New orally bioavailable 2-aminobenzamide-type histone deacetylase inhibitor possessing a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group

Article abstract of DOI:10.1016/j.bmc.2010.04.033

New 2-aminobenzamide-type histone deacetylase (HDAC) inhibitors were synthesized. They feature a sulfur-containing bicyclic arylmethyl moiety-a surface recognition domain introduced to increase in cellular uptake-and a substituted tert-amino group which affects physicochemical properties such as aqueous solubility. Compound 22 with a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group reduced the volume of human colon cancer HCT116 xenografts in nude mice to T/C 67% by oral administration at 45 mg/kg, which was comparable to the rate (T/C 62%) for a positive control, MS-275. Western blot analyses as well as cell cycle and TUNEL assays by flow cytometry suggested that the two compounds inhibited the growth of cancer cells via similar mechanisms.

Full text of DOI:10.1016/j.bmc.2010.04.033

Bioorganic & Medicinal Chemistry 18 (2010) 3925–3933
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New orally bioavailable 2-aminobenzamide-type histone deacetylase
inhibitor possessing a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group
Shingo Kiyokawa a, Yoshiyuki Hirata a, Yasuo Nagaoka a, Makio Shibano b, Masahiko Taniguchi b,
Masahide Yasuda b, Kimiye Baba b, Shinichi Uesato a,
*
a Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka 564-8680, Japan
b Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
New 2-aminobenzamide-type histone deacetylase (HDAC) inhibitors were synthesized. They feature a
sulfur-containing bicyclic arylmethyl moiety—a surface recognition domain introduced to increase in cel-
lular uptake—and a substituted tert-amino group which affects physicochemical properties such as aque-
ous solubility. Compound 22 with a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group reduced the
volume of human colon cancer HCT116 xenografts in nude mice to T/C 67% by oral administration at
45 mg/kg, which was comparable to the rate (T/C 62%) for a positive control, MS-275. Western blot anal-
yses as well as cell cycle and TUNEL assays by flow cytometry suggested that the two compounds inhib-
ited the growth of cancer cells via similar mechanisms.
Received 15 March 2010
Revised 12 April 2010
Accepted 13 April 2010
Available online 18 April 2010
Keywords:
Histone deacetylase inhibitor
2-Aminobenzamide-type
Sulfur-containing bicyclic arylmethyl
HCT116 xenograft
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
profile by, for example, enhancing cellular uptake.21 Additionally,
we expected the introduction of a 2-hydroxyethyl group at a
Histone deacetylase (HDAC) inhibitors induce gene expression,
differentiation, growth arrest, and apoptosis in cancer cells, and
represent a new, less toxic anti-cancer agent. A variety of HDAC
inhibitors possessing a hydroxamic acid or non-hydroxamic acid
moiety such as a 2-aminobenzamide group for binding a zinc ion
have been reported. Among them, vorinostat,1,2 romidepsin,3–5
MS-275,6–8 CI-994,9,10 LAQ824,11,12 PDX10113,14 and MGCD010315
are under clinical trials.16 Vorinostat and romidepsin are now
approved by US FDA for use in patients with relapsed cutaneous
T-cell lymphomas.17
In the present study, we have designed new orally active HDAC
inhibitors with a 2-aminobenzamide group as a Zn2+-chelating unit
and a 1,4-phenylene as a linker as well as a sulfur-containing (bicy-
clic arylmethyl)(2-hydroxyethyl)amino, an aryl sulfide, or an aryl-
methyl sulfide moiety as a surface recognition domain for the
following reasons: first, the 2-aminobenzamide group is metabol-
ically more stable than the hydroxamic acid group18 and renders
a molecule more orally-bioavailable6–8 and HDAC class I-selec-
tive.19,20 Second, in our previous study, replacement of a hydrocar-
bon chain with the 1,4-phenylene as a linker improved plasma
stability and HDAC inhibition.18 Third, insertion of a sulfur atom
in the surface recognition domain (a specificity element in the rec-
ognition of HDACs)21 could induce a change in the pharmacological
sec-amino group to improve physicochemical properties including
the aqueous solubility of the compound. Among the inhibitors syn-
thesized, 22 having a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)a-
mino moiety as a surface recognition domain showed marked
growth inhibition of human colorectal carcinoma HCT116 cells
and good solubility (Fig. 1). Anti-tumor tests with a HCT116 xeno-
graft model showed that 22 reduced the growth of tumors by 33%
(T/C, 67%) at 45 mg/kg/day by oral administration, which was sim-
ilar to the value (T/C, 62%) for a positive control, MS-275. Further-
more, the administration of MS-275 induced an increase in
running behavior as well as a slight loss of weight, neither of which
was observed in the mice treated with 22. The two compounds
accumulated acetylated-histone H3, stimulated p21/WAF1 protein
expression and induced G1-phase arrest and apoptosis to nearly
the same extent. Thus, 22 appears to inhibit the growth of cancer
cells via mechanisms similar to MS-275.6,7
O
O
N
H
NH2
NH2
N
H
N
H
N
N
S
O
O
OH
MS-275
22
* Corresponding author. Tel.: +81 6 6368 0834; fax: +81 6 6388 8609.
E-mail address: uesato@ipcku.kansai-u.ac.jp (S. Uesato).
Figure 1. Structures of MS-275 and 22.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.033

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S. Kiyokawa et al. / Bioorg. Med. Chem. 18 (2010) 3925–3933
in the presence of NaOH to give the carboxylic acid 5, which was
2. Chemistry
condensed to benzene-1,2-diamine in the same way as for 3, yield-
ing (naphthalene-2-yl)methyl sulfide 6. On the other hand, 9a and
9b were prepared starting with the formation of a thioether bond
between mercaptomethylbenzoic acid (7) and arylmethybromide
followed by condensation of the resulting products 8a and 8b
with benzene-1,2-diamine. Compounds 13a, 13b, 16 and 17, all
having a bicyclic arylmethyl-sec-amino group, were synthesized
starting from methyl 4-(aminomethyl)benzoate hydrochloride
(10) or 4-(aminomethyl)-N-(2-nitorophenyl))benzamide hydro-
chloride (14) (Scheme 2). Thus, 10 was subjected to reductive
Sulfides 3, 6, 9a and 9b were synthesized as illustrated in
Scheme 1. Methyl 4-(bromomethyl)benzoate (1) was reacted with
naphthalene-2-thiol using K2CO3 to yield the thioether 2a, which
was hydrolyzed to give the carboxylic acid 2b. The coupling of
2b to benzene-1,2-diamine utilizing 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide hydrochloride (WSC) and 1-hydroxybenzotri-
azole (HOBt) afforded (naphthalene-2-yl) sulfide 3. Arylmethyl
sulfides 6, 9a and 9b were prepared in two ways: bromomethyl-
benzoic acid (4) was treated with naphthalene-2-ylmethanethiol
b
a
S
NH2
Br
Br
S
H
N
OMe
OR1
O
O
O
O
O
2a: R1 = CH3
2b: R1 = H
3
1
d
c
S
S
NH2
H
N
OH
OH
O
O
5
6
4
f
e
NH2
Ar1
S
H
N
HS
Ar1
S
OH
OH
O
O
9a: Ar1 = 2,3-dihydrobenzo[
b][1,4]dioxin-2-yl
8a: Ar1 = 2,3-dihydrobenzo[
b][1,4]dioxin-2-yl
7
9b: Ar1 = 4-chlorophenyl
8b: Ar1 = 4-chlorophenyl
Scheme 1. Reagents and conditions: (a) (1) naphthalene-2-thiol, K2CO3, MeCN, 92% (2) LiOH, THF–H2O, 94%; (b) benzene-1,2-diamine, HOBt, WSC, DMF, 51%; (c)
naphthalene-2-ylmethanethiol, NaOH, EtOH, 75%; (d) benzene-1,2-diamine, HOBt, WSC, DMF, 20%; (e) Ar1CH2Br, NaOH, EtOH, 8a, 28%: 8b, 46%; (f) benzene-1,2-diamine,
HOBt, WSC, DMF, 9a, 84%: 9b, 82%.
ClH3N
OMe
N
H
NH2
H
N
O
10
S
a
O
c
13a
b
Ar2
N
H
Ar2
N
H
OH
OCH3
O
O
11a: Ar2 = 4-(thiophen-2-yl)phenyl
12a: Ar2 = 4-(thiophen-2-yl)phenyl
12b: Ar2 = isobenzothienyl
S
NH2
N
H
H
11b: Ar2 = isobenzothienyl
c
N
O
13b
S
N
H
NH2
H
N
e
O
16
ClH3N
NO2
H
Ar3
N
H
NO2
H
N
d
N
O
O
e
NH2
N
H
H
14
15a: Ar3 = 3-(thiophen-2-yl)phenyl
15b: Ar3 = biphenyl-4-yl
N
O
17
Scheme 2. Reagents and conditions: (a) Ar2CHO, NaBH(OAc)3, TEA, THF, 11a, 56%: 11b, 22%; (b) LiOH, THF–H2O, 12a, 95%: 12b, 89%; (c) benzene-1,2-diamine, HOBt, WSC,
DMF, 13a, 84%: 13b, 79%; (d) Ar3CHO, NaBH(OAc)3, THF, 15a, 51%: 15b, 40%; (e) SnCl2, NH4OAc, MeOH, 16, 32%: 17, 29%.

S. Kiyokawa et al. / Bioorg. Med. Chem. 18 (2010) 3925–3933
3927
Table 1
amination with arylaldehyde in the presence of NaBH(OAc)3 and
Effect of HDAC inhibitors on HCT116 cell growth and HDAC1
triethylamine (TEA) to give 11a or 11b. These compounds, after
their conversion into the carboxylic acids 12a and 12b, were con-
densed with benzene-1,2-diamine using WSC and HOBt to yield
13a and 13b, respectively.
Y
Ar
X
NH2
H
N
O
Meanwhile, 14 was subjected to reductive amination with aryl-
aldehyde in the presence of NaBH(OAc)3 and TEA to give 15a or
15b. The reduction of these products with SnCl2 and NH4OAc,
respectively, gave 16 and 17. Compounds 19, 20, 21, 22 and 23,
all possessing a bicyclic arylmethyl-tert-amino group, were syn-
thesized from 14 (Scheme 3). Compound 15b obtained from 14
was converted to 23 by sequential alkylation with 2-bromoethanol
and reduction with SnCl2 and NH4OAc. Furthermore, compound 18
prepared from 14 in the same way as 15b was converted to 19, 20
and 21 by N-methylation, N-methylsulfonylation and N-(N,N-
dimethylamino)carbonylation followed by reduction with SnCl2
and NH4OAc, respectively.
Compd
Ar
X
Y
HCT116
HDAC1
IC50 (lM)
a,b
c
IC50
2.6
3.6
(
l
M)
3
6
s
s
—
>100
>100
CH2
CH2
O
O
9a
9b
s
s
3.1
3.9
18
19
CH2
Cl
13a
13b
16
NH
NH
NH
CH2
CH2
CH2
2.3
3.4
5.0
1.0
1.1
3.3
3. Results and discussion
S
S
The sulfides 3, 6, 9a and 9b inhibited significantly the growth of
HCT116 cells (IC50s between 2.6 and 3.9
they showed greater IC50 values (18 to >100
than the other compounds synthesized as well as MS-275. In con-
trast, 13a and 13b possessing a S-containing bicyclic aryl group
exhibited lower IC50 rates both for the growth of HCT116 cells
l
M) (Table 1). However,
M) against HDAC1
l
S
(2.3 and 3.4 lM) and for HDAC1 (1.0 and 1.1 lM), respectively.
Compound 13a, having a (4-(thiophen-2-yl)benzyl)amino moiety
as a surface recognition domain, seemed especially promising. Its
17
NH
CH2
3.9
0.8
0.9
0.5
MS-275
regioisomer 16, possessing
a (3-(thiophen-2-yl)benzyl)amino
a
b
c
Measured after a 3-day incubation of test compounds with cells.
Assays were performed in triplicate.
Assays were performed in duplicate.
group, as well as non-sulfur-containing counterpart 17, possessing
a 4-phenylbenzylamino group, were inferior to 13a in terms of
inhibiting HCT116 and/or HDAC1. Thus, 13a was subjected to fur-
ther structural optimization by including a methyl (19), methylsul-
fonyl (20), N,N-dimethylaminocarbonyl (21), or hydroxyethyl (22)
group at the secondary amine.
was previously utilized for the design of LAQ824.11 Compound 22
showed the most promising profile in terms of inhibiting the
As shown in Table 2, introduction of the N,N-dimethylaminocar-
bonyl or hydroxyethyl group improved solubility. The latter group
growth of HCT116 cells (IC50 0.7 lM) and HDAC1 (IC50 0.8 lM),
being comparable to MS-275 (0.8 and 0.5
l
M, respectively).
ClH3N
NO2
H
N
O
14
N
NH2
H
NH2
N
H
N
N
O
a
CH3
19
S
S
OH
22
O
e
b
N
NH2
Ar4
H
N
H
NO2
H
N
c
SO2
N
S
O
CH3
O
20
18: Ar4 = 4-(thiophen-2-yl)phenyl
15b: Ar4 = biphenyl-4-yl
e
d
N
NH2
H
N
N
NH2
H
N
O
CO
N
S
O
OH
H3C
CH3
23
21
Scheme 3. Reagents and conditions: (a) Ar4CHO, NaBH(OAc)3, TEA, THF, 18, 54%; (b) (1) HCHO, AcOH, NaBH(OAc)3, CH2Cl2, 82% (2) SnCl2, NH4OAc, MeOH, 19%; (c) (1)
MeSO2Cl, TEA, CH2Cl2, 78% (2) SnCl2, NH4OAc, MeOH, 24%; (d) (1) (Me)2NCOCl, TEA, CH2Cl2, 94% (2) SnCl2, NH4OAc, MeOH, 38%; (e) (1) 2-bromoethanol, TEA, MeCN (2) SnCl2,
NH4OAc, MeOH, 22, 24%: 23, 20%.

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S. Kiyokawa et al. / Bioorg. Med. Chem. 18 (2010) 3925–3933
Table 2
0.9
Solubility and effects on HCT116 cell growth and HDAC1 of HDAC inhibitors
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
N
R
NH2
H
N
Ar
O
Compd
Ar
R
Solubility in
HCT 116 HDAC1
10% DMSO/H2O IC50
a,b
c
IC50 (lM)
(mg/mL)
0.1
(lM)
control
MS-275
22
13a
19
H
2.3
4.7
5.7
2.0
1.0
>10
1.2
0.8
0.8
S
S
S
S
S
Figure 3. Wet weights of tumor masses of mice fed 22 or MS-275. The cervical
spine was dislocated, and tumor masses were removed and weighed. Vertical bars
indicate standard errors. Significant differences (p <0.05) were obtained for tumor
volume with 22 or MS-275 versus the control.
CH3
0.2
0.1
20
CH3–SO2–
21
(CH3)2NCO– 0.8
22
a
b
control
MS-275
Acetylated H3
22
23
HOCH2CH2– 0.5
HOCH2CH2– 0.6
0.7
2.0
β Actin
0.7
0.5
MS-275
TSA
0.8
—
0.8
—
0.0099
a
b
c
p21/WAF1
Measured after a 3-day incubation of test compounds with cells.
Assays were performed in triplicate.
Assays were performed in duplicate.
β-Actin
Figure 4. Effect of 22 and MS-275 on nuclear histone (H3) acetylation and
p21WAF1 expression. (4a) Lysate (11 g) of HCT116 cells exposed to 22 or MS-275
at 10 M or 0.1% DMSO for 24 h was separated by polyacrylamide gel electropho-
resis (SDS–PAGE). (4b) Lysate (11 g) of SKBR3 cells exposed to 22 or MS-275 at
10 M or 0.1% DMSO for 24 h was examined using SDS–PAGE. The experiment was
l
Furthermore, in view of the lower value for HCT116 (IC50 0.7
lM)
l
of 22 relative to that (IC50 2.0 M) of 23 having the 4-phen-
l
l
ylbenzylamino unit, the thienyl group may contribute to the phar-
macological potency of 22.
l
repeated four times and representative blots are shown. Details are described in
Since 22 has a pharmacological profile comparable to MS-275,
we conducted comparative anti-tumor tests using xenografts of
HCT116 cells. The two compounds were administered orally to
mice at a dose of 45 mg/kg/day over 18 days according to the
schedules indicated in Figure 2a. Compound 22 suppressed the
growth of the xenografts to T/C: 67%, similar to MS-275 (62%).
The administration of MS-275 induced an increase in running
behavior as well as a slight loss of weight (Fig. 2b), these effects
being not observed in the mice treated with 22.
Figure 3 shows the wet weights of tumor masses removed from
the mice at day 18. The average weights of tumor masses with
MS-275 and 22 decreased to 61% and 63% of the control values,
respectively.
Section 5.
Both 22 and MS-275 promoted the hyperacetylation of core
histone H3 in the HCT 116 cells as detected by Western blotting
(Fig. 4a) and regulated the cells in the G0/G1 phase of the cell cycle
at 10 lM (Table 3). These compounds also induced apoptosis as
suggested by the appearance of a subG1 population, which was
confirmed with a TUNEL assay. The percentages of cells with posi-
tive TUNEL staining were 28.7% (22) and 27.6% (MS-275), respec-
tively (Fig. 5).
Furthermore, 22 induced the expression of p21/WAF1 protein in
SKBR-3 cells (p53-mutant) as did MS-275 (Fig. 4b). These findings
a
b1.1
1200
control
MS-275
22
1000
800
600
400
200
0
1
control
MS-275
22
0.9
0
5
10
15
20
0
5
10
15
20
day
day
Figure 2. Effects of 22 and MS-275 on HCT116 cell-inoculated xenografts in vivo. The effects of 22 (N) and MS-275 (j) on tumor volume (2a) as well as relative body weight
(RBW) changes (2b) at a dose of 45 mg/kg/day were examined as described in Section 5. (ꢀ) Represents the control group. Compounds were orally administered to mice on
days 0, 2, 4, 7, 9, 11, 14 and 16. Vertical bars indicate standard errors. Significant differences (p <0.01) were obtained at all time points except day0 for tumor volume with 22
or MS-275 vs. the control.

S. Kiyokawa et al. / Bioorg. Med. Chem. 18 (2010) 3925–3933
3929
diamine (6.1 g, 8.0 mmol) as reported:22 (1.8 g, 4.7 mmol, 68%).
Mp 155–156 °C. 1H NMR (DMSO-d6): d 4.28 (2H, d, J = 6.0 Hz,
CH2), 4.88 (2H, s, NH2), 5.10 (2H, s, CH2), 6.60 (1H, t, J = 8.0 Hz,
PhH), 6.78 (1H, dd, J = 8.0, 1.2 Hz, PhH), 6.97 (1H, ddd, J = 8.0, 8.0,
1.2 Hz, PhH), 7.17 (1H, d, J = 8.0 Hz, PhH), 7.36–7.43 (3H, m, ArH),
7.79 (1H, d, J = 7.6 Hz, ArH), 7.92–7.97 (3H, m, ArH), 8.54 (1H, d,
J = 3.6 Hz, PyrH), 8.60 (1H, s, PyrH), 9.61 (1H, s, CONH).
Table 3
Effects of 22 and MS-275 on the cell cycle in HCT116 cells
% of subG1
24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h
6.2 1.9 81.3 84.9 11.1 15.4 10.2 7.7
% of G0/G1
% of S
%of G2/M
Control (0.1%
DMSO)
22
15.3 20.6 65.0 64.9
4.7 17.4 75.6 76.1
6.9
5.8
5.1 12.7 9.4
2.2 13.9 4.1
MS-275
HCT116 cells (1.0 Â 106), after incubating for 24 h, were treated with 22 or MS-275
5.2.2. Methyl 4-((naphthalen-2-ylthio)methyl)benzoate (2a)
To a solution of 1 (0.70 g, 3.1 mmol) in MeCN (20 mL) was
added 2-naphthalenethiol (0.49 g, 3.1 mmol) followed by K2CO3
(0.21 g, 1.5 mmol), and the mixture stirred at room temperature
for 8 h. After the removal of insoluble materials, the solution was
concentrated, and the resulting solid was recrystallized from
CHCl3–n-hexane to yield 2a (0.87 g, 2.8 mmol, 92%). Mp 100–
(each 10 lM) for 24 or 48 h. The cells were then treated with a BD Cycle Test Plus
DNA reagent Kit. Data are representative of three independent experiments.
suggested that 22 inhibited the growth of the cancer cells via
mechanisms similar to MS-275.
4. Conclusion
101 °C. IR (KBr) 1725, 1434, 1283, 1095, 816 cmÀ1 1H NMR
.
(CDCl3): d 3.89 (3H, s, OCH3), 4.22 (2H, s, CH2Ph), 7.35–7.73 (9H,
m, ArH), 7.93 (2H, d, J = 8.4 Hz, ArH). FAB-MS m/z: 309 (M+H)+.
HR-FAB-MS m/z: (M+H)+ calcd for C19H17O2S, 309.0949; found,
309.0942.
A new orally bioavailable HDAC inhibitor, 22, comprising a
(2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group inhibited
the growth of HCT116 xenografts in nude mice more or less equiv-
alent to MS-275 (4) and showed a promising in vivo profile. Thus,
22 warrants further tests with other human cancer xenografts to
identify its effectiveness as an antitumor agent.
5.2.3. 4-((Naphthalen-2-ylthio)methyl)benzoic acid (2b)
1 M LiOH in H2O (4.5 mL) was added to a solution of 2a (0.35 g,
1.1 mmol) in tetrahydrofuran (THF) (5 mL), and the mixture stirred
at room temperature overnight. After the removal of THF, the mix-
ture was adjusted to pH 3 with 1 M HCl. The resulting precipitate
was collected by filtration, giving 2b (0.31 g, 1.1 mmol, 94%) as a
5. Experimental
5.1. General
Melting points were determined on a Yanagimoto MP-32 micro-
melting point apparatus and are uncorrected. IR spectra were re-
corded on a Shimadzu FTIR-8400 infrared spectrophotometer.
Low-resolution (LR)-FAB-MS spectra were measured on a JEOL
JMS-HX 100, and high-resolution (HR)- and LR-FAB-MS spectra, on
a JEOL Tandem MStation JMS-700. 1H NMR spectra were recorded
on a JEOL EX-400 (400 MHz) using tetramethylsilane as an internal
standard. Analytical TLC and PLC were performed using Silica Gel
60 F254 (Merck, 0.25 and 0.5 mm, respectively) glass plates. Column
chromatography was performed using Silica Gel 60 (70–230 mesh
ASTM). All solvents were dried over Na2SO4, and evaporated in
vacuo. The HCT 116 cell line was purchased from American Type
Culture Collection. The Cycle Test Plus DNA reagent Kit was
purchased from Becton Dickinson and Company. The TUNEL assay
reagent, DeadEnd™ Fluorometric TUNEL System, was obtained from
Promega. Results of flow cytometry were collected with a FACSCant
II flow cytometer (Becton Dickinson and Company).
solid. Mp >390 °C. IR (KBr) 3047, 1597, 1551, 1427 cmÀ1 1H NMR
.
(CDCl3): d 4.33 (2H, s, SCH2Ph), 7.27 (2H, d, J = 8.0 Hz, PhH), 7.26–
7.50 (3H, m, ArH), 7.75 (2H, d, J = 8.0 Hz, PhH), 7.79–7.85 (4H, m,
ArH). FAB-MS m/z: 293 (MÀH)À. HR-FAB-MS m/z: (MÀH)À calcd
for C18H13O2S, 293.0636; found, 293.0640.
5.2.4. N-(2-Aminophenyl)-4-((naphthalen-2-ylthio)ethyl)
benzamide (3)
To a solution of benzene-1,2-diamine (0.07 g, 0.68 mmol) in
N,N-dimethylformamide (DMF) (1 mL) was added 2b (0.10 g,
0.34 mmol), then HOBt (0.069 g, 0.51 mmol) and finally WSC
(0.098 g, 0.51 mmol), and the mixture stirred at room temperature
overnight. The reaction mixture was diluted with EtOAc, washed
with satd. NaHCO3 and brine, dried, and concentrated. The residue
was crystallized from CHCl3 to give 3 (0.066 g, 0.17 mmol, 51%) as a
solid. Mp 187–189 °C. IR (KBr) 3288, 1643, 1612, 1502, 1454,
1306 cmÀ1 1H NMR (DMSO-d6): d 4.45 (2H, s, SCH2Ph), 4.87 (2H,
.
5.2. Chemistry
s, PhNH2), 6.58 (1H, ddd, J = 7.6, 7.6, 1.2 Hz, PhH), 6.76 (1H, dd,
J = 7.6, 1.2 Hz, PhH), 6.96 (1H, ddd, J = 7.6, 7.6, 1.2 Hz, PhH), 7.13
(1H, d, J = 7.6 Hz, PhH), 7.44–7.54 (5H, m, ArH), 7.81–7.90 (6H, m,
ArH), 9.59 (1H, s, CONH). FAB-MS m/z: 385 (M+H)+. HR-FAB-MS
m/z: (M+H)+ calcd for, 385.1375; found, 385.1370.
5.2.1. Synthesis of MS-275
MS-275 was prepared from 4-(((pyridin-3-ylmethoxy)carbon-
ylamino)methyl)benzoic acid (2.0 g, 7.0 mmol) and benzene-1,2-
Figure 5. Flow cytometric analysis of FITC-TUNEL staining of HCT116 cells. Cells were incubated with 22, MS-275 (each 10
lM) or vehicle (no sample) for 24 h and then
treated as indicated in Section 5. Percentages of FITC-TUNEL-positive cells are indicated. They are results of one of two independent experiments.

3930
S. Kiyokawa et al. / Bioorg. Med. Chem. 18 (2010) 3925–3933
5.2.5. 4-((Naphthalen-2-ylmethylthio)methyl)benzoic acid (5)
To a suspension of 4 (0.25 g, 1.2 mmol) in EtOH (2 mL) were
added 2-naphthalenemethanethiol (0.20 g, 1.15 mmol) and a solu-
tion of NaOH (0.092 g, 2.3 mmol) in EtOH (3.7 mL), and the mixture
was stirred at room temperature over night. The solution, its pH ad-
justed to 3 with 1 M HCl, was concentrated. The resulting solid was
recrystallized from EtOAc–hexane to give 5 (0.27 g, 0.86 mmol,
7.88 (2H, d, J = 8.4 Hz, PhH). FAB-MS m/z: 291 (MÀH)À. HR-FAB-
MS m/z: (MÀH)À calcd for C15H12ClO2S, 291.0247; found, 291.0250.
5.2.10. N-(2-Aminophenyl)-4-((4-chlorobenzylthio)
methyl)benzamide (9b)
Compound 9b was prepared from 8b (0.14 g, 0.46 mmol)
according to the procedure used for 9a (0.145 g, 0.38 mmol, 82%).
75%). Mp 154–156 °C. IR (KBr) 3053, 1688, 1427 cmÀ1
.
1H NMR
Mp 120–122 °C. IR (KBr) 3300, 1632, 1601, 1489, 1230 cmÀ1 1H
.
(DMSO-d6): d 3.74 (2H, s, SCH2Ph), 3.84 (2H, s, ArCH2S), 7.41 (2H,
d, J = 8.4 Hz, PhH), 7.45–7.53 (4H, m, ArH), 7.76 (1H, s, ArH), 7.87–
7.90 (4H, m, ArH). FAB-MS m/z: 307 (MÀH)À. HR-FAB-MS m/z:
(MÀH)À calcd for C19H15O2S, 307.0793; found, 307.0800.
NMR (DMSO-d6): d 3.68 (2H, s, SCH2Ph), 3.74 (2H, s, PhCH2S), 4.90
(2H, s, PhNH2), 6.60 (1H, t, J = 7.2 Hz, PhH), 6.78 (1H, d, J = 8.0 Hz,
PhH), 6.97 (1H, t, J = 7.2 Hz, PhH), 7.16 (1H, d, J = 8.0 Hz, PhH),
7.33 (2H, d, J = 8.4 Hz, PhH), 7.39 (2H, d, J = 8.0 Hz, PhH), 7.41 (2H,
d, J = 8.0 Hz, PhH), 7.94 (2H, d, J = 8.0 Hz, PhH), 9.64 (1H, s, CONH).
FAB-MS m/z: 383 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for
C21H20ClN2OS, 383.0985; found, 383.0974.
5.2.6. N-(2-Aminophenyl)-4-((naphthalen-2-ylmethylthio)
methyl)benzamide (6)
The acid 5 (0.10 g, 0.32 mmol) was treated with benzene-1,2-
diamine (0.069 g, 0.64 mmol), DMF (1 mL), HOBt (0.065 g,
0.48 mmol) and WSC (0.092 g, 0.48 mmol) as for the preparation
of 3. The resulting solid was purified by PLC (CHCl3/MeOH = 10:1)
to give 6 (0.025 g, 0.063 mmol, 20%). Mp 145–147 °C. IR (KBr)
5.2.11. Methyl 4-((4-(thiophen-2-yl)benzylamino)
methyl)benzoate (11a)
A solution of 10 (0.21 g, 1.1 mmol) in THF (2 mL) was subjected
to reductive amination with 4-(2-thienyl)benzaldehyde (0.20 g,
1.1 mmol), NaBH(OAc)3 (0.34 g, 1.59 mmol) and TEA (0.22 mL,
1.59 mmol) in the conventional manner. The product was purified
by PLC (CHCl3) to give 11a (0.20 g, 0.59 mmol, 56%). Mp 71–72 °C.
3377, 3250, 1649, 1612, 1456, 1306 cmÀ1 1H NMR (DMSO-d6): d
.
3.76 (2H, s, SCH2Ph), 3.85 (2H, s, ArCH2S), 4.90 (2H, s, PhNH2),
6.60 (1H, t, J = 7.6 Hz, PhH), 6.78 (1H, d, J = 8.0 Hz, PhH), 6.98 (1H,
ddd, J = 8.0, 8.0, 1.2 Hz, PhH), 7.17 (1H, d, J = 8.0 Hz, PhH), 7.43
(2H, d, J = 8.4 Hz, PhH), 7.47–7.54 (3H, m, ArH), 7.77 (1H, s, ArH),
7.89–7.91 (3H, m, ArH), 7.95 (2H, d, J = 8.0 Hz, PhH), 9.65 (1H, s,
CONH). FAB-MS m/z: 399 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd
for C25H23N2OS, 399.1531; found, 399.1530.
IR (KBr) 3321, 1711, 1435, 1277 cmÀ1 1H NMR (DMSO-d6): d 3.69
.
(2H, s, NHCH2), 3.76 (2H, s, CH2NH), 3.84 (3H, s, OCH3), 7.13 (1H,
dd, J = 5.2, 3.6 Hz, thienylH), 7.38 (2H, d, J = 8.4 Hz, PhH),
7.48–7.53 (4H, m, ArH), 7.61 (2H, d, J = 8.4 Hz, PhH), 7.92 (2H, d,
J = 8.4 Hz, PhH). FAB-MS m/z: 338 (M+H)+. HR-FAB-MS m/z:
(M+H)+ calcd for C20H20NO2S, 338.1215; found, 338.1216.
5.2.7. 4-(((2,3-Dihydrobenzo[b][1,4]dioxin-2-yl)methylthio)
methyl)benzoic acid (8a)
5.2.12. 4-((4-(Thiophen-2-yl)benzylamino)methyl)benzoic acid
(12a)
To a suspension of 7 (0.048 g, 0.29 mmol) in EtOH (0.7 mL) was
added 2-bromomethyl-1,4-benzodioxane (0.043 mL, 0.29 mmol)
and then ethanolic NaOH (0.023 g, 0.58 mmol) (0.25 mL). After
being stirred at room temperature overnight, the mixture was ad-
justed to pH 3 with 1 M HCl and concentrated. The resulting crystal
was purified by PLC (CHCl3/MeOH = 10:1), yielding 8a (0.026 g,
0.082 mmol, 28%). Mp 120–122 °C. IR (KBr) 2918, 1686, 1429,
A solution of 11a (0.17 g, 0.52 mmol) in THF (2 mL) was treated
with 1 M LiOH in H2O (2.1 mL) in the conventional manner. The
mixture, after removal of THF, was adjusted to pH 3 with 1 M
HCl, and the precipitate was collected by filtration to give 12a
(0.16 g, 0.49 mmol, 95%). Mp 254–256 °C. IR (KBr) 2930, 1686,
1425 cmÀ1 1H NMR (DMSO-d6): d 4.17 (2H, s, NHCH2), 4.23 (2H,
.
1267 cmÀ1
.
1H NMR (DMSO-d6): d 2.69 (2H, d, J = 6.4 Hz, ArCH2S),
s, CH2NH), 7.16 (1H, dd, J = 5.2, 3.6 Hz, thienylH), 7.57 (2H, d,
J = 8.4 Hz, PhH), 7.58–7.6 (2H, m, thienylH), 7.66 (2H, d,
J = 8.4 Hz, PhH), 7.73 (2H, d, J = 8.4 Hz, phH), 7.99 (2H, d,
J = 8.4 Hz, PhH). FAB-MS m/z: 324 (M+H)+. HR-FAB-MS m/z:
(M+H)+ calcd for C19H18NO2S, 324.1058; found, 324.1063.
3.91 (2H, s, SCH2Ph), 3.93–3.98 (1H, m, CH), 4.25–4.31 (2H, m,
OCH2CH), 6.80–6.88 (4H, m, ArH), 7.43 (2H, d, J = 8.0 Hz, PhH),
7.87 (2H, d, J = 8.0 Hz, PhH). FAB-MS m/z: 315 (MÀH)À. HR-FAB-
MS m/z: (MÀH)À calcd for C17H15O4S, 315.0691; found, 315.0689.
5.2.8. N-(2-Aminophenyl)-4-(((2,3-dihydrobenzo[b][1,4]dioxin-
2-yl)methylthio) methyl)benzamide (9a)
5.2.13. N-(2-Aminophenyl)-4-((4-(thiophen-2-yl)benzylamino)
methyl)benzamide (13a)
Compound 8a (0.09 g, 0.28 mmol) was treated with benzene-
1,2-diamine (0.06 g, 0.56 mmol), DMF (1 mL), HOBt (0.057 g,
0.42 mmol) and WSC (0.081 g, 0.42 mmol) in the conventional
manner. The product was purified by PLC (CHCl3/MeOH = 10:1)
to give 9a (0.095 g, 0.23 mmol, 84%). Mp 125–127 °C. IR (KBr)
Compound 12a (0.10 g, 0.31 mmol) was treated with benzene-
1,2-diamine (0.067 g, 0.62 mmol), DMF (1 mL), HOBt (0.064 g,
0.47 mmol) and WSC (0.09 g, 0.47 mmol). The product was purified
by PLC (CHCl3/MeOH = 20:1) to give 13a (0.11 g, 0.26 mmol, 82%).
Mp 168–169 °C. IR (KBr) 3329, 3233, 1634, 1454, 1306 cmÀ1 1H
.
3381, 3217, 1630, 1458, 1304, 1265 cmÀ1
.
1H NMR (DMSO-d6): d
NMR (DMSO-d6): d 3.70 (2H, s, NHCH2), 3.77 (2H, s, CH2NH), 4.89
(2H, s, PhNH2), 6.60 (1H, ddd, J = 7.2, 7.2, 1.2 Hz, PhH), 6.78 (1H,
dd, J = 8.0, 1.2 Hz, PhH), 6.97 (1H, ddd, J = 7.2, 7.2, 1.2 Hz, PhH),
7.13 (1H, dd, J = 4.8, 3.2 Hz, thienylH), 7.17 (1H, d, J = 7.2 Hz,
PhH), 7.40 (2H, d, J = 8.4 Hz, PhH), 7.48–7.50 (4H, m, ArH), 7.52
(2H, dd, J = 4.8, 1.2 Hz, thienylH), 7.62 (2H, d, J = 8.4 Hz, PhH),
7.95 (2H, d, J = 8.4 Hz, PhH), 9.64 (1H, s, CONH). FAB-MS m/z: 414
(M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for C25H24N3OS, 414.1640;
found, 414.1637.
2.72 (2H, d, J = 6.0 Hz, ArCH2S), 3.93 (2H, s, SCH2Ph), 3.97–4.02
(1H, m, CH), 4.30–4.39 (2H, m, OCH2CH), 6.60 (1H, t, J = 7.6 Hz,
PhH), 6.78 (1H, d, J = 7.6 Hz, PhH), 6.83–6.91 (6H, m, ArH), 6.97
(1H, t, J = 7.6 Hz, PhH), 7.16 (1H, d, J = 7.6 Hz, PhH), 7.45 (2H, d,
J = 8.4 Hz, PhH), 7.94 (2H, d, J = 8.4 Hz, PhH), 9.65 (1H, s, CONH).
FAB-MS m/z: 407 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for
C23H23N2O3S, 407.1429; found, 407.1436.
5.2.9. 4-((4-Chlorobenzylthio)methyl)benzoic acid (8b)
Compound 8b was prepared from 7 (0.025 g, 0.12 mmol) accord-
ing to the procedure used for 8a (0.016 g, 0.055 mmol, 46%). Mp
5.2.14. Methyl 4-((benzo[b]thiophen-2-ylmethylamino)
methyl)benzoate (11b)
Compound 11b was prepared from 10 (0.25 g, 1.2 mmol) accord-
ing to the procedure used for 11a (0.086 g, 0.28 mmol, 22%). Mp 67–
68 °C. IR (KBr) 3327, 1715, 1429, 1279 cmÀ1. 1H NMR (DMSO-d6): d
158–159 °C. IR (KBr) 2939, 1692, 1427 cmÀ1
.
1H NMR (DMSO-d6):
d 3.66 (2H, s, SCH2Ph), 3.72 (2H, s, PhCH2S), 7.31 (2H, d, J = 8.4 Hz,
PhH), 7.37 (2H, d, J = 8.0 Hz, PhH), 7.39 (2H, d, J = 8.0 Hz, PhH),

S. Kiyokawa et al. / Bioorg. Med. Chem. 18 (2010) 3925–3933
3931
3.81 (2H, s, NHCH2), 3.85 (3H, s, CH3), 3.97 (2H, s, CH2NH), 7.26 (1H, s,
ArH), 7.27–7.35 (2H, m, ArH), 7.52 (2H, d, J = 8.4 Hz, PhH), 7.74 (1H, d,
J = 7.6 Hz, ArH), 7.90 (1H, d, J = 7.6 Hz, ArH), 7.93 (2H, d, J = 8.4 Hz,
PhH). FAB-MS m/z: 312 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for
C18H18NO2S, 312.1058; found, 312.1056.
Mp 75–77 °C. IR (KBr) 3366, 1666, 1504, 1337 cmÀ1
.
1H NMR
(CDCl3): d 3.86 (2H, s, NCH2Ph), 3.92 (2H, s, PhCH2N), 7.18–7.60
(12H, m, PhH), 7.70 (1H, t, J = 8.0 Hz, PhH), 7.97 (2H, d, J = 7.2 Hz,
PhH), 8.26 (1H, d, J = 8.8 Hz, PhH), 9.00 (1H, d, J = 8.8 Hz, PhH),
11.35 (1H, s, CONH). FAB-MS m/z: 438 (M+H)+. HR-FAB-MS m/z:
(M+H)+ calcd for C27H24N3O3, 438.1818; found, 438.1821.
5.2.15. 4-((Benzo[b]thiophen-2-ylmethylamino)methyl)benzoic
acid (12b)
5.2.20. N-(2-Aminophenyl)-4-((biphenyl-4-ylmethylamino)
methyl)benzamide (17)
Compound 12b was prepared from 11b (0.063 g, 0.20 mmol)
according to the procedure used for 12a (0.053 g, 0.18 mmol,
Compound 17 was prepared from 15b (0.073 g, 0.17 mmol)
according to the procedure used for 16 (0.020 g, 0.049 mmol,
29%). Mp 150–152 °C. IR (KBr) 3371, 3223, 1643, 1614, 1454,
89%). Mp 258–260 °C. IR (KBr) 2932, 1693, 1421 cmÀ1 1H NMR
.
(DMSO-d6): d 4.28 (2H, s, NHCH2), 4.51 (2H, s, CH2NH), 7.39–7.44
(2H, m, ArH), 7.66 (1H, s, ArH), 7.68 (2H, d, J = 8.4 Hz, PhH), 7.90
(1H, t, J = 4.4 Hz, ArH), 7.99 (2H, d, J = 8.4 Hz, PhH), 8.02 (1H, t,
J = 4. Hz, ArH). FAB-MS m/z: 298 (M+H)+. HR-FAB-MS m/z: (M+H)+
calcd for C17H16NO2S, 298.0902; found, 298.0904.
1306 cmÀ1 1H NMR (DMSO-d6): d 3.73 (2H, s, NHCH2), 3.79 (2H,
.
s, CH2NH), 4.90 (2H, s, PhNH2), 6.60 (1H, t, J = 6.8 Hz, PhH), 6.78
(1H, d, J = 7.6 Hz, PhH), 7.17 (1H, d, J = 7.6 Hz, PhH), 7.35–7.67
(10H, m, PhH), 7,95 (2H, d, J = 7.6 Hz, PhH), 9.64 (1H, s, CONH).
FAB-MS m/z: 408 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for
C27H26N3O, 408.2076; found, 408.2077.
5.2.16. N-(2-Aminophenyl)-4-((benzo[b]thiophen-2-ylmethyla-
mino)methyl) benzamide (13b)
Compound 13b was prepared from 12b (0.040 g, 0.13 mmol)
according to the procedure used for 13a (0.040 g, 0.10 mmol,
79%). Mp 149–150 °C. IR (KBr) 3250, 1649, 1612, 1454,
5.2.21. N-(2-Nitrophenyl)-4-((4-(thiophen-2-yl)benzylamino)
methyl)benzamide (18)
A suspension of 14 (0.41 g, 1.3 mmol) in THF (5 mL) was sub-
jected to reductive amination with 4-(2-Thienyl)benzaldeyde
(0.25 g, 1.33 mmol), NaBH(OAc)3 (0.42 g, 2.0 mmol) and TEA
(0.28 mL, 2.0 mmol). The product was purified by PLC (CHCl3) to
give 18 (0.32 g, 0.72 mmol, 54%). Mp 119–121 °C. IR (KBr) 3342,
1308 cmÀ1 1H NMR (DMSO-d6): d 3.82 (2H, s, NHCH2), 3.98 (2H,
.
s,CH2NH), 4.90 (2H, s, PhNH2), 6.60 (1H, t, J = 7.2 Hz, PhH), 6.78
(1H, dd, J = 7.2, 1.2 Hz, PhH), 6.97 (1H, ddd, J = 7.2, 7.2, 1.2 Hz,
PhH), 7.17 (1H, d, J =7.2 Hz, PhH), 7.28 (1H, s, ArH), 7.29–7.35
(2H, m, ArH), 7.50 (2H, d, J = 8.0 Hz, ArH), 7.75 (1H, d, J = 8.0 Hz,
ArH), 7.91 (1H, d, J = 8.0 Hz, ArH), 7.95 (2H, d, J = 8.0 Hz, PhH).
FAB-MS m/z: 388 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for
C23H22N3OS, 388.1484; found, 388.1474.
1678, 1501, 1431, 1335 cmÀ1 1H NMR (CDCl3): d 3.84 (2H, s,
.
NHCH2), 3.92 (2H, s, CH2NH), 7.08 (1H, dd, J = 5.2, 3.6 Hz, thienylH),
7.21–7.31 (3H, m, ArH), 7.37 (2H, d, J = 8.4 Hz, PhH), 7.54 (2H, d,
J = 8.4 Hz, PhH), 7.60 (2H, d, J = 8.4 Hz, PhH), 7.72 (1H, t,
J = 8.4 Hz, PhH), 7.98 (2H, d, J = 8.4 Hz, PhH), 8.29 (1H, dd, J = 8.4,
1.6 Hz, PhH), 9.02 (1H, d, J = 8.4 Hz, PhH), 11.36 (1H, s, CONH).
FAB-MS m/z: 444 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for
C25H22N3O3S, 444.1382; found, 444.1386.
5.2.17. N-(2-Nitrophenyl)-4-((3-(thiophen-2-yl)benzylamino)
methyl)benzamide (15a)
A suspension of 14 (0.47 g, 1.5 mmol) in THF (10 mL) was sub-
jected to reductive amination with 3-(2-thienyl)benzaldehyde
(0.29 mL, 1.53 mmol), NaBH(OAc)3 (0.49 g, 2.3 mmol) and TEA
(0.29 mL, 2.3 mmol). The product was purified by PLC (CHCl3) to
give 15a (0.34 g, 0.77 mmol, 51%). Mp 75–77 °C. IR (KBr) 3375,
5.2.22. N-(2-Aminophenyl)-4-((methyl(4-(thiophen-2-yl)
benzyl)amino)methyl)-benzamide (19)
A suspension of 18 (0.10 g, 0.23 mmol) in THF (3 mL) was sub-
jected to reductive amination with HCHO in H2O (36%) (0.024 mL,
0.23 mmol), AcOH (0.020 mL, 0.35 mmol) and NaBH(OAc)3
(0.074 g, 0.35 mmol). The mixture, after purification by PLC (CHCl3)
to a solid (0.05 g, 0.11 mmol, 82%), was reduced with NH4OAc
(0.091 g, 1.2 mmol), SnCl2Á2H2O (0.15 g, 0.66 mmol) and MeOH
(1 mL) at 50 °C for 5 h. The mixture was concentrated to a residue,
which was suspended with EtOAc. After the removal of insoluble
materials, the solution was treated with PLC (CHCl3/MeOH = 19:1)
to give 19 (0.009 g, 0.021 mmol, 19%). Mp 142–144 °C. IR (KBr)
1680, 1502, 1433, 1340 cmÀ1 1H NMR (CDCl3): d 3.86 (2H, s,
.
NHCH2), 3.92 (2H, s, CH2NH), 7.08 (1H, t, J = 3.6 Hz, thienylH),
7.21–7.35 (6H, m, ArH), 7.51–7.59 (4H, m, ArH), 7.71 (1H, ddd,
J = 8.4, 7.2, 1.6 Hz, PhH), 7.97 (2H, d, J = 8.4 Hz, PhH), 8.28 (1H,
dd, J = 8.4, 1.6 Hz, PhH), 9.01 (1H, d, J = 8.4 Hz, PhH), 11.35 (1H, s,
CONH). FAB-MS m/z: 444 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd
for C25H22N3O3S, 444.1382; found, 444.1378.
5.2.18. N-(2-Aminophenyl)-4-((3-(thiophen-2-yl)benzylamino)
methyl)benzamide (16)
3368, 3233, 1642, 1614, 1454, 1304 cmÀ1 1H NMR (CDCl3): d
.
Compound 15a (0.044 g, 0.099 mmol) was reduced with
NH4OAc (0.076 g, 0.99 mmol) and SnCl2Á2H2O (0.13 g, 0.59 mmol)
and MeOH (1 mL) at 60 °C for 5 h. The mixture was concentrated
to a residue, which was purified with PLC (CHCl3/MeOH = 20:1)
to give 16 (0.013 g, 0.031 mmol, 32%). Mp 144–146 °C. IR (KBr)
2.22 (3H, s, NCH3), 3.56 (2H, s, NCH2Ph), 3.60 (2H, s, PhCH2N),
6.83–6.86 (2H, m, PhH), 7.06–7.10 (2H, m, ArH), 7.27–7.34 (2H,
m, thienylH), 7.38 (2H, d, J = 8.0 Hz, PhH), 7.50 (2H, d, J = 8.0 Hz,
PhH), 7.59 (2H, d, J = 8.0 Hz, PhH), 7.84–7.88 (3H, m, ArH). FAB-
MS m/z: 428 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for
C26H26N3OS, 428.1797; found, 428.1793.
3321, 3231, 1634, 1456, 1296 cmÀ1 1H NMR (CD3OD): d 3.91
.
(2H, s, NHCH2), 3.97 (2H, s, CH2NH), 6.77 (1H, ddd, J = 8.0, 8.0,
1.2 Hz, PhH), 6.91 (1H, dd, J = 8.0, 1.2 Hz, PhH), 7.06–7.10 (2H, m,
ArH), 7.19 (1H, dd, J = 8.0, 1.2 Hz, PhH), 7.30 (1H, d, J = 8.0 Hz,
ArH), 7.37–7.42 (3H, m, ArH), 7.54 (2H, d, J = 8.0 Hz, ArH), 7.59
(1H, dt, J = 8.0, 1.2, 1.2 Hz, ArH), 7.68 (1H, s, PhH), 7.99 (2H, d,
J = 8.0 Hz, PhH). FAB-MS m/z: 414 (M+H)+. HR-FAB-MS m/z:
(M+H)+ calcd for C25H24N3OS, 414.1640; found, 414.1645.
5.2.23. N-(2-Aminophenyl)-4-((N-(4-(thiophen-2-yl)benzyl)
methylsulfonamido) methyl)benzamide (20)
A suspension of 18 (0.050 g, 0.11 mmol) in CH2Cl2 (0.5 mL) was
treated with methanesulfonyl chloride (8.5 lM, 0.35 mmol) and
TEA (0.03 mL, 0.22 mmol) at room temperature overnight. The
mixture, after purification by PLC (CHCl3) to an oily product
(0.045 g, 0.086 mmol, 78%), was reduced in a manner similar to
19, yielding 20 (0.010 g, 0.020 mmol, 24%). IR (KBr) 3335, 1649,
5.2.19. 4-((Biphenyl-4-ylmethylamino)methyl)-N-(2-nitro-
phenyl)benzamide (15b)
1450, 1315, 1146 cmÀ1 1H NMR (CDCl3): d 2.88 (3H, s, SO2CH3),
.
Compound 15b was prepared from 14 (0.41 g, 1.3 mmol)
according to the procedure used for 15a (0.32 g, 0.72 mmol, 54%).
4.37 (2H, s, NCH2Ph), 4.44 (2H, s, PhCH2N), 6.88 (1H, d, J = 4.0 Hz,
ArH), 7.08–7.34 (9H, m, ArH), 7.44 (2H, d, J = 8.0 Hz, ArH), 7.60

3932
S. Kiyokawa et al. / Bioorg. Med. Chem. 18 (2010) 3925–3933
(2H, d, J = 8.0 Hz, ArH), 7.93 (2H, d, J = 8.0 Hz, ArH). FAB-MS m/z:
492 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for C26H26N3O3S2,
492.1416; found, 492.1424.
were used at 6 weeks of age. Antibodies specific to p21/WAF1
and b-actin were purchased from Sigma–Aldrich, and anti-acetyl-
histone H3 was from Millipore.
5.2.24. N-(2-Aminophenyl)-4-((3,3-dimethyl-1-(4-(thiophen-2-
yl)benzyl)ureido) methyl)benzamide (21)
5.5. Evaluation of HDAC1 inhibition
A suspension of 18 (0.050 g, 0.11 mmol) in CH2Cl2 (0.5 mL) was
treated with dimethylcarbamyl chloride (0.010 mL, 0.11 mmol)
and TEA (0.03 mL, 0.22 mmol) at room temperature overnight.
The mixture, after purification by PLC (CHCl3) to an oily product
(0.053 g, 0.10 mmol, 94%), was reduced in a manner similar to
19, giving 21 (0.019 g, 0.039 mmol, 38%). IR (KBr) 3256, 1620,
The IC50 values of 3, 6, 9a and 9b were determined using a
SCADS inhibitor kit by the Screening Committee of New Anticancer
Agents.23,24 Those of other compounds were measured utilizing
HDAC1 Fluorimetric Drug Discovery Kit-AK-511 (BIOMOL).
5.6. Evaluation of growth inhibition in HCT116 cells
1454, 1393, 1315 cmÀ1 1H NMR (CDCl3): d 2.92 (6H, d, J = 2.4 Hz,
.
NCH3), 4.30 (2H, s, NCH2Ph), 4.35 (2H, s, PhCH2N) 7.09–7.32 (9H,
m, ArH), 7.58 (3H, m, ArH), 7.86 (2H, d, J = 7.6 Hz, ArH), 8.07 (1H,
s, ArH). FAB-MS m/z: 485 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd
for C28H29N4O2S, 485.2011; found, 485.2016.
HCT116 cells were cultured in McCoy’s 5a medium with 10%
FBS. The cells (1 Â 104 /mL) were inoculated onto standard 96-well
microtiter plates. Following 24 h of culture, serially diluted sam-
ples were added to the wells. After a 3-day culture, cell growth
was evaluated with the assay of WST-1 (Dojindo), and IC50 values
were calculated.
5.2.25. N-(2-Aminophenyl)-4-(((2-hydroxyethyl)(4-(thiophen-2-
yl)benzyl)amino) methyl)benzamide (22)
A solution of 18 (0.10 g, 0.23 mmol) in MeCN (1 mL) was re-
acted with 2-bromoethanol (0.033 mL, 0.46 mmol) and TEA
(0.064 mL, 0.46 mmol) at 60 °C for 12 h. The product was, after
purification with PLC (0.061 g, 0.13 mmol, 54%), was reduced in a
manner similar to 19, affording 22 (0.11 g, 0.24 mmol, 45%). Mp
5.7. Western blot analysis
HCT116 cells were plated onto 60-mm diameter dishes
(1.0 Â 106/dish). After incubating for 24 h, the cells were washed
twice with the serum-free medium (1 mL) and suspended with
the serum-free medium (5 mL) for 24 h. After removal of the med-
ium, the cells were washed twice with the serum-free medium
(1 mL) and incubated in the serum-free medium (5 mL) with a test
146–148 °C. IR (KBr) 3240, 1651, 1612, 1454, 1292 cmÀ1
.
1H NMR
2.71 (2H, t, J = 5.2 Hz, CH2CH2OH), 3.63 (2H, t,
(CDCl3):
d
J = 5.2 Hz, CH2CH2OH), 3.66 (2H, s, NCH2Ph), 3.71 (2H, s, PhCH2N),
6.85–6.87 (2H, m, PhH), 7.07–7.12 (2H, m, ArH), 7.28–7.33 (4H,
m, ArH), 7.45 (2H, d, J = 8.0 Hz, PhH), 7.59 (2H, d, J = 8.0 Hz, PhH),
7.84–7.89 (3H, m, ArH). FAB-MS m/z: 458 (M+H)+. HR-FAB-MS
m/z: (M+H)+ calcd for C27H28N3O2S, 458.1902; found, 458.1903.
compound (10
and floating and adherent cells were washed twice with PBS (each
1 mL), spraped, and resuspended in 200 L of modified Covance
lM) for another 24 h. The medium was discarded,
l
Laboratoes lysis buffer25 (pH 8.0 and 1% Nonidet P-40 were
adopted in place of pH 7.4 and 1% Triton-X 100, respectively).
The lysates were centrifuged at 14,000g for 15 min at 4 °C. The pro-
tein concentration was determined with a BCA protein assay kit
(Thermo Fisher Scientific). An equal amount of protein was then
resolved by SDS–PAGE and transferred to PVDF membrane. The
blots (1 Â Tris–HCl buffer saline, 5% non-fat milk and 0.05% Tween
20) were probed with an antibody specific to each protein and de-
tected using ECL chemiluminescence.
5.2.26. N-(2-Aminophenyl)-4-(((biphenyl-4-ylmethyl)(2-hydroxy-
ethyl)amino) methyl)benzamide (23)
Compound 23 was prepared from 15b (0.21 g, 0.48 mmol)
according to the procedure used for 22 (0.11 g, 0.23 mmol, 20%).
Mp 152–154 °C. IR (KBr) 3321, 3211, 1643, 1612, 1454,
1312 cmÀ1 1H NMR (DMSO-d6): d 2.53 (2H, m, CH2CH2OH), 3.51–
.
3.57 (2H, m, CH2CH2OH), 3.65 (2H, s, NCH2Ph), 3.71 (2H, s,
PhCH2N), 4.47 (1H, t, J = 5.2 Hz, CH2CH2OH), 4.89 (2H, s, PhNH2),
6.59 (1H, t, J = 7.6 Hz, PhH), 6.78 (1H, d, J = 7.6 Hz, PhH), 6.97 (1H,
t, J = 7.6 Hz, PhH), 7.16 (1H, d, J = 7.6 Hz, PhH), 7.35 (1H, t,
J = 7.6 Hz, PhH), 7.44–7.54 (6H, m, PhH), 7.65 (4H, dd, J = 9.2,
7.6 Hz, PhH), 7.95 (2H, d, J = 8.0 Hz, PhH), 9.63 (1H, s. CONH).
FAB-MS m/z: 452 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for
C29H30N3O2, 452.2338; found, 452.2334.
5.8. Examination of cell cycle regulation by flow cytometry
HCT116 cells were plated onto 60-mm diameter dishes
(1.0 Â 106/dish). After incubation for 24 h, the cells were treated
as for the Western blot analysis. The adherent cells were treated
with 0.25% trypsin (Invitrogen) and combined with the floating
cells. All the cells were treated with a Cycle Test Plus DNA reagent
Kit (Catalog No. 340242, Becton Dickinson). DNA content was mea-
sured with a FACS™Cant II.
5.3. Solubility tests of HDAC inhibitors in 10% DMSO
A solution (100 mg/mL) of each HDAC inhibitor in DMSO was
diluted with H2O to prepare a 10% DMSO suspension (10 mg/mL),
which was subsequently sonicated for 15 min, stirred with a Vor-
tex for 0.5 min and allowed to stand for 5 min. Dilution with 10%
DMSO followed by the same procedures was repeated until
residual fine particles which were suspended but not precipi-
tated, started to appear on observation with a magnifying glass
(Â10).
5.9. TUNEL assay by flow cytometry
HCT 116 cells (1.0 Â 106/dish) were treated as above. The col-
lected floating and adherent cells were washed twice with PBS
(each 1 mL). The cells were fixed with PBS (0.5 mL) (containing
BSA (2.5 mg) and 0.1% TRITON X-100) and 1% paraformaldehyde
(5 mL) for 20 min at 0 °C, washed twice with the above PBS (each
5 mL) and permeabilized by PBS (0.5 mL) (containing BSA
(2.5 mg) and 0.1% TRITON X-100) and 70% EtOH. The TUNEL reac-
5.4. Cells, animals and antibodies
tion was carried out by incubating cells (60 min; 37 °C) with 50 lM
HCT116 cells were cultured in McCoy’s 5A medium (Invitrogen)
of TUNEL mixture (DeadEnd™ Fluorometirc TUNEL System) and
treated with PI solution (Dojindo) and RNase (Wako) according
to the procedure (Catalog No. G3250, Promega). Rates of cell frag-
mentation were measured with FACS™Cant II.
supplemented with 10% fetal bovine serum (FBS), 50
lg/mL peni-
cillin G, and 50 g/mL streptomycin sulfate (Invitrogen) in a 5%
l
CO2 and 95% air atmosphere at 37 °C. Female BALB/c-nu/nu mice
(4 weeks of age) were purchased from Japan SLC Inc. The mice

S. Kiyokawa et al. / Bioorg. Med. Chem. 18 (2010) 3925–3933
3933
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l
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treated mice by tumor volume for control mice. At day18, the cer-
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5.11. Statistical analysis
The statistical significance of the effects of drugs virus control
was analyzed with Student’s t-test.
Acknowledgments
Part of this work was supported by the Strategic Project to Sup-
port the Formation of Research Bases at Private Universities. This is
a product of research financially supported in part by Kansai Uni-
versity Research Grants: Grant-in-aid for Joint Research, 2009–
2010. We are grateful to the Screening Committee for New Anti-
cancer Agents supported by a Grant-in-aid for Scientific Research
on the Priority Area ‘Cancer’ from The Ministry of Education, Cul-
ture, Sports, Science and Technology, Japan, for the evaluation of
HDAC1.
21. Marson, C. M.; Savy, P.; Rioja, A. S.; Mahadevan, T.; Mikol, C.; Veerupillai, A.;
Nsubuga, E.; Chahwan, A.; Joel, S. P. J. Med. Chem. 2006, 49, 800.
22. Gediya, L. K.; Belosay, A.; Khandelwal, A.; Purushottamachar, P.; Njar, V. C. O.
Bioorg. Med. Chem. 2008, 16, 3352.
23. Yoshida, M.; Furumai, R.; Nishiyama, M.; Komatsu, Y.; Nishino, N.; Horinouchi,
S. Cancer Chemother. Pharmacol. 2001, 48, S20.
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