Identification of a potent and stable antiproliferative agent by the prodrug formation of a thiolate histone deacetylase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2006.12.117

To identify prodrugs of a thiolate histone deacetylase inhibitor NCH-31 that show potent antiproliferative activity and are stable in human plasma, we synthesized several candidate prodrugs of NCH-31. Among these compounds, S-2-methyl-3-phenylpropanoyl compound 2 showed more potent antiproliferative activity and higher stability in human plasma than S-isobutyryl compound NCH-51.

Full text of DOI:10.1016/j.bmcl.2006.12.117

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1558–1561
Identification of a potent and stable antiproliferative agent by the
prodrug formation of a thiolate histone deacetylase inhibitor
Takayoshi Suzuki,a,* Shinya Hisakawa,a Yukihiro Itoh,a Sakiko Maruyama,b
Mineko Kurotaki,b Hidehiko Nakagawaa and Naoki Miyataa,*
aGraduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
bEvaluation Group, Drug Research Department, R&D Division, Pharmaceuticals Group, Nippon Kayaku Co., Ltd, 31-12,
Shimo 3-chome, Kita-ku, Tokyo 115-8588, Japan
Received 29 November 2006; revised 22 December 2006; accepted 29 December 2006
Available online 13 January 2007
Abstract—To identify prodrugs of a thiolate histone deacetylase inhibitor NCH-31 that show potent antiproliferative activity and
are stable in human plasma, we synthesized several candidate prodrugs of NCH-31. Among these compounds, S-2-methyl-3-phe-
nylpropanoyl compound 2 showed more potent antiproliferative activity and higher stability in human plasma than S-isobutyryl
compound NCH-51.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The dynamic homeostasis of the nuclear acetylation of
histones is regulated by the opposing activity of the en-
zymes histone acetyl transferases and histone deacety-
lases (HDACs).1,2 Deacetylation of histone lysine
residues is associated with a condensed chromatin struc-
ture resulting in transcriptional repression, whereas
acetylation of histones is associated with a more open
chromatin configuration and activation of transcrip-
tion.3 Aberrant activation of HDACs results in the tran-
scriptional repression of oncoprotein and is linked to the
malignant phenotypes of tumors.4,5 Inhibition of
HDACs causes histone hyperacetylation which leads
to the disruption of the chromatin structure and the
transcriptional activation of genes associated with can-
cer.3,6 In addition, it has recently been reported that
inhibition of an HDAC exerts antitumor effects through
the non-transcriptional pathway in addition to the inter-
vention of transcriptional regulation.7,8 Indeed, HDAC
inhibitors such as trichostatin A (TSA) and suberoylan-
ilide hydroxamic acid (SAHA) (Fig. 1) have potent anti-
cancer effects in vitro and in vivo.9,10
NCH-31 (Fig. 2), are potent HDAC inhibitors.11 Thiols
are thought to inhibit HDACs by coordinating the zinc
ion in the active site which is required for deacetylation
of the acetylated lysine substrate. Further, the S-isobu-
tyryl prodrug NCH-51 (Fig. 2), which is thought to be
hydrolyzed to free thiol within cells, showed antiprolif-
erative activity against cancer cells,12 however, NCH-
51 has stability problems. NCH-51 was vulnerable to
human plasma metabolism at the remaining rate of
approximately 50% after 24 h of incubation. We there-
fore decided to search for prodrugs that are more stable
in human plasma and exert more potent antiprolifera-
tive activity than NCH-51.
Since prodrugs with a less hindered acyl group tended
to exhibit less potent antiproliferative activity,12 we
designed S-acyl prodrugs 1–4 which bear an acyl group
more bulky than NCH-51. Four candidate prodrugs of
NCH-31 were synthesized as shown in Scheme 1. The
sulfhydryl group of NCH-31 was acylated with the
corresponding carboxylic acid to give the desired com-
pounds 1–4.
In the course of our study of HDAC inhibitors, we
found that a series of thiol-based analogues, including
The compounds synthesized in this study were initially
tested in antiproliferative activity assays using human
lung cancer NCI-H460 cells and human breast cancer
MDA-MB-231 cells (Table 1).13 Although S-2-meth-
oxy-2-phenylacetyl compound 3 and 3-phenyl-2-(phe-
nylmethyl)propanoyl compound 4 were less active than
thiol NCH-31 and its S-isobutyryl prodrug NCH-51,
Keywords: Histone deacetylase inhibitor; Antiproliferative agent;
Prodrug.
*
Corresponding authors. Tel./fax: +81 52 836 3407 (N. M.); e-mail
addresses: suzuki@phar.nagoya-cu.ac.jp; miyata-n@phar.nagoya-cu.
ac.jp
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.117

T. Suzuki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1558–1561
1559
O
O
O
H
N
OH
OH
N
H
N
H
O
Me2N
SAHA
TSA
Figure 1. Structures of TSA and SAHA.
Table 1. Proliferation inhibition data on NCI-H460 cells and MDA-
MB-231 cells for NCH-31, NCH-51, and compounds 1–4a
H
N
SH
N
S
O
O
H
N
N
S
6
R
NCH-31
S
O
H
N
S
N
Compound
R
EC50 (lM)
S
O
O
NCI-H460
7.6
MDA-MB-231
>20
NCH-31
NCH-51
–H
NCH-51
Me
Me
Ph
Me
2.1
3.2
1.5
11
4.4
Figure 2. Structures of NCH-31 and NCH-51.
1
2
3
>20
O
H
H
N
a or b
N
N
N
SH
S
R
6
Ph
S
O
6
S
O
1.9
Me
Ph
OMe
NCH-31
Ph
1: R =
2: R =
3: R =
4: R =
Me
>20
>20
Ph
Me
Ph
Ph
OMe
4
>20
Ph
a Values are means of at least two experiments.
Ph
Ph
Scheme 1. Reagents and conditions: (a) RCOCl (for 1 and 4), DMAP,
pyridine, 0 ꢁC to rt, 41% for 1, 54% for 4; (b) RCOOH (for 2 and 3),
EDCI, DMAP, DMF, rt, 63% for 2, 23% for 3.
(R)-10 and (S)-10 with NCH-31 in the presence of EDCI
and DMAP afforded optically active (R)-2 and (S)-2,
respectively. The enantiomeric excess of both (R)-2
and (S)-2 was determined to be 90% by chiral column
chromatography.15
S-2-phenylpropanoyl compound 1 showed similar activ-
ity against NCI-H460 cells and S-2-methyl-3-phenyl-
propanoyl compound 2 also displayed similar activity
against both NCI-H460 cells and MDA-MB-231 cells
when compared with NCH-51. Furthermore, we exam-
ined proliferation inhibition by NCH-51, compounds 1
and 2, against eight solid cancer cell lines. As can be seen
from Table 2, compound 2 showed potent proliferation
inhibition against various cancer cells representing a 2-
fold improvement over NCH-51 (average EC50 of 2
2.4 lM, NCH-51 4.9 lM).
We examined the antiproliferative activity of (R)-2 and
(S)-2 against four cancer cell lines, and there was not
much difference between the activities of the two stereo-
isomers (Table 3).
Next, we investigated the in vitro HDAC inhibitory
activity of compound 2 (Table 4).16 Although NCH-31
exhibited potent inhibitory activity against HDAC1
(IC50 = 0.048 lM), the S-2-methyl-3-phenylpropanoyl
prodrug 2 did not inhibit HDAC1 at a concentration
of 100 lM.
To investigate the difference in activity between (R)-2
and (S)-2, optically active (R)-2 and (S)-2 were prepared
from (S)-4,5,5-trimethyloxazolidin-2-one 514 as outlined
in Scheme 2. The chiral auxiliary 5 was converted to
compounds 6 and 7 by N-propionylation and N-3-phe-
nylpropionylation, respectively. Stereoselective enolate
benzylation of 6 and methylation of 7, and the subse-
quent hydrolysis of 8 and 9 gave optically active 2-
methyl-3-phenylpropanoic acids (R)-10 and (S)-10,
respectively. The condensation of carboxylic acids
Compound 2 was evaluated for the accumulation of
acetylated histone H4 using Western blot analysis
(Fig. 3).17 Treatment of HCT116 cells with compound
2 produced an increase in the accumulation of acetylat-
ed histone H4, which indicated that the antiproliferative
activity of compound 2 significantly correlates with the
inhibition of HDACs. Since S-2-methyl-3-phenylpropa-
noyl prodrug 2 was totally inactive in an enzyme assay

1560
T. Suzuki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1558–1561
Table 2. Proliferation inhibition data on eight human solid cancer cell lines for NCH-51, compounds 1 and 2a
Cell
NCH-51, EC50 (lM)
1, EC50 (lM)
2, EC50 (lM)
T-47D
Breast cancer
Breast cancer
Lung cancer
Renal cancer
Prostate cancer
Colon cancer
Colon cancer
Melanoma
8.3
4.4
2.1
6.8
9.5
2.3
1.3
4.3
>100
27
4.3
1.9
1.5
2.6
3.2
1.8
1.2
3.0
MDA-MB-231
NCI-H460
A498
3.2
26
PC-3
DLD-1
37
86
HCT116
MALME-3M
23
33
Mean
4.9
42
2.4
a Values are means of at least two experiments.
Table 4. HDAC1 inhibition data for TSA, NCH-31, and compound 2a
O
O
O
O
b
b
Compound
IC50 (lM)
N
N
O
O
O
TSA
NCH-31
2
0.026
0.048
>100b
a
O
Ph
NH
O
8
6
a Values are means of at least three experiments.
b 19% inhibition at 100 lM.
O
O
O
a
5
N
Ph
N
Ph
O
O
2
7
9
SAHA
(5 μM)
(5 μM)
control
O
O
O
H
c
c
d
d
N
N
N
Ac histone H4
8
9
S
Ph
HO
HO
Ph
Ph
6
S
S
O
Figure 3. Western blot analysis of histone hyperacetylation in
HCAT116 cells produced by compound 2 and by reference compound
SAHA.
(R)-10
(R)-2
O
H
N
S
Ph
Ph
Ph
6
was found to be tolerant to human plasma metabolism.
Nearly 100% of the intact compound was observed even
after 24-h incubation in human plasma.
O
(S)-10
(S)-2
Scheme 2. Reagents and conditions: (a) i—n-BuLi, THF, À78 ꢁC;
ii—propionyl chloride (for 6) or 3-phenylpropionyl chloride (for 7),
THF, À78 ꢁC, 71% for6, 83%for 7; (b)i—LDA, THF, À78 ꢁC;ii—BnBr
(for 8) or MeI (for 9), THF, À78 ꢁC to rt, 71% for 8, 63% for 9; (c)
LiOHÆH2O, THF, H2O, 0 ꢁC to rt, 77% for (R)-10, 78% for (S)-10; (d)
NCH-31, EDCI, DMAP, CH2Cl2, rt, 57% for (R)-2, 52% for (S)-2.
In summary, to identify potent prodrugs of a thiolate
HDAC inhibitor, NCH-31, that are more stable in
human plasma than NCH-51, we designed and pre-
pared several candidate prodrugs of NCH-31. Among
these compounds, S-2-methyl-3-phenylpropanoyl com-
pound 2 showed more potent antiproliferative activity
than S-isobutyryl compound NCH-51. Compound 2
also displayed high stability in human plasma. Fur-
ther study pertaining to compound 2 is underway to
investigate the therapeutic efficacy against human
cancers.
Table 3. Proliferation inhibition data on four human solid cancer cell
lines for (R)-2 and (S)-2a
Cell
(R)-2, EC50
(lM)
(S)-2 EC50
(lM)
MDA-MB-231
NCI-H460
PC-3
Breast cancer
Lung cancer
Prostate cancer
Colon cancer
3.1
3.2
9.9
0.75
4.0
3.0
4.4
0.62
HCT116
a Values are means of at least two experiments.
Table 5. Stability of NCH-51 and compound 2 in human plasmaa
Compound
%Rb
0h
2h
86
100
4h
8h
24h
(Table 4), it could possibly permeate the cell membrane
and be converted to active free thiol NCH-31 by enzy-
matic hydrolysis within the cell.
NCH-51
2
100
100
71
99
63
99
49
100
a Values are means of at least three experiments.
b The percentage of the parent compound remaining (%R) was mea-
sured after 2, 4, 8, and 24 h incubation at 37 ꢁC.
Finally, we assessed the stability of compound 2 in hu-
man plasma (Table 5).18 Interestingly, compound 2

T. Suzuki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1558–1561
1561
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13. The antiproliferative activity assay was performed as
follows. Cancer cells were plated in 96-well plates at initial
density of 1500 cells/well and incubated at 37 ꢁC. After 24 h,
cells were exposed to test compounds at various concen-
trations in 10% FBS-supplemented RPMI-1640 medium at
37 ꢁC in 5% CO2 for 48 h. The medium was removed and
replaced with 200 lL of 0.5 mg/mL of methylene blue in
RPMI-1640 medium, and cells were incubated at room
temperature for 30 min. Supernatants were removed from
the wells, and methylene blue dye was dissolved in 100 lL/
well of 3% aqueous HCl. Absorbance was determined on a
microplate reader (Bio-Rad) at 660 nm.
Acknowledgments
This research was partly supported by Grants-in Aid for
Young Scientists (B) from the Ministry of Education,
Science, Culture, Sports, Science and Technology, Ja-
pan, Grants-in Aid for Research in Nagoya City Uni-
versity, and grants from the Japan Securities
Scholarship Foundation, the Tokyo Biochemical Re-
search Foundation, Takeda Science Foundation, and
the TERUMO Lifescience Foundation. We thank the
Screening Committee of New Anticancer Agents, sup-
ported by a Grant-in-Aid for Scientific Research on Pri-
ority Area ‘Cancer’ from the Ministry of Education,
Culture, Sports, Science and Technology of Japan for
HDAC1 inhibition assay results.
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column: CHIRALCEL OA (Daicel Chemical Industries),
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rabbit IgG-horseradish peroxidase conjugates (diluted
1:5000) for 2 h at room temperature, and further washed
twice with water. The immunoblots were visualized by
enhanced chemiluminescence.
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