Synthesis and anticancer activity of some novel benzothiazole-thiazolidine derivatives

Article abstract of DOI:10.1080/10426507.2017.1395878

Sixteen new 2-(benzothiazol-2-ylthio)-N′-(3-substituted-4-(3,4-substitutedphenyl)thiazol-2(3H)-ylidene)acetohydrazide derivatives (4a-4p) were synthesized. The structures of the synthesized compounds were elucidated using FT-IR, ¹H-NMR, ¹³C-NMR, and HRMS spectral data. Anticancer activity of the compounds 4a-4p against C6 (rat brain glioma) and A549 (human lung adenocarcinoma) cell lines was evaluated by using MTT, inhibition of DNA synthesis, and flow cytometric analysis assays. According to MTT assay, 4a and 4d were found to be the most active compounds against C6 cell line with an IC₅₀ value of 0.03?mM. Moreover, IC₅₀ values of 4a (0.2 mM) and 4d (0.1 mM) against NIH3T3 (mouse embryo fibroblast cell line) were higher than their IC₅₀ values (0.03 mM) against C6 cell line. Accordingly, selectivity of compound 4a against C6 cell line was two-fold higher than that of compound 4d. Flow cytometry analysis showed that these compounds display anticancer activity by inducing apoptosis. As a result, compound 4a has a remarkable anticancer activity and a good selectivity towards C6 cell lines.

Full text of DOI:10.1080/10426507.2017.1395878

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: http://www.tandfonline.com/loi/gpss20
Synthesis and anticancer activity of some novel
benzothiazole-thiazolidine derivatives
Derya Osmaniye, Serkan Levent, Cankız Mina Ardıç, Özlem Atlı, Yusuf Özkay
& Zafer Asım Kaplancıklı
To cite this article: Derya Osmaniye, Serkan Levent, Cankız Mina Ardıç, Özlem Atlı, Yusuf Özkay
& Zafer Asım Kaplancıklı (2017): Synthesis and anticancer activity of some novel benzothiazole-
thiazolidine derivatives, Phosphorus, Sulfur, and Silicon and the Related Elements, DOI:
10.1080/10426507.2017.1395878
To link to this article: https://doi.org/10.1080/10426507.2017.1395878
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Date: 21 November 2017, At: 09:33

PHOSPHORUS, SULFUR, AND SILICON
https://doi.org/./..
Synthesis and anticancer activity of some novel benzothiazole-thiazolidine
derivatives
Derya Osmaniyea,b, Serkan Leventa,b, Cankız Mina Ardıçc, Özlem Atlıc, Yusuf Özkaya,b, and Zafer Asım Kaplancıklıa
aFaculty of Pharmacy, Department of Pharmaceutical Chemistry, Anadolu University, Eskis¸ehir, Turkey; bFaculty of Pharmacy, Doping and Narcotic
Compounds Analysis Laboratory, Anadolu University, Eskis¸ehir, Turkey; cFaculty of Pharmacy, Department of Pharmaceutical Toxicology, Anadolu
University, Eskis¸ehir, Turkey
ABSTRACT
ARTICLE HISTORY
Received  July 
Accepted  October 
Sixteen new 2-(benzothiazol-2-ylthio)-Nꢀ-(3-substituted-4-(3,4-substitutedphenyl)thiazol-2(3H)-ylidene)
acetohydrazide derivatives (4a-4p) were synthesized. The structures of the synthesized compounds were
elucidated using FT-IR, 1H-NMR, 13C-NMR, and HRMS spectral data. Anticancer activity of the compounds
4a-4p against C6 (rat brain glioma) and A549 (human lung adenocarcinoma) cell lines was evaluated by
using MTT, inhibition of DNA synthesis, and flow cytometric analysis assays. According to MTT assay, 4a
and 4d were found to be the most active compounds against C6 cell line with an IC50 value of 0.03 mM.
Moreover, IC50 values of 4a (0.2 mM) and 4d (0.1 mM) against NIH3T3 (mouse embryo fibroblast cell line)
were higher than their IC50 values (0.03 mM) against C6 cell line. Accordingly, selectivity of compound
4a against C6 cell line was two-fold higher than that of compound 4d. Flow cytometry analysis showed
that these compounds display anticancer activity by inducing apoptosis. As a result, compound 4a has a
remarkable anticancer activity and a good selectivity towards C6 cell lines.
KEYWORDS
Benzothiazole; Anticancer;
C cell line; A cell line;
Apoptosis
GRAPHICAL ABSTRACT
Introduction
immunity.[6] Thus, current situation highlights the need for the
discovery and improvement of new principal compounds of
simple structure, displaying optimal in vivo antitumor potency
and different mechanisms of action.
Global deaths caused by cancer are expected to carry on increas-
ing with a probable 12 million in the year 2030.[1] The effi-
cacy of conventional drugs in cancer treatment is adversely
affected by reasons such as their unfavourable pharmacologi-
cal and physico-chemical properties, involving in toxic effects
to healthy cells and tissues, poor water affinity, and the develop-
ment of multi-drug resistance.[2,3,4] Chemotherapy plays signif-
icant role in treatment of cancer.
In recent years, many new anticancer drugs have been used
in the treatment of various types of cancer.[5] However, at
present, anticancer chemotherapy still suffers from two major
restrictions. The first is the absence of selectivity of chemothera-
peutic agents, which carries side effects in the cancer treatment.
The second is the development of multiple-drug resistance by
cancer cells. In addition to this, cancer patients on chemother-
apy are vulnerable to bacterial attack resulting from their falling
Over the last five decades, a large number of benzothiazole
derivatives have been used to design of new bioactive com-
pounds because of their various pharmacological activities[7–12]
especially anticancer.[13,14] Cytotoxicity assays on a panel of
human cancer cell lines as well as preclinical and clinical stud-
ies of benzothiazoles showed that these molecules could be
developed as promising chemotherapeutics for the treatment
of cancer. Some of these new-generation benzothiazole con-
jugates indicated enhanced potency in comparison with the
most of the typical anticancer agents. Based on the notable effi-
cacy and potency data in animal models, many molecules of
this type are being evaluated in the clinic and are at differ-
ent phases. The rational drug design and development of these
CONTACT Zafer Asım Kaplancıklı
Turkey.
zakaplan@anadolu.edu.tr
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Anadolu University, Eskis¸ehir ,
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpss.
Supplemental data for this article can be accessed on the publisher’s website at https://doi.org/./...
©  Taylor & Francis Group, LLC

2
D. OSMANIYE ET AL.
via rich preclinical screenings or computer-aided drug design
need further detailed investigations for providing new leads with
higher efficacy and reduced toxicity. These methodologies, and
the development of benzothiazole based molecules therefore
provides a possibility of clinical trials.[15]
Benzothiazole analogs perform their anticancer activity
on different molecular targets. Some significant examples
of such biotargets are replication and mitosis inhibitors,[16]
topoisomerase II inhibitors,[17] tyrosine kinase inhibitors,[18]
inhibitors of thioredoxin signalling system,[19] cytochrome
P450 inhibitors,[20–22] heat shock protein 90 inhibitors,[23] epi-
dermal growth factor receptor inhibitors,[24] and cathepsin D
inhibitors.[25]
As a result of structure-based drug design approaches, the
3D structure data for therapeutic targets have enhanced the
chances for rapid replication of biologically active benzothia-
zoles. A structure activity relationship profile have been success-
fully developed for the newly synthesized compounds through
the systematic investigation of the functional groups in the
C2, C5, and C6 positions of benzothiazole.[26] Especially C2
substituted derivatives of benzothiazole have been studied for
their anticancer activity. However, while most investigators
set their sights on designing new benzothiazoles by substi-
tuting 2-aminobenzothiazoles or 2-arylbenzothiazoles, only a
few utilized 2-mercaptobenzothiazoles. Recent findings indi-
cated that 2-mercaptobenzothiazole has also emerged as an
important pharmacophore for the development of anticancer
agents.[23,25,27–30]
In addition to benzothiazole, thiazoline is another sul-
phur containing structure that is often subjected to new
anticancer development studies. In recent years, especially,
imino-thiazoline containing compounds have received con-
siderable attention owing to their diverse chemotherapeutic
potentials, including antitumor activity.[31–35]
Results and discussion
Chemistry
The compounds 4a-4p were synthesized as summarized in
Scheme 1. First, ethyl 2-(benzothiazol-2-ylthio)acetate was pre-
pared by the reaction of 2-mercaptobenzothiazole and ethyl 2-
chloroacetate in the presence of potassium carbonate. In the sec-
ond step, the reaction of ethyl 2-(benzothiazol-2-ylthio)acetate
(1) and excess of hydrazine hydrate afforded 2-(benzothiazol-
2-ylthio)acetohydrazide (2). Thiosemicarbazide derivatives
(3a and 3b) were obtained via reaction of 2-(benzothiazol-
2-ylthio)acetohydrazide (2) with cyclohexyl isothiocyanate
or phenyl isothiocyanate. In the last step, target com-
pounds (4a-4p) were prepared via ring closure reaction using
thiosemicarbazide derivatives (3a and 3b) and an appropriate
2-bromoacetophenone.
The structures of final compounds were elucidated by IR,
1H-NMR, 13C-NMR, and HRMS analysis. In the IR spectra,
the stretching absorptions, which were seen around 3036 cm−1
showed the N-H bond of hydrazide group. Signals belonging
to the carbonyl function appeared at 1651–1728 cm−1. The
stretching absorption at about 1203–1244 cm−1 were assigned
to the C-N single bond. The cyano group in compounds 4c
and 4k had signals at 2233 and 2231 cm−1, respectively. In the
1H-NMR spectra, aliphatic protons belonging to the cyclohexyl
ring were recorded between 0.97 ppm and 2.64 ppm. singlets
between 4.21 ppm and 4.35 ppm were assigned to the methy-
lene group and the protons of aromatic rings were recorded at
6.14-8.07 ppm. The peaks corresponding to hydrogens of the
benzothiazole overlapped in most cases, with the phenyl peaks.
The proton of hydrazide was recorded over 10 ppm. In addition,
in the 1H-NMR spectra of compounds 4a, 4b, 4c, and 4h peak
splitting indicated that these compounds were obtained as a
mixture of E and Z isomers. In the 13C-NMR spectra, aliphatic
carbons were recorded between 21.2 ppm and 59.3 ppm. Aro-
matic carbons gave peaks from 97.9 ppm to 165.6 ppm. C2 of
benzothiazole was recorded about 166 ppm. Carbonyl carbon
had a peak over 167 ppm. Carbon-fluorine splitting was also
Taking all the above findings into consideration in the
search for new lead compounds endowed with potent antitu-
mor activity, we report herein the synthesis of new a series
of benzothiazole-thiazoline derivatives and evaluation of their
antitumor activity.
Scheme . Synthesis of compounds 4a-4p.

PHOSPHORUS, SULFUR, AND SILICON
3
observed for compounds 4e and 4m. In addition, 2D NMR 58.82 and 71.04% DNA synthesis inhibition, whereas cisplatin
studies including, HMBC, HSQC, and NOESY were performed was found to have 18.20, 35.40, 48.49, 52.13 and 65.72% inhibi-
for compound 4a. Thus, all data observed in1H-NMR and tion at the same concentrations, respectively.
13C-NMR were also confirmed by 2D NMR spectra. In the MS
Tested compounds exhibited concentration-dependent
spectra, all masses matched well with the expected M+H values. inhibitory activity on DNA synthesis of C6 cell line. When the
Selected characterization spectra of compounds 3a, 3b, and 4a- DNA synthesis inhibitory activity of compounds is compared,
4p were presented in Figure S 1-S 75 (Supplemental Materials). it is clear that 4d has higher inhibition potency than cisplatin
at IC50, 2 x IC50, and 4 x IC50, whereas 4a has same inhibition
potency with cisplatin at 2 x IC50 and 4 x IC50. According
Biological evaluation
to these results, it can be suggested that compound 4d pos-
sesses higher antiproliferative activity against C6 cell line than
cisplatin.
Cytotoxicity test
In the research of discovering new anticancer agents, the most
common screening methods are cytotoxicity tests towards can-
Flow cytometric analysis
cer cell lines. These are high prevalent screening assays, exposing
compounds with the cytotoxic activity.[36] MTT assay, attached
Apoptosis is an important approach to express the cell death
to the capability of metabolically active cells that change the
pale yellow MTT colour to a spectrophotometrically measur-
able blue formazan salt, is one of the most favoured cytotoxic-
ity tests.[37] We assessed the antitumor potential of compounds
4a-4p on A549 and C6 cell lines with various concentrations
(1mM, 0.316 mM, 0.1 mM, 0.0316 mM, 0.01 mM, 0.00316 mM,
0.001 mM, 0.000316 mM). Besides, the cytotoxic activities of
these compounds were evaluated against healthy NIH3T3 cells
by virtue of showing the selectivity towards carcinogenic cells.
The IC50 values of the compounds against cell lines are presented
in Table S 1 (Supplemental Materials).
pathway since most of the anticancer drugs induce apoptosis in
cancer cells. Annexin V-FITC is a reagent that can detect and
measure quantity of apoptotic cells by flow cytometry. Colour-
ing cells with PI and Annexin V-FITC show the differences
between live, apoptotic, dead, and late apoptotic or necrotic
cells.[40] Thus, this method provides an evidence on the path-
way of the cell death.
Flow cytometry studies were carried out on C6 cells for com-
pounds 4a, 4d and cisplatin with the aim of determining the
stimulated cellular death pathway. Selected compounds were
used at concentrations of IC50/2, IC50, and 2 x IC50. Flow cyto-
metric analysis diagrams of compound 4a and 4d are presented
in Figure S 77 and Figure S 78 (Supplemental Materials),
respectively. At the IC50 concentration, compound 4a displayed
the highest population of apoptotic cells (28.48%), whereas cis-
platin displayed 20.90% apoptotic cells. Compound 4d also gen-
erated an analogous population of apoptotic cells (19.53) to that
of 20.57 at IC50. As a result, findings of flow cytometric analy-
sis revealed that compounds 4a and 4d provoked the apoptotic
induction in C6 cell line after 24 h treatment.
Compounds 4a, 4d were found to have the highest cytotoxic
activity against C6 cell line. These compounds indicated equal
IC50 value (0.03 mM) to cisplatin against C6 cell line.
One of the important criteria so as to being anticancer agent
candidate is to display minimum or no side-effects on healthy
cells. Hence, cytotoxicity of compounds 4a-4p against NIH3T3
cell line was evaluated. As seen in the Table S 1 (Supplemen-
tal Materials), IC50 values of compounds 4a, 4d against C6 cells
were lower than their IC50 values against NIH3T3 cell line.
Moreover, selectivity of compound 4a against C6 cells was two-
fold higher than that of compound 4d. Based on the obtained
results, it can be interpreted that compound 4a is a promising
anticancer agent. Compared to the A549 and C6 cell line, syn-
thesized compounds were found to be more effective on the C6
cell line. Therefore, C6 cell line was used in further tests, includ-
ing DNA synthesis inhibition and flow cytometric analysis.
Conclusion
A major challenge to medicinal chemists is determination
of new structures that may be useful in the design of novel,
selective and less-toxic anticancer agents. With this aim, we syn-
thesized new 2-(benzothiazol-2-ylthio)-Nꢀ-(3-substituted-4-
(3,4-substitutedphenyl)thiazol-2(3H)-ylidene)acetohydrazides
(4a-4p) and elucidated their anticancer activity against A549
and C6 cells. Cytotoxic activities of the synthesized benzothia-
DNA synthesis inhibition assay
This immunostaining assay is related to detect the attendance of zoles against healthy NIH3T3 cell line were also examined.
BrdU into nuclear DNA instead of thymidine during S-phase
It was determined that compounds 4a and 4d showed equal
of cell cycle using specific anti-BrdU antibodies.[38,39] Thus, cytotoxicity with cisplatin against C6 cell line. Moreover, com-
this study was performed to evaluate inhibitory effects of com- pound 4a showed more selective inhibition than 4d towards
pounds 4a and 4d on proliferation of C6 cell line. Test com- C6 cell line. Structures of the test compounds vary from each
pounds and reference agent cisplatin were used at the concen- other in terms of substituents on imino-thiazoline and phenyl
trations of IC50/4, IC50/2, IC50, 2 x IC50, and 4 x IC50. Figure S substructures. When the anticancer activity results are exam-
76 represents the DNA synthesis inhibitory effect of compounds ined, it is clearly seen that cyclohexyl substituted derivatives
4a and 4d on C6 cell line.
(4a-4h) are more effective than phenyl substituted compounds
Compound 4a indicated 4.24, 14.72, 18.37, 51.83 and 63.16% (4i-4p) against the C6 cell line. This finding suggests that
DNA synthesis inhibition at IC50/4 to 4 x IC50 concentrations, cyclohexyl group has a potency to induce anticancer activity.
respectively. Besides, compound 4d displayed 6.34, 16.87, 45.00, On the other hand, compound 4a, including a nonsubstituted

4
D. OSMANIYE ET AL.
phenyl group on the thiazolidine structure, possesses the most in EtOH (30 mL). After completion of the reaction the mix-
selective cytotoxic effects. This result shows that incorporation ture was cooled in an ice-bath, precipitated product was filtered,
of an electron withdrawing or donating substituent on phenyl dried, and recrystallized from EtOH. Yield 64%, m.p. = 192–
moiety decreases the selectivity. In conclusion, the anticancer 193°C (measured), m.p. = 193°C (reported.[41]
)
activity screening of novel benzothiazole-thiazoline derivatives
designated that compound 4a is the lead compound of the
series. According to flow cytometry analysis this compound has
cyclohexylhydrazine-1-carbothioamide 3a
2-(2-(Benzo[d]thiazol-2-ylthio)acetyl)-N-
a greater induction potential of apoptosis than cisplatin.
Yield: 88%, m.p. = 185–186°C, FTIR (ATR, cm−1): 3360 (N H),
–
1
Experimental
=
–
1691 (C O), 1230 (C N), 1006. H-NMR (300 MHz, DMSO-
1
d6): δ = H-NMR (300 MHz, DMSO-d6): δ = 1.03-1.23 (6H,
Chemistry
m, cyclohexyl), 1.52-1.77 (5H, m, cyclohexyl), 4.05 (1H, s, NH),
4.24 (2H, s, CH2), 7.38 (1H, td, J1 = 1.17 Hz, J2 = 8.13 Hz, ben-
zothiazole CH), 7.49 (1H, td, J1 = 1.23 Hz, J2 = 7.32 Hz, ben-
zothiazole CH), 7.87 (1H, d, J = 7.77 Hz, benzothiazole CH),
8.03 (1H, d, J = 7.23 Hz, benzothiazole CH), 9.29 (1H, s. NH),
10.17 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6): δ = 19.02,
25.25, 25.54, 32.13, 32.33, 35.62, 53.23, 121.67, 122.33, 125.08,
126.86, 135.27, 152.94, 166.41, 166.74, 180.81. ESI-MS (M+H):
C16H20N4OS3: 381.20.
All chemicals were obtained either from Sigma-Aldrich (Sigma-
Aldrich Corp., St. Louis, MO, USA) or Merck (Merck KGaA,
Darmstadt, Germany), and used without further chemical
purifications. Melting points of the compounds were measured
by using an automatic melting point determination instrument
(MP90, Mettler-Toledo, OH, USA) and were presented as uncor-
rected. 1H and 13C NMR spectra were recorded in DMSO-d6 by
a Bruker digital FT-NMR spectrometer (Bruker Bioscience, MA,
USA) at 300 MHz and 75 MHz, respectively. The IR spectra of
the compounds were recorded using an IRAffinity-1S Fourier
transform IR (FTIR) spectrometer (Shimadzu, Tokyo, Japan).
HRMS studies were performed on an LCMS-IT-TOF system
(Shimadzu, Tokyo, Japan). Chemical purities of the compounds
were checked by classical TLC applications performed on sil-
ica gel 60 F254 (Merck KGaA, Darmstadt, Germany). The Sup-
2-(2-(Benzothiazol-2-ylthio)acetyl)-N-phenylhydrazine-1-
carbothioamide 3b
Yield: 89%, m.p. = 163–164°C, FTIR (ATR, cm−1): 3323 (N H),
–
1
=
–
1653 (C O), 1232 (C N), 1004, 873. H-NMR (300 MHz,
1
plemental Materials contains sample H NMR, 13C NMR and
HRMS spectra of the products 4a – 4p (Figures S 1 – S 75).
1
DMSO-d6): δ = H-NMR (300 MHz, DMSO-d6): δ = 4.29 (2H,
s, CH2), 7.17 (1H, t, J = 7.26 Hz, phenyl CH), 7.30-7.47 (6H,
m, benzothiazole CH, phenyl CH), 7.82 (1H, d, J = 7.95 Hz,
benzothiazole CH), 8.01 (1H, d, J = 7.98 Hz, benzothiazole
CH), 9.63 (1H, s, NH), 9.77 (1H, s, NH), 10.44 (1H, s, NH).
13C-NMR (75 MHz, DMSO-d6): δ = 121.6, 122.3, 125.4, 125.7,
126.8, 128.6, 135.2, 139.4, 152.9, 166.4, 181.3. ESI-MS (M+H):
C16H14N4OS3: 375.15.
Materials and methods
Ethyl 2-(benzothiazol-2-ylthio)acetate 1
A mixture of benzothiazole-2-thiol (5.51 g, 33 mmol) and ethyl
2-chloroacetate (4.24 ml, 39 mmol) were refluxed in acetone
(100 mL) in the presence of potassium carbonate (4.55 g, 33 General procedure for the 2-(benzothiazol-2-ylthio)-Nꢀ
mmol) for 8 h. After completion of the reaction, acetone was -(3-substituted-4-(3,4-substitutedphenyl)thiazol-2(3H)-
removed under reduced pressure, the residue was washed with ylidene)acetohydrazide 4a-4p
water, dried, and recrystallized from EtOH. Yield 69%, M.P. =
58–59°C (measured), M.P. = 58°C (reported.[41]
)
A mixture of thiosemicarbazides (3a or 3b) (0.7 mmol) and
an appropriate 2-bromoacetophenone derivatives (0.7 mmol) in
EtOH (20 mL) were refluxed for 4 h. When reaction completed
water was added to the mixture for precipitation. The obtained
product was filtered, dried, and recrystallized from EtOH.
2-(benzothiazol-2-ylthio)acetohydrazide 2
Hydrazine hydrate (4.08 mL, 84 mmol) was added in por-
tions into a solution of ethyl 2-(benzothiazol-2-ylthio)acetate
(1) (7.18 g, 28 mmol) in EtOH and then the mixture was stirred
for 12 h at room temperature. The precipitated product was fil-
tered and the solid was washed with ethanol to remove the excess
of the hydrazine hydrate, dried, and recrystallized from EtOH.
2-(Benzothiazol -2-ylthio)-N-(3-cyclohexyl-4-phenylthiazol
-2(3H)-ylidene)acetohydrazide 4a
Yield: 79%, m.p. = 183–184°C, FTIR (ATR, cm−1): 3140 (N H),
–
1
=
=
=
–
1651 (C O), 1558-1427 (C N, C C), 1203 (C N). H-NMR
(300 MHz, DMSO-d6): δ = 0.97-1.05 (4H, m, cyclohexyl), 1.46-
1.67 (6H, m, cyclohexyl), 2.56-2.64 (1H, m, cyclohexyl), 4.21
(2H, s, CH2), 6.14 (1H, s, thiazolylidene CH), 7.35-7.45 (3H,
m, phenyl CH), 7.48-7.50 (4H, m, phenyl CH, benzothiazole
2-(2-(benzothiazol-2-ylthio)acetyl)-N-
substitutedhydrazine-1-carbothioamide 3a-3b
2-(benzothiazol-2-ylthio)acetohydrazide (2) (3.05 g, 13 mmol) CH), 7.87 (1H, d, J = 8.05 Hz, benzothiazole CH), 8.03 (1H,
and substituted isothiocyanate (13 mmol) were refluxed for 2 h d, J = 8.05 Hz, benzothiazole CH), 10.04-10.43 (1H, s, NH).

PHOSPHORUS, SULFUR, AND SILICON
5
13C-NMR (75 MHz, DMSO-d6): δ = 25.0 (cyclohexyl C4), 26.0
(cyclohexyl C3,3’), 27.9 (cyclohexyl C2,2’), 36.0 (-CH2), 58.9
(cyclohexyl C1), 97.9 (thiazolylidene C3), 121.6 (benzothia-
zole C6), 122.3 (benzothiazole C3), 125.0 (benzothiazole C5),
126.9 (benzothiazole C4), 129.2 (phenyl C2,2’), 129.3 (phenyl
C3,3’), 129.7 (phenyl C4), 132.2 (phenyl C1), 135.3 (benzothia-
zole C7), 141.5 (thiazolylidene C2), 153.1 (benzothiazole C2),
thiazolylidene CH), 7.64-7.68 (3H, m, phenyl CH, benzothia-
zole CH), 7.89-7.92 (3H, m, phenyl CH, benzothiazole CH),
10.35 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6): δ = 24.8,
25.1, 31.7, 34.8, 59.3, 105.6, 121.5, 122.2, 123.7, 125.2, 126.8,
129.8, 130.2, 135.1, 139.2, 148.0, 152.4, 165.2, 166.2, 167.0.
HRMS (m/z): [M+H]+ calcd for C24H23N5O3S3: 526.1036;
found: 526.1018.
=
162.5 (C O), 166.6 (benzothiazole C1), 167.9 (thiazolylidene
C1). HRMS (m/z): [M+H]+ calcd for C24H24N4OS3: 481.1185;
2-(Benzothiazol-2-ylthio)-N-(3-cyclohexyl-4-(4-
fluorophenyl)thiazol-2(3H)-ylidene)acetohydrazide 4e
found: 481.1174.
Yield: 85%, m.p. = 250–251°C, FTIR (ATR, cm−1): 3053 (N H),
2-(Benzothiazol-2-ylthio)-N-(3-cyclohexyl-4-(p-tolyl)
thiazol-2(3H)-ylidene)acetohydrazide 4b
–
1
=
=
=
–
1703 (C O), 1508-1487 (C N, C C), 1242 (C N). H-NMR
(300 MHz, DMSO-d6): δ = H-NMR (300 MHz, DMSO-d6):
1
Yield: 82%, m.p. = 257–258°C, FTIR (ATR, cm−1): 3049 (N H),
–
δ = 1.11-1.20 (1H, m, cyclohexyl), 1.29-1.41 (3H, m, cyclo-
hexyl), 1.61-1.65 (3H, m, cyclohexyl), 1.76-1.80 (2H, m, cyclo-
hexyl), 1.91 (2H, s, cyclohexyl), 4.33 (2H, s, CH2), 7.00-7.06
(2H, m, phenyl CH), 7.36-7.41 (2H, m, phenyl CH), 7.47-7.52
(3H, m, benzothiazole CH, thiazolylidene CH), 7.76 (1H, d,
J = 7.95 Hz, benzothiazole CH), 7.97 (1H, d, J = 8.04 Hz, ben-
zothiazole CH), 10.73 (1H, s, NH). 13C-NMR (75 MHz, DMSO-
d6): δ = 24.9, 25.1, 31.6, 35.1, 59.3, 102.8, 116.1 (d, J = 22.1
Hz), 121.7, 122.2, 125.1, 126.1 (d, J = 2.9 Hz), 126.8, 129.1 (d,
J = 9.4 Hz), 135.2, 152.8, 156.7, 162.1 (d, J = 248.9 Hz), 165.6,
166.8, 167.7. HRMS (m/z): [M+H]+ calcd for C24H23FN4OS3:
499.1091; found: 499.1075.
1
=
=
=
–
1714 (C O), 1583-1429 (C N, C C), 1238 (C N). H-NMR
(300 MHz, DMSO-d6): δ = 1.13-1.21 (1H, m, cyclohexyl), 1.32-
1.54 (4H, m, cyclohexyl), 1.63-1.67 (1H, m, cyclohexyl), 1.80-
2.01 (4H, m, cyclohexyl), 2.10 (3H, s, -CH3), 4.29 (2H, s, CH2),
6.95 (2H, d, J = 7.98 Hz, phenyl CH), 7.12 (1H, s, thiazolyli-
dene CH), 7.26 (2H, d, J = 8.10 Hz, phenyl CH), 7.37-7.43 (1H,
m, benzothiazole CH), 7.46-7.52 (1H, m, benzothiazole CH),
7.79 (1H, d, J = 8.01 Hz, benzothiazole CH), 8.01 (1H, d, J
= 8.01 Hz, benzothiazole CH), 10.13-10.15 (1H, s, NH). 13C-
NMR (75 MHz, DMSO-d6): δ = 21.2, 24.8, 25.1, 31.7, 34.8, 59.0,
102.1, 121.7, 122.3, 124.2, 125.1, 126.8, 128.8, 129.4, 135.3, 140.4,
141.4, 152.7, 165.4, 166.8, 167.8. HRMS (m/z): [M+H]+ calcd
for C25H26N4OS3: 495.1342; found: 495.1333.
2-(Benzothiazol-2-ylthio)-N-(4-(4-chlorophenyl)-3-
cyclohexylthiazol-2(3H)-ylidene)acetohydrazide 4f
2-(Benzothiazol-2-ylthio)-N-(4-(4-cyanophenyl)-3-
cyclohexylthiazol-2(3H)-ylidene)acetohydrazide 4c
Yield: 76%, m.p. = 257–258°C, FTIR (ATR, cm−1): 3057 (N H),
–
1
=
=
=
–
1716 (C O), 1591-1462 (C N, C C), 1238 (C N). H-NMR
Yield: 83%, m.p. = 221–222°C, FTIR (ATR, cm−1): 3059 (N H),
1
–
(300 MHz, DMSO-d6): δ = H-NMR (300 MHz, DMSO-d6): δ
=
=
=
2233 (CࣕN), 1728 (C O), 1582-1427 (C N, C C), 1238
(C N). H-NMR (300 MHz, DMSO-d6): δ = 1.13-1.21 (1H, m,
= 1.13-1.21 (1H, m, cyclohexyl), 1.31-1.54 (5H, m, cyclohexyl),
1.63-1.67 (1H, m, cyclohexyl), 1.80 (2H, s, cyclohexyl), 1.90-1.93
(1H, m, cyclohexyl), 2.00-2.04 (1H, m, cyclohexyl), 4.28 (2H, s,
CH2), 7.23-7.25 (3H, m, phenyl CH, thiazolylidene CH), 7.39-
7.42 (3H, m, phenyl CH, benzothiazole CH), 7.46-7.52 (1H, m,
benzothiazole CH), 7.78 (1H, d, J = 8.04 Hz, benzothiazole CH),
8.00 (1H, d, J = 7.83 Hz, benzothiazole CH), 10.20 (1H, s, NH).
13C-NMR (75 MHz, DMSO-d6): δ = 24.8, 25.1, 31.7, 34.8, 59.0,
103.4, 121.7, 122.3, 125.2, 125.9, 126.9, 129.0, 130.8, 135.3, 135.6,
140.1, 152.6, 165.3, 166.9, 167.8. HRMS (m/z): [M+H]+ calcd
for C24H23ClN4OS3: 515.0795; found: 515.0776.
1
–
cyclohexyl), 1.32-1.54 (4H, m, cyclohexyl), 1.63-1.67 (1H, m,
cyclohexyl), 1.80-2.01 (4H, m, cyclohexyl), 4.31 (2H, s, CH2),
6.95 (2H, d, J = 7.98 Hz, phenyl CH), 7.12 (1H, s, thiazolyli-
dene CH), 7.26 (2H, d, J = 8.1 Hz, phenyl CH), 7.37-7.43 (1H,
m, benzothiazole CH), 7.46-7.52 (1H, m, benzothiazole CH),
7.79 (1H, d, J = 8.01 Hz, benzothiazole CH), 8.01 (1H, d, J =
8.01 Hz, benzothiazole CH), 10.13-10.29 (1H, s, NH). 13C-NMR
(75 MHz, DMSO-d6): δ = 21.2, 25.1, 31.7, 34.8, 59.0, 101.7,
119.0, 121.4, 122.4, 124.3, 125.4, 127.0, 128.8, 129.5, 135.9, 140.4,
141.4, 152.4, 165.4, 166.1, 167.1. HRMS (m/z): [M+H]+ calcd
for C25H23N5OS3: 506.1137; found: 506.1132.
2-(Benzothiazol-2-ylthio)-N-(4-(4-bromophenyl)-3-
cyclohexylthiazol-2(3H)-ylidene)acetohydrazide 4g
2-(Benzothiazol-2-ylthio)-N-(3-cyclohexyl-4-(4-
nitrophenyl)thiazol-2(3H)-ylidene)acetohydrazide 4d
Yield: 89%, m.p. = 266–267°C, FTIR (ATR, cm−1): 3059 (N H),
–
1
=
=
=
–
1714 (C O), 1589-1475 (C N, C C), 1238 (C N). H-NMR
Yield: 77%, m.p. = 173–174°C, FTIR (ATR, cm−1): 3365 (N H), (300 MHz, DMSO-d6): δ = 1.13-1.21 (1H, m, cyclohexyl), 1.32-
–
1
=
=
=
–
1701 (C O), 1517-1427 (C N, C C), 1217 (C N). H-NMR 1.53 (5H, m, cyclohexyl), 1.63-1.67 (1H, m, cyclohexyl), 1.80
1
(300 MHz, DMSO-d6): δ = H-NMR (300 MHz, DMSO-d6): (2H, s, cyclohexyl), 1.90-2.04 (2H, m, cyclohexyl), 4.28 (2H, s,
δ = 1.13-1.21 (1H, m, cyclohexyl), 1.36-1.67 (5H, m, cyclo- CH2), 7.23 (1H, s, thiazolylidene CH), 7.32-7.35 (2H, m, phenyl
hexyl), 1.79-2.00 (4H, m, cyclohexyl), 2.44 (1H, s, cyclo- CH), 7.38-7.40 (2H, m, phenyl CH) 7.40-7.43 (1H, m, benzoth-
hexyl), 4.33 (2H, s, CH2), 7.30-7.44 (3H, m, benzothiazole CH, iazole CH), 7.47-7.52 (1H, m, benzothiazole CH), 7.79 (1H,

6
D. OSMANIYE ET AL.
135.3, 140.1, 141.0, 152.8, 165.5, 167.1. HRMS (m/z): [M+H]+
d, J = 7.59 Hz, benzothiazole CH), 8.01 (1H, d, J = 8.04 Hz,
benzothiazole CH), 10.19 (1H, s, NH). 13C-NMR (75 MHz,
DMSO-d6): δ = 24.8, 25.1, 31.7, 34.8, 59.0, 103.4, 121.7, 122.4,
124.4, 125.2, 126.3, 126.9, 131.0, 132.0, 135.3, 140.2, 152.6, 165.3,
166.9, 167.8. HRMS (m/z): [M+H]+ calcd for C24H23BrN4OS3:
559.0290; found: 559.0264.
calcd for C25H20N4OS3: 489.0872; found: 489.0866.
2-(Benzothiazol-2-ylthio)-N-(4-(4-cyanophenyl)-3-
phenylthiazol-2(3H)-ylidene)acetohydrazide 4k
Yield: 77%, m.p. = 254–255°C, FTIR (ATR, cm−1): 3049 (N H),
2-(Benzothiazol-2-ylthio)-N-(3-cyclohexyl-4-(3,4-
dichlorophenyl)thiazol-2(3H)-ylidene)acetohydrazide 4h
–
=
=
=
2231 (CࣕN), 1714 (C O), 1572-1425 (C N, C C), 1244
(C N). H-NMR (300 MHz, DMSO-d6): δ = 4.32 (2H, s, CH2),
1
–
6.93 (1H, s, thiazolylidene CH), 6.97 (2H, d, J = 8.10 Hz,
cyanophenyl CH), 7.35-7.40 (4H, m, phenyl CH, benzothiazole
CH), 7.45 (2H, d, J = 8.10 Hz, cyanophenyl CH), 7.50 (1H, dt,
J = 7.40 Hz – 1.30 Hz, benzothiazole CH), 7.58 (1H, d, J =
7.65 Hz, phenyl CH), 7.78 (1H, d, J = 7.40 Hz, benzothiazole
CH), 8.00 (1H, d, J = 7.40 Hz, benzothiazole CH), 12.03 (1H,
s, NH). 13C-NMR (75 MHz, DMSO-d6): δ = 34.9, 100.0, 118.8,
122.0, 122.4, 123.7, 123.9, 124.8, 124.9, 125.2, 126.9, 129.6, 131.1
135.7, 140.1, 141.5, 152.0, 165.6, 167.5. HRMS (m/z): [M+H]+
calcd for C25H17N5OS3: 500.0668; found: 500.0659.
Yield: 75%, m.p. = 277–278°C, FTIR (ATR, cm−1): 3057 (N H),
–
1
=
=
=
–
1703 (C O), 1577-1462 (C N, C C), 1240 (C N). H-NMR
(300 MHz, DMSO-d6): δ = 1.13-1.22 (1H, m), 1.32-1.56 (4H,
m, cyclohexyl), 1.63-1.67 (2H, m, cyclohexyl), 1.80 (2H, s, cyclo-
hexyl), 1.89-2.03 (2H, m, cyclohexyl), 4.27 (2H, s, CH2), 7.31
(1H, s, thiazolylidene CH), 7.35-7.41 (3H, m, dichlorophenyl
CH, benzothiazole CH), 7.44-7.49 (1H, m, benzothiazole CH),
7.71-7.73 (2H, m, dichlorophenyl CH, benzothiazole CH), 7.98
(1H, d, J = 7.86 Hz, benzothiazole CH), 10.26-10.29 (1H, s,
NH). 13C-NMR (75 MHz, DMSO-d6): δ = 24.8, 25.0, 31.7, 34.8,
59.1, 104.5, 121.6, 122.3, 125.2, 126.9, 127.4, 129.1, 130.9, 131.0,
132.0, 133.7, 135.2, 138.8, 152.6, 165.2, 167.0, 167.9. HRMS
(m/z): [M+H]+ calcd for C24H22Cl2N4OS3: 549.0406; found:
549.0381
2-(Benzothiazol-2-ylthio)-N-(4-(4-nitrophenyl)-3-
phenylthiazol-2(3H)-ylidene)acetohydrazide 4l
Yield: 75%, m.p. = 256–257°C, FTIR (ATR, cm−1): 3036 (N H),
–
2-(Benzothiazol-2-ylthio)-N-(3,4-diphenylthiazol-2(3H)-
ylidene)acetohydrazide 4i
1
=
=
=
–
1714 (C O), 1570-1427 (C N, C C), 1244 (C N). H-NMR
(300 MHz, DMSO-d6): δ = 4.32 (2H, s, CH2), 6.96 (1H, s,
thiazolylidene CH), 7.36-7.40 (4H, m, phenyl CH, benzothia-
zole CH), 7.50 (1H, dt, J = 7.40 Hz – 1.25 Hz, benzothiazole
CH), 7.60 (1H, d, J = 7.60 Hz, phenyl CH), 7.78 (1H, d, J =
7.40 Hz, benzothiazole CH), 8.00 (1H, d, J = 7.40 Hz, benzoth-
iazole CH), 8.07 (2H, d, J = 8.30 Hz, nitrophenyl CH), 8.38
(2H, d, J = 8.30 Hz, nitrophenyl CH), 12.01 (1H, s, NH). 13C-
NMR (75 MHz, DMSO-d6): δ = 34.8, 103.1, 121.2, 121.5, 122.1,
123.2, 123.8, 125.1, 126.7, 129.6, 129.9, 130.2, 130.7, 131.2, 135.1,
138.6, 147.8, 152.5, 165.3, 167.4. HRMS (m/z): [M+H]+ calcd
for C24H17N5O3S3: 520.0566; found: 520.0552.
Yield: 77%, m.p. = 233–234°C, FTIR (ATR, cm−1): 3059 (N H),
–
1
=
=
=
–
1705 (C O), 1564-1444 (C N, C C), 1219 (C N). H-NMR
(300 MHz, DMSO-d6): δ = 4.35 (2H, s, CH2), 7.03 (1H, s, thia-
zolylidene CH), 7.29 (2H, d, J = 7.50 Hz, phenyl CH), 7.30-7.36
(4H, m, phenyl CH), 7.39-7.47 (4H, m, phenyl CH, benzothia-
zole CH), 7.55 (2H, d, J = 7.50 Hz, phenyl CH), 7.78 (1H, d,
J = 7.50 Hz, benzothiazole CH), 7.99 (1H, d, J = 7.50 Hz, ben-
zothiazole CH), 12.18 (1H, s, NH). 13C-NMR (75 MHz, DMSO-
d6): δ = 34.6, 121.4, 121.7, 122.2, 124.7, 125.0, 126.7, 128.0,
128.8, 129.8, 130.1, 130.2, 130.8, 135.3, 138.5, 149.0, 152.9, 155.2,
165.8, 167.0. HRMS (m/z): [M+H]+ calcd for C24H18N4OS3:
475.0715; found: 475.0712.
2-(Benzothiazol-2-ylthio)-N-(4-(4-fluorophenyl)-3-
phenylthiazol-2(3H)-ylidene)acetohydrazide 4m
2-(Benzothiazol-2-ylthio)-N-(3-phenyl-4-(p-tolyl)thiazol-
2(3H)-ylidene)acetohydrazide 4j
Yield: 79%, m.p. = 226–227°C, FTIR (ATR, cm−1): 3049 (N H),
–
1
=
=
=
–
1714 (C O), 1572-1429 (C N, C C), 1227 (C N). H-NMR
Yield: 73%, m.p. = 233–234°C, FTIR (ATR, cm−1): 3049 (N H), (300 MHz, DMSO-d6): δ = 4.34 (2H, s, CH2), 6.88 (1H, s, thia-
–
1
=
=
=
–
1714 (C O), 1572-1427 (C N, C C), 1234 (C N). H-NMR zolylidene CH), 7.08 (2H, t, J = 8.85 Hz, fluorophenyl CH), 7.30-
(300 MHz, DMSO-d6): δ = 2.14 (3H, s, CH3), 4.32 (2H, s, 7.35 (2H, m, phenyl CH), 7.38 (1H, dt, J = 7.60 Hz – 1.90 Hz,
CH2), 6.93 (1H, s, thiazolylidene CH), 7.01 (2H, d, J = 8.05 Hz, benzothiazole CH), 7.45-7.53 (4H, m, phenyl CH, benzothiazole
methylphenyl CH), 7.31 (2H, d, J = 8.05 Hz, methylphenyl CH), CH), 7.76 (1H, d, J = 8.25 Hz, benzothiazole CH), 7.96 (1H, d,
7.37-7.42 (4H, m, phenyl CH, benzothiazole CH), 7.48 (1H, dt, J = 8.25 Hz, benzothiazole CH), 12.23 (1H, s, NH). 13C-NMR
J = 7.35 Hz – 1.30 Hz, benzothiazole CH), 7.55 (1H, d, J = (75 MHz, DMSO-d6): δ = 35.0, 103.1, 116.0 (d, J = 21.7 Hz),
7.65 Hz, Phenyl CH), 7.78 (1H, d, J = 7.35 Hz, benzothiazole 121.7, 122.2, 123.3, 123.4, 125.1, 126.8, 130.0, 130.2, 130.3, 130.6,
CH), 8.01 (1H, d, J = 7.35 Hz, benzothiazole CH), 12.06 (1H, 131.2 (d, J = 8.8 Hz), 135.2, 139.7, 152.8, 163.2 (d, J = 246.3 Hz),
s, NH). 13C-NMR (75 MHz, DMSO-d6): δ = 21.2, 34.9, 100.6, 165.6, 167.0. HRMS (m/z): [M+H]+ calcd for C24H17N4OFS3:
121.7, 122.3, 123.8, 123.9, 124.8, 124.9, 125.1, 126.8, 129.5, 130.8, 493.0621; found: 493.0607.

PHOSPHORUS, SULFUR, AND SILICON
7
2-(Benzothiazol-2-ylthio)-N-(4-(4-chlorophenyl)-3-
phenylthiazol-2(3H)-ylidene)acetohydrazide 4n
NIH3T3, A549, and C6 cells were used in the MTT assay.
NIH3T3 cells were incubated in DMEM medium (Sigma
Aldrich, St. Louis, MO, USA), added with fetal calf serum,
penicillin (100 IU/mL), streptomycin (100 mg/mL), and 7.5%
NaHCO3 at 37°C in a humidified atmosphere of 95% air and
5% CO2. A549 and C6 cells were incubated in RPMI (Hyclone,
Thermo Scientific, USA) medium, supplemented with fetal calf
serum, penicillin (100 IU/mL), streptomycin (100 mg/mL), and
7.5% NaHCO3 at 37°C in a humidified atmosphere of 95% air
and 5% CO2. NIH3T3, A549, and C6 cell lines were seeded into
the 96-well plates at a density of 1 × 104 cells. After 24 h of incu-
bating period, the culture mediums were removed and test com-
pounds were added at concentrations of 0.000316 – 1 mM. After
24 h incubation period, colorimetric measurements were per-
formed by a microplate reader (Biotek, USA) at 540 nm. Inhi-
bition % at all concentrations was determined using formula
below and the IC50 values were calculated from a dose-response
curve obtained by plotting the percentage inhibition versus the
log concentration with the use of Microsoft Excel 2013. The
results were displayed as mean standard deviation (SD).[43-46]
Cisplatin was used as a positive control.
Yield: 81%, m.p. = 248–249°C, FTIR (ATR, cm−1): 3048 (N H),
–
1
=
=
=
–
1717 (C O), 1572-1427 (C N, C C), 1203 (C N). H-NMR
(300 MHz, DMSO-d6): δ = 4.32 (2H, s, CH2), 6.92 (1H, s, thi-
azolylidene CH), 7.23 (2H, t, J = 8.70 Hz, chlorophenyl CH),
7.30-7.35 (2H, m, phenyl CH), 7.39 (1H, dt, J = 7.55 Hz –
1.90 Hz, benzothiazole CH), 7.45-7.53 (4H, m, phenyl CH, ben-
zothiazole CH), 7.78 (1H, d, J = 8.25 Hz, benzothiazole CH),
7.99 (1H, d, J = 8.25 Hz, benzothiazole CH), 12.16 (1H, s, NH).
13C-NMR (75 MHz, DMSO-d6): δ = 34.8, 101.2, 121.3, 121.7,
122.3, 123.5, 124.6, 125.1, 126.8, 129.0, 130.2, 130.5, 130.7, 131.6,
135.2, 137.4, 149.2, 152.7, 165.5, 167.1. HRMS (m/z): [M+H]+
calcd for C24H17N4OClS3: 509.0326; found: 509.0314.
2-(Benzothiazol-2-ylthio)-N-(4-(4-bromophenyl)-3-
phenylthiazol-2(3H)-ylidene)acetohydrazide 4o
Yield: 80%, m.p. = 249–250°C, FTIR (ATR, cm−1): 3048 (N H),
–
1
=
=
=
–
1717 (C O), 1572-1429 (C N, C C), 1246 (C N). H-NMR
(300 MHz, DMSO-d6): δ = 4.33 (2H, s, CH2), 6.99 (1H, s, thi-
azolylidene CH), 7.32-7.35 (3H, m, phenyl CH, benzothiazole
CH), 7.38 (2H, d, J = 8.55 Hz, bromophenyl CH), 7.45 (2H, d,
J = 8.55 Hz, bromophenyl CH), 7.48-7.53 (4H, m, Phenyl CH,
benzothiazole CH), 7.77 (1H, d, J = 7.50 Hz, benzothiazole CH),
8.00 (1H, d, J = 7.50 Hz, benzothiazole CH), 12.00 (1H, s, NH).
13C-NMR (75 MHz, DMSO-d6): δ = 34.8, 101.4, 121.3, 121.7,
122.3, 123.5, 124.0, 125.1, 126.9, 127.1, 127.6, 130.2, 130.7, 130.8,
132.0, 135.3, 139.6, 152.7, 165.5, 167.1. HRMS (m/z): [M+H]+
calcd for C24H17N4OBrS3: 552.9821; found: 552.9800.
% inhibition = 100 − (mean sample × 100/mean solvent)
DNA synthesis inhibition assay
The BrdU (5-bromo-20-deoxy-uridine) cell proliferation
method was performed to analyse the effects of compounds 4a
and 4d on proliferation of C6 cells. C6 cells were seeded into
the 96-well plates at a density of 1 × 104 cells. Compounds
were added into the each well at five different concentrations
(IC50/4, IC50/2, IC50, 2 x IC50, and 4 x IC50) and the plates were
incubated for 24 h. At the end of the incubation period, BrdU
solution was added and cells were reincubated for 2h at 37°C.
Anti-BrdU-POD (100 mL) was added and the mixture was
incubated for 90 min. Microplates were washed with phosphate
buffered saline for three times. After adding substrate solu-
tion, the mixture was incubated for 15 minutes. Colorimetric
measurements were performed at 492 nm. Proliferation of
control cells was assessed as percentage of normal and growth
inhibition % of cells, treated with 4a, 4d and cisplatin were
calculated.[47] The results were displayed as mean SD.
2-(Benzothiazol-2-ylthio)-N-(4-(3,4-dichlorophenyl)-3-
phenylthiazol-2(3H)-ylidene)acetohydrazide 4p
Yield: 79%, m.p. = 257–258°C, FTIR (ATR, cm−1): 3046 (N H),
–
1
=
=
=
–
1719 (C O), 1568-1427 (C N, C C), 1244 (C N). H-NMR
(300 MHz, DMSO-d6): δ = 4.30 (2H, s, CH2), 6.94 (1H, dd,
J = 7.50 Hz- 1.60 Hz, dichlorophenyl CH), 7.03 (1H, s, thia-
zolylidene CH), 7.32-7.35 (3H, m, phenyl CH, benzothiazole
CH), 7.48-7.53 (5H, m, phenyl CH, dichlorophenyl CH, ben-
zothiazole CH), 7.62 (1H, d, J = 1.60 Hz, dichlorophenyl CH),
7.72 (1H, d, J = 7.50 Hz, benzothiazole CH), 7.96 (1H, d,
J = 7.50 Hz, benzothiazole CH), 11.96 (1H, s, NH). 13C-NMR
(75 MHz, DMSO-d6): δ = 34.8, 101.8, 121.2, 121.6, 122.2, 123.3,
125.1, 126.8, 128.6, 130.0, 130.2, 130.5, 130.7, 131.0, 131.6, 131.9,
133.1, 135.2, 138.2, 152.7, 165.4, 167.3. HRMS (m/z): [M+H]+
calcd for C24H16N4OCl2S3: 542.9936; found: 542.9927.
Flow cytometric analysis
Death pathway of the carcinogenic cell lines was detected by
Annexin V-FITC Apoptosis Detection Kit (BD, Pharmingen) as
reported in the manufacturer’s instruction. Cisplatin and com-
pounds 4a and 4d, which possess the highest cytotoxic activ-
ity, were used at their IC50/2, IC50, and 2 x IC50 concentrations.
After 24 h incubation period, cells were harvested by centrifu-
gation at 1200 rpm for 5 min. at room temperature, rinsed with
cold water twice, and then suspended at 1 × 106 cells/mL con-
Cytotoxicity test
Metabolic activity of viable cells were measured by MTT centration in Annexin V-FITC binding buffer. Propidium iodide
assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)- (5 mL) and annexin V-FITC (5 mL) were added for staining the
2,5-diphenyltetrazolium salt to formazan product, which can cells, and the fluorescence measurements were performed using
be quantified spectrophotometrically to determine percent of a flow cytometer. FCSExpress software was used to display the
viable cells.[42]
percent of normal and apoptotic cells at different stages.[47] In

8
D. OSMANIYE ET AL.
the diagrams, Q1, Q2, Q3, and Q4 demonstrate the necrotic cells
(positive for PI and negative for annexin/ FITC), late apoptotic
or necrotic cells (positive for annexin and PI), live cells (nega-
tive for annexin and PI), and apoptotic cells (negative for PI and
positive for annexin), respectively. The experiments were car-
ried out in triplicates.
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well, A.D. Bioorg. Med. Chem. Lett. 2003, 13, 471-474.
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Acknowledgment
This study was financially supported by Anadolu University Scientific
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Conflicts of Interest
The authors declare no conflict of interest.
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