Synthesis and antileukemic activity of 1-((s)-2-amino-4,5,6,7- tetrahydrobenzo[d]thiazol-6-yl)-3-(substituted phenyl)urea derivatives

Article abstract of DOI:10.1246/bcsj.20090318

Heterocyclic urea derivatives play an important role as anticancer agents because of their good inhibitory activity against receptor tyrosine kinases (RTKs), raf kinases, protein tyrosine kinases (PTKs), and NADH oxidase, which play critical roles in many aspects of tumorigenesis. Benzothiazole moiety constitutes an important scaffold of drugs, possessing several pharmacological functions, mainly the anticancer activity. Based on these interesting properties of benzothiazolesand urea moiety to obtain new biologically active agents, we synthesized a series of novel 1-((S)-2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol- 6-yl)-3-(substituted phenyl)urea derivatives and evaluated for their efficacy as antileukemic agents against two human leukemic cell lines (K562 and Reh). These compounds showed good and moderate cytotoxic effect tocancer cell lines tested. Compounds with electron-withdrawing chloro and fluoro substituents on phenyl ring showed good activity and compounds with electron-donating methoxy group showed moderate activity. Compound with electronwithdrawing dichloro substitution on phenyl ring of aryl urea showed good activity. Further, lactate dehydrogenase (LDH) assay, flow cytometric analysis of annexin V-FITC/propidium iodide (PI) double staining and DNA fragmentation studies showed that compound with dichloro substitution on phenyl ring of aryl urea can induce apoptosis.

Full text of DOI:10.1246/bcsj.20090318

© 2010 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 83, No. 6, 689–697 (2010)
689
Synthesis and Antileukemic Activity of 1-((S)-2-Amino-4,5,6,7-
tetrahydrobenzo[d]thiazol-6-yl)-3-(substituted phenyl)urea Derivatives
Doddakunche S. Prasanna,1 Chandagirikoppal V. Kavitha,2 Kambappa Vinaya,1
Somasagara R. Ranganatha,1 Byregowda Raghava,1 Yelekere C. Sunil Kumar,1
Sathees C. Raghavan,2 and Kanchugarakoppal S. Rangappa*1
1Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore-570 006, India
2Department of Biochemistry, Indian Institute of Science, Bangalore-560 012, India
Received December 1, 2009; E-mail: rangappaks@gmail.com
Heterocyclic urea derivatives play an important role as anticancer agents because of their good inhibitory activity
against receptor tyrosine kinases (RTKs), raf kinases, protein tyrosine kinases (PTKs), and NADH oxidase, which play
critical roles in many aspects of tumorigenesis. Benzothiazole moiety constitutes an important scaffold of drugs, possessing
several pharmacological functions, mainly the anticancer activity. Based on these interesting properties of benzothiazoles
and urea moiety to obtain new biologically active agents, we synthesized a series of novel 1-((S)-2-amino-4,5,6,7-tetra-
hydrobenzo[d]thiazol-6-yl)-3-(substituted phenyl)urea derivatives and evaluated for their efficacy as antileukemic agents
against two human leukemic cell lines (K562 and Reh). These compounds showed good and moderate cytotoxic effect to
cancer cell lines tested. Compounds with electron-withdrawing chloro and fluoro substituents on phenyl ring showed good
activity and compounds with electron-donating methoxy group showed moderate activity. Compound with electron-
withdrawing dichloro substitution on phenyl ring of aryl urea showed good activity. Further, lactate dehydrogenase (LDH)
assay, flow cytometric analysis of annexin V-FITC/propidium iodide (PI) double staining and DNA fragmentation studies
showed that compound with dichloro substitution on phenyl ring of aryl urea can induce apoptosis.
Cancer is a group of disease having the hallmark of
uncontrolled cell proliferation leading to total collapse of
cellular homeostasis. Among different cancers, leukemia is one
of the major causes of cancer related deaths. The leukemias
account for the largest number of cases of childhood cancer and
are the primary cause of cancer related mortality of children.
Although tremendous improvement has occurred in the area of
cancer diagnostics and therapeutics in the past one decade, still
long way to go to cope with this deadly disease, especially
when the average life span of human being is increasing day by
day. The need of more targeted and relatively non toxic
chemotherapeutics is quite urgent.
of AhR pathway in sensitive tumor cells leads to induction of
the microsomal enzyme CYP1A1. This converts the drug into
highly reactive metabolites that form adducts with DNA
causing cell death.11 Further, the combinations of urea and
thiourea derivatives with benzothiazoles have produced DNA
topoisomerase12,13 or HIV reverse transcriptase inhibitors.14,15
In continuation of our research on heterocyclic antileukemic
agents16-19 and with the goal of discovering new antileukemic
agents, we synthesized a series of 1-((S)-2-amino-4,5,6,7-
tetrahydrobenzo[d]thiazol-6-yl)-3-(substituted phenyl)urea de-
rivatives 2a-2i and studied the effect of the novel derivatives
for their antileukemic activity.
Within the past ten years, a huge volume of research on the
synthesis, structure-activity relationships (SAR), and antican-
cer activities of the urea derivatives was reported. Heterocyclic
urea derivatives play an important role in anticancer agents
because of their good inhibitory activity against receptor
tyrosine kinases (RTKs),1-4 raf kinases,5,6 protein tyrosine
kinases (PTKs),7 and NADH oxidase,8 which play critical roles
in many aspects of tumorigenesis. Many aromatic urea
derivatives such as N-phenyl-N¤-(2-chloroethyl)ureas and ben-
zoylureas show good anticancer activity, and these compounds
have mainly been proved to be tubulin ligands that inhibit the
polymerization of tubulin.9,10
Currently, a benzothiazole derivative, 2-(4-amino-3-methyl-
phenyl)-5-fluorobenzothiazole, acting through a novel mecha-
nism, has entered the clinical trial phase I in U.K. It shows high
sensitivity toward the tumor cells by acting through aryl
hydrocarbon receptor (AhR) signaling pathway. The activation
Chemistry.
With the aim of obtaining new anticancer
agents and to study the cytotoxic effect of urea derivatives on
leukemia cells, we synthesized a series of 1-((S)-2-amino-
4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-3-(substituted phenyl)-
urea derivatives 2a-2i and evaluated the antileukemic activity
of these derivatives. Synthesis of 2a-2i was done as outlined in
Scheme 1. The key intermediate (S)-2,6-diamino-4,5,6,7-tetra-
hydrobenzo[d]thiazole (1) was synthesized using the earlier
reported method.20 Compound 1 on treatment with different
aryl-substituted isocyanates in presence of triethylamine using
dichloromethane as solvent gave compounds 2a-2i. The
synthesized novel compounds were characterized using the
melting point, optical rotation, IR, 1H NMR, 13C NMR, LCMS,
and elemental analysis data. The absence of -NH2 peak around
¤ 2.5 and the presence of a peak around ¤ 8.5 corresponding to
1
amide proton in the H NMR spectrum of the final compounds
and a strong absorption at 3395-3435 cm¹1 for -NH and 1670-
Published on the web May 28, 2010; doi:10.1246/bcsj.20090318

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Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
4,5,6,7-Tetrahydrobenzo[d]thiazole Derivatives
¹1
N
S
1690 cm for C=O in the IR spectrum of the compounds
along with the LCMS and elemental analysis data confirmed
the structure of the novel compounds 2a-2i.
N
S
O
NH2
i
NH2
R
N
H
N
H2N
H
Biological Study.
The human chronic myelogenous
2a−2i
1
leukemia (CML) K562 and pre B cell line Reh were selected
for the purpose of preliminary anticancer screening of newly
synthesized compounds. To assess the cytotoxicity, we em-
ployed 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 ¯M) of 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives 2a-
2i. In addition to this we also performed DNA fragmentation
assay which is an indicator of apoptosis. To quantify the
apoptotic cells formed upon treatment with 2e, we performed
Annexin V-FITC/PI double staining assay. Assays were carried
out in duplicate in at least two independent experiments.
R=
Cl
Cl
O
2e:
2f:
2a:
2b:
2c:
2d:
Cl
O
Cl
O
2g:
F
Cl
2h:
2i:
Results and Discussion
Newly synthesized urea derivatives 2a-2i containing
4,5,6,7-tetrahydrobenzo[d]thiazole pharmacophore were as-
sessed for cytotoxicity against two human leukemia cell lines
Scheme 1. Synthesis of compound 2. Reagents and
conditions: i) aryl isocyanates, triethylamine, dichloro-
methane, rt, 6-7 h.
2a
2b
2c
2d
2e
2f
2g
2h
2i
Figure 1. Dose- and time-dependent effect of 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives 2a-2i on K562 cell survival.
Approximately 0.75 © 105 cells/mL were cultured and treated with 2a-2i at a concentration of 10, 100, and 250 ¯M. In addition to
control cells (without compound), DMSO was also used as vehicle control. 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 represented as a graph.

D. S. Prasanna et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
691
2a
2b
2c
2d
2e
2f
2g
2h
2i
Figure 2. Dose- and time-dependent effect of 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives 2a-2i on Reh cell survival.
Approximately 0.75 © 105 cells/mL were cultured and treated with 2a-2i at a concentration of 10, 100, and 250 ¯M. In addition
to control cells (without compound), DMSO was also used as vehicle control. 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 represented as a
graph.
(K562 and Reh). The effective concentrations of these
derivatives required 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 Figures 1 and 3 (K562
cells) and Figures 2 and 4 (Reh cells), the exposure of these
derivatives for different time points decreased the number of
live cells in a time and concentration dependent manner. It was
observed that, the effect was improved linearly when incuba-
tion time was prolonged. Among the compounds 2a-2i,
compounds 2d, 2e, 2f, 2g, and 2h showed good inhibition
against K562 cells with IC50 values of 70.11, 40.06, 80.26,
48.03, and 42.00 ¯M, respectively and for Reh cells the
corresponding IC50 values were 90.32, 48.02, 90.21, 55.16, and
48.02 ¯M respectively (Table 1). The other compounds like 2a,
2b, 2c, and 2i exerted moderate inhibitory activity.
the first SAR, we observed that compounds 2d, 2e, 2f, 2g, and
2h which showed good inhibition against both the cell lines
are having electron-withdrawing chloro and fluoro groups at
different positions of phenyl ring of aryl urea. Compounds 2a,
2b, and 2c having electron-donating methoxy groups at
different positions of phenyl ring of aryl urea and compound
2i which is unsubstituted showed moderate inhibition. This
shows that electron-withdrawing groups on phenyl ring
increases the antiproliferative activity of compounds and
electron-donating groups on phenyl ring of the aryl urea
moiety decreases the activity. Second, we observed the effect
among the compounds having chloro group at ortho-, meta-,
and para-positions on the phenyl ring of the aryl urea moiety.
Compound 2h having chloro at para-position showed good
inhibition with IC50 values of 42.00 and 48.02 ¯M against
K562 and Reh cells respectively compared to compounds 2g
and 2f having chloro at meta- and ortho-positions with IC50
values 48.03 and 80.26 ¯M respectively against K562 cells and
55.16 and 90.21 ¯M respectively against Reh cells.
Comparing the IC50 values of the newly synthesized urea
derivatives 2a-2i, we were able to draw some of the SAR. In

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4,5,6,7-Tetrahydrobenzo[d]thiazole Derivatives
2c
2a
2b
2e
2d
2f
2i
2h
2g
Figure 3. Determination of the effect of 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives 2a-2i on cell proliferation by MTT assay.
After 48 and 72 h of exposure, K562 cells treated with 2a-2i at different concentrations (10, 100, and 250 ¯M) were incubated with
MTT (0.5 mg mL¹1) in duplicates and absorbance was measured at 570 nm. Results are presented as percentage of cell proliferation
(the cell viability of vehicle cells were considered as 100%). Error bars are represented in the figure.
When we compare the activity of compound 2e having a
ortho- and para-dichloro substitution and compound 2h having
a para-chloro substitution on the phenyl ring of arylurea moiety
with compounds 2f and 2g having monochloro substitution
at ortho- and meta-positions respectively, we observed that
compound 2e and 2h with chloro at para-position showed
most significant activity against both K562 and Reh cells.
We chose the dichloro substitution containing derivative 2e
for further studies. We performed LDH release assay to assess
the extent of cell damage induced by 2e. For this, K562 cells
were cultured with 10, 50, and 100 ¯M of 2e 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 on treatment with 2e (Figure 5).
K562 cells treated with different concentrations of 2e were
harvested after 72 h, the chromosomal DNA was extracted and
used for agarose gel electrophoresis as described earlier. The
results showed observed smear in the lanes 3 and 4 (Figure 7)
is due to the DNA breakage at multiple positions across the
chromosomal DNA. The intensity of smear is maximum at
100 ¯M. These results in conjunction with annexin V-FITC/PI
staining further suggest that compound 2e induces fragmenta-
tion of chromosomal DNA leading to apoptosis.
Further, to check the cytotoxicity of 2e on normal cells,
MTT assay was performed using 293T cells (human embryonic
kidney epithelial cells). Compound 2e did not show any
significant effect on proliferation of 293T cells (human
embryonic kidney epithelial cells) (Figure 8).
Further, with an interest of quantifying the early and late
apoptotic cells after treatment of cells with 2e, we performed
Annexin V-FITC/PI double staining assay. K562 cells were
taken for this assay. K562 cells were harvested after 48 h of
treatment with 2e (10, 50, and 100 ¯M) and were used for
double staining followed by FACS analysis. Results showed
that apoptosis induced by 2e is 3.5-fold higher than DMSO at
100 ¯M (Figure 6). These results suggest a total disruption of
cell membrane and further damage to the chromosomal DNA
upon treatment with compound 2e. To validate this we carried
out DNA fragmentation assay.
Experimental
Melting points were determined using SELACO-650 hot
stage melting point apparatus and were uncorrected. 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-d6 as a solvent and TMS as internal
standard (chemical shift in ¤). Spin multiplets are given as s
(singlet), d (doublet), t (triplet), and m (multiplet). 13C NMR
spectra were recorded on Shimadzu AMX 400-Bruker,
100 MHz spectrometer using DMSO-d6 as a solvent. Mass

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Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
693
2a
2b
2c
2d
2g
2e
2f
2i
2h
Figure 4. Determination of the effect of 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives 2a-2i on cell proliferation by MTT assay.
After 48 and 72 h of exposure, Reh cells treated with 2a-2i at different concentrations (10, 100, and 250 ¯M) were incubated with
MTT (0.5 mg mL¹1) in duplicates and absorbance was measured at 570 nm. Results are presented as percentage of cell proliferation
(the cell viability of vehicle cells were considered as 100%). Error bars are represented in the figure.
Table 1. IC50 Values of 4,5,6,7-Tetrahydrobenzo[d]thiazole
Derivatives as Determined based on MTT Assay
IC50
Compound
K562/¯M
Reh/¯M
2a
2b
2c
2d
2e
2f
2g
2h
2i
125.01 « 6.82
110.03 « 5.57
100.23 « 5.21
70.11 « 4.38
40.06 « 2.95
80.26 « 4.68
48.03 « 3.47
42.00 « 3.14
100.35 « 5.20
215.03 « 10.95
100.11 « 5.16
125.24 « 6.94
90.32 « 5.26
48.02 « 4.13
90.21 « 4.98
55.16 « 5.43
48.02 « 3.56
110.63 « 5.42
Figure 5. Time- and dose-dependent LDH release in K562
cells treated with 2e. K562 cells were incubated for 24 and
48 h with different concentrations of 2e. Release of LDH in
the medium was measured at 490 nm. Results are presented
as percentage of LDH release.
and purity were recorded on a LC-MSD-Trap-XCT. Elemental
(CHNS) analyses were obtained on Vario EL III Elementar.
Silica gel column chromatography 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 phen-
yl)urea Derivatives 2a-2i. To the solution of intermediate
compound 1 (1.0 equiv) in dichloromethane, triethylamine (3.0
equiv) was added and cooled to 0 °C, respective aryl isocyanate
(1.0 equiv) was 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 concen-
trated and water was added and extracted thrice using ethyl
acetate. The combined ethyl acetate layer was washed with
brine solution and dried over anhydrous sodium sulfate. Ethyl
acetate was evaporated under reduced pressure and the crude

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4,5,6,7-Tetrahydrobenzo[d]thiazole Derivatives
Figure 6. Detection of apoptosis induced by 2e in K562 cells by flow cytometry using Annexin V-FITC/PI double staining. K562
cells (0.75 © 105 cells/mL) were incubated with 2e (10, 50, and 100 ¯M) for 48 h and processed for annexin V-FITC/PI double
staining. The cells were then quantitatively or qualitatively 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 Vand PI positive, which are in the stage
of late apoptosis or necrosis. Panels shown are annexin V-FITC incubated with K562 cells, which are treated with DMSO (a), 10
(b), 50 (c), or 100 ¯M (d). In both the panels (e) is histogram showing comparison of early apoptotic and late apoptotic cells at
different doses of 2e.
product obtained was purified by silica gel (60-120 mesh)
column. The compounds 2a-2i were eluted at 70-80% ethyl
acetate in hexane.
methane (3 mL) using the general experimental procedure as
25
described was a pale blue solid. Mp: 168-170 °C; ½¡ꢀD ¹67.3
1
(c 1, CH3OH); H NMR (DMSO-d6, 400 MHz): ¤ 8.24 (s, 1H,
Synthesis of 1-((S)-2-Amino-4,5,6,7-tetrahydrobenzo[d]-
-NH), 8.07 (s, 1H, -NH), 7.38 (d, 1H, J = 10.62 Hz, 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-), 3.64 (s, 3H, -OCH3),
2.87 (dd, 1H, J = 4.96 Hz, J = 4.88 Hz, -CH), 2.55-2.49 (m,
2H, -CH2), 2.38 (dd, 1H, J = 5.92 Hz, J = 5.68 Hz, -CH),
thiazol-6-yl)-3-(2-methoxyphenyl)urea (2a):
The product
obtained (0.320 g, 83%) by the reaction of 1 (0.200 g,
1.2 mmol) with 2-methoxyphenyl isocyanate (0.176 g, 1.2
mmol) and triethylamine (0.360 g, 3.6 mmol) in dichloro-

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695
Figure 8. Effect of 2e on the growth of normal cells. After
48 and 72 h of exposure, 293T cells (human embryonic
kidney epithelial cells) treated with 2e at 10, 100, and
250 ¯M concentrations were incubated with MTT
(0.5 mg mL¹1) in duplicates and absorbance was measured
at 570 nm. Results are presented as percentage of cell
proliferation (the cell viability of vehicle cells were
considered as 100%).
Figure 7. Detection of DNA damage induced by 2e in
K562 cells. The chromosomal DNA was extracted from
K562 cells treated with different concentrations of 2e. The
purified DNA was then resolved on a 1% agarose gel at
30 V for 6 h. In both panels, Lane 1: DMSO; Lane 2-4:
K562 cells treated with 10, 50, and 100 ¯M, respectively.
M is Marker.
using the general experimental procedure as described was an
25
off-white solid. Mp: 174-176 °C; ½¡ꢀD ¹64.7 (c 1, CH3OH);
1H NMR (DMSO-d6, 400 MHz): ¤ 8.18 (s, 1H, -NH), 8.05 (s,
1H, -NH), 7.82 (s, 1H, Ar-H), 7.35-7.21 (m, 1H, Ar-H), 6.90-
6.86 (m, 2H, Ar-H), 6.70 (s, 2H, -NH2), 4.05-4.02 (m, 1H,
-CH-), 3.72 (s, 3H, -OCH3), 2.82 (dd, 1H, J = 4.90 Hz,
J = 4.96 Hz, -CH), 2.51-2.46 (m, 2H, -CH2), 2.39 (dd, 1H,
J = 5.86 Hz, J = 5.60 Hz, -CH), 1.88-1.81 (m, 1H, -CH2),
1.78-1.71 (m, 1H, -CH2); 13C NMR (DMSO-d6, 100 MHz): ¤
166.6, 158.8, 155.2, 144.5, 132.6, 122.6, 120.4, 116.9, 112.9,
108.8, 55.6, 45.2, 30.1, 28.7, 24.3; MS (ESI + ion): m/z =
319.2; IR (KBr, cm¹1): 3396, 3197, 1674; Elemental analysis:
Found: C, 56.64; H, 5.72; N, 17.64; S, 10.02%. Calculated for
C15H18N4O2S: C, 56.58; H, 5.70; N, 17.60; S, 10.07%.
1.90-1.84 (m, 1H, -CH2), 1.77-1.70 (m, 1H, -CH2); 13C NMR
(DMSO-d6, 100 MHz): ¤ 166.5, 155.8, 154.2, 144.4, 130.6,
128.6, 127.3, 120.5, 114.5, 112.8, 55.6, 45.3, 30.0, 28.7, 24.3;
MS (ESI + ion): m/z = 319.4; IR (KBr, cm¹1): 3403, 3211,
1679. Elemental analysis: Found: C, 56.66; H, 5.71; N, 17.58;
S, 10.09%. Calculated for C15H18N4O2S: C, 56.58; H, 5.70; N,
17.60; S, 10.07%.
Synthesis of 1-((S)-2-Amino-4,5,6,7-tetrahydrobenzo[d]-
thiazol-6-yl)-3-(4-methoxyphenyl)urea (2b):
The product
Synthesis of 1-((S)-2-Amino-4,5,6,7-tetrahydrobenzo[d]-
obtained (0.315 g, 82%) by the reaction of 1 (0.200 g, 1.2
mmol) with 4-methoxyphenyl isocyanate (0.176 g, 1.2 mmol)
and triethylamine (0.360 g, 3.6 mmol) in dichloromethane
(3 mL) using the general experimental procedure as described
thiazol-6-yl)-3-(4-fluorophenyl)urea (2d):
The product
obtained (0.305 g, 83%) by the reaction of 1 (0.200 g, 1.2 mmol)
with 4-fluorophenyl isocyanate (0.161 g, 1.2 mmol) and tri-
ethylamine (0.360 g, 3.6 mmol) in dichloromethane (3 mL)
using the general experimental procedure as described was a
25
was a white solid. Mp: 190-192 °C; ½¡ꢀD ¹66.6 (c 1, CH3OH);
25
1H NMR (DMSO-d6, 400 MHz): ¤ 8.20 (s, 1H, -NH), 8.06 (s,
1H, -NH), 7.30 (d, J = 9.24 Hz, 2H, Ar-H), 6.85 (d, 2H, J =
8.96 Hz, Ar-H), 6.70 (s, 2H, -NH2), 4.04-4.01 (m, 1H, -CH-),
3.70 (s, 3H, -OCH3), 2.82 (dd, 1H, J = 4.92 Hz, J = 4.98 Hz,
-CH), 2.51-2.46 (m, 2H, -CH2), 2.41-2.36 (dd, 1H, J = 5.90
Hz, J = 5.62 Hz, -CH), 1.89-1.82 (m, 1H, -CH2), 1.79-1.72
(m, 1H, -CH2); 13C NMR (DMSO-d6, 100 MHz); ¤ 166.5,
155.3, 154.3, 144.5, 134.1, 119.6 (2C), 114.4 (2C), 112.9, 55.6,
45.2, 30.0, 28.7, 24.2; MS (ESI + ion): m/z = 319.5; IR (KBr,
cm¹1): 3415, 3224, 1689; Elemental analysis: Found: C, 56.55;
H, 5.64; N, 17.54; S, 10.00%. Calculated for C15H18N4O2S: C,
56.58; H, 5.70; N, 17.60; S, 10.07%.
pale yellow solid. Mp: 185-187 °C; ½¡ꢀD ¹68.2 (c 1, CH3OH).
1H NMR (DMSO-d6, 400 MHz): ¤ 8.30 (s, 1H, -NH), 8.06 (s,
1H, -NH), 7.40 (d, 2H, J = 8.12 Hz, Ar-H), 7.11 (d, 2H, J =
8.96 Hz, Ar-H), 6.70 (s, 2H, -NH2), 4.04-4.01 (m, 1H, -CH-),
2.82 (dd, 1H, J = 4.92 Hz, J = 4.96 Hz, -CH), 2.51-2.46
(m, 2H, -CH2), 2.39 (dd, 1H, J = 5.86 Hz, J = 5.64 Hz, -CH),
1.89-1.82 (m, 1H, -CH2), 1.79-1.72 (m, 1H, -CH2); 13C NMR
(DMSO-d6, 100 MHz): ¤ 166.6, 155.0, 144.6, 142.4, 138.6,
129.6 (2C), 115.6 (2C), 45.3, 29.9, 28.7, 24.3; MS (ESI + ion):
m/z = 307.5; IR (KBr, cm¹1): 3415, 3220, 1682; Elemental
analysis: Found: C, 54.98; H, 4.89; N, 18.32; S, 10.44%.
Calculated for C14H15FN4OS: C, 54.89; H, 4.94; N, 18.29; S,
10.47%.
Synthesis of 1-((S)-2-Amino-4,5,6,7-tetrahydrobenzo[d]-
thiazol-6-yl)-3-(3-methoxyphenyl)urea (2c):
The product
Synthesis of 1-((S)-2-Amino-4,5,6,7-tetrahydrobenzo[d]-
thiazol-6-yl)-3-(2,4-dichlorophenyl)urea (2e): The product
obtained (0.335 g, 78%) by the reaction of 1 (0.200 g, 1.2
mmol) with 2,4-dichlorophenyl isocyanate (0.22 g, 1.2 mmol)
obtained (0.325 g, 85%) by the reaction of 1 (0.200 g, 1.2 mmol)
with 3-methoxyphenyl isocyanate (0.176 g, 1.2 mmol) and
triethylamine (0.360 g, 3.6 mmol) in dichloromethane (3 mL)

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Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
4,5,6,7-Tetrahydrobenzo[d]thiazole Derivatives
25
and triethylamine (0.360 g, 3.6 mmol) in dichloromethane
(3 mL) using the general experimental procedure as described
white solid. Mp: 221-223 °C. ½¡ꢀD ¹66.9 (c 1, CH3OH);
1H NMR (DMSO-d6, 400 MHz): ¤ 8.24 (s, 1H, -NH), 8.12 (s,
1H, -NH), 7.29 (d, 2H, J = 10.02 Hz, Ar-H), 6.86 (d, 2H,
J = 9.74 Hz, Ar-H), 6.72 (s, 2H, -NH2), 4.06-4.02 (m, 1H,
-CH-), 2.85 (dd, 1H, J = 4.88 Hz, J = 4.90 Hz, -CH), 2.53-
2.49 (m, 2H, -CH2), 2.40 (dd, 1H, J = 5.92 Hz, J = 5.64 Hz, -
CH), 1.88-1.81 (m, 1H, -CH2), 1.77-1.72 (m, 1H, -CH2).
13C NMR (DMSO-d6, 100 MHz): ¤ 166.6, 155.0, 140.0, 129.0
(2C), 124.9, 119.4 (2C), 112.8, 45.2, 29.9, 28.6, 24.2: MS
(ESI + ion): m/z = 339.6; IR (KBr, cm¹1): 3433, 3225, 1677,
1123; Elemental analysis: Found: C, 52.08; H, 4.70; N, 17.60;
S, 9.97%. Calculated for C14H15ClN4OS: C, 52.09; H, 4.68; N,
17.36; S, 9.93%.
25
was a white solid. Mp: 180-182 °C; ½¡ꢀD ¹66.4 (c 1, CH3OH);
1H NMR (DMSO-d6, 400 MHz): ¤ 8.28 (s, 1H, -NH), 8.05 (s,
1H, -NH), 7.36 (s, 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-), 2.81 (dd, 1H, J = 4.88 Hz, J = 4.96 Hz, -CH), 2.51-
2.46 (m, 2H, -CH2), 2.39 (dd, 1H, J = 5.94 Hz, J = 5.68 Hz,
-CH), 1.89-1.82 (m, 1H, -CH2), 1.78-1.71 (m, 1H, -CH2).
13C NMR (DMSO-d6, 100 MHz): ¤ 166.6, 154.5, 144.4, 136.5,
128.8, 128.0, 125.4, 121.9, 121.7, 112.7, 45.2, 29.8, 28.4, 23.9;
MS (ESI + ion): m/z = 358.1; IR (KBr, cm¹1): 3423, 3216,
1683; Elemental analysis: Found: C, 47.18; H, 4.02; N, 15.60;
S, 9.07%. Calculated for C14H14Cl2N4OS: C, 47.07; H, 3.95; N,
15.68; S, 8.98%.
Synthesis of 1-((S)-2-Amino-4,5,6,7-tetrahydrobenzo[d]-
thiazol-6-yl)-3-phenyl)urea (2i):
The product obtained
Synthesis of 1-((S)-2-Amino-4,5,6,7-tetrahydrobenzo[d]-
(0.31 g, 81%) by the reaction of 1 (0.200 g, 1.2 mmol) with
phenyl isocyanate (0.143 g, 1.2 mmol) and triethylamine
(0.360 g, 3.6 mmol) in dichloromethane (3 mL) using the
general experimental procedure as described was an off-white
thiazol-6-yl)-3-(2-chlorophenyl)urea (2f):
The product
obtained (0.325 g, 84%) by the reaction of 1 (0.200 g,
1.2 mmol) with 2-chlorophenyl isocyanate (0.181 g, 1.2 mmol)
and triethylamine (0.360 g, 3.6 mmol) in dichloromethane
(3 mL) using the general experimental procedure as described
25
1
solid. Mp: 164-166 °C; ½¡ꢀD ¹67.9 (c 1, CH3OH); H NMR
(DMSO-d6, 400 MHz): ¤ 8.22 (s, 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-), 2.81 (dd, 1H, J = 4.98 Hz, J = 4.96 Hz, -CH), 2.51-
2.46 (m, 2H, -CH2), 2.39 (dd, 1H, J = 5.86 Hz, J = 5.70 Hz,
-CH), 1.89-1.82 (m, 1H, -CH2), 1.75-1.70 (m, 1H, -CH2).
13C NMR (DMSO-d6, 100 MHz): ¤ 166.5, 155.2, 144.6, 132.6,
124.2 (2C), 121.6, 119.2 (2C), 112.8, 45.3, 30.1, 28.7, 24.1;
MS (ESI + ion): m/z = 305.6; IR (KBr, cm¹1): 3403, 3211,
1679, 1114; Elemental analysis: Found: C, 58.58; H, 5.62; N,
19.61; S, 11.07%. Calculated for C14H16N4OS: C, 58.31; H,
5.59; N, 19.43; S, 11.12%.
Antileukemic Activity. 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, USA.
Cells were grown in RPMI 1640 supplemented with 10% heat-
inactivated fetal bovine serum (FBS), 100 U mL¹1 of Penicillin,
and 100 ¯g of streptomycin/mL and incubated at 37 °C in a
humidified atmosphere containing 5% CO2.
25
was a pale pink solid. Mp: 176-178 °C; ½¡ꢀD ¹65.8 (c 1,
CH3OH); 1H NMR (DMSO-d6, 400 MHz): ¤ 8.34 (s, 1H, -NH),
8.08 (s, 1H, -NH), 7.36 (d, 1H, J = 10.06 Hz, Ar-H), 7.23-
7.19 (m, 2H, Ar-H), 6.94-6.89 (m, 1H, Ar-H), 6.72 (s, 2H,
-NH2), 4.06-4.03 (m, 1H, -CH-), 2.82 (dd, 1H, J = 4.92 Hz,
J = 4.96 Hz, -CH), 2.51-2.46 (m, 2H, -CH2), 2.38 (dd, 1H,
J = 5.88 Hz, J = 5.64 Hz, -CH), 1.88-1.82 (m, 1H, -CH2),
1.75-1.70 (m, 1H, -CH2). 13C NMR (DMSO-d6, 100 MHz): ¤
166.5, 154.7, 144.3, 132.5, 129.6, 127.2, 126.3, 122.1, 121.6,
113.0, 45.3, 29.8, 28.5, 24.1; MS (ESI + ion): m/z = 323.7;
IR (KBr, cm¹1): 3425, 3194, 1671; Elemental analysis: Found:
C, 52.18; H, 4.65; N, 17.41; S, 10.02%. Calculated for
C14H15ClN4OS: C, 52.09; H, 4.68; N, 17.36; S, 9.93%.
Synthesis of 1-((S)-2-Amino-4,5,6,7-tetrahydrobenzo[d]-
thiazol-6-yl)-3-(3-chlorophenyl)urea (2g):
The product
obtained (0.325 g, 84%) by the reaction of 1 (0.200 g,
1.2 mmol) with 3-chlorophenyl isocyanate (0.181 g, 1.2 mmol)
and triethylamine (0.360 g, 3.6 mmol) in dichloromethane
(3 mL) using the general experimental procedure as described
Trypan Blue Exclusion Assay:
Cell viability was
25
was an off-white solid. Mp: 147-149 °C; ½¡ꢀD ¹64.9 (c 1,
monitored by the Trypan blue exclusion assay as reported
earlier.18 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 and cells were exposed to different
concentrations (10, 100, and 250 ¯M) of 2a-2i. 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 hemocytometer chamber. The
results of these are represented as graphs as shown in Figures 1
and 2.
MTT Assay: Cell proliferation was further assessed by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay,18 which is based on the ability of viable cells
to metabolize a 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 ¯M of 2a-2i. Cells were harvested after 48 and 72 h of
treatment and incubated with MTT (0.5 mg mL¹1). The
percentage cell proliferation was calculated and IC50 values
CH3OH); 1H NMR (DMSO-d6, 400 MHz): ¤ 8.32 (s, 1H, -NH),
8.06 (s, 1H, -NH), 7.41 (s, 1H, Ar-H), 7.11 (d, 2H, J = 8.74
Hz, Ar-H), 6.70 (s, 2H, -NH2), 4.05-4.01 (m, 1H, -CH-),
2.83 (dd, 1H, J = 4.96 Hz, J = 4.94 Hz, -CH), 2.51-2.46 (m,
2H, -CH2), 2.39 (dd, 1H, J = 5.90 Hz, J = 5.68 Hz, -CH),
1.87-1.80 (m, 1H, -CH2), 1.75-1.69 (m, 1H, -CH2). 13C NMR
(DMSO-d6, 100 MHz): ¤ 166.5, 154.8, 144.4, 134.3, 131.8,
129.6, 125.4, 124.1, 122.6, 112.9, 45.3, 29.8, 28.4, 24.0; MS
(ESI + ion): m/z = 323.9; IR (KBr, cm¹1): 3437, 3241, 1687;
Elemental analysis: Found: C, 52.12; H, 4.75; N, 17.52; S,
9.99%. Calculated for C14H15ClN4OS: C, 52.09; H, 4.68; N,
17.36; S, 9.93%.
Synthesis of 1-((S)-2-Amino-4,5,6,7-tetrahydrobenzo[d]-
thiazol-6-yl)-3-(4-chlorophenyl)urea (2h):
The product
obtained (0.325 g, 84%) by the reaction of 1 (0.200 g, 1.2
mmol) with 4-chlorophenyl isocyanate (0.181 g, 1.2 mmol) and
triethylamine (0.360 g, 3.6 mmol) in dichloromethane (3 mL)
using the general experimental procedure as described was a

D. S. Prasanna et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
697
(concentration of compound causing 50% inhibition of cell
growth) were estimated after 72 h of compound treatment. The
histograms were plotted as shown in Figures 3 and 4.
References
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1
LDH Release Assay: The cytotoxicity of the compound
2e was further assessed by LDH release assay,18 which is an
indicator of membrane integrity and hence cell injury. LDH
assay was performed as per standard protocols to estimate the
LDH release in the culture media following the treatment of
2e (10, 50, and 100 ¯M) on K562 cells for 24 and 48 h. The
LDH release was measured. Percentage of LDH release was
calculated and plotted as a graph as shown in Figure 5.
Annexin V-FITC Flow Cytometric Analysis: The trans-
location of phosphatidyl serine from the inner to the outer leaf-
let of the plasma membrane is considered as one of the earliest
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apoptosis detection kit (Santacruz, USA). In brief, after 48 h of
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added and incubated in dark for 20 min. Cells were then sub-
jected to FACS (FACScan, BD Biosciences, USA) analysis. At
least 10000 events were recorded and represented as dot plots.
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induction of apoptosis accompanied the growth inhibition by
4,5,6,7-tetrahydrobenzo[d]thiazole derivative 2e, we analyzed
apoptotic DNA fragmentation by agarose gel electrophoresis
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100 ¯M 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
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Conclusion
In summary, we have described the synthesis of 1-((S)-
2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-3-(substituted
phenyl)urea derivatives 2a-2i, which showed moderate and
weak inhibitory activity toward human leukemic cells lines,
K562 and Reh. The derivatives 2d, 2e, 2f, 2g, and 2h with
electron-withdrawing chloro and fluoro substituents at different
positions showed good inhibitory effects. Compounds 2a, 2b,
2c, and 2i showed moderate inhibitory activity against both the
cell lines tested. Further investigations to understand the
mechanism by which these molecules induce apoptosis and
developing more potent molecules are under progress in our
laboratory.
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We are grateful to University Grants Commission (UGC),
Government of India for financial support to K.S.Rangappa
under the project UGC vide No. F. 540/14/DRS/2009 (SAP-I)
DRS-II Programme. Sathees C. Raghavan acknowledges
support from Lady Tata Memorial Trust international award
for leukemia research (London). D. S. Prasanna and K.
Vinaya 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.