Cytotoxic activities of some benzothiazole-piperazine derivatives

Article abstract of DOI:10.3109/14756366.2014.959513

Synthesis, characterization and cytotoxic activities of ten benzothiazole-piperazine derivatives were reported. In vitro cytotoxic activities of compounds were screened against hepatocellular (HUH-7), breast (MCF-7) and colorectal (HCT-116) cancer cell li

Full text of DOI:10.3109/14756366.2014.959513

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–6
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.959513
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RESEARCH ARTICLE
Cytotoxic activities of some benzothiazole-piperazine derivatives
1
1
2
2
Enise Ece Gurdal , Irem Durmaz , Rengul Cetin-Atalay , and Mine Yarim
1
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Yeditepe University, Kayisdagi, Istanbul, Turkey and Department of Molecular
Biology and Genetics, BilGen, Genetics and Biotechnology Research Center, Faculty of Science, Bilkent University, Bilkent, Ankara, Turkey
Abstract
Synthesis, characterization and cytotoxic activities of ten benzothiazole-piperazine derivatives Anticancer, benzothiazole, cytotoxicity,
were reported. In vitro cytotoxic activities of compounds were screened against hepatocellular
piperazine, sulphorodamine B
Keywords
(HUH-7), breast (MCF-7) and colorectal (HCT-116) cancer cell lines by sulphorhodamine B assay.
Based on the GI50 values of the compounds, most of the benzothiazole-piperazine derivatives
are active against HUH-7, MCF-7 and HCT-116 cancer cell lines. Compound 1d is highly
cytotoxic against all tested cancer cell lines. Further investigation of compound 1d by Hoechst
Staining and Fluorescence-Activated Cell Sorting Analysis (FACS) revealed that this compound
History
Received 21 August 2014
Accepted 21 August 2014
Published online 21 October 2014
1
causes apoptosis by cell cycle arrest at subG phase.
Introduction
were further analyzed to evaluate mechanism of action. Agents
caused induction of senescence-associated cell death through the
Cancer, remaining to be a major health problem today, is caused
by abnormal cell division without control. Cancer cells are able to
spread into other parts of the body through blood and lymph
system. Although there are many advances in treatment of cancer,
most of anticancer drugs still need to be improved because of
various limitations like emergence of drug resistance, low
therapeutic index and lack of selectivity. Thus, cancer studies
continue to develop new anticancer medicines, targeted therapies,
monoclonal antibodies, etc.
8
inhibition of some kinase proteins . In addition, Yarim and her
co-workers previously reported various piperazine derivatives
with high cytotoxic activity against liver (HUH-7, FOCUS,
MAHLAVU, HepG-2, Hep-3B), breast (MCF-7, BT20 and
T47D), colon (HCT-116), gastric (KATO-3), cervix (HeLa) and
9–11
endometrial (MFE-296) cancer cell lines
.
According to anticancer activity studies of benzothiazole-
piperazine backbone, arylsulphonamides and arylthiol derivatives
have potent cytotoxicity against a large scale of cancer cell
lines such as breast (MCF-7), hepatocellular (HepG-2), prostate
Benzothiazole derivatives were reported to be highly cytotoxic
1
against HCT-116 (colorectal cancer cell line) , MCF-7 (breast
+
2
3
(DU-145) cancers and CD4 human acute T-lymphoblastic
12,13
cancer cell line) , U937 and THP-1 (leukemia cancer cell lines) .
Isatin Mannich bases with benzothiazole moiety were reported as
potential anti-breast cancer drugs. Flow cell cytometry showed
that most active compound in series resulted in cell cycle arrest at
leukaemia (CCRF-CEM)
.
In this study, with the aid of aforementioned studies, we
reported the synthesis, purification and characterization of novel
compounds which contain benzothiazole-piperazine backbone in
their molecular structure. These compounds were tested for their
cytotoxic activities against hepatocellular (HUH-7), breast
(MCF-7) and colorectal (HCT-116) cancer cell lines with
sulphorhodamine B assay. The advanced analysis, Hoechst
staining and fluorescence-activated cell sorting analysis were
also done for compound 1d to understand the mechanism of
cytotoxicity.
4
G2/M phase . Thiourea derivatives bearing benzothiazole moiety
were reported to be cytotoxic against MCF-7 and HeLa breast
cancer cell lines. Most active derivatives of the series were
5
proven to act by damaging DNA with alkaline Comet assay .
2
MCF-7 (breast) and IGROV-1 (ovarian) cancer cell lines caused
-(4-Aminophenyl)benzothiazoles with cytotoxic activity against
6
generation of DNA adducts which induce apoptosis .
Piperazine ring is another value for anticancer drug candidates.
Piperazinobenzopyranones were reported as breast cancer resist-
ance protein inhibitors which are usually responsible with
demolished effect of anticancer medication . Piperazine ring Chemistry
carrying nucleoside analogues were evaluated for their cytotoxic
activities on various cancer cell lines and most active compounds
Methods and materials
7
All chemicals and reagents used in current study were of
analytical grade. The reactions were monitored by thin layer
chromatography (TLC) on Merck pre-coated silica GF254 plates.
ꢀ
Melting points ( C) of the compounds were determined by using a
Mettler Toledo FP62 capillary melting point apparatus (Mettler-
Toledo, Greifensee, Switzerland) and are uncorrected. Infrared
spectra were recorded on a Perkin-Elmer Spectrum One series FT-
IR apparatus Version 5.0.1 (Perkin Elmer, Norwalk, CT), using
Address for correspondence: Enise Ece Gurdal, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Yeditepe University,
34755 Kayisdagi, Istanbul, Turkey. Tel: +90 216 5780000 (ext. 1592).
Fax: +90 216 5780068. E-mail: egurdal@yeditepe.edu.tr

2
E. E. Gurdal et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
potassium bromide pellets, the frequencies were expressed in Cytotoxicity studies
ꢁ
1
cm . Elemental analyses were performed on LECO 932 CHNS
LECO-932, St. Joseph, MI) instrument. UV spectra were
The cytotoxic activity of the synthesized compounds was
investigated on liver (HUH-7), breast (MCF-7) and colon
(HCT-116) cancer cell lines, by means of sulphorhodamine B
(
recorded on EvolutionÔ 201/220 UV-visible spectrophotometer
and ethanol was used as solvent. The H-NMR spectra were
recorded with a Varian Mercury-400 FT-NMR spectrometer
(Varian Inc., Palo Alto, CA), using tetramethylsilane (TMS) as the
internal reference, with chloroform (CDCl ) as solvent, the
1
(SRB) assays in triplicate. Serial dilutions from 100 mM to 2.5 mM
were used, 5-fluorouracil (5-FU) was the reference compound.
3
Cell culture
chemical shifts were reported in parts per million (ppm).
Coupling constants were recorded in Hertz (Hz).
The human cancer cell lines were grown in Dulbecco’s Modified
Eagle’s Medium (DMEM) supplemented with 10% fetal bovine
serum (FBS) and 1% penicillin. Each cell line was maintained in
General procedure for synthesis of 2-chloro-N-(6-methylben-
zothiazol-2-yl)acetamide (1)
ꢀ
an incubator at 37 C supplied with 5% CO and 95% air.
2
2
in 42 ml benzene: triethlyamine mixture (20:1) . Later, acetyl-
-Amino-6-methylbenzothiazole (0.012 mol (2 g)) was dissolved
1
4
NCI-60 sulphorhodamine B (SRB) assay
ation was performed with 0.011 mol (0.87 ml) chloroacetyl Cancer cells (range of 2000 cell/well to 5000 cell/well) were
chloride in room temperature. Because of the highly reactant inoculated into 96-well plates in 200 ml of media and incubated in
ꢀ
nature of chloroacetyl chloride, it was added slowly in small 37 C incubators containing 5% CO and 95% air. After a 24 h
2
amounts. Reaction was monitored by TLC with silica gel plate incubation period, one plate for each cell line was fixed with
and benzene:methanol (9:1) mobile phase mixture. Reaction 100 ml 10% ice-cold trichloroacetic acid (TCA). This plate
completed in four days at room temperature. Precipitated crude represents the behaviour of the cells just prior to drug treatment
product was filtered, washed with benzene and dried. Ethanol and is accepted as the time-zero plate. The compounds to be
crystallization gave pure product.
4
General procedure for synthesis of N-(6-methylbenzothiazol-2-yl)- compounds, the corresponding volume of the compound was
tested were solubilized in DMSO to a final concentration of
ꢀ
0 mM and stored at +4 C. While treating the cells with the
2
-(4-substitued piperazinyl)acetamide derivatives (1a–f)
applied to the cell to achieve the desired drug concentration and
diluted through serial dilution. After drug treatment, the cells
were incubated in 37 C incubators containing 5% CO and 95%
2
N-(6-methylbenzothiazol-2-yl)-2-(4-substituedpiperazinyl)aceta-
mides were synthesized in acetone by the reaction of
ꢀ
air for 72 h. Following the termination of the incubation period
after drug treatment, the cells were fixed with 100 ml 10% ice-cold
TCA and incubated in the dark at +4 C for 1 h. Then the TCA
0
.0025 mol
(0.602 g)
2-chloro-N-(6-methylbenzothiazol-2-
yl)acetamide and 0.0025 mol of suitable piperazine, in presence
of 0.0025 mol (0.345 g) anhydrous K CO . Reactions were
ꢀ
2
3
was washed away with ddH O five times and the plates were left
2
to air dry. For the final step, the plates were stained with 100 ml of
monitored by TLC with silica gel plate and benzene:methanol
9:1) mobile phase mixture. Reactions completed in two days
(
0
.4% sulphorhodamine B (SRB) solution in 1% acetic acid
solution. Following staining, the plates were incubated in dark for
0 min at room temperature. The unbound dye was washed away
at room temperature. Potassium carbonate was removed by
filtration. After evaporation of acetone, precipitated products were
recrystallized from absolute ethanol or acetone:distilled water
mixture.
1
using 1% acetic acid and the plates were left to air dry. To
measure the absorbance results, the bound stain was then
solubilized using 200 ml of 10 mM Tris-Base. The OD values
were obtained at 515 nm.
General procedure for synthesis of 2-chloro-N-(6-ethoxyben-
zothiazol-2-yl)acetamide (2)
2
-Amino-6-ethoxybenzothiazole (0.012 mol (2.22 g)) was
Hoechst staining analysis
1
4
dissolved in 42 ml benzene: triethylamine mixture (20:1) .
Later, acetylation was performed with 0.013 mol (0.78 ml)
chloroacetyl chloride at room temperature. Because of the
highly reactant nature of chloroacetyl chloride, it was added
slowly in small amounts. Reaction was monitored by TLC with
silica gel plate and benzene:methanol (9:1) mobile phase mixture.
Reaction completed in four days at room temperature.
Precipitated crude product was filtered, washed with benzene
and dried. Ethanol crystallization gave pure product.
Cells were seeded on coverslips in 6-well plates. After
overnight culture, cells were exposed to compounds at a
concentration of their GI50 values for 72 h. To determine nuclear
condensation by Hoechst 33258 (Sigma-Aldrich) staining, cover-
slips were washed twice with icecold PBS, fixed in 1 ml of
cold methanol for 10 min, and then incubated with 3 Ig/ml of
Hoechst 33258 for 5 min in darkness. The coverslips were then
rinsed with distilled water, mounted on glass microscopic slides
using 50% glycerol, and examined under fluorescent microscopy
(
40ꢂ).
Fluorescence-activated cell sorting analysis
mides were synthesized in acetone by the reaction of 0.0025 mol Human cancer cell line (HUH-7) of interest were inoculated into
General procedure for synthesis of N-(6-ethoxybenzothiazol-2-yl)-
2
-(4-substitued-piperazinyl)acetamides (2a–d)
N-(6-ethoxybenzothiazol-2-yl)-2-(4-substituedpiperazinyl)aceta-
(
0
(
0.677 g) 2-chloro-N-(6-ethoxybenzothiazol-2-yl)acetamide and 100-mm culture dishes (300 000 cells/dish). Twenty-four hours
.0025 mol of suitable piperazine, in presence of 0.0025 mol later, cells were then treated with the desired compounds
0.345 g) anhydrous K CO . Reactions were monitored by according to their GI50 values and incubated for 72 h. Cells
2
3
TLC with silica gel plate and benzene:methanol (9:1) mobile were then collected by trypsinization and the pellets were fixed in
ꢀ
phase mixture. Reactions completed in two days at room ice-cold 70% ethanol and stored at ꢁ20 C. Before the analysis,
temperature. Potassium carbonate was removed by filtration. the samples were stained with MUSE cell cycle reagent (contains
After evaporation of acetone, precipitated products were propidium iodide solution) according to the manufacturer’s
recrystallized from absolute ethanol or acetone:distilled water protocol. Cell cycle analysis was conducted with MUSE cell
mixture.
cycle analyzer.

DOI: 10.3109/14756366.2014.959513
Cytotoxic benzothiazoles
3
Results
N-(6-methylbenzothiazol-2-yl)-2-[4-(3,4-dichlorophenyl)piperazi-
nyl]acetamide (1e)
Chemistry
ꢀ
ꢁ1
White powder. 16% (0.196 g), m.p. 243.8 C. UV (MeOH, lmax,
nm); 293 (log ": 4.25). FT-IR (KBr, cm ); 3278 (N–H), 3017
N-(6-methylbenzothiazol-2-yl)-2-[4-(2-methoxyphenyl)piperaziny-
l]acetamide (1a)
(
C–H, aromatic), 2893 (C–H, aliphatic), 1707 (C¼O, amide), 1605
ꢀ
1
White powder, 48% (0.417 g), m.p. 121.9 C (reported: (C¼C, aromatic), 1240 (C–N). H-NMR (CDCl , ppm); 2.48
3
ꢀ
1
22–124 C). UV (MeOH, lmax, nm); 291 (log ": 4.21). FT-IR (s, 3H, Ar–CH ), 2.80 (t, 4H, piperazine H , J ¼ 4.8 Hz), 3.27
3
2,6
ꢁ1
(
KBr, cm ); 3333 (N–H), 3058 (C–H, aromatic), 2908 (C–H, (t, 4H, piperazine H3,5, J ¼ 5.2 Hz), 3.35 (s, 2H, –COCH N–),
2
aliphatic), 1707 (C¼O, amide), 1605 (C¼C, aromatic), 1242 6.74–6.77 (dd, 1H, phenyl H , J ¼ 3.2 Hz, J ¼ 8.6 Hz), 6.98 (d,
2 1 2
1
5
1
(
C–N), 1056 (C–O) . H-NMR (CDCl , ppm); 2.48 (s, 3H, 1H, phenyl H , J ¼ 3.2 Hz), 7.25–7.26 (m, 1H, benzothiazole H ),
3
6
5
Ar–CH ), 2.86 (t, 4H, piperazine H , J ¼ 4.8 Hz), 3.18 (bs, 4H, 7.30 (d, 1H, phenyl H , J ¼ 9.2 Hz), 7.62 (s, 1H, benzothiazole H ),
3 2,6 5 7
H3,5), 3.36 (s, 2H, –COCH N–), 3.88 (s, 3H, –OCH ), 6.89 (d, 1H, 7.68 (d, 1H, benzothiazole H , J ¼ 8.4 Hz), 10.33 (bs, 1H,
2
3
4
phenyl H , J ¼ 9.2 Hz), 6.94–6.98 (m, 2H, phenyl H3,5), 7.02–7.06 –NHCOCH –). Anal Calcd for C H Cl N OS (435.37): C,
2
2
20 20
2 4
(m, 1H, phenyl H ), 7.25–7.28 (m, 1H, benzothiazole H ), 7.62 54.92; H, 5.07; N, 12.81; S, 7.33. Found: C, 54.91; H, 5.04; N,
4 5
(
s, 1H, benzothiazole H ), 7.68 (d, 1H, benzothiazole H , 12.83; S, 7.31.
7
4
J ¼ 8 Hz), 10.47 (bs, 1H, –NHCOCH –). Anal Calcd for
2
C H N O S (396.506): C, 63.61; H, 6.10; N, 14.13; S, 8.09. N-(6-methylbenzothiazol-2-yl)-2-[4-(4-chlorobenzyl)piperaziny-
2
1 24 4 2
Found: C, 63.52; H, 6.14; N, 14.25; S, 8.04.
l]acetamide (1f)
ꢀ
Cream-colored powder. 12% (0.115 g), m.p. 189.6 C. UV
(
(
N-(6-methylbenzothiazol-2-yl)-2-[4-(2-methoxyethyl)piperaziny-
l]acetamide (1b)
ꢁ1
MeOH, lmax, nm); 288 (log ": 4.27). FT-IR (KBr, cm ); 3100
N–H), 3038 (C–H, aromatic), 2929 (C–H, aliphatic), 1692
1
ꢀ
1
Cream colored, shiny powder. 12% (0.099 g), m.p. 81.1 C. UV (C¼O, amide), 1607 (C¼C, aromatic), 1271 (C–N). H-NMR
ꢁ
(
(
(
MeOH, lmax, nm); 292 (log ": 4.25). FT-IR (KBr, cm ); 3120 (CDCl , ppm); 2.47 (s, 3H, Ar–CH ), 2.54 (bs, 4H, piperazine
3
3
N–H), 3050 (C–H, aromatic), 2946 (C–H, aliphatic), 1697 H , ), 2.66 (bs, 4H, piperazine H , ), 3.27 (s, 2H, –N–CH –), 3.51
C¼O, amide) 1607 (C¼C, aromatic), 1259 (C–N), 1067 (C–O). (s, 2H, –COCH N–), 7.22–7.28 (m, 4H, phenyl), 7.29–7.31
3
5
2 6
2
2
1
H-NMR (CDCl , ppm); 2.46 (s, 3H, Ar-CH ), 2.62 (t, 2H, (m, 1H, benzothiazole H ), 7.61 (s, 1H, benzothiazole H ), 7.68
7
3
3
5
–
–
NCH CH –, J ¼ 5.2 Hz), 2.69 (bs, 8H, piperazine), 3.27 (s, 2H, (d, 1H, benzothiazole H , J ¼ 8.4 Hz), 10.4 (bs, 1H,
2 3 2 3 2 21 23 4
2
2
4
COCH N–), 3.36 (s, 3H, –OCH ), 3.51 (t, 2H, –CH OCH , –NHCOCH –). Anal. calcd. for C H ClN OS (414.952): C,
J ¼ 5.2 Hz), 7.23–7.26 (m, 1H, benzothiazole H ), 7.61 (s, 1H, 60.49; H, 6.04; N, 13.44; S, 7.69. Found: C, 60.41; H, 6.08; N,
5
benzothiazole H ), 7.67 (d, 1H, benzothiazole H , J ¼ 8 Hz), 13.41; S, 7.70.
7
4
1
0.39 (bs, 1H, –NHCOCH –). Anal Calcd for C H N O S
2 17 24 4 2
(348.463): C, 58.26; H, 7.48; N, 15.99; S, 9.15. Found: C, 58.22; N-(6-ethoxybenzothiazol-2-yl)-2-[4-(p-toluyl)piperazinyl]aceta-
H, 7.44; N, 15.97; S, 9.18.
mide (2a)
ꢀ
Honey colored irregular crystals. 36.5% (0.375 g), m.p. 146.7 C.
UV (MeOH, lmax, nm); 294 (log ": 4.18). FT-IR (KBr, cm );
3
N-(6-methylbenzothiazol-2-yl)-2-(4-cyclohexylpiperazinyl)aceta-
mide (1c)
ꢁ1
315 (N–H), 3006 (C–H; aromatic), 2980 (C–H; aliphatic), 1698
1
ꢀ
White powder. 2.8% (0.026 g), m.p. 181.3 C. UV (MeOH, lmax, (C¼O; amide), 1608 (C¼C; aromatic) and 1219 (C–N). H-NMR
ꢁ
1
nm); 289 (log ": 4.25). FT-IR (KBr, cm ); 3261 (N–H), 2932 (DMSO, ppm); 1.46 (t, 3H, –OCH CH , J ¼ 7.2 Hz), 2.29 (s, 3H,
2
3
(
C–H, aromatic), 2853 (C–H, aliphatic), 1703 (C¼O, amide), –Ph–CH ), 2.81 (t, 4H, piperazine H , J ¼ 5.2 Hz), 3.23 (t, 4H,
3
2,6
1
1
1
1
605 (C¼C, aromatic), 1262 (C¼N). H-NMR (CDCl , ppm); piperazine H3,5, J ¼ 5.2 Hz), 2.29 (s, 2H, –COCH N–), 4.06–4.12
3
2
.09–1.27 (m, 6H, cyclohexyl), 1.63–1.66 (m, 1H, cyclohexyl), (m, 2H, –OCH CH ), 6.86 (d, 2H, phenyl H , J ¼ 8.8 Hz), 7.03–
2 3 2,6
.80–1.88 (m, 4H, cyclohexyl), 2.48 (s, 4H, piperazine H2,6), 2.48 7.06 (dd, 2H, phenyl H3,5, J ¼ 2.4 Hz, J ¼ 8.8 Hz), 7.09–7.11
1
2
(
s, 3H, Ar–CH ), 2.67 (s, 4H, piperazine H3,5), 3.26 (s, 2H, (dd, 1H, benzothiazole H , J ¼ 8.8 Hz), 7.26–7.29 (m, 1H,
3
5
–
COCH N–), 7.25–7.27 (m, 1H, benzothiazole H ), 7.62 (s, 1H, benzothiazole H ), 7.67 (d, 1H, benzothiazol H , J ¼ 8.8 Hz),
7 4 2 22 26 4 2
2
5
7
4
benzothiazole H ), 7.68 (d, 1H, benzothiazole H , J ¼ 8 Hz), 10.36 (bs, 1H, –NHCOCH –). Anal Calcd for C H N O S
1
0.41 (bs, 1H, –NHCOCH –). Anal Calcd for C H N OS (410.532): C, 64.05; H, 6.84; N, 13.58; S, 7.77. Found: C, 64.04;
2 20 28 4
(372.528): C, 64.14; H, 8.07; N, 14.96; S, 8.56. Found: C, 64.12; H, 6.82; N, 13.55; S, 7.80.
H, 8.08; N, 14.97; S, 8.58.
N-(6-ethoxybenzothiazol-2-yl)-2-[4-(o-chlorophenyl)piperaziny-
l]acetamide (2b)
N-(6-methylbenzothiazol-2-yl)-2-[4-(pyridin-4-yl)piperazinyl]a-
cetamide (1d)
ꢀ
ꢁ1
Light brown colored powder. 47.5% (0.512 g), m.p. above 300 C.
White powder. 22% (0.195 g), m.p. 100.8 C. UV (MeOH, lmax, UV (MeOH, lmax, nm); 293 (log ": 4.16). FT-IR (KBr, cm );
nm); 293 (log ": 4.13). FT-IR (KBr, cm ); 3192 (N–H), 3050 3334 (N–H), 3065 (C–H; aromatic), 2971 (C–H; aliphatic), 1702
ꢀ
ꢁ
1
1
(
C–H, aromatic), 2842 (C–H, aliphatic), 1703 (C¼O, amide), (C¼O; amide), 1605 (C¼C; aromatic), 1226 (C–N). H-NMR
1
1
2
3
603 (C¼C, aromatic), 1212 (C–N). H-NMR (CDCl , ppm); (DMSO, ppm); 1.47 (t, 3H, –OCH CH , J ¼ 7.2 Hz), 2.85 (t, 4H,
3
2
3
.48 (s, 3H, Ar-CH ), 2.78 (t, 4H, piperazine H , J ¼ 5.4 Hz), piperazine H , J ¼ 4.4 Hz), 3.17 (bs, 4H, piperazine H ), 3.36
3 2,6 2,6 3,5
.45 (t, 4H, piperazine H3,5, J ¼ 4.8 Hz), 3.60 (s, 2H, –COCH N– (s, 2H, –COCH N–), 4.07–4.12 (m, 2H, –OCH CH ), 6.99–7.09
2
2
2
3
)
, 6.68–6.70 (dd, 2H, pyridine H , J ¼ 1.6 Hz, J ¼ 5 Hz), 7.26– (m, 3H, phenyl H4,5,6), 7.25 (d, 1H, phenyl H , J ¼ 1.2 Hz),
3
,5
1
2
3
7
.28 (m, 1H, benzothiazole H ), 7.63 (s, 1H, benzothiazole H ), 7.26–7.29 (m, 1H, benzothiazole H ), 7.38–7.39 (dd,
5 7 7
7
pyridine
.67 (d, 1H, benzothiazole H , J ¼ 8.4 Hz), 8.30–8.32 (dd, 2H, 1H, benzothiazole H , J ¼ 1.2 Hz, J ¼ 8 Hz), 7.68 (d, 1H,
2,6 1 2 4 2
4
5
1
2
H J ¼ 1.6 Hz, J ¼ 5.2 Hz), 10.37 (bs, 1H, benzothiazol H , J ¼ 8.8 Hz), 10.41 (bs, 1H, –NHCOCH –).
NHCOCH –). Anal Calcd for C H N OS (367.468): C, Anal Calcd for C H ClN O S (430.951): C, 58.25; H, 5.82;
19 21 5 21 23 4 2
,
–
6
1
2
1.76; H, 6.27; N, 18.95; S, 8.68. Found: C, 61.73; H, 6.29; N, N, 12.94; O, 7.39; S, 7.41. Found: C, 58.22; H, 5.82; N, 12.94;
8.96; S, 8.66. S, 7.40.

4
E. E. Gurdal et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
N-(6-ethoxybenzothiazol-2-yl)-2-[4-(p-cyanophenyl)piperaziny-
l]acetamide (2c)
Discussion
The synthesis of benzothiazole-piperazine derivatives is outlined
Beige colored powder. 42.7% (0.45 g), m.p. above 300 C. UV in Figure 1. The final compounds are obtained by two stages
ꢀ
1
ꢁ
(
(
(
MeOH, lmax, nm); 292 (log ": 4.21). FT-IR (KBr, cm ); 3194 which are N-acetylation of the primary amine and N-alkylation of
N–H), 3060 (C–H; aromatic), 2972 (C–H; aliphatic), 2215 the secondary amine. In order to obtain target compounds, firstly
CꢃN), 1699 (C¼O; amide), 1605 (C¼C; aromatic), 1225 (C–N). it is necessary to synthesize 2-chloro-N-(6-methylbenzothiazole-
1
H-NMR (DMSO, ppm); 1.46 (t, 3H, –OCH CH , J ¼ 7.2 Hz), 2-yl)acetamide (1) and 2-chloro-N-(6-ethoxybenzothiazole-2-
2
3
2
.80 (t, 4H, piperazine H , J ¼ 5.2 Hz), 3.42 (t, 4H, piperazine yl)acetamide (2) starting from 2-amino-6-methylbenzothiazole
2
2
,6
H3,5, J ¼ 5.2 Hz), 3.35 (s, 2H, –COCH N–), 4.06–4.12 (m, 2H, and 2-amino-6-ethoxybenzothiazole, respectively. Finally, com-
–
OCH CH ), 6.88 (d, 2H, phenyl H , J ¼ 8.8 Hz), 7.53 (d, 2H, pounds 1 and 2 are reacted with various piperazine derivatives
1 5
2
3
2,6
phenyl H3,5, J ¼ 8.8 Hz), 7.03–7.06 (dd, 1H, benzothiazole H , under basic conditions in which piperazines are N-alkylated to
J ¼ 2.4 Hz, J ¼ 8.6 Hz), 7.26–7.29 (m, 1H, benzothiazole H ), yield the final products. Structural and physical properties of the
1
2
7
7
–
6
1
.67 (d, 1H, benzothiazole H , J ¼ 8.8 Hz), 10.28 (bs, 1H, synthesized benzothiazole-piperazine derivatives are summarized
4
NHCOCH –). Anal Calcd for C H N O S (421.515): C, in Table 1.
22 23 5 2
2
2.39; H, 5.95; N, 16.54; S, 7.57. Found: C, 62.35; H, 5.96; N,
6.56; S, 7.58.
Synthesized compounds were identified with UV, IR and
H-NMR spectra. In UV spectrum of compounds there is one
1
significant band at 290 nm which represents n ! ꢀ* transition of
the series. In IR spectrum of benzothiazole-piperazine derivatives,
characteristic N–H stretching band was observed nearly at
N-(6-ethoxybenzothiazol-2-yl)-2-[4-(o-cyanophenyl)piperaziny-
l]acetamide (2d)
ꢁ1
3
at 3050 cm
330 cm . Other stretching bands were observed approximately
ꢁ1
ꢀ
1
Beige colored powder. 25.6% (0.27 g), m.p. above 300 C. UV
ꢁ1
ꢁ
(C–H; aromatic), 2980 cm
ꢁ1
(C–H; aliphatic),
(C¼C; aromatic) and
(
(
(
MeOH, lmax, nm); 290 (log ": 4.23). FT-IR (KBr, cm ); 3290
N–H), 3068 (C–H; aromatic), 2825 (C–H; aliphatic), 2221
CꢃN), 1703 (C¼O; amide), 1605 (C¼C; aromatic), 1225 (C–N).
ꢁ1
1
1
700 cm
240 cm (C–N). In H-NMR spectra of benzothiazole-pipera-
(C¼O; amide), 1605 cm
ꢁ1
1
1
zine derivatives, the protons of piperazine were seen approxi-
mately at 2.70 and 3.33 ppm as triplets (J ¼ 8.2 Hz). Amide N–H
gave broad singlet nearly at 10.33 ppm. Methylene protons of
–COCH N– gave singlet nearly at 3.35 ppm. The protons of
2
benzothiazole were seen approximately at 7.67 ppm as doublet
H-NMR (DMSO, ppm); 1.45 (t, 3H, –OCH CH , J ¼ 7.2 Hz),
2
3
2
.88 (t, 4H, piperazine H , J ¼ 4.4 Hz), 3.31 (t, 4H, piperazine
2
2
,6
H3,5, J ¼ 4.4 Hz), 3.38 (s, 2H, –COCH N–), 4.07–4.12 (m, 2H,
–
phenyl H ), 7.26–7.29 (m, 1H, benzothiazole H ), 7.58–7.61 (dd,
OCH CH ), 7.03–7.09 (m, 3H, phenyl H ), 7.51–7.56 (m, 1H,
2 3 4,5,6
3
7
1
H, benzothiazole H , J ¼ 1.6 Hz, J ¼ 8 Hz), 7.68 (d, 1H,
4 2
5
1
2
benzothiazol H , J ¼ 9.2 Hz), 10.36 (bs, 1H, –NHCOCH –). Table 1. Structural and physical properties of compounds 1a–f and 2a–d.
Anal Calcd for C H N O S (421.515): C, 62.39; H, 5.95; N,
2
2 23 5 2
ꢀ
Compounds
R
1
R
2
M.P. ( C) Yield % Log P*
1
6.54; S, 7.57. Found: C, 62.37; H, 5.96; N, 16.55; S, 7.58.
1
1b
a
Methyl 2-Methoxyphenyl
Methyl 2-Methoxyethyl
Methyl Cyclohexyl
121.9y
81.1
48
12
18
22
16
12
36.5
47.5
42.7
25.6
4.15
2.04
3.74
2.94
5.39
4.49
4.76
4.83
4.31
4.31
Biological activity
1
1
1
c
d
e
181.3
100.8
243.8
189.6
146.7
4300
4300
4300
The cytotoxic activity of the synthesized compounds 1a–f and
2
Methyl 4-Pyridyl
a–d was investigated on liver (HUH-7), breast (MCF-7) and
Methyl 3,4-Dichlorophenyl
Methyl 4-Chlorobenzyl
Ethoxy 4-Methylphenyl
Ethoxy 2-Chlorophenyl
Ethoxy 4-Cyanophenyl
Ethoxy 2-Cyanophenyl
colon (HCT-116) cancer cell lines, by means of sulphorhodamine 1f
B (SRB) assays in triplicate. As shown in Table 2, all tested 2a
2
2
2
b
c
d
compounds were screened with mean 50% growth inhibition
concentration (GI ) in micromolar concentration range. The
5
0
cytotoxicity results show that most of the substituted benzothia-
zole-piperazine derivatives are active against tested cancer cell
lines. Further investigation of compound 1d by Hoechst staining
*
Log P were calculated using ChemDraw Ultra V.9.0 (ChembridgeSoft
Corp., Cambridge, MA).
1
5
and FACS revealed that this compound causes apoptosis by cell yMelting point of compound 1a in the original reference is reported as
ꢀ
1
22–124 C.
cycle arrest at subG phase.
1
N
S
O
R1
N
O
Benzene:TEA
(20:1)
NH2
Cl
Cl
Cl
S
R1
N
H
R = Methyl (1)
1
R = Ethoxy (2)
1
R2
R2
R1
O
R1
O
N
N
K CO
3
N
N
2
Cl
N
HN
Acetone
S
N
S
N
H
H
R = Methyl (1a-f
)
1
R = Ethoxy (2a-d
)
1
Figure 1. Synthesis of compounds 1a–f and 2a–d.

DOI: 10.3109/14756366.2014.959513
Cytotoxic benzothiazoles
5
(
J ¼ 8 Hz) (H ) at 7.25–7.27 ppm as multiplets (H ) and 7.62 ppm methylene protons of Ar-OCH CH (2a–d) gave multiplets
4
5
2
3
as singlets (H ). Methyl protons of Ar-CH (1a–f) gave nearly at 4.06–4.12 ppm.
7
3
singlet nearly at 2.48 ppm. Methyl protons of Ar-OCH CH
(
In HCT-116 colorectal cancer cell line, the GI50 values for
2a–d) gave triplets nearly at 1.45 (J ¼ 7.2 Hz) ppm and benzothiazole-piperazine derivatives were in range of 4–25 mM.
Among series, 4-methylphenyl substituted compound 2a
2
3
(
GI : 4.5 mM) had the highest cytotoxicity. Cyclohexyl ring
5
0
(
1c, GI : 15.1 mM) lowered the activity of 6-methylbenzothiazole
5
0
Table 2. Cytotoxic activity data for compounds 1a–f and 2a–d.
Cancer cell line (GI50, mM)
derivatives. In addition, compounds 2c (GI : 6.3 mM) and 2d
5
0
(GI : 25.3 mM) showed that electron withdrawing cyano group
50
should be on para position rather than ortho position of phenyl
ring for higher cytotoxicity.
In MCF-7 breast cancer cell line, the GI50 values
2
R
2
2
Compounds HCT-116
MCF-7
R
HUH-7
R
1
1
1
1
1
1
2
2
2
2
5
a
b
c
d
e
f
a
b
c
d
-FU
4.8
7.7
15.1
7.9
8.0
5.0
4.5
5.8
1.0
0.8
0.9
0.9
0.9
0.8
1.0
0.9
0.8
0.9
15.1
no inh
26.9
9.2
16.6
no inh
61.4
20.4
29.8
19.8
3.51
1.0
–
0.8
0.9
0.9
–
0.9
0.9
0.9
0.9
5.7
10.7
7.9
3.1
9.7
7.0
9.4
6.5
7.4
1.0
0.8 for benzothiazole-piperazine derivatives were in range of
0.9
9
(
1
1
–60 mM. Among series, 4-pyridyl substituted compound 1d
0.9
1.0
0.9
1.0
0.9
0.8
GI : 9.2 mM) and 2-methoxyphenyl substituted compound
5
0
a (GI : 15.1 mM) had highest cytotoxicities. Also compounds
5
0
b (R : 2-methoxyethyl) and 1f (R : 4-chlorobenzyl) showed no
2
2
inhibition against MCF-7 cell line.
The activity data showed that all synthesized compounds have
6.3
25.3
30.66
6.7
18.67
0.9 good cytotoxic activity against HUH-7 liver cancer cell line in
range of 3–10 mM. The most active compound against this cell
a,b a
Figure 2. Fluorescence images of liver cancer (HUH7) cells . Cells were placed on cover slips and treated with GI50 concentration of compound 1d
b
or DMSO control for 72 h. Nuclear Hoechst 33258 stain was used to stain the cells.
100%
9
8
7
6
5
4
3
2
1
0%
0%
0%
0%
0%
0%
0%
0%
0%
G2/M
S
G1
subG1
0%
DMSO 1d
Figure 3. Cell cycle distribution analysis of compound 1d.

6
E. E. Gurdal et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
line was 1d (R : 4-pyridyl, GI : 3.1 mM). Compounds 1a References
2
50
(
R : 2-methoxyphenyl, GI : 5.7 mM) and 2b (R : 2-chlorophenyl,
2
50
2
1
.
Lion CJ, Matthews CS, Wells G, et al. Antitumour properties of
fluorinated
2
GI : 6.5 mM) were other highly active derivatives against HUH-7
5
0
benzothiazole-substituted hydroxycyclohexa-
,5-dienones (‘quinols’). Bioorg Med Chem Lett 2006;16:5005–8.
cell line.
We did not find any significant correlation between GI50
values and log P values of the compounds 1a–f and 2a–d
Table 1). Therefore, the difference in lipophilicity may not be a
2. Kashiyama E, Hutchinson I, Chua M, et al. Synthesis, metabolic
formation and biological properties of the C- and N-oxidation
products of antitumor 2-(4-aminophenyl)benzothiazoles. J Med
Chem 1999;42:4172–84.
(
significant factor for the difference in cytotoxicity of the series in
our study.
3
.
Kumbhare RM, Dadmal T, Kosurkar U, et al. Synthesis and
cytotoxic evaluation of thiourea and N-bis-benzothiazole deriva-
tives: a novel class of cytotoxic agents. Bioorg Med Chem Lett
Compound 1d (R ¼ methyl, R ¼ 4-pyridyl) was found highly
1
2
active against all tested cancer cell lines. Therefore, we carried
out Hoechst staining (Figure 2) and Fluorescence-Activated Cell
Sorting Analysis (FACS, Figure 3) for compound 1d to gain
insight into its mechanism of action.
To examine the type of cell death, induction of apoptosis
was investigated by Hoechst staining. Human liver cancer cell
line HUH-7 was treated with compound 1d. Hoechst staining
showed condensed nuclei that indicated apoptotic cells in
treated samples. Whereas in HUH-7 cell group treated with
DMSO, no apoptotic cells were present. This result indicated that
apoptosis is most likely to be the cell death type induced by
compound 1d.
2
012;22:453–5.
4. Solomon VR, Hu C, Lee H. Hybrid pharmacophore design and
synthesis of isatin–benzothiazole analogs for their anti-breast cancer
activity. Bioorg Med Chem Lett 2009;17:7585–92.
5
.
Saeed S, Rashid N, Jones PG, et al. Synthesis, characterization and
biological evaluation of some thiourea derivatives bearing ben-
zothiazole moiety as potential antimicrobial and anticancer agents.
Eur J Med Chem 2010;45:1323–31.
Leong CO, Gaskell M, Martin EA, et al. Antitumour 2-(4-
aminophenyl) benzothiazoles generate DNA adducts in sensitive
tumour cells in vitro and in vivo. Brit J Cancer 2003;88:470–7.
6
.
7. Boumendjel A, Nicolle E, Moraux T, et al. Piperazino-benzopyr-
anones and phenalkylaminobenzopyranones: potent inhibitors of
breast cancer resistance protein (ABCG2). J Med Chem 2005;48:
The effect of the compound 1d on the cell cycle was further
characterized by FACS analysis, using a propidium iodide stain.
This analysis revealed elevated subG phased cells indicating the
7
275–81.
8
.
Tuncbilek M, Guven EB, Onder T, Cetin Atalay R. Synthesis of
novel 6-(4-substituted piperazine-1-yl)-9-(b-D-ribofuranosyl) purine
derivatives, which lead to senescence-induced cell death in liver
cancer cells. J Med Chem 2012;55:3058–65.
1
subG cell cycle arrest compared to control cells treated with
1
DMSO. This result that indicates the presence of cells arrested at
subG phase supports the induction of apoptotic cell death in
those cells treated with compound 1d.
9. Yarim M, Koksal M, Durmaz I, Atalay R. Cancer cell cytotoxicities
of 1-(4-Substitutedbenzoyl)-4-(4-chlorobenzhydryl)piperazine
derivatives. Int J Mol Sci 2012;13:8071–85.
1
1
1
1
0. Gurdal EE, Yarim M, Durmaz I, Cetin-Atalay R. Cytotoxic activities
of some novel benzhydrylpiperazine derivatives. Drug Res 2013;63:
Conclusions
1
21–8.
In this study, ten benzothiazole-piperazine derivatives were
synthesized, purified and characterized by various analysis
methods. In vitro cytotoxic activities were screened against
colorectal (HCT-116), breast (MCF-7), and hepatocellular
(HUH-7) cancer cell lines by sulphorhodamine B assay. Most of
the compounds showed high cytotoxic activity against all tested
1. Gurdal EE, Durmaz I, Cetin-Atalay R, Yarim M. Synthesis and
cytotoxicity studies of novel benzhydrylpiperazine carboxamide and
thioamide derivatives. J Enzyme Inhib Med Chem 2014;29:205–14.
2. Al-Soud YA, Al-Sa’doni HH, Saeed B, et al. Synthesis and in vitro
antiproliferative activity of new benzothiazole derivatives. Arkivoc
2008;15:225–38.
cell lines. Hoechst staining and Fluorescence-activated cell 13. Al-Soud YA, Al-Sa’doni HH, Amajaour HA, et al. Synthesis,
characterization and anti-HIV and antitumor activities of new
coumarin derivatives. Z Naturforsch 2008;63b:83–9.
4. Kaplancikli ZA, Altintop MD, Turan-Zitouni G, et al. Synthesis,
antimicrobial activity and cytotoxicity of novel oxadiazole deriva-
tives. J Enzyme Inhib Med Chem 2012;27:51–7.
5. Abou El Ella DA, M u¨ ller FKU. A new scaffold for D3 dopaminergic
affinity containing arylpiperazine fragment: molecular modeling,
synthesis, in vitro and in vivo pharmacological evaluation.
Bull Pharm Sci 2007;30:259–73.
sorting analysis, were also performed with compound 1d to
understand the mechanism of cytotoxicity which revealed that this
compound causes apoptosis by cell cycle arrest at subG phase.
1
1
1
Declaration of interest
The authors have declared no conflict of interest. This work is supported
by The Scientific & Technological Research Council of Turkey
(
TUBITAK) (Project Number: 114S115).