Potent and selective inhibition of histone deacetylase 6 (HDAC6) does not require a surface-binding motif

Article abstract of DOI:10.1021/jm301355j

Hydroxamic acids were designed, synthesized, and evaluated for their ability to selectively inhibit human histone deacetylase 6 (HDAC6). Several inhibitors, including compound 14 (BRD9757), exhibited excellent potency and selectivity despite the absence of a surface-binding motif. The binding of these highly efficient ligands for HDAC6 is rationalized via structure-activity relationships. These results demonstrate that high selectivity and potent inhibition of HDAC6 can be achieved through careful choice of linker element only.

Full text of DOI:10.1021/jm301355j

Brief Article
pubs.acs.org/jmc
Potent and Selective Inhibition of Histone Deacetylase 6 (HDAC6)
Does Not Require a Surface-Binding Motif
Florence F. Wagner, David E. Olson, Jennifer P. Gale, Taner Kaya, Michel Weïwer, Nadia Aidoud,
Meryl Thomas, Emeline L. Davoine, Berenice C. Lemercier, Yan-Ling Zhang, and Edward B. Holson*
́
́
́
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142,
United States
S
* Supporting Information
ABSTRACT: Hydroxamic acids were designed, synthesized, and evaluated for their ability to selectively inhibit human histone
deacetylase 6 (HDAC6). Several inhibitors, including compound 14 (BRD9757), exhibited excellent potency and selectivity
despite the absence of a surface-binding motif. The binding of these highly efficient ligands for HDAC6 is rationalized via
structure−activity relationships. These results demonstrate that high selectivity and potent inhibition of HDAC6 can be achieved
through careful choice of linker element only.
Many HDAC inhibitors are designed as structural mimics of
acetyl-lysine and, as a result, often contain a zinc-binding group
(ZBG), a linker, and a cap group (Figure 1).
INTRODUCTION
■
Histone deacetylases (HDACs) are enzymes responsible for
catalyzing the hydrolysis of acetylated lysine residues located on
histone as well as nonhistone proteins.1 Such posttranslational
modifications are crucial for the regulation of many cellular
processes including gene transcription and protein function.2
Inhibition of the 11 zinc-dependent human HDACs has proven
to be a valuable strategy in the fight against cancer3 and other
human afflictions including psychiatric,4 metabolic,5 and
infectious6 diseases. These metal-dependent HDACs are
divided into classes and subclasses: class I (HDACs 1, 2, 3
and 8), class IIa (HDACs 4, 5, 7, 9), class IIb (HDACs 6, 10),
and class IV (HDAC11).1c The class IIb isoform HDAC6 is
primarily localized in the cytosol and features two independ-
ently active catalytic domains.2b,7 Functionally, HDAC6 is
unique as the only zinc-dependent HDAC that controls α-
tubulin acetylation.7 In recent years, HDAC6 has been
implicated in numerous disorders including several within the
central nervous system (CNS).8−10 Langley et al. reported that
selective HDAC6 inhibition could promote the survival and
regrowth of neurons following injury.9 This finding was
validated pharmacologically with tubastatin A, an HDAC6
selective compound, which was shown to confer protection in
primary cortical neuron cultures under oxidative stress.10
Intrigued by the opportunity for pharmacological inter-
vention in psychiatric diseases, we set out to identify selective
small molecule inhibitors of HDAC6. A major challenge in
CNS drug discovery is the efficient delivery of small molecules
across the blood−brain barrier (BBB). Because the ability of a
small molecule to cross the BBB is often inversely correlated
with its size, we set out to define the essential pharmacophoric
elements required for potent and selective binding of HDAC6.
We utilized the concept of ligand efficiency (ligE), which is
defined as biological activity per molecular size11 and has
become a beneficial tool in medicinal chemistry.12 Compounds
with high ligE (>0.50) often possess preferable lead-like
characteristics for the CNS such as a low molecular weight
(<350 g·mol−1) and lower hydrophobicity (cLogP < 4).13
Figure 1. Structures of acetylated lysine and known HDAC inhibitors:
SAHA, tubacin, tubastatin A, and “linkerless” compound 1.
These pharmacophoric elements are exemplified by the
structure of suberoylanilide hydroxamic acid (SAHA), a
nonselective inhibitor of HDACs 1, 2, 3, 6, and 8, which
possesses a 6-carbon alkyl chain linking a hydroxamic acid to a
capping moiety. The hydroxamic acid zinc binding group and a
large capping moiety are common features of selective HDAC6
inhibitors, and the latter is believed to be critical for selective
inhibition.14 Tubacin (Figure 1) was reported in 2003 by
Haggarty et al. as the first selective HDAC6 inhibitor.15 More
recently, nonpeptidic macrocycles were reported by Auzzas et
al. as HDAC6 selective inhibitors.16 While structurally similar
to SAHA in their ZBG and linker motifs, the selectivity was
attributed to specific interactions between the unique capping
Received: September 28, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm301355j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
motif and the surface topology of HDAC6.14b,16 While capping
motifs can impart HDAC6 selectivity, the overall physicochem-
ical properties and binding efficiency of these type of
compounds suffer as reflected in a ligand efficiency of 0.16
for tubacin. Subsequently, a highly selective HDAC6 inhibitor,
tubastatin A (Figure 1),10 with an alternative linker motif, a
substituted phenyl ring, was reported in 2010 by Butler et al.
Tubastatin A also exploits interactions between the cap group
and the HDAC enzyme rim and possesses a much more drug-
like structure as exemplified by its higher ligE of 0.31. While
these approaches focused on optimizing the surface binding
motifs or cap to impart potency and selectivity for HDAC6,
KrennHrubec et al. reported the design of “linkerless” or
“capless” selective HDAC inhibitor such as compound 1.17 This
compact (ligE of 0.38) HDAC8 selective inhibitor has >7-fold
selectivity versus HDAC6 but displays weak inhibitory activity
(14.0 μM). Similarly, for HDAC6, we chose to exploit close
contacts and structural differences between the various isoforms
within the catalytic binding domain. Herein, we describe our
efforts to identify and characterize these critical linker
interactions and to incorporate these design considerations
into novel, selective, and highly efficient small molecule
inhibitors of HDAC6.
fold). Next, we probed the effect of the length of the surface
group. While compounds 4−6 exhibited excellent potency for
HDAC6, the extra carbon spacers in 4 only provided marginal
improvement in selectivity for this isoform. In fact, the cap
group only appeared to have a modest effect on the relative
inhibition of HDACs 2, 4, 6, and 8. Strikingly, compound 7,
with a para-methyl amide, retained a high level of potency (28
nM) and selectivity (57-fold), demonstrating that a large
capping group is not critical to achieve potent and selective
inhibition of HDAC6.
Encouraged by the initial results with the para-substituted
phenyl hydroxamic acids, we set out to identify the minimal
pharmacophore that would confer potency and selectivity for
HDAC6 (Table 2). To our surprise, capless phenyl hydroxamic
Table 2. IC50s for HDACs 2, 4, 6, and 8
RESULTS AND DISCUSSION
We began our studies with amide linked para-substituted
phenyl hydroxamic acids (Table 1), as similar phenyl-linked
■
Table 1. IC50s for HDACs 2, 4, 6, and 8
a
Values are the mean of two experiments. Data are shown as IC50
values in μM
standard deviation. Compounds were tested in
duplicate in a 12-point dose curve with 3-fold serial dilution starting
from 33.33 μM.
acid, compound 8,21 exhibited good potency (115 nM) with a
remarkable ligE of 0.69. Moreover, the selectivity of compound
8 versus HDAC2 (70-fold) and HDAC8 (17-fold) remained
high.
We next turned our attention toward small molecules
containing a short linker between the ZBG and the aromatic
group, as several reported HDAC inhibitors contain an alkyl or
cinnamyl-linked hydroxamic acid motif.21 The results (shown
in Table 2, compounds 9−11) demonstrate that an sp2 carbon
at the α-position of the hydroxamic acid is crucial for
maintaining potent inhibition of HDAC6. Compounds 9 and
10, which possess an alkyl linker between the ZBG and the
aromatic group, demonstrated a reduced ability to inhibit
HDAC6.
However, cinnamyl linked compound 11 exhibited increased
potency for HDAC6, but the selectivity toward HDAC6 versus
HDAC2 (21-fold), while still remarkable, suffered slightly as
this compound became more potent on class I HDACs. The
importance of the sp2-hybridized carbon was further confirmed
by the potency of cyclohexenyl compound 12 (12 nM)
compared to the potency of cyclohexanyl compound 13 (376
nM). Moreover, compounds 11 and 12 illustrate that while the
sp2-carbon confers potency, substitution at the α-carbon
enhances selectivity for the HDAC6 isoform.
a
Values are the mean of two experiments. Data are shown as IC50
values in μM
standard deviation. Compounds were tested in
duplicate in a 12-point dose curve with 3-fold serial dilution starting
from 33.33 μM.
compounds have been previously shown to inhibit HDAC6
selectively.10,18 All compounds were tested against HDACs 1−
9 in a trypsin-free microfluidic lab-on-a-chip assay.19 IC50 values
for HDAC2 and HDAC8 (representative of class I), HDAC4
(representative of class IIa), and HDAC6 are reported.
Compound 2, with an ethylene piperazine capping element,
exhibited a 12-fold selectivity in favor of HDAC6 versus
HDAC2, however, its potency for HDAC6 was modest (1.36
μM). The more lipophilic compound 3 (BRD8148)20 proved
to be a potent (8 nM) and selective (42-fold vs HDAC2)
inhibitor of HDAC6; however, the pyridine nitrogen appears to
have no effect as it did not confer improved potency or
selectivity over the phenyl group in compound 4 (4 nM, 152-
B
dx.doi.org/10.1021/jm301355j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
Table 3. HDAC Inhibitors’ IC50s for HDACs 1−9
a
Values are the mean of two experiments. Data are shown as IC50 values in μM standard deviation. Compounds were tested in duplicate in a 12-
point dose curve with 3-fold serial dilution starting from 33.33 μM.
To probe the selectivity of these compounds versus the
broader family of HDAC isoforms, we profiled compounds 3, 7,
8, 12, and 14 against HDACs 1−9 (Table 3 and Supporting
Information).19 The cyclopentenyl linked compound 14
(BRD9757) represents one of the most concise inhibitors of
HDAC6 reported to date, with an IC50 of 30 nM and excellent
selectivity toward HDAC6 versus the class I (>20-fold) and
class II (>400-fold) HDACs tested (Table 3). Consistent with
this succinct design and remarkable potency, compound 14
displays a ligand efficiency of 0.84 for HDAC6. Potent and
selective inhibition of HDAC6 does not require a surface-
binding motif and can be achieved by relying on small linker
motifs coupled with the hydroxamic acid ZBG.
To further validate the selectivity of these compounds, we
investigated whether or not their biochemical selectivities
translated in cellular functional assays. HeLa cells were treated
with compounds at 10 and 30 μM for 24 h prior to measuring
the acetylation status of α-tubulin (HDAC6 dependent)7a
compared to total H3 acetylation (HDAC1, 2, 3 dependent)1c
(Figure 2). As expected, we observed a dose-dependent
efficient capless ligands were shown to have excellent potency
and selectivity toward HDAC6 versus HDAC1−9. The
selective inhibition of HDAC6 translated to a cell-based assay
in which selective increase in the level of Ac-tubulin, without
increasing histone acetylation, was observed upon treatment
with capless HDAC6 inhibitors. Furthermore, we demonstrated
that an sp2-carbon at the α-position of the hydroxamic acid
confers potency, while small capless cycloalkenyl motifs are
sufficient to achieve high selectivity for the HDAC6 isoform.
Ultimately, our studies should open the way for the design of
selective small molecule HDAC inhibitors with optimized
ligand efficiencies. These novel small molecule inhibitors can be
used as tools for probing the biological functions and relevance
of the different HDAC isoforms and serve as the basis for new
selective inhibitors of other HDAC isoforms.
EXPERIMENTAL SECTION
■
See Supporting Information for details. All final compounds were
confirmed to be of >95% purity based on HPLC LCMS analysis
(Alliance 2795, Waters, Milford, MA). Purity was measured by UV
absorbance at 210 nm. Identity was determined on a SQ mass
spectrometer by positive and negative electrospray ionization. All
reagents and solvents were purchased from commercial vendors and
used as received. 1H and 13C NMR spectra were recorded on a Bruker
300 MHz or Varian UNITY INOVA 500 MHz spectrometer as
indicated. Proton and carbon chemical shifts (δ) are reported in ppm
relative to tetramethylsilane (δ 0 for both H and 13C) and DMSO-d6
1
(1H δ 2.50, 13C δ 39.5). NMR data were collected at 25 °C. Flash
chromatography was performed using 40−60 μm silica gel (60 Å
mesh) on a Teledyne Isco Combiflash Rf system.
Representative Procedure for the Synthesis of Hydroxamic
Acid Derivatives.
Figure 2. Treatment of HeLa cells for 24h with compounds 7, 8, 12,
and 14 results in the increase of Ac-tubulin but not Ac-H3.
Concentrations in μM are shown in brackets.
Methyl 4-(methylcarbamoyl)benzoate (0.20 g, 1.04 mmol) was
dissolved in CH2Cl2 and methanol (1:2, 9 mL). The resulting
solution was cooled to 0 °C, and hydroxylamine (50 wt % in water,
0.952 mL, 31.1 mmol, 30 equiv) was added, followed by sodium
hydroxide (0.414 g, 10.35 mmol, 10.0 equiv). The reaction was
allowed to warm to room temperature and stirred for 12 h. The
solvent was then removed under reduced pressure, and the obtained
solid was dissolved in water. The pH was adjusted to 7 with a 1N
aqueous solution of HCl. The resulting precipitate was filtered and
dried under high vacuum to afford N-hydroxy-N-methylterephthala-
mide (7) (90 mg, 45% yield) as a white solid. ESI+ MS: m/z 195.3
increase in the level of Ac-tubulin when the cells were treated
with compounds 7, 8, 12, or 14 but did not observe a
significant increase in Ac-H3 even at 30 μM. In contrast, SAHA
and tubastatin A induced an increase in Ac-tubulin as well as a
moderate increase in Ac-H3. This confirms that cellular
HDAC6 is selectively inhibited by these highly ligand efficient
capless hydroxamic acids in a robust manner.
1
([M + H]+). H NMR (300 MHz, DMSO-d6): δ 11.33 (s, 1H), 9.14
CONCLUSIONS
■
(s, 1H), 8.55 (s, 1H), 7.95−7.75 (m, 4H), 2.79 (s, 3H). When
analogues did not precipitate, after adjusting the pH, the water was
removed under reduced pressure to afford white solids. The crude
material was dissolved in cold ethanol and filtered. The obtained solids
We demonstrated that large capping groups, such as those of
tubacin and tubastatin A, are not necessary to achieve potent
and selective inhibition of HDAC6. Several novel and highly
C
dx.doi.org/10.1021/jm301355j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
were then purified by reverse phase HPLC using a gradient of 0−20%
conditions. Trends Neurosci. 2009, 32, 591−601. (b) Gray, S. G.
Epigenetic treatment of neurological disease. Epigenomics 2011, 3,
431−450. (c) Grayson, D. R.; Kundakovic, M.; Sharma, R. P. Is there a
future for histone deacetylase inhibitors in the pharmacotherapy of
psychiatric disorders? Mol. Pharmacol. 2010, 77, 126−135. (d) Beurel,
E. HDAC6 regulates LPS-tolerance in astrocytes. PLoS One 2011, 6,
e25804. (e) McQuown, S. C.; Barrett, R. M.; Matheos, D. P.; Post, R.
J.; Rogge, G. A.; Alenghat, T.; Mullican, S. E.; Jones, S.; Rusche, J. R.;
Lazar, M. A.; Wood, M. A. HDAC3 is a critical negative regulator of
long-term memory formation. J. Neurosci. 2011, 31, 764−774.
(f) Guan, J.-S.; Haggarty, S. J.; Giacometti, E.; Dannenberg, J.-H.;
Joseph, N.; Gao, J.; Nieland, T. J. F.; Zhou, Y.; Wang, X.; Mazitschek,
R.; Bradner, J. E.; DePinho, R. A.; Jaenisch, R.; Tsai, L.-H. HDAC2
negatively regulates memory formation and synaptic plasticity. Nature
2009, 459, 55−60.
(5) (a) Feng, D.; Liu, T.; Sun, Z.; Bugge, A.; Mullican, S. E.;
Alenghat, T.; Liu, X. S.; Lazar, M. A. A circadian rhythm orchestrated
by histone deacetylase 3 controls hepatic lipid metabolism. Science
2011, 331, 1315−1319. (b) Gluckman, P. D.; Hanson, M. A.; Buklijas,
T.; Low, F. M.; Beedle, A. S. Epigenetic mechanisms that underpin
metabolic and cardiovascular diseases. Nature Rev. Endocrinol. 2009, 5,
401−408. (c) Mihaylova, M. M.; Vasquez, D. S.; Ravnskjaer, K.;
Denechaud, P.-D.; Yu, R. T.; Alvarez, J. G.; Downes, M.; Evans, R. M.;
Montminy, M.; Shaw, R. J. Class IIa histone deacetylases are hormone-
activated regulators of FOXO and mammalian glucose homeostasis.
Cell 2011, 145, 607−621. (d) Chou, D. H.; Holson, E. B.; Wagner, F.
F.; Tang, A. J.; Maglathlin, R. L.; Lewis, T. A.; Schreiber, S. L.; Wagner,
B. K. Inhibition of histone deacetylase 3 protects beta cells from
cytokine-induced apoptosis. Chem Biol. 2012, 19, 669−673.
(6) (a) Rotilli, D.; Simonetti, G.; Savarino, A.; Palamara, A. T.;
Migliaccio, A. R.; Mai, A. Non-cancer uses of histone deacetylase
inhibitors: effects on infectious diseases and beta-hemoglobinopathies.
Curr. Top. Med. Chem. 2009, 9, 272−291. (b) Andrews, K. T.; Haque,
A.; Jones, M. K. HDAC inhibitors in parasitic diseases. Immunol. Cell
Biol. 2012, 90, 66−77.
acetonitrile in water (0.1% TFA).
ASSOCIATED CONTENT
* Supporting Information
■
S
Spectroscopic characterization of the compounds in this paper;
details related to biochemical and cellular studies. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 617-714-7454. E-mail: edholson@broadinstitute.org.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Steve Johnston for analytical/purification support
and John McGrath for compound management support. We
also thank Joshua Ketterman for advice and assistance with cell
culture. This research was funded by the Stanley Medical
Research Institute.
ABBREVIATIONS USED
■
HDACs, histone deacetylases; CNS, central nervous system;
BBB, blood−brain barrier; LigE, ligand efficiency; ZBG, zinc-
binding group; SAHA, suberoylanilide hydroxamic acid; MD,
molecular dynamics
(7) (a) Hubbert, C.; Guardiola, A.; Shao, R.; Kawaguchi, Y.; Ito, A.;
Nixon, A.; Yoshida, M.; Wang, X.-F.; Yao, T.-P. HDAC6 is a
microtubule-associated deacetylase. Nature 2002, 417, 455−458.
(b) Zhang, Y.; Li, N.; Caron, C.; Matthias, G.; Hess, D.; Khochbin,
S.; Matthias, P. HDAC-6 interacts with and deacetylates tubulin and
microtubules in vivo. EMBO J. 2003, 22, 1168−1179. (c) Zou, H.; Wu,
Y.; Navre, M.; Sang, B.-C. Characterization of the two catalytic
domains in histone deacetylase 6. Biochem. Biophys. Res. Commun.
2006, 341, 45−50. (d) Zhang, Y.; Gilquin, B.; Khochbin, S.; Matthias,
P. Two catalytic domains are required for protein deacetylation. J. Biol.
Chem. 2006, 281, 2401−2404.
(8) (a) Dompierre, J. P.; Godin, J. D.; Charrin, B. C.; Cordelieres, F.
P.; King, S. J. Histone deacetylase 6 inhibition compensates for the
transport deficit in Huntington’s disease by increasing tubulin
acetylation. J. Neurosci. 2007, 27, 3571−3583. (b) Chen, S.; Owens,
G. C.; Makarenkova, H.; Edelman, D. B. HDAC6 regulates
mitochondrial transport in hippocampal neurons. PLoS One 2010, 5,
e10848.
(9) Rivieccio, A.; Brochier, C.; Willis, D.; Walker, D. E.; D’Annibale,
M. A.; McLaughlin, K.; Siddiq, A.; Kosikowski, A. P.; Jaffrey, S. R.;
Twiss, J. L.; Ratan, R. R.; Langley, B. HDAC6 is a target for protection
and regeneration following injury in the nervous system. Proc. Natl.
Acad. Sci. U. S. A. 2009, 106, 19599−19604.
(10) Butler, K. V.; Kalin, J.; Brochier, C.; Vistoli, G.; Langley, B.;
Kosikowski, A. P. Rational design and simple chemistry yield a
superior, neuroprotective HDAC6 inhibitor, tubastatin A. J. Am. Chem.
Soc. 2010, 132, 10842−10846.
(11) LigE was calculated using the following formula; ligE =
p(IC50 HDAC6)/(number of non-hydrogen atoms).
REFERENCES
■
(1) (a) Minucci, S.; Pelicci, P. G. Histone deacetylase inhibitors and
the promise of epigenetic (and more) treatments for cancer. Nature
Rev. Cancer 2006, 6, 38−51. (b) Haberland, M.; Montgomery, R. L.;
Olson, E. N. The many roles of histone deacetylases in development
and physiology: implications for disease and therapy. Nature Rev.
Genet. 2009, 10, 32−42. (c) Fass, D. M.; Kemp, M. M.; Schroeder, F.
A.; Wagner, F. F.; Wang, Q.; Holson, E. B. Histone Acetylation and
Deacetylation. In Epigenetic Regulation and Epigenomics: Advances in
Molecular Biology and Medicine; (Wiley-VCH Vrlag & Co. KGaA:
Weinheim, 2012; pp 515−561.
(2) (a) Marks, P. A.; Richon, V. M.; Miller, T.; Kelly, W. K. Histone
Deacetylase Inhibitors. Adv. Can. Res. 2004, 91, 137−168. (b) de
Ruijter, A. J. M.; Van Gennip, A. H.; Caron, H. N.; Kemp, S.; Van
Kuilenburg, A. B. P. Histone deacetylases (HDACs): characterization
of the classical HDAC family. Biochem. J. 2003, 370, 737−749.
(c) Glaser, K. B.; Staver, M. J.; Waring, J. F.; Stender, J.; Ulrich, R. G.;
Davidsen, S. K. Gene expression profiling of multiple histone
deacetylase (HDAC) inhibitors: defining a common gene set
produced by HDAC inhibition in T24 and MDA carcinoma cell
lines. Mol. Cancer Ther. 2003, 2, 151−163. (d) Spange, S.; Wagner, T.;
Heinzel, T.; Kramer, O. H. Acetylation of non-histone proteins
̈
modulates cellular signalling at multiple levels. Int. J. Biochem. Cell Biol.
2009, 41, 185−198.
(3) (a) Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Anticancer
activities of histone deacetylase inhibitors. Nature Rev. Drug Discovery
2006, 5, 769−784. (b) Khan, O.; La Thangue, N. B. HDAC inhibitors
in cancer biology: emerging mechanisms and clinical applications.
Immunol. Cell Biol. 2012, 90, 85−94. (c) Witt, O.; Deubzer, H. E.;
Milde, T.; Oehme, I. HDAC family: What are the cancer relevant
targets? Cancer Lett. 2009, 277, 8−21.
(12) (a) Abad-Zapatero, C.; Metz, J. T. Ligand efficiency indices as
guideposts for drug discovery. Drug Discovery Today 2005, 10, 464−
469. (b) Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A. The
maximal affinity of ligands. Proc. Natl. Acad. Sci. U. S. A. 1999, 96,
9997−10002. (c) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand
(4) (a) Chuang, D. M.; Leng, Y.; Marinova, Z.; Kim, H. J.; Chiu, C.
T. Multiple roles of HDAC inhibition in neurodegenerative
D
dx.doi.org/10.1021/jm301355j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
efficiency: a useful metric for lead selection. Drug Discovery Today
2004, 9, 430−431.
(13) Reynolds, C. H.; Bembenek, S. D.; Tounge, B. A. The role of
molecular size in ligand efficiency. Bioorg. Med. Chem. Lett. 2007, 17,
4258−4261.
(14) (a) Butler, K. V.; Kozikowski, A. P. Chemical origins of isoform
selectivity in histone deacetylase inhibitors. Curr. Pharm. Des. 2008, 14,
505−528. (b) Bieliauskas, A. V.; Pflum, M. K. H. Isoform-selective
histone deacetylase inhibitors. Chem. Soc. Rev. 2008, 37, 1402−1413.
(c) Estiu, G.; Greenberg, E.; Harrison, C. B.; Kwiatkowski, N. P.;
Mazitschek, R.; Bradner, J. E.; Wiest, O. On the structural origin of
selectivity in class II selective histone deacetylase Inhibitors. J. Med.
Chem. 2008, 51, 2898−2906. (d) Santo, L.; Hideshima, T.; Kung, A.
L.; Tseng, J.-C.; Tamang, D.; Yang, M.; Jarpe, M.; van Duzer, J. H.;
Mazitschek, R.; Ogier, W. C.; Cirstea, D.; Rodig, S.; Eda, H.; Scullen,
T.; Canavese, M.; Bradner, J.; Anderson, K. C.; Jones, S. S.; Raje, N.
Preclinical activity, pharmacodynamic, and pharmacokinetic properties
of a selective HDAC6 inhibitor, ACY-1215, in combination with
bortezomib in multiple myeloma. Blood 2012, 11, 2579−2589.
(e) Suzuki, T. Explorative study on isoform-selective histone
deacetylase inhibitors. Chem. Pharm. Bull. 2009, 57, 897−906.
(f) Dallavalle, S.; Pisano, C.; Zunino, F. Development and therapeutic
impact of HDAC6-selective inhibitors. Biochem. Pharmacol. 2012, 6,
759−765.
(15) Haggarty, S. J.; Koeller, K. M.; Wong, J. C.; Grozinger, C. M.;
Schreiber, S. L. Domain-selective small-molecule inhibitor of histone
deacetylase 6 (HDAC6)-mediated tubulin deacetylation. Proc. Natl.
Acad. Sci. U. S. A. 2003, 100, 4389−4394.
̂
(16) Auzzas, L.; Larsson, A.; Matera, R.; Baraldi, A.; Deschenes-
Simard, B.; Giannini, G.; Cabri, W.; Battistuzzi, G.; Gallo, G.; Ciacci,
A.; Vesci, L.; Pisano, C.; Hanessian, S. Non-natural macrocyclic
inhibitors of histone deacetylases: design, synthesis, and activity. J.
Med. Chem. 2010, 53, 8387−8399.
(17) KrennHrubec, K.; Marshall, B. L.; Hedglin, M.; Verdin, E.;
Ulrich, S. M. Design and evaluation of “linkerless” hydroxamic acids as
selective HDAC8 inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 2874−
2878.
(18) (a) Smil, D. V.; Manku, S.; Chantigny, Y. A.; Leit, S.; Wahhab,
A.; Yan, T. P.; Fournel, M.; Maroun, C.; Li, Z.; Lemieux, A.-M.;
Nicolescu, A.; Rahil, J.; Lefebvre, S.; Panetta, A.; Besterman, J. M.;
́
Deziel, R. Novel HDAC6 isoform selective chiral small molecule
histone deacetylase inhibitors. Bioorg. Med. Chem. Lett. 2009, 19, 688−
692. (b) Mazitscheck, R.; Bradner, J. E.; Grachan, M. Histone
deacetylase inhibitors and methods of use thereof. PCT US2012/
024868, Feb 2, 2012.
(19) (a) Holson, E.; Wagner, F.; Weïwer, M.; Zhang, Y. L.; Haggarty,
S. H.; Tsai, L. H. Inhibitors of histone deacetylases. WO2012149540
(A1), Nov 1, 2012. (b) Inhibition of HDAC10 and 11 was not
measured due to either low purity of the available recombinant HDAC
enzyme preparations and/or lack of activity of the enzymes and low
substrate conversion.
(20) Wilson, A. J.; Holson, E.; Wagner, F.; Zhang, Y.-L.; Fass, D. M.;
Haggarty, S. J.; Bhaskara, S.; Hiebert, S. W.; Schreiber, S. L.; Khabele,
D. The DNA damage mark pH2AX differentiates the cytotoxic effects
of small molecule HDAC inhibitors in ovarian cancer cells. Cancer Biol.
Ther. 2011, 12, 484−493.
(21) (a) Thaler, F.; Colombo, A.; Mai, A.; Amici, R.; Bigogno, C.;
Boggio, R.; Cappa, A.; Carrara, S.; Cataudella, T.; Fusar, F.; Gianti, E.;
Joppolo di Ventimiglia, S.; Moroni, M.; Munari, D.; Pain, F.; Regalia,
N.; Sartori, L.; Vultaggio, S.; Dondio, G.; Gagliardi, S.; Minucci, S.;
Mercurio, C.; Varasi, M. Synthesis and biological evaluation of N-
hydroxyphenylacrylamides and N-hydroxypyridin-2-ylacrylamides as
novel histone deacetylase inhibitors. J. Med. Chem. 2010, 53, 822−839.
(b) Siliphaivanh, P.; Harrington, P.; Witter, D. J.; Otte, K.; Tempest,
P.; Kattar, S.; Kral, A. M.; Fleming, J. C.; Deshmukh, S. V.; Harsch, A.;
Secrist, P. J.; Millera, T. A. Design of novel histone deacetylase
inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 4619−4624.
E
dx.doi.org/10.1021/jm301355j | J. Med. Chem. XXXX, XXX, XXX−XXX