Synthesis of amide and urea derivatives of benzothiazole as Raf-1 inhibitor

Article abstract of DOI:10.1016/j.ejmech.2007.10.008

A series of amide and urea derivatives of benzothiazole have been synthesized and evaluated for their antiproliferative profile in human SK-Hep-1 (liver), MDA-MB-231 (breast), and NUGC-3 (gastric) cell lines. Among them, compounds 1-2, 16-18, 23, and 25-26 had potent to moderate inhibitory activities. Further these compounds were investigated for their ability to inhibit Raf-1 activity.

Full text of DOI:10.1016/j.ejmech.2007.10.008

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1519e1524
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of amide and urea derivatives of benzothiazole as
Raf-1 inhibitor
Eun Young Song a, Navneet Kaur a, Mi-Young Park a, Yinglan Jin a, Kyeong Lee a,
*
,
Guncheol Kim b, Ki Youn Lee b, Jee Sun Yang c, Jae Hong Shin d, Ky-Youb Nam d,
Kyoung Tai No c,d, Gyoonhee Han
c,**
a Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Eoen-dong, Yuseong-gu, Daejeon 305-806, Republic of Korea
b Department of Chemistry, Chungnam National University, Yuseong-gu, Daejeon 305-764, Republic of Korea
c Department of Biotechnology, Yonsei University, Seodaemun-gu, Seoul 120-740, Republic of Korea
d Bioinformatics & Molecular Design Research Center (BMD), Yonsei Engineering Research Complex, Seodaemun-gu,
Seoul 120-740, Republic of Korea
Received 15 May 2007; received in revised form 28 September 2007; accepted 4 October 2007
Available online 11 October 2007
Abstract
A series of amide and urea derivatives of benzothiazole have been synthesized and evaluated for their antiproliferative profile in human
SK-Hep-1 (liver), MDA-MB-231 (breast), and NUGC-3 (gastric) cell lines. Among them, compounds 1e2, 16e18, 23, and 25e26 had potent
to moderate inhibitory activities. Further these compounds were investigated for their ability to inhibit Raf-1 activity.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Benzothiazole derivatives; Antiproliferative; Raf-1 activity
1. Introduction
be particularly useful, because Raf is the key activator of this
pathway, whereas other upstream targets such as growth factor
ligands, receptor tyrosine kinases or even Ras, have many other
potential effectors. Furthermore, dominant-negative mutants of
Raf can impair Ras transforming activity, confirming that
inhibition of Raf is a viable theraupeutic approach [4].
The design of new chemical entity (NCE) must comple-
ment the size, shape, and electronic properties of the molecu-
lar targets. Benzothiazole-type compounds have attracted
considerable attention to anticancer research [5e12], and
modified benzothiazole derivatives having additional func-
tional groups may enhance the biological activity. We herein
report our efforts toward the synthesis of a series of benzothia-
zole derivatives and their ability to inhibit Raf-1 kinase.
Accordingly, a series of benzothiazole derivatives (Fig. 1)
have been synthesized and investigated for their biological
activity. The structure of this series comprises benzothiazole
part, linker and phenyl ring. Depending upon the nature of
linker group between benzothiazole and phenyl ring the whole
The RaseRafeMEKeERK pathway plays a critical role in
many aspects of tumorigenesis. This Ras signal transduction
pathway normally function to transmit signals from growth
factors and cytokine receptors on cell surface to nucleus, re-
sulting in regulation of cell differentiation and division [1].
The highest incidence of Ras alteration is seen in pancreatic
cancer (90%), thyroid cancer (50%), colon cancer (50%),
lung cancer (30%), and acute myeloid leukemia (30%) [2,3].
Raf was the first identified and most characterized downstream
effector kinase of Ras. There are three members of the Raf
family of kinases, Raf-1 (C-raf), A-raf, and B-raf. Drugs target-
ing the Ras signal transduction pathway at the level of Raf may
* Corresponding author. Tel.: þ82 42 8604382; fax: þ82 42 8604595.
** Corresponding author. Tel.: þ82 2 21232882; fax: þ82 2 3627265.
E-mail addresses: kaylee@kribb.re.kr (K. Lee), gyoonhee@yonsei.ac.kr
(G. Han).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.10.008

1520
E.Y. Song et al. / European Journal of Medicinal Chemistry 43 (2008) 1519e1524
Table 1
In vitro cytotoxicity of amide derivatives of benzothiazole 1e18 {GI50 (mg/
mL)}
work has been divided into two parts, that is, compounds bear-
ing amide linkages (1e18) and the compounds bearing urea
linkage (19e26). The role of various substitutions on benzo-
thiazole and phenyl ring has been investigated.
Compds
X
Y
Z
SK-Hep-1 MDA-MB-231 NUGC-3
1
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
6-OMe
6-CF3
6-NO2
5-Me
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
2.01
2.11
0.86
1.25
>10
3.46
2
OMe
CN
2. Results and discussion
3
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
4
CF3 >10
NO2 >10
>10
>10
>10
>10
>10
>10
>10
>10
>10
Scheme 1 summarizes the general synthetic approach that
we employed for the synthesis of amide-linked compounds
1e18. Under this synthetic strategy, a solution of 2-aminoben-
zothiazole derivatives in dry DMF was stirred with NaH fol-
lowed by reaction with para derivative of benzoyl chloride
to afford desired urea derivatives 19e26.
5
6
Me
OMe >10
CN >10
>10
7
8
9
10
CF3 >10
NO2 >10
11
12
Me
NO2 >10
CF3 2.10
2.17
7-Me OMe >10
>10
All the compounds (1e18) were initially evaluated in
SRB assay using three cancer cell lines’ panel consisting
of SK-Hep-1 (liver), MDA-MB-231 (breast), and NUGC-3
(gastric) and their antiproliferative activity data (GI50) are
provided in Table 1. Adriamycin was used as a reference
compound. Compounds with 4-methoxy derivative of benzo-
thiazole (1e5) showed variable activity but this activity de-
pends upon the nature of para substitutent on phenyl ring.
Among these compounds, the highest activity was observed
when this para position bears methyl group, but moderate
activity was observed when this para position has methoxy
group. Absolutely no activity was observed if this position
has cyano, trifluoromethyl or nitro group. Further to establish
the role of methoxy group on benzothiazole ring, another set
of compounds (6e10) was studied which otherwise is similar
to compounds 1e5 but differs in position of methoxy group
on benzothiazole ring. From the comparative studies of these
two sets of compounds, we concluded that no activity was
observed if compounds bear methoxy group at the 5th posi-
tion of benzothiazole ring. Since among these compounds
(1e10), the highest activity was observed only with 1, com-
parison of 1 with other positional isomers was also necessary,
and thus we studied compound 11, which differs from com-
pound 1 only in the position of methoxy group, that is, in
former OMe group present at the 6th position while in later
it is present at the 4th position. Comparison showed that only
1 has the antiproliferative activity, while 11 and 6 have no
activity. Another compound 13 in this series with nitro group
on 6-position of benzothiazole and trifluoromethyl at the
para position of phenyl was synthesized utilizing the general
approach as shown in Scheme 1 and was found to be mod-
erately active with GI50 value of 1.65 mg/mL in NUGC-3
cell lines. However when the position of these two groups
was interchanged, that is, 12, the compound lost its activity.
In the final stage of these studies some di-substituted deriv-
atives were planned and synthesized with methyl groups at the
13
14
1.89
1.65
2.37
>10
7-Me Me
0.44
1.03
0.48
0.45
0.29
0.39
15
16
5-Me
5-Me
7-Me CN
7-Me CF3
7-Me NO2
1.18
0.62
1.40
1.32
0.11
17
18
5-Me
5-Me
1.36
1.74
0.07
Adriamycin
5th and 7th positions of benzothiazole ring (14e18) and vari-
able groups at para position of phenyl ring. In general, all
these compounds were potent inhibitors. Compound 18 was
the most cytotoxic in MDA-MD-231 cell lines. While amongst
all these compounds 16 has the highest antiproliferative activ-
ity in NUGC-3 cell line.
The general synthetic approach for the synthesis of com-
pounds 19e26 is shown in Scheme 2. The appropriate
substituted 2-aminobenzothiazole and commercially available
substituted phenyl isocyanate were reacted in the presence of
2,6-dimethylaminopyridine (5 mol%) to furnish urea deriva-
tives of benzothiazole (19e26) (Table 2).
The antiproliferative activity (GI50) for compounds 19e22
in SK-Hep-1 (liver), MDA-MB-231 (breast), and NUGC-3
(gastric) was not encouraging. The insertion of nitro group in
phenyl ring (compound 23) showed moderate antiproliferative
activity in MDA-MA-231 and NUGC-3 cell lines. The replace-
ment of fluoro group in 23 with methyl group at 6-position of
benzothiazole ring in 24 led to loss in activity. The probable
reason for this loss in activity is change in electronic parameter
at 6-position of benzothiazole ring, particularly, electron with-
drawing group (fluoro) in 23 while electron releasing group
(methyl) in 24. Further to validate the effect of electron with-
drawing group at 6-position of benzothiazole with the synergis-
tic effect of m-nitro group on phenyl ring, compound 25 was
synthesized (6-chloro) and as expected it was found to have
moderate antiproliferative activity compared to 23. Another
compound in this series, 26, has been previously [13] known
for its antiviral properties and in our studies it exhibited strong
inhibition against growth of all cancer cell lines.
Z
X
Y
X
Y
N
S
N
S
NH
NH
To investigate whether these benzothiazole compounds
inhibit Raf kinase activity, compounds 1e2, 16e17, 23, and
25e26 which exhibited potent antiproliferative activity in
cancer cell lines were further tested with respect to Raf inhibi-
tion. GW4054 was used as a positive control [14]. Inhibitory
Z
NH
19-26
Fig. 1. General structures of the synthesized benzothiazole derivatives.
O
O
1-18

E.Y. Song et al. / European Journal of Medicinal Chemistry 43 (2008) 1519e1524
1521
O
X
X
Y
N
S
N
S
a
Cl
NH
+
NH2
Z
Y
Z
O
1-18
27
28
Scheme 1. Reagents and conditions: (a) NaH, DMF (anhydrous), rt, 5 h, yield: 60e70%.
activity of the tested compounds was determined using a re-
ported assay protocol with modification [15]. The results
shown in Table 3 are presented as percentage inhibitions at
the tested concentrations. GW4054 inhibited Raf activity in
a dose-dependent manner, and complete inhibition was ob-
served at 1 mM. On the other hand, compounds 1e2, 23, and
26 showed moderate to poor inhibitory profiles ranging from
37 to 7.8 at% inhibition, and compounds 16e18 and 25
were inactive at 50 mM.
In summary, a series of amide and urea derivatives of ben-
zothiazole have been synthesized, and initial antiproliferative
activity screening using cancer cell lines showed that some an-
alogues had potent to moderate inhibitory activities. Based on
these results, further evaluations were conducted to investigate
the abilities of the selected analogues to inhibit Raf-1 kinase.
However, most of the tested compounds exhibited moderate to
poor inhibition. The further mechanism study of these series
will be the subject of future publications.
with brine two times and dried over MgSO4. After filtration
and concentration, the crude product was purified by column
chromatography (hexane:ethyl acetate) to afford the desired
amide in 60e70% yield.
3.1.1. N-(4-Methoxybenzo[d]thiazol-2-yl)-4-
methylbenzamide (1)
1H NMR (400 MHz, CDCl3) d 2.44 (s, 3H), 3.98 (s, 3H),
7.09 (d, J ¼ 1.60 Hz, 1H), 7.192 (dd, J ¼ 8.38 and 1.60 Hz,
1H), 7.30 (d, J ¼ 7.58 Hz, 2H), 7.78 (d, J ¼ 7.98 Hz, 2H),
8.55 (s, 1H), 8.63 (d, J ¼ 8.78 Hz, 1H); ESI-MS (m/z, %)
299 (MHþ, 100); HRMS (Mþ) calcd. for C16H14N2O2S
298.0776, found 298.0779.
3.1.2. 4-Methoxy-N-(4-methoxybenzo[d]thiazol-2-
yl)benzamide (2)
1H NMR (400 MHz, CDCl3) d 3.88 (s, 3H), 3.99 (s, 3H),
6.99 (d, J ¼ 8.78 Hz, 2H), 7.09 (d, J ¼ 2.39 Hz, 1H), 7.19
(dd, J ¼ 8.78 and 2.39 Hz, 1H), 7.85 (d, J ¼ 8.78 Hz, 2H),
8.51 (br s, 1H), 8.62 (d, J ¼ 8.78 Hz, 1H); ESI-MS (m/z, %)
315 (MHþ, 100); HRMS (Mþ) calcd. for C16H14N2O3S
314.0725, found 314.0729.
3. Experimental
All reactions were performed in oven-dried glassware with
magnetic stirring. Commercial grade reagents were used with-
out further purification. TLC analysis was performed using
glass plate precoated with silica gel 60F254. Flash column
chromatography was performed with 230e400 mesh silica
3.1.3. 4-Cyano-N-(4-methoxybenzo[d]thiazol-2-
yl)benzamide (3)
1
gel. H NMR spectra were obtained on 400 MHz spectrome-
1H NMR (400 MHz, CDCl3) d 4.00 (s, 3H), 7.12 (d,
J ¼ 2.00 Hz, 1H), 7.21 (dd, J ¼ 8.38 and 2.00 Hz, 1H), 7.82
(d, J ¼ 8.38 Hz, 2H), 7.99 (d, J ¼ 8.38 Hz, 2H), 8.55 (s, 1H),
8.59 (d, J ¼ 8.78 Hz, 1H); ESI-MS (m/z, %) 310 (MHþ, 22).
ters. 1H NMR spectra were recorded in parts per million
(ppm) relative to the peak for tetramethylsilane (d ¼ 0.00) as
an internal standard.
3.1. General procedure for compounds 1e18
3.1.4. N-(4-Methoxybenzo[d]thiazol-2-yl)-4-
To a solution of 2-amino-benzothiazole in DMF (0.15 M)
NaH (2 equiv) was added slowly and the mixture was stirred
vigorously for 5 min at room temperature. To the resulting
solution, benzoyl chloride (1.3 equiv) in 2 mL of DMF was
added, and the mixture was stirred for 5 h at room tempera-
ture. The reaction mixture was quenched by addition of water
and diluted with ethyl acetate. The organic layer was washed
(trifluoromethyl)benzamide (4)
1H NMR (400 MHz, CDCl3) d 4.00 (s, 3H), 7.11 (d,
J ¼ 1.60 Hz, 1H), 7.21 (dd, J ¼ 8.78 and 1.60 Hz, 1H), 7.78
(d, J ¼ 8.38 Hz, 2H), 7.99 (d, J ¼ 7.98 Hz, 2H), 8.57 (s, 1H),
8.61 (d, J ¼ 8.38 Hz, 1H); ESI-MS (m/z, %) 353 (MHþ, 17);
HRMS (Mþ) calcd. for C16H11N2O2SF3 352.0493, found
352.0476.
O
X
Y
Z
C
N
S
X
N
N
+
a
NH2
NH
S
NH
Y
Z
O
19-26
27
29
Scheme 2. Reagents and conditions: (a) 2,6-dimethylaminopyridine (5 mol%), Et3N, DMF, rt, 8 h, yield: 40e60%.

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E.Y. Song et al. / European Journal of Medicinal Chemistry 43 (2008) 1519e1524
Table 2
In vitro cytotoxicity of urea derivatives of benzothiazole 19e26 {GI50 (mg/
mL)}
8.04 (d, J ¼ 7.98 Hz, 2H), 8.10 (d, J ¼ 8.38 Hz, 2H), 10.71
(s, 1H); EI-MS (m/z, %) 309 (Mþ, 100). Anal. Calcd for
C16H11N3O2S: C: 62.12; H: 3.58; N: 13.58. Found: C:
63.19; H: 3.49; N: 13.64.
Compds
X
Y
Z
SK-Hep-1 MDA-MB-231 NUGC-3
19
20
21
22
23
24
25
26
6-F
6-F
H
H
H
H
H
H
H
o-Me >10
p-OMe >10
>10
>10
>10
>10
>10
>10
>10
>10
6-F
6-F
H
o-F
>10
>10
3.1.9. N-(5-Methoxybenzo[d]thiazol-2-yl)-4-
(trifluoromethyl)benzamide (9)
6-F
6-Me
6-Cl
m-NO2
1.96
0.89
>10
0.82
0.10
0.83
>10
1.03
0.18
1H NMR (400 MHz, CDCl3) d 3.97 (s, 3H), 7.01 (dd,
J ¼ 8.38 and 2.00 Hz, 1H), 7.51 (d, J ¼ 8.38 Hz, 1H), 7.78
(d, J ¼ 8.38 Hz, 2H), 7.81 (d, J ¼ 2.00 Hz, 1H), 7.99 (d,
J ¼ 8.38 Hz, 2H), 8.01 (br s, 1H); ESI-MS (m/z, %) 353
(MHþ, 42); HRMS (Mþ) calcd. for C16H11N2O2SF3
352.0493, found 352.0475.
m-NO2 >10
m-NO2 2.21
0.18
5-Me 6-Me o-Me
3.1.5. N-(4-Methoxybenzo[d]thiazol-2-yl)-4-
nitrobenzamide (5) [16]
1H NMR (400 MHz, CDCl3) d 4.01 (s, 3H), 7.12 (d,
J ¼ 2.39 Hz, 1H), 7.22 (dd, J ¼ 8.38 and 2.00 Hz, 1H), 8.05
(d, J ¼ 8.78 Hz, 2H), 8.37 (d, J ¼ 9.18 Hz, 2H), 8.58 (s, 1H),
8.60 (d, J ¼ 8.38 Hz, 1H); ESI-MS (m/z, %) 328 (Mþ ꢀ 1, 12).
3.1.10. N-(5-Methoxybenzo[d]thiazol-2-yl)-4-
nitrobenzamide (10)
1H NMR (400 MHz, CDCl3) d 3.99 (s, 3H), 7.02 (dd,
J ¼ 7.98 and 2.00 Hz, 1H), 7.55 (d, J ¼ 8.38 Hz, 1H), 7.80
(d, J ¼ 2.40 Hz, 1H), 7.92 (s, 1H), 8.05 (d, J ¼ 8.78, 2H),
8.38 (d, J ¼ 8.78 Hz, 2H); ESI-MS (m/z, %) 330 (MHþ, 5).
3.1.6. N-(5-Methoxybenzo[d]thiazol-2-yl)-4-
methylbenzamide (6)
1H NMR (400 MHz, DMSO-d6) d 2.38 (s, 3H), 3.90 (s,
3H), 7.34 (d, J ¼ 8.38 Hz, 2H), 7.55 (s, 2H), 7.76 (s, 1H),
7.87 (d, J ¼ 8.38 Hz, 2H), 10.41 (s, 1H); ESI-MS (m/z, %)
299 (MHþ, 100); HRMS (Mþ) calcd. for C16H14N2O2S
298.0776, found 298.0787.
3.1.11. N-(6-Methoxybenzo[d]thiazol-2-yl)-4-
methylbenzamide (11)
1H NMR (400 MHz, CDCl3) d 2.99 (s, 3H), 3.86 (s, 3H),
6.82 (d, J ¼ 8.8 Hz, 1H), 7.06 (d, J ¼ 8.8 Hz, 1H), 7.20 (d,
J ¼ 8.4 Hz, 2H), 7.30 (s, 1H), 7.88 (d, J ¼ 8.4 Hz, 2H), 11.7
(br s, 1H); 13C NMR (100 MHz, CDCl3) d 166.1, 164.8,
157.2, 146.2, 140.8, 132.2, 131.2, 129.4, 126.9, 118.3,
113.0, 105.2, 56.0, and 21.0; EI-MS (m/z, %) 298 (Mþ,
100). Anal. Calcd for C16H14N2O2S: C: 64.41; H: 4.73; N:
9.39. Found: C: 64.59; H: 4.79; N: 9.44.
3.1.7. 4-Methoxy-N-(5-methoxybenzo[d]thiazol-2-
yl)benzamide (7)
1H NMR (400 MHz, CDCl3) d 3.87 (s, 3H), 3.92 (s, 3H),
6.96 (d, J ¼ 8.78 Hz, 2H), 7.01 (dd, J ¼ 8.38 and 1.60 Hz,
1H), 7.45 (d, J ¼ 7.98 Hz, 1H), 7.81 (d, J ¼ 2.00 Hz, 1H),
7.85 (d, J ¼ 8.78 Hz, 2H), 8.19 (s, 1H); ESI-MS (m/z, %)
315 (MHþ, 100); HRMS (MHþ) calcd. for C16H15N2O3S
315.0798, found 315.0805.
3.1.12. 4-Nitro-N-(6-(trifluoromethyl)benzo[d]thiazol-2-
yl)benzamide (12)
1H NMR (400 MHz, CDCl3) d 7.84 (s, 2H), 8.15 (s, 1H),
8.23 (d, J ¼ 8.78 Hz, 2H), 8.41 (d, J ¼ 8.78 Hz, 2H), 10.64
(br s, 1H); ESI-MS (m/z, %) 368 (MHþ, 100); HRMS
(MHþ) calcd. for C15H9N3O3SF3 368.0311, found 368.0298.
3.1.8. 4-Cyano-N-(5-methoxybenzo[d]thiazol-2-
yl)benzamide (8)
1H NMR (400 MHz, CDCl3) d 3.91 (s, 3H), 7.54 (d,
J ¼ 1.20 Hz, 1H), 7.57 (s, 1H), 7.73 (d, J ¼ 1.60 Hz, 1H),
3.1.13. N-(6-Nitrobenzo[d]thiazol-2-yl)-4-
(trifluoromethyl)benzamide (13)
1H NMR (400 MHz, CDCl3) d 7.63 (s, 2H), 7.88 (s, 2H),
8.00 (s, 1H), 8.48 (d, J ¼ 8.48 Hz, 2H), 9.05 (s, 1H); ESI-MS
(m/z, %) 368 (MHþ, 35); HRMS (MHþ) calcd. for
C15H9N3O3SF3 368.0267, found 368.0264.
Table 3
In vitro inhibition of Raf-1 by the representative thiazoles and GW4054
Compds
Concentration (mM)
Raf kinase inhibition (%)a
1
50
50
37
2
16
7.8
na
50
50
17
18
na
na
50
50
23
25
7.9
na
3.1.14. N-(5,7-Dimethylbenzo[d]thiazol-2-yl)-4-
methylbenzamide (14)
50
50
26
GW4054
14.9
4.5
19.4
85.3
97.1
1H NMR (400 MHz, CDCl3) d 2.42 (s, 3H), 2.58 (s, 6H),
7.28 (d, J ¼ 7.98 Hz, 2H), 7.53 (s, 2H), 7.75 (d, J ¼ 8.38 Hz,
2H), 7.90 (s, 1H), 7.99 (d, J ¼ 8.38 Hz, 1H); ESI-MS (m/z,
%) 297 (MHþ, 100); HRMS (Mþ) calcd. for C17H16N2OS
296.0983, found 296.0999.
0.003
0.01
0.03
0.1
a
Values are means of three experiments, na ¼ not active.

E.Y. Song et al. / European Journal of Medicinal Chemistry 43 (2008) 1519e1524
1523
3.1.15. N-(5,7-Dimethylbenzo[d]thiazol-2-yl)-4-
methoxybenzamide (15)
3.2.3. 1-(6-Fluorobenzo[d]thiazol-2-yl)-3-phenylurea (21)
1H NMR (400 MHz, DMSO-d6) d 7.05 (t, J ¼ 7.18 Hz, 1H),
7.22 (td, J ¼ 9.18 and 2.39 Hz, 1H), 7.33 (t, J ¼ 7.98 Hz, 2H),
7.50 (d, J ¼ 7.98 Hz, 2H), 7.65 (dd, J ¼ 8.38 and 4.39 Hz, 1H),
7.82 (dd, J ¼ 8.78 and 2.39 Hz, 1H), 9.12 (s, 1H), 10.81 (br s,
1H); ESI-MS (m/z, %) 288 (MHþ, 100); HRMS (Mþ) calcd.
for C14H10N3OSF2 287.0529, found 287.0518.
1H NMR (400 MHz, CDCl3) d 2.57 (s, 6H), 3.87 (s, 3H),
6.96 (d, J ¼ 8.78 Hz, 2H), 7.52 (s, 2H), 7.82 (d, J ¼ 8.78 Hz,
2H), 7.91 (s, 1H); ESI-MS (m/z, %) 313 (MHþ, 100);
HRMS (Mþ) calcd. for C17H16N2O2S 312.0932, found
312.0934.
3.2.4. 1-(6-Fluorobenzo[d]thiazol-2-yl)-3-
(2-fluorophenyl)urea (22)
1H NMR (400 MHz, DMSO-d6) d 7.09 (m, 1H), 7.23 (m,
3H), 7.67 (dd, J ¼ 8.78 and 4.39 Hz, 1H), 7.84 (dd, J ¼ 8.38
and 2.39 Hz, 1H), 8.10 (t, J ¼ 7.98 Hz, 1H), 9.10 (s, 1H),
11.04 (br s, 1H); ESI-MS (m/z, %) 306 (MHþ, 100).
3.1.16. 4-Cyano-N-(5,7-dimethyl-benzothiazol-2-
yl)-benzamide (16)
1H NMR (400 MHz, DMSO-d6) d 2.54 (s, 6H), 7.73 (s,
2H), 8.03 (d, J ¼ 7.9 Hz, 2H), 8.10 (d, J ¼ 7.9 Hz, 2H),
10.59 (s, 1H); EI-MS (m/z, %) 308 (MHþ, 100), 292 (40),
177 (100), 162 (30); HRMS (Mþ) calcd. for C17H13N3OS
307.0779, found 307.0785.
3.2.5. 1-(6-Fluoro-benzothiazol-2-yl)-3-
(3-nitro-phenyl)-urea (23)
3.1.17. N-(5,7-Dimethylbenzo[d]thiazol-2-yl)-4-
(trifluoromethyl)benzamide (17)
1H NMR (400 MHz, CDCl3) d 2.58 (s, 6H), 7.53 (s, 2H),
7.73 (d, J ¼ 8.38 Hz, 2H), 7.96 (d, J ¼ 8.38 Hz, 2H), 8.038
(s, 1H); ESI-MS (m/z, %) 351 (MHþ, 67); HRMS (Mþ) calcd.
for C17H13N2OSF3 350.0701, found 350.0688.
1H NMR (400 MHz, DMSO-d6) d 7.09 (m, 1H), 7.23 (m,
3H), 7.66 (dd, J ¼ 8.78 and 4.39 Hz, 1H), 7.83 (dd, J ¼ 8.38
and 2.39 Hz, 1H), 8.10 (t, J ¼ 7.98 Hz, 1H), 9.10 (br s, 1H),
11.03 (br s, 1H); ESI (m/z) 333 (MHþ); HRMS (MHþ) calcd.
for C14H10N4O3SF 333.0458, found 333.0467.
3.2.6. 1-(6-Methylbenzo[d]thiazol-2-yl)-3-
(3-nitrophenyl)urea (24)
3.1.18. N-(5,7-Dimethylbenzo[d]thiazol-2-yl)-4-
nitrobenzamide (18)
1H NMR (400 MHz, DMSO-d6) d 2.54 (s, 6H), 7.73 (s,
2H), 8.17 (d, J ¼ 8.78 Hz, 2H), 8.37 (d, J ¼ 8.78 Hz, 2H),
10.68 (s, 1H); ESI-MS (m/z, %) 328 (MHþ, 77); HRMS
(Mþ) calcd. for C16H13N3O3S 327.0678, found 327.0680.
1H NMR (400 MHz, DMSO-d6) d 2.37 (s, 3H), 7.19 (d,
J ¼ 7.98 Hz, 1H), 7.48 (d, J ¼ 7.98 Hz, 1H), 7.58 (t,
J ¼ 7.98 Hz, 1H), 7.66 (s, 1H), 7.85 (t, J ¼ 9.58 Hz, 2H),
8.60 (s, 1H), 9.71 (s, 1H), 11.37 (br s, 1H); ESI-MS (m/z,
%) 329 (MHþ, 100); HRMS (MHþ) calcd. for C15H13N4O3S
329.0708, found 329.0698.
3.2. General procedure for compounds 19e26
3.2.7. 1-(6-Chlorobenzo[d]thiazol-2-yl)-3-
(3-nitrophenyl)urea (25)
1H NMR (400 MHz, DMSO-d6) d 7.41 (dd, J ¼ 8.38 and
2.00 Hz, 1H), 7.60 (t, J ¼ 8.38 Hz, 2H), 7.83 (d, J ¼ 7.58 Hz,
1H), 7.89 (d, J ¼ 7.98 Hz, 1H), 8.05 (s, 1H), 9.77 (s, 1H),
11.36 (br s, 1H); ESI-MS (m/z, %) 349 (MHþ, 42).
To a solution of 2-amino-benzothiazole (1 equiv) in 10 mL of
DMF (0.1 M) Et3N (1.6 equiv) and 2,6-dimethyaminopyridine
(5 mol%) were added. Isocyanate (1.6 equiv) was added, and
the mixturewas stirred for 8 h at room temperature. The reaction
mixture was quenched by the addition of water diluted with
ethyl acetate. The organic layer was washed with brine and dried
over MgSO4. After filtration and concentration, the crude prod-
uct was purified by column chromatography (hexane:ethyl
acetate) to afford the desired urea in 40e60% yield.
3.2.8. 1-(5,6-Dimethylbenzo[d]thiazol-2-yl)-3-
o-tolylurea (26) [13]
1H NMR (400 MHz, DMSO-d6) d 2.28 (s, 3H), 2.29 (s,
3H), 2.31 (s, 3H), 7.03 (td, J ¼ 7.58 and 1.20 Hz, 1H), 7.22
(t, J ¼ 7.98 Hz, 2H), 7.46 (s, 1H), 7.65 (s, 1H), 7.85 (d,
J ¼ 7.58 Hz, 1H), 8.73 (s, 1H), 11.11 (br s, 1H); 13C NMR
(100 MHz, CDCl3) d 175.2, 153.1, 146.3, 122.0, 134.9,
134.0, 133.3, 131.5, 130.1, 127.2, 125.3, 121.5, 120.0, 19.0,
18.2, 15.1; EI-MS (m/z, %) 311 (Mþ, 100).
3.2.1. 1-(6-Fluorobenzo[d]thiazol-2-yl)-3-o-tolylurea (19)
1H NMR (400 MHz, DMSO-d6) d 2.26 (s, 3H), 7.02 (t,
J ¼ 7.18 Hz, 1H), 7.20 (m, 3H), 7.67 (dd, J ¼ 8.78 and
1.20 Hz, 1H), 7.83 (d, J ¼ 7.18 Hz, 2H), 8.56 (s, 1H), 11.13
(br s, 1H); ESI-MS (m/z, %) 302 (MHþ, 100); HRMS
(MHþ) calcd. for C15H13N3OSF 302.0763, found 302.0768.
3.3. Biology
3.2.2. 1-(6-Fluorobenzo[d]thiazol-2-yl)-3-
(4-methoxyphenyl)urea (20)
3.3.1. For SRB assay (sulforhodamine B assay) for GI50
Cells were harvested from exponential phase cultures by
trypsinization, counted and plated in 96-well plates. Optimal
seeding densities for the MDA-MB-231 cell lines were deter-
mined to ensure exponential growth during a 5-day assay. The
SRB assay was performed according to the method of Skehan
1H NMR (400 MHz, DMSO-d6) d 3.72 (s, 3H), 6.90 (d,
J ¼ 7.98 Hz, 2H), 7.21 (m, 1H), 7.40 (d, J ¼ 7.98 Hz, 2H),
7.63 (s, 1H), 7.81 (d, J ¼ 7.58 Hz, 1H), 8.94 (s, 1H), 10.77
(br s, 1H); ESI-MS (m/z, %) 318 (MHþ, 100); HRMS (Mþ)
calcd. for C15H12N3O2SF 317.0634, found 317.0647.

1524
E.Y. Song et al. / European Journal of Medicinal Chemistry 43 (2008) 1519e1524
et al. and Papazisis et al., with minor modifications [17,18].
The culture medium was aspirated prior to fixation of the cells
by the addition of 200 mL 10% cold trichloroacetic acid. After
1-h incubation at 4 ꢁC, cells were washed five times with
deionized water. The cells were then stained with 200 mL
0.1% SRB (SigmaeAldrich) dissolved in 1% acetic acid for
at least 15 min and subsequently washed four times with 1%
acetic acid to remove unbound stain. The plates were left to
dry at room temperature, bound protein stain was solubilized
with 200 mL 10 mM unbuffered Tris base, and optical density
(OD) was read at 540 nm.
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This research was supported by the Korea Science and
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