The structural requirements of histone deacetylase inhibitors: Suberoylanilide hydroxamic acid analogs modified at the C6 position

Article abstract of DOI:10.1016/j.bmcl.2012.09.093

Suberoylanilide hydroxamic acid (SAHA, Vorinostat), the first FDA-approved histone deacetylase (HDAC) inhibitor drug, was modified at the C6 position to study the structural requirements for high potency and selectivity. Substituents on the C6 position only modestly influenced inhibitor potency, with poorer activity observed as substituent size increased. Interestingly, C6 substituents also modestly influenced selectivity compared to the parent compound, SAHA. This systematic study documenting the influence of substituents on the SAHA linker region will aid development of anti-cancer drugs targeting HDAC proteins.

Full text of DOI:10.1016/j.bmcl.2012.09.093

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7084–7086
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The structural requirements of histone deacetylase inhibitors: Suberoylanilide
hydroxamic acid analogs modified at the C6 position
⇑
Sun Ea Choi, Mary Kay H. Pflum
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 June 2012
Revised 20 September 2012
Accepted 25 September 2012
Available online 2 October 2012
Suberoylanilide hydroxamic acid (SAHA, Vorinostat), the first FDA-approved histone deacetylase (HDAC)
inhibitor drug, was modified at the C6 position to study the structural requirements for high potency and
selectivity. Substituents on the C6 position only modestly influenced inhibitor potency, with poorer activ-
ity observed as substituent size increased. Interestingly, C6 substituents also modestly influenced selec-
tivity compared to the parent compound, SAHA. This systematic study documenting the influence of
substituents on the SAHA linker region will aid development of anti-cancer drugs targeting HDAC
proteins.
Keywords:
Histone deacetylase
HDAC inhibitor
Isoform selectivity
Vorinostat
Ó 2012 Elsevier Ltd. All rights reserved.
Histone deacetylase (HDAC) proteins are transcription factors
that influence gene expression by altering the acetylation status
of lysine residues on nucleosomal histones. The HDAC protein fam-
ily consists of 18 members and is divided into four classes based on
size, cellular localization, number of catalytic active sites, and
homology to yeast HDAC proteins.1 The eleven class I, II, and IV
HDAC proteins are metal ion-dependant proteins and sensitive to
the inhibitors discussed here.2
HDAC proteins are over-expressed in many cancers, making
them attractive targets of anti-cancer drugs.3 In fact, two HDAC
inhibitors, suberoylanilide hydroxamic acid (SAHA, Vorinostat,
Fig, 1) and Romidepsin, were approved for treatment of cutaneous
T-cell lymphoma.4 In addition to lymphoma, HDAC activity has
been associated with a variety of other cancers. Over-expression
of class I HDAC proteins (HDAC1, 2, and 3) was observed in ovarian
cancer.5 Altered HDAC2 activity in gastric cancers,6 mutations of
HDAC1 and HDAC3 in lung cancers,7 and abnormal HDAC8 protein
activity in acute myeloid leukemia tissues have been reported for
the class I proteins.8 Overproduction of class II HDAC6 was ob-
served in breast cancer tissues.9 Because individual HDAC isoforms
play independent roles in specific cancers, development of iso-
form-selective inhibitors as anti-cancer drugs has been proposed.
However, most HDAC inhibitors nonspecifically target all eleven
metal ion-dependent HDAC proteins, including SAHA.10 It has been
proposed that the non-selectivity of HDAC inhibitors may cause
cancer patients in the clinic to suffer from the side effects, such
as fatigue, anorexia, diarrhea, and cardiac arrhythmia.11
Unfortunately, the clinical toxicity of HDAC inhibitors is poorly
characterized because few isoform-selective molecules have been
reported. In addition, sequence similarity in the active site of the
isoforms has challenged selective inhibitor design.12
Most HDAC inhibitors, including SAHA (Fig. 1), have a similar
construction consisting of a capping group that is solvent-exposed,
a carbon linker that is surrounded by a hydrophobic tunnel, and a
metal binding moiety that is buried in the protein active site.13
While the capping group and metal binding moiety have been
modified extensively, few structure–activity relationship studies
focusing on the linker area of SAHA have been reported.12 How-
ever, MS275 (Fig. 1) contains an intra-linker aryl group and dis-
plays nanomolar class I selectivity,10,14 while a recently reported
nanomolar HDAC6-selective inhibitor (1, Fig. 1) contains a cyclic
substituent at the C7 position of SAHA.15 These examples highlight
the possible role of the linker region in selectivity and the need for
a systematic study of linker substituents.
To study the linker region, SAHA analogs containing hydro-
phobic substituents on the C2 and C3 positions were previously
reported (Fig. 1).16 Positioning substituents near the hydroxamic
acid reduced inhibitor potency, with IC50 values in the
lM range.
However, the C3-ethyl SAHA analog displayed 12-fold selectivity
for HDAC6 over HDAC3. Combining the data from compound 1
and the C2- and C3-SAHA analogs, we theorized that SAHA analogs
with substituents positioned nearer to the solvent-exposed cap-
ping group might display potent inhibition with HDAC isoform
selectivity.
We report here the synthesis and evaluation of SAHA analogs
with substituents attached at the C6 position (Fig. 1). To our
knowledge, C6-modified SAHA analogs have not been reported
⇑
Corresponding author. Tel.: +1 313 577 1515; fax: +1 313 577 8822.
E-mail address: pflum@chem.wayne.edu (M.K.H. Pflum).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.093

S. E. Choi, M.K.H. Pflum / Bioorg. Med. Chem. Lett. 22 (2012) 7084–7086
7085
Cu(I)I,
R
PhNH2,
Pd(OH)2/C,
H2,
RLi,
TBTU, DIPEA,
BnO2C
OMe
O
6
TMSCl,
THF
CH3CN
EtOAc
7b-d
O
c: 58% over 2 steps
d: 76% over 2 steps
R: b phenyl
c t-butyl: 89%
d 2-ethylhexyl: 81%
O
R
OMe
OMe
O
PhHN
PhHN
O
8b-d
8b'
O
R
NH2OH,
KOH
NHOH
PhHN
MeOH
O
2b-d
b: 60% over 4 steps
c: 99%
d: 58%
Scheme 2. Modified synthesis of C6-SAHA analog (1b–d).
Figure 1. Structures of HDAC inhibitors discussed in the text.
Table 1
HDAC inhibition by SAHA, MS-275, and the C6-SAHA analogs 2a–d using HeLa cell
lysates
O
DMSO,
MeOH,
H2SO4,
Oxalyl Chloride,
TEA,
ª
O
Compounds
R
IC50 (nM)
OMe
HO
CH2Cl2,
THF,
99%
SAHA
MS-275
2a
2b
2c
86
4
O
4
3
3160 160
349 28
O
Methyl
OBn
H
MeO
P
MeO
Phenyl
344 44
OMe
BnO2C
O
OMe
t-Butyl
2-Ethylhexyl
1940 300
456 28
O
NaH, THF
92% over 2 steps
2d
O
5
6
O
ª
Values are the mean of at least three experiments with standard error given.
Cu(I)I,
MeLi,
Pd(OH)2/C,
PhNH2,
H2,
TBTU, DIPEA,
BnO2C
OMe
TMSCl,
THF
EtOAc
CH3CN
53% over 3 steps
O
7a
unsaturated ester 6 was present even after purification of phenyl
ester 7b due to similar polarity. The mixture of unsaturated ester
6 and phenyl ester 7b were carried through the reaction series of
hydrogenolysis and aniline coupling to give a mixture of anilide
8b and unsaturated anilide compound 8b0. Fortunately, after instal-
lation of the hydroxamic acid, final compound 2b was isolated.
HDAC inhibitory activities of the C6-SAHA analogs were mea-
sured using the Fluor de LysÒ in vitro fluorescence activity assay
kit (Enzo) using HeLa cell lysates as the source of HDAC activity
(Table 1), as previously reported.16,17 The methyl and phenyl vari-
ants 2a and 2b were the most potent, displaying nanomolar IC50
values, which were only four-fold reduced compared to SAHA. In
addition, the 2-ethylhexyl variant 2d, which contained the longest
substituent of the series, displayed potent inhibitory activity in the
nanomolar range. The potent inhibition of these C6 analogs is in
contrast to the C3-phenyl SAHA variant (Fig. 1, IC50 of
73,000 nM), which displayed 811-fold reduced activity versus
SAHA.16b The results indicate that the active site of HDAC proteins
can accommodate a bulky substituent at the C6 position. Interest-
ingly, the t-butyl variant 2c, which contains the bulkiest substitu-
O
O
NH2OH,
KOH
NHOH
OMe
PhHN
PhHN
MeOH, 58%
O
O
2a
8a
Scheme 1. Initial synthesis of C6-SAHA methyl analog (2a).
despite the fact that SAHA analogs with substituents at the C7
position, such as 1, showed potency and selectivity. Due to the
symmetrical relationship of the C6 and C3 positions on SAHA, the
C6-SAHA analog synthesis had similarities to that of the C3-SAHA
analogs, previously reported.16b The main difference between the
two syntheses was the order of installation of the terminal amide
groups. Similar to the C2 and C3-SAHA analogs, we selected hydro-
phobic substituents since the carbon linker is surrounded by a
hydrophobic channel in the HDAC structure.13
We initially synthesized the C6-methyl SAHA analog 2a, as out-
lined in Scheme 1. Under Fisher conditions, commercially available
-caprolactone 3 was opened to give alcohol 4, which was
e
subjected to Swern oxidation to give aldehyde 5. For the Horner–
Wadsworth–Emmons reaction, benzyl dimethyl phosphono-
acetate was added to crude compound 5 to give the corresponding
ent with methyl groups on the
potency, which was 20-fold reduced compared to SAHA. In sum-
mary, the inhibition data show that most C6-SAHA analogs
a-carbon, displayed the weakest
a
,b-unsturated benzyl ester 6. A mixture of (E) and (Z)-isomers of
maintain nanomolar potency, but substitution at the
may decrease inhibitory activity.
a-carbon
ester 6 was treated with a copper (I) iodide and methyl lithium
to give the C6-methyl substituted benzyl ester 7a. Without purifi-
cation, ester 7a was deprotected by hydrogenolysis and coupled
with aniline to give anilide 8a, which was directly converted to
the methyl hydroxamic acid final product 2a.
To create the remaining C6-SAHA analogs, purification by col-
umn chromatography was required after 1,4-addition since the
mixture of (E) and (Z)-isomers 6 incompletely converted to ester
7 (Scheme 2). Using this additional purification step, the C3-t-butyl
2c and C3-2-ethylhexyl 2d SAHA analogs were synthesized as de-
scribed for the methyl variant. Unfortunately, contaminating
The C6-SAHA analogs were next evaluated for potency against
individual HDAC isoforms- HDAC1 and HDAC3 representing class
I and HDAC6 representing class II. All compounds were tested at
a single concentration near their IC50 values using the Fluor de
Lys™ kit (Fig. 2). Consistent with previous data,10,16a SAHA exhib-
ited roughly equal inhibition against HDAC1, HDAC3, and HDAC6.
The phenyl variant 2b also similarly inhibited HDAC1, HDAC3,
and HDAC6. In contrast, the methyl variant 2a showed modest
dual-preference for HDAC1 and HDAC3 over HDAC6 at 500 nM.
The 2-ethylhexyl variant 2d also showed preference for HDAC3

7086
S. E. Choi, M.K.H. Pflum / Bioorg. Med. Chem. Lett. 22 (2012) 7084–7086
substituted on the C2 and C3 positions (Fig. 1),16 the data suggest
that the linker region of HDAC inhibitors, particularly near the
capping group, is an interesting yet underexplored area of future
drug design.
100
80
60
40
20
0
HDAC1
HDAC3
HDAC6
Acknowledgments
We thank the National Institute of Health (GM067657) and
Wayne State University for funding, S.V.W. Weerasinghe, P. P.
Das, B. B. Parida, and Z. Wu for technical assistance, and G. Padige
and M. Wambua for comments on the manuscript.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
09.093.
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over HDAC1 and HDAC6. However, the bulkiest analog, the t-butyl
variant 2c, displayed preference for HDAC1 and HDAC6 over
HDAC3. The data indicate that the methyl, t-butyl, and 2-ethyl-
hexyl variants (2a, 2c, and 2d) display modestly different prefer-
ences for each HDAC isoform while still maintaining nanomolar
or low micromolar potency.
To more thoroughly assess the selectivity observed in the initial
screen, we determined the IC50 values of the C6-t-butyl variant 2c
against HDAC1, HDAC3, and HDAC6. We selected the t-butyl
analog because it showed the most potential to create a dual
HDAC1/HDAC6-selective inhibitor, which would be useful for the
treatment and study of acute myeloid leukemia.18 As expected
based on the initial screen, the C6-t-butyl analog 2c displayed
modest preference for HDAC1 and HDAC6 compared to HDAC3
(six-fold and two-fold, respectively, Table 2). As a control, SAHA
showed no selectivity, as expected (Table 2).10 The analysis shows
that substituents on the C6 position modestly influence inhibitor
selectivity and may promote creation of dual selective inhibitors.
In conclusion, SAHA analogs containing substituents on the C6
position in the linker region can display nanomolar IC50 values,
indicating that subsitutents near the solvent-exposed capping
group are accommodated in the HDAC active site. In addition,
C6-substituents can also modestly influence selectivity for individ-
ual HDAC isoforms. Combined with earlier studies of SAHA analogs