Synthesis and Identification of a new class of antileukemic agents containing 2-(arylcarboxamide)-(S)-6-amino-4,5,6,7-tetrahydrobenzo[d]thiazole

Article abstract of DOI:10.1016/j.ejmech.2010.08.056

Recently we have reported the effect of (S)-6-aryl urea/thiourea substituted-2-amino-4,5,6,7-tetrahydrobenzo[d]thiazole derivatives as potent anti-leukemic agents. To elucidate further the Structure Activity Relationship (SAR) studies on the anti-leukemic activity of (S)-2,6-diamino-4,5,6,7- tetrahydrobenzo[d]thiazole moiety, a series of 2-arlycarboxamide substituted-(S)-6-amino-4,5,6,7-tetrahydrobenzo[d]thiazole were designed, synthesized and evaluated for their anti-leukemic activity by trypan blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) assays and cell cycle analysis. Results suggest that the position, number and bulkiness of the substituent on the phenyl ring of aryl carboxamide moiety at 2nd position of 6-amino-4,5,6,7-tetrhydrobenzo[d] thiazole play a key role in inhibiting the proliferation of leukemia cells. Compounds with ortho substitution showed poor activity and with meta and para substitution showed good activity.

Full text of DOI:10.1016/j.ejmech.2010.08.056

European Journal of Medicinal Chemistry 45 (2010) 5331e5336
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and Identification of a new class of antileukemic agents containing
2-(arylcarboxamide)-(S)-6-amino-4,5,6,7-tetrahydrobenzo[d]thiazole
,
,
,
*
D.S. Prasanna a 1, C.V. Kavitha b 1, K. Vinaya a, S.R. Ranganatha b, Sathees C. Raghavan b, K.S. Rangappa a
a Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore-570 006, India
b Department of Biochemistry, Indian Institute of Science, Bangalore- 560 012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently we have reported the effect of (S)-6-aryl urea/thiourea substituted-2-amino-4,5,6,7-tetrahy-
drobenzo[d]thiazole derivatives as potent anti-leukemic agents. To elucidate further the Structure
Activity Relationship (SAR) studies on the anti-leukemic activity of (S)-2,6-diamino-4,5,6,7-tetrahy-
drobenzo[d]thiazole moiety, a series of 2-arlycarboxamide substitutede(S)-6-amino-4,5,6,7-tetrahy-
drobenzo[d]thiazole were designed, synthesized and evaluated for their anti-leukemic activity by trypan
blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydro-
genase (LDH) assays and cell cycle analysis. Results suggest that the position, number and bulkiness of
the substituent on the phenyl ring of aryl carboxamide moiety at 2nd position of 6-amino-4,5,6,7-tetr-
hydrobenzo[d]thiazole play a key role in inhibiting the proliferation of leukemia cells. Compounds with
ortho substitution showed poor activity and with meta and para substitution showed good activity.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 27 July 2010
Received in revised form
20 August 2010
Accepted 25 August 2010
Available online 19 September 2010
Keywords:
Tetrahydrobenzo[d]thiazole
Cytotoxicity
Apoptosis
Cancer therapeutics
Leukemia
1. Introduction
potency than imatinib mesylate (GleevecÒ) and inhibits the
majority of kinase mutations in imatinib-resistant CML [18e21].
Leukemias are uniformly fatal diseases of unknown aetiology
characterized by excessive and abnormal proliferation of primitive
white blood cells and their precursors with infiltration into the
various tissues of the body. Leukemias occur in people of all ages.
The leukemias of childhood are the more common one and these
are the cancers of the hematopoietic system, involving in most
cases, malignant transformation of lymphoid progenitor cells and
less commonly transformation of myeloid progenitor cells.
Thiazole moieties are having various pharmacological activities, in
general like cyclin dependent kinase inhibitors [1,2], CCR antagonists
[3,4], adenosine receptor antagonists [5e8], anti-inflammatory [9]
and antibacterial activities [10]. There are many reports available on
the anti-cancer activity of 2-aminothiazole and benzothiazole
In our laboratory, we have synthesized a series of 1-((S)-2-
amino-4,5,6,7-tetrahydrobenzo[d] thiazol-6-yl)- 3-(substituted
phenyl)-thiourea and urea derivatives by coupling amine at posi-
tion 6 of (S)-2,6-diaminotetrahydrobenzo[d]thiazole with different
substituted aryl isothiocyanates and isocyanates and identified
them as potent anti-leukemic agents [22,23]. We continued to seek
alternative templates by converting the amine at position 2 of (S)-
2,6-diaminotetrahydrobenzo[d]thiazole to amide functionality and
to check their efficacy as anti-leukemic agents.
2. Chemistry
We synthesized a series of novel class of 2-N-(substituted
phenyl) carbonyl substituted 2,6-diamino-4,5,6,7-tetrahydrobenzo
[d]thiazol hydrochloride derivatives 4(aeh) and evaluated the anti-
leukemic activity of these derivatives. Synthesis of 4(aeh) was done
as outlined in Scheme 1. The key intermediate (S)-2,6-diamino-
4,5,6,7-tetrahydrobenzo[d]thiazole 1 was synthesized using the
earlier reported method [24]. Compound 1 on treatment with Boc
anhydride in presence of potassium carbonate using tetrahydro-
furan as solvent at ꢀ5 to 0 ꢁC yielded the mono amino protected
compound 2. This mono protected compound on treatment with
different aryl substituted acid chlorides in presence of triethylamine
using dichloromethane as solvent gave compounds 3(aeh). These
derivatives [11e17].
N-(2-Chloro-6-methylphenyl)-2-[[6-[4-(2-
hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]-amino)]-1,3-
thiazole-5-carboxamide (BMS-354825, dasatinib, SPRYCELÒ) is
a
novel multi-targeted kinase inhibitor recently approved in
several countries for the treatment of chronic myelogenous
leukemia (CML) as well as Philadelphia chromosome-positive
acute lymphocytic leukemia (ALL). Dasatinib exhibits greater
* Corresponding author. Tel.: þ91 821 2515149; fax: þ91 821 2500846.
E-mail addresses: rangappaks@chemistry.uni-mysore.ac.in, rangappaks@gmail.
com (K.S. Rangappa).
1
Both the authors contributed equally.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.056

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D.S. Prasanna et al. / European Journal of Medicinal Chemistry 45 (2010) 5331e5336
O
R
N
N
S
N
S
O
O
ii
NH2
i
NH
NH2
S
O
N
H
O
N
H
H2N
2
3(a-h)
1
O
R
iii
N
S
NH
H2N
HCl
4(a-h)
Reagents and conditions: i) Boc anhydride, K2CO3, THF, -5 to 0 oC; ii)Acid chloride, Triethyl amine,
Dichloromethane: iii) HCl in Ether
R=
e =
a =
F
O
f =
b =
NO2
F
F
g =
h=
c =
d=
Br
Cl
NO2
Cl
Scheme 1. Synthesis of 2-(arylcarboxamide)-(S)-6-amino-4,5,6,7-tetrahydrobenzothiazole derivatives.
compounds on deprotection of boc protection using HCl in ether
yielded the hydrochloride salts of the desired compounds 4(aeh).
The synthesized novel compounds were characterized using the IR,
1H NMR, LCMS and elemental analysis data.
4.2. Biology
Newly synthesized 4,5,6,7-tetrahydrobenzo[d]thiazole deriva-
tives 4(aeh) were assessed for cytotoxicity against two human
leukemia cell lines, K562 and CEM. The effective concentrations of
these derivatives required to inhibit K562 or CEM cell growth and
survival were determined first by carrying out dose response
experiments using trypan blue dye exclusion assay. In this assay,
the cells were counted at intervals of 24 h till the control cells
attained stationary phase (data not shown). The cell viability was
further assessed by MTT assay. The exposure of these derivatives at
various concentrations for different time points decreased the
number of live cells in a concentration and time dependent manner.
Among the compounds 4(aeh), compounds 4b, 4d, 4e, 4g and 4h
showed good inhibition against K562 cells with IC50 values of
3. Pharmocology
The human leukemia cells, K562 and CEM were selected for the
purpose of preliminary anti-cancer screening of newly synthesized
compounds. To assess the cytotoxicity, we employed trypan blue
dye exclusion assay, MTT assay, LDH assay and cell cycle analysis.
For this, cells growing in log phase were treated with different
concentrations (10, 50 and 100 mM) of 4,5,6,7-tetrahydrobenzo[d]
thiazole derivatives 4(aeh). Further, cytotoxicity compound 4e on
the growth of normal cells was assessed using 293T cells (human
embryonic kidney epithelial cells) using MTT assay.
17.88
for CEM cells the corresponding IC50 values were 13.78
22.82 M, 4.52 M, 9.02 M and 8.72 M respectively (Table 1). The
mM, 21.01
m
M, 4.52
mM, 8.16
m
M and 7.72
m
M, respectively and
mM,
m
m
m
m
4. Results and discussion
other compounds namely 4a, 4c and 4f exerted moderate inhibi-
tory activity.
4.1. Chemistry
The structure of compound 2 was confirmed by missing of peak
corresponding to amine at position 6 of tetrahydrobenzothiazole
and the presence of peak corresponding to Boc protected eNH and
a singlet peak corresponding to 9 tert-butyl protons of Boc in the 1H
NMR spectrum of compound 2. The structures of compounds 4
(aeh) were confirmed by the absence of eNH2 peak around
Table 1
IC50 values of tetrahydrobenzo[d]thiazole derivatives determined by MTT assay.
IC50 (in
K562
mM)
Compound
CEM
4a
4b
4c
4d
4e
4f
65.73
17.88
57.23
21.01
4.52
>100
8.16
7.72
61.35
13.78
56.62
22.82
4.52
81.24
9.02
8.72
d
6.5 ppm corresponding to the amine at position 2 of tetrahy-
drobenzo[d]thiazole and the presence of a peak around d 12.5 ppm
corresponding to amide proton in the 1H NMR spectrum of the final
compounds and a strong absorption at 3395e3435 cmꢀ1 for eNH
and 1670e1690 cmꢀ1 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 4(aeh).
4g
4h

D.S. Prasanna et al. / European Journal of Medicinal Chemistry 45 (2010) 5331e5336
5333
In our previous studies we reported that the nature of the
substituent on the phenyl group of 1-((S)-2-amino-4,5,6,7-tetra-
hydrobenzo[d]thiazol-6-yl)-3-(substituted phenyl)-thiourea and
urea derivatives playing a key role in the anti-leukemic activity of
these compounds [22,23]. Electron withdrawing groups on the
phenyl ring of the aryl thiourea/urea moiety showed potent
activity. Here, in these compounds 4(aeh), we observed that the
position and size of the substituent on the phenyl ring of aryl car-
boxamide playing the key role. Comparing the IC50 values of the
newly synthesized derivatives 4(aeh), we were able to draw some
of the structure activity relationships (SAR). Compound 4a, which is
having unsubstituted phenyl ring showed moderate activity with
an IC50 value of 65.73
CEM cells. Compound 4c with 2-fluoro substitution on phenyl ring
also showed moderate activity with an IC50 value of 57.23
against K562 cells and 56.62 M against CEM cells. Compound 4f
having 2,6-difluoro substitution showed poor activity with IC50
values of >100 M for K562 cells and 81.24 M for CEM cells.
mM against K562 cells and 61.35 mM against
mM
Fig. 1. Time- and dose-dependent LDH release in K562 cells treated with 4e. K562 cells
were incubated for 24 h with different concentrations of 4e. Release of LDH in the
medium was measured at 490 nm. Results are presented as percentage of LDH release.
m
m
m
Compounds 4b and 4d having methoxy and bromo substitution
Further, to determine the cytotoxic effect of 4e on normal cells,
respectively at the meta position on the phenyl ring showed good
293T cells (human embryonic kidney epithelial cells) were treated
with 4e at different concentrations (1e5 mM) and viability of cells
activity. The corresponding IC50 values are 17.88
respectively for K562 cells and 13.78 and 22.82
m
M and 21.02
mM
m
M respectively for
was determined by MTT assay. Data reveals that 4e has no signifi-
cant effect on normal cells at tested concentrations (Fig. 3).
CEM cells. Comparing the activity of 4b and 4d with that of
compounds 4c and 4f, we observed that, the position of the
substitution is playing a key role in the inhibitory activity.
Compound 4g having a meta dinitro (3,5-dinitro) substitution
5. Conclusions
showed good activity with IC50 values of 8.16
mM and 9.02 mM
From the structure activity relationship studies, we observed
that the position and number of the substituents on the phenyl ring
of aryl carboxamide moiety in the tetrahydrobenzothiazole deriv-
atives 4(aeh) are important in reasoning the activity. Compounds
with no substitution and/or ortho substitution are shown poor
inhibition of proliferation of leukemia cells, whereas the
compounds with meta and para substitution the reasonable
inhibiting the proliferation of leukemia cells. Bulky substituents at
the meta and ortho position also play a dominant role in inhibiting
the leukemic cells proliferation.
respectively for K562 and CEM cells. The activity of compound 4g is
good compared to that of compounds 4b and 4d with mono
methoxy and mono bromo substitution at meta positions respec-
tively. This may be due to the substitution at meta position and also
the presence of the bulkiness of nitro group.
Compound 4h having ortho and para dichloro substitution also
showed good activity with IC50 values of 7.72
mM and 8.72 mM
respectively for K562 and CEM cells which is comparable with that
of compound 4g with meta dinitro substitution. Even though the
chloro group is small compared to nitro groups, the activity may be
due to the presence of chloro at ortho position.
Compound 4e having a tert-butyl substitution at para position
showed more potent and better activity compared to all other
molecules in the series. Since we observed the complete death of
6. Experimental Section
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 stan-
cells at 10
mM of 4e, we tested at low concentrations to determine
the IC50 value and it was found to be 4.52
mM for both the cell lines
(data not shown). The effect of 4e on cell viability was also
a concentration and time dependent manner like other molecules
in the series tested. The potency of this molecule could be due to
the bulkiness of the tert-butyl group. Further, to investigate the
cytotoxicity of 4e we carried out LDH release assay, which is an
indicator of cell death at early time point of 24 h at low concen-
trations (Fig. 1). The data showed that 4e induces the release of LDH
into the media in a concentration dependent manner.
In addition, the results from trypan blue exclusion assay and MTT
assay showed that 4e significantly reduced the number of live cells.
Hence, to explore the effect of 4e on cell cycle distribution of K562
cells, we studied cell cycle distribution by fluorescence activated cell
sorting (FACS). The histogram of vehicle control (DMSO) treated cells
showed a standard cell cycle pattern, which include G1 and G2 peaks
separated by S phase peak (Fig. 2). G0/G1 peak (mostly dead cells)
was either absent or not prominent. Although we were not able to
visualize the prominent arrest at any stage of the cell cycle, we noted
the accumulation of subploid cells, the subG1 phase (G0/G1), and
dard (chemical shift in d ppm). Spin multiplets are given as s
(singlet), d (doublet), t (triplet) and m (multiplet). Mass and purity
were recorded on an LCeMSD-Trap-XCT. Elemental (CHNS) anal-
yses were obtained on Vario EL III Elementar. Silica gel column
chromatography was performed using Merck 7734 silica gel
(60e120 mesh) and Merck made TLC plates.
6.1. Synthesis of 2-(arylcarboxamide)-(S)-6-amino-4,5,6,7-
tetrahydrobenzothiazole derivatives
6.1.1. Synthesis of (S)-tert-butyl 2-amino-4,5,6,7-tetrahydrobenzo
[d]thiazol-6-ylcarbamate 2
To the solution of compound (S)-2,6-diamino-4,5,6,7-tetrahy-
drobenzo[d]thiazol 1 (3 g, 0.018 mol) in tetrahydrofuran, potassium
carbonate (4.9 g, 0.035 mol) was added followed by Boc anhydride
(3.8 g, 0.0175 mol) dropwise at ꢀ5 ꢁC and stirred at ꢀ5 to 0 ꢁC for 6 h.
Completion of reactionwas monitored using TLC. After completion of
reaction, tetrahydrofuran was removed under reduced pressure.
Water was added and the reaction mixture was extracted thrice using
ethyl acetate (3 ꢂ 50 mL). The combined organic layer was dried over
anhydrous sodium sulphate. Ethyl acetate was removed under
reduced pressure and purified using silica gel (60e120 mesh) using
decline of both G1 and G2/M (Fig. 2A and B) phases at 10 mM when
compared with untreated cells. We did not see prominent changes in
any phase of cell cycle at low concentrations and our studies suggest
that significant growth inhibition by 4e could be due to apoptosis.

5334
D.S. Prasanna et al. / European Journal of Medicinal Chemistry 45 (2010) 5331e5336
Fig. 2. Cell cycle analysis of K562 cells treated with 4e. K562 cells (0.75 ꢂ 105 cells/ml) were incubated at 37 ꢁC with 4e (1, 5 and 10
mM). Following 48 h of incubation, cells were
fixed and stained with propidium iodide and subjected to FACS analysis. Panel A and B show histograms comparing the effect of 4e at specific cell cycle stages. In the Panel A, the
first histogram represents DMSO treated cells. Panel B show the quantification of cells in different stages of cell cycle followed by treatment with 4e.
1:1 hexane: ethyl acetate as eluent. Yield 4.5 g (95%).1H NMR (DMSO-
d6, 400 MHz) :6.94(d,1H, NH), 6.65(s, 2H, NH2), 3.63(bs,1H, eCHe),
2.70 (m, 1H, eCH2e), 2.56 (m, 2H, eCH2e), 2.37 (m,1H, eCH2e),1.86
(t, 1H, eCH2e), 1.37 (s, 9H, e(CH3)3). Elemental Analysis: Found: C,
53.63; H, 7.06; N,15.52; S,12.01; Calculated for C12H19N3O2S: C, 53.51;
H, 7.11; N, 15.60; S, 11.90.
(1eq) were added at cold condition and stirred at room temperature
for 6e8 h (completion of reaction was confirmed by TLC). After
completion of the reaction, the reaction mixture was concentrated
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 sulphate. Ethyl acetate was evapo-
rated under reduced pressure and the crude product obtained was
purified by silica gel (60e120 mesh) column to get the compounds 3
(aeh). To the solution of compound 3(aeh) in dry ether (3 mL) at
d
6.1.2. General procedure for the synthesis 2-N-aryl carbonyl
substituted 2,6-diamino-4,5,6,7-tetrahydrobenzo[d]thiazol
hydrochloride 4(aeh)
To a solution of tert-butyl (S)-2-amino-4,5,6,7-tetrahydrobenzo
[d]thiazol-6-ylcarbamate 2 (1 eq) in dichloromethane, triethyl-
amine (3 eq) was added and cooled to 0 ꢁC, respective acid chlorides
0
ꢁC saturated HCl in ether (1 ml) was added slowly and stirred at
0 ꢁC for 3 h, white solid formed was filtered and dried under vaccum
to get the compounds 4(aeh).
6.1.2.1. Synthesis of (S)-N-(6-amino-4,5,6,7-tetrahydrobenzo[d]thia-
zol-2-yl)benzamide hydrochloride 4a. The product 4a was obtained
by reaction of compound 2 (0.40 g, 1.5 mmol) with benzoyl chloride
(0.21 g, 1.5 mmol) and triethylamine (0.45 g, 4.5 mol) in dichloro-
methane (5 ml) and then cleaving the Boc protection using HCl in
ether using the general experimental procedure as described. Yield
0.35 g (76%). IR (KBr, cmꢀ1): 1688. 1H NMR (DMSO-d6, 400 MHz)
d:
12.41 (bs,1H, NH), 8.44 (d, 3H, NHþ3 salt), 7.65 (m, 5H, Ar-H), 3.53 (bs,
1H, eCHe), 3.14e3.09 (m, 1H, eCH2e), 2.83e2.75 (m, 1H, eCH2e),
2.73e2.65 (m, 2H, eCH2e), 2.19 (t, 1H, eCH2e), 1.98 (t, 1H, eCH2e).
MS (ESI þ ion): m/z ¼ 310.2. Elemental Analysis: Found: C, 54.34; H,
5.27; N, 13.65; S, 10.22; Calculated for C14H16ClN3OS: C, 54.27; H,
5.21; N, 13.56; S, 10.35.
6.1.2.2. Synthesis of (S)-N-(6-amino-4,5,6,7-tetrahydrobenzo[d]thia-
zol-2-yl)-3-methoxy benzamide hydrochloride 4b. The product 4b
was obtained by reaction of compound 2 (0.40 g, 1.5 mmol) with 3-
methoxy benzoyl chloride (0.26 g, 1.5 mmol) and triethylamine
(0.45 g, 4.5 mol) in dichloromethane (5 ml) and then cleaving the Boc
Fig. 3. Effect of 4e on the growth of normal cells. 293T cells exposed to different
concentrations of 4e were incubated with MTT and absorbance was measured at
570 nm.

D.S. Prasanna et al. / European Journal of Medicinal Chemistry 45 (2010) 5331e5336
5335
protection using HCl in ether using the general experimental proce-
dure as described. Yield 0.40 g (78%). IR (KBr, cmꢀ1): 1690,1237,1112.
2.77e2.67 (m, 2H, eCH2e), 2.17 (t, 1H, eCH2e), 1.98 (t, 1H, eCH2e).
MS (ESI þ ion): m/z ¼ 346.1. Elemental Analysis: Found: C, 48.52; H,
3.97; N, 12.11; S, 9.19; Calculated for C14H14ClF2N3OS: C, 48.63; H,
4.08; N, 12.15; S, 9.27.
1H NMR (DMSO-d6, 400 MHz) : 12.48 (bs, 1H, NH), 8.49 (d, 3H, NH3þ
d
salt), 7.65 (m, 1H, Ar-H), 7.44 (m, 2H, Ar-H), 7.12 (s, 1H, Ar-H), 3.92 (s,
3H, eOCH3), 3.57 (bs,1H, eCHe), 3.13e3.09(m,1H, eCH2e), 2.8e2.76
(m, 1H, eCH2e), 2.71e2.64 (m, 2H, eCH2e), 2.18 (t, 1H, eCH2e), 1.96
(t, 1H, eCH2e). MS (ESI þ ion): m/z ¼ 340.3. Elemental Analysis:
Found: C, 52.91; H, 5.36; N, 12.42; S, 9.37; Calculated for
C15H18ClN3O2S: C, 53.01; H, 5.34; N, 12.36; S, 9.44.
6.1.2.7. Synthesis of (S)-N-(6-amino-4,5,6,7-tetrahydrobenzo[d]thia-
zol-2-yl)-3,5-dinitro benzamide hydrochloride 4g. The product 4g
was obtained by reaction of compound 2 (0.40 g, 1.5 mmol) with
3,5-dinitro benzoyl chloride (0.34 g, 1.5 mmol) and triethylamine
(0.45 g, 4.5 mol) in dichloromethane (5 ml) and then cleaving the
Boc protection using HCl in ether using the general experimental
procedure as described. Yield 0.47 g (78%). IR (KBr, cmꢀ1): 1690,
6.1.2.3. Synthesis of (S)-N-(6-amino-4,5,6,7-tetrahydrobenzo[d]thia-
zol-2-yl)-2-fluorobenzamide hydrochloride 4c. The product 4c was
obtained by reaction of compound 2 (0.40 g, 1.5 mmol) with 2-
fluoro benzoyl chloride (0.24 g, 1.5 mmol) and triethylamine
(0.45 g, 4.5 mmol) in dichloromethane (5 ml) and then cleaving the
Boc protection using HCl in ether using the general experimental
procedure as described. Yield 0.37 g (75%).
1545,1338. 1H NMR (DMSO-d6, 400 MHz)
d: 12.49 (bs, 1H, NH), 8.59
(s, 2H, Ar-H), 7.48 (s, 1H, Ar-H), 8.39 (d, 3H, NHþ3 salt), 3.53 (bs,
1H, eCHe), 3.19e3.12 (m, 1H, eCH2e), 2.85e2.78 (m, 1H, eCH2e),
2.74e2.66 (m, 2H, eCH2e), 2.18 (t, 1H, eCH2e), 1.97 (t, 1H, eCH2e).
MS (ESI þ ion): m/z ¼ 400.1. Elemental Analysis: Found: C, 42.014 H,
3.47; N, 17.58; S, 7.93.; Calculated for C14H14ClN5O5S: C, 42.06; H,
3.53; N, 17.52; S, 8.02.
IR (KBr, cmꢀ1): 1688, 1033. 1H NMR (DMSO-d6, 400 MHz)
d: 12.41
(bs,1H, NH), 8.41 (d, 3H, NHþ3 salt), 7.74e7.69 (m, 1H, Ar-H), 7.65e7.59
(m,1H, Ar-H), 7.38e7.32 (m, 2H, Ar-H), 3.53(bs,1H, eCHe), 3.15e3.09
(m, 1H, eCH2e), 2.83e2.77 (m, 1H, eCH2e), 2.73e2.64 (m,
2H, eCH2e), 2.17 (t, 1H, eCH2e), 1.97 (t, 1H, eCH2e). MS (ESI þ ion):
m/z ¼ 328.1. Elemental Analysis: Found: C, 51.36; H, 4.54; N,12.88; S,
9.75; Calculated for C14H15ClFN3OS: C, 51.30; H, 4.61; N,12.82; S, 9.78.
6.1.2.8. Synthesis of (S)-N-(6-amino-4,5,6,7-tetrahydrobenzo[d]thia-
zol-2-yl)-2,4-dichloro benzamide hydrochloride 4h. The product 4h
was obtained by reaction of compound 2 (0.40 g, 1.5 mmol) with
2,4-dichloro benzoyl chloride (0.31 g, 1.5 mmol) and triethylamine
(0.45 g, 4.5 mmol) in dichloromethane (5 ml) and then cleaving
the Boc protection using HCl in ether as given in general proce-
dure. Yield 0.39 g (68%). IR (KBr, cmꢀ1): 1686, 722. 1H NMR
6.1.2.4. Synthesis of (S)-N-(6-amino-4,5,6,7-tetrahydrobenzo[d]thia-
zol-2-yl)-3-bromobenzamide hydrochloride 4d. The product 4d was
obtained by reaction of compound 2 (0.40 g, 1.5 mmol) with 3-
bromo benzoyl chloride (0.33 g, 1.5 mmol) and triethylamine
(0.45 g, 4.5 mmol) in dichloromethane (5 ml) and then cleaving the
Boc protection using HCl in ether using the general experimental
procedure as described. Yield 0.38 g (67%).
(DMSO-d6, 400 MHz) d
: 12.47 (bs, 1H, NH), 8.39 (d, 3H, NHþ3 salt),
7.39 (s, 1H, Ar-H), 7.23 (m, 2H, Ar-H), 3.49 (bs, 1H, eCHe),
3.16e3.11 (m, 1H, eCH2e), 2.84e2.75 (m, 1H, eCH2e), 2.75e2.67
(m, 2H, eCH2e), 2.14 (t, 1H, eCH2e), 1.99 (t, 1H, eCH2e). MS
(ESI þ ion): m/z ¼ 379.0. Elemental Analysis: Found: C, 44.37; H,
3.60; N, 11.21; S, 8.41; Calculated for C14H14Cl3N3OS: C, 44.40; H,
3.73; N, 11.10; S, 8.47.
IR (KBr, cmꢀ1): 1686, 688. 1H NMR (DMSO-d6, 400 MHz)
d: 12.41
(bs, 1H, NH), 8.41 (d, 3H, NHþ3 salt), 7.76 (m, 1H, Ar-H), 7.52 (m, 2H,
Ar-H), 7.34 (s, 1H, Ar-H), 3.50 (bs, 1H, eCHe), 3.17e3.11 (m, 1H,
eCH2e), 2.83e2.77 (m,1H, eCH2e), 2.75e2.65 (m, 2H, eCH2e), 2.19
(t, 1H, eCH2e), 1.95 (t, 1H, eCH2e). MS (ESI þ ion): m/z ¼ 389.0.
Elemental Analysis: Found: C, 43.29; H, 3.86; N, 10.89; S, 8.15;
Calculated for C14H15BrClN3OS: C, 43.26; H, 3.89; N, 10.81; S, 8.25.
6.2. Biological study
The human chronic myelogenous leukemia (CML) K562 and
CEM were selected for the purpose of preliminary anti-cancer
screening of newly synthesized compounds. To assess the cyto-
toxicity, we employed trypan blue dye exclusion assay, MTT assay,
LDH assay and FACS analysis. For this, cells growing in log phase
were treated with different concentrations (as mentioned in
respective assays) of 4,5,6,7-tetrahydrobenzo[d]thiazole deriva-
tives 4(aeh). Assays were carried out in duplicate in at least three
independent experiments.
6.1.2.5. Synthesis of (S)-N-(6-amino-4,5,6,7-tetrahydrobenzo[d]thia-
zol-2-yl)-4-tert-butylbenzamide hydrochloride 4e. The product 4e
was obtained by reaction of compound 2 (0.40 g, 1.5 mmol) with 4-
tert-butyl benzoyl chloride (0.30 g, 1.5 mmol) and triethylamine
(0.45 g, 4.5 mmol) in dichloromethane (5 ml) and then cleaving the
Boc protection using HCl in ether using the general experimental
procedure as described. Yield 0.44 g (79%).
IR (KBr, cmꢀ1): 1684,1399.1H NMR (DMSO-d6, 400 MHz)
d: 12.41
(bs,1H, NH), 8.41 (d, 3H, NHþ3 salt), 7.79 (d, 2H, Ar-H), 7.54 (d, 2H, Ar-
H), 3.53 (bs, 1H, eCHe), 3.14e3.09 (m, 1H, eCH2e), 2.84e2.76 (m,
1H, eCH2e), 2.71e2.62 (m, 2H,eCH2e), 2.15 (t, 1H, eCH2e), 1.99 (t,
1H, eCH2e), 1.33 (s, 9H, (eCH3)3). MS (ESI þ ion): m/z ¼ 366.2.
Elemental Analysis: Found: C, 59.14; H, 6.66; N, 11.42; S, 8.69.;
Calculated for C18H24ClN3OS: C, 59.08; H, 6.61; N, 11.48; S, 8.76.
6.2.1. Cell lines and culture
Human cell lines, K562 and CEM were purchased from National
Center for Cell Science, Pune, India. Cells were grown in RPMI 1640
supplemented with 10% heat-inactivated fetal bovine serum (FBS),
100 U/mL of Penicillin, and 100 mg of streptomycin/ml and incu-
bated at 37 ꢁC in a humidified atmosphere containing 5% CO2.
6.1.2.6. Synthesis of (S)-N-(6-amino-4,5,6,7-tetrahydrobenzo[d]thia-
zol-2-yl)-2,6-difluoro benzamide hydrochloride 4f. The product 4f
was obtained by reaction of compound 2 (0.40 g, 1.5 mmol) with
2,6-difluoro benzoyl chloride (0.27 g, 1.5 mmol) and triethylamine
(0.45 g, 4.5 mmol) in dichloromethane (5 ml) and then cleaving the
Boc protection using HCl in ether using the general experimental
procedure as described. Yield 0.41 g (78%). IR (KBr, cmꢀ1): 1689,
6.2.2. Trypan blue exclusion assay
Cell viability was monitored by the Trypan blue exclusion
assay as reported earlier [25]. Briefly, K562 or CEM cells, growing
in exponential phase were seeded at a density of 0.75 ꢂ 105 cells/
ml in a 6-well tissue culture plate for 24 h and cells were
exposed to a different concentrations (10, 50, and 100 mM) of 4
(aeh). 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.
1167. 1H NMR (DMSO-d6, 400 MHz)
d: 12.45 (bs, 1H, NH), 8.46 (d,
3H, NHþ3 salt), 7.65 (m, 1H, Ar-H), 7.26 (t, 2H, Ar-H), 3.53 (bs, 1H,
eCHe), 3.16e3.10 (m, 1H, eCH2e), 2.85e2.79 (m, 1H, eCH2e),

5336
D.S. Prasanna et al. / European Journal of Medicinal Chemistry 45 (2010) 5331e5336
6.2.3. MTT assay
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Acknowledgements
We are grateful to UGC, Govt. of India for financial support to
KSR under the projects vide no. F. 31-143/2005(SR), UGC-SAP
(Phase II) vide No.F. 540/10/2004-05 (SAP eDRS-II). SCR acknowl-
edges support from Lady Tata Memorial Trust international award
for leukemia research (London). DSP is grateful to Council of
Scientific and Industrial Research, New Delhi for financial support
under CSIR-SRF order No. 09/119(0173)2K8 EMR-I
.