Identification of novel, selective, and stable inhibitors of class II histone deacetylases. Validation studies of the inhibition of the enzymatic activity of HDAC4 by small molecules as a novel approach for cancer therapy

Article abstract of DOI:10.1021/jm900555u

5-Aryl-2-(trifluoroacetyl)thiophenes were identified as a new series of class II HDAC inhibitors (HDACi). Further development of this new series led to compounds such as 6h, a potent inhibitor of HDAC4 and HDAC6 (HDAC4 WT IC ₅₀ = 310 nM, HDAC6

Full text of DOI:10.1021/jm900555u

6782 J. Med. Chem. 2009, 52, 6782–6789
DOI: 10.1021/jm900555u
Identification of Novel, Selective, and Stable Inhibitors of Class II Histone Deacetylases. Validation
Studies of the Inhibition of the Enzymatic Activity of HDAC4 by Small Molecules as a Novel Approach
for Cancer Therapy
Jesus M. Ontoria,* Sergio Altamura, Annalise Di Marco, Federica Ferrigno, Ralph Laufer, Ester Muraglia,
Maria Cecilia Palumbi, Michael Rowley, Rita Scarpelli, Carsten Schultz-Fademrecht, Sergio Serafini, Christian Steinkuhler,
and Philip Jones
€
Istituto Di Ricerche Di Biologia Molecolare, P. Angeletti SpA (IRBM-MRL, Rome), Via Pontina Km 30,600, I-00040 Pomezia, Italy
Received April 30, 2009
5-Aryl-2-(trifluoroacetyl)thiophenes were identified as a new series of class II HDAC inhibitors
(HDACi). Further development of this new series led to compounds such as 6h, a potent inhibitor of
HDAC4 and HDAC6 (HDAC4 WT IC50 = 310 nM, HDAC6 IC50 = 70 nM) that displays 40-fold
selectivity over HDAC1 and improved stability in HCT116 cancer cells (t1/2=11 h). Compounds 6h and
2 show inhibition of R-tubulin deacetylation in HCT116 cells at 1 μM concentration and antiprolifera-
tion effects only at concentrations where inhibition of histone H3 deacetylation is observed.
Introduction
panobinostat9 and nonhydroxamic acid-based HDACi such
as the 2-aminophenylamides MS-27510 and MGCD-010311 or
the dithiol FK22812 are currently in the clinic. Most of them
hit a subset of both class I and class II HDACs or, as in the
case of the 2-aminophenylamides, show selectivity versus class
I HDACs. All of them elicit similar adverse effects, mainly
fatigue, nausea, vomiting, and diarrhea, which become dose-
limiting in clinical trials. Elucidation of the function of each
HDAC subtype would allow the identification of second
generation inhibitors to be developed, which could target
individual HDAC isoforms and thereby achieve a wider
therapeutic index. Consequently, there remains a widely
recognized need to identify selective HDACi.13
In an effort to develop a second generation HDACi with
enhanced efficacy and a larger therapeutic window, we were
interested in targeting a more restricted set of HDAC iso-
forms. RNAi knock-down experiments were performed
against all 11 class I and II HDACs in three different cancer
cell lines (HeLa, HCT116, and A549). In these experiments, it
was observed that ablation of HDAC4 expression led to cell
growth inhibition in all three human tumor cell lines but no
detectable effects in human fibroblast or myelopoietic pro-
genitors. Moreover, knockdown of HDAC4 in HeLa cells
produced mitotic arrest followed by caspase-dependent apop-
totic cell death.14 In parallel, given the problems associated
with the isolation and purification of class II HDACs from
mammalian cells, two enzymatic assays for the measurement
of the inhibitory activity of small molecules against HDAC4
were developed in our laboratory.15 These two assays use
HDAC4 expressed in bacteria and the enzymatic efficiency of
this protein was enhanced either by an H-Y mutation in the
active site to give a constitutively active mutant (CAM) or by
use of the wild-type (WT) enzyme with an “unnatural”
trifluoroacetyl-lysine substrate.15 As the natural substrate of
HDAC4 is unknown, there is currently no cellular target
engagement marker for HDAC4. However, R-tubulin is
Histone deacetylases (HDACsa) are involved in the deace-
tylation of histones, transcription factors, and other proteins
in eukaryotic cells.1 HDAC inhibition causes hyperacetyla-
tion of histones, which leads to a more open chromatin
structure, thereby facilitating gene transcription and ulti-
mately leading to cell-growth arrest, differentiation, and
apoptosis.1b The development of specific HDACi represents
a new strategy in cancer therapy.2
There are currently 18 known mammalian HDACs,
grouped into four classes based on sequence similarity.3 The
class I, II, and IV HDACs are zinc-dependent enzymes,
whereas class III HDACs (SIRT1-SIRT7) are structurally
unrelated and requireNADþ for activity.4 The class I HDACs
(HDAC1, 2, 3, and 8) are ∼400 residues long and are generally
nuclear, whereas the class II HDACs exhibit nucleocytoplas-
mic shuttling. In turn, class II HDACs are subdivided into
class IIa (HDAC4, 5, 7, and 9) and IIb (HDAC6 and 10).
Class IIa enzymes are characterized by the presence of an
N-terminal extension of ∼600 residues with distinct regula-
tory and functional properties, whereas the class IIb enzymes
contain two catalytic domains.5 Class IV currently includes
only HDAC11, which shares greatest sequence similarity to
the class I enzymes.6
The cellular function of each individual HDAC isoform is
not fully understood and neither is the specific role each plays
in tumorogenesis. Notwithstanding, the urgency for alterna-
tive therapies for cancer has allowed a rapid development of
broad spectrum HDACi. For instance, hydroxamic acid-
based HDACi including vorinostat (SAHA),7 belinostat,8 or
*To whom correspondence should be addressed. Phone and Fax:
þ39-069121632. E-mail: jmontoria@gmail.com.
a Abbreviations: HDAC, histone deacetylase; NADþ, nicotinamide
adenine dinucleotide; WT, wild type; CAM, constitutively active
mutant; TFMK, trifluoromethylketone.
r
pubs.acs.org/jmc
Published on Web 10/16/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6783
Scheme 1. Synthesis of 2,2,2-Trifluoro-1-(5-arylthiophen-2-yl)-
ethanones 6a-l
Figure 1. Class II HDAC inhibitors 1 and 2.
deacetylated by HDAC616 and given that the compounds we
have developed inhibit both HDAC4 and 6, inhibition of
HDAC6 can be used as a surrogate marker to measure
inhibition of HDAC4 in cells. With these tools, we initiated
a target validation program to identify potent and selective
class II HDACi, with the aim to show if inhibition of the
deacetylase activity of HDAC4 by a small molecule would
recapitulate the growth inhibitory phenotype observed in the
RNAi experiments.
a Reagents and conditions: (a) TMSCF3, CsF, DME, room temp
(97%); (b) Dess-Martin periodinane, CH2Cl2, room temp (70%); (c)
ArB(OH)2, Na2CO3, Pd(Ph3P)4, DMF, 90 °C, o/n.
Scheme 2. Synthesis of 2,2,2-Trifluoro-1-(4-phenylthiophen-2-yl)-
8
ethanone
ethanone 10
and 2,2,2-Trifluoro-1-(2-aryl-1,3-thiazol-5-yl)-
As previously reported, a series of 5-(trifluoroacetyl)-
thiophene-2-carboxamides was identified as a novel class of
potent and selective class II HDACi.17 These molecules,
exemplified by compound 1 (Figure 1), are active site binders
and typically display around 10-fold selectivity for class II
HDACs (HDAC4, 6) over their class I HDACs (HDAC1, 3)
counterparts. Unfortunately, most of these derivatives lacked
cell-based activity as they failed to show inhibition of either
histone H3 or R-tubulin deacetylation in HCT116 cells, the
cellular substrates of HDAC1 and 6, respectively.18 The lack
of cellular activity displayed by these inhibitors was demon-
strated to be due to the instability of the trifluoromethylk-
etone (TFMK) moiety, which is readily reduced in cells to the
corresponding inactive alcohol, with half-lives depending on
the nature of the amide substituents and to be in the range of
20 min to 2 h.
a Reagents and conditions: (a) TMSCF3, CsF, DME, room temp;
(b) Dess-Martin periodinane, CH2Cl2 or DMF, room temp.
2-phenyl-5-trifluoroacetyl-1,3-thiazole 10 from the corre-
sponding commercially available carbaldehydes 7 and 9 as
illustrated in Scheme 2.
The modest selectivity and the instability in cells prompted
us to seek alternatives structures, aiming to obtain more
potent, selective, and stable HDAC4 inhibitors. One ap-
proach was based on structural modifications that could
address the metabolic instability issue by reducing molecular
recognition by the intracellular carbonyl reductases, thus
decreasing the subsceptibility of the TFMK group toward
reduction. In a recent paper, we reported the identification of
compounds where the amide bond was replaced by hetero-
aromatic bioisosteres such as 1,2,4- and 1,3,4-oxadiazoles and
1,3-thiazoles such as 2.19 Herein, we report a novel series of
5-aryl-2-(trifluoroacetyl)thiophenes that are potent and selec-
tive class II HDACi and display improved stability in
HCT116 cancer cells. Both classes of inhibitor have been used
to evaluate the antiproliferative effect of the inhibition of the
deacetylase activity HDAC4 by small molecules.
Results and Discussion
Analysis of the X-ray structure of HDAC4 with a
5-(trifluoroacetyl)thiophene-2-carboxamide inhibitor showed
that the central thiophene-2-carboxamide portion of the
inhibitor binds in the lipophilic channel connecting the zinc
binding site to the surface of the enzyme.22 We hypothesized
that replacement of the carboxamide moiety by a lipophilic
substituent could be tolerated and may potentially give addi-
tional binding interactions with the enzyme. First, we
explored the replacement of the carboxamide linker by aro-
matic six-membered rings. Introduction of a phenyl ring in
position 5 of the thiophene gave a modestly active compound,
6a, that displayed low micromolar activity in both HDAC4
WT and HDAC4 CAM assays with around 10-fold selectivity
against HDAC1. Compound 6a also showed submicromolar
potency against HDAC6. On the other hand, when the
aromatic substituent was moved to position 4, as in com-
pound 8, a significant loss of potency was observed against
HDAC4 and 6. With the aim to improve activity of 6a against
class II HDACs, substituents were added to the phenyl ring.
Introduction of a methoxy group in the para position as in 6b
led toanincreasein activity againstHDAC4 WT, maintaining
high selectivity against HDAC1. However, 6b was much less
active against HDAC6. When the methoxy group was mi-
grated to positions meta, 6c, or ortho, 6d, a significant loss of
activity against HDAC4 WT was observed. This fact can
be explained by a possible clash of the substituent with
the channel that connects the active site with the surface of
the enzyme.22 When we introduced an electron-withdrawing
group such as cyano in the para position of the phenyl ring
Chemistry
5-Aryl substituted 2-(trifluoroacetyl)thiophenes were pre-
pared by the method shown in Scheme 1. Reaction of
5-bromothiophene-2-carbaldehyde 3 with Ruppert’s reagent
in the presence of cesium fluoride afforded the secondary
alcohol 4.20 Subsequent oxidation of (4) with Dess-Martin
periodinane produced the trifluomethylketone 5.21 Reaction
of the key intermediate 5 with arylboronic acids under stan-
dard Suzukicross-coupling conditions gave the desired 5-aryl-
2-trifluoroacetylthiophenes 6a-l. Similar methodology as for
the addition of the trifluoromethyl group and subsequent
oxidation of the secondary alcohol intermediate was applied
to the synthesis of 5-phenyl-2-trifluoroacetylthiophene 8 and

6784 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Table 1. Enzymatic Activity of Compounds 1, 2, 6a-l, 8, and 10-13
Ontoria et al.
a IC50 values are the mean ( standard error of three independent determinations unless indicated in brackets.
as in compound 6e, a dramatic improvement in the inhibitory
activity against all the HDAC isoforms was observed, and in
particular class II HDACs. We have recently shown that
trifluoroacetyl thiophene derivatives are active site binders
with the hydrated trifluoromethylketone chelating the active
site zinc in a bidentate manner.22 Introduction of electron-
withdrawing groups in the substituents of the thiophene ring
would favor the displacement of the trifluoromethylketone
equilibra to the hydrated form, leading to an increase of
activity in the enzymatic assays. An improvement in both
activity and selectivity over class I HDAC was also observed
in the 4-acetyl analogue 6f, which displayed double digit
nanomolar activity against HDAC4 and 6 with more than
100-fold selectivity against HDAC1. Moreover, introduction
of a carboxylic acid as in 6g afforded potent class II HDAC
inhibitors although a minor loss in class I HDAC selectivity
was observed. A slight improvement of selectivity over class I
HDACwas observedwhen the carboxylicgroup was placed in

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6785
Table 2. Stability of 2-Aryl-5-(trifluoroacetyl)thiophenes in the S9 Subcellular Fraction of HCT116 Cells and in Whole HCT116 Cells
a N.D.: not determined.
the meta position, and compound 6h displayed 40-fold selec-
tivity for HDAC4 WT, and 180-fold for HDAC6, against
HDAC1. Next, we explored the introduction of heteroaro-
matic systems as substitutents of the thiophene core. The 2-
pyridyl analogue 6i was essentially inactive in the HDAC4
CAM assay and against HDAC6. However, the electron-
deficient quinoxaline-6-yl analogue 6j showed a significant
improvement in activity against class II HDACs but displayed
also submicromolar activity against HDAC1. On the other
hand, the electron-rich indol-5-yl analogue 6k was essentially
inactive against HDAC1 and 4, similarly, a loss of activity was
observed in the 2,3-dihydro-1,4-benzodioxin-6-yl analogue 6l,
although this compound showed good HDAC6 activity.
Replacement of the thiophene core by 1,3-thiazole was also
examined, as in compound 10, and this structural modifica-
tion led to an improvement of activity against all the isoforms
tested. Encouragingly, 10 displayed IC50=130 and 210 nM
against HDAC4 WT and HDAC6 and around 25-fold selec-
tivity over HDAC1.
Having identified potent and selective class II HDAC
inhibitors, we sought to identify the most stable derivatives
for use in validation studies in cells. Accordingly, the inhibi-
tors were profiled for stability in the S9 subcellular fraction of
HCT116 cells and also in HCT116 cells.18 As a reference
compound, we used our initial lead from the carboxamide
series 1, which displayed a short half-life in the S9 fraction
(t1/2=15 min) and in HCT116 cells (t1/2=3.5 h). Interestingly,
introduction of the aromatic ring with an electron-withdraw-
ing group in position para, as in compounds 6e and 6f, led to a
stabilization of the molecules, showing half-lives of more than
2 h in the S9 fraction assay and 8 h in HCT116 cells. This result
could be rationalized by the electron-withdrawing group
favoring the displacement of the trifluoromethylketone equi-
libra to the hydrated form, making it less proned to the
reduction. A further stabilization in cells was displayed by
compound 6h (t1/2=11 h) that bears a carboxylate group in
the meta position. However, compound 6j, which possesses
a heterocyclic system, was unstable and not investigated
further. Similarly, the 1,3-thiazole analogue 10, which dis-
played good potency against class II HDACs, showed a short
half-life (t1/2 =15 min), which prevented its use in cells. In
parallel, we also tested some analogues bearing a 1,2,4-
oxadiazole ring linked to the thiophene core (Table 1).19
Interestingly, compound 2 showed a remarkable stability
in the S9 subcellular fraction (t1/2 > 120 min) and also in cells
(t1/2 = 13 h). The stability of these compounds could be
affected by modification of the sulphone substituent. Elonga-
tion of the alkyl chain as in compound 11 resulted in a modest
reduction in t1/2, while the addition of a phenyl ring as in
benzyl sulfone 12 led to a further decrease of the half-life in
cells with t1/2=5.5 h. Given the fact that the validation studies
for HDAC4 would be carried out in HCT116 cells, 2 and 6h
appeared to be suitable probe compounds to evaluate the
phenotype of HDAC4 inhibition by a small molecule in
HCT116 cancer cells (Table 2).
Although compounds 2 and 6h had longer half-lives than
other analogues, these were still shorter than the duration of
the HCT116 cell proliferation assay and the histone H3 and
R-tubulin deacetylation assays in HCT116 cells, 72 and 24 h,
respectively. To ensure that sufficient quantities of our in-
hibitors were present for the duration of the experiments, the
medium was changed and fresh HDACi was added every 12 h.
Compound 2 is a potent inhibitor of HDAC4 WT (IC50=
24 nM) that displays IC50=230 nM on HDAC6 and more
than 100-fold selectivity over HDAC1 (IC50=2700 nM). This
compound showed inhibition of R-tubulin deacetylation
in HCT116 cells at submicromolar concentrations (EC50
=
640 nM) (Table 3), demonstrating that the compound entered
cells and was stable enough to inhibit HDAC6 in cells, and by
association also HDAC4. Moreover, compound 2 showed
inhibition of H3 deacetylation due to HDAC1 inhibition at
14-fold higher concentration, EC50 = 9000 nM. However,
despite inhibiting HDAC6 and by correlation HDAC4 at
submicromolar concentrations, this compound showed anti-
proliferative activity in HCT116 cells at much higher concen-
trations, EC50=12.8 μM. This is similar to the concentration

6786 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Table 3. Enzymatic and Cell-Based Activity of Compounds 2 and 6h
Ontoria et al.
HDAC1 IC50
(nM)a
HDAC4 CAM IC50
(nM)a
HDAC4 WT IC50
(nM)a
HDAC6 IC50
(nM)a
Tub-Ac EC50
(nM)b
H3 EC50
(nM)b
PRO EC50
(nM)b
compd
2
6h
2,700 ( 300
12,800 ( 1,200
780 ( 48
96 ( 5.7
24 ( 2.6
310 ( 23
230 ( 23
70 ( 3.5
640
1000
9,000
>50,000
12,800
>50,000
a IC50 values are the mean ( standard error of five independent determinations. b EC50 values are the mean of two independent determinations.
needed to inhibit deacetylation of histone H3, suggesting that
inhibition of histone deacetylation, and therefore inhibition of
HDAC1, correlates to antiproliferation effects. Similarly,
compound 6h is a potent inhibitor of HDAC4 CAM (IC50=
96 nM) that inhibits HDAC6 at comparable concentrations,
IC50=70 nM, and shows no inhibition of HDAC1 at 10 μM.
This compound showed inhibition of R-tubulin deacetylation
in HCT116 cells with EC50=1 μM; on the basis of this it is
expected that HDAC4 would also be inhibited in cells at
similar concentration. In contrast, no inhibition of histone
H3 deacetylation or inhibition of HCT116 cell growth was
observed at 50 μM.
results strongly suggest that inhibition of the catalytic activity
of HDAC4 by small molecules is not a suitable strategy for
cancer therapy.
Experimental Section
Solvents and reagents were obtained from commercial suppli-
ers and were used without further purification. Organics extracts
were dried over anhydrous sodium sulfate (Merck). Flash chro-
matography purifications were performed on Merck silica gel
(200-400 mesh) as the stationary phase or were conducted using
prepacked cartridges on a Biotage system, eluting with petroleum
ether/ethyl acetate. Purity of the compounds was determined by
analytical RP-HPLC, data was obtained by two methods on an
Acquity Waters UPLC using a flow rate of 0.5 mL/min, an
Acquity UPLC BEH C18, 1.7 μm, 2.1 mm ꢀ 50 mm column as
the stationary phase, and mobile phase comprised of MeCN þ
0.1% HCO2H (solvent A) and H2O þ 0.1% HCO2H (solvent B).
Method 1: 10% solvent A (0.2 min) to 100% solvent A (2.0 min)
then 100% solvent A (0.8 min). Method 2: 10% solvent A
(0.2 min) to 100% solvent A (2.5 min) then 100% solvent A
(0.2 min). All the compounds described in this article showed
purities higher than 95% in both analytical methods. Nuclear
magnetic resonance spectra (1H NMR) were recorded at 300 K in
the indicated solvent on Bruker AM series spectrometers operat-
ing at (reported) frequencies between 300 and 400 MHz. Chemi-
cal shifts (δ) for signals corresponding to nonexchangeable
protons (and exchangeable protons where visible) were recorded
in parts per million (ppm) relative to tetramethylsilane and were
measured using the residual solvent peak as reference. Signals are
tabulated in the order: multiplicity (s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet; br, broad, and combinations thereof);
coupling constant(s) in Hz; number of protons. HPLC-MS were
performed on a Waters Alliance 2795 apparatus, equipped with a
diode array and a ZQ mass spectrometer using an X-Terra C18
column (5 μm, 4.6 mm ꢀ 50 mm). Mobile phase comprised a
lineargradient ofbinary mixtures ofH2O containing 0.1% formic
acid (solvent A) and MeCN containing 0.1% formic acid
(solvent B). Compounds were purified by RP-HPLC (Waters
Xterra C18, 7 μm, 19 mm ꢀ 100 mm; flow: 20 mL/min; gradient:
A: H2O (þ 0.1% TFA); B: MeCN (þ 0.1% TFA); 20% A linear
to 80% A in 12 min). High resolving power accurate mass
measurement electrospray (ES) mass spectral data were acquired
by use of a Bruker Daltonics apex-Qe or Waters Synapt Q-TOF
Fourier transform ion cyclotron resonance mass spetrometer
(FT-ICR MS). External calibration was accomplished with
oligomers of polypropylene.
These results strongly suggest that inhibition of the
deacetylase activity of HDAC4 by small molecules does
not cause inhibition of HCT116 cell growth and therefore
does not recapitulate the growth inhibitory phenotype
observed in the RNAi experiments. These results suggest
that inhibition of the catalytic activity of HDAC4 by small
molecules is not a viable approach for cancer therapy.
Indeed, these results were sufficient to terminate our re-
search around this target. A possible explanation for the
apparent contradiction with the silencing experiments with
RNA can be based on the fact that, although the role of
class IIa HDACs in tissue-specific gene regulation is well-
documented,23 the contribution of the catalytic domain to
this activity is not understood. A substantial body of
evidence illustrates that class IIa HDACs exert transcrip-
tional repression through protein-protein interactions
using the N-terminal domain, and while these protein-
protein interactions would be abolished by the RNA inter-
ference, the inhibition of the catalytic activity of HDAC4 by
a small molecule may have no influence on the protein-
protein interaction.5b,c,24 Similarly, it should be remem-
bered that currently there is no cellular HDAC4 target
engagement assay, and although we have developed two
in vitro assays, these may not be a true representation of the
physiological conditions. It should also be remembered that
when comparing small-molecule versus RNAi inhibition,
we need to consider the kinetic, mechanistic, and potential
off-target differences of each treatment,25 and these subtle
differences may lead to profound discrepancies between the
outcome of both techniques.
1-(5-Bromo-2-thienyl)-2,2,2-trifluoroethanol (4). A solution of
5-bromothiophene-2-carboxaldehyde 3 (1.0 g, 5.3 mmol) and
CsF (80 mg, 0.53 mmol) in DME (20 mL) was treated with
TMSCF3 (0.92 mL, 6.3 mmol) and then stirred for 2 h at RT.
The reaction mixture was quenched by adding 3N HCl and
stirred for 30 min. The mixture was diluted with CH2Cl2, and the
organic phase was separated, washed with brine, and dried.
Evaporation of the solvent under reduced pressure gave a crude,
which was purified by silica gel chromatography (1-10%
EtOAc/petroleum ether gradient) to give 4 (1.3 g, 95%) as an
oil. 1H NMR (300 MHz, CDCl3) δ 7.00 (d, J=3.7 Hz, 1H), 6.95
(d, J=3.7 Hz, 1H), 5.20 (q, J=6.2 Hz, 1H), 3.62 (br s, 1H). MS
(ES) C6H4F3BrOS requires 260, 262; found 243, 245 (M-OH)þ.
1-(5-Bromo-2-thienyl)-2,2,2-trifluoroethanone (5). To a solu-
tion of the alcohol 4 (0.5 g, 1.92 mmol) in CH2Cl2 (10 mL) at RT
Conclusions
We have identified a novel series of 5-aryl-2-(trifluoroace-
tyl)thiophene that are potent class II HDAC inhibitors. Some
analogues from this new class of compounds inhibit HDAC4
WT, HDAC4 CAM, and HDAC6 at nanomolar concentra-
tions and display 100-fold selectivity over HDAC1. Introduc-
tion of substituents in the aryl ring or replacement of this aryl
ring by 1,2,4-oxadiazole led to compounds with improved
stability in HCT116 cells. One of these compounds, 6h,
together with a 5-(1,2,4-oxadiazole)-2-(trifluoroacetyl)thi-
ophene analogue, 2, were used to demonstrate that inhibition
of the deacetylase activity of HDAC4 by small molecules does
not cause inhibition of cell growth in HCT116 cells. These

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6787
was added Dess-Martin periodinane (0.8 g, 1.92 mmol) and the
reaction mixture was stirred for 3 h and then quenched by
pouring into aqueous NaHCO3 (saturated solution) containing
7-fold excess of Na2S2O3. The mixture is stirred for 30 min, and
then the layers were separated and the organic layer was washed
with water and brine and dried. Evaporation of the solvent gave
the crude product, which was purified by silica gel chromatog-
raphy (1-10% EtOAc/petroleum ether gradient) to give 5
(0.35 g, 70%) as an oil. 1H NMR (300 MHz, CDCl3) δ
7.74-7.68 (m, 1H), 7.23 (d, J=4.2 Hz, 1H).
1H), 8.09-8.05 (m, 1H), 7.98 (d, J=7.7 Hz, 1H), 7.90 (t, J=
7.7 Hz, 1H), 7.84 (d, J=4.2 Hz, 1H), 7.42 (dd, J=7.2, 5.5 Hz,
1H). HRMS (ESI) m/z calcd for C11H7F3NOS 258.0200, found
258.0205. RP-HPLC method 1, tR=1.23 min; method 2, tR=
1.34 min.
2,2,2-Trifluoro-1-[5-(quinoxalin-6-yl)thiophen-2-yl]ethanone
(6j). Yield: 4%. 1H NMR (400 MHz, CD3CN) δ 8.95-8.90
(m, 2H), 8.53 (d, J=1.7 Hz, 1H), 8.25-8.19 (m, 2H), 8.15-8.12
(m, 1H), 7.88 (d, J=4.3 Hz, 1H). HRMS (ESI) m/z calcd for
C14H8F3N2OS 309.0309, found 309.0308. RP-HPLC method 1,
tR=1.78 min; method 2, tR=2.02 min.
2,2,2-Trifluoro-1-[5-(1H-indol-5-yl)thiophen-2-yl]ethanone (6k).
Yield: 25%. 1H NMR (400 MHz, DMSO-d6) δ 11.42 (br s,
1H), 8.12 (s, 1H), 8.12-8.09 (m, 1H), 7.75 (d, J=4.2 Hz, 1H),
7.59 (d, J=8.6 Hz, 1H), 7.52 (d, J=8.6 Hz, 1H), 7.47-7.44 (m,
1H), 6.57-6.54 (m, 1H). HRMS (ESI) m/z calcd for
C14H9F3NOS 296.0351, found 296.0359. RP-HPLC method 1,
tR=1.91 min; method 2, tR=2.19 min.
1-[5-(2,3-Dihydro-1,4-benzodioxin-6-yl)thiophen-2-yl]-2,2,2-
trifluoroethanone (6l). Yield: 69%. 1H NMR (400 MHz, DMSO-
d6) δ 8.10-8.07 (m, 1H), 7.72 (d, J=4.2 Hz, 1H), 7.40 (d, J=2.2
Hz, 1H), 7.35 (dd, J=8.4, 2.2 Hz, 1H), 6.98 (d, J=8.4 Hz, 1H),
4.35-4.28 (m, 4H). HRMS (ESI) m/z calcd for C14H10F3O3S
315.0297, found 315.0303. RP-HPLC method 1, tR=1.98 min;
method 2, tR=2.28 min.
General Procedure for the Synthesis of 6a-l. A mixture of 5
(25 mg, 0.1 mmol) and the appropriate boronic acid (1.3 equiv)
in DMF (2 mL), together with 2N Na2CO3 aqueous solution
(0.1 mL, 0.2 mmol) was degassed with a stream of Ar for 10 min.
Pd(PPh3)4 (6 mg, 0.005 mmol) was added and the reaction
heated overnight at 90 °C. The reaction mixture was concen-
trated under reduced pressure and CH2Cl2 was added. The
organic phase was washed with 1N NaOH, brine, dried, and
concentrated under reduced pressure. The crude material was
purified by preparative RP-HPLC, and the desired fractions
were freeze-dried to give 6a-l.
2,2,2-Trifluoro-1-(5-phenylthiophen-2-yl)ethanone (6a). Yield:
1
17%. H NMR (300 MHz, DMSO-d6) δ 8.20-8.10 (m, 1H),
7.92-7.82 (m, 3H), 7.57-7.47 (m, 3H). HRMS (ESI) m/z calcd
for C12H8F3OS 257.0242, found 257.0247. RP-HPLC method 1,
tR=2.04 min; method 2, tR=2.35 min.
2,2,2-Trifluoro-1-(4-phenylthiophen-2-yl)ethanone (8). A solu-
tion of 4-phenylthiophene-2-carboxaldehyde 7 (150 mg,
2,2,2-Trifluoro-1-[5-(4-methoxyphenyl)thiophen-2-yl]ethanone
(6b). Yield: 14%. 1H NMR (400 MHz, DMSO-d6)
δ 8.12-8.08 (m, 1H), 7.84 (d, J=8.8 Hz, 2H), 7.73 (d, J=4.4
Hz, 1H), 7.08 (d, J=8.8 Hz, 2H), 3.84 (s, 3H). HRMS (ESI) m/z
calcd for C13H10F3O2S 287.0348, found 287.0354. RP-HPLC
method 1, tR=2.02 min; method 2, tR=2.33 min.
2,2,2-Trifluoro-1-[5-(3-methoxyphenyl)thiophen-2-yl]ethanone
(6c). Yield: 17%. 1H NMR (300 MHz, DMSO-d6) δ 8.16-8.12
(m, 1H), 7.87 (d, J=4.2 Hz, 1H), 7.46-7.42 (m, 2H), 7.40-7.38
(m, 1H), 7.10-7.06 (m, 1H), 3.84 (s, 3H). HRMS (ESI) m/z calcd
for C13H10F3O2S 287.0348, found 287.0354. RP-HPLC method
1, tR=2.04 min; method 2, tR=2.34 min.
2,2,2-Trifluoro-1-[5-(2-methoxyphenyl)thiophen-2-yl]ethanone
(6d). Yield: 39%. 1H NMR (400 MHz, DMSO-d6) δ 8.12-8.08
(m, 1H), 8.04-8.00 (d, J=7.8 Hz, 1H), 7.96-7.92 (d, J=4.4 Hz,
1H), 7.49 (t, J=8.4 Hz, 1H), 7.26 (d, J=8.4 Hz, 1H), 7.12 (d, J=
7.6 Hz, 1H), 4.01 (s, 3H). HRMS (ESI) m/z calcd for
C13H10F3O2S 287.0348, found 287.0353. RP-HPLC method 1,
tR=2.03 min; method 2, tR=2.33 min.
0.8 mmol) and CsF (12 mg, 0.08 mmol) in DME (10 mL) was
treated with TMSCF3 (153 μL, 0.96 mmol) and then stirred for 2
h at RT. The reaction mixture was quenched by adding 3N HCl
and stirred for 30 min. The mixture was diluted with CH2Cl2,
and the organic phase was separated and washed with brine and
dried. Evaporation of the solvent under reduced pressure gave a
crude that was purified by silica gel chromatography (10-90%
EtOAc/petroleum ether gradient) to give an oil that was diluted
in CH2Cl2 (10 mL). The resulting solution was treated with
Dess-Martin periodinane (340 mg, 0.8 mmol) and stirred for 3
h at RT. The reaction mixture was quenched by addition of
aqueous sodium thiosulfate (saturated solution) and extracted
with CH2Cl2. The combined organic extracts were dried. Eva-
poration of the solvent under reduced pressure gave a crude that
was purified by silica gel chromatography (50% EtOAc/petro-
1
leum ether) to give 8 (35 mg, 17%) as a solid. H NMR (300
MHz, CDCl3) δ 8.54-8.49 (m, 1H), 8.23-8.20 (m, 1H), 7.70 (d,
J=7.2 Hz, 2H), 7.53 (t, J=7.2 Hz, 2H), 7.43 (t, J=7.2 Hz, 1H).
HRMS (ESI) m/z calcd for C12H8F3OS 257.0242, found
257.0246. RP-HPLC method 1, tR=1.99 min; method 2, tR=
2.29 min.
4-[5-(Trifluoroacetyl)thiophen-2-yl]benzonitrile (6e). Yield:
1
26%. H NMR (400 MHz, DMSO-d6) δ 8.22-8.18 (m, 1H),
8.12-8.04 (m, 3H), 8.02-7.96 (m, 2H). HRMS (ESI) m/z calcd
for C13H7F3NOS 282.0195, found 282.0200. RP-HPLC method
1, tR=1.88 min; method 2, tR=2.15 min.
2,2,2-Trifluoro-1-(2-phenyl-1,3-thiazol-5-yl)ethanone (10). A
solution of 2-phenyl-1,3-thiazole-5-carbaldehyde 9 (50 mg,
0.26 mmol) and CsF (8 mg, 0.052 mmol) in DME (3 mL) was
treated with TMSCF3 (0.26 mL, 0.52 mmol, 2 N solution in
THF) and then stirred for 3 h at RT. The reaction mixture
was quenched by adding 1N HCl and stirred for 30 min.
The mixture was diluted with EtOAc, and the organic phase
was separated, washed with aqueous NaHCO3 (saturated
solution), and dried. Evaporation of the solvent under reduced
pressure gave a residue, which was diluted in CH2Cl2 (5 mL).
The resulting solution was treated with Dess-Martin period-
inane (165 mg, 0.39 mmol) and stirred for 2 h at RT. The
reaction mixture was quenched by addition of aqueous sodium
thiosulfate (saturated solution) and extracted with CH2Cl2.
The combined organic extracts were dried. Evaporation of the
solvent under reduced pressure gave a crude that was puri
fied by RP-HPLC (Waters SYMMETRY C18, 7 μm, 19 mm ꢀ
300 mm; flow: 20 mL/min; gradient: A: H2O (þ 0.1% TFA);
B: MeCN (þ 0.1% TFA); 60% A linear to 10% A in 14 min)
to give 10 (7.5 mg, 10%) as a solid. 1H NMR (300 MHz,
CDCl3) δ 8.60 (s, 1H), 8.07-8.03 (m, 2H), 7.59-7.47 (m, 3H).
HRMS (ESI) m/z calcd for C11H7F3NOS 258.0200, found
1-[5-(4-Acetylphenyl)thiophen-2-yl]-2,2,2-trifluoroethanone
1
(6f). Yield: 7%. H NMR (400 MHz, DMSO-d6) δ 8.22-8.18
(m, 1H), 8.10-8.02 (m, 4H), 8.02-7.98 (m, 1H), 2.63 (s, 3H).
HRMS (ESI) m/z calcd for C14H10F3O2S 299.0348, found
299.0354. RP-HPLC method 1, tR=1.89 min; method 2, tR=
2.16 min.
4-[5-(Trifluoroacetyl)thiophen-2-yl]benzoic Acid (6g). Yield:
12%. 1H NMR (400 MHz, DMSO-d6) δ 13.14 (br s, 1H),
8.20-8.16 (m, 1H), 8.05 (d, J=8.4 Hz, 2H), 8.01 (d, J=8.4
Hz, 2H), 7.95 (d, J=4.0 Hz, 1H). HRMS (ESI) m/z calcd for
C13H8F3O3S 301.0141, found 301.0147. RP-HPLC method 1,
tR=1.72 min; method 2, tR=1.96 min.
3-[5-(Trifluoroacetyl)thiophen-2-yl]benzoic Acid (6h). Yield:
12%. 1H NMR (300 MHz, DMSO-d6) δ 8.31 (br s, 1H),
8.18-8.15 (m, 1H), 8.13 (d, J = 7.4 Hz, 1H), 8.04 (d, J =
8.0 Hz, 1H), 7.94 (d, J=4.2 Hz, 1H), 7.66 (t, J=7.4 Hz, 1H).
HRMS (ESI) m/z calcd for C13H8F3O3S 301.0141, found 301.0146.
RP-HPLC method 1, tR=1.70 min; method 2, tR=1.93 min.
2,2,2-Trifluoro-1-[5-(pyridin-2-yl)thiophen-2-yl]ethanone (6i).
Yield: 4%. 1H NMR (400 MHz, CD3CN) δ 8.63 (d, J=4.6 Hz,

6788 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Ontoria et al.
258.0200. RP-HPLC method 1, tR=1.50 min; method 2, tR=
1.69 min.
F. The Histone Deacetylase Inhibitor LBH589 Is a Potent Anti-
myeloma Agent that Overcomes Drug Resistance. Cancer Res.
2006, 66, 5781–5789.
(10) Ryan, Q. C.; Headlee, D.; Acharya, M.; Sparreboom, A.; Trepel, J.
B.; Ye, J.; Figg, W. D.; Hwang, K.; Chung, E. J.; Murgo, A.;
Melillo, G.; Elsayed, Y.; Monga, M.; Kalnitskiy, M.; Zwiebel, J.;
Sausville, E. A. Phase I and Pharmacokinetic Study of MS-275, a
Histone Deacetylase Inhibitor, in Patients with Advanced and
Refractory Solid Tumors or Lymphoma. J. Clin. Oncol. 2005, 23,
3912–3922.
Acknowledgment. We thank Ottavia Cecchetti for biologi-
cal testing, Dr. Silvia Pesci for NMR spectrometry, Dr. Edith
Monteagudo and Maria V. Orsale for metabolic stability
studies, Vincenzo Pucci for performing LC-MS/MS analysis,
and Dr. Charles W. Ross and Claudio Giuliano for HRMS
determinations. This work was supported by a grant from the
MIUR.
(11) (a) Siu, L. L.; Pili, R.; Duran, I.; Messersmith, W. A.; Chen, E. X.;
Sullivan, R.; MacLean, M.; King, S.; Brown, S.; Reid, G. K.; Li, Z.;
Kalita, A. M.; Laille, E. J.; Besterman, J. M.; Martell, R. E.;
Carducci, M. A. Phase I Study of MGCD0103 Given as a Three-
Times-per-Week Oral Dose in Patients with Advanced Solid
Tumors. J. Clin. Oncol. 2008, 26, 1940–1947. (b) Kell, J. Drug
Evaluation: MGCD-0103, a Histone Deacetylase Inhibitor for the
Treatment of Cancer. Curr. Opin. Invest. Drugs 2007, 8, 485–492.
(12) (a) Byrd, J. C.; Marcucci, G.; Parthum, M. R.; Xiao, J. J.; Klisovic,
R. B.; Moran, M.; Lin, T. S.; Liu, S.; Sklenar, A. R.; Davis, M. E.;
Lucas, D. M.; Fisher, B.; Shank, R.; Tejaswi, S. L.; Binkley, P.;
Wright, J.; Chan, K. K.; Grever, M. R. A Phase 1 and Pharmaco-
dynamic Study of Depsipeptide (FK228) in Chronic Lymphocytic
Leukemia and Acute Myeloid Leukemia. Blood 2005, 105, 959–
967. (b) Piekarz, R. L.; Frye, A. R.; Wright, J. J.; Seth, M.; Steinberg, S.
M.; Liewehr, D. J.; Rosing, D. R.; Sachdev, V.; Fojo, T.; Bates, S. E.
Cardiac Studies in Patients Treated with Depsipeptide, FK228, in a
Phase II Trial for T-Cell Lymphoma. Clin. Cancer Res. 2006, 12, 3762–
3773.
Supporting Information Available: Enzyme and cell-based
assays conditions, stability experiments conditions. Experi-
mental procedures and analytical data for compounds 2, 11,
12, and 13. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) (a) Brittain, D.; Weinmann, H.; Ottow, E. Recent Advances in the
Medicinal Chemistry of Histone Deacetylase Inhibitors. Annu.
Rep. Med. Chem. 2007, 42, 337–348. (b) Minucci, S.; Pelicci, P. G.
Histone Deacetylase Inhibitors and the Promise of Epigenetic
(and More) Treatments for Cancer. Nat. Rev. Cancer 2006, 6, 38–51.
(c) Rodriquez, M.; Aquino, M.; Bruno, I.; De Martino, G.;
Taddei, M.; Gomez-Paloma, L. Chemistry and Biology of Chromatin
Remodeling Agents: State of Art and Future Perspectives of HDAC
Inhibitors. Curr. Med. Chem. 2006, 13, 1119–1139. (d) Jones, P.;
Steink€uhler, C. From Natural Products to Small Molecule Ketone
Histone Deacetylase Inhibitors: Development of New Class Specific
Agents. Curr. Pharm. Des. 2008, 14, 545–561.
(2) Mai, A.; Massa, S.; Pezzi, R.; Simeoni, S.; Rotili, D.; Nebbioso, A.;
Scognamiglio, A.; Altucci, L.; Loidl, P.; Brosch, G. Class II (IIa)-
Selective Histone Deacetylase Inhibitors. 1. Synthesis and Biolo-
gical Evaluation of Novel (Aryloxopropenyl)pyrrolyl Hydroxya-
mides. J. Med. Chem. 2005, 48, 3344–3353.
(13) Itoh, Y.; Suzuki, T.; Miyata, N. Isoform-Selective Histone Deace-
tylase Inhibitors. Curr. Pharm. Des. 2008, 14, 529–544.
(14) Cadot, B.; Brunetti, M.; Coppari, S.; Fedeli, S.; Dello Russo, C.
€
Gallinari, P.; De Francesco, R.; Steinkuhler, C.; Filocamo, G. Loss
of HDAC4 Causes Segregation Defects During Mitosis of p53-
Deficient Human Tumor Cells. Cancer Res. 2009, 69, 6074–6082.
(15) (a) Lahm, A.; Paolini, C.; Pallaoro, M.; Nardi, M. C.; Jones, P.;
Neddermann, P.; Sambucini, S.; Bottomley, M. J.; Lo Surdo, P.;
ꢀ
€
Carfi, A.; Koch, U.; De Francesco, R.; Steinkuhler, C.; Gallinari,
P. Unraveling the Hidden Catalytic Activity of Vertebrate Class IIa
Histone Deacetylases. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
17335–17340. (b) Jones, P.; Altamura, S.; De Francesco, R.; Gallinari,
P.; Lahm, A.; Neddermann, P.; Rowley, M.; Serafini, S.; Steink€uhler, C.
Probing the Elusive Catalytic Activity of Vertebrate Class IIa Histone
Deacetylases. Bioorg. Med. Chem. Lett. 2008, 18, 1814–1819.
(3) De Ruijter, A. J.; van Gennip, A. H.; Caron, H. N.; Kemp, S.;
van Kuilenburg, A. B. Histone Deacetylases (HDACs): Characteriza-
tion of the Classical HDAC Family. Biochem. J. 2003, 370, 737–
749.
(4) Michan, S.; Sinclair, D. Sirtuins in Mammals: Insights into their
(16) 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.
Biological Function. Biochem. J. 2007, 404, 1–13.
(5) (a) Guo, L.; Han, A.; Bates, D. L.; Cao, J.; Chen, L. Crystal
Structure of a Conserved N-Terminal Domain of Histone Deace-
tylase 4 Reveals Functional Insights into Glutamine-Rich Do-
mains. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 4297–4302. (b)
Verdin, E.; Dequiedt, F.; Kasler, H. G. Class II Histone Deacetylases:
Versatile Regulators. Trends Genet. 2003, 19, 286–293. (c) Yang, X. J.;
Gregoire, S. Class II Histone Deacetylases: From Sequence to Function,
Regulation, and Clinical Implication. Mol. Cell. Biol. 2005, 25, 2873–
2884.
ꢀ
(17) Jones, P.; Bottomley, M. J.; Carfi, A.; Cecchetti, O.; Ferrigno, F.;
Lo Surdo, P.; Ontoria, J. M.; Rowley, M.; Scarpelli, R.; Schultz-
€
Fademrecht, C.; Steinkuhler, C. 2-Trifluoroacetylthiophenes, a
Novel Series of Potent and Selective Class II Histone Deacetylase
Inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 3456–3461.
(18) Scarpelli, R.; Di Marco, A.; Ferrigno, F.; Laufer, R.; Marcucci, I.;
€
Muraglia, E.; Ontoria, J. M.; Rowley, M.; Serafini, S.; Steinkuhler,
C.; Jones, P. Studies of the Metabolic Stability in Cells of
5-(Trifluoroacetyl)thiophene-2-carboxamides and Identification
of More Stable Class II Histone Deacetylase (HDAC) Inhibitors.
Bioorg. Med. Chem. Lett. 2008, 18, 6078–6082.
(6) Voelter-Mahlknecht, S.; Ho, A. D.; Mahlknecht, U. Chromosomal
Organization and Localization of the Novel Class IV Human
Histone Deacetylase 11 Gene. Int. J. Mol. Med. 2005, 16, 589–598.
(7) (a) Kelly, W. K.; Richon, V. M.; O’Connor, O.; Curley, T.;
McGregor-Curtelli, B.; Tong, W.; Klang, M.; Schwartz, L.; Ri-
chardson, S.; Rosa, E.; Drobnjak, M.; Cordon-Cordo, C.; Chiao, J.
H.; Rifkind, R.; Marks, P. A.; Scher, H. Phase I Clinical Trial of
Histone Deacetylase Inhibitor: Suberoylanilide Hydroxamic Acid
Administered Intravenously. Clin. Cancer Res. 2003, 9, 3578–3588.
(b) Kelly, W. K.; O'Connor, O.; Krug, L. M.; Chiao, J. H.; Heaney, M.;
Curley, T.; McGregor-Cuterlli, B.; Tong, W.; Secrist, J. P.; Schwartz, L.;
Richardson, S.; Olgac, S.; Marks, P. A.; Scher, H.; Richon, V. M. Phase I
Study of an Oral Histone Deacetylase Inhibitor, Suberoylanilide Hydro-
xamic Acid, in Patients with Advanced Cancer. J. Clin. Oncol. 2005,
23, 3923–3931. (c) Duvic, M.; Talpur, R.; Ni, X.; Zhang, C.; Hazarika,
P.; Kelly, C.; Chiao, J. H.; Reilly, J. F.; Ricker, J. L.; Richon, V. M.;
Frankel, S. R. Phase 2 Trial of Oral Vorinostat (Suberoylanilide
Hydroxamic Acid, SAHA) for Refractory Cutaneous T-Cell Lymphoma
(CTCL). Blood 2007, 109, 31–39.
(19) Muraglia, E.; Altamura, S.; Branca, D.; Cecchetti, O.; Ferrigno, F.;
Orsale, M. V.; Palumbi, M. C.; Rowley, M.; Scarpelli, R.;
€
Steinkuhler, C.; Jones, P. 2-Trifluoroacetylthiophene Oxadiazoles
as Potent and Selective Class II Human Histone Deacetylase
Inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 6083–6087.
(20) Singh, R. P.; Chakraborty, D.; Shreeve, J. Nucleophilic Trifluor-
omethylation and Difluorination of Substituted Aromatic Alde-
hydes with Ruppert’s and Deoxofluor Reagents. J. Fluorine Chem.
2001, 111, 153–160.
(21) Linderman, R. J.; Graves, D. M. Oxidation of Fluoroalkyl-Sub-
stituted Carbinols by the Dess-Martin Reagent. J. Org. Chem.
1989, 54, 661–668.
(22) Bottomley, M. J.; Lo Surdo, P.; Di Giovine, P.; Cirillo, A.;
Scarpelli, R.; Ferrigno, F.; Jones, P.; Neddermann, P.; De Fran-
ꢀ
€
cesco, R.; Steinkuhler, C.; Gallinari, P.; Carfi, A. Structural and
Functional Analysis of the Human HDAC4 Catalytic Domain
Reveals a Regulatory Structural Zinc-Binding Domain. J. Biol.
Chem. 2008, 283, 26694–26704.
(8) Giles, F.; Fischer, T.; Cortes, J.; Garcia-Manero, G.; Beck, J.;
Ravandi, F.; Masson, E.; Rae, P.; Laird, G.; Sharma, S.; Kantar-
jian, H.; Dugan, M.; Albitar, M.; Bhalla, K. A Phase I Study of
Intravenous LBH589, a Novel Cinnamic Hydroxamic Acid Ana-
logue Histone Deacetylase Inhibitor, in Patients with Refractory
Hematologic Malignancies. Clin. Cancer Res. 2006, 12, 4628–
4635.
(23) (a) Zhang, C. L.; McKinsey, T. A.; Chang, S.; Antos, C. L.; Hill, J.
A.; Olson, E. N. Class II Histone Deacetylases Act as Signal-
Responsive Repressors of Cardiac Hypertrophy. Cell 2002, 110,
479–488. (b) Vega, R. B.; Matsuda, K.; Oh, J.; Barbosa, A. C.; Yang, X.;
Meadows, E.; McAnally, J.; Pomajzl, C.; Shelton, J. M.; Richardson, J.
A.; Karsenty, G.; Olson, E. N. Histone Deacetylase 4 Controls Chon-
ꢀ
ꢁ
(9) Maiso, P.; Carvajal-Vergara, X.; Ocio, E. M.; Lopez-Perez, R.;
ꢁ
Mateo, G.; Gutierrez, N.; Atadja, P.; Pandiella, A.; San Miguel, J.

Article
drocyte Hypertrophy During Skeletogenesis. Cell 2004, 119, 555–566.
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6789
Histone Deacetylase, Regulates Nur77 Transcription and TCR-
Mediated Apoptosis. Immunity 2003, 18, 687–698.
(c) Chang, S.; McKinsey, T. A.; Zhang, C. L.; Richardson, J. A.; Hill, J.
A.; Olson, E. N. Histone Deacetylases 5 and 9 Govern Responsiveness
of the Heart to a Subset of Stress Signals and Play Redundant Roles in
Heart Development. Mol. Cell. Biol. 2004, 24, 8467–8476. (d)
Dequiedt, F.; Kasler, H.; Fischle, W.; Kiermer, V.; Weinstein, M.;
Herndier, B. G.; Verdin, E. HDAC7, a Thymus-Specific Class II
(24) Martin, M.; Kettmann, R.; Dequiedt, F. Class IIa Histone Deace-
tylases: Regulating the Regulators. Oncogene 2007, 26, 5450–5467.
(25) Fitzgerald, K. RNAi versus Small Molecules: Different Mechan-
isms and Specificities can lead to Different Outcomes. Curr. Opin.
Drug Discovery Dev. 2005, 8, 557–566.