Synthesis and antitumor activity of optically active thiourea and their 2-aminobenzothiazole derivatives: A novel class of anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2008.12.002

A novel series of optically active 2-aminobenzothiazole derivatives were synthesized by reaction of optically active amine (I) with thiophosgene to obtain optically active isothiocyanates (IIa-h) which on condensation with 4-fluoro-3-chloro aniline (III)

Full text of DOI:10.1016/j.ejmech.2008.12.002

European Journal of Medicinal Chemistry 44 (2009) 2923–2929
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antitumor activity of optically active thiourea and their
2-aminobenzothiazole derivatives: A novel class of anticancer agents
S.N. Manjula a, N. Malleshappa Noolvi b, K. Vipan Parihar a, S.A. Manohara Reddy a, Vijay Ramani a,
,
Andanappa K. Gadad c, Gurdial Singh d, N. Gopalan Kutty a, C. Mallikarjuna Rao a
*
a Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Madhav Nagar, Manipal 576104, Karnataka, India
b Department of Pharmaceutical Chemistry, KLE’s College of Pharmacy, Hubli 580031, Karnataka, India
c School of Pharmacy, Faculty of Medical Sciences, The University of the West Indies, (St. Augustine), Champs Fleurs, Mount Hope, Trinidad and Tobago, WI, USA
d Department of Chemistry, University of the West Indies, Trinidad and Tobago, WI, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of optically active 2-aminobenzothiazole derivatives were synthesized by reaction of
optically active amine (I) with thiophosgene to obtain optically active isothiocyanates (IIa–h) which on
condensation with 4-fluoro-3-chloro aniline (III) yielded various optically active thioureas (IVa–h).
Further oxidative cyclisation in the presence of bromine and chloroform yielded title compounds (Va–h).
The structures of these compounds were established by IR, 1H NMR, 13C NMR, Mass and HRMS. The
compounds (IVa–h and Va–h) were evaluated for in vitro cytotoxicity against mouse Ehrlich Ascites
Carcinoma (EAC) and two human cancer cell lines (MCF-7 and HeLa). In preliminary MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cytotoxicity studies the optically active thiourea
derivatives (IVe, IVf and IVh) were found most effective. In EAC cells the IC50 values for IVe, IVf, IVh and
Received 15 May 2008
Received in revised form
7 November 2008
Accepted 8 December 2008
Available online 16 December 2008
Keywords:
2-Aminobenzothiazoles
Thiourea
Vg were found in the range of 10–24
in the range of 15–30 M and 33–48
IVf) showed dose-dependent DNA damaging activity.
m
M, whereas in MCF-7 and HeLa cells the IC50 values were observed
Comet assay
MTT assay
DNA damage
MCF-7
m
mM, respectively. In alkaline comet assay the compounds (IVe and
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
signaling pathway. The activation 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 [8]. Further, the combinations
of urea and thiourea derivatives with benzothiazoles have produced
DNA topoisomerase [9,10] or HIV reverse transcriptase inhibitors
[11,12]. Based on these reports, we herein report the syntheses and
in vitro evaluation of antitumor activity of a series of novel optically
active thioureas and their derivatives 2-aminobenzothiazole.
The present study involves the syntheses of optically active
thioureas and 2-aminobenzothiazole followed by preliminary
cytotoxicity screening against mouse Ehrlich Ascites Carcinoma
(EAC) and two human cancer cell lines (MCF-7 and HeLa) using MTT
assay at 24 h of exposure. The promising molecules were subjected
to cytotoxicity assay at different time points along with the DNA
damaging potential of two compounds in sensitive cells (MCF-7).
Genotoxicity and cytotoxicity of anticancer drugs to the normal
cells are major problems in cancer therapy and engender the risk of
inducing secondary malignancy [1]. A dose of anticancer drug
sufficient to kill tumor cells is often toxic to the normal tissue and
leads to many side effects, which in turn, limits its treatment effi-
cacy. In recent years, there has been a concerted search for the
discovery and development of novel selective antitumor agents,
devoid of many of the unpleasant side effects of conventional
antitumor agents.
In the efforts to develop drugs with such capabilities, scientists
have focused upon many different aspects of cancer biology during
their research. Among the antitumor drugs discovered in the recent
years, various benzothiazoles [2–4] as well as urea and thiourea
derivatives [5–7] possess potent anticancer properties. Currently,
a benzothiazole derivative, 2-(4-amino-3-methylphenyl)-5-fluo-
robenzthiazole, acting through a novel mechanism, has entered the
clinical trial phase I in UK. It shows high sensitivity towards the
tumor cells by acting through aryl hydrocarbon receptor (AhR)
2. Results and discussion
2.1. Chemistry
We have synthesized newer optically active 2-amino-
benzothiazoles with chiral carbon attached in the side chain shown
* Corresponding author. Tel.: þ91 820 2571201x22482.
E-mail address: cmallikin@yahoo.co.in (C. Mallikarjuna Rao).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.12.002

2924
S.N. Manjula et al. / European Journal of Medicinal Chemistry 44 (2009) 2923–2929
in Scheme 1. The compounds were synthesized using various
optically active isothiocynates (IIa–h), which were prepared from
different optically active aromatic and aliphatic primary amines
[13]. Prepared isothiocynates yielded thioureas (IVa–h) on
condensation with 4-fluoro-3-chloro-aniline (III). Oxidative cycli-
sation by bromine resulted in the synthesis of proposed
compounds (Va–h). The derivatives were characterized by spectral
studies using IR, 1H NMR, 13C NMR, DIPMS and HRMS. Optical
rotation was measured by using polarimeter.
–NH–CS–NH– by a peak at d 181–179 ppm corresponding to >C]S.
The mass spectra showed Mþ peak at respective molecular weights
of the compounds. Some of them were subjected to HRMS to obtain
respective molecular weights.
Optically active novel 2-aminobenzothiazoles showed IR
absorption band at 3309, 2867,1629 cmꢀ1. 1H NMR spectra revealed
all the corresponding peaks at
4.60 ppm, proton of chiral carbon, broad peak at
–NH. Aromatic proton showed peaks at
7.30–7.00 ppm. 13C NMR
d
1.25–1.00 ppm, –CH3,
d 4.85–
d
6.90–5.99 ppm,
d
The structures of optically active thiocynates were confirmed
through the following spectral data. IR absorption peak at 2000–
2200 cmꢀ1 corresponding to –SCN, 1H NMR showing a doublet at
gave valuable information to confirm cyclisation of substituted
thioureas to respective substituted 2-aminobenzothiazoles with
characteristic peak at d 165–155 ppm for C-2. Further HRMS gave all
d
2.00–1.40 ppm corresponding to –CH3 on chiral carbon,
4.65 ppm corresponding proton on same chiral carbon,
3.84 ppm for –OCH3 and
d
4.90–
3.90–
the Mþ ion peaks corresponding to molecular weight of confirmed
novel compounds.
d
d
7.33–6.90 ppm for aromatic protons.
Thioureas were confirmed by the absence of characteristic IR
absorption peak at 2000–2200 cmꢀ1 –SCN group. Bands at 1600–
1610 cmꢀ1 confirmed formation of thioureas –NH–CS–NH–. 1H
NMR spectra elaborated the up field shift in resonance of proton
2.2. Preliminary cytotoxicity and time course MTT assay
In vitro cytotoxicity of synthesized compounds was assessed by
standard MTT bioassay in different cancer cells at 24 h of drug
exposure. In EAC cells the optically active thioureas were found to
be more effective when compared with benzothiazole derivatives.
To assess the efficacy and to select the promising compounds
human cancer cells (MCF-7 and HeLa cells) were used.
attached to chiral carbon atom from
d
4.90–4.65 ppm to
d 6.00–
5.00 ppm. Further occurrence of two broad peaks at
d
8.31 and
6.23 ppm corresponding two –NH groups substantiated the
formation of thioureas. 13C NMR confirmed conversion of –SCN to
Scheme 1. Synthetic pathway of novel optically active thioureas and their derivatives, 2-aminobenzothiazoles.

S.N. Manjula et al. / European Journal of Medicinal Chemistry 44 (2009) 2923–2929
2925
Table 1
Table 1 (continued)
Characteristics and preliminary cytotoxicity screening of synthesized compounds by
MTT assay at 24 h of drug exposure.
Compounds
R
R0
IC50
EAC
(mM)
MCF-7
29.80
HeLa
Cl
H
Cl
N
S
S
C
H
N
H
N
R
H
Vg
–CH3
24.62
48.01
F
N
H
F
R
Vh
20.63
4.18
40.56
2.62
60.81
3.24
R'
Cl
R'
Doxorubicin
S
IV a-g
V a-g
NH C NH
F
Compounds
R
R0
IC50
EAC
(mM)
O
MCF-7
26.08
HeLa
The known numbers of cells (1.0 ꢁ 104) were incubated for 24 h in a 5% CO2 incu-
bator at 37 ꢂC in the presence of different concentrations of test compounds. After
24 h of drug incubation the MTT solution was added and supernatant was discarded
IVa
IVb
IVc
IVd
IVe
–CH3
23.56
80.25
45.64
29.94
17.32
66.21
and 100 ml DMSO was added in each well and absorbance was recorded at 540 nm by
ELISA reader.
–CH2CH3
>100
31.39
34.15
23.23
m
M
NTa
Concentration required to inhibit the 50% cell growth. Experiment was performed in
triplicate.
a
NT: Not tested.
OCH3
–CH3
77.53
71.43
42.99
In MCF-7 cells, the IC50 values for benzothiazole and thiourea
derivatives were in the range of 29.80–42.73 and 20.56–
34.15
values for benzothiazole and thiourea derivatives were found in
the range of 48.01–72.54 and 33.81–77.53 M, respectively
(Table 1). In MCF-7 cells, the compounds Vc and IVb did not
show 50% inhibition even at a concentration of 100 M; hence
mM, respectively (Table 1). Similarly in HeLa cells, the IC50
F
–CH3
m
m
Cl
–CH3
they were not evaluated in HeLa cells. Present study reveals that
among the human cancer cell lines tested, MCF-7 cells are more
sensitive to all the tested compounds than HeLa cells. Many
anticancer drugs are effective against MCF-7 and HeLa cells by
causing apoptosis through the expression of caspase-3, gener-
ating reactive oxygen species (ROS) and damaging DNA [14].
Cisplatin causes cytotoxicity in MCF-7 and HeLa cells by a
similar mechanism [15] Chemotherapeutic agents such as
doxorubicin, mitoxantrone and bleomycin cause cytotoxicity by
generating ROS [16]. Hence like other cytotoxic drugs (doxoru-
bicin, mitoxantrone, bleomycin and cisplatin), the synthesized
compounds may act as effective anticancer drugs by similar
mechanism. Previous studies have shown that strong electro-
negative atom substitution such as chloro/bromo at the para
position of the aromatic ring increases the lipophilicity of
molecules and is responsible for enhanced cytotoxicity in MTT
model [17]. Similar substitutions are present in the compounds
IVe, IVf, IVh and Vg. We have also observed enhanced cyto-
toxicity in these molecules. Hence these molecules were taken
up to assess the cytotoxic potency at different intervals in MCF-7
cells.
IVf
IVg
–CH3
–CH3
15.11
25.04
25.71
45.75
49.09
25.48
Cl
F
S
NH C NH
O
IVh
10.63
20.56
33.81
Va
Vb
–CH3
36.64
32.22
42.73
39.30
58.93
53.26
–CH2CH3
In the MTT time course study, the selected compounds (IVe,
IVf, IVh and Vg) showed dose-dependent and time-dependent
activities. The previous study reported that the most potent
fluorinated benzothiazole, 5F 203, produced apoptosis and DNA
damage in MCF-7 cells, which is characteristic of cytotoxic activity
[18,19] In our present study also MTT assays revealed substantial
cytotoxicity in MCF-7 cells with increasing exposure to drug
concentration, the IC50 values of promising compounds at 48 and
72 h were significantly reduced as compared with 24 h values
(Table 2).
OCH3
Vc
–CH3
–CH3
75.44
35.35
>100
mM
NTa
F
Vd
42.32
61.93
Cl
Ve
Vf
–CH3
–CH3
25.47
29.78
33.00
32.90
69.69
72.54
2.3. Effect on DNA damage
A number of cytotoxic compounds (cisplatin, doxorubicin, etc.) act
as anticancer drugs by causing DNA damage and subsequently

2926
S.N. Manjula et al. / European Journal of Medicinal Chemistry 44 (2009) 2923–2929
Table 2
Table 4
Cytotoxic activity of promising compounds in MCF-7 cells at different time points of
drug exposure by MTT assay.
The DNA damaging activity of IVf in MCF-7 cells at 24 h of drug exposure by comet
assay.a
Compounds
IC50 (
m
m)
Compounds
Control
Tail moment
OTM
Tail length
Tail DNA
7.18 ꢃ 1.05
18.91 ꢃ 1.45
35.24 ꢃ 2.03b
43.45 ꢃ 2.47c
8.83 ꢃ 0.83
65.56 ꢃ 3.23
130.02 ꢃ 4.47b
202.92 ꢃ 6.35c
210.31 ꢃ 7.12c
9.75 ꢃ 1.16
14.11 ꢃ 0.75
17.12 ꢃ 0.72b
20.41 ꢃ 0.72c
24 h
48 h
72 h
5
mM
13.31 ꢃ 0.59
18.53 ꢃ 0.99
22.13 ꢃ 1.34
IVh
IVe
IVf
Vg
20.56
23.23
25.04
29.80
12.80
16.27
18.91
24.84
8.61
11.40
13.98
15.00
10
25
50
m
m
m
M
M
M
121.88 ꢃ 15.55c 146.48 ꢃ 26.28c 319.90 ꢃ 33.40c 37.63 ꢃ 2.68c
DO ꢁ 100 nm
47.67 ꢃ 2.74c
36.55 ꢃ 1.61
149.04 ꢃ 4.61c
31.14 ꢃ 1.15c
The IC50 values of promising compounds at 48 h and 72 h were significantly reduced
in comparison with 24 h values. The most promising compound (IVh) causes 1.66
and 2.5 time reduction in IC50 at 48 h and 72 h.
a
All values are means ꢃ SEM.
P < 0.01 compared to control.
P < 0.001 compared to control.
b
c
inducing apoptosis in cancerous cells. To determine the DNA
damaging activity of synthesized compounds, alkaline comet assay
was performed in MCF-7 cells. Comet assay is a rapid and inexpensive
method for measuring DNA single-strand breaks (SSBs). It also has an
advantage over the other DNA damage-detecting methods, such as
sister chromatid exchange, alkali elution, and micronucleus assay,
because of its high sensitivity [20]. 24 h treatment with IVe and IVf
produced a dose-dependent increase in tail moment. The damage
was that the intermediate thioureas (IVe, IVf and IVh) were more
active than corresponding title compounds. The promising
compounds, IVe and IVf showed significant DNA damage in MCF-
7cells. The DNA damaging activity of the molecules might be due to
the generation of ROS/highly reactive intermediates. Further
studies are in progress to elucidate the molecular mechanism
responsible for their cytotoxicity and DNA damaging effect.
produced by IVf was quite prominent as at 50 mM it produced
4. Experimental section
apoptotic bodies and tail moment above 100. Elongated tail length
and reduced DNA content in head are sufficient indicators of DNA
damage, and higher degrees of damage would result in greater
number of smaller fragments, ending up with longer tails. The SSB in
DNA results from a number of different reaction types including base
and nucleotide excision repairs, direct scission of the DNA backbone
by chemical or radical attack, strand breakage following binding of
intercalating agents and alkali-labile DNA adducts [21–23]. Both the
studied compounds significantly increased the tail moment and tail
length in MCF-7 cells. Previous studies have shown that cytotoxicity
of benzothiazoles is mediated via activation of the AhR signaling
pathway in sensitive MCF-7 cells [18,24,25]. The activation of AhR
pathway leads to generation of reactive electrophilic species by
inducing CYP1A1 expression. The generated highly reactive inter-
mediates cause DNA damage, ultimately resulting in cell death by
activation of apoptotic machinery [8,26]. Hence our compounds may
be acting by similar mechanism(s) which, however, need to be
confirmed by gene expression studies. (Tables 3 and 4).
4.1. Chemistry
1H NMR spectra were measured at 300 MHz with a JEOL GSX
270FT NMR spectrometer. Chemical shifts were measured relative
to internal standard TMS (
67.8 MHz on the same instrument with internal TMS (
d
: 0). 13C NMR spectra were recorded at
d
: 0, CDCl3).
IR spectra were recorded on an UNICAM series FT-instrument. Mass
spectra were recorded on AEI MS 902 or VG ZAB-E-instruments.
Microanalyses were performed by MEDAC Ltd., Surrey. Melting
points were determined on Gallen Kamp capillary melting point
apparatus and are uncorrected. Optical rotations were measured in
chloroform solution using a Bellingham and Stanley ADP 220
polarimeter. Flash chromatography was performed using Fluka
silica gel 60 (230–400 mesh). Thin layer chromatography was
carried out using pre-coated aluminum plates (Merck Kieselghur
60 F254) which were visualized under UV light and then with either
phosphomolybdic acid or basic aqueous potassium permanganate
as appropriate. All anhydrous reactions were carried out under
argon or nitrogen. Anhydrous transfers were done with standard
syringe techniques: all glasswares were pre-dried overnight.
Dichloromethane was distilled from calcium hydride and stored
over 4 Å molecular sieves.
3. Conclusion
In the present study, we have described the syntheses and in
vitro cytotoxicity screening of optically active thioureas and their
derivatives, 2-aminobenzothiazoles. Various aromatic, alicylic and
aliphatic substitutions were considered for synthesis to draw the
structure–activity relationship studies in relation to cytotoxic
activity. An interesting observation made during cytotoxic study
4.1.1. General procedure for the synthesis of optically active
substituted isothiocyanates (IIa–h, Scheme 1) [13]
To the solution of thiophosgene (0.01 mol) in CH2Cl2, optically
active substituted primary amine (Ia–h) (0.01 mol) was added
quickly at room temperature under stirring. Then a 25 ml satu-
rated solution of NaHCO3 was added slowly and stirred for 3 h. The
CH2Cl2 layer was dried over anhydrous sodium sulphate and
removed under reduced pressure. The residue was purified by
flash chromatography using silica gel and petroleum ether:ether
(40:1).
Table 3
The DNA damaging activity of IVe in MCF-7 cells at 24 h of drug exposure by comet
assay.a
Compounds
Control
Tail moment
OTM
Tail length
Tail DNA
7.18 ꢃ 1.05
8.31 ꢃ 0.98
8.83 ꢃ 0.83
65.56 ꢃ 3.23
71.89 ꢃ 4.16
9.75 ꢃ 1.16
11.21 ꢃ 0.94
16.90 ꢃ 1.23c
18.80 ꢃ 1.38d
29.89 ꢃ 1.62d
31.14 ꢃ 1.15d
5
m
M
10.23 ꢃ 0.72
22.46 ꢃ 5.67
25.71 ꢃ 7.29
37.12 ꢃ 2.57d
36.55 ꢃ 1.61d
10
25
50
m
m
m
M
M
M
18.62 ꢃ 2.58
22.00 ꢃ 3.10b
64.04 ꢃ 5.27d
47.67 ꢃ 2.74d
102.56 ꢃ 6.42d
110.70 ꢃ 7.51d
205.23 ꢃ 7.09d
149.04 ꢃ 4.61d
4.1.2. General procedure for the synthesis of optically active
substituted thioureas (IVa–h, Scheme 1)
To a solution of II (0.01 mol) in dry MeOH (10 ml), an equimolar
quantity of aromatic primary amine (0.01 mol) was added with
stirring. The reaction mixture was heated on a steam bath at 70 ꢂC
for about 1 h and then the solvent was distilled off. The solid
residue that separated was washed with water and dried at
DOX 100 nm
a
All values are means ꢃ SEM.
P < 0.05 compared to control.
P < 0.01 compared to control.
b
c
d
P < 0.001 compared to control.

S.N. Manjula et al. / European Journal of Medicinal Chemistry 44 (2009) 2923–2929
2927
40 ꢂC. The resulting compounds were purified over silica gel
using CHCl3:EtOAc (7:3) to yield pure optically active thiourea
derivatives.
4.1.3.6. 1-(3-Chloro-4-fluorophenyl)-3-((S)-1-cyclohexylethyl)thiourea
(IVf). The compound was prepared as per the general procedure
mentioned above purified and isolated as colourless solid; yield
20
75%; mp 74–75 ꢂC; [
a]
¼ ꢀ45.7 (c1.05 CHCl3); IR (KBr) vmax 3237,
D
4.1.3. General procedure for the synthesis of optically active
substituted 2-aminobenzothiazoles (Va–h, Scheme 1)
3052, 2933, 2850,1608,1544 cmꢀ1; 1H NMR (CDCl3)
s, 1H, NH), 7.33–7.14 (m, 3H, Ar-H), 5.78 (br s, 1H, NH), 4.37 (br s,
1H), 1.77–1.05 (m, 14H); 13C NMR (CDCl3)
ppm; 180.0, 158.4, 155.0,
d ppm; 8.25 (br
Optically active substituted thioureas (0.01 mol) were dissolved
in chloroform (15 ml), the reaction mixture was cooled in an ice
bath and then bromine:chloroform (1:9) mixture was added drop
wise. The reaction was monitored by TLC and after an hour, was
poured on to crushed ice. The solid that separated was filtered,
dried in each case and then purified by column chromatography
using silica gel. It was eluted with CHCl3:EtOAc (7:3) and the
eluents on evaporation and crystallization yielded pure optically
active substituted 2-aminobenzothiazoles.
d
133.0, 127.8, 125.3, 117.6, 55.7, 42.9, 29.2, 29.0, 26.3, 26.1, 26.0, 17.2;
DIPMS 315.1 (Mþ).
4.1.3.7. 1-(3-Chloro-4-fluorophenyl)-3-((R)-octan-2-yl)thiourea
(IVg). The compound was prepared as per the general procedure
mentioned above purified and isolated as colourless solid; yield
20
74%; mp 60–61 ꢂC; [
a
]
¼ ꢀ41.1 (c1.24 CHCl3); IR (KBr) vmax 3224,
D
3045, 2927, 2856,1600,1544 cmꢀ1; 1H NMR (CDCl3)
d ppm; 8.45 (br
s, 1H, NH), 7.35–7.18 (m, 3H, Ar-H), 5.75 (br s, 1H, NH), 4.47 (br s,
4.1.3.1. 1-(3-Chloro-4-fluorophenyl)-3-((R)-1-phenylethyl)thiourea
(IVa). This compound was prepared as per the above-mentioned
1H), 1.31–1.24 (m, 10H), 1.21 (d, 3H, J ¼ 6.4 Hz), 0.91 (m, 3H); 13C
NMR (CDCl3)
d 179.7, 158.4, 133.0, 127.8, 125.3, 122.2, 117.5, 51.5,
procedure purified and isolated as pale yellow liquid: yield 64%;
36.4, 31.6, 29.0, 25.8, 22.5, 20.3, 14.0 ppm; DIPMS 317.1 (Mþ).
20
mp 110 ꢂC [
a
]
D
¼ þ4.2(c1.20 CHCl3); IR (KBr) vmax 3130, 2928,
2831, 2120, 1610 cmꢀ1
;
1H NMR (CDCl3)
d
ppm; 7.70 (br s, 1H,
4.1.3.8. 1-(2-(Benzyloxy)cyclohexyl)-3-(3-chloro-4-fluorophenyl)thiourea
NH), 7.62–7.29 (m, 5H, Ar-H), 6.25 (br s, 1H, NH), 4.65 (m, 1H), 3.62
(IVh). The compound was prepared as per the general procedure
(m, 1H), 3.44 (m, 2H), 1.76–1.25 (m, 8H); 13C NMR (CDCl3)
mentioned above purified and isolated as colourless solid: yield
20
d
ppm; 138.5, 132.0, 129.0, 128.0, 128.2, 80.9, 60.5, 32.0, 31.3, 30.0,
61%; mp 112–113 ꢂC; [
a]
¼ þ75.4 (c1.18 CHCl3); IR (KBr) vmax
D
23.7, 23.4.
3232, 3035, 2944,1594,1556 cmꢀ1; 1H NMR (CDCl3)
d ppm; 9.39 (br
s, 1H, NH), 7.37–7.21 (m, 3H, Ar-H), 7.79–7.95 (m, 5H, Ar-H), 6.25 (br
s, 1H, NH), 4.76 (d, 3H, J ¼ 6.0 Hz, 1H), 4.44 (d, 3H, J ¼ 6.2 Hz, 1H),
3.43 (m, 2H), 2.36–1.27 (m, 8H); HRMS (EI) m/z calculated for
C20H22ClFN2OS: 393.1198; found: 393.1198.
4.1.3.2. 1-(3-Chloro-4-fluorophenyl)-3-((R)-1-phenylpropyl)thiourea
(IVb). The compound was prepared as per the general procedure
mentioned above purified and isolated as colourless solid; yield
20
60%; mp 210–211 ꢂC; [
a
]
D
¼ ꢀ50.4 (c1.30 CHCl3); IR (KBr) vmax
3224, 3041, 2967, 1600, 1542 cmꢀ1; 1H NMR (CDCl3)
d
ppm; 7.77 (br
4.1.3.9. 5-Chloro-6-fluoro-N-((R)-1-phenylethyl)benzo[d]thiazol-2-
amine (Va). The compound was prepared as per the general
s, 1H, NH) 7.38–7.28, (m, 3H, Ar-H), 7.15–7.07, (m, 5H, Ar-H), 6.30 (br
s, 1H, NH), 5.26 (1H), 1.09 (m, 2H), 0.92 (t,3H, J ¼ 7.25 Hz,
procedure mentioned above purified and isolated as colourless
20
J ¼ 7.23 Hz); 13C NMR (CDCl3)
d
ppm; 185.5, 155.2, 140.6, 133.1,
solid; yield 55%; mp 109–110 ꢂC; [
a
]
D
¼ þ11.2 (c1.06 CHCl3); IR
129.0, 128.0, 127.7, 126.5, 125.3, 125.2, 117.7, 60.6, 29.8, 10.5.
(KBr) vmax 3303, 2975, 1629, 1563 cmꢀ1; 1H NMR (DMSO-d6)
d ppm;
7.62–7.04 (m, 7H, Ar-H) 6.88, (br s, 1H, NH), 4.89, (m, 1H), 1.41 (d,
4.1.3.3. 1-(3-Chloro-4-fluorophenyl)-3-((R)-1-(4-methoxyphenyl)ethyl)
thiourea (IVc). The compound was prepared as per the general
procedure mentioned above purified and isolated as colourless solid;
yield 68%; mp 29–30 ꢂC; IR (KBr) vmax 3326, 2969, 1637, 1598, 1563,
3H, J ¼ 6.9 Hz); 13C NMR (DMSO-d6)
d ppm; 156.2, 155.3, 154.8,
145.0, 137.7, 129.0, 127.4, 126.4, 120.5, 117.9, 116.7, 49.5, 23.2; HRMS
(EI) m/z calcd for C15H12ClFN2S: 307.0467; found: 307.0462.
1496 cmꢀ1; 1H NMR (CDCl3)
d
ppm; 7.30–7.27 (m, 3H, Ar-H), 7.14–6.82
(m, 4H, Ar-H), 6.31 (br s, 1H, NH), 5.50 (b, 1H), 3.84 (s, 3H), 1.54 (d, 3H),
3.43; 13C NMR (CDCl3)
ppm; 180.1, 160.0, 158.3, 155.0, 143.5, 133.2,
4.1.3.10. 5-Chloro-6-fluoro-N-((R)-1-phenylpropyl)benzo[d]thiazol-2-
amine (Vb). The compound was prepared as per the general
d
procedure mentioned above purified and isolated as colourless
20
130.0, 127.7, 125.2, 118.2, 113.0, 55.3, 21.7; HRMS (EI) m/z calcd for
C16H16ClFN2OS: 339.0729; found: 339.0730.
solid; yield 40%; mp 159–160 ꢂC; [
a
]
¼ þ10.5 (c1.04 CHCl3); IR
D
(KBr) vmax 3153, 3070, 2929, 1619, 1519 cmꢀ1
ppm; 9.20 (br s, 1H, NH), 7.90–7.32, (2H, Ar-H), 7.29–7.01, (5H, Ar-
H), 4.90, (1H), 1.98, (m, 2H), 0.99, (3H); 13C NMR (DMSO-d6)
ppm;
167.0, 153.7, 142.5, 130.6, 128.5, 127.3, 126.6, 117.7, 116.7, 114.4, 110.0,
60.3, 29.7, 10.9; 19F NMR
;
1H NMR (DMSO-d6)
d
4.1.3.4. 1-(3-Chloro-4-fluorophenyl)-3-((R)-1-(4-fluorophenyl)ethyl)
thiourea (IVd). The compound was prepared as per the general
d
procedure mentioned above purified and isolated as colourless
d
ꢀ124.3, 126.6 ppm; HRMS (EI) m/z calcd
20
solid; yield 70%; mp 55–56 ꢂC; [
a
]
D
¼ ꢀ52.2 (c1.18 CHCl3); IR (KBr)
for C16H14ClFN2S: 321.0623; found: 321.0618.
vmax 3224, 3033, 2969, 1602, 1535 cmꢀ1 1H NMR (CDCl3)
; d ppm;
8.31 (br s, 1H, NH), 7.31–7.00 (m, 7H, Ar-H), 6.23 (br s,1H, NH), 5.56
4.1.3.11. 5-Chloro-6-fluoro-N-((R)-1-(4-methoxyphenyl)ethyl)benzo[d]
thiazol-2-amine (Vc). The compound was prepared as per the
(br s, 1H), 1.55 (d, 3H, J ¼ 6.7 Hz); 13C NMR (CDCl3)
d ppm; 179.9,
163.8, 160.5, 158.4, 137.7, 127.7, 125.1, 117.8, 117.5, 115.5, 53.7, 21.6;
general procedure mentioned above purified and isolated as col-
20
DIPMS 327.0 (Mþ).
ourless solid; yield 38%; mp 119–120 ꢂC; [
a
]
¼ þ11.9 (c1.00
D
CHCl3); IR (KBr) vmax 3421, 3153, 3077, 2850, 1627, 1525 cmꢀ1
;
1H
4.1.3.5. 1-(3-Chloro-4-fluorophenyl)-3-((R)-1-(4-chlorophenyl)ethyl)
thiourea (IVe). The compound was prepared as per the general
NMR (DMSO-d6) ppm; 7.82–7.22 (6H, Ar-H), 5.70 (br s, 1H, NH),
4.63 (br s, 1H), 3.98 (s, 3H, OCH3), 1.62 (d, 3H, J ¼ 6.8 Hz, 3H); HRMS
(EI) m/z calcd for C16H14ClFN2OS: 336.0499; found: 336.0478.
d
procedure mentioned above purified and isolated as colourless
20
solid; yield 55%; mp 58–60 ꢂC; [
a
]
¼ ꢀ77.2 (c1.23 CHCl3); IR (KBr)
D
vmax 3218, 3035, 1598, 1542, 1496 cmꢀ1
;
1H NMR (CDCl3)
d
ppm;
4.1.3.12. 5-Chloro-6-fluoro-N-((R)-1-(4-fluorophenyl)ethyl)benzo[d]
thiazol-2-amine (Vd). The compound was prepared as per the
8.20 (br s, 1H, NH), 7.33–7.28 (m, 3H, Ar-H), 7.23–7.09 (m, 4H, Ar-H),
6.18 (br s, 1H, NH), 5.57 (1H), 1.51 (d, 3H, J ¼ 6.7 Hz); 13C NMR
general procedure mentioned above and isolated as colourless solid;
20
(CDCl3)
d
ppm; 180.1, 158.5, 155.2, 140.5, 133.5, 129.0, 127.5, 125.3,
yield 45%; mp 124–125 ꢂC; [
a]
¼ þ20.8 (c1.25 CHCl3); IR (KBr) vmax
D
125.2, 117.8, 117.5, 53.7, 21.5; DIPMS 343.0 (Mþ).
3405, 2915, 1627, 1511 cmꢀ1; 1H NMR (DMSO-d6)
d ppm; 7.88–6.96

2928
S.N. Manjula et al. / European Journal of Medicinal Chemistry 44 (2009) 2923–2929
(6H, Ar-H), 6.85 (br s, 1H, NH), 4.84 (m, 1H), 1.64 (d, 3H, J ¼ 6.6 Hz,
3H); 13C NMR (DMSO-d6)
ppm; 155.5, 136.5, 135.0, 130.0, 129.0,
128.0, 127.9, 120.1, 118.0, 116.9, 116.3, 116.0, 115.6, 111.1, 49.0, 23.5;
for 12–14 days in the peritoneal cavity of Swiss albino mice. The
tumor cell cultures were started from mouse Ehrlich Ascites with at
least one passage in vitro prior to use.
d
HRMS (EI) m/z calcd for C15H11ClF2N2S: 325.0372; found: 325.0381.
4.2.2. In vitro cytotoxic activities (MTT assay)
4.1.3.13. 5-Chloro-N-((R)-1-(4-chlorophenyl)ethyl)-6-fluorobenzo[d]
thiazol-2-amine (Ve). The compound was prepared as per the
In vitro cytotoxicity was determined using a standard MTT assay
[27] with protocol appropriate for the individual test system. In
brief, exponentially growing cells were plated in 96-well plates (104
cells/well in 100 ml of medium) and incubated for 24 h for attach-
ment. Test compounds were prepared prior to the experiment by
dissolving in 0.1% DMSO and diluted with medium. The cells were
general procedure mentioned above purified and isolated as col-
20
ourless solid; yield 35%; mp 149–150 ꢂC; [
a]
¼ þ14.9 (c1.00
D
CHCl3); IR (KBr) vmax 3421, 3153, 3077, 2852, 1623, 1525 cmꢀ1
;
1H
NMR (DMSO-d6) ppm; 7.82–7.22 (6H, Ar-H), 5.70, (br s, 1H, NH),
d
4.63, (br s, 1H), 1.62, (d, 3H, J ¼ 6.7 Hz, 3H); HRMS (EI) m/z calcd for
then exposed to different concentrations of the drugs (1–100
in the volume of 100 l/well. Cells in the control wells received the
same volume of medium containing 0.1% DMSO. After 24 h, the
medium was removed and cell cultures were incubated with 100
MTT reagent (1 mg/ml) for 4 h at 37 ꢂC. The formazan produced by
the viable cells was solubilized by addition of 100 l DMSO. The
suspension was placed on micro-vibrator for 5 min and absorbance
was recorded at 540 nm by the ELISA reader. The experiment was
performed in triplicate. The percentage cytotoxicity was calculated
using the formula
mM)
C15H11Cl2FN2S: 341.0077; found: 341.0078.
m
4.1.3.14. 5-Chloro-N-((R)-1-cyclohexylethyl)-6-fluorobenzo[d]thiazol-
2-amine (Vf). The compound was prepared as per the general
ml
procedure mentioned above purified and isolated as colourless
m
20
solid; yield 51%; mp 134–135 ꢂC; [
a]
¼ þ26.4 (c1.06 CHCl3); IR
D
(KBr) vmax 3424, 3158, 3091, 2927, 2852, 1629 cmꢀ1
;
1H NMR
(DMSO-d6) ppm; 9.50 (br s, 1H, NH), 7.78 (1H, Ar-H), 7.54 (1H,
d
Ar-H), 3.90 (1H), 1.79–1.50 (m, 3H), 1.25 (d, 3H, J ¼ 6.7 Hz, 3H),
ðControl abs ꢀ Blank absÞ ꢀ ðTest abs ꢀ Blank absÞ
% Cytotoxicity ¼
ꢁ 100
ðControl abs ꢀ Blank absÞ
1.23–0.99 (m, 8H); 13C NMR (DMSO-d6)
115.9, 114.6, 111.3, 110.5, 56.0, 28.6, 25.2, 25.5, 17.0,16.9; 19F NMR
ppm; 126.3; HRMS (EI) m/z calcd for C15H18ClFN2S: 313.0936;
d
ppm; 166.7, 145.6, 129.3,
For MTT time course study, MCF-7 cells (5 ꢁ103cells/well)
seeded in 96-well plates were exposed to different concentrations
d
of test compounds (1–50 mM). The percentage cytotoxicity and IC50
found: 313.0940.
values were determined at 24, 48 and 72 h of drug incubation.
4.1.3.15. 5-Chloro-6-fluoro-N-(R)-(octan-2-yl)benzo[d]thiazol-2-amine
(Vg). The compound was prepared as per the general procedure
4.2.3. DNA damaging activity (alkaline comet assay)
The effect of promising compounds on DNA was assessed by
comet assay as described by protocol [28] with slight modifications.
Briefly, after the drug treatment, cells were harvested from each
culture flask and counted by trypan blue exclusion method. About
mentioned above purified and isolated as colourless solid; yield
20
35%; mp 119–120 ꢂC; [
a
]
D
¼ þ41.4 (c1.11 CHCl3)IR (KBr) vmax 3154,
3068, 2925, 1627, 1523, 1471 cmꢀ1; 1H NMR (DMSO-d6)
(br s, 1H, NH), 7.37 (s, 1H, Ar-H), 6.93 (s, 1H, Ar-H), 5.54 (m, 1H), 2.97
(3H), 1.53–0.77 (m, 13H); 13C NMR(DMSO-d6)
ppm; 155.6, 137.8,
d ppm; 8.04
20,000 cells in 50 ml medium was suspended in 150 ml of low
d
melting agarose (0.75%) and layered onto slides pre-coated with an
earlier layer of agarose (1.5%). Slides were kept in lysing solution
(2.5 M NaCl, 100 mM EDTA, 10 mM Tris; pH 10–10.5; 1% Triton X-
100 and 10% DMSO) overnight at 4 ꢂC in dark, these were subjected
to unwinding under alkaline conditions (pH 13) for 1 h to allow
DNA supercoils to relax and express DNA single-strand breaks and
alkali-labile sites. Electrophoresis was then carried out under
highly alkaline (pH 13) conditions for 20 min at 16 V and 300 mA.
After neutralization, staining was done using ethidium bromide.
Two slides were prepared for each concentration. About 50 cells
were captured per slide using fluorescent microscope and the
images were analyzed using Komet 5.5 software (Kinetic imaging
systems, UK). A variety of objective measurements like Head DNA,
Tail DNA, and Olive Tail Moment (OTM) were made. Since the Tail
moment has been regarded as one of the best indices of DNA
damage among the other parameters, it was calculated as follows:
120.7, 120.5, 118.9, 117.9, 116.6, 52.1, 40.5, 39.6, 32.1, 29.5, 26.3, 21.1,
14.5; HRMS (EI) m/z calcd for C15H20ClFN2S: 315.1093; found:
315.1085.
4.1.3.16. N-(1R,2S)-2-(Benzyloxycyclohexyl)-5-chloro-6-fluorobenzo[d]
thiazol-2-amine (Vh). The compound was prepared as per the
general procedure mentioned above purified and isolated as col-
20
ourless solid: yield 41%; mp 102–103 ꢂC; [
a
]
¼ þ70.4 (c1.19
D
CHCl3); IR (KBr) vmax 3232, 3035, 2944, 1594, 1556 cmꢀ1
(CDCl3) ppm; 9.39 (br s, 1H, NH), 7.82–7.22 (m, 2H, Ar-H), 7.19 (m,
5H, Ar-H), 4.70 (d, 3H, J ¼ 4.7 Hz, 1H), 4.40 (d, 3H, J ¼ 4.6 Hz, 1H),
3.42 (m, 2H), 2.36–1.27 (m, 8H); 13C NMR (DMSO-d6)
ppm; 168.6,
;
1H NMR
d
d
150.8, 146.7, 137.5, 128.9, 127.8, 124.6, 122.6, 117.6, 107.6, 75.1, 72.5,
56.6, 29.1, 28.5, 23.3, 22.1; HRMS (EI) m/z calculated for
C20H20ClFN2OS: 390.1198; found: 390.1194.
Tail length ꢁ %Tail DNA
Tail moment ¼
4.2. Pharmacology
100
4.2.1. Cell lines
Acknowledgement
Human cancer cell lines, MCF-7 and HeLa cells, procured from
National Centre for Cell Sciences, Pune, India, were cultured in
One of the authors, S.N. Manjula, is grateful to the Department of
Science and Technology, Goverment of India for providing financial
support to carry out this project (Research grant no. SSR/WOS-A/
LS-37/2007) and also to Dr. B.S. Satish Rao, Professor and Head,
MEM medium supplemented with 10% FBS, 1%
50 g/ml gentamicin sulphate in a CO2 incubator in a humidified
atmosphere of 5% CO2 and 95% air. The EAC cells were maintained
L glutamine and
m

S.N. Manjula et al. / European Journal of Medicinal Chemistry 44 (2009) 2923–2929
2929
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