Design, synthesis, and evaluation of isoindolinone-hydroxamic acid derivatives as histone deacetylase (HDAC) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.06.038

We designed and synthesized hydroxamic acid derivatives bearing a 4-(3-pyridyl)phenyl group as a cap structure, and found that they exhibit potent histone deacetylase (HDAC) inhibitory activity. A representative compound, 17a, showed more potent growth-in

Full text of DOI:10.1016/j.bmcl.2007.06.038

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4895–4900
Design, synthesis, and evaluation of isoindolinone-hydroxamic
acid derivatives as histone deacetylase (HDAC) inhibitors
Shoukou Lee,a Chihiro Shinji,a Kiyoshi Ogura,b Motomu Shimizu,b Satoko Maeda,c
Mayumi Sato,b Minoru Yoshida,c Yuichi Hashimotoa and Hiroyuki Miyachia,*
aInstitute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
bTumor Therapy Project, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization
for Medical Research, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan
cRIKEN, Hirosawa, Wako, Saitama 351-0198, Japan
Received 21 May 2007; revised 7 June 2007; accepted 11 June 2007
Available online 13 June 2007
Abstract—We designed and synthesized hydroxamic acid derivatives bearing a 4-(3-pyridyl)phenyl group as a cap structure, and
found that they exhibit potent histone deacetylase (HDAC) inhibitory activity. A representative compound, 17a, showed more potent
growth-inhibitory activity against pancreatic cancer cells and greater upregulation of p21WAF1/CIP1 expression than the clinically used
HDAC inhibitor suberoylanilide hydroxamic acid (ZolinzaTM).
Ó 2007 Elsevier Ltd. All rights reserved.
Epigenetic regulation of specific gene expression is med-
iated by several mechanisms, among which one of the
most important is post-translational acetylation of the
side-chain amino groups of specific histone lysine resi-
dues. The acetylation status of histones is modulated
by histone acetyltransferases (HAT) and histone deacet-
ylases (HDAC).1,2 HAT is generally considered as a
transcriptional activator, and HDAC is considered as
a transcriptional inhibitor, because histone acetylation
is associated with transcriptionally active chromatin,
whereas histone deacetylation is associated with tran-
scription repression. Many recent studies have shown
that inhibition of HDAC elicits anticancer effects in sev-
eral lines of tumor cells by inhibiting cell growth and
inducing apoptosis.3–5 Therefore, compounds that inhi-
bit HDAC activity may depress the expression of certain
genes, resulting in antiproliferative and antitumor
effects.6 Natural and synthetic HDAC inhibitors have
been studied extensively (Fig. 1), and suberoylanilide
hydroxamic acid (SAHA; ZolinzaTM) has been approved
by the FDA for once-daily oral treatment of advanced
cutaneous T-cell lymphoma (CTCL), and further clini-
cal studies of ZolinzaTM for the treatment of various solid
tumors are in progress.
Numerous biological studies have indicated that
HDACs are heterogeneous, consisting of 18 isozymes,
which can be categorized into four classes (class I, class
IIa, class IIb, and class III). Class I and class II HDACs
are zinc-containing amidehydrolases, and class III
HDAC consists of NAD-dependent amidehydrolase.
The biological function and distribution of each class
of HDACs have been extensively studied from a molec-
ular-pharmacological viewpoint, but much remains to
be learnt.
We have been engaged in structural development studies
of the multi-drug template thalidomide for the creation
of structurally novel drug leads,7–12 and have already
reported the design and synthesis of potent HDAC inhib-
itors with cyclic amide structure (isoindolinone), such as
compounds 8 and 9.13,14 In this paper, we report the
design, synthesis, and biological activity of novel HDAC
inhibitors with a pyridylphenyl group as a cap structure.
Synthetic routes to the present series of cyclic amide
derivatives are outlined in Chart 1.
Keywords: HDAC; HDAC inhibitor; Isoindoklinone; Hydroxamic
acid; Pancreatic cancer; p21.
4-Iodobenzoic acid (10) was reduced with BH3ÆTHF to
afford 4-iodobenzyl alcohol (11). This alcohol was
*
Corresponding author. E-mail: miyachi@iam.u-tokyo.ac.jp
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.038

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S. Lee et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4895–4900
O
H
N
HN
OH
O
O
O
O
N
N
H
HN
O
N
N
H
NH2
H
N
H
O
O
O
N
O
1
S
O
O
O
N
HO
2
5
3
O
O
O
O
H
N
OH
N
H
OH
HN
O
N
H
H
N
SH
N
O
O
4
6
O
O
O
OH
O
H
N
N
H
N
N
H
N
7
OH
O
N
N
H
8
Figure 1. Structures of representative natural HDAC inhibitors (1 (trapoxin A), 4 (TSA)), and synthetic HDAC inhibitors (2 (tubacin), 3 (MS-275), 5
(SAHA: ZolinzaTM), 6 and 7 (ADS102550), and our lead compound 8).
O
O
I
OMe
O
O
N
N
I
I
a)
I
b)
c)
d)
O
NH2 HCl
OH
OH
O
I
10
11
12
13
14
O
O
O
O
O
OH
OH
N
H
N
N
OMe
N
f)
g)
e)
15a; pyridin-3-yl
15b; pyridin-4-yl
16a; pyridin-3-yl
16b; pyridin-4-yl
17a; pyridin-3-yl
17b; pyridin-4-yl
N
N
N
Chart 1. Reagents and conditions: (a) BH3ÆTHF, THF, 0 °C, 96%; (b) phthalimide, DEAD, P(Ph)3, THF/toluene, rt, 84%; (c) NH2NH2ÆH2O,
MeOH, HCl, reflux, quant.; (d) methyl 4-bromomethyl-3-methoxycarbonylcinnamate, TEA, MeOH, reflux, 45%; (e) 3-pyridylboronic acid (or 4-
pyridylboronic acid), Pd(P(Ph)3)4, Na2CO3, DME, reflux, 26–74%; (f) NaOH, MeOH, reflux, 18–54%; (g) NH2OHÆHCl, EDCI, HOBt, i-Pr2Net,
DMF, rt, 12–39%.
treated with phthalimide under Mitsunobu condition to
afford N-benzylphthalimide (12). Compound 12 was
hydrazinolyzed to afford 4-iodobenzylamineÆHCl (13).
Compound 13 was reacted with methyl 4-bromometh-
yl-3-methoxycarbonylcinnamate in the presence of tri-
ethylamine as a base to afford the cyclic amide (14).
Suzuki coupling of 14 with 3-pyridylboronic acid (or
4-pyridylboronic acid) in the presence of Pd(P(Ph)3)4
afforded the pyridylphenyl derivatives 15a and 15b,
respectively. Alkaline hydrolysis of 15a and 15b afforded
cinnamic acids 16a and 16b, respectively. Hydroxyl-
amineÆHCl was condensed with 16a and 16b, in the pres-
ence of EDCI, to give the final hydroxamic acid
derivatives, 17a and 17b, respectively.15
Recent X-ray crystallographic analyses of histone deace-
tylase-like protein (HDLP),16 and HDAC 8,17 com-
plexed with trichostatin A (TSA, 4)18 indicate that the
HDAC catalytic domain consists of a narrow tube-like
pocket, with the zinc ion buried near the bottom of
the active site. Therefore, the structural requirements
for potent HDAC inhibitors involve three key regions,

S. Lee et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4895–4900
4897
that is, (1) a zinc-binding domain, which interacts
with the active site zinc, (2) a linking domain, which
occupies the channel, and (3) a cap domain, which inter-
acts with the residues on the rim of the active site. Pre-
viously we have reported novel HDAC inhibitors based
on a new structural scaffold.13,14 We used a N-benzyliso-
indolinone structure as a novel linker/cap domain that
might be suitable to occupy the narrow channel of the
HDAC active site. An SAR study indicated that the
introduction of a bulky substituent at the 4-position of
the distal benzene ring tended to potentiate inhibitory
activity toward HDAC, especially HDAC 4, and a phe-
nyl group was the best substituent (compound 9).14
Our previous molecular modeling study indicated that
the backbone-benzene ring of our lead compound (8),
fused to the cyclic amide skeleton, exhibits a p–p stack-
ing interaction with the side-chain benzyl group of
208Phe of HDAC 8.14 The environment of the cap
structure benzyl group of compound (8) contains some
important amino acid residues which may be involved
in electrostatic interactions, such as Tyr100 and
Glu148 (Fig. 2). We expected that the introduction of
basic character into the cap structure of 8 might en-
hance the interaction with the HDAC ligand binding
pocket, and further strengthen the HDAC inhibitory
activity. Therefore, we planned to synthesize hybrid-
type compounds 17a and 17b (Fig. 3). The results are
summarized in Table 1, together with those for the lead
compounds 8, 9, and the positive controls ZolinzaTM (5)
Figure 2. Docking model structures of compound 8 into the HDAC 8
binding pocket.
O
O
OH
O
N
H
O
N
OH
N
N
H
9
8
O
O
OH
N
N
H
17a: pyridin-3-yl
17b: pyridin-4-yl
N
Figure 3. Structural development of our HDAC inhibitors.
Table 1. HDAC inhibitory activities of the prepared compounds
O
O
OH
N
R1
N
H
Compound
R1
HDAC inhibitory activity IC50 (nM)
HDAC 4
HDAC 1
HDAC 6
230 70
8
H
4-t-Bu
4-Ph
250 20
230 20
220 30
220
180
98
4
4
6
7
18
9
990 20
750 11
280 10
17a
17b
4-(Pyridin-3-yl)
4-(Pyridin-4-yl)
ZolinzaTM(5)a
TSA (4)
92
6
56
3300 200
290 50a
2100 200
340 30a
15,000 1800
200 10a
16
1
29
5
53
9
a ZolinzaTM was prepared by Prof. Norikazu Nishino’s group (Graduate School of Life Science and Systems Engineering, Kyushu Institute of
Technology). The HDAC inhibitory activity of ZolinzaTM was assayed by Prof. Nishino and two of the present authors, Satoko Maeda and Minoru
Yoshida, and we thank Prof. Nishino for disclosure of the results.

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S. Lee et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4895–4900
and TSA (4). TSA is well known as a classical (or rep-
resentative) potent HDAC inhibitor, which exhibited
some toxicity and biological instability. We selected
HDAC 1, HDAC 4, and HDAC 6 as representatives
of class I, class IIa, and class IIb, respectively, because
sufficient amounts of these isozymes were available for
our purpose.
genic transformation, resulting in altered gene(s) tran-
scription and increased proliferation.20
Considering these points, we examined the growth-
inhibiting effects of our compounds 17a, 9, and the po-
sitive control ZolinzaTM (5) on two human pancreatic
cancer cell lines, PANC-1 and PT-45 (Fig. 4). Both cell
lines contain a mutation in the Smad4 gene, and PT-45 is
more chemoresistant. As shown in Figure 4, ZolinzaTM
dose-dependently decreased the proliferation of
PANC-1 and PT-45 cells, with IC50 values of 450 and
4000 nM, respectively. These results indicate that an
HDAC inhibitor can inhibit pancreatic cancer cell
growth. Compounds 17a and 9 also caused dose-depen-
dent decreases in the proliferation of PANC-1 and PT-
45 cells. The IC50 values of 17a and 9 for PANC-1 cells
were 35 and 150 nM, respectively, and those for PT-45
cells were 200 and 300 nM, respectively. Both 17a and
9 exhibited more potent antiproliferative activity than
ZolinzaTM, presumably reflecting their more potent
HDAC inhibitory activities.
Our earlier SAR study indicated that the introduction
of a bulky substituent at the 4-position of the distal
phenyl group enhanced HDAC 4 inhibitory activity,
and decreased HDAC 6 inhibitory activity, while
HDAC 1 inhibitory activity was apparently not chan-
ged.14 However, we found here that the appropriate
introduction of a basic functionality at the distal ben-
zene ring dramatically affected the HDAC inhibitory
activity. The 3-pyridyl derivative (17a) exhibited about
2-fold greater HDAC 4 inhibitory activity than the 4-
phenyl derivative (9), having activity comparable with
that of the naturally occurring HDAC inhibitor TSA.
As for HDAC 6 inhibitory activity, the introduction of
a 3-pyridyl group instead of a phenyl group restored
the activity to that of the non-substituted compound
(compare 8, 9, 17a). In the case of HDAC 1, the intro-
duction of a 3-pyridyl group also increased the inhib-
itory activity to some extent. This is interesting,
because the introduction of a phenyl group (9) did
not cause any clear effect. All these data clearly indi-
cated that the introduction of a 3-pyridyl group
increases the inhibitory activity toward the HDAC iso-
zymes tested. The reason for this might be that the
pyridyl nitrogen atom interacts favorably with acidic
amino acid residues located near the cap structure.
The 3-pyridyl group of MS-275 (3) might also contrib-
ute to the positive interaction with acidic amino acid
residues located near the cap structure, though the
HDAC inhibitory activity of MS-275 is not so high
(micromolar order).
HDAC inhibitors have been reported to upregulate the
expression of the tumor repressor, p21WAF1/CIP1, and
to downregulate cyclin D1 in many types of tumor cells,
in parallel with cell cycle arrest in the G1 phase. Activa-
tion of the p21WAF1/CIP1 gene is associated with the inhi-
bition of proliferation and induction of differentiation
and/or apoptosis of tumor cells, both in vitro and
in vivo.5,6 Therefore, we examined the ability of repre-
sentative compounds to upregulate the expression of
We also examined the effects of these
p21WAF1/CIP1
HDAC inhibitors on the expression of another impor-
.
21
kip1
tant gene, p27kip1
and it plays a central role in the suppression of tumori-
.
p27
is a cyclin kinase inhibitor,
22
ID50 (nM)
compd.
17a
PANC-1
35
PT-45
200
It is of interest to note that the position of the pyridyl
nitrogen is important for potent HDAC inhibitory
activity, because the 4-pyridyl derivative (17b) exhibited
a more than 30-fold decrease in HDAC inhibitory
activity, being far less potent than the non-substituted
compound (8). Although the reason for this is not yet
clear, there might be electronic and/or steric interactions
between the 4-position substituent and amino acids
adjacent to the binding site.
9
150
300
ZolinzaTM
450
4000
PANC-1
ZolinzaTM
100
75
50
25
0
9
Thus, we obtained the HDAC inhibitor 17a, which is
more potent than Zolinza (5), and as potent as TSA
(4). We therefore thought it worthwhile to examine the
potential of 17a as an anticancer agent.
17a
Pancreatic cancer is the intractable solid tumor, and the
fifth leading cause of cancer death in Japan and the Uni-
ted States. The incidence is virtually equal to the mortal-
ity, and the 5-year survival rate is only a few percent.19
The treatment options remain unsatisfactory, although
our understanding of the molecular biology of pancre-
atic cancer has advanced. In general, HDAC activity is
increased in cancer cells, including pancreatic cancer
cells, and this increase has been shown to induce onco-
-10
-9
-8
-7
-6
Drug concentration (M)
Figure 4. Effect of 9, 17a, and ZolinzaTM on pancreatic cancer cell
growth.

S. Lee et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4895–4900
4899
genesis in a variety of human cancers by inhibiting the
activity of cyclin E- or A-containing cdk2 (cyclin depen-
dent kinase 2) complexes.
Acknowledgments
The work described in this paper was partially sup-
ported by a Grant-in-Aid for Scientific Research from
The Ministry of Education, Culture, Sports, Science
and Technology, Japan. The authors thank Prof.
Nishino, Graduate School of Life Science and Sys-
tems Engineering, Kyushu Institute of Technology,
for data on the HDAC inhibitory activity of Zolinza,
and Dr. Keisuke Imai, Lead Discovery Research
Labs., Astellas Pharma Inc., for computational
docking studies.
As shown in Figure 5, the p21WAF1/CIP1 gene expression
of PANC-1 cells was augmented 3-fold as compared to
that of the vehicle control after exposure of the cells to
1 lM ZolinzaTM for 24 h, and the effect remained for
more than 48 h. These results strongly suggest that the
enhancement of p21WAF1/CIP1 expression is involved in
the initial inhibition of PANC-1 cell growth by HDAC
inhibitors. Compound 17a exhibited highly potent
p21WAF1/CIP1 expression-enhancing activity: 0.1 lM
17a exhibited almost the same activity as 1 lM Zolin-
zaTM, and 1 lM 17a augmented the p21WAF1/CIP1 gene
expression 8-fold over the vehicle control in cells
exposed for 48 h.
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hydroxamic acid derivatives bearing
a
4-(3-pyr-
idyl)phenyl group as a cap structure, which exhibit
more potent HDAC inhibitory activity than the clini-
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hydroxyacrylamide (17a). H NMR (500 MHz, CDCl3) d
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FAB MS: (M+H)+ Calcd for C23H19N3O3: 385.1426.
Found: 385.1464.
Figure 5. Effects of 17a and ZolinzaTM on mRNA expression of p21
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quantitative real-time PCR. The ratios of p21 and p27 mRNA levels
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treated cells.
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