Solid phase combinatorial synthesis of benzothiazoles and evaluation of topoisomerase II inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2005.09.051

To investigate one possible mechanism of action of the cytotoxic activity of benzothiazoles, we synthesized 2-(substituted-phenyl)benzothiazoles and evaluated their ability to inhibit topoisomerase II activities. Solid phase combinatorial method using tri

Full text of DOI:10.1016/j.bmc.2005.09.051

Bioorganic & Medicinal Chemistry 14 (2006) 1229–1235
Solid phase combinatorial synthesis of benzothiazoles
and evaluation of topoisomerase II inhibitory activity
a
a
a
b
Suk-June Choi, Hyen Joo Park, Sang Kook Lee, Sang Woong Kim,
c
a,
*
Gyoonhee Han and Hea-Young Park Choo
a
Ewha Womans University, School of Pharmacy, Seoul 120-750, Republic of Korea
Leadgenex, Inc. Venture Town Dasan, 1687-2 Shinil Dong, Daedeok Gu, Deajon 306-230, Republic of Korea
b
c
Yonsei University, Department of Biotechnology, Seoul 120-749, Republic of Korea
Received 24 June 2005; accepted 21 September 2005
Available online 18 October 2005
Abstract—To investigate one possible mechanism of action of the cytotoxic activity of benzothiazoles, we synthesized 2-(substituted-
phenyl)benzothiazoles and evaluated their ability to inhibit topoisomerase II activities. Solid phase combinatorial method using
0
0
0
trityl resin was employed and benzothiazole derivatives with various substitution on 2 -, 3 -, or 4 -position of phenyl group
were obtained in ca. 30 mg scale (7–96% yield). Most of the compounds synthesized exhibited topoisomerase II inhibitory activity
at 100 lM. 2-(3-Amino-4-methylphenyl)benzothiazole showed high activity (IC50 = 71.7 lM), comparable to etoposide
(
ꢀ
IC50 = 78.4 lM).
2005 Elsevier Ltd. All rights reserved.
1
. Introduction
densation, and the maintenance of genome stability
by catalyzing the passage of individual DNA strands
(topoisomerase I) or double helices (topoisomerase
II) through one another. In accordance, topoisomerase
activities are activated in cancer cell growths, and thus
are good targets for antineoplastic drugs. Many anti-
tumor drugs such as camptothecin family target topo-
isomerase I (topo I), while doxorubicin and etoposide
are representative topoisomerase II (topo II) inhibi-
Simple 2-(4-aminophenyl)benzothiazoles display potent
and selective antitumor activity against breast, ovarian,
colon, and renal cell lines, but their mechanism of action
has not yet to be elucidated. These compounds are ac-
tive only against certain human cancer cell lines such
as breast MCF-7, MDA 468, renal TK 10, and ovarian
1
IGROV1. The biological profile is unlike any known
6
anticancer agents, and is the focus of extensive study.
The activities were considered to be related to the
metabolism, since the sensitive cell lines efficiently
retained and metabolized 2-(4-aminophenyl)benzothiaz-
oles to acetylated and 6-hydroxylated derivatives. In
addition to 2-(4-aminophenyl)benzothiazoles, 4-meth-
oxy-2-(4-trifluoromethyl/t-butyl phenyl)benzothiazoles
were reported effective against B-12 melanoma and
tors. Many antitumor topo II inhibitors act as a
result of interactions with both the enzyme and
DNA, for example, hypericin interacts with DNA at
7
,8
the N sites of purine residues. The decatenation as-
7
2
–4
say is specific for measuring topo II activity because it
is based on the conversion of catenated DNA to its
decatenated form, which requires DNA double strand
breakage followed by strand rotation and ligation
5
9
A-NK cells.
activities uniquely done by topo II. The removal of
these KDNA by the enzyme can be seen in agarose
gels.
DNA topoisomerases are ubiquitous enzymes that
control and modify the topological states of DNA.
They are considered to play important roles in replica-
tion, recombination, transcription, chromosome con-
In this study, to investigate one possible mechanism
of action of the cytotoxic activity of benzothiazoles,
we synthesized 2-(substituted-phenyl)benzothiazoles
and evaluated their cytotoxicity and ability to inhibit
topo II activities. The reported method by Mourtas
et al. was employed for the preparation of these
Keywords: Benzothiazoles; Solid phase synthesis; Topoisomerase II
inhibitory activity; Cytotoxicity.
*
Corresponding author. Tel.: +82 2 3277 3042; fax: +82 3277 2851;
e-mail: hypark@ewha.ac.kr
1
0
compounds.
0
968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.051

1230
S.-J. Choi et al. / Bioorg. Med. Chem. 14 (2006) 1229–1235
2
. Results
In the literature, the structure–activity relationship anal-
ysis on 2-(4-aminophenyl)benzothiazoles reported
that the benzothiazole nucleus was essential for potent
2
.1. Solid phase synthesis of benzothiazoles
0
cytotoxicity, and the substitution at 3 -position of the
phenyl ring with alkyl or halogen groups increased the
The procedure developed by Mourtas and co-workers
was very useful for the synthesis of various benzothiazoles
4
cytotoxicity of antitumor benzothiazoles.
(
Scheme 1). The yield was 7–96% after column chroma-
tography. This method had difficulties in preparation of
compounds in large quantities, but was very effective for
the synthesis of diverse compounds having various func-
tional groups in small scale (30 mg scale). Compounds
2.3. Topo II inhibition
A plasmid cleavage assay was used to investigate the
compounds on human DNA. The reference drug, etopo-
side, is a potent inhibitor stabilizing specifically DNA–
with alkyl, alkoxy, halogen, CF , amino, nitro, and
3
1
2
ethylthio phenyl/pyridine were prepared. However, the
reaction of solid bound-2-aminobenzothiol with 1-pyr-
rolidine carbonyl chloride or benzo[b]thiophene-2-car-
bonyl chloride was not successful. The structure and
yield of the synthesized compounds are given in Table 1.
topo II covalent complexes.
All the synthesized
compounds showed topo II inhibitory activity (9–89%)
at 100 lM as shown in Table 3. Especially, the cytotoxic
compounds, 4 and 18, showed relatively high topo II
inhibitory activity. The IC50 values of 4 and 18 were
7
1.7 and 107.8 lM, respectively, and were comparable
2
.2. Cytotoxicity
to etoposide (IC50 = 78.4 lM). The electrophotogram
with the inhibitory effects of test compounds are shown
in Figure 1.
The in vitro cytotoxic activities were evaluated using
1
1
SRB assay method. The following human solid tumor
cell lines were used: A549 (lung cancer), Col2 (colon
cancer), SNU-638 (stomach cancer), HT1080 (fibrosar-
coma cancer), and HL-60 (myeloid leukemia). The
IC50 values evaluated were compared with those of ellip-
ticine, doxorubicin, and etoposide.
Recently, some fused heterocyclic compounds such as
benzoxazole, benzimidazole, benzothiazole, and oxazolo-
pyridine derivatives are reported to have eukaryotic
topo II inhibitor activities. Especially, 2-phenoxy-
methylbenzothiazole was reported to have very high topo
II inhibitor activity.
1
3
All the synthesized compounds exhibited less cytotoxicity
than ellipticine or doxorubicin, clinically used agents for
the treatment of solid tumor. Only compounds 4 and
3. Discussion
1
8, with the high topo II inhibitory activity, showed a sig-
nificant cytotoxicity on HL-60 human leukemia cell line,
comparable to higher than that of doxorubicin. In addi-
tion, relatively cytotoxic compounds such as 4, 18, and
We designed and synthesized a series of 2-(4-aminophe-
nyl)benzothiazole, based on the antitumor activity of
benzothiazoles. Cytotoxic effects of the compounds were
measured by the SRB protein-staining method using
cultured human lung (A549), colon (Col2), stomach
(SNU-638), fibrosarcoma (HT1080), and myeloid leuke-
mia (HL-60) cancer cells. As shown in Table 2, the com-
pounds tested exhibited moderate cytotoxic activity, and
compounds 4 had a potential selective cytotoxicity
0 0
1
7 have an amino at the 3 - or 4 -position of phenyl ring.
However, their nitro analogs (23, 24, 25, and 11) and dim-
ethylamino analog (2) showed weak activities. Fluoro-
0
containing compounds, including 3 -trifluoromethoxy
0
(8) and 3 -difluoromethylthio (16) substituted analogs,
also showed weak cytotoxic activities.
NH2
SH
NH2
S
DIPEA
MC
Cl
P
=
Cl
OCH3
H
N
NH2
S
DIPEA
DMF
COCl
O
S
-
H O
2
N
S
TFA
TES
10
Scheme 1. Solid-phase synthesis of benzothiazoles.

S.-J. Choi et al. / Bioorg. Med. Chem. 14 (2006) 1229–1235
Table 1. The structure and yield of benzothiazoles
1231
R1
R2
N
S
A
Y
R3
R5
R4
Compound
A
Y
R
1
R
2
R
3
R
4
R
5
Yield (%)
1
2
3
4
5
6
7
8
9
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
—
—
SC
H
H
H
H
H
H
H
H
H
H
H
2
H
5
H
H
N(CH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CF
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH
H
H
H
H
H
H
H
H
H
H
H
H
92
18
13
40
11
15
12
13
8
H
3 2
)
–CH@CH–
Cl
NH
H
H
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2
CH
CH
3
3
H
t-Butyl
CH
2
Cl
H
H
OCF
3
OCH
H
3
OCH
n-Heptyl
NO
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
20
40
13
7
H
2
OCH
H
3
OCH
H
3
OCH
3
3
OC
F
2
H
5
H
H
15
10
9
H
H
3
H
SCHF
H
2
H
a
a
Cl
Cl
H
NH
H
2
42
19
86
48
50
75
20
40
96
H
NH
Cl
H
2
Cl
Cl
H
H
H
Cl
H
SCHF
H
2
H
H
CN
H
H
Cl
Cl
H
NO
H
2
H
NO
NO
CH
2
H
2
3
H
a
2
The yield after Pd-C/H reduction.
A
A
A
B
C
D
D
E
F
1
2
3
4
5
6
7
8
9
10 11 12
allowing for the passage of double-stranded DNA
9
followed by a resealing of the DNA. Relaxation of
KDNA
DNA supercoils by topo II is considered crucial to its
role in DNA replication and transcription. Further-
more, topo II plays a critical role in chromosome
condensation and separation during mitosis, and in the
attachment of DNA loops to the nuclear matrix and
B
B
C
E 13 14 15 16 17 18 19 20 21 22 23 24 25
KDNA
1
4
chromosomal scaffold.
Based on this information, we evaluated the benzothiaz-
oles for the catalytic activity of topo II. With respect to
topo II, the decatenation of KDNA was induced by topo
II. In this case, compounds 4 and 20 showed the stron-
gest inhibitory activity with the IC50 of 71.7 and
Figure 1. Effects of the synthesized analogs on the decatenation of
KDNA by topoisomerase II. (A) Lane A, KDNA without enzyme
(catenated form); lane B, KDNA with 1 U topoisomerase II (decat-
enated form); lane C, decatenated KDNA marker; lane D, linear
KDNA marker; lane E, etoposide, 200 lM; lane F, etoposide, 100 lM;
lanes 1–12, KDNA with 1 U topoisomerase II in the presence of
compounds 1–12 at a concentration of 100 lM, respectively. (B) Lane
A, KDNA without enzyme (catenated form); lane B, KDNA with 1 U
topoisomerase II (decatenated form); lane C, decatenated KDNA
marker; lane D, linear KDNA marker; lane E, etoposide, 200 lM;
lanes 13–25, KDNA with 1 U topoisomerase II in the presence of
compounds 13–25 at a concentration of 100 lM, respectively.
70.5 lM, respectively, and were comparable with the
antitumor agent etoposide with IC50 of 78.4 lM. Com-
pounds 18 and 2 caused moderate inhibition of 70%
and 54% at 100 lM, respectively, but others showed
weak inhibition (Table 3). No considerable functional
groups were taken into consideration for topo II inhibi-
tory activity among the tested compounds. Cytotoxic
compounds 4 and 18 possess amino substitution and
showed high topo II activity. However, another amino-
containing cytotoxic compound 17 showed low topo II
activity.
against myeloid leukemic cancer cells (IC50 = 0.02 lM)
compared to other cancer cell lines.
Topo II is an important nuclear enzyme controlling
DNA topology through catalysis of a transient breakage
of double-stranded DNA in an ATP-dependent fashion,
In view of the inhibitory activity of topo II by benzo-
thiazoles, the intercalating potential of an individual

1232
S.-J. Choi et al. / Bioorg. Med. Chem. 14 (2006) 1229–1235
Table 2. Effects of benzothiazole derivatives on cytotoxicity in human
a
cancer cells
compound, because of the planar structure of com-
pounds, might be one of the factors for the catalytic
activity, and further study is needed to clarify the addi-
tional factors. No obvious correlation was observed be-
tween the cytotoxicity of the individual compound and
the inhibitory activity of DNA decatenation by topo
II, in vitro. However, it is not plausible to exclude the
possibility that differences in compound-uptake into
cells exist, or that compounds are metabolized or
sequestered in different ways, or that other mechanisms
of actions might be included.
a
b
Compound
Cytotoxicity (IC50, lM)
A549
SNU-638 Col2 HT1080 HL-60
1
2
3
4
5
6
7
8
9
>50
>50
>50
>50
>50
>50
>50
>50
43.49
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
46.36
>50
0.02
>50
>50
43.79
>50
>50
>50
>50
>50
33.30
>50
27.19
23.54
13.09
>50
48.02
17.56
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
In summary, new structural modifications of benzo-
thiazoles were suggested for cytotoxic agents against hu-
man cancer cells, some compounds inhibited the
catalytic activity of topo II, but further study for the
mechanism of action by the compounds might be
needed.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
19.73 >50
21.39
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
31.48
>50
28.53
29.95
>50
36.54
>50
40.48
47.12
>50
33.94
38.68
0.05
>50
>50
>50
4. Conclusion
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
49.78
>50
>50
>50
>50
>50
>50
When a series of 2-(4-aminophenyl)benzothiazole were
synthesized and topo II inhibitory activity was evalu-
ated, most of compounds showed moderate inhibition
at 100 lM. The compound 4 had the strongest inhib-
itory activity with IC50 of 71.7 lM, which were com-
parable with the antitumor agent etoposide with
IC50 of 78.4 lM. Also the compound 4 showed a po-
tential selective cytotoxicity against myeloid leukemic
cancer cells (IC50 = 0.02 lM) compared to other can-
cer cells.
>50
>50
>50
>50
>50
27.91
33.11
1.86
21.72 >50
Ellipticine
Doxorubicin
Etoposide
1.15
0.15
2.44
2.87
0.063
6.19
1.34
0.064
020
3.42
0.041
0.14
0.093
1.24
a
11
Cytotoxicity was measured as described previously.
The IC50 values were determined from triplicate tests.
b
Table 3. Effects of benzothiazoles (100 lM) on topo II activities
5
. Experimental
a
Compound
Decatenation activity
topo II % inhibition)
(
5
.1. Materials and methods
1
2
3
4
5
6
7
8
9
41
54
39
89
30
32
34
28
32
31
23
20
21
21
27
24
37
70
16
84
28
26
14
34
9
All melting points were taken in Pyrex capillaries using
electrothermal digital melting point apparatus (B u¨ chi).
The IR spectra were recorded on a FT-Infrared spec-
1
trometer (Bio-Rad Co., USA) using KBr pellet.
H
NMR spectra were recorded on a 400 MHz Varian
FT-NMR spectrometer using trimethylsilane as an
internal standard. Samples were dissolved in acetone-
d6. Mass spectra were obtained on a Tandem Mass
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
spectrometer JMS-HX110/110A (Jeol).
5.2. General procedure for solid phase synthesis
After 0.3 g 4-methoxytrityl resin (1.8 mmol/g) with an
equimolar amount of 2-aminothiophenol and 0.7 equiv
diisopropylethylamine (DIPEA) was reacted in dichlo-
romethane (DCM) for 0.5–4 h at rt, the resin was
washed and filtered with the mixture of DCM/metha-
nol/DIPEA (85:10:5). The resin was swelled in DMF.
Then, threefold equivalents of benzoyl chloride and DI-
PEA were added, the mixture was shaken for 3 h at rt,
and the mixture was washed and filtered with DCM
and DMF, alternatively. Washed resin on the filter pa-
per was filtrated with 7 · 6 ml of 1.1% TFA in DCM/
TES (95:5). The filtrates of seven treatments were con-
centrated in vacuum to an oily residue and dissolved
Etoposide
58
a
Decantation assay for topo II catalytic activity was done by the
protocol by TopoGen, Inc.

S.-J. Choi et al. / Bioorg. Med. Chem. 14 (2006) 1229–1235
1233
in 6–10 ml of DMF:methanol (9:1) which contained 0.1–
.2 mmole DTT. After 3 h of standing at rt, the mixture
was extracted with ethyl acetate, washed with water,
dried and concentrated in vacuum, and purified by the
column chromatography (hexane:ethyl acetate = 3:1).
5.2.8. 2-(3-(Trifluoromethoxy)phenyl)benzo[d]thiazole (8).
1
0
Pale yellow solid, mp 99–102 ꢁC, H NMR (acetone-d ,
6
400 MHz) d 8.27–8.31 (2H, m), 8.13 (1H, dd, J = 1.2 and
8.0 Hz), 8.08 (1H, dd, J = 1.2 and 8.0 Hz), 7.53–7.61
(3H, m), 7.49 (1H, dt, J = 1.2 and 8.0 Hz). FAB-
+
HRMS(NBA-CsI) m/z 296.0357 (M Cs, C H F NOS
9 3
1
4
5
.2.1. 2-(2-(Ethylthio)pyridin-3-yl)benzo[d]thiazole (1).
requires 296.0357).
1
Yellow oil, H NMR (acetone-d , 400 MHz) d 8.59
6
(
1H, dd, J = 1.2 and 8.0 Hz), 8.26 (1H, dd, J = 1.2 and
5.2.9. 2-(3,4-Dimethoxyphenyl)benzo[d]thiazole (9). Pale
yellow solid, mp 132–133 ꢁC, H NMR (acetone-d6,
1
8
8
.0 Hz), 8.06–8.15 (2H, m), 7.58 (1H, dt, J = 1.2 and
.4 Hz), 7.50 (1H, dt, J = 1.2 and 8.4 Hz), 7.29 (1H, d,
J = 8.0 Hz). FABHRMS(NBA-CsI) m/z 273.0522
400 MHz) d 7.91(1H, dd, J = 1.2 and 8.4 Hz), 7.87
(1H, dd, J = 1.2 and 8.4 Hz), 7.40 (1H, d, J = 2.0 Hz),
7.30 (1H, dd, J = 2.0 and 8.4 Hz), 7.39 (1H, dt, J = 1.2
and 8.4 Hz), 7.29 (1H, dt, J = 1.2 and 8.4 Hz), 6.99
(1H, d, J = 8.4 Hz), 3.82 (3H, s), 3.78 (3H, s). FAB-
+
(
M Cs, C H N S requires 273.0520).
14 13 2 2
5
.2.2. 4-(Benzo[d]thiazol-2-yl)-N,N-dimethylbenzenamine
1
+
(
2). Dark brown oil, H NMR (acetone-d , 400 MHz) d
HRMS(NBA-CsI) m/z 271.9800 (M Cs, C H NO S
15 13
6
2
7
.93–8.00 (3H, m), 7.91 (1H, dd, J = 1.2 and 8.0 Hz),
.46 (1H, dt, J = 1.2 and 8.0 Hz), 7.35 (1H, dt, J = 1.2
requires 271.9803).
7
and 8.0 Hz), 6.83–6.87 (2H, m), 3.18 (3H, s), 2.78 (3H,
5.2.10. 2-(4-Heptylphenyl)benzo[d]thiazole (10). White
solid, mp 46–48 ꢁC, H NMR (acetone-d , 400 MHz)
d 8.06 (1H, d, J = 8.0 Hz), 7.90–8.02 (2H, m), 7.90
+
s). FABHRMS(NBA-CsI) m/z 255.0961 (M Cs,
1
6
C H N S requires 255.0956).
1
5
14
2
(1H, d, J = 8.0 Hz), 7.48 (1H, dt, J = 1.6 and 8.4 Hz),
5
.2.3. 2-(2-Chlorostyryl)benzo[d]thiazole (3). Pale yellow
7.37 (1H, dt, J = 1.6 and 8.4 Hz), 7.29–7.32 (2H, m),
2.68 (2H, t, J = 8.0 Hz), 1.61–1.70 (2H, m), 1.25–1.35
(10H, m), 0.89 (3H, t, J = 5.8 Hz). FABHRMS(NBA-
1
solid, mp 116–118 ꢁC,
00 MHz) 8.31–8.35 (2H, m), 8.21 (1H, d,
J = 8.4 Hz), 8.15 (1H, dd, J = 0.8 and 8.0 Hz), 8.11
H
NMR (acetone-d6,
4
d
+
CsI) m/z 310.1625 (M Cs, C H NS requires
2
0
24
(
(
1H, dd, J = 0.8 and 8.0 Hz), 7.97–8.00 (2H, m), 7.94
1H, d, J = 8.4 Hz), 7.61 (1H, dt, J = 1.2 and 8.0 Hz),
310.1629).
7
CsI) m/z 272.0302 (M Cs, C H ClNS requires
.53 (1H, dt, J = 1.2 and 8.0 Hz). FABHRMS(NBA-
5.2.11. 2-(4-Nitrophenyl)benzo[d]thiazole (11). Pale yel-
low solid, mp 225–227 ꢁC, H NMR (acetone-d6,
400 MHz) d 8.35–8.38 (2H, m), 8.27–8.30 (2H, m),
+
1
1
5
11
2
72.0301).
8.14 (1H, d, J = 8.0 Hz), 7.97 (1H, d, J = 8.0 Hz), 7.56
(1H, dt, J = 0.8 and 8.0 Hz), 7.47 (1H, dd, J = 0.8 and
5
.2.4. 5-(Benzo[d]thiazol-2-yl)-2-methylbenzenamine (4).
1
+
8.0 Hz). FABHRMS(NBA-CsI) m/z 257.0385 (M Cs,
White solid, mp 72–74 ꢁC, H NMR (acetone-d6,
4
00 MHz) d 8.30 (1H, dd, J = 0.8 and 8.0 Hz), 7.97
C H N O S requires 257.0385).
1
3
9
2
2
(
(
1H, dd, J = 0.8 and 8.0 Hz), 7.48–7.52 (2H, m), 7.41
1H, dt, J = 1.2 and 8.0 Hz), 7.30 (1H, dd, J = 1.6 and
5.2.12. 2-(3,4,5-Trimethoxyphenyl)benzo[d]thiazole (12).
1
7.6 Hz), 7.13 (1H, d, J = 7.6 Hz). FABHRMS(NBA-
CsI) m/z 241.0798 (M Cs, C H N S requires
White solid, mp 141–144 ꢁC, H NMR (acetone-d6,
400 MHz) d 7.93 (1H, dd, J = 0.8 and 8.0 Hz), 7.89
(1H, dd, J = 0.8 and 8.0 Hz), 7.41 (1H, dt, J = 1.2 and
+
1
4
13
2
2
41.0799).
8.0 Hz), 7.31 (1H, dt, J = 1.2 and 8.0 Hz), 7.27–7.28
(2H, m), 3.84 (6H, s), 3.70 (3H, s). FABHRMS(NBA-
5
6
.2.5. 2-p-Tolylbenzo[d]thiazole (5). White solid, mp 62–
1
+
CsI) m/z 302.0855 (M Cs, C H NO S requires
302.0851).
6 ꢁC, H NMR (acetone-d , 400 MHz) d 8.07 (1H, dd,
6
16 16
3
J = 0.8 and 8.4 Hz), 8.00–8.05 (3H, m), 7.54 (1H, dt,
J = 1.2 and 8.0 Hz), 7.44 (1H, dt, J = 1.2 and 8.0 Hz),
2
(
.43 (3H, s). FABHRMS(NBA-CsNa) m/z 248.0505
M Cs, C H NSNa requires 248.0510).
5.2.13. 2-(2,6-Dimethoxyphenyl)benzo[d]thiazole (13).
+
1
Pale yellow solid, mp 117–119 ꢁC, H NMR (acetone-
1
4
11
d , 400 MHz) d 8.06 (1H, dd, J = 0.8 and 8.0 Hz), 8.03
6
5
.2.6. 2-(4-tert-Butylphenyl)benzo[d]thiazole (6). White
H
00 MHz) d 8.05–8.09 (3H, m), 8.03 (1H, dd, J = 1.2
(1H, dd, J = 0.8 and 8.0 Hz), 7.52 (1H, dt, J = 1.2 and
8.0 Hz), 7.42–7.49 (2H, m), 6.79–6.82 (2H, m), 3.77
1
solid, mp 104–105 ꢁC,
4
and 8.8 Hz), 7.60–7.64 (2H, m), 7.54 (1H, dt, J = 1.2
NMR (acetone-d6,
+
(6H, s). FABHRMS(NBA-CsI) m/z 272.0746 (M Cs,
C H NO S requires 272.0745).
1
5
14
2
and 7.2 Hz), 7.44 (1H, dt, J = 1.2 and 7.2 Hz), 1.39
(
C H NS requires 268.1160).
+
9H s). FABHRMS(NBA-CsI) m/z 268.1155 (M Cs,
5.2.14. 2-(2-Ethoxyphenyl)benzo[d]thiazole (14). Brown
1
solid, mp 58–61 ꢁC, H NMR (acetone-d , 400 MHz) d
1
7
18
6
8.54 (1H, dd, J = 1.6 and 8.0 Hz), 8.09 (1H, dd,
5
.2.7. 2-(3-(Chloromethyl)phenyl)benzo[d]thiazole (7).
J = 1.2 and 8.0 Hz), 7.94 (1H, dd, J = 1.2 and 8.0 Hz),
7.49 (1H, dt, J = 1.2 and 8.0 Hz), 7.45 (1H, dt, J = 1.2
and 8.0 Hz), 7.38 (1H, dt, J = 1.2 and 8.0 Hz), 7.12
(1H, dt, J = 1.2 and 8.0 Hz), 7.05 (1H, dd, J = 1.6 and
8.0 Hz), 4.31 (2H, q, J = 7.2 Hz), 1.66 (3H, t,
1
Pale yellow solid, mp 82–83 ꢁC, H NMR (acetone-d ,
4
7
6
00 MHz) d 7.94 (1H, dd J = 1.2 and 7.6 Hz), 7.92–
.88 (2H, m), 7.41 (1H, dt, J = 1.2 and 8.0 Hz), 7.31
(1H, dt, J = 1.2 and 8.0 Hz), 7.29–7.24 (2H, m), 2.30
(2H, s). FABHRMS(NBA-CsI) m/z 260.0305 (M Cs,
+
J = 7.2 Hz). FABHRMS(NBA-CsI) m/z 256.0797
(M Cs, C H NOS requires 256.0796).
+
C H ClNS requires 260.0301).
4
1
11
15 14

1234
S.-J. Choi et al. / Bioorg. Med. Chem. 14 (2006) 1229–1235
5
.2.15.
thiazole (15). Brown solid, mp 101–104 ꢁC, H NMR
acetone-d , 400 MHz) d 8.79 (1H, dd, J = 2.4 and
2-(2-Fluoro-5-(trifluoromethyl)phenyl)benzo[d]-
7.59 (1H, dt, J = 1.2 and 8.0 Hz), 7.51 (1H, dt, J = 1.2
and 8.0 Hz), 7.14–7.18 (2H, m). FABHRMS(NBA-
1
+
CsI) m/z 237.0489 (M Cs, C H N S requires 237.0486).
(
6
14
9
2
6
.4 Hz), 8.16 (1H, d, J = 8.4 Hz), 7.97 (1H, d,
J = 8.4 Hz), 7.72–7.76 (1H, m), 7.56 (1H, dt, J = 1.2
and 8.4 Hz), 7.46 (1H, dt, J = 1.2 and 8.4 Hz), 7.38
5.2.23. 2-(2-Chloro-5-nitrophenyl)benzo[d]thiazole (23).
1
Pale yellow solid, mp 190–193 ꢁC, H NMR (acetone-
(1H, dd, J = 6.4 and 9.2 Hz). FABHRMS(NBA-CsI)
m/z 298.0316 (M Cs, C H F NS requires 298.0314).
d , 400 MHz) d 9.23 (1H, d, J = 2.8 Hz), 8.41 (1H, dd,
6
+
J = 2.8 and 8.8 Hz), 8.20–8.24 (2H, m), 8.00 (1H, d,
J = 8.8 Hz), 7.67 (1H, dt, J = 1.2 and 7.2 Hz), 7.59 (1H,
dt, J = 1.2 and 7.2 Hz). FABHRMS(NBA-CsI) m/z
1
4
8 4
5
.2.16. 2-(3-(Difluoromethylthio)phenyl)benzo[d]thiazole
1
+
(
16). White solid, mp 74–76 ꢁC, H NMR (acetone-d ,
290.9993 (M Cs, C H ClN O S requires 290.9995).
13
6
7
2
2
4
00 MHz) d 8.37–8.39 (1H, m), 8.21 (1H, dd, J = 0.8
and 8.0 Hz), 8.12 (1H, dd, J = 0.8 and 8.0 Hz), 8.09
1H, dd, J = 0.8 and 8.0 Hz), 7.80 (1H, d, J = 8.0 Hz),
5.2.24. 2-(2-Chloro-4-nitrophenyl)benzo[d]thiazole (24).
1
(
Yellow solid, mp 80–82 ꢁC, H NMR (acetone-d6,
7
.68 (1H, t, J = 8.0 Hz), 7.58 (1H, dt, J = 1.2 and
400 MHz) d 8.67 (1H, d, J = 8.8 Hz), 8.51 (1H, d,
J = 2.0 Hz), 8.41 (1H, dd, J = 2.0 and 8.8 Hz), 8.23
(1H, dd, J = 0.8 and 8.0 Hz), 8.23 (1H, dd, J = 0.8 and
8.0 Hz), 7.67 (1H, dt, J = 0.8 and 8.4 Hz), 7.59 (1H,
dt, J = 0.8 and 8.4 Hz). FABHRMS(NBA-CsI) m/z
8
FABHRMS(NBA-CsI)
.0 Hz), 7.50 (1H, dt, J = 1.2 and 8.0 Hz), 7.41 (1H, s).
294.0223
+
(M Cs,
m/z
C H F NS requires 294.0223).
1
4
10
2
2
+
5
.2.17. 4-(Benzo[d]thiazol-2-yl)-3-chlorobenzenamine (17).
290.9994 (M Cs, C H ClN O S requires 290.9995).
1
3
7
2
2
1
Yellow solid, mp 220–224 ꢁC, H NMR (acetone-d ,
6
4
00 MHz) d 8.03 (1H, d, J = 8.4 Hz), 7.91 (1H, d,
J = 8.0 Hz), 7.85 (1H, dd, J = 0.8 and 8.0 Hz), 7.38
1H, dt, J = 1.2 and 8.0 Hz), 7.28 (1H, dt, J = 1.2 and
.0 Hz), 6.75 (1H, d, J = 2.4 Hz), 6.66 (1H, dd, J = 2.4
and 8.4 Hz). FABHRMS(NBA-CsI) m/z 261.0252
5.2.25. 2-(4-Methyl-3-nitrophenyl)benzo[d]thiazole (25).
1
Yellow solid, mp 183–185 ꢁC, H NMR (acetone-d6,
(
8
400 MHz) d 8.70 (1H, s), 8.32 (1H, s), 8.13 (2H, d,
J = 8.0 Hz), 7.72 (1H, s), 7.61 (1H, t, J = 12.0 Hz),
7.52 (1H, t, J = 12.0 Hz). FABMS m/z 271.3 (M +1).
+
+
(
M Cs, C H ClN S requires 261.0253).
13 10 2
5
.3. Cytotoxic activity
5
.2.18. 3-(Benzo[d]thiazol-2-yl)-4-chlorobenzenamine (18).
1
Brown solid, mp 96–98 ꢁC, H NMR (acetone-d6,
4
A human lung carcinoma (A549, ATCC), colon carci-
noma (Col2, University of Illinois at Chicago), stomach
carcinoma (SNU-638, Korea Cell Bank at Seoul
National University), fibrosarcoma cancer (HT1080),
00 MHz) d 8.03 (1H, d, J = 8.0 Hz), 7.84–7.96 (2H,
m), 7.36 (1H, dt, J = 1.2 and 8.0 Hz), 7.28 (1H, dt,
J = 1.2 and 8.0 Hz), 6.75 (1H, d, J = 2.0 Hz), 6.66 (1H,
dd, J = 0.8 and 8.0 Hz). FABHRMS(NBA-CsI) m/z
4
and myeloid leukemia (HL-60) cells (5 · 10 cells/ml)
+
2
61.0257 (M Cs, C H ClN S requires 261.0253).
1
were treated with different concentrations of the test
agent for 3 days. After treatment, cells were fixed with
TCA and cell viability was measured using the sulforho-
3
10
2
5
.2.19. 2-(3,5-Dichlorophenyl)benzo[d]thiazole (19). White
1
11
solid, mp 120–121 ꢁC, H NMR (acetone-d , 400 MHz) d
damine B (SRB) protein-staining method. The result
6
8
8
(
.19 (1H, d, J = 2.4 Hz), 8.00 (1H, dd, J = 0.8 and
.0 Hz), 7.94–7.98 (2H, m), 7.66 (1H, d, J = 8.4), 7.47
1H, dt, J = 1.2 and 8.0 Hz), 7.38 (1H, dt, J = 1.2 and
was expressed in percentage, relative to solvent-treated
control incubations, and the IC50 values were calculated
using non-linear regression analysis (percent survival
versus concentration).
+
8.0 Hz). FABHRMS(NBA-CsI) m/z 279.9756 (M Cs,
C H Cl NS requires 279.9755).
1
3
7
2
5
.4. KDNA decatenation activity of topo II
5
.2.20. 2-(3,4-Dichlorophenyl)benzo[d]thiazole (20). Brown
1
solid, mp 150–155 ꢁC, H NMR (acetone-d , 400 MHz)
d 8.32 (1H, d, J = 2.4 Hz), 8.14 (1H, dd, J = 1.6 and
The topo II assay was done by the protocol provided by
TopoGEN, Inc. The total reaction volume of the topo
II-mediated cleavage reaction was fixed at 20 ll. Briefly,
assay buffer [50 mM Tris–HCl, pH 8, 120 mM KCl,
10 mM MgCl , 0.5 mM ATP, 0.5 mM dithiothreitol,
2
and 30 lg/ml bovine serum albumin (BSA)] containing
6
7
.2 Hz), 8.07–8.11 (2H, m), 7.79 (1H, d, J = 8.8 Hz), 7.60
(
1H, dt, J = 1.2 and 8.4 Hz), 7.51 (1H, dt, J = 1.2 and
+
.4 Hz). FABHRMS(NBA-CsI) m/z 279.9753 (M Cs,
8
C H Cl NS requires 279.9755).
13
7
2
2
00 ng of KDNA (TopoGen), and a solution of the test
5
.2.21. 2-(2-(Difluoromethylthio)phenyl)benzo[d]thiazole
drugs were added to 1 U of the human recombinant
topo II (the amount of enzyme, which resulted in the
complete decatenation of 200 ng of KDNA). After
10 min of incubation at 37 ꢁC, the reaction was stopped
by the addition of 5 ll of stop buffer containing the
loading dye (1% sarkosyl, 0.025% bromophenol blue,
and 5% glycerol), and then the reaction mixture was
analyzed on a 1% agarose gel by running at 40 V for
3.5 h in TBE buffer (89 mM Tris, 89 mM borate, and
2 mM Na-EDTA, pH 8.3). Gels were stained with
SYBR Green I (Molecular Probes, Eugene, OR), and
1
(
21). White solid, mp 188–190 ꢁC, H NMR (acetone-
d , 400 MHz) d 8.07 (1H, s), 8.00 (1H, dd, J = 0.8 and
6
8
.0 Hz), 7.94–7.90 (2H, m), 7.87 (1H, J = 0.8 and
8.0 Hz), 7.45 (1H, dt, J = 1.2 and 8.0 Hz). FAB-
HRMS(NBA-CsI) m/z 294.0225 (M Cs, C H NO S
requires 294.0223).
+
1
5
13
2
5
.2.22. 4-(Benzo[d]thiazol-2-yl)benzonitrile (22). Pale yel-
1
low solid, mp 120–123 ꢁC, H NMR (acetone-d6,
00 MHz) d 8.08–8.19 (2H, m), 7.87–7.90 (2H, m),
4

S.-J. Choi et al. / Bioorg. Med. Chem. 14 (2006) 1229–1235
1235
observed under UV illumination. For the quantitative
determination of topo II activity, photographic nega-
tives were densitometrically scanned using AlphaImager
3. Kashiyama, E.; Huctchison, I.; Chua, M.-S.; Stinson, F.
S.; Phillips, L. R.; Kaur, G.; Sausville, E. A.; Bradshaw, T.
D.; Westwell, A. D.; Stevens, M. F. G. J. Med. Chem.
1
999, 42, 4172.
2
200 (AlphaEase version 5.5). The inhibition of topo II
4
5
. Huctchison, I.; Chua, M.-S.; Browne, H. L.; Trapani, V.;
Bradshaw, T. D.; Westwell, A. D.; Stevens, M. F. G.
J. Med. Chem. 2001, 44, 1446.
was calculated from the equation: % inhibition = [inten-
sity of sample-treated DNA/intensity of vehicle-treated
control DNA] · 100.
. Goldfarb, R. H.; Ohashi, M.; Yoshino, K. U.S. Patent
6
,013,659 2000.
. Walker, J.; Nitiss, J. L. Cancer Invest. 2002, 20, 570.
. Burden, D. A.; Osheroff, N. Biochim. Biophys. Acta 1998,
6
7
Acknowledgments
1
400, 139.
This work was supported by Grant No. 2000-0-215-001-3
from the Basic Research Program of the Korea Science
and Engineering Foundation and M10310200002-
03B4620-00200 from Korea Institute of Science & Tech-
nology Evaluation and Planning.
8
. Muller, S. O.; Eckert, I.; Lutz, W. K.; Stopper, H. Mutat.
Res. 1996, 371, 165.
. Pommier, Y.; Pourquier, P.; Fan, Y.; Strumberg, D.
Biochim. Biophys. Acta 1998, 1400, 83.
9
10. Mourtas, S.; Gatos, D.; Barlos, K. Tetrahedron Lett. 2001,
2, 2201.
4
1
1. Lee, S. K.; Cui, B.; Mehta, R. R.; Kinghorn, A. D.;
Pezzuto, J. M. Chem. Biol. Interact. 1998, 115, 215.
2. Joseph, B.; Facompre, M.; Da Costa, H.; Routier, S.;
Merour, J.-Y.; Colson, P.; Houssier, C.; Bailly, C. Bioorg.
Med. Chem. 2001, 9, 1533.
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