Design, synthesis, and biological evaluation of novel 1,3,4-thiadiazole derivatives as potential antitumor agents against chronic myelogenous leukemia: Striking effect of nitrothiazole moiety

Article abstract of DOI:10.3390/molecules23010059

In an attempt to develop potent antitumor agents, new 1,3,4-thiadiazole derivatives were synthesized and evaluated for their cytotoxic effects on multiple human cancer cell lines, including the K562 chronic myelogenous leukemia cell line that expresses the Bcr-Abl tyrosine kinase. N-(5-Nitrothiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)acetamide (2) inhibited the Abl protein kinase with an IC₅₀ value of 7.4 μM and showed selective activity against the Bcr-Abl positive K562 cell line. Furthermore, a Bcr-Abl-compound 2 molecular modelling simulation highlighted the anchoring role of the nitrothiazole moiety in bonding and hydrophobic interaction with the key amino acid residues. These results provide promising starting points for further development of novel kinase inhibitors.

Full text of DOI:10.3390/molecules23010059

molecules
Article
Design, Synthesis, and Biological Evaluation of
Novel 1,3,4-Thiadiazole Derivatives as Potential
Antitumor Agents against Chronic Myelogenous
Leukemia: Striking Effect of Nitrothiazole Moiety
Mehlika Dilek Altıntop 1, ,†
*
ID , Halil Ibrahim Ciftci 2,3,† ID , Mohamed O. Radwan 2,4
,
ID
Belgin Sever 1 ID , Zafer Asım Kaplancıklı 1 ID , Taha F. S. Ali 2,5, Ryoko Koga 2, Mikako Fujita 6,
Masami Otsuka 2,* and Ahmet Özdemir 1
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskis¸ehir 26470, Turkey;
belginsever@anadolu.edu.tr (B.S.); zakaplan@anadolu.edu.tr (Z.A.K.); ahmeto@anadolu.edu.tr (A.Ö.)
Department of Bioorganic Medicinal Chemistry, School of Pharmacy, Kumamoto University,
2
Kumamoto 862-0973, Japan; hiciftci@kumamoto-u.ac.jp (H.I.C.); mohamedosman251@gmail.com (M.O.R.);
tahafarouk1@yahoo.com (T.F.S.A.); kk1205@kumamoto-u.ac.jp (R.K.)
Stanford PULSE Institute, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA
Department of Chemistry of Natural Compounds, National Research Center, Dokki, 12622 Cairo, Egypt
Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt
3
4
5
6
Research Institute for Drug Discovery, School of Pharmacy, Kumamoto University,
Kumamoto 862-0973, Japan; mfujita@kumamoto-u.ac.jp
*
Correspondence: mdaltintop@anadolu.edu.tr (M.D.A.); motsuka@gpo.kumamoto-u.ac.jp (M.O.);
Tel.: +90-222-335-0580 (ext. 3772) (M.D.A.)
†
These authors contributed equally to this work.
Received: 24 October 2017; Accepted: 25 December 2017; Published: 27 December 2017
Abstract: In an attempt to develop potent antitumor agents, new 1,3,4-thiadiazole derivatives
were synthesized and evaluated for their cytotoxic effects on multiple human cancer cell lines,
including the K562 chronic myelogenous leukemia cell line that expresses the Bcr-Abl tyrosine kinase.
N-(5-Nitrothiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)acetamide
(
2) inhibited the Abl protein kinase with an IC50 value of 7.4
µM and showed selective activity
against the Bcr-Abl positive K562 cell line. Furthermore, a Bcr-Abl-compound
2
molecular modelling
simulation highlighted the anchoring role of the nitrothiazole moiety in bonding and hydrophobic
interaction with the key amino acid residues. These results provide promising starting points for
further development of novel kinase inhibitors.
Keywords: thiadiazole; thiazole; benzothiazole; Bcr-Abl; kinase inhibitor; leukemia
1. Introduction
Cancer is the fastest growing disease throughout the world. In developed countries, cancer has
become the leading cause of death, whereas in developing countries, it is the second leading cause of
death after cardiovascular disorders. By 2030, the annual number of new cancer diagnoses is estimated
to be 21 million worldwide, with 17 million patients dying of cancer every year and 75 million people
living with cancer diagnoses. Due to the increasing global burden of cancer, extensive efforts have
been devoted to the discovery of new potent and selective anticancer agents which destroy tumor cells
or at least limit their proliferation [1–5].
Protein kinases have emerged as one of the most frequently targeted families of proteins in
anticancer drug discovery owing to their role in the regulation of cellular pathways, particularly those
Molecules 2018, 23, 59; doi:10.3390/molecules23010059
www.mdpi.com/journal/molecules

Molecules 2018, 23, 59
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involved in signal transduction through the catalysis of phosphorylation reactions. Kinase inhibitors
have played an ascendant role in the treatment of cancer and other diseases [ 11]. In particular,
6–
imatinib is the first approved tyrosine kinase inhibitor that binds to the kinase domain of Bcr-Abl
observed in 95% of chronic myelogenous leukemia (CML) patients [12]. After the milestone approval of
imatinib, more than 25 anticancer drugs that target kinases have been approved, and several promising
candidates are in various stages of clinical evaluation [6–12].
Thiadiazole, the bioisostere of pyrimidine and oxadiazole, has been studied extensively for more
than one hundred years due to its outstanding pharmacological applications. The sulfur atom of the
thiadiazole ring imparts improved liposolubility, and the mesoionic nature of 1,3,4-thiadiazoles also
allows these compounds to cross cellular membranes and interact with biological targets with distinct
affinities. 1,3,4-Thiadiazoles display a broad spectrum of biological activities, including anticancer,
antibacterial, antifungal, antiviral, antiepileptic, antidiabetic, analgesic, and anti-inflammatory
activities [13–19]. In particular, recent studies have pointed out the significance of 1,3,4-thiadiazole as
a promising scaffold for antitumor drug discovery and development against different cancer cell lines
through the inhibition of diverse molecular targets, including histone deacetylase (HDAC), c-Src/Abl
tyrosine kinase, focal adhesion kinase (FAK), and tubulin polymerization [13
the diverse therapeutic applications of thiazoles and benzothiazoles have encouraged medicinal
chemists to synthesize a large number of thiazole/benzothiazole-based therapeutic agents [30 39].
In particular, the clinical efficacy of tiazofurin, bleomycins (BLMs), and dasatinib has pointed out
the pivotal role of the thiazole scaffold in the field of current cancer research [30 33]. Recent patents
–29]. On the other hand,
–
–
have indicated that thiazole derivatives show potent antitumor activity against different cancer cell
lines through the inhibition of kinases, pro-matrix metalloproteinase activation, signal transducer and
activator of transcription 3 (STAT3), the Bcl-2 family, and HDACs [32]. In recent years, benzothiazole
has also emerged as a privileged scaffold for anticancer drug discovery. Therefore, antitumor effects of
benzothiazole derivatives on different cancer cells have been investigated and these studies have led
to the discovery of clinical candidates such as Phortress [33–39].
Prompted by the aforementioned findings, herein we explored the possibility of 1,3,4-thiadiazole
as a pivotal scaffold for tyrosine kinase inhibitors. Figure 1a illustrates the imatinib-Bcr-Abl
kinase domain interaction; the Met318, Thr315, Asp381, Glu286, His361, and Ile360 hydrogen
bonding or CH-π interaction reported [12]; and the Tyr253 and Val256 interactions calculated (see
Supplementary Material) together with hydrophobic residues within the van der Waals radii of Leu248,
Leu370, Phe317, Val289, Val299, and Ile313 [12]. We reasoned that the introduction of a hydrogen
bonding substituent and a hydrophobic substituent into the thiadiazole scaffold would provide
promising inhibitors of Bcr-Abl tyrosine kinase (Figure 1b), as will be demonstrated in the next section.
(a)
(b)
Figure 1.
(a) Schematic illustration of interaction between imatinib and the kinase domain. Reported
interacting amino acids [12] are colored red. Additional interacting amino acids, suggested by MOE
calculation, are colored blue. The hydrophobic residues within van der Waals contact reported in [12
]
are colored black; (b) Design of 1,3,4-thiadiazole-based inhibitors.

Molecules 2018, 23, 59
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2. Results
In the present study, we designed compounds
1–10 by means of the introduction
of
a
4-(trifluoromethyl)phenylamino group as hydrophobic moiety and N-(thiazol-2-yl)-
a
2-mercaptoacetamide as a hydrogen-bonding moiety into 1,3,4-thiadiazole.
The synthesis of the hitherto unreported compounds
outlined in Scheme 1. Initially, 4-(4-(trifluoromethyl)phenyl)thiosemicarbazide
(
1
–
10
)
was performed as
was
obtained by the reaction of 4-(trifluoromethyl)phenyl isothiocyanate with hydrazine
hydrate.Then, 5-(4-(Trifluoromethyl)phenyl)amino-1,3,4-thiadiazole-2(3H)-thione ( ) was synthesized
via the ring closure reaction of 4-(4-(trifluoromethyl)phenyl)thiosemicarbazide ( ) with carbon
disulfide in the presence of potassium hydroxide. Finally, the reaction of compound with
N-(aryl)-2-chloroacetamides in the presence of potassium carbonate afforded new 1,3,4-thiadiazole
derivatives ( 10). The structures of compounds 10 were confirmed by IR, 1H NMR, 13C NMR,
and mass spectral data.
(A)
B
A
B
1–
1–
Scheme 1. The synthetic route for the preparation of compounds
(i) NH2NH2 H2O, ethanol, room temperature (r.t.), 4 h; (ii) (1) CS2/KOH, ethanol, reflux, 10 h; (2) HCl,
pH 4–5; (iii) RNHCOCH2Cl, K2CO3, acetone, r.t., 8 h.
1–10. Reagents and conditions:
·
MTT assay was carried out to determine the cytotoxic effects of compounds 1–10 on K562 CML
and other leukemia cell lines (Jurkat and MT-2) and the HeLa human cervical carcinoma cell line after
48 h of incubation. Imatinib was chosen as a positive control. The results summarized in Table 1 are
expressed as concentration-dependent activity in all cases. In general, compounds
notable antitumor activity against cancer cells except the K562 CML cell line. Compound
to be the most effective cytotoxic agent on the MT-2 cell line with an IC50 value of 38.3 M. Compounds
were found to possess IC50 values lower than 35 M against the HeLa cell line. In particular,
the most active agent was found to be compound with an IC50 value of 12.4 M, followed by
compound with an IC50 value of 14.1 M (Figure 2a). Conversely, compounds , and 10 showed
no significant activity against the HeLa cell line. Interestingly, compound showed anticancer activity
against the K562 cell line with an IC50 value of 33 M, whereas other compounds were inactive even
5
and
6
exhibited
6
was found
µ
2
–
8
µ
2
µ
3
µ
1, 9
2
µ

Molecules 2018, 23, 59
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at 300
against the imatinib-sensitive K562 cell line. Compound
peripheral blood mononuclear cells (PBMC) (IC50 = 141.3
µ
M (Figure 2b). It was striking that compound
2
was the sole active compound in this series
2
exhibited ~5 times lower cytotoxicity on
M) than imatinib (IC50 = 28.3 M) as
µ
µ
shown in Figure 2c. These results suggest that compound
cytotoxicity towards the K562 cell line.
2
exhibits a significant degree of selective
Compound 2
Compound 3
Imatinib
HeLa
100
80
60
40
20
0
0
10
30
100
300
Concentration (µM)
(a)
Compound 2
Compound 6
Imatinib
K562
100
80
60
40
20
0
0
10
30
100
300
Concentration (µM)
(b)
120
Compound 2
Imatinib
PBMC
100
80
60
40
20
0
0
10
30
100
300
1000
Concentration (µM)
(c)
Figure 2. Anticancer effects of tested compounds and imatinib on (
and (c) PBMC after 48 h of treatment.
a) HeLa cells; (b) K562 cells;

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Table 1. The cytotoxic effects of compounds 1–10 and imatinib after 48 h of incubation.
IC50 (µM) *
Compound
K562
MT-2
Jurkat
HeLa
PBMC
1
2
3
4
5
6
7
8
>300
33.0 ± 4
>300
>300
>300
>300
>300
>300
>300
>300
166.8 ± 8
>300
267.3 ± 14
78.5 ± 12
38.3 ± 2
197.9 ± 11
>300
>300
>300
9.7 ± 3
>300
17.9 ± 4
129.4 ± 6
>300
97.9 ± 10
54.4 ± 8
>300
135.1 ± 12
12.4 ± 2
14.1 ± 5
32.9 ± 2
17.3 ± 8
28.7 ± 10
30.2 ± 4
30.9 ± 4
69.4 ± 7
75.3 ± 9
15.2 ± 6
-
141.3 ± 16
-
-
-
-
-
-
-
-
>300
9
10
Imatinib
96.1 ± 7
>300
5.0 ± 2
>300
6.7 ± 2
28.3 ± 6
* The reported values represent the mean ± SD for each compound based on three independent experiments.
Anticancer screening results indicated that compound
and it was chosen for the evaluation of apoptosis and necrosis in HeLa cells, which was carried
out with the annexin V/ethidium homodimer III staining method. Compound and imatinib at
2 was the most active anticancer agent,
2
the IC50 concentrations were incubated with HeLa cells and then were stained and observed by a
fluorescence microscope. If the cells are colored red with ethidium homodimer III, and not stained
green, the cells are judged to be in necrosis. On the contrary, the completely opposite results indicate
apoptosis. The apoptotic and necrotic effects of compound
48 h, and the results showed that compound and imatinib both induced late apoptosis or necrosis
(data not shown). The apoptotic/necrotic effects of compound and imatinib were also tested on HeLa
cells at an earlier time (3 h) at IC50 concentrations, and the results indicated that compound showed
96% apoptotic activity at 3 h as illustrated in Figure 3. In contrast, HeLa cells treated with imatinib
2 were compared with imatinib at 24 h and
2
2
2
at 3 h showed 78% apoptotic activity. The results revealed that compound
strongly than imatinib.
2
induced apoptosis more
(a)
ethidium
homodimer III
bright
annexin V
hoechst33342
overlap
(b)
ethidium
homodimer III
bright
annexin V
hoechst33342
overlap
Figure 3. Cont.

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(c)
ethidium
homodimer III
bright
annexin V
hoechst33342
overlap
(d)
120
100
80
60
40
20
0
Necrosis
Late apoptosis/necrosis
Apoptosis
Compound 2
Imatinib
Figure 3. Cellular changes of HeLa cells following exposure to IC50 concentrations of control.
) imatinib; ( ) compound ; ( ) for 3 h; ( ) a total of approximately 100 stained cells randomly
(
a
b
2
c
d
selected in each experiment of (b,c) were categorized into three types: “apoptosis”, “late apoptosis or
necrosis”, and “necrosis”.
In order to explore the inhibitory potency of compound 2 against kinase enzymes, eight kinases
(ABL1, BRK, BTK, CSK, FYN A, LCK, LYN B, and SRC) were selected from a large panel of kinases.
Multipoint dose-response experiments using a selected kinase family were tested for inhibition by
compound
enzyme with an IC50 value of 7.4
with IC50 values in the micromolar range. On the other hand, compound
LYN B, and SRC as shown in Table 2. MT-2 has high expression levels of LYN [40], and thus it may be
one reason that compound was less effective than imatinib against the MT-2 cell line. These results
indicate that compound targets not only ABL, but also inhibits multiple kinases, such as CSK, BTK,
2
. Compound
2
displayed the most potent inhibitory activity against the ABL1 kinase
M (Figure 4), and significantly inhibited BTK, CSK, FYN A, and LCK
was inactive against BRK,
µ
2
2
2
and LCK, in the panel of kinases. It can be concluded that compound
profile than imatinib (Figure 5).
2 has a different kinase inhibitor
Table 2. The protein kinase inhibition of compound 2 and imatinib.
IC50 (µM)
Kinase
Compound 2
Imatinib
ABL1
BRK
BTK
CSK
FYN A
LCK
7.4
0.2
19
>100
16.4
10
0.3
5.8
99
>100
30.2
18.4
70.8
40.1
>100
>100
LYN B
SRC

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120
100
80
60
40
20
0
Compound 2
Imatinib
0
3
10
30
100
300
Concentration (µM)
Figure 4. ABL1 kinase inhibition of compound
30 µM, and 100 µM).
2
and imatinib at varying concentrations (3 µM, 10 µM,
Compound 2
Imatinib
100
80
60
40
20
0
ABL1
Figure 5. Inhibition of protein kinases by compound 2 and imatinib.
In order to optimize our promising antiproliferative lead compound and investigate the binding
BRK
BTK
CSK
FYN A
LCK
LYN B
SRC
2
modes of the synthesized compounds with the Bcr-Abl tyrosine kinase, a molecular docking simulation
study was conducted. The co-crystal structure of imatinib with the Bcr-Abl tyrosine kinase was selected
as the docking model (PDB ID code: 1IEP [12]).
Accordingly, after the validation of the applied computational protocol, compounds
1, 2, and 10
were docked into (1IEP) [12 41]. The resultant binding mode of compound reveals the role of the
,
2
distal nitro group in the formation of a crucial hydrogen bond with Met318. It seems that the presence
of the nitro group induces a reorientation of the thiazole moiety to allow a significant hydrogen bond
formation with Thr315. One more critical hydrogen bond is established between the sulfur bridge
atom and Asp381 residue. The trifluoromethylphenyl group is embedded in the hydrophobic region
of the pocket including Ile293, Leu298, and Leu354. Moreover, the CH-
stabilize and lock the Abl kinase into its inactive conformation. Based on the docking study, it can
be speculated that the high IC50 value of compound compared to imatinib can be attributed to the
missed interactions with Ile360 and His361. Additionally, the polar thiazole ring of compound has
π bond with Tyr253 can help
2
2
lower affinity than the pyridine moiety of imatinib towards the surrounding hydrophobic residues
Phe317, Tyr253, Leu248, and Leu370 (Figure 6).

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Figure 6. Comparison of binding mode of imatinib (left) and compound
site of Abl kinase (1IEP). Docking scores are 11.3 and 9.2 kcal/mol, respectively. The key amino acid
residues are depicted as cyan sticks. Hydrogen bonds and the CH- bond are shown as white dashes.
2 (right) into the ATP binding
−
−
π
Unlike compound 2, the docked conformations of compounds 1 and 10 did not form the pivotal
interactions with the key amino acid residues, such as Met318 and Tyr253. Both compounds formed
only two hydrogen bonds with the carboxylate group of Glu286 and van der Waals interactions between
the trifluoromethylphenyl group and the hydrophobic region of the pocket (Figure 7). In conclusion,
the absence of or a shift in the distal nitro group abolishes the Abl-kinase inhibitory activity.
Figure 7. Binding conformations of compound
1 (left) and compound 10 (right) into the ATP binding
site of Abl kinase (1IEP). The key amino acid residues are depicted as cyan sticks. Hydrogen bonds
are shown as black dashes. For clarity, the receptor Connolly surface is represented as follows (pink:
hydrophilic; green: hydrophobic; white: neutral).
3. Materials and Methods
3.1. Chemistry
All reagents were purchased from commercial suppliers and were used without further
purification. Melting points (m.p.) were determined on an Electrothermal 9100 melting point apparatus
(Weiss-Gallenkamp, Loughborough, UK) and are uncorrected. IR spectra were recorded on an
1
IRPrestige-21 Fourier Transform Infrared spectrophotometer (Shimadzu, Tokyo, Japan). H NMR and
13C NMR spectra were recorded on a Varian Mercury-400 FT-NMR spectrometer (Agilent, Palo Alto,
CA, USA). Mass spectra were recorded on a Shimadzu LCMS-IT-TOF system (Shimadzu, Kyoto, Japan).
Thin Layer Chromatography (TLC) was performed on TLC Silica gel 60 F254 aluminium sheets (Merck,
Darmstadt, Germany) to check the purity of the compounds.

Molecules 2018, 23, 59
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3.1.1. General Procedure for the Synthesis of the Compounds
4-(4-(Trifluoromethyl)phenyl)thiosemicarbazide (A)
A mixture of 4-(trifluoromethyl)phenyl isothiocyanate (0.1 mol) and hydrazine hydrate (0.2 mol) in
ethanol (30 mL) was stirred at room temperature for 4 h and then filtered. The residue was crystallized
from ethanol [42].
5-(4-(Trifluoromethyl)phenyl)amino-1,3,4-thiadiazole-2(3H)-thione (B)
4-(4-Trifluoromethylphenyl)thiosemicarbazide (A) was dissolved in a solution of potassium
hydroxide in ethanol. Carbon disulfide was then added while stirring, and the reaction mixture was
heated under reflux for 10 h. The solution was cooled and acidified to pH 4–5 with a hydrochloric acid
solution and crystallized from ethanol [43].
N-(Aryl)-2-chloroacetamides
Chloroacetyl chloride (0.1 mol) was added dropwise with stirring to a mixture of arylamine
(0.1 mol) and triethylamine in toluene at 0–5 ◦C. The solvent was evaporated under reduced pressure.
The residue was washed with water to remove triethylamine hydrochloride and crystallized from
ethanol [44].
N-(Aryl)-2-[(5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio]acetamide
Derivatives (1–10)
A
mixture of N-(aryl)-2-chloroacetamide (2 mmol) and 5-(4-trifluoromethylphenyl)
amino-1,3,4-thiadiazole-2(3H)-thione (2 mmol) in acetone was stirred at room
in the presence of potassium carbonate. The reaction mixture
The residue was washed with water and crystallized from ethanol [43].
). Yield:
(B)
temperature for
was filtered.
8 h
N-(Thiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)acetamide (
1
◦
78%. m.p.: 271.4–272.5 C. IR
ν
(cm−1): 3257.77, 3194.12 (N-H stretching), 3076.46 (Aromatic
max
C-H stretching), 2920.23, 2821.86, 2740.85 (Aliphatic C-H stretching), 1674.21 (C=O stretching),
1618.28, 1593.20, 1570.06, 1519.91, 1450.47 (N-H bending, C=N and C=C stretching), 1413.82,
1384.89 (C-H bending), 1330.88, 1267.23, 1238.30, 1178.51, 1159.22, 1107.14, 1068.56, 1047.35,
1008.77 (C-N stretching and aromatic C-H in plane bending), 968.27, 873.75, 831.32, 813.96, 773.46,
717.52, 684.73, 661.58, 626.87 (Aromatic C-H out of plane bending and C-S stretching). 1H NMR
(400 MHz, DMSO-d6)
δ (ppm): 4.25 (s, 2H), 7.25 (d, J = 3.6 Hz, 1H), 7.50 (d, J = 3.2 Hz, 1H),
7.68 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 8.8 Hz, 2H), 10.79 (brs, 1H), 12.43 (brs, 1H). 13C NMR (100
MHz, DMSO-d6): 36.79 (CH2), 114.29 (CH), 117.65 (2CH), 122.18 (d, J = 31.4 Hz, C), 126.30
(C), 126.89 (d, J = 3.9 Hz, 2CH), 138.24 (CH), 143.94 (C), 153.40 (C), 157.75 (C), 164.36 (C),
165.93 (C). HRMS (ESI) (m/z): [M + H]+ calcd. for C14H10F3N5OS3: 418.0072, found: 418.0077.
N-(5-Nitrothiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)acetamide
(
2).
◦
Yield: 88%. m.p.: 275.0–276.5 C. IR
ν
max
(cm−1): 3354.21, 3197.98 (N-H stretching), 3142.04 (Aromatic
C-H stretching), 2933.73, 2864.29 (Aliphatic C-H stretching), 1681.93 (C=O stretching), 1614.42, 1558.48,
1508.33, 1469.76 (N-H bending, C=N and C=C stretching), 1411.89, 1381.03, 1352.10 (C-H bending),
1321.24, 1298.09, 1269.16, 1238.30, 1180.44, 1159.22, 1114.86, 1093.64, 1066.64 (C-N stretching and
aromatic C-H in plane bending), 968.27, 840.96, 812.03, 779.24, 767.67, 738.74, 723.31, 682.80, 663.51
1
(Aromatic C-H out of plane bending and C-S stretching). H NMR (400 MHz, DMSO-d6)
δ (ppm):
4.10 (s, 2H), 7.68 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H), 8.45 (s, 1H), 10.74 (s, 1H), 13.11 (brs,
1H). 13C NMR (100 MHz, DMSO-d6): 41.50 (CH2), 117.07 (2CH), 121.51 (d, J = 31.4 Hz, C), 125.87
(C), 126.39 (d, J = 3.9 Hz, 2CH), 136.65 (C), 143.64 (C), 145.80 (CH), 155.41 (C), 163.95 (2C), 174.18
(C). HRMS (ESI) (m/z): [M + H]+ calcd. for C14H9F3N6O3S3: 462.9923, found: 462.9926. Ethyl

Molecules 2018, 23, 59
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2-(2-(2-((5-((4-(trifluoromethyl◦)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)acetamido)thiazol-4-yl)acetate (
(cm−1): 3246.20, 3196.05 (N-H stretching), 3140.11 (Aromatic
max
3).
Yield: 87%. m.p.: 222.3–224.1 C. IR
ν
C-H stretching), 2922.16, 2848.86, 2719.63 (Aliphatic C-H stretching), 1716.65 (C=O stretching), 1685.79
(C=O stretching), 1618.28, 1560.41, 1521.84, 1498.69 (N-H bending, C=N and C=C stretching), 1413.82,
1367.53 (C-H bending), 1317.38, 1259.52, 1207.44, 1161.15, 1114.86, 1066.64, 1058.92, 1028.06 (C-N, C-O
stretching and aromatic C-H in plane bending), 958.62, 937.40, 877.61, 840.96, 792.74, 744.52, 729.09,
1
682.80, 632.65 (Aromatic C-H out of plane bending and C-S stretching). H NMR (400 MHz, DMSO-d6)
δ
(ppm): 1.18 (t, J = 6.8 Hz, 7.2 Hz, 3H), 3.69 (s, 2H), 4.08 (q, J = 6.8 Hz, 2H), 4.21 (s, 2H), 7.01 (s, 1H),
7.68 (d, J = 9.2 Hz, 2H), 7.76 (d, J = 8.8 Hz, 2H), 10.79 (brs, 1H), 12.48 (brs, 1H). 13C NMR (100 MHz,
DMSO-d6): 14.07 (CH3), 36.61 (CH2), 36.85 (CH2), 60.31 (CH2), 110.72 (CH), 117.19 (2CH), 121.70 (d, J =
32.1 Hz, C), 125.92 (C), 126.42 (d, J = 3.2 Hz, 2CH), 143.50 (C), 153.39 (C), 157.49 (C), 164.35 (C), 166.02
(C), 169.98 (2C). HRMS (ESI) (m/z): [M + H]+ calcd. for C18H16F3N5O3S3: 504.0440, found: 504.0445.
N-(Benzothiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)acetamide (
4). Yield:
◦
85%. m.p.: 293.8–294.5 C. IR
ν
max
(cm−1): 3232.70, 3182.55 (N-H stretching), 3138.18, 3066.82, 3037.89
(Aromatic C-H stretching), 2953.02, 2916.37, 2831.50, 2729.27 (Aliphatic C-H stretching), 1681.93 (C=O
stretching), 1602.85, 1566.20, 1444.68 (N-H bending, C=N and C=C stretching), 1417.68, 1382.96 (C-H
bending), 1323.17, 1267.23, 1232.51, 1163.08, 1109.07, 1068.56, 1047.35, 1012.63 (C-N stretching and
aromatic C-H in plane bending), 983.70, 873.75, 846.75, 833.25, 802.39, 756.10, 727.16, 678.94, 657.73
1
(Aromatic C-H out of plane bending and C-S stretching). H NMR (400 MHz, DMSO-d6)
δ (ppm):
4.32 (s, 2H), 7.32 (t, J = 7.6 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.67 (d, J = 8.8 Hz, 2H), 7.74–7.78 (m,
3H), 7.98 (d, J = 7.6 Hz, 1H), 10.79 (brs, 1H), 12.68 (brs, 1H). 13C NMR (100 MHz, DMSO-d6): 37.03
(CH2), 117.17 (2CH), 120.63 (CH), 121.71 (d, J = 32.1 Hz, C), 121.75 (CH), 123.68 (CH), 125.81 (CH),
126.18 (C), 126.41 (d, J = 3.9 Hz, 2CH), 131.44 (C), 143.46 (C), 148.48 (C), 153.30 (C), 157.77 (C), 164.38
(C), 167.10 (C). HRMS (ESI) (m/z): [M + H]+ calcd. for C18H12F3N5OS3: 468.0229, found: 468.0213.
N-(6-Fluorobenzothiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)acetamide
◦
(5
). Yield: 87%. m.p.: 309.9–310.8 C. IR
ν
(cm−1): 3238.48, 3186.40 (N-H stretching), 3082.25
max
(Aromatic C-H stretching), 2951.09, 2916.37, 2854.65, 2792.93, 2723.49 (Aliphatic C-H stretching),
1678.07 (C=O stretching), 1608.63, 1589.34, 1566.20, 1450.47 (N-H bending, C=N and C=C stretching),
1419.61, 1382.96 (C-H bending), 1327.03, 1249.87, 1168.86, 1112.93, 1068.56, 1051.20, 1012.63 (C-N
stretching and aromatic C-H in plane bending), 983.70, 918.12, 873.75, 835.18, 817.82, 804.32,
748.38, 707.88, 673.16, 659.66 (Aromatic C-H out of plane bending and C-S stretching). 1H NMR
(400 MHz, DMSO-d6)
δ (ppm): 4.31 (s, 2H), 7.30 (td, J = 2.8 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H),
7.74–7.79 (m, 3H), 7.90 (dd, J = 2.4, 8.8 Hz, 1H), 10.79 (s, 1H), 12.70 (brs, 1H). 13C NMR (100 MHz,
DMSO-d6): 36.99 (CH2), 108.21 (d, J = 26.9 Hz, CH), 114.31 (d, J = 24.4 Hz, CH), 117.17 (2CH),
121.72 (d, J = 32.1 Hz, C), 121.77 (d, J = 9.6 Hz, CH), 125.82 (C), 126.41 (d, J = 3.8 Hz, 2CH),
132.70 (d, J = 10.9 Hz, C), 143.47 (C), 145.22 (C), 153.27 (C), 157.50 (C), 159.88 (C), 164.39 (C),
167.22 (C). HRMS (ESI) (m/z): [M + H]+ calcd. for C18H11F4N5OS3: 486.0135, found: 486.0131.
N-(6-Chlorobenzothiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)acetamide
◦
(6
). Yield: 90%. m.p.: 317.3–318.1 C. IR
ν
(cm−1): 3232.70, 3180.62 (N-H stretching), 3136.25,
max
3072.60, 3035.96 (Aromatic C-H stretching), 2951.09, 2914.44, 2825.72, 2713.84 (Aliphatic C-H
stretching), 1678.07 (C=O stretching), 1602.85, 1566.20, 1448.54 (N-H bending, C=N and C=C
stretching), 1417.68, 1382.96 (C-H bending), 1325.10, 1265.30, 1236.37, 1165.00, 1111.00, 1099.43,
1068.56, 1051.20, 1012.63 (C-N stretching and aromatic C-H in plane bending), 981.77, 879.54,
846.75, 817.82, 806.25, 765.74, 746.45, 692.44, 659.66 (Aromatic C-H out of plane bending and
C-S stretching). 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 4.32 (s, 2H), 7.47 (d, J = 8.8 Hz, 1H),
7.66–7.77 (m, 5H), 8.14 (s, 1H), 10.80 (s, 1H), 12.79 (brs, 1H). 13C NMR (100 MHz, DMSO-d6):
37.01 (CH2), 117.17 (2CH), 121.48 (CH), 121.56 (C), 121.85 (CH), 126.41 (d, J = 3.8 Hz, 2CH),
126.54 (C), 127.72 (CH), 129.02 (C), 133.16 (C), 143.46 (C), 147.39 (C), 153.26 (C), 158.70 (C), 164.39
(C), 167.36 (C). HRMS (ESI) (m/z): [M + H]+ calcd. for C18H11ClF3N5OS3: 501.9839, found:

Molecules 2018, 23, 59
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501.9864.
acetamide (
N-(6-Methylbenzothiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)
◦
7
). Yield: 81%. m.p.: 294.3–295.1 C. IR
ν
(cm−1): 3232.70, 3184.48 (N-H stretching),
max
3138.18, 3068.75, 3034.03 (Aromatic C-H stretching), 2954.95, 2916.37, 2829.57, 2789.07, 2729.27
(Aliphatic C-H stretching), 1681.93 (C=O stretching), 1606.70, 1587.42, 1566.20, 1450.47 (N-H
bending, C=N and C=C stretching), 1417.68, 1384.89 (C-H bending), 1325.10, 1269.16, 1234.44,
1165.00, 1111.00, 1070.49, 1047.35, 1012.63 (C-N stretching and aromatic C-H in plane bending),
983.70, 815.89, 748.38, 675.09, 657.73 (Aromatic C-H out of plane bending and C-S stretching). 1H
NMR (400 MHz, DMSO-d6)
δ (ppm): 2.41 (s, 3H), 4.30 (s, 2H), 7.26 (d, J = 8.4 Hz, 1H), 7.64–7.76
(m, 6H), 10.79 (s, 1H), 12.59 (brs, 1H). 13C NMR (100 MHz, DMSO-d6): 20.98 (CH3), 37.04 (CH2),
117.18 (2CH), 120.29 (CH), 121.35 (CH), 121.72 (d, J = 32.1 Hz, C), 125.83 (C), 126.43 (d, J = 3.9 Hz,
2CH), 127.53 (CH), 131.59 (C), 133.18 (C), 143.48 (C), 146.45 (C), 153.35 (C), 156.92 (C), 164.39 (C),
166.96 (C). HRMS (ESI) (m/z): [M + H]+ calcd. for C19H14F3N5OS3: 482.0385, found: 482.0366.
N-(6-Methoxybenzothiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)
◦
acetamide (
8
). Yield: 83%. m.p.: 278.7–280.1 C. IR
ν
(cm−1): 3230.77, 3182.55 (N-H stretching),
max
3072.60 (Aromatic C-H stretching), 2953.02, 2914.44, 2835.36, 2733.13 (Aliphatic C-H stretching),
1681.93 (C=O stretching), 1608.63, 1591.27, 1564.27, 1462.04 (N-H bending, C=N and C=C stretching),
1417.68, 1384.89 (C-H bending), 1327.03, 1265.30, 1170.79, 1163.08, 1109.07, 1068.56, 1033.85,
1012.63 (C-N, C-O stretching and aromatic C-H in plane bending), 983.70, 873.75, 837.11, 819.75,
796.60, 748.38, 707.88, 659.66 (Aromatic C-H out of plane bending and C-S stretching). 1H NMR
(400 MHz, DMSO-d6)
δ (ppm): 3.81 (s, 3H), 4.30 (s, 2H), 7.04 (d, J = 8.4 Hz, 1H), 7.57–7.77 (m,
6H), 10.80 (s, 1H), 12.55 (s, 1H). 13C NMR (100 MHz, DMSO-d6): 37.45 (CH2), 56.08 (CH3), 105.20
(CH), 115.48 (CH), 117.64 (2CH), 121.73 (CH), 122.18 (d, J = 32.0 Hz, C), 126.29 (C), 126.88 (d,
J = 3.8 Hz, 2CH), 133.24 (C), 143.04 (C), 143.94 (C), 153.81 (C), 156.14 (C), 156.69 (C), 164.85
(C), 167.22 (C). HRMS (ESI) (m/z): [M + H]+ calcd. for C19H14F3N5O2S3: 498.0334, found:
498.0328. N-(6-Ethoxybenzothiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)
acetamide (
9
). Yield: 82%. m.p.: 270.5–271.9 ◦C. IR
ν
(cm−1): 3180.62 (N-H stretching),
max
3070.68 (Aromatic C-H stretching), 2914.44, 2835.36, 2738.92 (Aliphatic C-H stretching), 1683.86
(C=O stretching), 1606.70, 1591.27, 1560.41, 1456.26 (N-H bending, C=N and C=C stretching), 1413.82,
1386.82 (C-H bending), 1323.17, 1259.52, 1211.30, 1170.79, 1109.07, 1066.64, 1041.56, 1012.63 (C-N,
C-O stretching and aromatic C-H in plane bending), 983.70, 943.19, 869.90, 840.96, 817.82, 792.74,
1
750.31, 673.16, 659.66 (aromatic C-H out of plane bending and C-S stretching). H NMR (400 MHz,
DMSO-d6)
δ (ppm): 1.35 (t, J = 6.8 Hz, 7.2 Hz, 3H), 4.07 (q, J = 7.2 Hz, 2H), 4.29 (s, 2H), 7.03 (dd,
J = 2.4, 8.8 Hz, 1H), 7.56 (d, J = 2.4 Hz, 1H), 7.64–7.68 (m, 3H), 7.76 (d, J = 8.8 Hz, 2H), 10.79 (s,
1H), 12.55 (s, 1H). 13C NMR (100 MHz, DMSO-d6): 14.65 (CH3), 36.97 (CH2), 63.58 (CH2), 105.36
(CH), 115.36 (CH), 117.16 (2CH), 121.26 (CH), 121.70 (d, J = 32.1 Hz, C), 125.81 (C), 126.41 (d,
J = 3.9 Hz, 2CH), 132.76 (C), 142.49 (C), 143.45 (C), 153.31 (C), 155.44 (C), 155.59 (C), 164.37 (C),
166.71 (C). HRMS (ESI) (m/z): [M + H]+ calcd. for C20H16F3N5O2S3: 512.0491, found: 512.0494.
N-(6-Nitrobenzothiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)acetamide
(
10). Yield: 92%. m.p.: 311.8–312.5 ◦C. IR
ν
(cm−1): 3184.48 (N-H stretching), 3068.75,
max
3034.03 (Aromatic C-H stretching), 2949.16, 2912.51, 2794.85 (Aliphatic C-H stretching), 1683.86
(C=O stretching), 1608.63, 1583.56, 1564.27, 1519.91, 1448.54 (N-H bending, C=N and C=C stretching),
1415.75, 1384.89 (C-H bending), 1325.10, 1286.52, 1238.30, 1172.72, 1109.07, 1070.49, 1045.42, 1012.63
(C-N, C-O stretching and aromatic C-H in plane bending), 981.77, 904.61, 873.75, 846.75, 825.53, 752.24,
1
719.45, 688.59, 659.66 (Aromatic C-H out of plane bending and C-S stretching). H NMR (400 MHz,
DMSO-d6)
δ (ppm): 4.36 (s, 2H), 7.67 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 9.2 Hz, 1H),
8.29 (d, J = 8.8 Hz, 1H), 9.06 (s, 1H), 10.80 (s, 1H), 13.11 (brs, 1H). 13C NMR (100 MHz, DMSO-d6):
37.04 (CH2), 117.17 (2CH), 119.09 (CH), 120.71 (CH), 121.71 (d, J = 32.0 Hz, C), 121.80 (CH), 125.80 (C),

Molecules 2018, 23, 59
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126.41 (d, J = 3.9 Hz, 2CH), 132.20 (C), 143.05 (C), 143.44 (C), 153.17 (C), 153.43 (C), 163.44 (C), 164.40
(C), 167.98 (C). HRMS (ESI) (m/z): [M + H]+ calcd. for C18H11F3N6O3S3: 513.0080, found: 513.0056.
3.2. Biochemistry
3.2.1. Cell Cultures
HeLa human cervical carcinoma cells were incubated in Dulbecco’s modified Eagle’s medium
(DMEM) (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum
(FBS) (Equitech-Bio, Kerrville, TX, USA). MT-2, Jurkat, and K562 human leukemic cells were incubated
in RPMI 1640 (Wako Pure Chemical Industries) supplemented with 10% FBS. Peripheral blood
mononuclear cells (PBMC) were incubated in RPMI 1640, and supplemented with 10% fetal bovine
serum (FBS) (Biosera, Kansas City, MO, USA) [45]. All media were supplemented with 89
streptomycin (Meiji Seika Pharma, Tokyo, Japan), and cells were incubated at 37 ◦C in a humidified
atmosphere of 95% air and 5% CO2. Growing cells were plated at 2
104 cells/mL into 24-well
µg/mL
×
microtiter tissue culture plates (Iwaki brand Asahi Glass Co., Chiba, Japan) and incubated for 48 h
before the addition of the drugs (the optimal cell number for cytotoxicity assays was determined in
preliminary experiments). Stock solutions (1 mM, 3 mM, 10 mM, and 30 mM) of the compounds and
imatinib (Wako Pure Chemical Industries) were prepared in dimethyl sulfoxide (DMSO; Wako Pure
Chemical Industries) and then added to fresh culture medium. The concentration of DMSO in the final
culture medium was 1%.
3.2.2. MTT Assay
The level of cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (Dojindo Molecular Technologies, Kumamoto, Japan) was quantified as previously described
in the literature with small modifications [46]. The tested compounds were incubated with cells in
various concentrations for 48 h. The cells were then stained with MTT solution and incubated further
◦
for 4 h at 37 C. The solution was removed, and the formazan crystals were solubilized by an addition
of 100 µL DMSO. The absorbance of the converted dye in the living cells was measured at a wavelength
of 550 nm using a microplate spectrophotometer Infinitive M1000 (Tecan, Groding, Austria). Cell
viability was calculated as a percentage of the viable control cells. All experiments were performed in
triplicate, and IC50 values were defined as the drug concentrations which reduced absorbance to 50%
of control values.
3.2.3. Apoptotic/Necrotic/Healthy Cells Detection Assay
HeLa cells were incubated with compound
2 and imatinib at IC50 concentration for 3 h, 24 h,
and 48 h. Then, detection of apoptotic/necrotic/healthy cells was performed according to the
manufacturer’s instructions (PromoKine, Heidelberg, Germany). After the cells were washed
twice with 1
FITC-Annexin V solution, 2
was added and the cells were incubated for 15 min at room temperature (RT), protected from light.
The cells were washed with 1 binding buffer, analyzed by the all-in-one fluorescence microscope
×
binding buffer, a staining solution containing 50
µ
L of 1
×
binding buffer, 2 µL of
µL of ethidium homodimer III solution, and 2
µL of Hoechst 33,342 solution
×
Biorevo Fluorescence BZ-9000 (Keyence, Osaka, Japan), and counted as described previously in [47].
3.2.4. Kinase Inhibition Assay
Kinase selectivity profiling system (TK-2) assays were performed according to the manufacturer’s
instructions (Promega Corporation, Madison, WI, USA). Multipoint dose-response experiments were
performed using eight kinases, namely: ABL1, BRK, BTK, CSK, FYN A, LCK, LYN B, and SRC. Briefly,
the kinase and substrate strips were diluted with 95
ATP solution, respectively. The kinase reaction was performed using 1
varying concentrations (15 µM, 30 µM, 150 µM, and 500 µM), 2 µL of kinase working stock, and 2 µL
µL of 2.5
×
Kinase Buffer and 15
µL of 100 µM
µL of compound solution at

Molecules 2018, 23, 59
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of ATP/substrate working stock. After 1 h of incubation at room temperature, kinase activity was
quantified using the ADP-Glo Kinase Assay (Promega Corporation). Kinase inhibition was quantified
using an Infinitive M1000 luminescence microplate spectrophotometer (Tecan, Groding, Austria).
The concentration of test compounds required to decrease the kinase activity by 50% was determined
using ImageJ software and identified as the IC50.
3.3. Molecular Modelling
The software MOE 2015.10 (Chemical Computing Group, Montreal, Canada) was employed for
all molecular docking and visualization procedures. 1IEP was retrieved from the RCSB Brookhaven
Protein Data Bank. Before the docking simulations, ligands and the target protein were prepared
with the standard protocol of the MOE 2015.10 software, including the addition of hydrogens,
the assignment of bond order, and the assessment of the correct protonation state. All docking
calculations were performed using default settings implemented in MOE 2015.10 [48–50].
4. Conclusions
In the current work, new 1,3,4-thiadiazole compounds were synthesized and evaluated for their
cytotoxic effects on the K562 CML cell line and other leukemia cell lines (Jurkat and MT-2) and the
HeLa human cervical carcinoma cell line. Compound
2 was the most potent anticancer agent and
exhibited tumor selectivity. Compound specifically inhibited Abl kinase, and showed preferential
2
antiproliferative activity against K562 cell line expressing the oncogenic kinase Bcr-Abl. Furthermore,
this compound exhibited higher BTK kinase inhibitory activity than imatinib. These results suggest
that compound 2 has a different kinase inhibitor profile than imatinib.
Figure 8 shows the Bcr-Abl binding mode of compounds
2, 1, and 10. As shown in Figure 8a,
compound forms hydrogen bonding between the nitrothiazole group and Met318 in addition to
2
Tyr253-thiazole, and Thr315–thiazole interactions and the trifluoromethylphenyl group are surrounded
by hydrophobic Val289, Val299, and Ile313 residues. This outcome indicates that our original “hydrogen
bonding–thiadiazole–hydrophobic interaction” concept (Figure 1b) is workable. In contrast, compound
1
without the nitro group does not have interaction with Met318, Tyr253, or Thr315 residues and is
inactive against the K562 cell line, suggesting the anchoring role of the nitro group. Compound 10 has
an intervening benzene ring between the nitro substituent and the thiazole ring, misplacing the nitro
group away from the Met318.
(a)
(b)
Figure 8. Cont.

Molecules 2018, 23, 59
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(c)
Figure 8. Illustrated binding mode between Bcr-Abl and compound
10 (c) based on the docking study.
2 (a); compound 1 (b); compound
According to the obtained biological results and the molecular docking study, the introduction of a
nitro substituent into the thiazole scaffold represents an important lead for the discovery of new protein
kinase inhibitors, especially because of the emerging resistance to existing drugs. Further research can
be carried out on the development of novel effective inhibitors by the optimization of compound
enhance its potency against kinase enzymes and cancer cell lines.
2 to
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: This study was supported by Anadolu University Scientific Research Projects Commission
under grant No. 1605S198 and the Bilateral Joint Research Project from the Japanese Society for the Promotion of
Science (the grant No. 16039901-000867).
Author Contributions: A.Ö. and M.D.A. designed the research, and A.Ö., M.D.A., B.S. and Z.A.K. performed
the synthetic work. M.F. and H.I.C. coordinated the biological research, H.I.C. undertook the main biological
experiments, R.K. carried out the cellular studies, and M.O.R. and T.F.S.A. were responsible for the molecular
modelling. M.O., M.D.A., and H.I.C. mainly wrote the manuscript. M.O. and M.D.A. were also responsible for the
correspondence of the manuscript. All authors discussed, edited, and approved the final version.
Conflicts of Interest: The authors report no conflicts of interest.
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Sample Availability: Samples of the compounds 1–10 are available from the authors.
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