.2 (C=O), 161.8 (C), 154.1 (C), 152.2
(C), 136.0 (C), 131.5 (C), 130.5 (C), 127.0 (CH), 127.0 (CH), 125.3 (CH), 115.8 (CH), 115.6
(CH), 114.8 (CH). MS (EI) m/z 344.00 (M+). Anal. calcd. for: C15H9FN4OS2: C, 52.32; H, 2.63;
N, 16.27. Found: C, 52.54; H, 2.61; N, 16.11.
2.1.1.6. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-(4-nitrophenyl)carbamothioate (11d).
The title compound was prepared using the general procedure A. Compound 11d was obtained
1
as yellow solid, m.p. 193-197 °C, yield 64%. H NMR (400 MHz, DMSO-d6): δ (ppm) 8.99 (s,
1H, NH), 8.59 (d, J = 7.9 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 7.7 Hz, 2H), 7.16-7.41
(m, 4H).13C NMR (101 MHz, DMSO-d6): δ (ppm) 164.4 (C=O), 160.0 (C), 154.2 (C), 152.5 (C),
137.3 (C), 131.5 (C), 129.0 (C), 127.0 (CH), 126.7 (CH), 125.5 (CH), 118.6 (CH), 115.1 (CH),
114.0 (CH). MS (EI) m/z 371.00 (M+). Anal. calcd. for: C15H9N5O3S2: C, 48.51; H, 2.44; N,
18.86. Found: C, 48.76; H, 2.54; N, 18.51.
2.1.1.7. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-2-chloroethanethioate (12).
9

Chloroacetyl chloride (3.5 gm, 31.6 mmol) was added slowly to a cold solution of
benzo[4,5]thiazolo[2,3-c][1,2,4]triazole-3-thiol 8 (5 gm, 24.3 mmol) and triethylamine (7.3 g,
o
72.81 mmol) in CH2Cl2. The reaction mixture was stirred for 1 h at 0 C then for 5 h at room
temperature. The organic layer was then separated and evaporated in vacuo. The residue
obtained was used directly for the next coupling step without purification. MS (EI) m/z 283.00
(M+).
2.1.2. General procedure (B) for the preparation of compounds 13a-i.
The appropriate amine (3.3 mmol) and K2CO3 (1.4 g, 9.9 mmol) were added, under mechanical
stirring, to a solution of S-benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl 2-chloroethanethioate 12
o
(0.9 g, 3.3 mmol) in 30 mL DMF. The reaction mixture was heated at 60 C for 4 h. After
cooling, the mixture was poured onto 100 mL H2O, extracted with ethyl acetate (3 x 50 mL),
washed with brine and dried. The solvent was removed in vacuo, and the residue was
chromatographed on a silica gel column using methylene chloride/methanol (9.5:0.5) as eluent to
afford the final compounds 13a-i in good yields.
2.1.2.1. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-2-(pyrrolidin-1-yl)ethanethioate (13a).
The title compound was prepared using the general procedure B. Compound 13a was obtained
1
as yellow oil. H NMR (400 MHz, DMSO-d6): δ (ppm) 8.16 (d, J = 7.9 Hz, 1H), 8.08 (d, J = 8.2
Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 4.23 (s, 2H, COCH2), 2.11-1.65 (m,
8H). 13C NMR (101 MHz, DMSO-d6): δ (ppm) 165.1 (C=O), 156.5 (C), 143.3 (C), 131.9 (C),
130.0 (C), 127.5 (CH), 126.9 (CH), 126.0 (CH), 114.9 (CH), 52.9 (CH2), 46.3 (CH2), 25.7 (CH2).
MS (EI) m/z 318.00 (M+). Anal. calcd. for: C14H14N4OS2: C, 52.81; H, 4.43; N, 17.60. Found: C,
52.69; H, 4.34; N, 17.48.
2.1.2.2. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-2-(piperidin-1-yl)ethanethioate (13b).
10

The title compound was prepared using the general procedure B. Compound 13b was obtained
1
as off-white solid, m.p. 195-198 °C, yield 62%. H NMR (400 MHz, DMSO-d6): δ (ppm) 8.16
(d, J = 8.1 Hz, 1H), 7.52-8.08 (m, 3H), 4.35 (s, 1H, COCH2), 1.62-1.36 (m, 10H).13C NMR (101
MHz, DMSO-d6): δ (ppm) 165.2 (C=O), 156.6 (C), 143.2 (C), 131.9 (C), 130.0 (C), 127.6 (CH),
126.9 (CH), 126.0 (CH), 114.8 (CH), 46.9 (CH2), 42.9 (CH2), 26.3 (CH2), 24.2 (CH2). MS (EI)
m/z 332.00 (M+). Anal. calcd. for: C15H16N4OS2: C, 54.19; H, 4.85; N, 16.85. Found: C, 54.61;
H, 4.57; N, 17.13.
2.1.2.3. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-2-(azepan-1-yl)ethanethioate (13c).
The title compound was prepared using the general procedure B. Compound 13c was obtained
1
as orange solid, m.p. 186-189 °C, yield 66%. H NMR (400 MHz, DMSO-d6): δ (ppm) 8.16 (d,
J = 8.0 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.53 (t, J = 7.8 Hz, 1H), 4.36
(s, 1H, COCH2), 1.74-1.49 (m, 12H). 13C NMR (101 MHz, DMSO-d6): δ (ppm) 166.5 (C=O),
153.2 (C), 143.4 (C), 131.9 (C), 130.0 (C), 127.6 (CH), 126.9 (CH), 126.0 (CH), 114.8 (CH),
48.0 (CH2), 45.6 (CH2), 28.7 (CH2), 24.9 (CH2). MS (EI) m/z 346.00 (M+). Anal. calcd. for:
C16H18N4OS2: C, 55.47; H, 5.24; N, 16.17. Found: C, 55.15; H, 5.37; N, 15.94.
2.1.2.4. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-2-(cyclohexylamino)ethanethioate (13d).
The title compound was prepared using the general procedure B. Compound 13d was obtained
1
as light brown solid, m.p. 194-197 °C, yield 57%. H NMR (400 MHz, DMSO-d6): δ (ppm)
8.21 (d, J = 8.3 Hz, 1H), 8.03-7.51 (m, 3H), 7.10 (s, 1H, NH), 3.83 (s, 2H, COCH2), 3.02-2.94
(m, 1H), 1.94-1.47 (m, 4H), 1.41-0.91 (m, 6H). 13C NMR (101 MHz, DMSO-d6): δ (ppm) 166.0
(C=O), 158.8 (C), 149.0 (C), 131.9 (C), 127.4 (C), 126.9 (CH), 125.9 (CH), 115.0 (CH), 113.2
(CH), 48.3 (CH2), 47.5 (CH), 32.8 (CH2), 25.0 (CH2), 24.2 (CH2). MS (EI) m/z 346.00 (M+).
Anal. calcd. for: C16H18N4OS2: C, 55.47; H, 5.24; N, 16.17. Found: C, 55.19; H, 5.64; N, 15.87.
2.1.2.5. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-2-morpholinoethanethioate (13e).
11

The title compound was prepared using the general procedure B. Compound 13e was obtained
1
as brown solid, m.p. 187-190 °C, yield 61%. H NMR (400 MHz, DMSO-d6): δ (ppm) 8.18 (d,
J = 8.3 Hz, 1H), 8.10-7.54 (m, 3H), 4.35 (s, 2H, COCH2), 4.09 (t, J = 8.0 Hz, 4H), 3.09 (t, J =
8.0 Hz, 4H). 13C NMR (101 MHz, DMSO-d6): δ (ppm) 166.0 (C=O), 155.8 (C), 143.0 (C), 139.8
(C), 131.9 (C), 129.7 (CH), 127.6 (CH), 119.6 (CH), 114.9 (CH), 66.7 (CH2), 59.6 (2CH2), 46.2
(2CH2). MS (EI) m/z 334.00 (M+). Anal. calcd. for: C14H14N4O2S2: C, 50.28; H, 4.22; N, 16.75.
Found: C, 50.63; H, 3.99; N, 16.35.
2.1.2.6. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-2-(4-methylpiperazin-1-yl)ethanethioate
(13f).
The title compound was prepared using the general procedure B. Compound 13f was obtained
1
as brown oil. H NMR (400 MHz, DMSO-d6): δ (ppm) 8.20 (d, J = 8.1 Hz, 1H), 8.11-7.50 (m,
3H), 4.15 (s, 2H, COCH2), 2.97 (t, J = 8.0 Hz, 4H), 2.72 (t, J = 7.8 Hz, 4H), 2.31 (s, 3H, CH3).
13C NMR (101 MHz, DMSO-d6): δ (ppm) 167.5 (C=O), 153.9 (C), 143.4 (C), 138.7 (C), 131.9
(C), 129.1 (CH), 126.4 (CH), 120.0 (CH), 114.9 (CH), 66.7 (CH2), 60.8 (2CH2), 59.6 (2CH2),
46.2 (CH3). MS (EI) m/z 347.00 (M+). Anal. calcd. for: C15H17N5OS2: C, 51.85; H, 4.93; N,
20.16. Found: C, 51.71; H, 5.18; N, 20.39.
2.1.2.7.
S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-2-
(hexylamino)ethanethioate (13g).
The title compound was prepared using the general procedure B. Compound 13g was obtained
1
as light brown solid, m.p. 234-237 °C, yield 52%. H NMR (400 MHz, DMSO-d6): δ (ppm)
8.21 (d, J = 8.2 Hz, 1H), 8.09-7.53 (m, 3H), 3.87 (s, 2H, COCH2), 3.12 (s, 1H, NH), 2.93 (t, J =
8.0 Hz, 2H), 1.30-1.10 (m, 5H), 0.86-0.81 (m, 5H). 13C NMR (101 MHz, DMSO-d6): δ (ppm)
166.9 (C=O), 156.7 (C), 142.9 (C), 131.9 (C), 130.0 (C), 127.4 (CH), 126.9 (CH), 126.0 (CH),
115.0 (CH), 56.4 (CH2), 38.2 (CH2), 31.4 (CH2), 29.2 (CH2), 26.4 (CH2), 22.5 (CH2), 14.4 (CH-
12

2). MS (EI) m/z 348.00 (M+). Anal. calcd. for: C16H20N4OS2: C, 55.15; H, 5.79; N, 16.08. Found:
C, 55.34; H, 6.12; N, 15.87.
2.1.2.8.
S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-2-((1R,2S,5S)-adamantan-2-
ylamino)ethanethioate (13h).
The title compound was prepared using the general procedure B. Compound 13h was obtained
as white solid, m.p. 210-213 °C, yield 59%. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.13 (d, J
= 8.1 Hz, 1H), 8.09 (d, J = 8.1 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 4.31 (s,
13
2H, COCH2), 4.18 (s, 1H, NH), 2.02-1.58 (m, 15H). C NMR (101 MHz, DMSO-d6): δ (ppm)
169.1 (C=O), 156.8 (C), 142.6 (C), 131.9 (C), 129.8 (C), 127.6 (CH), 127.0 (CH), 126.1 (CH),
114.8 (CH), 57.1 (CH2), 53.0 (C), 37.3 (3CH2), 36.0 (3CH2), 27.2 (3CH). MS (EI) m/z 398.00
(M+). Anal. calcd. for: C20H22N4OS2: C, 60.27; H, 5.56; N, 14.06. Found: C, 59.98; H, 5.37; N,
13.89.
2.1.2.8.
S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-2-(benzo[d]thiazol-2-
ylamino)ethanethioate (13i).
The title compound was prepared using the general procedure B. Compound 13i was obtained
1
as light brown solid, m.p. 189-191 °C, yield 56%. H NMR (400 MHz, DMSO-d6): δ (ppm)
7.64 (d, J = 7.8 Hz, 1H), 7.60-7.41 (m, 4H), 7.34 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H),
7.00 (t, J = 7.5 Hz, 1H), 5.11 (s, 1H, NH), 3.55 (s, 2H, COCH2).13C NMR (101 MHz, DMSO-
d6): δ (ppm) 167.0 (C=O), 162.8 (C), 153.1 (C), 151.2 (C), 149.3 (C), 148.4 (C), 142.3 (C), 141.0
(C), 139.0 (CH), 131.3 (CH), 130.3 (CH), 125.9 (CH), 121.4 (CH), 119.3 (CH), 118.2 (CH),
114.5 (CH), 58.6 (CH2). MS (EI) m/z 397.00 (M+). Anal. calcd. for: C17H11N5OS3: C, 51.37; H,
2.79; N, 17.62. Found: C, 51.26; H, 3.01; N, 17.95.
2.1.3. General procedure (C) for the preparation of compounds 14a-d.
13

A solution of an appropriate acid chloride (2.41 mmol) in 10 mL methylene chloride or
tetrahydrofuran was added dropwise to a mixture of benzo[4,5]thiazolo[2,3-c][1,2,4]triazole-3-
thiol 8 (0.5 g, 2.41 mmol) and triethylamine (0.3 g, 3.1 mmol) in 20 mL methylene chloride or
tetrahydrofuran. The reaction mixture was heated for 5 h while monitoring by TLC until
completion. The reaction mixture was then evaporated, and 50 mL of H2O was added followed
by extraction with ethyl acetate (3 x 20 mL). The combined organic layer was washed with brine
and dried over anhydrous Na2SO4. The solvent was removed in vacuo, and the residue was
purified by flash column chromatography using ethyl acetate/hexane (2:8) as eluent to give the
final compounds 14a-d.
2.1.3.1. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-benzothioate (14a).
The title compound was prepared using the general procedure C. Compound 14a was obtained
1
as yellow solid, m.p. 211-214 °C, yield 67%. H NMR (400 MHz, DMSO-d6): δ (ppm) 8.51-
13
7.97 (m, 4H), 7.89-7.44 (m, 5H). C NMR (101 MHz, DMSO-d6): δ (ppm) 166.4 (C=O), 163.3
(C), 153.4 (C), 134.2 (C), 132.2 (C), 131.2 (C), 130.5 (CH), 129.7 (CH), 128.9 (CH), 127.8
(CH), 127.2 (CH), 125.5 (CH), 115.1 (CH). MS (EI) m/z 311.00 (M+). Anal. calcd. for:
C15H9N3OS2: C, 57.86; H, 2.91; N, 13.49. Found: C, 58.23; H, 3.11; N, 13.67.
2.1.3.2. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-3,4,5-trimethoxybenzothioate (14b).
The title compound was prepared using the general procedure C. Compound 14b was obtained
1
as yellow solid, m.p. 237-239 °C, yield 59%. H NMR (400 MHz, DMSO-d6): δ (ppm) 8.20-
7.79 (m, 2H), 7.63-7.45 (m, 2H), 7.23 (s, 2H), 3.82 (s, 9H, 3OCH3). 13C NMR (101 MHz,
DMSO-d6): δ (ppm) 167.4 (C=O), 165.8 (C), 153.1 (C), 130.6 (C), 127.8 (C), 127.2 (C), 126.4
(C), 125.5 (C), 115.1 (CH), 114.4 (CH), 109.4 (CH), 108.2 (CH), 107.0 (CH), 60.9 (2CH3), 56.5
(CH3). MS (EI) m/z 401.00 (M+). Anal. calcd. for: C18H15N3O4S2: C, 53.85; H, 3.77; N, 10.47.
Found: C, 53.91; H, 3.57; N, 10.81.
14

2.1.3.3. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-3,5-dinitrobenzothioate (14c).
The title compound was prepared using the general procedure C. Compound 14c was obtained
1
as yellow solid, m.p. 246-249 °C, yield 73%. H NMR (400 MHz, DMSO-d6): δ (ppm) 8.99 (s,
1H), 8.87 (s, 2H), 8.13-7.86 (m, 2H), 7.64-7.33 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ
(ppm) 164.37 (C=O), 161.7 (C), 152.1 (C), 148.7 (C), 134.5 (C), 131.5 (C), 130.6 (C), 129.3
(CH), 127.1 (CH), 127.0 (CH), 125.3 (CH), 122.5 (CH), 114.7 (CH). MS (EI) m/z 401.00 (M+).
Anal. calcd. for: C15H7N5O5S2: C, 44.89; H, 1.76; N, 17.45. Found: C, 45.11; H, 2.15; N, 17.67.
2.1.3.4. S-Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl-adamantane-1-carbothioate (14d).
The title compound was prepared using the general procedure C. Compound 14d was obtained
1
as white solid, m.p. 187-190 °C, yield 61%. H NMR (400 MHz, DMSO-d6): δ (ppm) 8.06 (d,
J = 6.3 Hz, 1H), 7.87-7.40 (m, 3H), 1.95 (s, 3H), 1.80-1.73 (m, 6H), 1.71-1.59 (m, 6H). 13C
NMR (101 MHz, DMSO-d6): δ (ppm) 166.8 (C=O), 161.8 (C), 158.5 (C), 131.6 (C), 129.0 (C),
127.2 (CH), 116.2 (CH), 114.8 (CH), 113.1 (CH), 43.5 (C), 39.5 (3CH2), 36.5 (3CH2), 27.8
(3CH). MS (EI) m/z 369.00 (M+). Anal. calcd. for: C19H19N3OS2: C, 61.76; H, 5.18; N, 11.37.
Found: C, 61.89; H, 5.36; N, 11.31.
2.2. Pharmacological Screening
2.2.1. Anticancer activity
2.2.1.1. Evaluation of growth inhibitory activity by NCI
Three of the new compounds (11b, 13b, and 13h) were evaluated for their growth inhibitory
activity against 60 cancer cell lines in the drug development program (DDP) by the NCI-USA.
The test was performed using the SRB assay [30,31]. The most relevant results were presented in
Table 1.
15

2.2.1.2. Cell Viability Assay
Three of the American Type human (MCF-7, A549, and Hep3B) cancer cell lines (ATCC,
Manassas, VA) were used in the cell viability (MTT) assay. The cancer cells were cultured in
Dulbecco’s modified Eagle’s medium/F12 medium (DMEM/F-12) in humidified incubator
containing 5% CO2 at 37 °C according to the previous report [32]. The MTT was used to
evaluate the effect of the new compounds 9-14 on the viability of the three cancer cell lines
according to the previous reports [33-35]. After seeding the cancer cells in 96-well plates, the
latter were incubated and incubated overnight then treated with the test compounds for 72 h.
Following this treatment, cells were treated with the MTT for 2 h then the reduced MTT was
dissolved in DMSO and its absorbance was determined using Varioskan™ Flash Multimode
Reader (Thermo Scientific, USA), Table 2.
2.2.2. Flow Cytometric analysis
Flow cytometry was used for the determination of cell cycle perturbations and the ability of
test compounds to induce apoptosis as mentioned before [36]. The A549 cells were
exposed to compound 13i at two concentrations, 5 and 10 μM for 48 h then the cells were
stained with propidium iodide, and flow cytometry was performed. Data analysis was
performed using BD FACSDiva 6.0 software (BD Pharmingen, San Diego, CA, USA). The
results were presented in Fig. 5.
2.2.3. Caspase-3/7 Assay
The ability of the novel compounds to induce apoptosis was tested using the Caspase-Glo 3/7
assay kit (Promega, Madison, WI) according to the manufacturer’s instructions and as mentioned
before [37,38]. Following the incubation process, the luminescence was measured using
16

Varioskan™ Flash Multimode Reader (Thermo Scientific, USA). The results were presented in
Fig. 6.
2.2.4. Kinase profiling assay protocol
Kinase profiling test was done at Reaction Biology Corp., PA, USA, using their kinases analysis
services [39]. Kinases activity is expressed as the percent remaining kinase activity in test
samples compared to vehicle (DMSO) reactions, Tables 3 and 4.
2.3. Molecular Docking Study
AutoDock 4.2 [40] was used to perform the docking study of the new compounds into CDK2
kinase. The crystal structures of CDK-2 [41] was obtained the Protein Data Bank
(https://www.rcsb.org/, PDB code: 3TNW). This study was carried out using the previously used
docking protocol [33,42]. The PDB format of the test compounds were prepared according to the
previous reports [33,42]. During the preparation of the protein structure, water molecules and
bound ligands were removed to avoid interference with the study. The 2/3D binding modes of
the new compounds were generated using Discovery studio visualizer (DSV, v16.1.0.15350)
[43]. The results were presented in Table 5 and Figs. 7-9.
3. Results and Discussion
3.1 Chemistry
Synthesis of the starting material benzo[4,5]thiazolo[2,3-c][1,2,4]triazole-3-thiol derivative 8
was described in Schemes 1. It was achieved through the cyclization of 2-
hydrazinobenzothiazole 7, obtained from refluxing 2-mercaptobenzothiazole 6 with hydrazine
hydrate in ethanol, with carbon disulfide in alcoholic potassium hydroxide as previously reported
[28,29]. Subsequently, the reaction of thiol intermediate 8 with various isocyanate derivatives in
17

the presence of trimethylamine afforded the final S-thiocarbamate (9, 10, 11a-d) derivatives in
good yield (56-68%).
S
N
c
N
N
H
(62%)
O
S
N
S
9
O
O
CH3
S
S
8
N
N
S
N
S
N
a
N
N
b
d
NHNH2
N
SH
N
(58%)
H
S
N
SH
6
10
S
7
O
11a, R = CH3
11b, R = Cl
11c, R = F
N
N
e
N
(56-68%)
H
N
11d, R = NO2
S
O
11a-d
R
Scheme 1. Reagents and reaction conditions: a) N2H4.H2O, reflux, EtOH, 20 h; b) i. CS2, KOH,
EtOH, reflux; ii. Conc. HCl; c) p-toluenesulfonyl isocyanate, DCM, TEA, reflux, 12 h; d) 1-
Adamantyl isocyanate, DCM, TEA, reflux, 12 h; e) appropriate substituted-phenyl isocyanate,
DCM, TEA, reflux, 12 h.
In addition, compound 12 was prepared from the S-alkylation of thiol intermediate 8 with
chloroacetyl chloride. Compounds 13a-i were obtained from the reaction of primary/secondary
amines with compound 12, Scheme 2. This reaction takes place through nucleophilic substitution
with elimination of hydrochloride acid. The anhydrous potassium carbonate was used as acid
removing agent.
18

Scheme 2. Reagents and reaction conditions: f) ClCH2COCl, TEA, DCM, 0 ○C, 1 h, rt, 5 h; g)
appropriate amine, K2CO3, DMF, 60 ○C, 3 h.
Similarly, compounds 14a-d were prepared by acylation of the thiol group in compound 8 with
different acid chlorides including benzoyl, 3,4,5-trimethoxybenzoyl, 3,5-dinitrobenzoyl and
adamantly chloride, Scheme 3. The reaction was performed in DCM in the presence of TEA as a
catalyst. Quantitative elemental and spectral (mass, 1H-NMR, and 13C-NMR) analyses were used
to confirm the chemical structures of all new compounds.
Scheme 3. Reagents and reaction conditions: h) Appropriate acyl chloride, TEA, DCM, 2 h.
3.2. Pharmacological screening
3.2.1. Anticancer activity
3.2.1.1. Structure activity relationship (SAR)
19

The relationship between chemical structure and cytotoxicity of the new compounds (SAR)
against the selected cancer lines was presented in Fig. 4. Among the carbamate derivatives 9-11,
compound 11d bearing 4-nitophenyl substituent was the most active as cytotoxic agent. In
addition, replacement of 4-nitrophenyl in compound 11d moiety with 4-fluoro or 4-methyl
groups resulted in a noticeable decrease in the cytotoxic activity. SAR study of compounds 13a-i
revealed the importance of the size of the cyclic amine on cytotoxic activity. A significant
increase in cytotoxic activity on the increase in ring size from five-membered to seven-
membered ring. Compounds 13c bearing the bulky azepine ring exhibited higher cytotoxicity
compared to compounds 13a and 13b, , Fig. 4.
replace NO2 by CH3, F or Cl
Th most active
decrease activity
S
N
N
N
N
replace CH2 by NH
R = 4-NO2- Ph
N
N
Activity increases with the
increase in the size of the
alicyclic amine
S
R = cyclic amine
Scaffold A
R
O
removal of -CH2-
decrease
activity
S
N
H
R attached
N
replacement of cyclohexylamino
moiety in comp. 13d with the open
hexyl chain in comp. 13g resulted in
an increase in cytotoxic activity
against the three cancer cell lines.
R =
directly to C=O
increase cytotoxic activity
Fig. 4. SAR of cytotoxic activity of compounds 9-14.
Moreover, it was found that the replacement of the cyclohexylamino moiety in compound 13d
with the open hexyl chain in compound 13g resulted in increase in cytotoxicity. The high activity
of compound 13i might be explained by the well-known anticancer activity of the attached 2-
aminobenzothiazole moiety [20]. Finally, the thioesters 14a-d were also less active than
compounds 13a-i. Among the four derivatives, compound 14c bearing the 3,5-dinitrophenyl
moiety was the most active as cytotoxic agent. Cytotoxic activity of compounds 14 decreased on
20

removal/replacement of the dinitro groups with 3,4,5-trimethoxy or adamantly groups, Fig. 4.
Obviously, these results were in concordance to some extent with the results of NCI screening
assay, where compound 13h exhibited high cytotoxic activity (IC50 in the range of 4.83-10.68
μM), while compounds 11b and 13b showed weaker or no cytotoxic activity (IC50 in the range of
9.23-31.55 μM).
2.2.1.2. NCI Anticancer Screening
The one-stage screening process started with the selection of three of the obtained compounds
(i.e. 11b, 13b and 13h) by the Drug Evaluation Branch of National Cancer Institute (NCI-USA).
The selected compounds were subjected to a primary in vitro evaluation against 57 cell lines at a
single dose of 10 µM (it is referred to as one-dose assay). The 57 cell panel is derived from nine
different cancer strains: Leukemia, Lung, Melanoma, Colon, CNS, Ovary, Renal, Breast, and
Prostate cancers [30,31]. The output from the single-dose screening was reported as a mean
graph available for analysis by the COMPARE program, (Supplementary data, Table S1).
Interestingly, the three compounds (11b, 13b, and 13h) exhibited nearly similar antiproliferative
activity against SNB-75 cell line with growth inhibition in the range of 23.05-25.41% (i.e. 74.59-
76.95% growth). In addition, compounds 11b and 13h showed moderate growth inhibitory
activities towards several breast (MCF-7, MDA-MB-231/ATCC, T-47D) and prostate (PC-3)
cancer cell lines. On the other hand, the tested compounds (11b, 13b, and 13h) displayed no or
very low growth inhibitory activities against leukemia, colon cancer, and melanoma cell lines.
Moreover, the results of the one-dose assays revealed that compound 13h has the highest
antiproliferative activity against HOP-92 (Non-Small Cell Lung Cancer) cells with growth
inhibition of 53.76% (i.e. 46.24% growth), Table S1. Moreover, compound 13h caused 33.48%
inhibition (i.e. 76.52% growth) in the growth of SNB-75 (CNS cancer) cell line, Table 1.
21

Table 1. Selected NCI anticancer screening data at single dose assay (10 μM) as percentage cell
growth against selected human cancer cell line.
Percentage cell growth
Percentage cell growth
Cell lines
Cell lines
11b
13b
13h
11b
13b
13h
Non-Small Cell Lung Cancer
Renal Cancer
CAKI-1
HOP-62
99.65
87.8
89.87
84.92
104.06
91.31
80.01
74.83
86.23
68.29
76.41
78.30
NCI-H226
Leukemia
CCRF-CEM
CNS Cancer
SNB-75
UO-31
Prostate Cancer
PC-3
99.80
76.95
103.54 86.05
81.39
92.97
77.45
Breast Cancer
MCF-7
74.59
76.52
88.02
93.68
87.84
92.86
109.77
92.22
88.72
89.46
87.22
HS 578T
Ovarian Cancer
OVCAR-4
98.6
104.73 87.62
144.13 100.03
T-47D
OVCAR-5
89.08
MDA-MB-468
87.06
97.09
101.97
Bold values represent growth% < 90.00%.
Compound 11b exhibited the highest growth inhibitory activity against UO-31 (renal cancer)cell
line with growth inhibition of 25.17 (i.e. 74.83% growth), while compound 13b showed the
highest effect against UO-31 (renal cancer) cell line with growth inhibition of 31.71% (i.e.
68.29% growth).
3.2.1.3. MTT assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to
evaluate the anti-proliferative potential of the new compounds 9-14. The MTT assay was
performed against three (Hep3B, A549, and MCF-7) cancer cell lines according to the previous
reports [33-35]. The cytotoxic activity of the new compounds expressed as IC50 values (the
concentration that cause inhibition of 50%) compared to doxorubicin was presented in Table 2.
22

The carbamates 9-11 exhibited cytotoxic activity against Hep3B cells with IC50 in the range of
5.05-35.25 μM. Among the six derivatives, compound 11d was the most active against the three
cancer cell lines. On the other hand, compounds 9, 10, and 11b were inactive against A549 cells.
The results revealed higher anticancer potential for the compounds 13a-i than both the thioesters
14a-d the thiocarbamates 9, 10, and 11a-d. Among these derivatives, compounds 13c and 13f-
13i exhibited the highest cytotoxic activity (IC50 in the range of 3.17-14.18 μM). Compound 13c
was more active than compounds 13a and 13b. In addition, the compound 13f was more active
than the morpholino 13e and hexylamino 13g analogs. Among the nine derivatives, compound
13i showed the highest cytotoxicity against A549 and MCF-7 cells, while compound 13f
exhibited the highest growth inhibitory activity against the Hpe3B cells.
The thioesters 14a-d showed weaker cytotoxic activity than compounds 13a-i. However,
compounds 14a-d displayed the highest cytotoxic activity against Hep3B cells (IC50 values in the
range of 6.95-1885 μM). Compound 14c was the most active among the four derivatives against
the three cancer cell lines.
Table 2. Anticancer activity of compound 9-14 against A549, MCF-7 and Hep3B cancer cell
lines
a
IC50
Comp. No
X
R
A549
MCF-7
Hep3B
NH
NH
>60
20.33±1.84 16.25±1.32
9
>60
>60
35.25±2.27
14.23±1.93
10
11a
11b
11c
11d
NH
NH
NH
NH
34.56±2.19 9.28±1.03
>60
17.42±1.72 9.83±1.18
7.88±0.66 5.84±0.41
31.55±2.76 22.72±2.23
11.42±1.12
5.05±0.62
23

CH2
CH2
CH2
CH2
CH2
43.56±4.17 26.82±2.39 28.75±3.29
13a
13b
13c
13d
13e
11.25±1.11 9.23±1.06
4.62±0.85 5.65±1.02
15.69±1.76
4.98±0.73
38.56±3.41 19.23±1.23 16.25±1.56
24.57±2.52 16.61±1.88 20.27±1.93
CH2
CH2
6.05±1.03
7.55±0.96
3.37±0.36
13f
14.18±1.33 8.28±0.61
10.68±1.28 4.83±0.56
13.82±1.43
13g
CH2
8.23±1.16
13h
CH2
-
3.55±0.37
3.17±0.92
4.32±0.66
13i
37.50±3.19 9.74±0.42
18.51±1.79
14a
-
-
14.05±1.22 13.08±1.26 11.38±1.10
14b
14c
13.81±1.37 8.13±0.92
6.95±0.74
-
-
>60
36.12±3.31 18.85±1.52
2.4 ± 0.17 1.7 ± 0.31
14d
-
2.3 ± 0.15
Doxorubicin
Results were presented as mean ± S.D. (n = 3), after 72 h treatment with test compounds or
vehicle (control).
3.2.2. Flow Cytometric analysis
In identify the potential mechanism of the newly benzothiazolotriazole derivatives and to
determine whether the inhibition of cancer cell growth was related to cell cycle arrest, the most
potent compound 13i was selected to be examined using flow cytometry to analyze the effect on
the cell cycle in A549 cell line [36]. A549 cells were treated with compound 13i at two
concentrations of 5 and 10 μM for 48 h. While there was no significant change in the cell cycle
distribution, the results revealed the ability of 13i to induce apoptosis in A549 cells as indicated
by the increase of the fraction of apoptotic cells to 8.23% and 14.9% at 5 and 10 μM
concentrations, respectively compared to control (2.34%), Fig. 5.
24

Fig. 5. Cell cycle analysis. Cells were treated with test compound or vehicle for 48 h, stained
with propidium iodide, and analyzed by flow cytometry.
We did not observe a significant cell cycle arrest at that time point which could be due to the
extensive cell death at that late stage. These findings suggested that compound 13i can efficiently
induce A549 cells apoptosis in a dose-dependent manner.
3.2.3. Caspase 3/7 Assay
It is well known that caspases, a family of cysteine proteases play a pivotal role in the induction
of apoptosis especially, caspase-3/7. To further evidence the apoptotic inducing mechanism, the
25

activity of caspase-3/7 was determined for some of the most potent compounds (13f-i) in cell
viability analysis. After treatment with the test compounds 13f-i, the activation of caspase-3/7 in
A549 cells was determined according to the previous reports [37,38]. The results revealed
induction of caspase-3/7 activity in A549 cells after treatment with 5-20 μM of compounds 13f,g
and 13i, Fig. 5. The three compounds increase the activity caspase-3/7 by nearly 3- to 4-fold. On
the other hand, compound 13h exhibited only weak induction in the activity of caspase-3/7.
Interestingly, compound 13i was the most active compound which confirms the consistency of
these results with that of the cell viability analysis and flow cytometry as well. These results
indicated that the tested compounds have the ability to induce apoptosis through the activation of
effector caspase-3/7 family.
Fig. 6. The effect of test compounds 13h-g and 13f on caspases 3/7 activity in A549 cells.
Caspase-3/7activities were measured using Caspase-Glo Assay Kit after the indicated treatment
for 48 h. All data are depicted as mean ± S.D (n=4). Significant difference from untreated
control condition at *p < 0.05, #p<0.01.
3.2.4. Kinase Profiling Assay
To identify the possible molecular anticancer targets which could mediate the anticancer
potential of the new
S-benzo[4,5]thiazolo[2,3-c][1,2,4]triazoles, kinase profiling assay was
performed. 13f, 13g and 13i were selected for their high cytotoxic activity, Table 2. The
26

profiling assay was performed against ten different kinases of different families and types. The
kinase profiling assays were performed at Reaction Biology Corp., PA, USA using the
radiolabeled ATP determination method as reported [39]. The compounds were tested in single
dose duplicate mode at a conc. of 10
positive controls.
μM using staurosporine, GW5074 and SB202190 as
The results revealed that the three tested compounds 13f, 13g and 13i exhibited weak to
moderate inhibition against ten kinases with inhibition percentages up to 29.67%, Table 3.
Among the tested kinases, CDK2/Cyclin A1 was the most sensitive kinase to the three selected
compounds with enzyme activity percentages of 70.33 to 74.91%. Compound 13g displayed the
highest inhibitory activity against CDK2/Cyclin A1. Moreover, compounds 13g and 13i showed
good activity against ABL1 kinase as well with enzyme activity of 79.78 and 76.24%,
respectively.
Table 3. Kinase profiling results (enzyme activity percentages) of compounds 13f, 13g and 13i
against ten different kinases relative to DMSO control.
Kinase activity %
Positive
Control
IC50
(nM)
Kinase\Compd.
13i
13g
13f
76.24
93.22
87.79
104.92
79.78
82.37
86.24
87.44
70.33
87.13
94.66
94.56
84.83
95.06
80.97
89.33
88.48
73.41
84.63
93.29
83.81
85.28
Staurosporine 17.1
Staurosporine 2.58
ABL1
AKT1
B-RAF
c-Src
GW5074
21.9
Staurosporine 1.51
Staurosporine 1.31
Staurosporine 30.4
Staurosporine 1.76
Staurosporine 2.28
Staurosporine 1.51
CDK2/Cyclin A1 74.91
90.01
96.46
89.33
93.64
EGFR
FGFR1
KDR/VEGFR2
LCK
27