Synthesis and identification of a new class of (S)-2,6-diamino-4,5,6,7- tetrahydrobenzo[d]thiazole derivatives as potent antileukemic agents

Article abstract of DOI:10.1007/s10637-009-9276-y

Benzothiazoles are multitarget agents with broad spectrum of biological activity. Among the antitumor agents discovered in recent years, the identification of various 2-(4-aminophenyl) benzothiazoles as potent and selective antitumor drugs against different cancer cell lines has stimulated remarkable interest. Some of the benzothiazoles are known to induce cell cycle arrest, activation of caspases and interaction with DNA molecule. Based on these interesting properties of benzothiazoles and to obtain new biologically active agents, a series of novel 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives 5(a-i) were synthesized and evaluated for their efficacy as antileukemic agents in human leukemia cells (K562 and Reh). The chemical structures of the synthesized compounds were confirmed by 1H NMR, LCMS and IR analysis. The cytotoxicity of these compounds were determined using trypan blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Results showed that, these compounds mediate a significant cytotoxic response to cancer cell lines tested. We found that the compounds having electron withdrawing groups at different positions of the phenyl ring of the thiourea moiety displayed significant cytotoxic effect with IC50 value less than 60 μM. To rationalize the role of electron withdrawing group in the induction of cytotoxicity, we have chosen molecule 5g (IC ₅₀ ～15 μM) which is having chloro substitution at ortho and para positions. Flow cytometric analysis of annexin V-FITC/propidium iodide (PI) double staining and DNA fragmentation suggest that 5g can induce apoptosis. Springer Science + Business Media, LLC 2009.

Full text of DOI:10.1007/s10637-009-9276-y

Invest New Drugs (2010) 28:454–465
DOI 10.1007/s10637-009-9276-y
PRECLINICAL STUDIES
Synthesis and identification of a new class
of (S)-2,6-diamino-4,5,6,7-tetrahydrobenzo[d]thiazole
derivatives as potent antileukemic agents
D. S. Prasanna & C. V. Kavitha & B. Raghava &
K. Vinaya & S. R. Ranganatha & Sathees C. Raghavan &
K. S. Rangappa
Received: 19 May 2009 /Accepted: 22 May 2009 /Published online: 10 June 2009
#
Springer Science + Business Media, LLC 2009
Summary Benzothiazoles are multitarget agents with
broad spectrum of biological activity. Among the antitumor
agents discovered in recent years, the identification of
various 2-(4-aminophenyl) benzothiazoles as potent and
selective antitumor drugs against different cancer cell lines
has stimulated remarkable interest. Some of the benzothia-
zoles are known to induce cell cycle arrest, activation of
caspases and interaction with DNA molecule. Based on
these interesting properties of benzothiazoles and to obtain
new biologically active agents, a series of novel 4,5,6,7-
tetrahydrobenzo[d]thiazole derivatives 5(a–i) were synthe-
sized and evaluated for their efficacy as antileukemic agents
in human leukemia cells (K562 and Reh). The chemical
structures of the synthesized compounds were confirmed by
1H NMR, LCMS and IR analysis. The cytotoxicity of these
compounds were determined using trypan blue exclusion,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) and lactate dehydrogenase (LDH) assays.
Results showed that, these compounds mediate a significant
cytotoxic response to cancer cell lines tested. We found that
the compounds having electron withdrawing groups at
different positions of the phenyl ring of the thiourea moiety
displayed significant cytotoxic effect with IC50 value less
than 60 μM. To rationalize the role of electron withdrawing
group in the induction of cytotoxicity, we have chosen
molecule 5g (IC50 ~15 μM) which is having chloro
substitution at ortho and para positions. Flow cytometric
analysis of annexin V-FITC/ propidium iodide (PI) double
staining and DNA fragmentation suggest that 5g can induce
apoptosis.
.
.
Keywords Tetrahydrobenzo[d]thiazole Cytotoxicity
.
.
.
Apoptosis DNA fragmentation Cancer therapeutics
Leukemia
Introduction
Cancer chemotherapy targeting tumor progression repre-
sents one of the most relevant challenges of chemists and
oncologist. Leukemia is one of the major types of cancer
affecting a significant segment of the population, especially
children. The American Cancer Society (ACS) estimated
that 44,790 new cases of leukemia would be diagnosed in
the United States in 2009, whereas about 21,870 adults and
children would die of leukemia during 2009 [1]. In order to
gain new insight into the complexity of the disease, robust
screening methods for evaluating different natural or
synthetic drugs have been carried out by the scientific
community. Although the incidence rate of this disease
remains almost unchanged, some success has been reported
in its treatment. But even if the success of clinical trials in
identifying new agents and treatment modalities has been
significant, current treatments have many limitations related
to their side effects and the development of acquired drug
resistance [2].
:
:
:
:
D. S. Prasanna B. Raghava K. Vinaya S. R. Ranganatha
K. S. Rangappa (*)
Department of Studies in Chemistry, University of Mysore,
Manasagangotri,
Mysore 570 006, India
e-mail: rangappaks@chemistry.uni-mysore.ac.in
e-mail: rangappaks@gmail.com
:
C. V. Kavitha S. C. Raghavan
Department of Biochemistry, Indian Institute of Science,
Bangalore 560 012, India

Invest New Drugs (2010) 28:454–465
455
Thiazole is an important heterocyclic ring widely used in
anticancer drug research area. The interest in the antineo-
plastic activity of thiazoles increased due to the presence of
thiazole moiety in natural chemotherapeutic agents like
tiazofurin [3, 4], distamycin [5], bleomycin [6, 7], netropsin
and thianetropsin [8]. Benzothiazole derivatives have been
studied extensively followed by the use of riluzole (6-
trifluoromethoxy-2-benzothiazolamine) [9], which was
found to interfere with glutamate neurotransmission. Some
of the 2-aminobenzothiazole derivatives possess cytotoxicity
on cancer cells which is comparable to that of cisplatin [10]. In
this respect, the benzothiazole constitutes an important
scaffold of drugs, possessing several pharmacological func-
tions. The interesting anticancer properties of these com-
pounds take the initiative of synthesis and anticancer studies
of many new benzothiazole derivatives [11-15]. In addition,
these derivatives are used as neurotransmission blocker [16-
18], calmodulin (CaM) antagonists [19] and neuroprotective
agent [20, 21]. Cantharidin containing benzothiazoles pos-
sess antitumor properties in hepatocellular carcinoma, breast
cancer, acute myelogenous leukemia and non-small cell lung
carcinoma. It is reported that compounds having substituted
amine groups at position 2 and 6 of benzothiazole moiety
show good antitumor activity [22]. Tetrahydrobenzothiazoles
which are similar in structure to benzothiazoles also claim
various biological activities [23-29]. Pramipexol, a 2,6-
diamino-4,5,6,7-tetrahydrobenzo[d]thiazole derivative with
(S) configuration at position 6 is being used as a drug for
schizophrenia and Parkinson’s disease.
Based on the results reported for the benzothiazole
derivatives, biological importance of the 4,5,6,7-tetrahy-
drobenzo[d]thiazole moiety with substitution at position 6
and also from our own experience in the synthesis and
evaluation of anticancer property of substituted heterocyclic
compounds [30-37], we designed and synthesised a new
series of 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives
with arylthiourea substitution at position 6. To the best of
our knowledge, all 6-amine substituted derivatives of (S)-
4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine are novel,
and here we report the synthesis and cytotoxic activities
of novel (S)-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-di-
amine derivatives on human leukemia cells.
phy was performed using Merck 7734 silica gel (60–
120 mesh) and Merck made TLC plates.
General procedure for the synthesis 1-((S)-2-amino-
4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-3-(substituted phe-
nyl)thiourea derivatives 5(a–i)
To the solution of intermediate compound 4 (1 eq) in
dichloromethane, triethylamine (3 eq) was added and
cooled to 0 °C, respective aryl isothiocyanate (1 eq) were
added at cold condition and stirred at room temperature for
6–7 h. Progress of the reaction was monitored by TLC.
Upon completion, the reaction mixture was concentrated,
water was added and extracted thrice using ethyl acetate.
The combined ethyl acetate layer was washed with brine
solution and dried over anhydrous sodium sulphate. Ethyl
acetate was evaporated under reduced pressure and the
crude product obtained was purified by silica gel (60–120
mesh) column. The compounds 5(a–i) were eluted at 70–
80% ethyl acetate in hexane.
Synthesis of 1-((S)-2-amino-4,5,6,7-tetrahydrobenzo[d]
thiazol-6-yl)-3-(4-methoxy phenyl)thiourea (5a)
The product obtained (0.33 g, 83%) by the reaction of 4
(0.2 g, 1.2 mmol) with 4-methoxy phenyl isothiocyanate
(0.195 g, 1.2 mmol) and triethylamine (0.36 g, 3.6 mmol)
in dichloromethane (3 ml) using the general experimental
1
procedure described was white solid. H NMR (DMSO-d6,
400 MHz) δ: 8.20–8.16 (dd, 1H, -NH), 8.06 (s, 1H, -NH),
7.31–7.29 (d, 2H, Ar-H), 6.85 (d, 2H, Ar-H), 6.70 (s, 2H,
-NH2), 4.04–4.01 (m, 1H, -CH-), 3.70 (s, 3H, -OCH3),
2.84–2.79 (dd, 1H, -CH), 2.51–2.46 (m, 2H, -CH2), 2.41–
2.36 (dd, 1H, -CH), 1.89–1.82 (m, 1H, -CH2), 1.79–1.72
(m, 1H, -CH2). MS (ESI + ion): m/z = 335.57. IR (KBr, cm−1):
3411, 3207, 1371, 1106.
Synthesis of 1-((S)-2-amino-4,5,6,7-tetrahydrobenzo[d]
thiazol-6-yl)-3-(4-fluoro phenyl)thiourea (5b)
The product obtained (0.32 g, 84%) by the reaction of 4
(0.2 g, 1.2 mmol) with 4-fluoro phenyl isothiocyanate
(0.181 g, 1.2 mmol) and triethylamine (0.36 g, 3.6 mmol)
in dichloromethane (3 ml) using the general experimental
1
procedure described was white solid. H NMR (DMSO-d6,
400 MHz) δ: 8.16–8.12 (dd, 1H, -NH), 8.01 (s, 1H, -NH),
7.41–7.40 (d, 2H, Ar-H), 7.10–7.12 (d, 2H, Ar-H), 6.65
(s, 2H, -NH2), 4.0–3.95 (m, 1H, -CH-), 2.77–2.73 (dd, 1H,
-CH), 2.45–2.39 (m, 2H, -CH2), 2.29–2.23 (dd, 1H, -CH),
1.80–1.72 (m, 1H, -CH2), 1.77–1.71 (m, 1H, -CH2). MS
(ESI + ion): m/z = 323.52. IR (KBr, cm−1): 3407, 3224,
1369, 1118.
Experimental
IR spectra were recorded using a Jasco FTIR-2008 series.
1H NMR spectra were recorded on Shimadzu AMX
400-Bruker, 400 MHz spectrometer using DMSO as a
solvent and TMS as internal standard (chemical shift in
δ ppm). Spin multiplets are given as s (singlet), d (doublet),
t (triplet) and m (multiplet). Mass and purity were recorded
on a LC–MSD-Trap-XCT. Silica gel column chromatogra-
Synthesis of 1-((S)-2-amino-4,5,6,7-tetrahydrobenzo[d]
thiazol-6-yl)-3-(3-chloro phenyl)thiourea (5c)
The product obtained (0.325 g, 81%) by the reaction of
4 (0.2 g, 1.2 mmol) with 3-chloro phenyl isothiocyanate

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Invest New Drugs (2010) 28:454–465
(0.2 g, 1.2 mmol) and triethylamine (0.36 g, 3.6 mmol) in
dichloromethane (3 ml) using the general experimental
procedure described was white solid. H NMR (DMSO-d6,
400 MHz) δ: 8.26–8.23 (dd, 1H, -NH), 8.10 (s, 1H, -NH),
7.41 (s, 1H, Ar-H), 7.10–7.12 (m, 3H, Ar-H), 6.77 (s, 2H,
-NH2), 3.94–3.91 (m, 1H, -CH-), 2.94–2.89 (dd, 1H, -CH),
2.56–2.49 (m, 2H, -CH2), 2.4–2.37 (dd, 1H, -CH), 1.79–
1.73 (m, 1H, -CH2), 1.69–1.62 (m, 1H, -CH2). MS (ESI +
ion): m/z = 339.94. IR (KBr, cm−1): 3423, 3233, 1376,
1096.
8.02 (s, 1H, Ar-H), 7.35–7.21 (m, 1H, Ar-H), 6.95–6.91 (d,
2H, Ar-H), 6.70 (s, 2H, -NH2), 4.04–4.01 (m, 1H, -CH-),
3.70 (s, 3H, -OCH3), 2.84–2.79 (dd, 1H, -CH), 2.51–2.46
(m, 2H, -CH2), 2.41–2.36 (dd, 1H, -CH), 1.89–1.82 (m, 1H,
-CH2), 1.79–1.72 (m, 1H, -CH2). MS (ESI + ion): m/z =
335.40. IR (KBr, cm−1): 3442, 3237, 1379, 1132.
1
Synthesis of 1-((S)-2-amino-4,5,6,7-tetrahydrobenzo[d]
thiazol-6-yl)-3-(2-fluoro phenyl)thiourea (5e)
The product obtained (0.32 g, 84%) by the reaction of 4
(0.2 g, 1.2 mmol) with 2-fluorophenyl isothiocyanate
(0.181 g, 1.2 mmol) and triethylamine (0.36 g, 3.6 mmol)
in dichloromethane (3 ml) using the general experimental
Synthesis of 1-((S)-2-amino-4,5,6,7-tetrahydrobenzo[d]
thiazol-6-yl)-3-(3-methoxy phenyl)thiourea (5d)
1
The product obtained (0.33 g, 83%) by the reaction of 4
(0.2 g, 1.2 mmol) with 3-methoxyphenyl isothiocyanate
(0.195 g, 1.2 mmol) and triethylamine (0.36 g, 3.6 mmol)
in dichloromethane (3 ml) using the general experimental
procedure described was white solid. H NMR (DMSO-d6,
400 MHz) δ: 8.20–8.18 (dd, 1H, -NH), 8.07 (s, 1H, -NH),
7.40–7.37 (d, 1H, Ar-H), 7.20–7.16 (m, 2H, Ar-H), 6.90–
6.85 (m, 1H, Ar-H), 6.75 (s, 2H, -NH2), 4.09–4.05 (m, 1H,
-CH-), 2.89–2.74 (dd, 1H, -CH), 2.55–2.49 (m, 2H, -CH2),
2.40–2.35 (dd, 1H, -CH), 1.90–1.84 (m, 1H, -CH2), 1.77–
1
procedure described was white solid. H NMR (DMSO-d6,
400 MHz) δ: 8.18–8.16 (dd, 1H, -NH), 8.06 (s, 1H, -NH),
Fig. 1 Dose- and time-
dependent effect of 4,5,6,7-tet-
rahydrobenzo[d]thiazole
derivatives 5(a–i) on K562 (a)
and Reh (b) cell survival. Ap-
proximately, 0.75×105 cells/ml
were cultured and treated with 5
(a–i) at a concentration of 10,
100 and 250 μM. Cell viability
was determined by trypan blue
exclusion assay. Viable cells
were counted everyday till the
control cells reached stationary
phase and the data was repre-
sented as a graph

Invest New Drugs (2010) 28:454–465
457
1.70 (m, 1H, -CH2). MS (ESI + ion): m/z = 323.61. IR
(KBr, cm−1): 3404, 3212 1381, 1116.
Synthesis of 1-((S)-2-amino-4,5,6,7-tetrahydrobenzo[d]
thiazol-6-yl)-3-(2,4-dichloro phenyl)thiourea (5g)
Synthesis of 1-((S)-2-amino-4,5,6,7-tetrahydrobenzo[d]
thiazol-6-yl)-3-(2-chloro phenyl)thiourea (5f)
The product obtained (0.345 g, 78%) by the reaction of 4
(0.2 g, 1.2 mmol) with 2,4-dichlorophenyl isothiocyanate
(0.241 g, 1.2 mmol) and triethylamine (0.36 g, 3.6 mmol)
in dichloromethane (3 ml) using the general experimental
The product obtained (0.325 g, 81%) by the reaction of 4
(0.2 g, 1.2 mmol) with 2-chlorophenyl isothiocyanate
(0.2 g, 1.2 mmol) and triethylamine (0.36 g, 3.6 mmol) in
dichloromethane (3 ml) using the general experimental
1
procedure described was white solid. H NMR (DMSO-d6,
400 MHz) δ: 8.18–8.16 (dd, 1H, -NH), 8.07 (s, 1H, -NH),
7.38 (s, 1H, Ar-H), 7.23–7.19 (m, 2H, Ar-H), 6.70 (s, 2H,
-NH2), 4.04–4.01 (m, 1H, -CH-), 2.84–2.79 (dd, 1H, -CH),
2.51–2.46 (m, 2H, -CH2), 2.41–2.36 (dd, 1H, -CH), 1.89–
1.82 (m, 1H, -CH2), 1.79–1.72 (m, 1H, -CH2). MS (ESI +
ion): m/z = 374.50. IR (KBr, cm−1): 3452, 3246, 1392,
1133.
1
procedure described was white solid. H NMR (DMSO-d6,
400 MHz) δ: 8.18–8.16 (dd, 1H, -NH), 8.06 (s, 1H, -NH),
7.38–7.35 (d, 1H, Ar-H), 7.23–7.19 (m, 2H, Ar-H), 6.94–
6.89 (m, 1H, Ar-H), 6.70 (s, 2H, -NH2), 4.04–4.01 (m, 1H,
-CH-), 2.84–2.79 (dd, 1H, -CH), 2.51–2.46 (m, 2H, -CH2),
2.41–2.36 (dd, 1H, -CH), 1.89–1.82 (m, 1H, -CH2), 1.79–
1.72 (m, 1H, -CH2). MS (ESI + ion): m/z = 339.77. IR
(KBr, cm−1): 3397, 3186, 1361, 1092.
Synthesis of 1-((S)-2-amino-4,5,6,7-tetrahydrobenzo[d]
thiazol-6-yl)-3-(4-chloro phenyl)thiourea (5h)
Fig. 1 (continued)

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Invest New Drugs (2010) 28:454–465
The product obtained (0.325 g, 81%) by the reaction of 4
1.2 mmol) and triethylamine (0.36 g, 3.6 mmol) in
dichloromethane (3 ml) using the general experimental
procedure described was white solid. H NMR (DMSO-d6,
400 MHz) δ: 8.22–8.19 (dd, 1H, -NH), 8.07 (s, 1H, -NH),
7.30 (m, 5H, Ar-H), 6.70 (s, 2H, -NH2), 4.04–4.01 (m, 1H,
-CH-), 2.84–2.79 (dd, 1H, -CH), 2.51–2.46 (m, 2H, -CH2),
2.41–2.36 (dd, 1H, -CH), 1.89–1.82 (m, 1H, -CH2), 1.79–
1.72 (m, 1H, -CH2). MS (ESI + ion): m/z = 305.63. IR
(KBr, cm−1): 3403, 3211, 1379, 1114.
(0.2 g, 1.2 mmol) with 4-chlorophenyl isothiocyanate
(0.2 g, 1.2 mmol) and triethylamine (0.36 g, 3.6 mmol) in
dichloromethane (3 ml) using the general experimental
1
1
procedure described was white solid. H NMR (DMSO-d6,
400 MHz) δ: 8.20–8.16 (dd, 1H, -NH), 8.06 (s, 1H, -NH),
7.31–7.29 (d, 2H, Ar-H), 6.86–6.84 (d, 2H, Ar-H), 6.70 (s,
2H, -NH2), 4.04–4.01 (m, 1H, -CH-), 2.84–2.79 (dd, 1H,
-CH), 2.51–2.46 (m, 2H, -CH2), 2.41–2.36 (dd, 1H, -CH),
1.89–1.82 (m, 1H, -CH2), 1.79–1.72 (m, 1H, -CH2). MS
(ESI + ion): m/z = 339.68. IR (KBr, cm−1): 3433, 3225,
1377, 1123.
Biology
Synthesis of 1-((S)-2-amino-4,5,6,7-tetrahydrobenzo[d]
thiazol-6-yl)-3-phenyl) thiourea (5i)
Human leukemia cells K562 (chronic myelogenous leuke-
mia) and Reh (B-cell leukemia) were selected for the
purpose of preliminary anti-cancer screening of newly
synthesized compounds. The cytotoxicity was assessed by
The product obtained (0.31 g, 83%) by the reaction of 4
(0.2 g, 1.2 mmol) with phenyl isothiocyanate (0.160 g,
Fig. 2 Determination of cell proliferation by MTT assay. After 48 h
and 72 h of exposure, K562 cells (a) or Reh cells (b) treated with 5(a–
i) at different concentrations (10, 100 and 250 μM) were incubated
with MTT (0.5 mg/ml) in duplicates and absorbance was measured at
570 nm. Results are presented as percentage of cell proliferation. Error
bars are represented in the figure

Invest New Drugs (2010) 28:454–465
459
employing trypan blue dye exclusion assay, MTT assay,
LDH assay and FACS analysis. For this, cells growing in
log phase were treated with different concentrations (10,
100 and 250 μM) of 4,5,6,7-tetrahydrobenzo[d]thiazole
derivatives 5(a–i). Besides, DNA fragmentation assay was
performed to assess whether the potent molecule 5g is able
to damage DNA which is an indicator of apoptosis.
Annexin V-FITC/ propidium iodide (PI) double staining
assay was done to quantify the apoptotic cells formed upon
treatment with 5g. All the assays were carried out in
duplicate in two independent experiments.
USA. Cells were grown in RPMI 1640 supplemented with
10% heat-inactivated fetal bovine serum, 100 U/ml of
penicillin, and 100 μg of streptomycin/ml and incubated at
37°C in a humidified atmosphere containing 5% CO2.
Trypan blue exclusion assay
Cell viability was monitored by the trypan blue exclusion
assay. Cells (K562 or Reh) growing in exponential phase
were seeded at a density of 0.75×105 cells/ml in a 6-well
tissue culture plate for 24 h prior to the experiments and
cells were exposed to 10, 100 and 250 μM concentrations
of 5(a–i). Cells were collected at intervals of 24 h and
resuspended in 0.4% trypan blue and further incubated for
5 min after which the number of viable cells was estimated
in a haemocytometer chamber. The results are presented as
graphs in Fig. 1.
Cell lines and culture
Human cell line, K562 was purchased from National Center
for Cell Science, Pune, India and Reh cell line was a kind gift
from Prof. Michael Lieber, University of Southern California,
Fig. 2 (continued)

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Invest New Drugs (2010) 28:454–465
Table 1 IC50 values of 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives
5(a–i) as determined based on MTT assay
Compound
IC50
K562 (in µM)
Reh (in µM)
5a
5b
5c
5d
5e
5f
65 5.5
42 3.5
20 4.0
95 5.1
30 3.2
45 3.5
11 3.5
30 6.0
50 7.4
125 7.5
45 3.5
30 2.5
90 6.2
34 5.5
55 4.3
20 3.6
30 3.5
185 7.5
Fig. 3 Time- and dose-dependent LDH release in K562 cells treated
with 5g. K562 cells were incubated for 24 h and 48 h with different
concentrations of 5g. Release of LDH in the medium was measured at
490 nm. Results are presented as percentage of LDH release
5g
5h
5i
yellow tetrazolium salt to violet formazan. Exponentially
growing K562 or Reh cells (1×104 cells /well) were plated
in duplicates and incubated with 10, 100 and 250 μM of 5
(a–i). Cells were harvested after 48 and 72 h of treatment
MTT assay
Cell proliferation was further assessed by MTT assay,
which is based on the ability of viable cells to metabolize a
N
S
O
N
O
O
NH2
(ii)
NH2
(i)
N
H
S
H2N
N
H
2
1
3
N
N
S
(iii)
NH2
(iv)
NH2
R
S
S
N
H
N
H
H2N
4
5(a-i)
F
R=
O
a =
e =
Cl
F
b =
c =
d =
f =
Cl
Cl
g =
h =
Cl
O
Cl
i=
Scheme 1 Reagents and conditions: i) (a) Br2, AcOH, 60 °C, 1 h; (b) thiourea, 1 h; ii) 47% aq. HBr; reflux, 15 h; iii) (a). L-(+)-tartaric acid,
75 °C; (b). KOH; iv) aryl isothiocyanates, triethyl amine, dichloromethane, rt, 6–7 h

Invest New Drugs (2010) 28:454–465
461
and incubated with MTT (0.5 mg/ml) as described earlier
[31]. The percentage cell proliferation was calculated and
IC50 values (concentration of compound causing 50%
inhibition of cell growth) were estimated after 72 h of
5(a–i) treatment. The histograms are plotted as shown in
Fig. 2.
LDH release assay
The cytotoxicity of 5g was further assessed by LDH release
assay, which is an indicator of membrane integrity and
hence cell injury. LDH assay was performed as per standard
protocol to estimate the release of LDH in the culture media
Fig. 4 Detection of apoptosis induced by 5g in K562 cells by flow
cytometry using Annexin V-FITC/PI double staining. K562 cells
(0.75×105 cells/ml) were incubated with 5g (10, 50 and 100 μM) for
48 h and processed for annexin V-FITC/PI double staining. The cells
were then quantitatively monitored. In each panel, lower left quadrant
shows cells which are negative for both annexin V-FITC and PI, lower
right shows annexin V positive cells which are in the early stage of
apoptosis, upper left shows only PI positive cells which are dead, and
upper right shows both annexin V and PI positive, which are in the
stage of late apoptosis or necrosis. Panels shown are annexin V-FITC/
PI incubated with K562 cells, which are treated with DMSO (a),
10 μM (b), 50 μM (c) or 100 μM (d). e is the histogram showing
comparison of early apoptotic and late apoptotic cells at different
doses of 5g

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Invest New Drugs (2010) 28:454–465
upon treatment with 10, 50 and 100 μM of 5g in K562
cells. After 24 and 48 h of compound exposure, the LDH
release was measured [31]. The percentage of LDH release
was calculated and plotted as shown in Fig. 3.
2,6-diamino-4,5,6,7-tetrahydrobenzo[d]thiazole 3. Optical
resolution of 3 with L-(+)-tartaric acid afforded the (S)
isomer 4. This (S) isomer when treated with different aryl
substituted isothiocyanates gave compounds 5(a–i).
Annexin V-FITC flow cytometric analysis
Cytotoxic effect of 4,5,6,7-tetrahydrobenzo[d]thiazole
derivatives 5(a–i) on human leukemia cells
The translocation of phosphatidyl serine from the inner to
the outer leaflet of the plasma membrane is considered as
one of the earliest events in apoptosis and can be measured
by annexin V-FITC/PI double staining. In brief, after 48 h
of treatment with 10, 50 and 100 μM of 5g, K562 cells
were washed in PBS and resuspended in binding buffer
(HEPES–NaOH 10 mM pH 7.4, 144 mM NaCl and 25 mM
CaCl2). Annexin V-FITC (0.2 mg/ml) and PI (0.05 mg/ml)
were added and incubated in dark for 20 min. Cells were
then subjected to FACS (FACScan, BD Biosciences, USA)
analysis. At least 10,000 events were recorded and
represented as dot plots.
The antiproliferative activity of all the newly synthesized 5
(a–i) compounds has been evaluated in vitro against human
leukemia cells (K562 and Reh). The effective concentra-
tions of 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives re-
quired to inhibit K562 or Reh cell growth and survival were
determined first by carrying out dose response experiments
using trypan blue dye exclusion and MTT assays. The cells
were counted at intervals of 24 h till the control cells
attained stationary phase. As shown in Fig. 1a & 2a (K562
cells) and Fig. 1b & 2b (Reh cells), the exposure of these
derivatives for different durations decreased the number of
live cells in a time and concentration dependent manner. It
was observed that, the effect was improved linearly when
incubation time was prolonged. Among the compounds 5
(a–i), compounds 5b, 5c, 5e, 5f, 5g and 5h showed strong
inhibition against K562 cells with IC50 values of 42 μM,
20 μM, 30 μM, 45 μM, 11 μM and 30 μM, respectively
DNA fragmentation assay
To determine the apoptotic response induced by 5g, we
analyzed apoptotic DNA fragmentation which is considered
as hallmark of apoptosis using agarose gel electrophoresis.
Briefly, K562 cells were cultured in absence or presence of
5g at 10, 50 and 100 μM for 72 h. Cells were harvested and
genomic DNA was extracted using standard protocol. The
DNA samples were run on 1% agarose gel and visualized
by ethidium bromide staining and photographed.
D
10
50
100
M
(µM)
10.0 Kb
5.0
Results and discussion
3.0
The chemistry of 4,5,6,7-tetrahydrobenzo[d]thiazole moiety
has long attracted the interest of synthetic chemists due to
its wide range of biological activity. Benzothiazole deriv-
atives have been shown to be useful for treatment of
various diseases including neurodegenerative disorders,
local brain ischemia, Huntingtons’s disease and cancer
[38]. With the aim of obtaining new anticancer agents, a
series of 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives 5
(a–i) were synthesised. Synthesis of 5(a–i) was done as
outlined in Scheme 1. The intermediate (S)-2,6-diamino-
4,5,6,7-tetrahydrobenzo[d]thiazole was synthesized using
the earlier reported method [39]. The bromination of 4-
acetamidocyclohexanone 1 with bromine in hot acetic acid
produced 2-bromo-4-acetamidocyclohexanone and cyclisa-
tion of bromo compound with thiourea gave 6-acetamido-2-
amino-4,5,6,7-tetrahydrobenzo[d]thiazole 2, which on
hydrolysis with refluxing aqueous HBr yielded racemic
2.0
0.5
0.3
Fig. 5 Detection of DNA damage induced by 5g in K562 cells. The
chromosomal DNA was extracted from K562 cells treated with
different concentrations of 5g. The purified DNA was then resolved
on a 1% agarose gel at 30 V for 6 h. In this panel, D represents
DMSO; M represents Marker

Invest New Drugs (2010) 28:454–465
463
and for Reh cells the corresponding IC50 values were
45 μM, 30 μM, 34 μM, 55 μM, 20 μM and 30 μM
respectively (Table 1). The other compounds like 5a, 5d
and 5i exhibited moderate inhibition. The inhibition shown
by 5b, 5c, 5e, 5f, 5g, and 5h could be attributed to the
presence of electron withdrawing halogen group on the
phenyl ring. However, it is noteworthy to mention that (S)-
6-(substituted arylthiourea)-2-amino-4,5,6,7-tetrahydro-
benzo[d]thiazole molecules with chloro as substituent on
the phenyl ring showed significant antiproliferation against
leukemia cells. Similar results were shown in our previous
reports where halogen substituted analogs of different
heterocycles possessed potent antitumor activity [30,
31]. To confirm the above statement, molecule 5g with
ortho, para dichloro substitution was selected for further
studies.
LDH release assay was performed to test the cell damage
induced by 5g. For this, K562 cells were cultured with 10,
50 and 100 μM of 5g for 24 and 48 h, the release of LDH
(an indicator of membrane integrity) was measured. Results
showed a dose- and time-dependent increase in LDH
release (Fig. 3), which further confirmed our results.
From the SAR studies, it reveals that the substituents on
phenyl ring of arylthiourea play a key role in the
antiproliferative activity. Further, the number of halogen
atoms also plays a role in the determination of cytotoxicity
of these molecules. The investigations of these structural
modifications and preliminary SAR would help to further
design and develop more potent compounds.
DNA fragmentation and strand breakage induced by 5g
in K562 cells
To elucidate the mode of action of the investigated
compounds DNA fragmentation was performed, which is
a characteristic feature of the programmed cell death or
apoptosis [40, 41]. K562 cells treated with different
concentrations of 5g were harvested after 72 h, the
chromosomal DNA was extracted and used for agarose
gel electrophoresis. The result showed fragmentation of
DNA leading to a smear in the lanes in which cells were
treated with 5g (Fig. 5). The observed smear was the result
of DNA breakage at multiple positions across the chromo-
somal DNA. The intensity of smear was maximum at 50
and 100 μM. These results in conjunction with annexin V-
FITC/PI staining further suggested that 5g induces frag-
mentation of chromosomal DNA leading to apoptosis.
Conclusion
In this study, we have described the synthesis of 4,5,6,7-
tetrahydrobenzo[d]thiazole derivatives 5(a–i), which
showed strong and moderate growth inhibitory activity
against human leukemic cell lines, K562 and Reh. The
derivatives 5b, 5c, 5e, 5f, 5g and 5h with chloro and fluoro
substituents at different positions showed significant inhib-
itory effects whereas 5a, 5d and 5i showed moderate
inhibitory activity. The best results were obtained for
compound 5g having a dichloro group on the phenyl ring
of arylthiourea at the 2nd and 4th position. Further
investigations to understand the mechanism by which these
molecules induce apoptosis is under progress in our
laboratory.
Treatment of 5g in K562 cells led to translocation
of phosphatidyl serine to the outer membrane
The annexin V has a strong affinity for phosphatidyl
serine which is externalized in the membranes of apoptotic
cells. Data from cytotoxic studies showed that 5g is more
potent and able to induce apoptosis. Our interest was to
quantify the different types of apoptotic cells induced by
5g. In order to detect and quantify the apoptosis induced
by 5g, we used annexin V-FITC/PI double staining. K562
cells harvested after 48 h of treatment with 5g (10, 50 and
100 μM) were used for double staining followed by
Fluorescence-activated cell sorting (FACS) analysis.
Results showed that apoptosis induced by 5g treatment
was remarkable at 50 μM compared to 10 μM and the
effect was retained even at 100 μM, which is 5 fold higher
than dimethyl sulfoxide (DMSO) treated cells (Fig. 4).
These results suggest the total disruption of cell membrane
and further damage to the chromosomal DNA upon
treatment with 5g. This was further confirmed by DNA
fragmentation assay.
Acknowledgements We are grateful to University Grants Commis-
sion (UGC), Government of India for financial support to KSR under
the projects vide no. F. 31-143/2005(SR), UGC-SAP (Phase I) vide
No. F. 540/10/2004–05 (SAP I) Programme. SCR acknowledges
support from Lady Tata Memorial Trust international award for
leukemia research (London). DSP and KV are grateful to Council of
Scientific and Industrial Research, New Delhi for financial support
under CSIR-SRF order No. 09/119(0173)2K8 EMR-I and order No.
09/119(0172)2K8 EMR-I, respectively.
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