Anti-tumor activity of new orally bioavailable 2-amino-5-(thiophen-2-yl) benzamide-series histone deacetylase inhibitors, possessing an aqueous soluble functional group as a surface recognition domain

Article abstract of DOI:10.1016/j.bmcl.2012.01.053

New orally bioavailable 5-(thiophen-2-yl)-substituted 2-aminobenzamide- series histone deacetylase inhibitors were synthesized. These compounds possess a morpholine or piperadine-derived moiety as an aqueous soluble functional group. Among them, 8b, havin

Full text of DOI:10.1016/j.bmcl.2012.01.053

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1926–1930
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Bioorganic & Medicinal Chemistry Letters
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Anti-tumor activity of new orally bioavailable 2-amino-5-(thiophen-2-yl)
benzamide-series histone deacetylase inhibitors, possessing an aqueous
soluble functional group as a surface recognition domain
Yoshiyuki Hirata a, Masahiko Hirata b, Yasuyuki Kawaratani a, Makio Shibano b, Masahiko Taniguchi b,
Masahide Yasuda b, Yoshiro Ohmomo b, Yasuo Nagaoka a, 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 orally bioavailable 5-(thiophen-2-yl)-substituted 2-aminobenzamide-series histone deacetylase
inhibitors were synthesized. These compounds possess a morpholine or piperadine-derived moiety as
an aqueous soluble functional group. Among them, 8b, having a 4-ethyl-2,3-dioxopiperazine-1-carbox-
amide group as a surface recognition domain, showed promising inhibitory activities against HCT116 cell
growth and HDAC1/2. Notably, unlike MS-275, this compound did not induce apoptosis in the cell cycle
tests. We therefore conducted antitumor tests of 8b and MS-275 against HCT116 cell xenografts in nude
mice. Compound 8b reduced the volume of tumor mass to T/C: 60% and 47% at 45 and 80 mg/kg over
16 days, respectively. These values were comparable to the rate (T/C: 51% at 45 mg/kg) for MS-275. Fur-
thermore, 8b, at neither 45 nor 80 mg/kg, induced the weight loss which was observed in the mice given
MS-275 at 45 mg/kg.
Received 26 December 2011
Revised 14 January 2012
Accepted 14 January 2012
Available online 26 January 2012
Keywords:
Histone deacetylase inhibitor
Antitumor activity
2-Amino-5-(thiophen-2-yl)benzamide
HCT116 xenograft
Ó 2012 Elsevier Ltd. All rights reserved.
Histone deacetylases (HDACs) have drawn attention as biologi-
cal molecules, and their overexpression is closely associated with
the incidence and growth of carcinomas.1–5 HDAC inhibitors regu-
late the growth of cancer cells through the epigenetic activation
of gene transcription leading to cell cycle regulation as well as
apoptosis. HDACs are grouped into four classes, which are further
classified into 18 isoforms on the basis of their size, cellular locali-
zation, number of catalytic active sites, protein homology, etc.6,7
Most of these enzymes contain a zinc ion at the active site, and a
number of papers have reported on the docking of these enzymes
and HDAC inhibitors, utilizing the X-ray crystal structure of histone
deacetylase-like protein (HDLP),8 HDAC8,9,10 and HDAC2.11
Most HDAC inhibitors comprise a hydroxamic acid or 2-amino-
benzamide moiety as a zinc ion-chelating domain. The former
compounds, typified by TSA12,13 and SAHA8,14 (Zolinza), are broad-
ranging inhibitors of HDACs, whereas the latter, typified by MS-
27515–17 and MGCD-0103,18 are orally available inhibitors that
target HDAC 1, 2 and 3 (Fig. 1).
HDAC1/2-selective inhibition by projecting the 2-thienyl ring into
the extensive internal cavity adjacent to the zinc-binding site within
the HDAC1/2 pocket.11,19–24 Since both HDAC1 and 2 are deeply in-
volved in canceration,25–30 the inhibitors selective for these iso-
forms may provide potential utility as anti-cancer therapeutics.
The Merck & Co. group prepared 5-(thiophen-2-yl)-substituted
2-aminobenzamide derivatives with an aqueous soluble functional
group such as a phosphonate/phosphinate or phenylglycine/phen-
ylalanine derivative. Although representative compounds showed
inhibitory activity against tumor growth in a HCT116 xenograft
mouse model, the efficacy did not seem to reflect the increased
selectivity toward HDAC1/2.
In the course of searching for orally bioavailable 2-aminobenz-
amide-series HDAC inhibitors, we prepared the 5-(thiophen-
2-yl)-substituted compound 1a which had
a (benzo[d][1,3]
dioxol-5-ylmethyl)(2-hydroxyethyl)amino group as a surface rec-
ognition domain (Fig. 2). Compound 1a showed stronger HDAC1/2
inhibitory activities31 (HDAC1 IC50: 0.06
lM; HDAC2 IC50: 0.74
lM)
Recently, diverse 2-amino-5-(thiophen-2-yl)benzamide-series
HDAC inhibitors were synthesized by the groups of Merck & Co.,
Takeda Pharmaceuticals and MethylGene. These compounds exert
than the 5-(thiophen-2-yl)-unsubstituted counterpart (1b)
(HDAC1 IC50: 1.17 lM; HDAC2 IC50: 3.36 lM) as was the case in
MS-275 and its 2-thienyl-substituted derivative,19 though it had
a problem with aqueous solubility (<0.1 ml for 10% DMSO) (Table
1). This finding prompted us to synthesize new potential 5-(thio-
phen-2-yl)-substituted 2-aminobenzamide-series compounds
with aqueous soluble functionality. In the present study, we tried
⇑
Corresponding author. Tel.: +81 6 6368 0834; fax: +81 6 6388 8609.
E-mail address: uesato@ipcku.kansai-u.ac.jp (S. Uesato).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.053

Y. Hirata et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1926–1930
1927
CH3
N
O
H
N
CH3
CH3
H3C
H
N
OH
N
H
OH
O
O
O
SAHA
TSA
O
N
O
N
H
NH2
N
N
H
N
N
H
NH2
H
N
N
O
O
MGCD-0103
MS-275
Figure 1. Structure of HDAC inhibitors.
O
O
N
NH2
R
H3C
H
N
N
H
NH2
O
H
N
OH
O
O
1a: R = thiophen-2-yl
1b: R = H
S
2
Figure 2. Structure of 5-(thiophen-2-yl)-substituted 2-aminobenzamides.
Table 1
Solubility and effect of HDAC inhibitors against HCT116 cell growth and HDAC1/2 activities
Compound
IC50
(l
M)a
IC50
(l
M)b
10% DMSO/H2O (mg/mL)
HCT116
HDAC1
HDAC2
1a
1b
2
8a
8b
8c
MS-275
TSA
0.49
1.20
0.56
0.44
0.51
0.53
0.76
0.06
1.17
0.04
0.10
0.05
0.08
0.48
0.006
0.74
3.36
0.47
0.88
0.67
0.80
1.51
0.018
<0.1
0.2
0.5
c
—
0.6
1.0
0.8
a
b
c
Measured after a 3-day incubation of test compounds with cells. Assays were performed in triplicate.
Assays were performed in duplicate.
Compound 8a was an oil substance.
to produce the new HDAC inhibitors in which the 4-
(((benzo[d][1,3]dioxol-5-ylmethyl)(2-hydroxyethyl)amino)methyl)
benzamide moiety in 1a was replaced with a 4-((N-substituted
ureido)methyl)benzamide moiety.
and 4-ethyl-2,3-dioxopiperazine-1-carbonyl chloride followed by
the reduction of the resulting product 7b. Compound 8c was syn-
thesized utilizing 6 and 4-methylpiperazine-1-carbonyl chloride
in the conventional way.
New 5-(thiophen-2-yl)-substituted 2-aminobenzamide-series
histone deacetylase inhibitors 8a, 8b and 8c were synthesized
as illustrated in Scheme 1. Thus, 4-((tert-butoxycarbonylami-
no)methyl)benzoic acid (3) was reacted with oxalyl choride to
yield the carbonyl chloride 4. This compound was treated with 2-
nitro-5-thiophen-2-yl-phenylamine to yield the condensation
product 5. Deblocking of a Boc group with 15% HCl afforded the
amine hydrochloride salt 6. Compound 6 was reacted with 4-mor-
pholinecarbonyl chloride in the presence of TEA to give the nitro-
phenyl derivative 7a, which was reduced to 8a with SnCl2 and
NH4OAc. Similarly, 8b was prepared through condensation of 6
We evaluated the antiproliferative activities (IC50s) of the com-
pounds synthesized using a WST-1assay against human colon can-
cer HCT116 cells. We also measured their inhibitory activities
against HDAC1/2 as well as aqueous solubility in 10% DMSO/H2O
(Table 1). In the present experiments, we selected MS-275 and
compound 223 (Fig. 2) as reference compounds because the former
is a typical 5-(thiophen-2-yl)-unsubstituted 2-aminobenzamide-
type HDAC inhibitor, whereas the latter is a 5-(thiophen-2-yl)-
substituted 2-aminobenzamide-type HDAC inhibitor exhibiting a
highly selective HDAC1/2 inhibition. Compounds 8a, 8b and 8c,
as well as 2, exhibited slightly stronger inhibition than MS-275

1928
Boc
Y. Hirata et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1926–1930
Boc
Boc
ClH3N
NO2
S
N
H
a
N
H
N
H
NO2
b
c
H
N
H
N
COOH
COCl
O
O
3
4
6
5
S
O
O
g
d, e or f
R
N
NH2
H
N
R
N
H
NO2
H
6
H
N
O
O
S
S
N
O
N
O
8a: R =
8b: R =
7a: R =
N
O
N
N
N
7b: R =
CH3
CH3
O
O
N
O
N
N CH3
N CH3
8c: R =
7c: R =
Scheme 1. Reagents: (a) oxalyl chloride, DMF, Py; (b) 2-nitro-5-(thiophen-2-yl)-benzeneamine, DMF, Py; (c) 15% HCl, THF, MeOH; (d) 4-morpholinecarbonyl chloride, THF,
TEA, DMF; (e) 4-ethyl-2,3-dioxo-1-piperazinecarbonyl chloride, CH2Cl2, TEA, DMF; (f) 4-methylpiperazine-1-carbonyl chloride, CH2Cl2, TEA, DMF; (g) SnCl2, NH4OAc, THF,
MeOH.
unsubstituted derivative) though these compounds suppressed
the growth of HCT116 cells to the same extent.
8b
2
control
MS-275
Compound 8b was moderately soluble (0.6 mg/ml) in 10%
DMSO and had a pharmacokinetic profile (Cmax: 8.3 lM; T1/2: 2.2
acetylated-histone H3
histone H3
h)32 similar to HDAC inhibitors33–37 under development. We thus
conducted comparative anti-tumor tests of 8b and MS-275 using
xenografts of HCT116 cells in nude mice.32 Mice were divided into
the following four groups: vehicle (0.1% Tween 80), n = 6; MS-275
(45 mg/kg), n = 7; 8b (45 mg/kg), n = 7; 8b (80 mg/kg), n = 7. Each
compound was administered orally to nude mice with tumor sizes
of 90–110 mm3 on days 0, 2, 4, 6, 8, 10, 12 and 14, and tumor vol-
ume and body weight were monitored over 16 days.
Figure 3. Effects of 8b, 2 and MS-275 on the histone H3 acetylation. HCT116 cells
were treated with 8b, 2 or MS-275 at 10 M for 24 h. Cells were lysed to extract
crude histone proteins. The Western blot analysis was performed with crude
histone samples using antibodies specific to hisotne H3 and total acetylated histone
H3. Results are representative of five independent experiments.
l
Compound 8b suppressed the growth of tumor xenografts to
T/C: 60% at 45 mg/kg and 47% at 80 mg/kg. These values were close
to the rate (T/C: 51% at 45 mg/kg) for MS-275 (Fig. 4A). Further-
more, a loss of weight was induced by MS-275 at 45 mg/kg, though
not by 8b at either 45 or 80 mg/kg (Fig. 4B).
against the growth of HCT116 cells. Compound 8b suppressed the
HDAC1/2 activities to nearly the same extent as 2 and promoted
the hyperacetylation of core histone H3 as did
2
and
MS-275 in the HCT116 cells (Fig. 3). Noticeably, in the cell cycle
tests (Table 2), the G2/M phase cells were more accumulated on
A new orally bioavailable 2-amino-5-(thiophen-2-yl)benzam-
ide-series HDAC inhibitor, 8b, comprising a 4-ethyl-2,3-dioxopip-
erazine-1-carboxamide group, had a promising pharmacological
profile, including the tumor-shrinking activity comparable to that
of MS-275 as well as no weight loss in nude mice. This in vivo effi-
cacy could be attributed to the cytostatic inhibitory effect of 8b on
exposure of the cells to 8b or 2 than to MS-275 at 10 lM for
48 h. Additionally, MS-275 induced apoptosis as suggested by the
appearance of subG1 population (30.2% in 48 h), whereas neither
8b nor 2 did (1.4% and 1.4%, respectively). This result suggests that
both 8b and 2 (2-thienyl-substituted derivatives) might have a
more cytostatic effect on HCT116 cells than MS-275 (2-thienyl-
Table 2
Effect of HDAC inhibitors on the cell cycle in HCT116 cells
% of subG1
% of G0/G1
% of S
% of G2/M
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
Control (0.1%) DMSO
8b
2
5.7
2.3
4.6
8.8
2.3
1.4
1.4
74.2
71.5
79.5
72.7
81.9
75.3
75.0
62.1
10.6
3.8
5.2
10.4
2.8
2.8
9.5
22.4
10.7
13.6
5.4
20.5
20.8
5.5
MS-275
30.2
4.9
2.2
HCT116 cells (1.0 Â 106), after incubating for 24 h, were treated with 8b, 2 or MS-275 (each 10 M) for 24 or 48 h. The cells were then treated with a BD Cycletest Plus DNA
reagent Kit. Data are representative of three independent experiments.

Y. Hirata et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1926–1930
1929
1.1
A
B
1200
1100
1000
900
800
700
600
500
400
300
200
100
0
1.05
control
MS-275 (45 mg/kg)
1
0.95
0.9
8b (45 mg/kg)
8b (80 mg/kg)
control
MS-275 (45 mg/kg)
86b((4455mmgg//kkgg))
0.85
0.8
6 (80 mg/kg)
8b (80 mg/kg)
0
2
4
6
8
10 12 14 16 18
0
2
4
6
8
10 12 14 16 18
day
day
Figure 4. Effects of 8b and MS-275 on HCT116 cell-inoculated xenografts in vivo. The effects of 8b and MS-275 on tumor volume (4A) as well as relative body weight (RBW)
changes (4B) were examined. Compounds were orally administered to mice at the indicated time points. Vertical bars indicate standard errors. Significant differences
between tumor volumes: p < 0.05 (by two-tailed Student t-test) for 8b or MS-275 versus control at all the time points. Significant differences between body weights: p < 0.05
for MS-275 versus control, 8b (45 mg/kg) or 8b (80 mg/kg) at all the time points except for day16 as well as for MS-275 versus 8b (80 mg/kg) at day16.
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