Synthesis and cytotoxic evaluation of thiourea and N-bis-benzothiazole derivatives: A novel class of cytotoxic agents

Article abstract of DOI:10.1016/j.bmcl.2011.10.106

Benzothiazolyl thiocarbamides has been achieved using a catalytic amount of 4-dimethylaminopyridine (DMAP) followed by its chemoselective oxidative cyclization with 1,3-di-n-butylimidazolium tribromide[bbim][Br₃] to afford the N-bis-benzothiazole derivatives. All the synthesized compounds were evaluated for cytotoxic activity against two human monocytic cell lines (U 937, THP-1) and a mouse melanoma cell line (B16-F10). Based on their IC₅₀ values, the majority of the benzothiazolyl thiocarbamides and N-bis-benzothiazoles had significant antiproliferative activity on U 937 and B16-F10 cells, the compounds 3b, 3e, 3f, 3k, 6c and 6h were found to be the most active. The present findings indicate clearly that the compound 3e exhibited more antiproliferative activity on U 937 cells than the standard molecule, etoposide. Nevertheless, these compounds have shown comparatively less cytotoxicity towards THP-1 cells.

Full text of DOI:10.1016/j.bmcl.2011.10.106

Bioorganic & Medicinal Chemistry Letters 22 (2012) 453–455
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and cytotoxic evaluation of thiourea and N-bis-benzothiazole
derivatives: A novel class of cytotoxic agents
Ravindra M. Kumbhare a, , Tulshiram Dadmal a, Umesh Kosurkar a, V. Sridhar b, J. Venkateswara Rao b,
⇑
⇑
a Fluoroorganic Division, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India
b Biology Division, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Benzothiazolyl thiocarbamides has been achieved using a catalytic amount of 4-dimethylaminopyridine
(DMAP) followed by its chemoselective oxidative cyclization with 1,3-di-n-butylimidazolium
tribromide[bbim][Br3] to afford the N-bis-benzothiazole derivatives. All the synthesized compounds were
evaluated for cytotoxic activity against two human monocytic cell lines (U 937, THP-1) and a mouse mel-
anoma cell line (B16-F10). Based on their IC50 values, the majority of the benzothiazolyl thiocarbamides and
N-bis-benzothiazoles had significant antiproliferative activity on U 937 and B16-F10 cells, the compounds
3b, 3e, 3f, 3k, 6c and 6h were found to be the most active. The present findings indicate clearly that the com-
pound 3e exhibited more antiproliferative activity on U 937 cells than the standard molecule, etoposide.
Nevertheless, these compounds have shown comparatively less cytotoxicity towards THP-1 cells.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 12 July 2011
Revised 4 October 2011
Accepted 29 October 2011
Available online 6 November 2011
Keywords:
Benzothiazolyl thiocarbamide
N-Bis-benzothiazole
Chemoselective oxidative cyclization
1,3-Di-n-butylimidazolium
tribromide[bbim][Br3]
Cytotoxic activity
Benzothiazoles are an important class of heterocycles, which
can serve as unique and versatile scaffolds for experimental drug
design.1 2-Aminobenzothiazoles have received considerable atten-
tion because of their interesting pharmacological activities includ-
ing anticonvulsant,2 analgesic,3 anti-tumor,4,5 antibacterial,6,7
antimicrobial8,9 and muscle relaxant agents.10 The combinations
of urea and thiourea derivatives with benzothiazole have produced
DNA topoisomerase11,12 or HIV reverse transcriptase inhibitors13,14
for example frentizole (1) (Fig. 1) is a nontoxic antiviral and immu-
nosuppressive agent used clinically in rheumatoid arthritis and
systemic lupus erythematosus15 and thiourea derivatives YH3945
( 2) (Fig. 1), a selective and potent inhibitor of farnesyl-protein
transferase, is being developed for the treatment of cancer.16
Similarly, substituted benzothiazoles such as 2-(3,4-dimethoxy-
phenyl)-5-fluorobenzothiazole (PMX 610) (3) (Fig. 1) has been
shown to exhibit exquisitely potent (GI50 <0.1 nM) and selective
in vitro antitumor properties in human cancer cell (e.g., colon,
nonsmall-cell lung and breast subpanels) lines of the National Can-
cer Institute (NCI) 60 human cancer cell line screen.17 Other benzo-
thiazoles such as, 2-(4-amino-3-methylphenyl) benzothiazole (DF
203) and the 2-(4-amino-3-methylphenyl)-5-fluoro benzothiazole
(5F 203) (4 and 5 of Fig. 1) were found to activate on arylhydrocar-
bon receptor (AhR) via translocation from the cytosol to the
nucleus. Therein the induced cytochrome P450 CYP1A1 enzyme
activity was determined, which subsequently leads to the genera-
tion of a reactive chemical intermediate that selectively generates
DNA adducts only in sensitive tumor types (e.g., mammary and
ovarian tumor cell lines).18 Especially interesting are bis-benzothi-
azole derivatives which exhibit their importance in amyloid-
imaging,19 vulcanization accelerators20 and starting materials for
various pharmaceutical industries.21 In continuation of our
previous studies for the design of novel anticancer agents22–24
and importance to keep in mind the application of thiourea and
N
H
N
H
N
F
N
F
F
S
O
N
H
N
H3CO
N
N
Frentizole (1)
N
S
O
YH 3945 (2)
CH3
NH2
OCH3
OCH3
F
N
S
R
N
S
PMX610 (3)
R = H (DF 203) 4
R = F (5F 203) 5
⇑
Corresponding authors. Tel.: +91 40 27191776; fax: +91 40 27193185.
E-mail addresses: kumbhare@iict.res.in (R.M. Kumbhare), jv@iict.res.in (J.V. Rao).
Figure 1. Chemical structures of biologically important compounds.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.106

454
R. M. Kumbhare et al. / Bioorg. Med. Chem. Lett. 22 (2012) 453–455
-
Br-
benzothiazole derivatives, a new series of benzothiazolyl thiocar-
bamide (3a–3l) and N-bis-benzothiazole (6a–6l) motifs has been
synthesized as anticancer agents.
Br3
No Solvent
0 ºC
N
N
N
N
Br2
4
5
Thiazole ring in benzothiazole moiety is frequently constructed
via an oxidative cyclization of thiobenzanilides using various oxi-
dants, including Jacobson’s and traditional Hugershoff’s meth-
ods.25–27
Scheme 2. Preparation of 1,3-di-n-butylimidazolium tribromide[bbim][Br3].
R2
Methods for synthesis of N-bis-benzothiazoles are scarce in lit-
erature. Chemoselective oxidative cyclization of unsymmetrical
thioureas with N-bromosuccinimide (NBS) as an oxidizing agent
for the synthesis of N-bis-benzothiazole is also effective28 but, this
method is not explored much for different functionalities. Still,
there have been no reports on ionic liquid mediated chemoselec-
tive oxidative cyclization of benzothiazolyl thiocarbamides. In
the view of this, there remains a wide scope to develop a general,
efficient and green method for the synthesis of more functionalized
N-bis-benzothiazol-2-yl amine derivatives. Recently ionic liquid
has gained more attention in synthetic organic chemistry as an
alternative green reaction media.29 Hence, a total of 24 derivatives
were synthesized and evaluated for their in vitro cytotoxic activi-
ties against two human monocytic cell lines (U 937, THP-1) and
a mouse melanoma cell line (B16-F10).
R2
[bbim] [Br3]
N
S
N
S
R1
R1
S
70 ºC,
30-40 min.
N
H
N
H
N
S
N
H
6
3
6a: R1 = H; R2 = H
6c: R1 = 6-F; R2 = F
6e: R1 = 4-Cl; R2 = F
6g: R1 = 6-OEt; R2 = H 6h: R1 = 6-OEt; R2 = F
6i: R1 = 6-F; R2 = H
6k: R1 = H; R2 = F
6b: R1 = H; R2 = Cl
6d: R1 = 6-F; R2 = Cl
6f: R1 = 6-Me; R2 = H
6j: R1 = 4-Cl; R2 = H
6l: R1 = 6-Me; R2 = F
Scheme 3. Chemoselective oxidative cyclization of thiourea using ionic liquid.
The appropriate substituted 2-aminobenzothiazole 1 and com-
mercially available substituted phenyl isothiocyanate 2 were re-
acted in the presence of 4-dimethylaminopyridine (5 mol %) in
N,N-dimethyl formamide to furnish substituted benzothiazolyl thi-
ocarbamides derivative 3 Scheme 1.
Next, we examined the oxidative cyclization of benzothiazolyl
thiocarbamides using new tribromide-based ionic liquid 1,3-di-n-
butylimidazolium tribromide[bbim][Br3] as a reagent of choice.
1,3-Di-n-butylimidazolium bromide 4 on drop wise addition of
molecular bromine under stirring formed exothermally red liquid
1,3-di-n-butylimidazolium tribromide 5 Scheme 2.
Subsequently, the reaction of benzothiazolyl thiocarbamide 3a
was performed in 1,3-di-n-butylimidazolium tribromide[bbim][Br3]
5. The reaction was sluggish at room temperature. Surprisingly reac-
tion proceeded well at 70 °C giving N-bis-benzothiazole 6a exclu-
sively in 92% yield via C–S bond formation. Thus the oxidative
cyclization was found to be chemoselective Scheme 3.
derivatives have shown significant decrease in cell viability in all
the test cell lines on concentration dependent manner (Table 1).
In summary, we have developed a novel protocol for the syn-
thesis of symmetrical as well as unsymmetrical N-bis-ben-
zothiazol-2-yl amines derivatives. An ionic liquid, 1,3-di-n-
butylimidazolium tribromide[bbim][Br3] is found to be an efficient
chemoselective reagent for the oxidative cyclization of benzothiaz-
olyl thiocarbamides. This method can be classified as ‘green’ as it
utilizes mild reaction conditions, eliminates toxic bromine va-
pours, and avoids the use of organic solvents.
Comparison with the IC50 values of positive control (etoposide)
indicated that the THP-1 cells are more sensitive (approximately 9
times) than the other two (U 937 and B16-F10) cell lines. However,
several of these benzothiazolyl thiocarbamides (3a–3l) and N-bis-
benzothiazole (6a–6l) exhibited relatively high activities against
these drug-resistant cancerous cell lines. Comparatively, about
75% of the test compounds (3 and 6 series) have shown cytotoxic
The structure of product 6a was determined on the basis of 1H
NMR spectrum. It shows only two doublets and two triplets with
same coupling constant (J = 7.5 Hz) which are consistent with
structure of 6a. Other benzothiazolyl thiocarbamides also partici-
pated well in this reaction. The scope of above reaction is illus-
trated with respect to different functionalities.
The biological activities of benzothiazolyl thiocarbamide
(3a–3l) and N-bis-benzothiazole (6a–6l) were evaluated to investi-
gate their antiproliferative activities in different types of human
leukemia (THP-1 and U 937) and mouse melanoma (B16-F10) cell
lines. It is evident from the results that the several of the test
activity with IC50 less than 50 lM against THP-1 cells, followed by
46% against U 937 and 30% against B16-F10 cells. However, consid-
ering the IC50 values of the standard molecule, the order of sensitiv-
ity of these cell lines towards benzothiazolyl thiocarbamides and N-
bis-benzothiazole derivatives is U 937 > B16-F10 > THP-1.
This study provides a convenient green method for synthesis of
various benzothiazolyl thiocarbamide (3a–3l) and N-bis-benzothi-
azole (6a–6l) as novel anticancer agents. Further lead optimization
is ongoing.
S
N
N
S
N
4-DMAP (5 mol%)
DMF, r.t., 2-4 h.
N
H
R2
C
NH2
N
H
S
S
R1
R2
R1
1
2
3
3a: R1 = H; R2 = H
3c: R1 = H; R2 = Cl
3e: R1 = 6-F; R2 = F
3g: R1 = 4-Cl; R2 = H
3i: R1 = 6-Me; R2 = H
R1 = H, 6-F, 6-OMe
6-OC2H5, 4-Cl
R2 = H, F, Cl
3b: R1 = H; R2 = F
3d: R1 = 6-F; R2 = H
3f: R1 = 6-F; R2 = Cl
3h: R1 = 4-Cl; R2 = F
3j: R1 = 6-Me; R2 = F
3l: R1 = 6-OEt; R2 = F
3k: R1 = 6-OEt; R2 = H
Scheme 1. Synthesis of benzothiazolyl thiocarbamides.

R. M. Kumbhare et al. / Bioorg. Med. Chem. Lett. 22 (2012) 453–455
455
Table 1
In vitro cytotoxicity of benzothiazolyl thiocarbamides (3a–3l) and N-bis-benzothiazole (6a–6l) derivatives (structural analogues) against U 937, THP-1 and B16-F10 cancer cells
b
b
Compounds
IC50 values in micro molar
Compounds
IC50 values in micro molar
U 937
B16-F10
THP-1
U 937
B16-F10
THP-1
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
—
—
—
—
—
36.49 1.83
26.73 1.34
38.97 1.95
46.60 2.33
34.58 1.73
31.16 1.56
31.79 1.59
—
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
40.04 2.0
—
—
28.76 1.44
45.43 2.27
17.46 0.87
41.01 2.05
36.18 1.81
34.18 1.71
37.09 1.86
38.99 1.95
—
32.05 1.61
—
—
—
27.40 1.37
47.46 2.37
41.94 2.09
—
—
16.23 0.81
18.69 0.94
47.73 2.39
48.13 2.41
—
—
—
48.42 2.42
—
26.58 1.33
40.63 2.03
—
43.52 2.18
—
17.94 0.89
—
—
—
—
—
—
—
—
—
—
33.16 1.66
—
29.46 1.47
—
29.59 1.48
18.69 0.94
—
36.91 1.85
43.77 2.18
39.54 1.98
2.16 0.11
—
—
—
37.72 1.89
28.13 1.41
17.94 0.89
Etoposidea
18.69 0.94
Etoposidea
2.16 0.11
—
Indicates IC50 valves are more than 50
l
M.
a
Standard drug (positive control).
b
IC50 is defined as the concentration, which results in a 50% decrease in cell number as compared with that of the control cultures in the absence of an inhibitor. The values
represent the mean SE of four individual observations.
12. Choi, S.-J.; Park, H. J.; Lee, S. K.; Kim, S. W.; Han, G.; Choo, H.-Y. P. Bioorg. Med.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.106.
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