Design of novel histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.05.080

Histone deacetylase (HDAC) inhibitors that target Class I and Class II HDACs are of synthetic and therapeutic interest and ongoing clinical studies indicate that they show great promise for the treatment of cancer. Moreover, Zolinza^R (vorinostat) was recently approved by the FDA for the treatment of the cutaneous manifestations of cutaneous T-cell lymphoma [Nat. Rev. Drug Disc. 2007, 6, 21]. As part of a broader effort to more fully explore the structure-activity relationships (SAR) of HDAC inhibitors, we sought to identify novel HDAC inhibitor structures through iterative design by utilizing low affinity ligands as synthetic starting points for SAR development. Novel and potent HDAC inhibitors have been identified using this approach and herein we report the optimization of the recognition elements of a novel series of malonyl-derived HDAC inhibitors.

Full text of DOI:10.1016/j.bmcl.2007.05.080

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4619–4624
Design of novel histone deacetylase inhibitors
Phieng Siliphaivanh,a,* Paul Harrington,a, David J. Witter,a Karin Otte,a Paul Tempest,a
Sam Kattar,a Astrid M. Kral,b Judith C. Fleming,b Sujal V. Deshmukh,a Andreas Harsch,a
Paul J. Secristb and Thomas A. Millera
aDepartment of Drug Design & Optimization, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA
bDepartment of Cancer and Biology Therapeutics, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA
Received 30 April 2007; revised 22 May 2007; accepted 24 May 2007
Available online 27 May 2007
Abstract—Histone deacetylase (HDAC) inhibitors that target Class I and Class II HDACs are of synthetic and therapeutic interest
and ongoing clinical studies indicate that they show great promise for the treatment of cancer. Moreover, ZolinzaR (vorinostat) was
recently approved by the FDA for the treatment of the cutaneous manifestations of cutaneous T-cell lymphoma [Nat. Rev. Drug
Disc. 2007, 6, 21]. As part of a broader effort to more fully explore the structure–activity relationships (SAR) of HDAC inhibitors,
we sought to identify novel HDAC inhibitor structures through iterative design by utilizing low affinity ligands as synthetic starting
points for SAR development. Novel and potent HDAC inhibitors have been identified using this approach and herein we report the
optimization of the recognition elements of a novel series of malonyl-derived HDAC inhibitors.
ꢀ 2007 Elsevier Ltd. All rights reserved.
HDAC inhibitors possess diverse biological activities
O
and many of these agents have been demonstrated to
be effective inhibitors of tumor growth in animal models
of cancer.1–5 With few exceptions, HDAC inhibitors can
be broadly characterized by a common pharmacophore
that summarizes key elements of inhibitor–enzyme inter-
actions (Fig. 1).1,3,6–10 Because most of the original
HDAC inhibitors have been derived from relatively
complex and potent natural products, a large number
of efforts have focused on the refinement of the existing
pharmacophore using designs that mimic those found in
nature.11–15 In contrast, few efforts have been under-
taken to design HDAC inhibitors de novo using less
complex, low-affinity synthetic starting points. Impor-
tantly, an inverse correlation between molecular com-
plexity and lead tractability/quality has been noted in
the literature.16 This report details how less complex,
lower affinity lead structures can represent unique start-
ing points for SAR development. Moreover, this study
demonstrates how iterative evolution of low-affinity
leads can afford potent scaffolds with structures unique
H
N
OH
N
H
ZolinzaR
Linker
O
Surface
Recognition
Metal
Binding
Figure 1. Summary of HDAC inhibitor structural characteristics.
from those derived from traditional optimization of
structurally complex, high-affinity lead structures culmi-
nating in a novel, potent HDAC inhibitor.
As part of a broader effort to more fully explore the
SAR of HDAC inhibitors, we have identified novel
HDAC inhibitor structures through iterative design
using acetohydroxamic acid 1 as a synthetic starting
point for SAR development. We chose to use
compound 1 because it represents a minimum struc-
tural requirement for HDAC inhibition through a
well-defined interaction (IC50 = 625 lM). Namely, the
hydroxamic acid moiety found in existing HDAC
inhibitors has been shown to interact directly with
the active-site zinc of the HDAC enzyme.17 Having
established the metal binding domain of the HDAC
inhibitor pharmacophore (Fig. 1) as a hydroxamic acid
for our initial efforts, we sought to iteratively optimize
Keywords: Histone deacetylases; Malonyl benzamides.
*
Corresponding author. Tel.: +1 617 992 2055; fax: +1 617 992
2043; e-mail: Phieng_siliphaivanh@merck.com
Present address: Department of Medicinal Chemistry, Amgen Inc.,
One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.080

4620
P. Siliphaivanh et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4619–4624
the linker and surface recognition domains to obtain
novel and potent HDAC inhibitors. The first round
of optimization included the incorporation of alkyl,
cyclic, and aromatic linker domain followed by recog-
nition domains’ incorporation and optimization. These
studies revealed that hydrophobic aryl moieties are
preferred over acyclic alkyl linker groups (Fig. 2).
Thus, a potency increase of ꢀ30-fold was observed
for benzoyl hydroxamic acid 2 over alkyl-hydroxa-
mates such as acetohydroxamic acid 1.
Having established 4 as a viable and potent lead struc-
ture, we sought to explore the application of this core
scaffold with other potential zinc binding domains since
hydroxamates are known to have short half-lives and
generally poor pharmacokinetic properties. HDAC
inhibitors containing an N-(2-aminophenyl) moiety in
the metal binding domain are of significant clinical
4,5,18,19
importance.
As such, to further explore the
SAR of the malonyl-derived HDAC inhibitor 4, the
corresponding 2-aminophenyl benzamide analogue
5a20 became the focus of our synthetic efforts.
Further modifications of the surface recognition domain
by substitution on the aryl linker with tert-butyl (3) and
then diphenylmalonamide (4) further enhanced activity
to nanomolar levels (Fig. 2).
The synthesis of 2-aminophenyl benzamide analogues of
4 is shown in Scheme 1. tert-Butyl ester 6 was obtained
by esterification of benzoyl chloride 7 with potassium
O
O
O
OH
OH
N
H
OH
N
N
H
H
1
2
3
IC50 = 625 μM
IC50 = 20 μM
IC50 = 715 nM
O
O
OH
NH
O
N
NH
O
N
H
H
NH2
O
O
NH
NH
4
5a
IC50 = 5 nM
Figure 2. Evolution of HDAC inhibitors.
O
O
MeO
OMe
-Bu)3
1) Pd2(dba)3, P(
t
K3PO4, PhMe
2) RNH2, 200 °C
O
O
KO
or
Cl
O
O
O
Br
Br
1)
RHN
Pd2(dba)3, P(
K3PO4, PhMe
NHR
6
7
t
-Bu)3
2) TFA
O
O
H2N
NHR
OH
NH2
NHR
O
N
H
NH2
O
HOBT, EDC
O
O
NHR
8
NHR
5
Scheme 1. Synthesis of phenyl benzamide.

P. Siliphaivanh et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4619–4624
4621
Table 1. Structure–activity relationship of N,N0 diarylmalonamides
tert-butoxide. Palladium mediated coupling21 between
dimethyl malonate and phenyl bromide 7, followed by
thermal displacement of the esters and tert-butyl ester
cleavage, provided the carboxylic acid 8. An alternate
route to obtain 8 was through palladium mediated cou-
pling with the malonyl diamide followed by tert-butyl
ester cleavage. Finally, EDC coupling of carboxylic acid
8 with phenylene diamine provided the desired benz-
amides 5.
O
O
N
H
R
NH2
N
H
HN
R
O
Compound
R
HDAC1
IC50 (nM)
HCT116-72
IC50 (nM)
Compound 5a, the N-(2-aminophenyl)amide analogue
of 4, displayed good HDAC inhibitory activity, with
an IC50 = 41 nM against HDAC1 and an IC50 = 171 nM
in HCT116-72 h cell proliferation assays.22
5a
5b
41
34
171
N
F
500a
Encouraged by the activity of benzamide 5a, our atten-
tion was directed toward the synthesis of analogues with
diverse substitution around the phenyl ring in the
surface recognition domain. Representative analogues
(Table 1) demonstrate that a wide array of functionality
can be tolerated in the malonyl–phenyl rings including
nitrile, methoxy, and morpholine moieties. A reduction
in cellular potency can be seen in biaryl 5i, possibly
due to its low hydrophilicity. The clogP23 of 5i is greater
than that of the phenyl amide 5a by two log units. Nota-
bly, ortho-substitution of the malonyl–phenyl rings was
tolerated in contrast to ortho-substitution of the phenyl
ring within vorinostat, which leads to a marked decrease
in activity.
5c
63
222a
5d
5e
56
35
607
383
F
O
H3C
CH3
O
5f
47
21
394
220
Moreover, both amide NH moieties were essential for
significant enzymatic and cellular potency (Table 2).
Incorporation of a single methyl group on the malonyl
sidechains to give 5j resulted in a 5-fold loss of potency.
Similarly, the dimethyl derivative 5k was 100-fold less
potent indicating that hydrogen bonding, either inter-
or intra-molecular, may play an important role in the
recognition of the HDAC active site. Similarly, it was
shown that malonyl di-ester analogues possessed signif-
icant reduction in HDAC enzymatic activity as well
(data not shown).
O
5g
5h
O
O
N
N
45
542
5i
103
1200
In addition the aniline-amide proved to be optimal for
HDAC1 activity (Table 3). The one-carbon homologue,
benzylamine 5l, was less active against HDAC1 and this
trend continued to 5m, which was approximately 5-fold
less active in the HDAC-enzyme assay and almost 10-
fold less active in the cell proliferation assay.
a HCT116-96 h.
Table 2. SAR of 5a, 5j, and 5k
O
Having explored the SAR in the surface recognition do-
main, we set out to iteratively re-optimize potency by
further exploring the linker domain. Substitution at
the benzylic position with a fluoride 5n or methyl 5p
was assessed in an effort to minimize potential metabo-
lism issues since the methine unit is easily oxidized. Fur-
thermore, a hydroxyl group 5o was installed with the
hope of improving the compound solubility. Addition-
ally, we investigated the effect of inserting a methylene,
thus allowing further flexibility of the sidechain (9a–
9b). However, data demonstrated that these derivatives
suffer attenuated cell potency (Table 4) and did not offer
advantages in subsequent physiochemical and pharma-
cokinetic analyses (data not shown).
O
N
H
NH2
N
R1
R2N
O
Compound
R1
R2
H
HDAC1
IC50 (nM)
HCT116-72
IC50 (nM)
5a
5j
H
41
171
H
CH3
CH3
CH3
196
7195
1145
NA
5k

4622
P. Siliphaivanh et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4619–4624
Table 3. SAR of malonyl sidechain extension compound
The synthesis of a-substituted analogues 5n–5p was
achieved through a common intermediate carboxylic
acid 7 (Scheme 2). EDC coupling with monoprotected
o-phenylenediamine provided amide 10. Deprotonation
of the malonate and trapping with an appropriate elec-
trophile followed by TFA deprotection of the Boc moi-
ety furnished the necessary analogues 12. The hydroxyl
substituted compound 5o was obtained by stirring the
carboxylic acid 7 in aqueous LiOH in air followed by
EDC coupling with phenylene diamine (Scheme 3).
O
O
N
H
NH2
N
n
H
HN
O
n
Compound
n
HDAC1
IC50 (nM)
HCT116-72
IC50 (nM)
Synthesis of the benzyl derivatives was achieved through
alkylation of the dialkyl malonamide anion with benzyl
bromide 13 (Scheme 4). Hydrolysis of the methyl ester
followed by EDC coupling furnished the benzamides
9a and 9b.
5a
5l
0
1
3
41
115
596
987
199
229
5m
Table 4. SAR of substituted diarylmalonamide on benzyl analogues
Having established that 5a exhibited good activity in
enzymatic and excellent cellular assays, 5a was evaluated
against other HDAC subtypes.24 Compound 5a was
found to be inactive against HDAC6 and HDAC8,
almost 10-fold selective for HDAC1 over HDAC2,
and 20-fold selective over HDAC3 (Table 5). Having a
Class I selective compound may offer significant advan-
tages over less selective HDAC inhibitors in terms of tol-
erability and efficacy since Class I and Class II do have
different biological function.
O
HN
O
N
H
H
N
NH2
n
R
O
Compound
R
n
HDAC1
IC50 (nM)
HCT116-72
IC50 (nM)
The pharmacokinetic profile of 5a in several animal spe-
cies was determined (Table 6). Good oral bioavailability
was seen in rat and dog (F = 88% for both species). In
addition, the compound exhibited low plasma clearance
in higher order species and an acceptable half-life across
species.
5a
5n
5o
5p
9a
9b
H
0
0
0
0
1
1
41
48
171
425
F
OH
Me
H
94
773
198
47
2125
579
Me
200
2519
O
O
H2N
1. KOt-Bu
NHBoc
NHR
O
OH
NHR
O
N
H
NHBoc
2. MeI or
selectfluorTM
HOBT, EDCI
O
O
7
10
NHR
NHR
O
O
TFA
NHR
N
NHR
N
H
H
NHBoc
NH2
O
O
E
E
O
NHR
O
NHR
11
12
Scheme 2. Incorporation of substitution at the methine position.
O
O
H2N
NH2
LiOH
NHR
O
OH
NHR
N
H
NH2
oxygen
HOBT, EDCI
O
O
HO
NHR
O
NHR
7
5o
Scheme 3. Synthesis of hydroxyl derivative.

P. Siliphaivanh et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4619–4624
4623
O
O
O
O
1)
NaH, THF
R'NH2
R'
R'
O
O
N
N
O
H
H
R
R
O
Br
R'
O
13
HN
R
O
N
2) KOH, THF, MeOH
3) EDC, HOBT, DMF
H
O
NH2
NH
R'
9a: R = H
9b: R = Me
H2N
NH2
Scheme 4. Synthesis of benzyl derivatives.
Table 5. HDAC selectivity profile for 5a
HDAC1
IC50 (nM)
HDAC2
IC50 (nM)
HDAC3
IC50 (nM)
HDAC6
IC50 (nM)
HDAC8
IC50 (nM)
36
313
697
>10,000
>10,000
Table 6. Pharmacokinetic profile for 5a
IV dose
(mg/kg)
CL
(ml/min/kg)
Vd
(L/kg)
po dose
(mg/kg)
AUC
(Norm) (lM h kg/mg)
F (%)
T 1/2 (h)
Rat
Dog
2.0
0.6
1.0
55
3
12
4.0
1.2
2.0
0.48
10.6
3.88
88
88
27
3.8
8.5
1.8
1.7
Rhesus
2.5
10.4
9. Yoshida, M.; Horinouchi, S.; Beppu, T. Bioessays 1995,
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better understand the HDAC inhibitor pharmacophore
beyond historical findings, we have utilized an iterative
approach to HDAC inhibitor design. These efforts have
yielded novel, potent HDAC inhibitor structures that
extend the current HDAC inhibitor pharmacophore to
include agents with sterically bulky branched functional-
ities at the linker/surface recognition domain interface.
Further profiling of 5a will be reported in due course
along with other novel HDAC inhibitors.
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development solution for HDACs 1, 2, 3, and 6 was
167·-diluted 20· Developer Concentrate (BIOMOL: KI-
105) plus 10 lM SAHA. For HDAC8, the development
solution was 100·-diluted 5· Developer Concentrate
(BIOMOL: KI-176) plus 10 lM SAHA. The assay was
read in a VictorV plate reader (Perkin-Elmer, Wellesley,
MA) at Ex 360 nm/Em 460 nm.
21. Beare, N. A.; Hartwig, J. F. J. Org. Chem. 2002, 67, 541.
22. HDAC cell-based proliferation results were determined by
the following procedure: HCT116 cells were plated in 96-
well plates at density of 1000 cells/well. The next day, cells
were treated with either 0.2% DMSO or increasing
concentrations of 7i dissolved in DMSO (final concentra-
tion of DMSO 0.2%). After 72 h incubation at 37 ꢁC with
5% CO2, viable cells were quantitated using Vialight Plus
(Cambrex) according to manufacturer’s instructions.
23. clogP was calculated using ACD lab software.
24. HDAC enzymatic activities were determined by the
following procedure: 3· serial dilutions of a 10 mM
solution of inhibitor were performed in DMSO followed
by a 20· dilution into assay buffer. Twenty microliters of
HDAC was preincubated with 5 ll diluted compound at
RT for 10 min. The reaction was initiated by the addition
of 25 ll of the appropriate substrate (HDACs 1, 2, 3, and
6: Fluor-de-Lys substrate KI-104; HDAC8: Fluor-de-Lys
HDAC8 substrate KI-178), incubated 15 (HDAC8) or 60
(HDACs 1, 2, 3, and 6) minutes at 37 ꢁC, before adding
50 ll of the appropriate development solution. The
The substrate for HDAC1, HDAC2, HDAC3, and
HDAC6 was 30 lM Fluor-de-Lys substrate (BIOMOL:
KI-104) and for HDAC8, 30 lM Fluor-de-Lys HDAC8
substrate (BIOMOL: KI-178). The development solution
for HDAC1, HDAC2, HDAC3, and HDAC6 was 167·-
diluted 20· Developer Concentrate (BIOMOL: KI-105)
plus 10 lM SAHA. For HDAC8, the development solu-
tion was 100·-diluted 5· Developer Concentrate (BIO-
MOL: KI-176) plus 10 lM SAHA. HDAC final
concentration in the reaction was 1–30 nM. HDAC8 was
a generous gift of Paola Gallinari from Institute for
Research in Molecular Biology, Pomezia, Italy. Carboxy-
terminal FLAG-tagged human HDACs 1, 2, 3 (co-
expressed with the domain of SMRT), and 6 were
overexpressed in mammalian cells and affinity-purified
using an anti-Flag antibody matrix, eluted from the matrix
with 100 lg/ml of a competing FLAG peptide in 20 mM
Tris–Cl, pH 8.0, 150 mM NaCl, 10% glycerol, and
protease inhibitor cocktail (Roche cat. No. 1836153).