Conformational refinement of hydroxamate-based histone deacetylase inhibitors and exploration of 3-piperidin-3-ylindole analogues of dacinostat (LAQ824)

Article abstract of DOI:10.1021/jm100007m

Inspired by natural product HDAC inhibitors, we prepared a series of conformationally restrained HDAC inhibitors based on the hydroxamic acid dacinostat (LAQ824, 7). Several scaffolds with improved biochemical and cellular potency, as well as attenuated hERG inhibition, were identified, suggesting that the introduction of molecular rigidity is a viable strategy to enhance HDAC binding and mitigate hERG liability. Further SAR studies around a 3-piperidin-3-ylindole moiety resulted in the discovery of compound 30, for which a unique conformation was speculated to contribute to overcoming increased lipophilicity and attenuating hERG binding. Separation of racemate 30 afforded 32, the R enantiomer, which demonstrated improved potency in both enzyme and cellular assays compared to dacinostat.

Full text of DOI:10.1021/jm100007m

2952 J. Med. Chem. 2010, 53, 2952–2963
DOI: 10.1021/jm100007m
Conformational Refinement of Hydroxamate-Based Histone Deacetylase Inhibitors and Exploration of
3-Piperidin-3-ylindole Analogues of Dacinostat (LAQ824)
†
†
†
†
‡
‡
Young Shin Cho,*,† Lewis Whitehead, Jianke Li, Christine H.-T. Chen, Lei Jiang, Markus Vogtle, Eric Francotte,
Paul Richert,‡ Trixie Wagner,‡ Martin Traebert,‡ Qiang Lu,† Xueying Cao,† Berengere Dumotier,‡ Jasna Fejzo,†
Srinivasan Rajan,† Ping Wang,† Yan Yan-Neale,† Wenlin Shao,† Peter Atadja,† and Michael Shultz†
€
†Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, and ‡Werk Klybeck,
Klybeckstrasse 141, CH-4002 Basel, Switzerland
Received January 6, 2010
Inspired by natural product HDAC inhibitors, we prepared a series of conformationally restrained
HDAC inhibitors based on the hydroxamic acid dacinostat (LAQ824, 7). Several scaffolds with
improved biochemical and cellular potency, as well as attenuated hERG inhibition, were identified,
suggesting that the introduction of molecular rigidity is a viable strategy to enhance HDAC binding and
mitigate hERG liability. Further SAR studies around a 3-piperidin-3-ylindole moiety resulted in the
discovery of compound 30, for which a unique conformation was speculated to contribute to over-
coming increased lipophilicity and attenuating hERG binding. Separation of racemate 30 afforded 32,
the R enantiomer, which demonstrated improved potency in both enzyme and cellular assays compared
to dacinostat.
Histone acetylation/deacetylation is one of the few enzy-
matic activities implicated in the unpacking/packing of
chromatin and subsequent regulation of gene transcrip-
tion.1 Histone deacetylases (HDACs) catalyze the removal
of acetyl groups from lysine residues at the N-terminal tails
of histones, which results in condensation of chromosomal
DNA and transcriptional repression.2 These enzymes also
regulate the acetylation levels of non-histone proteins
involved in cell growth and survival pathways such as
R-tubulin, hsp90, and p53.3 There are four identified classes
of HDACs (classes I-IV), which are characterized by their
different substrate specificities and subcellular localiza-
tion.4 Many research groups are active in elucidating the
physiological role of different HDACs in cells, and there
has been great effort to develop HDAC inhibitors as novel
cancer therapeutics.5
Small molecule inhibitors of HDACs have been identified
both synthetically and from natural sources, and several
inhibitors are at various stages of clinical development. On
the basis of their chemical structures, HDAC inhibitors can
be divided into several different chemical classes including
hydroxamic acids, benzamides, cyclic peptides, and aliphatic
acids. The hydroxamic acidvorinostat (SAHA(1), Figure 1) is
the first HDAC inhibitor approved by the FDA for the
treatment of advanced cutaneous T-cell lymphoma (CTCL),6
and ITF-2357 (2) and belinostat (PXD-101 (3)) are in phase II
clinical trials for the treatment of hematological tumors.
Benzamide-based HDAC inhibitors MS-275 (4) and MGCD-
0103 (5) are in phase II clinical trials against a variety of
hematological and solid tumors. Naturally occurring HDAC
inhibitor romidepsin (FK-228 (6)),7 a bicyclic depsipeptide, has
recently been approved by FDA for treatment of patients with
CTCL. Our initial efforts in this area culminated in the
discovery of the hydroxamic acid dacinostat (LAQ824, 7).8
Herein, we report our strategy to identify analogues with
improved potency and safety profiles and progress made in
the 3-piperidin-3-ylindole series.
While examining different classes of HDAC inhibitors, we
were intrigued by the HDAC inhibitory profile of the natural
products such as romidepsin (6), spiruchostatin A (8), and
apicidin (10) (Figures 1 and 2). In 2004, Yurek-George and
co-workers reported the total synthesis of spiruchostatin A
(8), a potent HDAC inhibitor that causes the accumulation of
acetylated histone-H4.9 Like romidepsin (6),10 spiruchostatin
A is presumed to be reduced intracellularly to release a zinc-
binding thiol. Reduced spiruchostatin A is a potent inhibitor
of HDAC1 (IC50 = 0.62 nM) and inhibits the growth of
several cancer cell lines with IC50 values of 1-10 nM. How-
ever, epi-spiruchostatin A (9), prepared by the same group,
was inactive in the same cancer cell lines even at 10 μM. (S)-
Stereochemistry at C(1) of spiruchostatin A appears to be
critical to garner a favorable interaction between the cyclic
depsipeptide “cap” moiety and the amino acid residues
around the rim of HDAC1’s binding channel. Such impact
of a single stereocenter on potency was also demonstrated
during the medicinal chemistry campaigns based on apicidin
(10). In this case, chirality at C(12) was critical to obtain an
optimal spatial relationship between the “cap” and the rest of
the molecule, thereby promoting HDACinhibition.11 Clearly,
these natural product HDAC inhibitors utilize intrinsic con-
formational constraints to ensure optimal enzyme binding.
We recognized several structural similarities between apicidin
and our synthetic HDAC inhibitor, dacinostat (7), such as
indole moieties and zinc-binding carbonyl moieties separated
bydifferentspacers. Anin silicoalignment ofcomputationally
*To whom correspondence should be addressed. Phone: 617-871-
3495. Fax: 617-871-4081. E-mail: youngshin.cho@novartis.com.
r
pubs.acs.org/jmc
Published on Web 03/05/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2953
Figure 3. Apicidin (10, green) crystal structure superimposed with
dacinostat (7, gray) using FieldAlign.13
Figure 1. Structures of small molecule HDAC inhibitors.
spatial relationships between the indole of the “cap” and the
HDAC “channel binder”. To this end, the flexible dacinostat
spacer was rigidified (11-18) to access subtle conformational
changes in analogue structures (Figure 5). In addition, we
expected to improve HDAC1 binding affinity by reducing
rotational entropy. For direct comparison among these con-
formationally constrained analogues, we synthesized a series
of compounds, albeit as racemates, with the “cap” fixed as
2-methylindole.
Chemistry
Compounds 11-13 were assembled from the secondary
amines 19-21, which were prepared following procedures
outlined in Scheme 1. Reaction of 2-methylindole with
N-benzyl-3-piperidone under acidic conditions resulted in
a regioisomeric mixture of condensation products which
was subsequently reduced to piperidinylindole 19 under
hydrogenation conditions.15 2-Methylindole and malei-
mide were heated to reflux in glacial acetic acid to provide
2,5-pyrrolidinedione, which was then converted to pyrroli-
dinylindole 20 via LiAlH4 reduction.16 Synthesis of 21
commenced with the condensation reaction of the magne-
sium salt of 2-methylindole with the acid chloride of N-Boc-
2-piperidinecarboxylic acid to afford a 3-ketoindole; these
conditions also removed the Boc protecting group. Reduc-
tion with LiAlH4 gave piperidinylmethylindole 21.17 The
amines 19 and 21 were converted to the final products 11
and 13, respectively, via reductive amination with (E)-3-(4-
formylphenyl)acrylic acid methyl ester 22 and subsequent
conversion of the methyl esters to the hydroxamic acids.8
Alternatively, compound 12 was prepared via reaction of
the amine 20 with 4-bromobenzyl bromide followed by
a Heck cross-coupling with methyl acrylate18 and hydro-
xamic acid conversion. Synthesis of 14 started with reductive
amination between the known compounds (2-methyl-
1H-indol-3-yl)acetaldehyde 23 and 2-(4-bromophenyl)-
pyrrolidine 24 (Scheme 1).19 Heck reaction followed by
treatment of the resulting methyl ester with aqueous
hydroxyamine solution in the presence of sodium methoxide
provided 14. Syntheses of compounds 15-18 were reported
previously.20
Figure 2. Structures of spiruchostatin A (8), epi-spiruchostatin A
(9), and apicidin (10).
determined dacinostat conformations with a published X-ray
crystal structureof apicidin12 showed good overlap(Figure3),
suggesting that the binding mode of natural product HDAC
inhibitors might be mimicked by restraining the conforma-
tional flexibility of dacinostat.
To systematically incorporate conformational constraint
into dacinostat (7), we generated an HDAC1 homology
model based on an in-house HDAC8 X-ray crystal struc-
ture.14 When dacinostat (7) was docked into the HDAC1
enzyme, the “cap” of 7, roughly defined as the aminoalkyl-
indole region, was found to interact with the rim of the
HDAC binding channel and the hydrophobic surface
(Figure 4). The benzene ring of 7 docked in proximity to
the three phenyl rings of residues Phe150, Tyr204, and
Phe205 to form favorable hydrophobic interactions. This
conformation also allowed the protonated benzylamine to
contact Asp99 through a salt bridge. The hydroxyethyl unit
(-CH2CH2OH) was found to interact with either the
primary or secondary hydration shell of Asp99 and Glu98
and with their methylene groups. The indole moiety was
localized above Phe205, providing another hydrophobic
contact. This indicated to us that substitutions off the
indole ring might be used to explore additional interactions
with the HDAC1 hydrophobic surface.
On the basis of this model, we probed the interaction of
dacinostat-like analogues with HDACs by introducing different

2954 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Cho et al.
Figure 4. Dacinostat (7) docked within an HDAC1 homology model: (left) side view; (right) top view.
Results and Discussion
The biochemical activity of each compound was assessed
using purified HDAC1.21a The antiproliferative activity of
these compounds was determined in the HCT116 human
colon cancer cell line and the H1299 human lung cancer
line.21a As shown in Table 1, the introduction of conforma-
tional restrictions to dacinostat (7) has varying effects on
biological activity. 3-Piperidin-3-ylindole 11 and 3-pyrrolidin-
3-ylindole 12 demonstrate improved biochemical and cellular
potency over dacinostat (7), while 3-piperidin-2-ylmethylin-
dole 13 exhibits a slight loss of potency. Enzymatic and
cellular activity also improves when the pyrrolidine is shifted
further from the indole, as seen in 14. Compounds 15-18, in
which the amine is fused to the benzene ring, do not display
any improvements in potency in the enzymatic or cellular
assays.
Compounds 11 and 18 were docked in our HDAC1 homol-
ogy model in an attempt to understand the wide range of
in vitro activity observed (Figure 6). We speculate that the
improved potency of 3-piperidin-3-ylindole 11 over dacino-
stat 7 results from the loss of rotational degrees of freedom by
rigidifying the linker region while retaining most of the
favorable interactions with HDAC1 observed with 7. The
model also suggests that the same key interactions are main-
tained in docking poses of both R and S enantiomers,
although the R enantiomer seems to be more optimal in
HDAC binding on visual inspection. On the other hand, 18,
which conformationally locks the amine with the benzene
ring, appears to introduce steric hindrance at the edge of the
HDAC1channel. Thisresults in a shiftof the benzenering and
loss of directionality of the protonated amine to the Asp99salt
bridge, causing the relative loss in potency.
The presence of a basic tertiary amine and two flanking
aromatic rings in this compound series was speculated to be
Figure 5. Rigidified analogues.
the potential source of the observed hERG binding.22
Blockage of the hERG ion channel has been implicated in
drug-induced QT interval prolongation, which may lead to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2955
Scheme 1a
Table 1. IC50 Values for Compounds in the HDAC1 Enzyme Assay,
HCT116 and H1299 Cellular Assays, and the Patch Clamp Assaya
IC50 (nM)
HDAC1b
HCT116c
H1299c
Q-patch clampd
7
9.0 ( 1.4
5.0 ( 1.2
2.8 ( 0.4
24 ( 1
3 ( 0
27 ( 4
41 ( 1
13 ( 1
76 ( 4
13 ( 2
5.6
3.2
161 ( 7
31 ( 1
12200
27600
13400
9100
11
12
13
14
15
16
17
18
14 ( 10
153 ( 1
4.3 ( 0.7
232 ( 127
725 ( 52
165 ( 13
>1,000
27 ( 1
1
19400
4800
41 ( 12
119 ( 18
42 ( 1
545 ( 25
14800
2700
3200
a See Experimental Section and Supporting Information for detailed
descriptions of each assay. b Results expressed as the mean ( standard
deviation of two to five separate IC50 determinations. For each deter-
mination, a single concentration-inhibition curve was obtained to
afford an IC50 value. c Results expressed as the mean ( standard
deviation of two separate IC50 determinations. For each determination,
concentration-inhibition curves were obtained in triplicate and then
averaged to afford a single IC50 curve with a g95% confidence interval.
d Results are from single IC50 determination. For each determination,
concentration-inhibition curves were obtained in triplicate and then
averaged to afford a single IC50 curve with a g95% confidence interval.
those results will be reported in due course. Herein, we focus
our discussion on the 3-piperidin-3-ylindole series based
on 11.
Several analogues of 11 were prepared to understand the
effect of substitution on the C(2) position of the indole.21a
Biochemical and cell viability assay results suggest that
a hydrophobic substituent at C(2) is required to improve
potency (Table 2);8,25 however, the increasing lipophilicity
also increases hERG inhibition. At this juncture, we intro-
duced a fluorine substituent on the benzene ring in an attempt
to modulate the basicity and/or molecular conformation,
thereby attenuating the hERG affinity.26 Despite reduced
pKa values, the incorporation of fluorine resulted in enhanced
hERG inhibitory activity in all analogues of the 3-piperidin-3-
ylindole series, with the exception of compound 30 (Table 3).
This suggests that increased lipophilicity resulting from fluor-
ine substitution may have a dominant role in controlling
hERG activity in this series. Compounds 28 and 29 display
increased hERG inhibition as well as loss of potency in
HDAC enzyme and cellular assays compared to the non-
fluorinated analogues 25 and 26, respectively. Interestingly,
compound 30 exhibits about a 2-fold reduction in hERG
inhibition compared to 27; however, we also observe a 3-fold
decrease in HDAC enzyme and cell antiproliferation activity.
Addition of chlorine instead of fluorine results in more potent
hERG inhibition, as illustrated by 31. We surmised that
unique conformational constraints in 30 might be the origin
of its reduced hERG affinity, and therefore, we performed
density functional calculations on the truncated structure of
30 (Figure 7a).27 The ortho-fluoro substituent in 30 is pro-
posed to be in proximity to the protonated benzylic amine
a (a) (1) 2-Methyl-1H-indole, aqueous H3PO4, AcOH, 100 °C, 72%;
(2) Pd(OH)2/C, H2, MeOH, 83%; (b) (1) 2-methyl-1H-indole, AcOH,
reflux; (2) LAH, THF, 31% over two steps; (c) (1) oxalyl chloride,
CH2Cl2; (2) 2-methyl-1H-indole, EtMgBr, benzene; (3) LAH, THF,
15% over three steps; (d) (1) TiCl4, NaBH3CN, NEt3, CH2Cl2, 50%;
(2) Pd2 (dba)3, HP(tBu)3 BF4, methyl acrylate, Cy2NMe, dioxane, 97%;
3
(3) aqueous NH2OH, NaOMe, MeOH, 0 °C, 65%.
fatal torsades de pointes.23 Structurally, dacinostat (7)
satisfies all three key determinants of hERG blockers as
described by Farid and co-workers: (1) substituents form
extensive ring stacking and/or hydrophobic interactions
with the crown-shaped hydrophobic interior of the pore;
(2) a basic center interacts with the propeller-shaped hydro-
philic field within the pore; (3) the molecule has an ability to
assume multiple poses when bound to hERG under the
constraints of points 1 and 2.21b,24 The hERG channel IC50
values of 11-18 were determined in an automated electro-
physiology assay (Q-patch clamp assay).21c 11 and 14
demonstrated a trend of increasing potency against HDAC
and reduced hERG inhibition relative to dacinostat. The
difference in hERG inhibitory activity between 7 and 11
cannot be explained by either decreased lipophilicity
(cLogP of 2.1 for 7 and 3.8 for 11) or reduced basicity
(measured pKa 21c of 7.5 for 7 and 7.8 for 11); therefore, it is
plausible that our strategy of introducing rigidity into the
dacinostat framework affected the hERG profile by redu-
cing the number of possible binding poses in the hERG
channel. Favorable effects of rigidification on HDAC
potency and hERG inhibition in several scaffolds prompted
us to initiate chemistry efforts to investigate the structure-
activity relationships around each constrained spacer, and
˚
and methylene hydrogens on the piperidine (X = 1.7 A, Y =
˚
2.6 A, respectively). This conformation creates a steric shield
around the protonated benzylic amine, which may mitigate
external influence, such as solvent or the hERG channel’s
preferred region for accommodating a cationic center. To test
our hypothesis, NMR experiments (19F/1H 2D HOSEY) with
the hydrogen chloride salt of compound 30 in DMSO-d6 were
performed (Figure 7b).21c Indeed, we observed spatial proxi-
mity of the fluorine and several hydrogens predicted by the
model.21d

2956 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Cho et al.
Figure 6. Compounds 11 (gray R, pink S) and 18 (gray) aligned with dacinostat (7, blue) in an HDAC1 homology model.
Figure 7. (a) Density functional minimized geometries of 30. N-Hydroxyacrylamide functional group was removed to facilitate in silico
calculation. (b) Observed “through-space” cross-peaks from the single 19F atom in compound 30 to the certain protons (13, 14, 15, 20, and 23)
in the 19F/1H 2D HOSEY NMR spectrum.
Table 2. IC50 Values for Compounds in the HDAC1 Enzyme Assay,
HCT116 and H1299 Cellular Assays, and the Patch Clamp Assay, pKa of
the Benzylic Amine,21c and cLogPa
Table 3. IC50 Values for Compounds in the HDAC1 Enzyme Assay,
HCT116 and H1299 Cellular Assays, and the Patch Clamp Assay, pKa of
the Benzylic Amine,21c and cLogPa
IC50 (nM)
IC50 (nM)
R, X HDAC1b HCT 116c H 1299c Q-patch clampd pKa cLogP
R
HDAC1b HCT 116c H 1299c Q-patch clampd pKa cLogP
28 H, F 136 ( 31 332 ( 82 3520
15300
19300
30000
9900
nde 3.5
7.4 5.3
7.3 5.2
7.4 5.8
25 H 11 ( 2
11 Me 5.0 ( 1.2 5.6
26 Ph 11 ( 3 6.9 ( 1.1 31 ( 1
27 tBu 3.0 ( 1.4 0.9
6.3 ( 1
25 ( 10 141 ( 74
29300
27600
28400
13500
7.3
7.8
7.7
7.8
3.3
3.8
5.1
5.1
29 Ph, F 16 ( 1
30 Bu, F 10 ( 1
11 ( 1
3.8
106 ( 26
20 ( 5
t
31 ( 1
t
31 Bu, Cl 19 ( 5
5.2 ( 1.0 60 ( 20
a See Experimental Section and Supporting Information for detailed
descriptions of each assay. b Results expressed as the mean ( standard
deviation of two to five separate IC50 determinations. For each deter-
mination, a single concentration-inhibition curve was obtained to
afford an IC50 value. c Results expressed as the mean ( standard
deviation of two separate IC50 determinations. For each determination,
concentration-inhibition curves were obtained in triplicate and then
averaged to afford a single IC50 curve with a g95% confidence interval.
d Results are from single IC50 determination. For each determination,
concentration-inhibition curves were obtained in triplicate and then
averaged to afford a single IC50 curve with a g95% confidence interval.
e nd: not determined.
a See Experimental Section and Supporting Information for detailed
descriptions of each assay. b Results expressed as the mean ( standard
deviation of two to five separate IC50 determinations. For each deter-
mination, a single concentration-inhibition curve was obtained to
afford an IC50 value. c Results expressed as the mean ( standard
deviation of two separate IC50 determinations. For each determination,
concentration-inhibition curves were obtained in triplicate and then
averaged to afford a single IC50 curve with a g95% confidence interval.
d Results are from single IC50 determination. For each determination,
concentration-inhibition curves were obtained in triplicate and then
averaged to afford a single IC50 curve with a g95% confidence interval.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2957
Table 4. IC50 Values for Compounds in the HDAC1 Enzyme Assay and in the HCT116 and H1299 Cellular Assays and % Inhibition in the Manual
Patch Clamp Assaya
a See Experimental Section and Supporting Information for detailed descriptions of each assay. b Results expressed as the mean ( standard
deviation of two to five separate IC50 determinations. For each determination, a single concentration-inhibition curve was obtained to afford an IC50
value. c Results expressed as the mean ( standard deviation of two separate IC50 determinations. For each determination, concentration-inhibition
curves were obtained in triplicate and then averaged to afford a single IC50 curve with a g95% confidence interval. d Values are mean ( SEM for n =
3 (32) and 4 (33).
Having identified a racemic HDAC inhibitor 30 that
demonstrates an improved balance of antitumor activity
and hERG inhibition, we turned our attention to preparing
its enantiomers. The R and S enantiomers of 30, 32, and 33
were prepared from chiral intermediates obtained by chro-
matographic resolution, using simulated moving bed (SMB)
technology and a proprietary chiral stationary phase.21a,28
Stereochemistry was determined by single-crystal X-ray ana-
lysis of one of the intermediates.21a,c The more potent HDAC
inhibitor of the two enantiomers, 32, turned out to also be less
active in the hERG manual patch clamp assay (Table 4).21c,29
Such separation of hERG activity between enantiomers has
been observed previously.30 Itwas alsoobserved that32shows
significantly improved cellular potency as measured by its
inhibitory effect on cancer cell proliferation, even though its
increased activity in the enzyme assay is less pronounced.31
Compared to the parent compound dacinostat (7), 32 is 2-fold
more potent at HDAC1 inhibition and 5-fold more potent
against both HCT116 and H1299 cell lines. Isoform selectivity
of 32 and 33 was also examined, proving both enantiomers
to be pan-HDAC inhibitors with weaker activity against
HDAC6 and HDAC8.21e
expression system. HDAC activity was measured in a fluores-
cent assay in which deacetylation of the substrate, bis-Boc-
(Ac)Lys-rhodamine 110, generates a fluorophore that can be
detected on a fluorometric plate reader.
Monolayer Cell Proliferation Assay. Cells were plated at
5000-10000 cells per well in 96-well plates and treated with
eight serial compound dilutions. Cell viability following 72 h of
compound treatment was measured using the CellTiter-Glo or
MTS assay. Assays were performed following the manufacture’s
protocol. XLfit 4 was used for plotting of the growth curves and
calculation of IC50 values.
Chemistry. All nonaqueous reactions were carried out under
a nitrogen atmosphere unless otherwise noted. All solvents
employed were commercially available “anhydrous” grade,
and reagents were used as received unless otherwise noted. A
Biotage Initiator Sixty system was used for microwave heating.
Flash column chromatography was performed either on an
Analogix Intelliflash 280 using Si 50 columns (32-63 μm,
˚
230-400 mesh, 60 A) or on a Biotage SP1 system (32-63 μm
˚
particle size, KP-Sil, 60 A pore size). Preparative high pressure
liquid chromatography (HPLC) was performed using a Waters
2525 pump with 2487 dual wavelength detector and 2767 sample
manager. Columns were Waters C18 OBD 5 μm, either 50 mm ꢀ
100 mm Xbridge or 30 mm ꢀ 100 mm Sunfire. Systems were run
with either a 5-95% or 10-90% ACN/H2O gradient with either
a 0.1% TFA or 0.1% NH4OH modifier. NMR spectra were
recorded on a Bruker AV400 (Avance 400 MHz) or AV500
(Avance 500 MHz) instruments. Analytical LC-MS was con-
ducted using an Agilent 1100 series with UV detection at 214 and
254 nm and an electrospray mode (ESI) coupled with a Waters
ZQ single quadrupole mass detector. One of two methods was
used: (method A) 5-95% ACN/H2O with 5 mM ammonium
formate with a 2 min run, 3 μL injection through an inertisil
C8 3 cm ꢀ 5 mm ꢀ 3 μm; (method B) 20-95% ACN/H2O with
10 mM ammonium formate with a 2 min run, 3 μL injection
through an inertisil C8 3 cm ꢀ 5 mm ꢀ 3 μm.
Conclusion
In summary, rigidified analogues of dacinostat have been
prepared to identify several novel scaffolds that display a
combination of improved HDAC inhibition and reduced
hERG inhibition. One such scaffold, based on 3-piperidin-3-
ylindole, was investigated and led to the discovery of a potent
HDAC inhibitor 30 with attenuated hERG inhibition. We
proposed that this reduced inhibition is the result of the
molecule’s unique conformation, making interactions with
the hERG channel less favorable. Separation of racemate 30
afforded the more potent HDAC inhibitor 32, which exhibits
reduced potency in the hERG manual patch clamp assay. Our
approach of refining the three-dimensional structure of
HDAC binding analogues was shown to benefit both HDAC
and hERG potency profiles.
Analytical HPLC UV purity was assessed using an Agilent
1100 HPLC system and one of the following methods. For
method 1 (at 214 nm), an Inertsil ODS3 3 μm, 3.0 mm ꢀ 100 mm
C18 column was used with a flow rate of 1.5 mL/min and a
gradient of 10-95% acetonitrile/water with 0.1% TFA over
15 min. For method 2 (at both 254 and 214 nm), an Inertsil
ODS3 3 μm, 3.0 mm ꢀ 100 mm C18 column was used with a flow
rate of 1.0 mL/min and a gradient of 5-95% acetonitrile/water
with 0.1% TFA over 7.75 min. For method 3 (at both 254 and
215 nm), a Nova-Pak 4 μm, 3.9 mm ꢀ 150 mm C18 column was
used with a flow rate of 2.0 mL/min and a gradient of 10-90%
acetonitrile/water with 0.1% TFA over 5.0 min. LC/ESI-MS
data were recorded using a Waters LCT Premier mass spectro-
meter with dual electrospray ionization source and Agilent 1100
Experimental Section
HDAC Enzyme Assay. The HDAC enzymatic assay measures
compound activity in inhibiting purified HDAC isoforms.
HDACs 1, 3, and 6 were immunopurified from 293 cells stably
expressing the FLAG-tagged HDAC isoform, whereas HDACs
2, 4, 5, 7, 8, 9, 10, and 11 were purified from the baculovirus

2958 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Cho et al.
liquid chromatograph. The resolution of the MS system was
approximately 12 000 (fwhm definition). HPLC separation was
performed at 1.0 mL/min flow rate with a gradient from 10% to
95% in 2.5 min. Ammonia formate (10 mM) was used as the
modifier additive in the aqueous phase. Sulfadimethoxine
(Sigma; protonated molecule m/z 311.0814) was used as a
reference and acquired through the LockSpray channel every
third scan.
PREP instrument equipped with 16 columns (7.5 cm ꢀ 2.12 cm
from Princeton Chromatography Corporation) containing the
stationary phase FA-2392/6 (own phase, immobilized Amylose
tris-(S)-methylbenzyl carbamate coated on 12 μm silica gel):
(mobile phase) hexane/2-propanol/EtOH, 75:15:10; (SMB para-
meters) feed concentration of 0.5% in the mobile phase (1.5 mL/
min); (mobile phase) 12.7 mL/min; (extract rate) 3.0 mL/min;
(cycle time) 60 min; (column configuration) 6-6-3-1. Chiral
purity of the extract and raffinate was assessed by chiral HPLC
using a Agilent 1200 HPLC system and a Chiralpak AS (Chiral
Technologies, Illkirch, France) column (4.6 mm ꢀ 250 mm,
20 μm material) at a flow rate of 1 mL/min, with a mixture of
heptane/ethanol 95:5 (v/v) as the mobile phase tR1(raffinate) =
13.76 min and tR2(extract) = 23.61 min.
General Procedure A for Preparation of 3-Piperidin-3-yl-1H-
indoles. 2-Methyl-3-piperidin-3-yl-1H-indole (19). A mixture of
2-methyl-1H-indole (3.0 g, 22.6 mmol), 1-benzylpiperidin-3-one
(11.3 g, 2.0 equiv), and 85% H3PO4 in water (2.61 mL, 10 equiv)
in glacial acetic acid (30 mL) was heated at 100 °C for 4 h. After
cooling, the reaction mixture was diluted with EtOAc, washed
with water and brine, dried over Na2SO4, filtered, and concen-
trated in vacuo to give a crude mixture of 3-(1-benzyl-1,2,5,6-
tetrahydropyridin-3-yl)-2-methyl-1H-indole and 3-(1-benzyl-
1,4,5,6-tetrahydropyridin-3-yl)-2-methyl-1H-indole (4.9 g, 72%
yield). The crude mixture was diluted with MeOH (40 mL) and
treated with Pd(OH)2/C (20 wt %, wet, 2.0 g). The reaction bottle
was evacuated and flushed with H2 three times and shaken under
50psiina Parrhydrogenator overnight. The reaction mixturewas
filtered through a pad of Celite and concentrated in vacuo to give
the crude product 2-methyl-3-piperidin-3-yl-1H-indole (2.9 g,
83% yield). 1H NMR (400 MHz, CD3OD) δ 7.46 (d, J =
7.7 HZ, 1 H), 7.23 (m, 1 H), 7.15 (m, 1 H), 7.04-6.94 (m, 1 H),
3.01(m, 1 H), 2.75 (m, 2 H), 2.56 (m, 1 H), 2.30 (s, 3 H), 2.35 (m,
1 H), 1.83-1.21 (m, 6 H); MS m/z 215.9 (M þ H)þ.
(1) The enantiomeric excess of enantiomer/raffinate was
>99%. The X-ray crystal structure was determined for (S)-
3-(2-tert-butyl-1H-indol-3-yl)piperidine-1-carboxylic acid benzyl
ester.
(2) The enantiomeric excess of enantiomer/extract was 97%:
(R)-3-(2-tert-butyl-1H-indol-3-yl)piperidine-1-carboxylic acid
benzyl ester.
3-Piperidin-3-yl-1H-indole. The compound was synthesized
from 1H-indole following general procedure A in 54% yield
1
over two steps. H NMR (400 MHz, CD3OD) δ 7.58 (d, J =
(S)-3-(2-tert-Butyl-1H-indol-3-yl)piperidine-1-carboxylic acid
benzyl ester (enantiomer/raffinate, 3 g, 7.68 mmol) was dissolved
in MeOH (80 mL) and treated with HCl (32%, 10 M, 0.85 mL,
1.1 equiv) and Pd/C (10%, 600 mg). The mixture was degassed
and subjected to hydrogenation in a Parr shaker for 2 h. The
reaction mixture was filtered through a pad of Celite and
concentrated. The crude product was partitioned between aqu-
eous1 M NaOH and CH2Cl2. The aqueous phase was reextracted
with CH2Cl2. The organic phase was dried over MgSO4, filtered,
and concentrated to afford 2-tert-butyl-3-(S)-piperidin-3-yl-1H-
indole in 83% yield.
2-Methyl-3-pyrrolidin-3-yl-1H-indole (20). A solution of mal-
eimide (5.63 g, 56.9 mmol) and 2-methyl-1H-indole (7.70 g,
1.02 equiv) in glacial acetic acid (50 mL) was heated to reflux for
3 days. After cooling, the reaction mixture was concentrated in
vacuo to remove acetic acid and diluted with EtOAc (300 mL).
The organic phase was washed with water (2 ꢀ 100 mL),
saturated aqueous NaHCO3 (3 ꢀ 150 mL), dried over MgSO4,
filtered, and concentrated in vacuo. Column chromatography
(EtOAc/hexanes, 20-60%) gave 3-(2-methyl-1H-indol-3-yl)-
pyrrolidine-2,5-dione (4.04 g) in 31% yield. 1H NMR (400 MHz,
CDCl3) δ 7.95 (br s, 1H), 7.30 (m, 2 H), 7.20-7.09 (m, 2 H), 4.32
(dd, J = 9.8, 5.7 Hz, 1H), 3.25 (dd, J = 19, 9.8 Hz, 1H), 3.05 (dd,
J = 19, 5.7 Hz, 1H), 2.07 (s, 3 H).
To a solution of 3-(2-methyl-1H-indol-3-yl)pyrrolidine-2,5-
dione (2.22 g, 9.73 mmol) and THF (100 mL) was added LiAlH4
(3.85 g, 10 equiv) portionwise, and the resulting mixture was
heated to reflux for 20 h. The reaction mixture was cooled to
0 °C, treated carefully with EtOAc (7 mL) and water (3.5 mL),
and stirred at room temperature for 20 min. The mixture was
treated with 8.0 mL of 1 N aqueous solution of NaOH and
8.0 mL of water and then heated to reflux for 2 h. The mixture
was cooled to room temperature and filtered. The filtrate was
concentrated in vacuo. The remaining water was removed
azeotropically with toluene to give the crude product 2-methyl-
3-pyrrolidin-3-yl-1H-indole (1.95 g) in quantitative yield. 1H
NMR (400 MHz, CD3OD) δ 7.55 (m, 1 H), 7.25 (m, 1 H),
7.05-6.91 (m,2 H), 3.65 (m, 1 H), 3.20-3.03 (m, 2 H), 2.70 (m,
2 H), 2.39 (s, 3 H), 2,24 (m, 2 H); MS m/z 201.1 (M þ H)þ.
8.0 Hz, 1 H), 7.32 (d, J = 8.0 Hz, 1 H), 7.07 (t, J = 8.0 Hz, 1 H),
7.00 (s, 1 H), 6.97 (d, J = 8.0 Hz, 1 H), 3.09-2.99 (m, 2 H), 2.63
(m, 2 H), 2.16 (d, J = 12 Hz, 1 H), 2.14 (m, 1 H), 1.80 (m, 1 H),
1.70 (m, 2 H).
2-Phenyl-3-piperidin-3-yl-1H-indole. The compound was
synthesized from 2-phenyl-1H-indole following general proce-
dure A in 96% yield over two steps. 1H NMR (400 MHz,
DMSO-d6) δ 11.2 (s, 1 H), 7.80 (d, J = 4.0 Hz, 1 H), 7.53 (m,
4 H), 7.42 (m, 1 H), 7.35 (d, J = 8.0 Hz, 1 H), 7.08 (t, J = 8.0 Hz,
1 H), 6.98 (t, J = 8.0 Hz, 1 H), 3.08 (m, 1 H), 2.99 (m, 2 H), 2.70
(m, 1 H), 2.15 (m, 1 H), 1.82 (m, 1 H), 1.70 (m, 1 H); MS m/z
277.2 (M þ H)þ.
2-tert-Butyl-3-piperidine-3-yl-1H-indole. The compound was
synthesized from 2-tert-butyl-1H-indole following general pro-
cedure A in 73% yield over two steps. H NMR (400 MHz,
1
CDCl3) δ 8.05 (br s, 1 H), 7.53 (d, J = 8.0 Hz, 1 H), 7.35 (d, J =
8.0 Hz, 1 H), 7.13 (t, J = 8.0 Hz, 1 H), 7.06 (t, J = 8.0 Hz, 1 H),
3.84 (m, 1 H), 3.58 (br d, J = 12 Hz, 1 H), 3.49 (m, 1 H), 3.41 (m,
1 H), 2.97 (m, 1 H), 2.96 (m, 2 H), 2.02 (br d, J = 12 Hz, 2 H),
1.51 (s, 9 H); MS m/z 257.2 (M þ H)þ.
2-tert-Butyl-3-(S)-piperidin-3-yl-1H-indole and 2-tert-Butyl-
3-(R)-piperidin-3-yl-1H-indole. To a solution of 2-tert-butyl-3-
piperidine-3-yl-1H-indole (5 g, 19.5 mmol) in CH2Cl2 (200 mL)
and N-methylmorpholine (10 mL) was added benzyl chlorofor-
mate (3.76 mL, 25.4 mmol, 1.3 equiv) dropwise over 10 min. The
resulting mixture was stirred at 30 °C for 12 h. The reaction
mixture was treated with saturated aqueous NaHCO3 (50 mL),
and the phases were separated. The organic phase was dried over
MgSO4, filtered, and concentrated. Column chromatography
(EtOAc/heptanes, 0-25%) afforded 3-(2-tert-butyl-1H-indol-
3-yl)piperidine-1-carboxylic acid benzyl ester in 70% yield.
1H NMR (400 MHz, DMSO-d6) δ 10.36 (br s, 1H), 7.65 (d,
J = 8.0 Hz, 1 H), 7.45-7.25 (m, 6 H), 6.85-7.00 (m, 2 H),
4.95-5.15 (br m, 2 H), 3.90-4.15 (br m, 2 H), 3.35-3.65 (br m,
1 H), 3.15 (m, 2 H), 2.20 (m, 1 H), 1.77 (d, J = 12.0 Hz, 2 H),
1.45-1.55(m,1H),1.44-1.25 (br m, 9 H); MS m/z391.1 (M þ H)þ.
An amount of 147 g of racemic 3-(2-tert-butyl-1H-indol-3-yl)-
piperidine-1-carboxylic acid benzyl ester was resolved by simulat-
ing moving bed (SMB) chromatography on a UOP-SORBEX

Article
2-Methyl-3-piperidin-2-ylmethyl-1H-indole (21). To a solu-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2959
General Procedure C for Preparation of Methyl Esters. (E)-3-
{4-[3-(2-Phenyl-1H-indol-3-yl)piperidin-1-ylmethyl]phenyl}acrylic
Acid Methyl Ester. 2-Phenyl-3-piperidin-3-yl-1H-indole (2.0 g,
7.16 mmol) was dissolved in CH2Cl2 (20 mL) and treated with
triethylamine (3.0 mL, 3.0 equiv) and 4-bromobenzyl bromide
(1.97 g, 1.1 equiv). After being stirred for 4 h, the reaction mixture
was diluted with EtOAc, washed with water and brine, dried over
MgSO4, filtered, and concentrated in vacuo. Column chromato-
graphy (EtOAc/hexanes, 0-90%) provided 3-[1-(4-bromobenzyl)-
piperidin-3-yl]-2-phenyl-1H-indole (2.1 g) in 66% yield. 1H NMR
(400 MHz, CDCl3) δ 8.00 (br s, 1 H), 7.86 (d, J = 8.0 Hz, 1 H),
7.51-7.40 (m, 7 H), 7.37 (d, J = 8.0 Hz, 1 H), 7.23-7.11 (m, 4 H),
3.51 (dd, J = 20, 12 Hz, 2 H), 3.31 (m, 1 H), 2.96 (m, 2 H), 2.08 (m,
2 H), 1.92 (m, 1 H), 1.81-1.66 (m, 2 H); MS m/z 447.1 (M þ H)þ.
A microwave vial was charged with a solution of 3-[1-(4-
bromobenzyl)piperidin-3-yl]-2-phenyl-1H-indole (500 mg, 1.11
mmol) in 1,4-dioxane (10 mL) and tri-tert-butylphosphonium
tetrafluoroborate (13 mg, 0.04 equiv) and Pd2(dba)3 (10 mg, 0.01
equiv), then flushed with nitrogen three times. The resulting
mixture was treated with Cy2NMe (0.28 mL, 1.2 equiv) and
methyl acrylate (0.20 mL, 2.0 equiv), then heated at 100 °C by
microwave for 1 h. The reaction mixture was diluted with
EtOAc, washed with water and brine, dried over MgSO4,
filtered, and concentrated in vacuo. Column chromatography
(MeOH/CH2Cl2, 0-15%) gave (E)-3-{4-[3-(2-phenyl-1H-in-
dol-3-yl)piperidin-1-ylmethyl]phenyl}acrylic acid methyl ester
(310 mg) in 62% yield. 1H NMR (400 MHz, CDCl3) δ 8.01 (br s,
1H), 7.86 (d, J = 8.0 Hz, 1 H), 7.69 (d, J = 16 Hz, 1 H),
7.50-7.34 (m, 10 H), 7.20-7.10 (m, 2 H), 6.42 (d, J = 16 Hz,
1 H), 3.81 (s, 3 H), 3.57 (dd, J = 20, 12 Hz, 2 H), 3.31 (m, 1 H),
2.97 (m, 2 H), 2.68 (t, J = 10 Hz, 1 H), 2.15-2.04 (m, 2 H), 1.92
(br d, J = 8.0 Hz, 1 H), 1.76 (m, 2 H); MS m/z 451.0 (M þ H)þ.
(E)-3-{4-[3-(2-tert-Butyl-1H-indol-3-yl)piperidin-1-ylmethyl]-
phenyl}acrylic Acid Methyl Ester. The compound was synthe-
sized from 2-tert-butyl-3-piperidine-3-yl-1H-indole following
general procedure C in 66% yield over two steps.
tion of N-Boc-2-piperidinecarboxylic acid (3.18 g, 13.6 mmol)
in CH2Cl2 (32 mL) with a trace of DMF (0.7 mL) was added
oxalyl chloride (1.8 mL, 1.5 equiv), and the resulting mixture
was stirred at room temperature for 1 h. The reaction mixture
was concentrated in vacuo and further dried azeotropically with
benzene. At the same time, a solution of EtMgBr in Et2O (3.0 M,
9.0 mL, 2.1 equiv) was added dropwise to a cooled (0 °C)
solution of 2-methyl-1H-indole (3.25 g, 1.8 equiv) in benzene
(37 mL), and the resulting mixture was stirred at 0 °C for 9 min.
Then the reaction mixture was treated with a solution of N-Boc-
2-piperidinecarboxylic acid chloride in benzene (15 mL) drop-
wise with vigorous stirring. The resulting mixture was stirred at
0 °C for 1 h and treated with EtOAc (40 mL) and saturated
aqueous NaHCO3 (35 mL), then stirred for 1 h. After separa-
tion, the aqueous phase was extracted with EtOAc. Combined
organics were dried over MgSO4, filtered, and concentrated
in vacuo. Column chromatography (MeOH/CH2Cl2, 5-40%)
gave (2-methyl-1H-indol-3-yl)piperidin-2-ylmethanone (0.550 g)
1
in 17% yield. H NMR (400 MHz, CD3OD) δ 7.86-7.84 (m,
1 H), 7.40-7.37 (m, 1 H), 7.21 (ddd, J = 13.0, 7.4, 1.5 Hz, 1 H),
7.20 (ddd, J = 12.9, 7.5, 1.5 Hz, 1 H), 4.26 (dd, J = 11.6, 2.7 Hz,
1 H), 3.25-3.21 (m, 1 H), 2.83 (td, J = 12.8, 3.0 Hz, 1 H), 2.74 (s,
3 H), 2.15-2.11 (m, 1 H), 2.00-1.95 (m, 1 H), 1.81-1.70 (m, 2 H),
1.58-1.47 (m, 1 H), 1.39 (ddd, J = 24.8, 12.3, 3.8 Hz, 1 H); MS
m/z 243.1 (M þ H)þ.
To a solution of (2-methyl-1H-indol-3-yl)-piperidin-2-yl-
methanone (491 mg, 2.03 mmol) in THF (20 mL) was added
LiAlH4 (278 mg, 3.5 equiv), and the resulting mixture was stirred
at room temperature for 2 h. The reaction mixture was quenched
with 1 N NaOH (7.2 mL), stirred for 10 min, filtered, and
concentrated in vacuo. The crude 2-methyl-3-piperidin-2-yl-
methyl-1H-indole (0.47 g, quantitative yield) was used for the
next reaction without purification. 1H NMR (400 MHz,
CD3OD) δ 7.46 (d, J = 7.7 Hz, 1 H), 7.25-7.21 (m, 1 H),
7.18-7.11 (m, 1 H), 7.04-6.94 (m, 1 H), 3.03-2.99 (m, 1 H),
2.78-2.75 (m, 2 H), 2.60-2.53 (m, 1 H), 2.39 (s, 3 H), 2.35-2.34
(m, 1 H), 1.83-1.21 (m, 6 H); MS m/z 229.1 (M þ H)þ.
1
3-[1-(4-Bromobenzyl)piperidin-3-yl]-2-tert-butyl-1H-indole: H
NMR (400 MHz, CDCl3) δ 7.87 (br s, 1 H), 7.75 (d, J = 8.0 Hz,
1 H), 7.41 (d, J = 8.0 Hz,, 2 H), 7.31 (d, J = 8.0 Hz, 1 H), 7.24 (d,
J = 8.0 Hz, 2H), 7.12-7.03 (m, 2 H), 3.53 (dd, J = 44, 12 Hz,
2 H), 3.40 (tt, J = 12, 3.7 Hz, 1 H), 2.99 (br d, J = 11 Hz, 1 H),
2.88 (br d, J = 11 Hz, 1 H), 2.63 (t, J = 12 Hz, 1 H), 2.20 -2.05
(m, 2 H), 1.83 (m, 3 H), 1.47 (s, 9 H); MS m/z 426.8 (M þ H)þ.
(E)-3-{4-[3-(2-tert-Butyl-1H-indol-3-yl)piperidin-1-ylmethyl]-
phenyl}acrylic acid methyl ester: 1H NMR (400 MHz, CDCl3) δ
7.88 (br s, 1 H), 7.75 (d, J = 8.0 Hz, 1 H), 7.69 (d, J = 16 Hz,
1 H), 7.46 (d, J = 8.0 Hz, 2 H), 7.39 (d, J = 8.0 Hz, 2 H), 7.30 (d,
J = 8.0 Hz, 1 H), 7.11-7.02 (m, 2 H), 6.43 (d, J = 16 Hz, 1 H),
3.82 (s, 3 H), 3.60 (dd, J = 50, 14 Hz, 2 H), 3.42 (m, 1 H), 3.02
(br d, J = 12 Hz, 1 H), 2.89 (br d, J = 12 Hz, 1 H), 2.64 (t, J =
10 Hz, 1 H), 2.16 (m, 2 H), 1.84 (m, 3 H), 1.46 (s, 9 H); MS m/z
430.9 (M þ H)þ.
General Procedure B for Preparation of Methyl Esters. (E)-3-
{4-[3-(2-Methyl-1H-indol-3-yl)piperidin-1-ylmethyl]phenyl}acrylic
Acid Methyl Ester. 2-Methyl-3-piperidin-3-yl-1H-indole (19) (300
mg, 1.40 mmol) was diluted in CH2Cl2 (20 mL) and treated with
methyl 4-formylcinnamate (266 mg, 1 equiv) and triethylamine
(0.58 mL, 3.0 equiv). The resulting mixture was treated with TiCl4
(0.67 mL, 0.48 equiv) dropwise. Once 2-methyl-3-piperidin-3-yl-
1H-indole was consumed, the reaction mixture was treated with
NaBH3CN (278 mg, 3.0 equiv). After 2 h, the reaction mixture
was basified to pH 13 with 5 N NaOH and extracted with EtOAc.
Combined organic phase was dried over Na2SO4, filtered, and
concentrated in vacuo. Column chromatography afforded (E)-3-
{4-[3-(2-methyl-1H-indol-3-yl)piperidin-1-ylmethyl]phenyl}acrylic
acid methyl ester (140 mg) in 26% yield. 1H NMR (400 MHz, CDCl3)
δ7.75(brs,1H),7.70(m,2H),7.48(d,J= 8.0 Hz, 2 H), 7.38 (d, J=
8.0 Hz, 2 H), 7.27 (d, J = 8.0 Hz, 1 H), 7.12-7.04 (m, 2 H), 6.44 (d,
J = 16 Hz, 1 H), 3.82 (s, 3 H), 3.58 (s, 2 H), 3.11 (m, 1 H), 2.96 (m,
2 H), 2.54 (t, J = 8.0 Hz, 1 H), 2.53 (m, 1 H), 2.41 (s, 3 H), 2.09 (m,
1 H), 1.92 (m, 1 H), 1.64 (m, 2 H); MS m/z 389.0 (M þ H)þ.
(E)-3-{4-[3-(1H-Indol-3-yl)piperidin-1-ylmethyl]phenyl}acrylic
Acid Methyl Ester. The compound was synthesized from
3-piperidin-3-yl-1H-indole following general procedure B in
45% yield. 1H NMR (400 MHz, CDCl3) δ 7.97 (br s, 1 H), 7.70
(d, J = 16 Hz, 1 H), 7.64 (d, J = 8.0 Hz, 1 H), 7.48 (d, J = 8.0 Hz,
2 H), 7.38 (d, J = 8.0 Hz, 2 H), 7.36 (d, J = 8.0 Hz, 1H), 7.18 (m,
1H), 7.10 (m, 1H), 7.01(brd, J= 2.0 Hz, 1 H), 6.43 (d, J= 16Hz,
1H), 3.82(s, 3 H), 3.58(brs, 2H), 3.19(m,2H), 2.91 (m, 1H), 2.11
(m, 2 H), 1.79 (m, 2 H), 1.56 (m, 2 H); MS m/z 374.9 (M þ H)þ.
(E)-3-{4-[2-(2-Methyl-1H-indol-3-ylmethyl)piperidin-1-ylmethyl]-
phenyl}acrylic Acid Methyl Ester. The compound was synthesized
from 2-methyl-3-piperidin-2-ylmethyl-1H-indole following general
procedure B in 47% yield. MS m/z 403.9 (M þ H)þ.
(E)-3-{4-[2-(2-Methyl-1H-indol-3-yl)pyrrolidin-1-ylmethyl]-
phenyl}acrylic Acid Methyl Ester. The compound was synthe-
sized from 2-methyl-3-pyrrolidin-3-yl-1H-indole following
general procedure C in 12% yield over two steps.
3-[1-(4-Bromobenzyl)pyrrolidin-2-yl]-2-methyl-1H-indole: 1H
NMR (400 MHz, CDCl3) δ 8.15 (s, 1 H), 7.98 (m, 1 H), 7.58
(d, J = 7.8 Hz, 2 H), 7.39 (d, J = 8.4 Hz, 2 H), 7.31 (m, 3 H), 3.81
(s, 2 H), 3.76 (m, 1 H), 3.10-2.91 (m, 4 H), 2.43-2.39 (m, 2 H),
2.38 (s, 3 H); MS m/z 371.0 (M þ H)þ.
(E)-3-{4-[2-(2-Methyl-1H-indol-3-yl)pyrrolidin-1-ylmethyl]-
phenyl}acrylic Acid Methyl Ester: 1H NMR (400 MHz, CD3OD)
δ 7.58 (m, 2 H), 7.34 (d, J = 7.6 Hz, 2 H), 7.22 (m, 3 H), 6.99 (m,
2 H), 6.39 (d, J = 16.1 Hz, 1 H), 3.72 (s, 3 H), 3.54 (m, 1 H), 3.50
(s, 2 H), 2.83-2.73 (m, 2 H), 2.68-2.58 (m, 2 H), 2.27 (s, 3 H),
2.13-2.06 (m, 2 H); MS m/z 375.1 (M þ H)þ.
(E)-3-{3-Fluoro-4-[3-(1H-indol-3-yl)piperidin-1-ylmethyl]phe-
nyl}acrylic Acid Methyl Ester. The compound was synthesized
from 3-piperidin-3-yl-1H-indole and 4-bromo-2-fluorobenzyl

2960 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Cho et al.
bromide following general procedure C in 10% yield over
two steps.
3.3 mL). The resulting mixture was stirred at room temperature
for 4 h. To the reaction mixture was added MeOH (5 mL)
dropwise followed by addition of 1 N aqueous solution of HCl
(10 mL). The resulting mixture was heated to reflux for 3 h. After
cooling, the reaction mixture was diluted with EtOAc, washed
with water and brine, dried over Na2SO4, filtered, and concen-
trated in vacuo. Column chromatography (EtOAc/hexanes,
10-100%) gave 3-[1-(4-bromo-2-chlorobenzyl)piperidin-3-yl]-
2-tert-butyl-1H-indole (270 mg) in 47% yield over two steps.
1H NMR (400 MHz,, CDCl3) δ 8.02 (br s, 1 H), 7.94 (d, J =
8.0 Hz, 1 H), 7.64-7.60 (m, 2 H), 7.47 (dd, J = 8.0 Hz, 2.0 Hz,
1 H), 7.43 (d, J = 8.0 Hz, 1 H), 7.24 (qd, J = 8.0, 2.0 Hz, 1 H),
3.76 (s, 2 H), 3.59 (tt, J = 12, 4.0 Hz, 1 H), 3.14 (br d, J = 12 Hz,
1 H), 3.07 (m, 1 H), 2.92 (t, J = 12 Hz, 1 H), 2.42 (td, J = 10,
2.0 Hz, 1 H), 2.27 (m, 1 H), 2.05-1.92 (m, 3 H), 1.61 (s, 9 H); MS
m/z 459.0 (M þ H)þ.
3-[1-(4-Bromobenzyl)piperidin-3-yl]-1H-indole: 1H NMR (400
MHz, CDCl3) δ 8.04 (br s, 1 H), 7.66 (d, J = 8.0 Hz, 1 H),
7.38-7.11 (m, 6 H), 7.02 (d, J = 4.0 Hz, 1 H), 3.63 (br d, J =
4.0 Hz, 2 H), 3.21 (m, 2 H), 2.93 (m, 1 H), 2.69 (t, J = 8.0 Hz, 1 H),
2.38 (t, J = 8.0 Hz, 1 H), 2.12 (m, 1 H), 1.98 (m, 1 H), 1.80 (m,
2 H), 1.53 (m, 1 H); MS m/z 388.8 (M þ H)þ.
(E)-3-{3-Fluoro-4-[3-(1H-indol-3-yl)piperidin-1-ylmethyl]phe-
nyl}acrylic acid methyl ester: 1H NMR (400 MHz, CDCl3) δ 8.10
(br s, 1 H), 7.65 (m, 2 H), 7.48 (t, J = 8.0 Hz, 1 H), 7.37 (d, J =
8.0 Hz, 1 H), 7.29 (m, 1 H), 7.19 (m, 2 H), 711 (m, 1 H), 7.03 (m,
1 H), 6.43 (d, J = 16 Hz, 1 H), 3.83 (s, 3 H), 3.66 (br s, 2 H), 3.22
(m, 2 H), 2.94 (m, 1 H), 2.54 (m, 1 H), 2.34-2.07 (m, 3 H),
1.65-1.50 (m, 2 H).
(E)-3-{3-Fluoro-4-[3-(2-phenyl-1H-indol-3-yl)piperidin-1-yl-
methyl]phenyl}acrylic Acid Methyl Ester. The compound was
synthesized from 2-phenyl-3-piperidin-3-yl-1H-indole and
4-bromo-2-fluorobenzyl bromide following general procedure
C in 26% yield over two steps.
Following the second part of general procedure C, (E)-3-
{4-[3-(2-tert-butyl-1H-indol-3-yl)piperidin-1-ylmethyl]-3-chloro-
phenyl}acrylic acid methyl ester was prepared. H NMR (400
1
MHz, CDCl3) δ 7.81 (br s, 1 H), 7.68 (d, J = 8.0 Hz, 1 H), 7.53 (d,
J = 8.0 Hz, 1 H), 7.52 (d, J = 16 Hz, 1 H), 7.39 (d, J = 1.6 Hz,
1 H), 7.2 (dd, J = 8.0, 1.5 Hz, 1 H), 7.19 (d, J = 8.0 Hz, 1 H), 7.00
(m, 1 H), 6.95 (m, 1 H), 6.32 (d, J = 16 Hz, 1 H), 3.73 (s, 3 H), 3.58
(s, 2 H), 3.34(m, 1 H), 2.92 (br d, J =11Hz, 1 H), 2.82 (brdd, J =
11, 3.7 Hz, 1 H), 2.68 (t, J = 11 Hz, 1 H), 2.19 (m, 1 H), 2.02 (m,
1 H), 1.76 (m, 3 H), 1.38 (s, 9 H); MS m/z 465.1 (M þ H)þ.
(E)-3-(4-{1-[2-(2-Methyl-1H-indol-3-yl)ethyl]pyrrolidin-2-yl}-
phenyl)acrylic Acid Methyl Ester (14). (2-Methyl-1H-indol-3-yl)-
acetaldehyde (23, 495 mg, 1.5 equiv) was diluted in CH2Cl2 (20mL)
and treated with 2-(4-bromophenyl)pyrrolidine (24, 430 mg, 1.904
mmol) and triethylamine (0.42 mL, 3.0 equiv). The resulting
mixture was treated with TiCl4 (0.72 mL, 0.50 equiv) dropwise.
Once 2-(4-bromophenyl)pyrrolidine was consumed, the reaction
mixture was treated with NaBH3CN (286 mg, 3.0 equiv). After 2 h,
the reaction mixture was basified to pH 13 with 5 N NaOH and
extracted with EtOAc. Combined organic phase was dried over
Na2SO4, filtered, and concentrated in vacuo. Column chromato-
graphy (EtOAc/heptanes, 0-40%) afforded 3-{2-[2-(4-bromophe-
nyl)pyrrolidin-1-yl]ethyl}-2-methyl-1H-indole (365 mg) in 50%
3-[1-(2-Fluoro-4-methylbenzyl)piperidin-3-yl]-2-phenyl-1H-in-
dole: 1H NMR (400 MHz, CDCl3) δ 7.99 (br s, 1 H), 7.85 (d, J =
8.0 Hz, 1 H), 7.51-7.38 (m, 6 H), 7.33-7.11 (m, 5 H), 3.59 (br s,
2 H), 3.31 (m, 1 H), 2.97 (m, 2 H), 2.74 (t, J = 10 Hz, 1 H), 2.17
(m, 1 H), 2.95 (m, 1 H), 1.93 (m, 1 H), 1.78 (m, 2 H); MS m/z
462.7 (M þ H)þ.
(E)-3-{3-Fluoro-4-[3-(2-phenyl-1H-indol-3-yl)piperidin-1-yl-
1
methyl]phenyl}acrylic acid methyl ester: H NMR (400 MHz,
CDCl3) δ 8.00 (br s, 1 H), 7.84 (d, J = 8.0 Hz, 1 H), 7.63 (d, J =
16 Hz, 1 H), 7.48-7.36 (m, 7 H), 7.26 -7.09 (m, 4 H), 6.41 (d,
J = 16 Hz, 1 H), 3.81 (s, 3 H), 3.64 (s, 2 H), 3.31 (m, 1 H), 2.99 (m,
2 H), 2.74 (t, J=12Hz, 1 H), 2.18 (m, 1 H), 2.05 (m, 1 H), 1.91 (m,
1 H), 1.71 (m, 1 H), 1.62 (m, 1 H); MS m/z 468.8 (M þ H)þ.
(E)-3-{4-[3-(2-tert-Butyl-1H-indol-3-yl)piperidin-1-ylmethyl]-
3-fluorophenyl}acrylic Acid Methyl Ester. The compound was
synthesized from 2-tert-butyl-3-piperidine-3-yl-1H-indole and
4-bromo-2-fluorobenzyl bromide following general procedure
C in 67% yield over two steps.
3-[1-(4-Bromo-2-fluorobenzyl)piperidin-3-yl]-2-tert-butyl-1H-
indole: 1H NMR (400 MHz, CDCl3) δ 7.88 (br s, 1 H), 7.74 (d,
J = 8.0 Hz, 1 H), 7.36 (t, J = 8.0 Hz, 1 H), 7.31 (d, J = 8.0 Hz,
1 H), 7.23 (d, J = 8.0 Hz, 1 H), 7.20 (d, J = 8.0 Hz, 1 H), 7.10 (t,
J = 8.0 Hz, 1 H), 7.05 (t, J = 8.0 Hz, 1 H), 3.61 (br s, 2 H), 3.41
(m, 1 H), 3.01 (br d, J = 12 Hz, 1 H), 2.90 (br d J = 12 Hz, 1 H),
2.72 (t, J = 10 Hz, 1 H), 2.25-2.04 (m, 2 H), 1.84 (m, 3 H), 1.47
(s, 9 H).
1
yield. H NMR (400 MHz, CDCl3) δ 7.70 (br s, 1 H), 7.40 (d,
J = 8.0 Hz, 1 H), 7.34 (d, J = 8.0 Hz, 1 H), 7.25 (d, J = 8.0 Hz,
1 H), 7.21 (d, J = 8.0 Hz, 1 H), 7.11-7.01 (m, 2 H), 3.55 (m, 1 H),
3.27 (m, 1 H), 2.92-2.70 (m, 3 H), 2.42 (m, 1H), 2.33 (m, 2 H), 2.30
(s, 3 H), 2.17 (m, 1 H), 2.00 (m, 1 H), 1.88 (m, 1 H); MS m/z 384.8
(M þ H)þ.
A microwave vial was charged with a solution of 3-{2-[2-(4-
bromophenyl)pyrrolidin-1-yl]ethyl}-2-methyl-1H-indole (290 mg,
0.757 mmol) in 1,4-dioxane (10 mL), tri-tert-butylphosphonium
tetrafluoroborate (8.8 mg, 0.04 equiv), and Pd2(dba)3 (6.9 mg, 0.01
equiv), then flushed with nitrogen three times. The resulting
mixture was treated with Cy2NMe (0.19 mL, 1.2 equiv) and
methyl acrylate (0.14 mL, 2.0 equiv), then heated at 100 °C by
microwave for 1 h. The reaction mixture was diluted with
EtOAc, washed with water and brine, dried over MgSO4, filtered,
and concentrated in vacuo. Column chromatography (EtOAc/
heptanes, 0-50%) gave (E)-3-(4-{1-[2-(2-methyl-1H-indol-3-yl)-
ethyl]pyrrolidin-2-yl}phenyl)acrylic acid methyl ester (285 mg) in
97% yield. 1H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 16 Hz,
1 H), 7.92 (br s, 1 H), 7.66 (m, 2 H), 7.56 (m, 3 H), 7.47 (d, J =
8.0 Hz, 1 H), 7.33-7.21 (m, 2 H), 6.65 (d, J = 16 Hz, 1 H), 4.05 (s,
3 H), 3.79 (m, 1 H), 3.55 (m, 1 H), 3.10 (m, 1 H), 2.99 (m, 2 H), 2.65
(m, 1 H), 2.57 (m, 1 H), 2.52 (s, 3 H), 2.42 (m, 1 H), 2.23 (m, 1 H),
2.12 (m, 1 H), 1.94 (m, 1 H); MS m/z 389.3 (M þ H)þ.
(E)-3-{4-[3-(2-tert-Butyl-1H-indol-3-yl)piperidin-1-ylmethyl]-
3-fluorophenyl}acrylic acid methyl ester: H NMR (400 MHz,
1
CDCl3) δ 7.88 (br s, 1 H), 7.74, (d, J = 4.0 Hz, 1 H), 7.64 (d, J =
16 Hz, 1 H), 7.50 (t, J = 8.0 Hz, 1 H), 7.31 (d, J = 8.0 Hz, 1 H),
7.25 (d, J = 8.0 Hz, 1 H), 7.18 (d, J = 12 Hz, 1 H), 7.10 (t, J =
8.0 Hz, 1 H), 7.04 (t, J = 8.0 Hz, 1 H), 6.42 (d, J = 16 Hz, 1 H),
3.83 (s, 3 H), 3.66 (br s, 2 H), 3.42 (m, 1 H), 3.03 (br d, J = 12 Hz,
1 H), 2.92 (br d, J = 12 Hz, 1 H), 2.73 (t, J = 12 Hz, 1 H), 2.24
(m, 1 H), 2.09 (m, 1 H), 1.85 (m, 3 H), 1.47 (s, 9 H); MS m/z 449.1
(M þ H)þ.
(E)-3-{4-[3-(2-tert-Butyl-1H-indol-3-yl)piperidin-1-ylmethyl]-
3-chlorophenyl}acrylic Acid Methyl Ester. To a solution of
4-bromo-2-chlorobenzoic acid (300 mg, 1.26 mmol) in DMF
(3 mL) were added HBTU (717 mg, 1.5 equiv), HOBt (255 mg,
1.5 equiv), and DIPEA (0.88 mL, 4.0 equiv), and the resulting
mixture was stirred at room temperature for 20 min. The
reaction mixture was treated with 2-tert-butyl-3-piperidine-
3-yl-1H-indole (400 mg, 1.2 equiv) and stirred for additional
4 h. The reaction mixture was quenched with water, diluted
with EtOAc, washed with water and brine, dried over Na2SO4,
filtered, and concentrated in vacuo. The crude product (4-bromo-
2-chlorophenyl)-[3-(2-tert-butyl-1H-indol-3-yl)piperidin-1-yl]-
methanone was dissolved in THF (10 mL) and treated with
a solution of borane tetrahydrofuran complex in THF (1.0 M,
General Procedure D for Preparation of Hydroxamic Acids. (E)-
N-Hydroxy-3-{4-[3-(2-methyl-1H-indol-3-yl)piperidin-1-ylmethyl]-
phenyl}acrylamide(11). Toa cooled (0°C)solutionof(E)-3-{4-[3-
(2-methyl-1H-indol-3-yl)piperidin-1-ylmethyl]phenyl}acrylicacid
methyl ester (640 mg, 1.63 mmol) in MeOH (3 mL) was added a
solution of NH2OH in water (50%, 1.08 mL, 10 equiv) followed
by a solution of NaOMe in MeOH (25%, 1.76 mL, 5 equiv), and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2961
the resulting mixture was stirred at 0 °C for 2 1/2 h. The reaction
mixture was neutralized with a 1 N aqueous solution of HCl until
the pH was 7-8. Precipitate was collected, washed with Et2O, and
dried to provide (E)-N-hydroxy-3-{4-[3-(2-methyl-1H-indol-3-
yl)piperidin-1-ylmethyl]phenyl}acrylamide (461 mg) in 73% yield.
1H NMR (400 MHz, CD3OD) δ 7.57-7.47 (m, 4 H), 7.33 (d, J =
8.0 Hz, 2 H), 7.22 (d, J = 8.0 Hz, 1 H), 6.98 (t, J = 8.0 Hz, 1 H),
6.92 (t, J= 8.0Hz, 1 H), 6.45 (d, J =16 Hz, 1 H), 3.55 (dd, J= 20,
12 Hz, 2 H), 3.11 (m, 1 H), 2.99 (br d, J = 12 Hz, 1 H), 2.86 (br d,
J = 12 Hz, 1 H), 2.53 (t, J = 12 Hz, 1 H), 2.35 (s, 3 H), 2.12 (m,
1 H), 1.95 (m, 1 H), 1.80 (m, 3 H). Anal. RP-HPLC tR = 2.62 min
(method 3, purity 100.00%/100.00%). HR-MS m/z (M þ H)þ:
measd 390.2175, calcd 390.2182.
(E)-N-Hydroxy-3-{4-[3-(2-methyl-1H-indol-3-yl)pyrrolidin-1-
ylmethyl]phenyl}acrylamide (12). 12 was synthesized from (E)-
3-{4-[2-(2-methyl-1H-indol-3-yl)pyrrolidin-1-ylmethyl]phenyl}-
acrylic acid methyl ester following general procedure D in 87%
yield. 1H NMR (400 MHz, CD3OD) δ 7.60 (d, J = 8.0 Hz, 2 H),
7.56 (d, J = 8.0 Hz, 2 H), 7.47 (d, J = 8.0 Hz, 2 H), 7.24 (d, J =
8.0 Hz, 1 H), 7.01 (m, 1 H), 6.94 (m, 1 H), 6.48 (d, J = 16 Hz,
1 H), 3.84 (dd, J = 20, 12 Hz, 2 H), 3.67 (q, J = 8.0 Hz, 1 H),
3.08-2.80 (m, 4 H), 2.37 (s, 3 H), 2.24 (q, J = 8.0 Hz, 2 H). Anal.
RP-HPLC tR = 3.23 min (method 1, purity 100.00%). HR-MS
m/z (M þ H)þ: measd 376.2014, calcd 376.2025.
82% yield. 1H NMR (400 MHz, CD3OD) δ 7.62 (d, J = 8.0 Hz,
2 H), 7.56 (m, 3 H), 7.45 (d, J = 8.0 Hz, 2 H), 7.31 (d, J = 8.0 Hz,
1 H), 6.99-6.89 (m, 2 H), 6.48 (d, J = 16 Hz, 1H), 3.88 (br s, 2 H),
3.43 (m, 1 H), 3.18 (m, 1 H), 3.04 (m, 1 H), 2.53 (m, 1 H), 2.18 (m,
1 H), 1.95-1.82(m, 4 H), 1.44 (s,9 H). Anal. RP-HPLCtR =3.11
min (method 1, purity 94.83%). HR-MS m/z (M þ H)þ: measd
432.2655, calcd 432.2651.
(E)-3-{3-Fluoro-4-[3-(1H-indol-3-yl)piperidin-1-ylmethyl]-
phenyl}-N-hydroxyacrylamide (28). 28 was synthesized from (E)-
3-{4-[3-(1H-indol-3-yl)piperidin-1-ylmethyl]phenyl}acrylic acid
methyl ester following general procedure D in 9% yield. 1H
NMR (400 MHz, CD3OD) δ 7.54-7.45 (m, 3 H), 7.36-7.30
(m, 3 H), 7.06 (t, J = 8.0 Hz, 1 H), 7.01 (s, 1 H), 6.95 (t, J =
8.0 Hz, 1 H), 6.48 (d, J = 16 Hz, 1 H), 3.68 (s, 2 H), 3.17 (m, 2 H),
3.00 (m, 1 H), 2.22-2.02 (m, 3 H), 1.83 (m, 2 H), 1.55 (m, 1 H).
Anal. RP-HPLC tR = 3.02 min (method 1, purity 100.00%). MS
m/z 393.83 (M þ H)þ.
(E)-3-{3-Fluoro-4-[3-(2-phenyl-1H-indol-3-yl)piperidin-1-
ylmethyl]phenyl}-N-hydroxyacrylamide (29). 29 was synthesized
from (E)-3-{3-fluoro-4-[3-(2-phenyl-1H-indol-3-yl)piperidin-1-yl-
methyl]phenyl}acrylic acid methyl ester following general proce-
dure D in 81% yield. 1H NMR (400 MHz, CD3OD) δ 7.70 (d, J =
8.0 Hz, 1 H), 7.51-7.27 (m, 10 H), 7.07 (t, J = 8.0 Hz, 1 H), 6.97 (t,
J = 8.0 Hz, 1 H), 6.47 (d, J = 16 Hz, 1 H), 3.65 (br s, 2 H), 2.96 (m,
2 H), 2.77 (t, J = 10 Hz, 1 H), 2.22 (m, 1 H), 2.08 (m, 1 H),
1.93-1.65 (m, 3 H), 1.33 (m, 1 H). Anal. RP-HPLC tR = 5.43 min
(method 1, purity 100.00%). HR-MS m/z (M þ H)þ: measd
470.2226, calcd 470.2244.
(E)-N-Hydroxy-3-{4-[2-(2-methyl-1H-indol-3-ylmethyl)piperidin-
1-ylmethyl]phenyl}acrylamide (13). 13 was synthesized from (E)-
3-{4-[2-(2-methyl-1H-indol-3-ylmethyl)piperidin-1-ylmethyl]-
phenyl}acrylic acid methyl ester following general procedure D in
17% yield. 1H NMR (400 MHz, CD3OD) δ 7.60-7.42 (m, 5 H),
7.25 (d, J = 8.0 Hz, 2 H), 7.20 (d, J = 8.0 Hz, 2 H), 6.96 (t, J =
8.0 Hz, 1 H), 6.87 (t, J = 8.0 Hz, 1 H), 6.49 (d, J = 16 Hz, 1 H),
4.29 (d, J = 12 Hz, 1 H), 3.59 (d, J = 12 Hz, 1 H), 2.89 (m, 2 H),
2.72 (m, 3 H), 2.35 (s, 3 H), 1.68 (m, 1 H), 1.54 (m, 2 H), 1.28 (m,
2 H). Anal. RP-HPLC tR = 3.45 min (method 1, purity 100.00%).
HR-MS m/z (M þ H)þ: measd 404.2338, calcd 404.2338.
(E)-N-Hydroxy-3-(4-{1-[2-(2-methyl-1H-indol-3-yl)ethyl]-
pyrrolidin-2-yl}phenyl)acrylamide (14). 14 was synthesized from
(E)-3-(4-{1-[2-(2-methyl-1H-indol-3-yl)ethyl]pyrrolidin-2-yl}-
phenyl)acrylic acid methyl ester following general procedure D in
65% yield. 1H NMR (400 MHz, CD3OD) δ 7.54 (d, J = 16 Hz,
1 H), 7.44 (d, J = 8.0 Hz, 2 H), 7.31 (d, J = 8.0 Hz, 2 H), 7.18 (d,
J = 8.0 Hz, 2 H), 6.93 (m, 1 H), 6.83 (m, 1 H), 6.44 (d, J = 16 Hz,
1 H), 3.51 (m, 1 H), 2.83 (m, 1 H), 2.72 (m, 2 H), 2.43 (m, 2 H),
2.30-2.15 (m, 2 H), 2.23 (s, 3 H), 1.93 (m, 2 H), 1.71 (m, 1 H)).
Anal. RP-HPLC tR = 3.16 min (method 1, purity 93.70%). HR-
MS m/z (M þ H)þ: measd 390.2174, calcd 390.2182.
(E)-3-{4-[3-(2-tert-Butyl-1H-indol-3-yl)piperidin-1-ylmethyl]-
3-fluorophenyl}-N-hydroxyacrylamide (30). 30 was synthesized
from (E)-3-{4-[3-(2-tert-butyl-1H-indol-3-yl)piperidin-1-ylmethyl]-
3-fluorophenyl}acrylic acid methyl ester following general proce-
dure D in 11% yield. 1H NMR (400 MHz, CD3OD) δ 7.56 (d, J =
8.0 Hz, 1H), 7.50(d, J=16 Hz, 1 H), 7.41 (t, J= 8.0 Hz, 1 H), 7.28
(m, 4 H), 6.95 (t, J = 8.0 Hz, 1 H), 6.87 (t, J = 8.0 Hz, 1 H), 6.47
(d, J = 16 Hz, 1 H), 3.64 (br s, 2 H), 3.43 (m, 1 H), 3.01 (br d, J =
12 Hz, 1 H), 2.88 (m, 1 H), 2.75 (t, J = 12 Hz, 1 H), 2.21 (m, 1 H),
2.05 (m, 1 H), 1.79 (m, 3 H), 1.49 (s, 9 H). Anal. RP-HPLC tR =
2.90 min (method 3, purity 98.48%/95.39%). HR-MS m/z (M þ
H)þ: measd 450.2559, calcd 450.2557.
(E)-3-{4-[3-(2-tert-Butyl-1H-indol-3-yl)piperidin-1-ylmethyl]-
3-chlorophenyl}-N-hydroxyacrylamide (31). 31 was synthesized
from (E)-3-{4-[3-(2-tert-butyl-1H-indol-3-yl)piperidin-1-ylmethyl]-
3-chlorophenyl}acrylic acid methyl ester following general proce-
dure D in 53% yield. 1H NMR (400 MHz, CD3OD) δ 7.65-7.47
(m, 5H), 7.30(d, J= 8.0 Hz, 1 H), 6.98-6.89 (m, 2 H), 6.48 (d, J=
16 Hz, 1 H), 3.81 (br s, 2 H), 3.48 (m, 1 H), 3.11 (m, 1 H), 2.94 (m,
2 H), 2.41 (m, 1 H), 2.15 (m, 1 H), 1.83 (m, 3 H), 1.43 (s, 9 H). Anal.
RP-HPLC tR = 8.33 min (method 2, purity 100.00%/93.81%).
HR-MS m/z (M þ H)þ: measd 466.2247, calcd 466.2261.
(E)-N-Hydroxy-3-{4-[3-(1H-indol-3-yl)piperidin-1-ylmethyl]-
phenyl}acrylamide (25). 25 was synthesized from (E)-3-{4-[3-(1H-
indol-3-yl)piperidin-1-ylmethyl]phenyl}acrylic acid methyl ester
following general procedureD in38% yield. 1H NMR (400 MHz,
CD3OD) δ 7.38 (m, 4 H), 7.22 (m, 3 H), 6.95 (m, 1 H), 6.89 (s,
1 H), 6.85 (m, 1 H), 6.39 (d, J = 16 Hz, 1 H), 3.43 (s, 2 H), 3.05 (m,
2H), 2.83(m, 1H),1.95(m,3H),1.68(m, 2H),1.43(m, 1H).Anal.
RP-HPLC tR = 2.74 min (method 2, purity 100.00%/100.00%).
HR-MS m/z (M þ H)þ: measd 376.2029, calcd 376.2025.
(E)-3-{4-[(R)-3-(2-tert-Butyl-1H-indol-3-yl)piperidin-1-ylmethyl]-
3-fluorophenyl}-N-hydroxyacrylamide (32). 32was synthesized from
2-tert-butyl-3-(R)-piperidin-3-yl-1H-indole following general pro-
cedures C and D in 45% yield. 1H NMR (500 MHz, CD3OD) δ7.59
(d, J = 10 Hz, 1 H), 7.49 (d, J = 15 Hz, 1 H), 7.46 (d, J = 10 Hz,
1 H), 7.32 (m, 4 H), 6.97 (t, J = 7.0 Hz, 1 H), 6.89 (t, J = 7.0 Hz,
1H),6.50(d, J=15Hz,1H),3.70(brs, 2H),3.46(m, 1H),3.05(br
d, J = 10 Hz, 1 H), 2.91 (br d, J = 10 Hz, 1 H), 2.79 (t, J = 10 Hz,
1 H), 2.26 (m, 1 H), 2.11 (m, 1 H), 1.82 (m, 3 H), 1.45 (s, 9 H). Anal.
RP-HPLC tR = 9.36 min (method 2, purity 100.00%/96.46%).
HR-MS m/z (M þ H)þ: measd 450.2565, calcd 450.2557.
(E)-N-Hydroxy-3-{4-[3-(2-phenyl-1H-indol-3-yl)piperidin-1-yl-
methyl]phenyl}acrylamide (26). 26 was synthesized from (E)-3-
{4-[3-(2-phenyl-1H-indol-3-yl)piperidin-1-ylmethyl]phenyl}-
acrylic acid methyl ester following general procedure D in 78%
yield. 1H NMR (400 MHz, CD3OD) δ 7.73 (d, J = 8.0 Hz, 1 H),
7.47 (m, 7 H), 7.35 (m, 4 H), 7.07 (t, J = 8.0 Hz, 1 H), 6.98 (t, J =
8.0 Hz, 1 H), 6.45 (d, J = 16 Hz, 1 H), 3.57 (dd, J = 16, 13 Hz,
2H), 3.31 (m, 1 H), 2.94 (m, 2H), 2.70 (t, J = 10 Hz, 1 H), 2.13 (m,
2 H), 1.90-1.68 (m, 3 H). Anal. RP-HPLC tR = 2.22 min
(method 3, purity 100.00%/98.07%). HR-MS m/z (M þ H)þ:
measd 452.2332, calcd. 452.2338.
(E)-3-{4-[(S)-3-(2-tert-Butyl-1H-indol-3-yl)piperidin-1-ylmethyl]-
3-fluorophenyl}-N-hydroxyacrylamide (33). 33 was synthesized
from 2-tert-butyl-3-(S)-piperidin-3-yl-1H-indole following general
procedures C and D in 34% yield. 1H NMR (500 MHz, CD3OD) δ
7.59 (d, J = 10.0 Hz, 1 H), 7.44 (d, J = 15 Hz, 1 H), 7.44 (t, J =
7 Hz, 1 H), 7.42 (d, J = 5.0 Hz, 1 H), 7.32 (d, J = 10 Hz, 2 H), 7.28
(d, J = 10 Hz, 1 H), 6.97 (t, J = 7 Hz, 1 H), 6.89 (t, J = 7 Hz, 1 H),
6.51 (d, J = 15 Hz, 1 H), 3.69 (br s, 2 H), 3.46 (m, 1 H), 3.05 (br d,
J = 10 Hz, 1 H), 2.91 (br d, J = 10 Hz, 1 H), 2.79 (t, J = 10 Hz,
(E)-3-{4-[3-(2-tert-Butyl-1H-indol-3-yl)piperidin-1-ylmethyl]-
phenyl}-N-hydroxyacrylamide (27). 27 was synthesized from
(E)-3-{4-[3-(2-tert-butyl-1H-indol-3-yl)piperidin-1-ylmethyl]-
phenyl}acrylic acid methyl ester following general procedure D in

2962 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Cho et al.
1 H), 2.25 (m, 1 H), 2.09 (m, 1 H), 1.82 (m, 3 H), 1.45 (s, 9 H). Anal.
RP-HPLC tR = 9.37 min (method 2, purity 95.69%/94.57%).
HR-MS m/z (M þ H)þ: measd 450.2555, calcd 450.2557.
inhibitors. Bioorg. Med. Chem. Lett. 2006, 16, 5948–5952. (b) Jones,
P.; Altamura, S.; De Francesco, R.; Gonzalez Paz, O.; Kinzel, O.;
Mesiti, G.; Monteagudo, E.; Pescatore, G.; Rowley, M.; Verdirame, M.;
Steinkuhler, C. A novel series of potent and selective ketone histone
deacetylase inhibitors with antitumor activity in vivo. J. Med. Chem.
2008, 51, 2350–2353.
Acknowledgment. The authors thank Weijia Zheng for
preparation of crystalline material for single-crystal X-ray
analysis, Travis Stams for access to HDAC8 X-ray crystal
structure, Robert Pearlstein for helpful advice on hERG
modeling, and Karen Miller-Moslin, Christopher Brain, and
Timothy Ramsey for helpful suggestions in the preparation of
the manuscript.
(12) (a) Kranz, M.; Murray, P. J.; Taylor, S.; Upton, R. J.; Clegg, W.;
Elsegood, M. R. J. Solution, solid phase and computational
structures of apicidin and its backbone-reduced analogs. J. Pept.
Sci. 2006, 12, 383–388. (b) The apicidin geometry was retrieved from
the Cambridge Crystallographic Database, code number HEWGOG.
Atomic coordinates were assumed to be at, or close to, a “global”
minimum and subsequently held rigid in all calculations. Dacinostat
conformations were generated as part of the alignment procedures
used in FieldView. The aligned atomic coordinates of dacinostat in
Figure 3 were inspected visually for strain and torsions using MOGUL
(CCDC).
Supporting Information Available: Figures A and B, Tables A
and B, methods of automated Q-patch clamp studies, manual
patch clamp electrophysiology and high-throughput determina-
tion of pKa, procedure of NMR experiments for compound 30
and spectra, and X-ray structure data of (S)-3-(2-tert-butyl-1H-
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(13) (a) Nonpolar hydrogens were removed for a clear image. (b) Field-
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˚
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(31) (a) The apparent discrepancy may be due to both the sensitivity
limitation of the enzyme assay and the different assay durations.
The enzyme assay measures the direct inhibition of compound on
HDAC enzyme within 1 h of assay time, whereas the cellular
proliferation assay measures the continuous compound effect over
the course of 3 days which could lead to enhanced activity. (b) To
ensure that the observed increase in the inhibition of cellular
proliferation was based on improved HDAC inhibition and not a
result of off-target activities, we screened 32 against over 60
GPCRs, enzymes, and transporters. Most of the IC50 values are
over 10 μM, whereas the lowest IC50 values are observed with
histamine H2, cyclooxygenase 2, and adrenergic R1a receptor at
1-4 μM, suggesting a relatively clean safety profile of 32.