Benzothiazole thiourea derivatives as anticancer agents: Design, synthesis, and biological screening

Article abstract of DOI:10.1134/S1068162017050065

In a systematic effort to identify a potent anticancer agent, we synthesized benzothiazole thiourea derivatives and examined their cytotoxic activity against five different human and animal cancer cell lines. Benzothiazolylthiocarbamides have been prepared in excellent yields by reaction of substituted 2-amino benzothiazoles with carbon disulfide and dimethyl sulfate followed by their ammonolysis. Cytotoxicity of the four compounds were screened for antitumor activity against human breast cancer cells (MCF-7), human cervix epithelial carcinoma (HeLa), human colon cancer cell line (HT-29), human leukemia cell line (K-562), and mouse neuroblastoma cell line (Neuro-2a) using cisplatin as a reference by MTT assay. Our results presented herein provide experimental evidence that benzothiazolylthiocarbamides induce apoptosis in cancer cell lines. According to flow cytometry results, treatment of HT-29 cells with 1-(6-ethoxy-1,3-benzothiazol- 2-yl)thiourea produced a large population of apoptotic cell (79.45%), which was 1.2-fold higher than that produced by cisplatin (65.28%) at the same concentration.

Full text of DOI:10.1134/S1068162017050065

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2017, Vol. 43, No. 5, pp. 576–582. © Pleiades Publishing, Ltd., 2017.
Benzothiazole Thiourea Derivatives as Anticancer Agents:
Design, Synthesis, and Biological Screening1
Fatemeh Eshkil, Hossein Eshghi2, Amir. Sh. Saljooghi,
Mehdi Bakavoli, and Mohammad Rahimizadeh
Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, 91775-1436 Iran
Received January 9, 2017; in final form, April 4, 2017
Abstract—In a systematic effort to identify a potent anticancer agent, we synthesized benzothiazole thiourea
derivatives and examined their cytotoxic activity against five different human and animal cancer cell lines.
Benzothiazolylthiocarbamides have been prepared in excellent yields by reaction of substituted 2-amino ben-
zothiazoles with carbon disulfide and dimethyl sulfate followed by their ammonolysis. Cytotoxicity of the
four compounds were screened for antitumor activity against human breast cancer cells (MCF-7), human
cervix epithelial carcinoma (HeLa), human colon cancer cell line (HT-29), human leukemia cell line
(K-562), and mouse neuroblastoma cell line (Neuro-2a) using cisplatin as a reference by MTT assay. Our
results presented herein provide experimental evidence that benzothiazolylthiocarbamides induce apoptosis
in cancer cell lines. According to flow cytometry results, treatment of HT-29 cells with 1-(6-ethoxy-1,3-ben-
zothiazol-2-yl)thiourea produced a large population of apoptotic cell (79.45%), which was 1.2-fold higher
than that produced by cisplatin (65.28%) at the same concentration.
Keywords: anticancer activity, benzothiazolylthiocarbamides, MTT assay, flow cytometry
DOI: 10.1134/S1068162017050065
INTRODUCTION
thiazoles have received considerable attention because
of their interesting pharmacological activities, includ-
ing anticonvulsant [4], analgesic [5], anti-tumor
[6, 7], antibacterial [8, 9], antimicrobial [10, 11], and
muscle relaxant activities [12]. The combinations of
urea and thiourea derivatives with benzothiazole have
produced DNA topoisomerase [13, 14] or HIV reverse
transcriptase inhibitors [15, 16]. Frentizole (I) (Fig. 1)
is a non-toxic antiviral and immunosuppressive agent
used clinically in rheumatoid arthritis and systemic
lupus erythematosus [17] and thiourea derivative
YH3945 (II) (Fig. 1), a selective and potent inhibitor
of farnesylprotein transferase, is being developed for
the treatment of cancer [18].
Thiourea and its derivatives have been widely used
in research and technological applications such as in
the pharmaceutical industry [13, 19], as catalysts in
chemical reactions [20–22], and for extraction of toxic
metals using a solid supported liquid membrane sys-
tem [23]. The antiviral [24], cytotoxic [13, 19], and
antifungal [25] activities of these compounds are well
known. In addition, the corrosion of some metals can
be determined using thiourea and its derivatives [26].
Cancer is a mass of cells with malignant transfor-
mation, which divide uncontrollably, invade and
spread to distant organs, and develop secondary dis-
ease. Cancer is among the four major non-communi-
cable diseases (heart disease, cancers, lung disease,
and diabetes) that are the leading causes of deaths
worldwide. A 2013 survey by the World Health Organi-
zation (WHO) estimated that non-communicable dis-
eases (NCDs) kill more than 36 million people each
year. Cancer is second in the list; killed approximately
21% of these 36 million people, so that 8.2 million
people worldwide died from cancer in 2012. Even
though the incidence of cancers is comparatively
higher than other diseases, improvements in cancer
therapy have significantly reduced the number of
deaths due to cancer. Also improvement of quality of
life and survival of cancer patients will be greatly
enhanced by the development of highly effective drugs
to selectively kill malignant cells. In the current study
we tried to synthesize derivatives of benzothiazole
thiourea and then investigate their anticancer activity.
Benzothiazoles are an important class of heterocy-
cles, which can serve as unique and versatile scaffolds
for experimental drug design [1–3]. 2-Aminobenzo-
Our results presented herein provide experimental
evidence that benzothiazolethiourea derivatives
induce apoptosis in human cancer cells. These com-
pounds, especially the lead compound (Vd), may be
useful in the treatment of cancer.
1
2
The article is published in the original.
Corresponding author: e-mail: heshghi@um.ac.ir.
576

BENZOTHIAZOLE THIOUREA DERIVATIVES AS ANTICANCER AGENTS
577
N
F
F
F
N
N
S
N
NH
N
NH
NH
N
O
S
O
O
Frentizole (I)
YH 3945 (II)
Fig. 1. Structures of frentizole and YH3945, two derivatives of benzothiazoles.
RESULTS AND DISCUSSION
Benzothiazole thiourea derivatives were prepared
by an easy protocol, as shown in Scheme. Treatment of
substituted 2-amino benzothiazole (III) with carbon
disulfide in alkali media gave an intermediate, which
was methylated with dimethyl sulfate in excellent
yields. Ammonolysis of compounds (ꢀVa–d) in etha-
nol under reflux conditions gave benzothiazole
thiourea derivatives (Va–d) in excellent yields. Struc-
tures of synthesized title compounds (Va–d) were con-
firmed by FT-IR, NMR, and mass spectrometry tech-
niques.
In this study, the potential of four benzothiazole-
thiourea derivatives as effective anticancer compounds
in vitro were investigated. The interesting pharmaco-
logical activities of 2-aminobenzothiazoles, especially
as anti-tumor agents, provide a strong rationale for
continued research into the development of new com-
pounds containing this structure for improved thera-
peutic performance.
S–Na+
S
S–
S
N
CS2, NaOH (20 M)
N
S
N
(CH3)2SO4
NH2
NH
NH
DMSO, r.t
S
R
S
R
R
(III)
(IV)
(a) R = H
a) R = H
(b) R = CH3
(c) R = OCH3
(d) R = OEt
b) R = CH3
c) R = OCH3
d) R = OEt
NH2
S
N
NH3, EtOH
NH
reflux
S
R
(V)
(a) R = H
(b) R = CH3
(c) R = OCH3
(d) R = OEt
Scheme 1. General synthesis of the benzothiazole thiourea derivatives.
Newly synthesized four compounds were screened epithelial carcinoma (HeLa), human colon cancer cell
for their in vitro growth inhibitory activities against line (HT-29), human leukemia cell line (K-562), and
five human and animal cultured cell lines, namely, mouse neuroblastoma cell line (Neuro-2a) by MTT
human breast cancer cells (MCF-7), human cervix assay using cisplatin as a comparative standard.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 5 2017

578
ESHKIL et al.
Table 1. Cytotoxic activities of compounds (Va–d) against MCF-7, HeLa, HT-29, K-562, and Neuro-2a cancer cell lines
after 72 h of continuous exposure
Cell lines
Compound
(Va)
(Vb)
(Vc)
(Vd)
MCF-7
HeLa
HT-29
K-562
Neuro-2a
L-929
8.55 2.33
11.9 2.75
12.8 3.25
6.72 3.00
6.25 2.13
21.7 3.15
47.3 4.98
45.2 5.17
4.97 1.89
0.39 0.11
8.1 2.44
14.8 2.23
15.0 3.11
3.90 2.05
16.2 2.97
69.7 8.18
85.3 9.45
98.1 12.5
40.5 5.59
25.8 2.93
57.4 6.47
235.3 16.5
200.5 18.7
22.7 2.90
211.0 20.9
134.1 9.5
135
6
230.7 18.2
107.6 11.7
0.7 0.2
Cisplatin
a
The concentration of the complex required to inhibit cell growth by 50%. The experiments were done in triplicate. Data were expressed
as the mean of the triplicate. The agent with IC > 100 μM is considered to be inactive.
50
Table 2. Percentages of the cell death pathways observed by the flow cytometry assay
Late apoptotic/necrotic
cells, %
Treatment
Vital cells, %
Apoptotic cells, %
Necrotic cells, %
Control
Cisplatin
Compound (Vd)
81.13
33.74
19.73
8.68
34.65
43.91
9.45
30.63
35.54
0.74
0.98
0.82
Results are shown as IC50 after 72 h of continuous and apoptosis cells (Q4, right square at the bottom),
respectively. As it follows from Table 2 and Fig. 2,
compound (Vd) produced a large population of apop-
totic cell (79.45%), which is nearly 1.2-fold larger than
that produced by cisplatin (65.28%) at the same con-
centration. Therefore, the newly synthesized com-
pounds, particularly the lead compound (Vd), could
induce apoptosis of HT-29 cancer cells. But the
proapoptotic property needs further investigation to
better understand the precise mechanism of action of
these compounds and basic pre-clinical research is
needed before they could be recommended for human
administration.
exposure (Table 1).
For compounds (Va–d) and cisplatin as a compar-
ative standard, we have measured their IC50 values in
all cell lines at concentrations in the range between
20 nM and 200 μM. For different cell lines, the values
determined for these compounds spanned the range
from 3.90 to 98.1 μM (IC50 over 200 μM was ignored),
while those found for the comparative standard ranged
between 0.39 an up to 200 μM (Table 1). These values
confirmed that we prepared weakly-to-moderately
cytotoxic compounds, with compound (Vd) showing
the strongest cytotoxicity in both cell lines. If we set
aside some cases in Table 1, cytotoxicity in this assay
In this work the synthesis, spectroscopic character-
followed the general trend: (Vd) > (Va) > (Vb), (Vc). ization, and anticancer activity of benzothiazole
The highest cytotoxic activity was observed for com- thiourea derivatives, are reported. Because of the
pound (Vd), which was approximately the same and
four times more potent than that of cisplatin against the
two examined cancer cell lines (MCF-7 and HT-29,
respectively). The effect of synthesized compound on
mouse fibroblast cell line (L-929) was evaluated as
control, simultaneously. Our results confirmed that
the synthesized compound produced no cytotoxic
effects on L-929 cells. The compounds exhibited no
cytotoxicity effects upon 24 and 48 h of exposure.
interesting pharmacological activities of this import-
ant class of heterocycles, we used four novel com-
pounds of this family in this investigation. The results
obtained can be summarized as follows:
(1) In addition to anticonvulsant, analgesic, anti-
bacterial, antimicrobial, and muscle relaxing proper-
ties, for which they are now so well known, benzothi-
azoles also exhibit a number of other therapeutic
effects including anti-cancer activities, which our
results confirm by the excellent cytotoxic activities of
the newbenzothiazole thiourea derivatives, particu-
larly against human colon cancer cell line (HT-29).
Based on the results of in vitro cytotoxicity studies
of newly synthesized benzothiazole thiourea deriva-
tives, the highly active compound was selected for
apoptosis assay by flow cytometry. The results are pre-
sented in Table 2 and Fig. 2. Four areas in the dia-
grams stand for necrotic cells (Q1, left square on the ity of these compounds belongs to compound (Vd),
top), late apoptosis or necrosis cells (Q2, right square which reflects the higher electron donating nature of
on the top), live cells (Q3, left square at the bottom), OEt group in compound (Vd) and its steric effects.
(2) Our results predict that the highest cytotoxic activ-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 43
No. 5
2017

BENZOTHIAZOLE THIOUREA DERIVATIVES AS ANTICANCER AGENTS
579
FI
FI
FI
104
Cisplatin
Compound (Vd)
Control
104
103
102
101
100
104
103
102
101
100
Q2
Q1
Q2
Q1
Q2
Q1
103
102
101
100
Q4
Q3
Q4
Q3
Q4
Q3
103
-FITC
104
100
101
102
103
104 100
101
102
103
104 100
101
102
Annexin V
Annexin V-FITC
Annexin V-FITC
Fig. 2. Flow cytometry results after the exposure of HT-29 cancer cells to the active compound (Vd) and cisplatin. Four areas in
the diagrams represent four different cell states: necrotic cells (Q1), late apoptotic or necrotic cells (Q2), living cells (Q3), and
apoptotic cells (Q4).
(3) Our results confirm that compound (Vd) pro- vigorously. To this carbon disulfide (1.6 mL) and
duces a higher population of apoptotic cells than other
investigated compounds in this study, even higher than
cisplatin. This is the desirable death mechanism for a
cytotoxic drug.
1.2 mL of 20 M aqueous sodium hydroxide were added
dropwise during 30 min with stirring. Dimethyl sulfate
(0.02 mol) was added gradually, keeping the reaction
mixture stirring in freezing mixture for 2 h. The reac-
tion mixture was then poured into ice water. The solid
obtained was filtered, washed with water, dried under
high vacuum, and recrystallized from ethanol.
EXPERIMENTAL
All solvents, reagents, and compounds were pur-
chased from Merck and Fluka companies. RPMI-
1640 medium, Dulbecco’s modified Eagle’s medium
(DMEM), and fetal bovine serum (FBS) were pur-
chased from GIBCO (Gaithersburg, USA). Penicillin
and streptomycin were purchased from Biochrom AG
(Berlin, Germany). MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide, a yellow tetra-
zole) was purchased from Sigma Co., Ltd. Cisplatin
was purchased from Sigma Aldrich.
Methyl-1,3-benzothiazol-2-ylcarbamodithioate (ꢀVa).
C9H8N2S3. Yellow solid (85% yield), mp 196–200°C;
IR: 3230, 3188, 3050, 1605, 1525, 1448, 748; 1H NMR
(CDCl3): 2.75 (s, 3H, CH3), 4.9 (br s, 1H, NH), 7.45–
7.49 (t, J = 7.6 Hz, 1H), 7.53–7.58 (t, J = 7.2 Hz, 1H),
7.68–7.88 (d, J = 8.0 Hz, 1H), 7.83–7.85 (d, J = 8.0
Hz, 1H);13C NMR: 199.9, 174.5, 153.2, 130.8, 125.3,
124.5, 121.8, 118.3, 19.0; MS (m/z): 240 (M+), 238,
191, 165, 134, 90.
NMR spectra were recorded on Avance Brucker-
400 MHz spectrometers; H spectra were scanned at
Methyl(6-methyl-1,3-benzothiazol-2-yl)carba-
modithioate (ꢀVb). C10H10N2S3. Yellow solid (78%
yield), mp 197–199°C; IR: 3247, 3199, 2987, 3060,
2971, 1681, 1581, 1527, 1358, 743; 1H NMR (DMSO-
d6): 2.39 (s, 3H, CH3), 2.56 (s, 3H, CH3), 3.38 (br s,
1H, NH) 7.30–7.32(d, J = 8.4 Hz, 1H), 7.40–7.42 (d,
J = 8.0 Hz, 1H),7.71 (s, 1H); 13C NMR (DMSO):
185.2, 134.5, 129.1, 126.5, 123.1, 114.6, 21.4, 18.4; MS
(m/z): 254 (M+), 252, 206, 163, 147, 91, 43.
1
400 MHz and 13C, at 100 MHz. All chemical shifts in
NMR experiments are reported as δ, ppm, and were
referenced to residual solvent. FT-IR spectra (KBr; ν,
cm–1) were recorded on an AVATAR-370-FTIR Ther-
moNicolet. All mass spectra were scanned on a Varian
Mat CH-7 at 70 eV. Reaction was monitored by TLC
using silica gel plates and the products were identified
by comparison of their spectra and physical data with
those of the authentic samples. Melting points were
measured on an Electrothermal 9100 apparatus.
Methyl(6-methoxy-1,3-benzothiazol-2-yl)carba-
modithioate (ꢀVc). C10H10N2OS3. Yellow solid (81%
yeild), mp 197°C; IR: 3145, 3071, 2987, 2826, 1603,
1
1573, 1330, 834; H NMR (CDCl3): 2.70 (s, 3H,
General Synthesis of Methyl-1,3-Benzothiazol-2-
ylcarbamodithioateDerivatives (ꢀVa–d)
CH3), 3.88 (s, 3H, CH3), 7.07–7.09 (d, J = 9.2 Hz,
1H), 7.27 (s, 1H), 7.69–7.71 (d, J = 7.6 Hz, 1H), 12.30
(br s, 1H, NH); 13C NMR (CDCl3): 157.2, 153.9,
A solution of 2-aminobenzothiazole derivative (III)
(0.02 mol) in dimethylsulfoxide (10 mL) was stirred
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 5 2017

580
ESHKIL et al.
(m/z): 239 (M+), 235, 221, 204, 196, 181, 164, 137, 95,
69, 29.
146.1, 142.2, 130.9, 120.0, 115.7, 104.4, 55.8, 18.6, MS
(m/z): 270 (M+), 268, 220, 196, 178, 134, 91, 28.
Methyl(6-ethoxy-1,3-benzothiazol-2-yl)carba-
modithioate (ꢀVd). C11H12N2OS3. Yellow solid (73%
yeild), mp 197°C; IR: 3186, 3068, 2975, 2913, 2880,
1-(6-Ethoxy-1,3-benzothiazol-2-yl)thiourea (Vd).
C10H11N3OS2. Yellow solid (64% yield), mp 220–
225°C; IR: 3450, 3205, 3158, 3037, 2970, 2931, 1610,
1
1
1602, 1568, 1323, 988, 816; H NMR (DMSO-d6):
1592, 1531, 1212, 1059, 804; H NMR (DMSO-d6):
1.32–1.36 (t, J = 8.0 Hz, 3H, CH3), 2.55 (s, 3H, CH3),
4.03–4.08 (q, J = 6.8 Hz, 2H, CH2), 7.07–7.09 (d, J =
8.0 Hz, 1H), 7.44 (s,1H), 7.53 (d, 1H), 13.76 (br s, 1H,
NH); 13C NMR (DMSO): 156.3, 128.2, 116.7, 116.3,
107.1, 64.2, 18.3, 15.0, MS (m/z): 284 (M+), 282, 235,
206, 178, 164, 57.
1.32–1.35 (t, J = 12.0 Hz, 3H, CH3), 4.02–4.07 (q,
J = 20.0 Hz, 2H, CH2), 6.97–7.00 (d, J = 12.0 Hz,
1H), 7.50 (s, 1H), 7.58–7.60 (d, J = 8.0 Hz, 1H), 9.03
(s, 2H, NH2), 11.59 (br s, 1H, NH); 13C NMR
(DMSO): 179.9, 159.5, 155.9, 131.9, 121.0, 115.4,
107.9, 106.1, 64.0, 15.1; MS (m/z): 253 (M+), 250,
234, 218, 193, 165, 138, 95, 69, 60, 43, 28.
General Synthesis of 1-(1,3-Benzothiazol-
2-yl)thiourea Derivatives (Va–d)
Biological Studies
Methyl
benzo[d]thiazol-2-ylcarbamodithioate
Cell culture methods. Human breast cancer cells
MCF-7 (ATCC HTB-22), human cervix epithelial
carcinoma HeLa (ATCC CCL-2), human colon can-
cer cell line HT-29 (ATCC HTB-38), human leuke-
derivatives (ꢀV) (0.01 mol) were dissolved in ethanol
(25 mL). To this ammonia (0.1 mol) was added and
refluxed for 4 h. The reaction mixture was cooled and
poured into ice water. The solid obtained was filtered, mia cell line K-562 (ATCC CCL-243), mouse neuro-
washed with water, dried under high vacuum, and
recrystallized from ethanol.
blastoma cell line Neuro-2a (ATCC CCL-131), and
mouse fibroblast L-929 cell line (ATCC CCL-1) were
obtained from the American Type Culture Collection
(ATCC; Manassas, VA, USA) and cultured at 37°C in
a humidified atmosphere of 5% CO2 in air. HeLa cells
were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) with 0.1 mM nonessential amino
acids, 2 mM L-glutamine, 1.0 mM sodium pyruvate
and 5% fetal bovine serum, at 37°C in an atmosphere
of 5% CO2. Cells were plated in 24-well sterile plates at
1-(1,3-Benzothiazol-2-yl)thiourea (Va). C8H7N3S2.
Light yellow solid (68% yield), mp 215–220°C; IR:
3271, 3185, 3121, 3024, 1614, 1567, 1523, 1187, 754;
1H NMR (DMSO-d6): 7.14–7.18 (t, J = 16 Hz, 1H),
7.27–7.31 ( t, J = 16 Hz, 1H), 7.56–7.58 (d, J = 8.0 Hz,
1H), 7.61–7.63 (d, J = 8.0 Hz, 1H), 9.48–9.54 (br s,
2H, NH2), 10.50–10.63 (br s, 1H, NH); 13C NMR
(DMSO): 181.4, 174.5, 153.2, 130.8, 125.3, 124.5,
121.8, 118.3; MS (m/z): 209 (M+), 207, 149, 122, 95,
60; Anal. calcd for C8H7N3S2: C, 45.91; H, 3.37; N,
20.08; S, 30.64. Found: C, 45.71; H, 3.40; N, 19.00; S,
30.96.
a density of 1 × 104 cells/well in 100 μL of medium and
incubated for 24 h. Also MCF-7 and HT-29 cells were
cultured in DMEM containing 10% fetal bovine serum,
100 units/mL of penicillin, and 100 μg/mL of streptomy-
cin. K-562 cells were cultured in RPMI-1640 containing
10% fetal bovine serum, 100 units/mL of penicillin,
and 100 μg/mL of streptomycin.
1-(6-Methyl-1,3-benzothiazol-2-yl)thiourea (Vb).
C9H9N3S2. Light green solid (65% yield), mp 215°C;
IR: 3269, 3182, 3113, 2974, 1617, 1558, 1527, 1182, 812;
MTT assay in human cancer cell lines. Compounds
(Va–d) were screened for antitumor activity against
1H NMR (DMSO-d6): 2.25 (s, 3H, CH3), 7.24– human cell lines MCF-7, HeLa, HT-29, and K-562
and mouse cell line Neuro-2a using cisplatin as a com-
parative standard. Cell viability was evaluated using a
colorimetric method based on atetrazolium salt MTT
([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide]), which is reduced by living cells to
yield purple formazan crystals. Cells were seeded in
96-well plates at a density of (2–5) × 104 MCF-7,
HeLa, HT-29, K-562, and Neuro-2a cells per well in
200 μL of culture medium and left to incubate over-
night for optimal adherence. After careful removal of
the medium, 200 μL of a dilution series of the com-
pounds in fresh medium were added and incubation
was performed at 37°C/5% CO2 for 24 h or 72 h. Com-
7.22(d, J = 8.0 Hz, 1H), 7.60–7.56 (d, J = 16.0 Hz,
1H), 7.72 (s, 1H, NH), 9.10 (s, 2H, NH2), 11.79 (br s,
1H, NH); 13C NMR (DMSO): 181.4, 174.5, 150.2,
134.1, 130.7, 126.6, 121.3, 117.1, 20.9; MS (m/z): 223
(M+), 221, 204, 163, 135, 91, 76, 60, 28.
1-(6-Methoxy-1,3-benzothiazol-2-yl)thiourea (Vc).
C9H9N3OS2. Light yellow solid (60% yield), mp
160°C; IR: 3272, 3177, 3125, 3025, 2965, 2835, 1600,
1529, 1487, 1225, 830; 1H NMR (DMSO-d6): 3.83 (s,
3H, CH3), 7.07–7.09 (d, J = 9.2 Hz, 1H), 7.27 (s, 1H),
7.69–7.71(d, J = 7.6 Hz, 1H), 9.08 (s, 2H, NH2), 11.70
(br s, 1H, NH); 13C NMR (DMSO):181.4, 174.5,
156.7, 145.5, 131.9, 118.2, 114.6, 104.9, 55.8; MS pounds (Va–d) were first solubilized in DMSO,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 43
No. 5
2017

BENZOTHIAZOLE THIOUREA DERIVATIVES AS ANTICANCER AGENTS
581
diluted in medium, and added to the cells at final con- dent experiments. Statistical tests, including one-way
centrations between 20 nM and 200 μM. The percent- ANOVA, Tukey multiple comparison, or unpaired
age of DMSO in cell culture medium did not exceed Student’s t-tests, were performed using SPSS, ver.17
1%. Cisplatin was first solubilized in saline and then software. A p-value of less than 0.05 was considered as
added at the same concentrations used for the other significant.
compounds. At the end of the incubation period, the
compounds were removed and the cells were incu-
bated with 200 μL of MTT solution (500 μg/mL). After
ACKNOWLEDGMENTS
3–4 h at 37°C/5% CO2, the medium was removed and
the purple formazan crystals were dissolved in 200 μL
of DMSO by shaking. The cell viability was evaluated
by measurement of the absorbance at 570 nm by using
a STAT FAX-2100 microplate reader (Awareness
Technology, Palm City, FL, USA).The cell viability
was calculated dividing the absorbance of each well by
that of the control wells (cells treated with medium
containing 1% DMSO). Each experiment was
repeated at least three times and each point was deter-
mined in at least three replicates.
This work was supported by a grant number
3.29734, from Ferdowsi University of Mashhad,
Mashhad, Iran. We are grateful for help provided by
Buali Research Institute, Faculty of Pharmacy, Mash-
had University of Medical Sciences, Mashhad, Iran.
REFERENCES
1. Kok, S.H.L., Gambari, R., Chui, C.H., Yuen, M.C.W.,
Lin, E., Wong, R.S.M., Lau, F.Y., Cheng, G.Y.M.,
Lam, W.S., Chan, S.H., Lam, K.H., Cheng, C.H.,
Lai, P.B.S., Yu, M.W.Y., Cheung, F., Tang, J.C.O.,
and Chan, A.S.C., Bioorg. Med. Chem., 2008, vol. 16,
pp. 3626–3631.
Apoptosis assay for compound (Vd) by flow cytome-
try. Evaluation of apoptosis by flow cytometry is gen-
erally accomplished by methods that use annexin
V-FITC as vital dye, which accesses phosphatidylser-
ine exposed on the external membrane at the begin-
ning of apoptosisprocess. The differentiation between
apoptotic and necrotic cells can be performed by
simultaneous staining with propidium iodide (PI).
Therefore, annexin V-FITC was used as a marker of
phosphatidylserine exposure and PI as a marker for
dead cells. This combination allows differentiation among
early apoptotic cells (annexin V-positive, PI-negative),
late apoptotic/necrotic cells (annexin V-positive,
PI-positive), and viable cells (annexin V-negative,
PI-negative) [27].
Cells (5 × 105) were seeded and treated with com-
pound (Vd )and incubated for 24 h at a concentration
close to the IC50 at 37°C. Following treatment, the
cells were harvested by trypsinization and centrifuga-
tion at 1000 rpm for 5 min. The supernatant was
removed and the cell pellet was washed in PBS fol-
lowed by two washes in binding buffer (10 mM
HEPES, 150 mM NaCl, 5 mM KCl, 1.8 mM CaCl2,
1 mM MgCl2). The cells were incubated with an
annexin V-FITC antibody (5 mL in 100 mL binding
buffer) and incubated at 4°C for 15 min in the dark.
Samples were washed in binding buffer and the super-
natant was discarded. The pellet was resuspended in
490 mL binding buffer and 10 mL propidium iodide
(10 mg/mL PBS) was added to the samples before
analysis by flow cytometry. Flow cytometry was done
using Partec PAS flow cytometer (Partec GmbH,
Germany) with cisplatin as a reference.
2. Liu, C., Lin, J., Pitt, S., Zhang, R.F., Sack, J.S., Kiefer, S.E.,
Kish, K., Doweyko, A. M., Zhang, H., Marathe, P.H.,
Trzaskos, J., Mckinnon, M., Dodd, J.H., Barrish, J.C.,
Schieven, G.L., and Leftheris, K., Bioorg. Med. Chem.
Lett., 2008, vol. 18, pp. 1874–1879.
3. Henriksen, G., Hauser, A.I., Westwell, A.D., Yousefi, B.H.,
Schwaiger, M., Drzega, A., and Wester, H.J., J. Med.
Chem., 2007, vol. 50, pp. 1087–1089.
4. Amnerkar, N.D. andBhusari, K.P., Eur. J. Med.
Chem., 2010, vol. 45, pp. 149–159.
5. Duggan, P.J., Lewis, R.J., Lok, Y.P., Lumsden, N.G.,
Tuck, K.L., and Yang, A., Bioorg. Med. Chem. Lett.,
2009, vol. 19, pp. 2763–2765.
6. Kashiyama, E., Hutchinson, I., Chua, M-S., Stinson, S.F.,
Phillips, L.R., Kaur, G., Sausville, E.A., Bradshaw, T.D.,
Westwell, A.D., and Stevens, M.F.G., J. Med. Chem.,
1999, vol. 42, pp. 4172–4184.
7. Hutchinson, I., Jennings, S.A., Vishnuvajjala, B.R.,
Westwell, A.D., and Stevens, M.F.G., J. Med. Chem.,
2002, vol. 45, pp. 744–747.
8. Kumbhare, R.M. and Ingle, V.N., Ind. J. Chem., 2009,
vol. 48, pp. 996–1000.
9. Charris, J., Monasterios, M., Dominguez, J., Infante, W.,
and De Castro, N., Heterocycl. Commun., 2002, vol. 8,
pp. 275–280.
10. Bondock, S., Fadaly, W., and Metwally, M.A., Eur. J.
Med. Chem., 2009, vol. 44, pp. 4813–4818.
11. Bondock, S., Fadaly, W., and Metwally, M.A., Eur. J.
Med. Chem., 2010, vol. 45, pp. 3692–3701.
12. Gurupadayya, B.M., Manohara, Y.N., and Gopal, M.,
Indian J. Heterocycl. Chem., 2006, vol. 15, pp. 113–116.
13. Esteves-Souza, A., Pissinate, K., Nascimento, M.G.,
Grynberg, N.F., and Echevarria, A., Bioorg. Med.
Chem., 2006, vol. 14, pp. 492–499.
Statistical analysis. IC50 values expressed as mean
standard deviation (SD) from at least three indepen-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 5 2017

582
ESHKIL et al.
14. Choi, S.-J., Park, H.J., Lee, S.K., Kim, S.W., Han, G., 21. Wenzel, A.G. and Jacobsen, E.N., J. Am. Chem. Soc.,
and Choo, H.-Y.P., Bioorg. Med. Chem., 2006, vol. 14,
2002, vol. 124, pp. 12964–12965.
pp. 1229–1235.
22. Tang, Y., Deng, L., Zhang, Y., Dong, G., Jiahua, J.,
Chen, J., and Yang, Z., Org. Lett., 2005, vol. 7,
pp. 1657–1659.
15. Venkatachalam, T.K., Mao, C., and Uckun, F.M., Bio-
org. Med. Chem., 2004, vol. 12, pp. 4275–4284.
16. Lind, P.T., Morin, J.M., Noreen, R.J., and Ternansky, R.J.,
23. Fontas, C., Hidalgo, M., Salvado, V., and Antico, E.,
WO 9303022 CA, vol. 119, p. 160110.
Anal. Chim. Acta, 2005, vol. 547, pp. 255–261.
17. Scheetz, M.E., Carlson, D.G., and Schinitsky, M.R.,
24. Venkatachalam, T.K., Sudbeck, E., and Uckun, F.M.,
Virus. Infect. Immun., 1977, vol. 15, pp. 145–148.
J. Mol. Struct., 2005, vol. 751, pp. 41–54.
18. Lee, J., La, S., Ahn, B.R., Jeong, T.C., and Kim, D.H.,
Rapid Commun. Mass Spectrom., 2004, vol. 18,
pp. 1901–1910.
25. Rodriguez-Fernandez, E., Manzano, J.L., Benito, J.J.,
Hermosa, R., Monte, E., and Criado, J.J., J. Inorg.
Biochem., 2005, vol. 99, pp. 1558–1572.
19. Ziessel, R., Bonardi, L., Retailleau, P., and Ulrich, G.,
26. Abdel-Rehim, S.S., Khaled, K.F., and Abd-Elshafi, N.S.,
Org. Chem., 2006, vol. 71, pp. 3093–3102.
Electrochim. Acta, 2006, vol. 51, pp. 3269–3275.
20. Fuerst, D.E. and Jacobsen, E.N., J. Am. Chem. Soc.,
2005, vol. 127, pp. 8964–8965.
27. Krishan, A., J. Cell. Biol., 1975, vol. 66, pp. 188–193.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 43
No. 5
2017