Optimization of the in vitro cardiac safety of hydroxamate-based histone deacetylase inhibitors

Article abstract of DOI:10.1021/jm200388e

Histone deacetylase (HDAC) inhibitors have shown promise in treating various forms of cancer. However, many HDAC inhibitors from diverse structural classes have been associated with QT prolongation in humans. Inhibition of the human ether a-go-go related gene (hERG) channel has been associated with QT prolongation and fatal arrhythmias. To determine if the observed cardiac effects of HDAC inhibitors in humans is due to hERG blockade, a highly potent HDAC inhibitor devoid of hERG activity was required. Starting with dacinostat (LAQ824), a highly potent HDAC inhibitor, we explored the SAR to determine the pharmacophores required for HDAC and hERG inhibition. We disclose here the results of these efforts where a high degree of pharmacophore homology between these two targets was discovered. This similarity prevented traditional strategies for mitigating hERG binding/modulation from being successful and novel approaches for reducing hERG inhibition were required. Using a hERG homology model, two compounds, 11r and 25i, were discovered to be highly efficacious with weak affinity for the hERG and other ion channels.

Full text of DOI:10.1021/jm200388e

ARTICLE
pubs.acs.org/jmc
Optimization of the in Vitro Cardiac Safety of
Hydroxamate-Based Histone Deacetylase Inhibitors
Michael D. Shultz,* Xueying Cao, Christine H. Chen, Young Shin Cho, Nicole R. Davis,†
Joe Eckman, Jianmei Fan, Alex Fekete, Brant Firestone, Julie Flynn,‡ Jack Green, Joseph D. Growney,
Mats Holmqvist, Meier Hsu,§ Daniel Jansson, Lei Jiang, Paul Kwon, Gang Liu, Franco Lombardo,
Qiang Lu,|| Dyuti Majumdar, Christopher Meta, Lawrence Perez, Minying Pu, Tim Ramsey,
Stacy Remiszewski, Suzanne Skolnik, Martin Traebert, Laszlo Urban, Vinita Uttamsingh,^ Ping Wang,
Steven Whitebread, Lewis Whitehead, Yan Yan-Neale, Yung-Mae Yao, Liping Zhou, and Peter Atadja
Novartis Institutes for Biomedical Research, Inc., 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
S Supporting Information
b
ABSTRACT: Histone deacetylase (HDAC) inhibitors have shown
promise in treating various forms of cancer. However, many HDAC
inhibitors from diverse structural classes have been associated with
QT prolongation in humans. Inhibition of the human ether a-go-go
related gene (hERG) channel has been associated with QT prolon-
gation and fatal arrhythmias. To determine if the observed cardiac
effects of HDAC inhibitors in humans is due to hERG blockade, a
highly potent HDAC inhibitor devoid of hERG activity was
required. Starting with dacinostat (LAQ824), a highly potent
HDAC inhibitor, we explored the SAR to determine the pharma-
cophores required for HDAC and hERG inhibition. We disclose
here the results of these efforts where a high degree of pharmaco-
phore homology between these two targets was discovered. This
similarity prevented traditional strategies for mitigating hERG binding/modulation from being successful and novel approaches for
reducing hERG inhibition were required. Using a hERG homology model, two compounds, 11r and 25i, were discovered to be
highly efficacious with weak affinity for the hERG and other ion channels.
’ INTRODUCTION
hydroxamates such as vorinostat 1 (SAHA),24 belinostat 2
(PXD-101),25 dacinostat 3 (LAQ824),26 4 (SB-939),27 and ben-
zamides such as mocetinostat 5 (MGCD-0103)28 and entinostat 6
(SNDX-275 formerly MS-275),29 while Romidepsin (FK228),24,30,31
a natural product not belonging to either class, has also received
approval from regulatory agencies (Figure 1). There have been
several excellent reviews detailing additional compounds under-
going clinical investigation in this highly competitive field.32ꢀ40
Although these agents hold great promise for a variety of
indications, there has been concern about dose limiting toxicities
observed in human trials including fatigue, thrombocytopenia,
gastrointestinal toxicity, and QT prolongation.41ꢀ43 Episodes of
QT prolongation have been reported for 2, 3, 4, romidepsin,
givinostat, and JNJ-26481585, while atrial fibrillation and peri-
carditis were observed with CHR-3996 and 5, respectively.28
Because of the wide variety of chemical classes where QT
prolongation, ventricular fibrillation, and/or other cardiac
toxicities have been reported, there is speculation that the
The post-translational modification of proteins via acetylation
and deacetylation is a powerful mechanism of regulating protein
function, stability, activity, and subcellular localization.1ꢀ12 In
cancer and other diseases, enzymes that control posttranslational
modifications have altered levels of activity due to genetic and
epigenetic changes that accrue on a cellular level. One such
mechanism of posttranslational modification is the acetylation of
the terminal amino group on lysine side chains, catalyzed by
histone acetyl transferases and the deacetylation step that is
catalyzed by a family of enzymes known as histone deacetylases
(HDACs).13 Pharmacological inhibition of HDACs has been
shown to be a successful strategy to treat certain forms of cancer,
as evidenced by their approved use by regulatory agencies.14,15 In
addition to their promise as antineoplastic agents, HDAC inhibi-
tors are under extensive study to treat a wide range of disorders
including Huntington’s disease, fibrosis, cardiac hypertrophy,
multiple sclerosis, spinal muscle atrophy, and as anti-infective
agents.16ꢀ23 Several HDAC inhibitors have entered clinical devel-
opment as anticancer treatments and studies with these and other
agents toward nononcology indications are gaining momentum.
The two most prominent classes of HDAC inhibitors are
Received: April 1, 2011
Published: June 08, 2011
r
2011 American Chemical Society
4752
dx.doi.org/10.1021/jm200388e J. Med. Chem. 2011, 54, 4752–4772
|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. Select clinical and FDA approved HDAC inhibitors.
cardiovascular events are an on-target effect, but this hypothesis
had not yet been proven experimentally.44ꢀ46
low oral and cardiac exposure with this compound specifically
rather than the behavior of HDAC inhibitors in general.59 Our
efforts were primarily focused at the hERG channel, but ad-
vanced compounds were also profiled against sodium and
calcium ion channels.60 To guide our efforts, we employed
homology models of both HDAC-1 and the hERG channel
when previously reported approaches to resolve hERG binding
were not successful. Herein we report the results of these efforts
which led to the identification of several promising compounds
including 11r and 25i, which demonstrated superior antitumor
activity than both 1 and 3.
Of the HDAC inhibitors to enter late stage clinical trials, 3 has
the greatest in vitro potency against multiple HDAC isoforms as
well as the hERG ion channel.47ꢀ52 Following a number of drug
withdrawals from the market due to QT prolongation, torsade de
pointes, and sudden death, the hERG channel has become linked
with QT prolongation.53ꢀ57 As part of our effort to develop a
second-generation HDAC inhibitor, we initiated a program with
the goal of enhancing the in vitro potency and in vivo efficacy of a
series of hydroxamate based HDAC inhibitors while simulta-
neously eliminating the interaction with the cardiac ion channels
that have been linked to QT prolongation.51 We hypothesized
that a highly potent HDAC inhibitor devoid of activity against
the hERG or other ion channels would either provide a superior
clinical candidate or help determine if the observed cardiac
effects were a consequence of HDAC inhibition. None of the
active clinical compounds demonstrated a substantial (>5000
fold) window between HDAC inhibition and hERG inhibition
due to their limited HDAC potency and/or solubility. Com-
pound 1, for which the most data is publicly available, reaches
plasma exposures of only 0.96 μM following a 150 mg/kg oral
dose in dog and 1.58 μM following an 800 mg oral dose in
human, only marginally surpassing cellular IC50 values for cells
sensitive to 1.58,59 The lack of cardiac findings in the preclinical
and clinical telemetry studies of 1 therefore speaks more to the
’ CHEMISTRY
The N-hydroxy phenylacrylamide scaffold (2ꢀ4) has been
employed extensively by many groups to generate potent HDAC
inhibitors and has resulted in at least four clinical programs. This
scaffold was further explored by a variety of N-alkyl derivatives
based on the general structure 11. Structure-based drug design,
physicochemical property modulation, and a matched molecular
pair approaches were used in the design of these analogues.
Scheme 1 outlines methods where R1-NH2, typically a trypta-
mine or tryptamine analogue, was reacted with methyl 4-formyl-
cinnamate (7) via reductive amination to generate substituted
methyl 4-aminomethyl cinnamates (8). Subsequent alkylation of
the secondary amines via reductive amination or reaction with
4753
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
alkyl halides or epoxides yielded tertiary amines (9). Alterna-
tively, when R1 precursors were more readily available as an
aldehyde, they were reacted with methyl 4-aminomethylcinna-
mate (8) under similar reductive amination conditions and could
be further elaborated as described above. For some compounds,
it was necessary to construct the scaffold using similar reactions
but where sequence of the synthetic sequence was altered (e.g.,
11r). Reductive amination with the 2-H indole moiety was not
efficient under all circumstances, therefore the 2-methyl indole
moiety was optionally used due to higher yields under typical
reaction conditions (e.g., the low yielding reductive amination
reactions between fluorinated alkyl amines and 1H-indole-3-
acetaldehyde was dramatically improved by switching to 2-meth-
yl-indole-3-acetaldehyde). The secondary (8) or tertiary amines
(9) were then converted to the corresponding hydroxamates (11)
via aqueous hydroxylamine in methanolic sodium methoxide.
The synthesis of 2-heteroaryl indoles was accomplished by N-
bromosuccinimide mediated bromination of indole-3-acetoni-
trile followed by Suzuki coupling to form 2-aryl and 2-heteroaryl
indoles (12). Nitrile hydrogenation was accomplished with
rhodium on alumina or boraneꢀTHF to yield the modified
tryptamine 13 that was used in the synthesis of target compounds
(Scheme 2).
Scheme 1. General Synthesis of Hydroxamate-Based HDAC
Inhibitorsa
3-(2-Amino-ethyl)-1H-indole-2-carboxylic acid ethyl ester 14
was synthesized using the JappꢀKlingemann reaction as pre-
viously described (Scheme 3).61 Reductive amination with
4-bromo benzaldehyde proceeded smoothly using SiliaBond
cyanoborohydride. To incorporate the 2-(1H-indol-2-yl)-pro-
pan-2-ol motif, several approaches were tried, however under
basic conditions, the tryptamine nitrogen formed a lactam
with the indole 2-ethyl ester intermediate. The cyclization
reaction was prevented by alkylation of the linker nitrogen.
a Reagents: (a) NaBH(OAc)3, Et3N, THF (optional) (method A); (b)
SiliaBond cyanoborohydride, (optional); (c) alkyl halide, base; (d) alkyl
epoxide, base; (e) aldehyde, method A or SiliaBond cyanoborohydride
(f) NH2OH/H2O, NaOMe, MeOH (method B).
Scheme 2. Synthesis of 2-Substituted Tryptamine Intermediatesa
a Reagents: (a) NBS, DCM (67% yield); (b) Pd(PPh3)4, K2CO3; (c) H2 (50 psi), RhꢀAl2O3, EtOHꢀNH4OH (optional) (method C); (d) BH3-THF
(optional) (method D).
Scheme 3. Synthesis of (E)-N-Hydroxy-3-{4-[({2-[2-(1-hydroxy-1-methyl-ethyl)-1H-indol-3-yl]-ethyl}-isopropyl-amino)-
methyl]-phenyl}-acrylamide 11ra
a Reagents: (a) (i) KOH, EtOH; (ii) benzenediazonium; (b) method A; (c) isopropyl iodide, Et3N, CH3CN; (d) methyllithium, THF; (E) methyl
acrylate, Pd2dba3, 1 equiv H2O; (f) method B.
4754
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
Scheme 4. General Synthesis of Azatryptaminesa
a
3
Reagents: (a) (CH2O)n, DMA:butanol, dimethyl amine (method C); (b) (i) MeOSO Me, THF; (ii) NaCN, H2O (method D); (c) B2H6, BF3, THF
Scheme 5. Synthesis of 2-tert-Butyl-1H-indole 17fa
telemetry, measurement of myocardium concentrations, ex vivo
cardiac preparations, isolated canine Purkinje fiber, etc.).62,63
The accepted definition of the cardiac safety index (CSI) is the
ratio of hERG IC50 value (or another measure of adverse cardiac
event) and the Cmax of the dose required to generate the desired
in vivo effect.57 To allow for decision making earlier in the lead
optimization process, we introduced an in vitro cardiac safety
a Reagents: (a) (CH3)3COCl, K2CO3, toluene; (b) n-buLi, THF.
index (iCSI) parameter defined as the ratio of the hERG IC50,
either radioligand binding or the cellular patch clamp, and the
cellular IC50 used for compound profiling (antiproliferative
activity in the HCT116 cell line). Other levels of inhibition, such
as hERG IC20 or a cellular IC90, can be used and may be even
more ideal. Similar to other off-target selectivity measurements,
this approach helps identify discrete structural modifications that
modulate the desired safety window and does not provide false
comfort when compounds have weak in vitro activity.
Following N-alkylation with isopropyl iodide, methyllithium
addition resulted in 15. A Heck reaction with one equivalent of
water yielded 16a, which was converted to the desired hydro-
xamate 16b using aqueous hydroxylamine in methanolic sodium
methoxide.
The synthesis of several azaindole derivatives began with
functionalization of the 3-position in a straightforward manner
via a Mannich reaction (method E) between commercially
available azaindoles (17aꢀf) and dimethylamine in the presence
of paraformaldehyde. Conversion of 18 to the quaternary ammo-
nium salt followed by a retro-Michael addition reaction with
sodium cyanide (method F) resulted in 19 in good yields. Nitrile
reduction using rhodium on alumina led to the desired aza-
tryptamine analogues 20 (Scheme 4). Conversion to the desired
hydroxamates was accomplished as described in Scheme 1.
Several azaindoles that were not commercially available were
synthesized as described below. The synthesis of 2-tert-butyl-1H-
pyrrolo[2,3-b]pyridine 17f (Scheme 5) was accomplished using
modifications of previously reported methods. Subsequent ela-
boration to the azatryptamine 20g was performed as described
above. Reaction of 1-amino pyridinium iodide with a variety of
substituted propiolic acid ethyl esters provided 2-substituted
pyrazolo[1,5-a]pyrimidines 21aꢀc (Scheme 6). Reduction of
the ester with lithium aluminum hydride followed by manganese
dioxide oxidation to the aldehyde proceeded in good to excellent
yields. Conversion to the nitroethylene 23 was high yielding,
however, diborane reduction to azatryptamines 24 were low
yielding but scalable, so sufficient quantities of the azatryptamine
could be prepared. Elaboration to 25hꢀ25n proceeded as
described in Scheme 1.
hERG IC50
CSI ¼
CmaxðunboundÞ
hERG IC50
hERG IC50
iCSI ¼
¼
HCT116 IC50
cellular IC50
It has been recommended that, at minimum, the hERG IC50
should be at least 30-fold higher than the free fraction Cmax of a
given compound in vivo and a 100-fold margin is generally
preferred.57 Because it is likely that efficacious concentrations at
Cmax will be several orders of magnitude higher than the cellular
antiproliferative IC50s, an iCSI goal of 5000 was set to provide
sufficient margin of safety prior to the initiation of in vivo studies.
In part, this ratio was chosen to be approximately 10-fold higher
than the iCSI calculated for 3. We profiled our compounds
against the hERG channel in radioligand binding, manual patch
clamp, and automated Q-Patch assays.64,65
hERG activity has been reported to be dependent on several
calculable properties including lipophilicity (clogP), amine basi-
city (pKa), and three-dimensional fit into a pharmacophore
model. A large number of HDAC inhibitors from our earlier
efforts were screened for hERG activity. For these compounds,
the pKas were determined experimentally to determine how well
hERG potency correlated with clogP and pKa. Whereas clogP
and hERG activity appeared to be only weakly correlated
(Supporting Information Figure 1), the basicity of the amine
appeared to have some correlation with hERG patch clamp
activity (data not shown). The interpretation of clogP SAR is
complicated by potential errors in calculation as well as the
inherent challenge of modulating this single parameter without
affecting other factors known to affect affinity for the hERG
’ RESULTS AND DISCUSSION
The determination of cardiac safety risk with preclinical and
clinical compounds has been an area of intense efforts and
debate.57 Unfortunately, the current standard for assessing the
cardiac safety of a particular compound requires information
available only after the initiation of clinical development (e.g.,
unbound concentrations at Cmax in human) or from an integrated
risk assessment approach with extremely low throughput (in vivo
4755
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
Scheme 6. Synthesis of 2-Substituted Pyrazolo[1,5-a]pyridin-3-yl-ethylamine Analoguesa
a Reagents: (a) K2CO3, DMF; (b) LiAlH4, THF; (c) MnO2, THF; (d) NH4OAc, MeNO2; (e) B2H6, BF3, THF.
channel such as distance between the amine and a hydrophobe,
zwitterions, and the introduction of additional substituents such
as β-carbonyl, β-hydroxyl, or other moieties that attenuate amine
basicity. On the basis of the apparent pKa correlation and
previously reported successes against hERG achieved by mod-
ulating amine basicity, we decided to more closely investigate the
role the linker amine basicity of 11 played in affecting HDAC and
hERG activity. The interpretation of pKa SAR may not be
straightforward due to multiple molecular parameters that are
concomitantly altered. To aid our investigation, we made a series
of N-ethyl and N-propyl derivatives (Table 1, compounds
11bꢀh) where the hydrogen atoms on the terminal methyl
groups were replaced with fluorine atoms in a stepwise
manner. The inductive effects of the fluorine atoms decrease
the basicity while having negligible effects on the ligand size,
flexibility, clogP, or the overall pharmacophore. The amine
basicity was thus modulated in relative isolation from other
variables that may obfuscate the HDAC and hERG SAR
interpretations.
Within analogues of the general formula 11, an increase in
hERG and HDAC binding potency is observed upon conversion
to a tertiary amine (Table 1). The ethyl substituent of 11b
increased both the HCT116 cellular and hERG potency nearly
5-fold relative to 11a. A single fluorine atom on the terminal
methyl group (11c) had no appreciable affect on HDAC inhibi-
tion or hERG affinity. When a difluoromethyl (11d) or triflu-
oromethyl group (11e) was incorporated, hERG binding was
completely abolished (<5% inhibition at 30 μM), and there was
also a 100ꢀ200-fold decrease of HCT116 antiproliferative
activity. With two or three fluorine atoms incorporated, the
amine basicity is more significantly reduced and thus the equi-
librium at physiological pH lies entirely on the side of the neutral
amine species. In an attempt to attenuate the inductive effects in a
more subtle manner, an additional methylene group was intro-
duced to insulate the amine from the di- and trifluoromethyl
groups (11g and 11h). There was also a sharp reduction of both
hERG and HDAC inhibition with both of these homologated
analogues relative to 11f. Although it was predicted that the 3,3,3-
trifluoropropyl group would adjust the pKa of 11h between that
of 11c and 11d, the measured pKas of the ethyl series were higher
than predicted and the measured pKas of the propyl series were
lower than predicted (Table 1). The modifications at R2 do not
significantly affect the measured hydroxamate pKa (data not
shown), therefore all changes in affinity were attributed to the
alterations in protonated amine equilibrium. The ammonium
ion species distributions in this series was calculated (Figure 2),
and only the ethyl and monofluoro analogues (11b and 11c,
respectively) were predicted to have any ammonium species
present at physiological pH.
Although correlations between pKa and hERG binding have
been well documented, we are unaware of a similar relationship
with regard to HDAC inhibition. To better understand why
HDAC inhibition was so highly sensitive to the basicity of the
amine linker, an HDAC homology model was analyzed. The
docked structure of 11a places Asp99 and the central amine of
this scaffold within 3.4 Å, suggesting an ionic interaction
(Figure 3). The dacinostat scaffold (11) has demonstrably
greater potency than other hydroxamate and benzamide based
HDAC inhibitors47 that lack a linker with a basic amine. The
importance of this interaction is further illustrated when the
measured pKa values were plotted against the HDAC-1 enzy-
matic and HCT116 cellular IC50 values (Figure 4). The effect of
lipophilicity is negligible as a factor in HDAC potency between
11b and 11e (ΔclogP = 0.264), however a nearly 200-fold
difference in cell growth inhibition is observed. It is interesting
to note that when the amine basicity was reduced to effectively
eradicate the ammonium species at physiological pH, the anti-
proliferative activity was indistinguishable from 1. The correla-
tion between pKa, hERG, and HDAC inhibition indicates that
modulation of amine basicity would be an unsuccessful approach
for improving the iCSI within this series.
Another proven approach for reducing hERG activity is the
formation of zwitterions by the incorporation of a carboxylic acid
or similar motif (e.g., fexofenadine).66 The 2-position of the
indole ring position was chosen for its ease of substitution and
exposure to solvent in our docking models (Figure 3). The
HDAC-1 enzymatic potency of 11j was slightly reduced com-
pared to 11a, however in several cellular assays, no appreciable
activity was observed, presumably due to decreased cellular and
nuclear penetration (Table 2). It appeared that incorporation of
4756
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
Table 1. Inhibitory Activity and iCSI of Compounds against HDAC-1, HCT116, and hERG in Relation to the pKa of the Linker
Amine
a Radioligand binding assay. b Manual patch clamp assay.
zwitterions would not be a successful approach, thus alternate
approaches were considered.
our favor with the introduction of amphiphilic modifications.
The 2-phenyl indole analogue 11k improves HCT116 cell
growth inhibition by an order of magnitude relative to 11a,
however the iCSI is reduced due to a more dramatic increase in
hERG inhibition. The 2-pyridyl analogue 11l has a lower iCSI
than the phenyl analogue 11k, but the potential to form an
internal hydrogen bond between the pyridyl nitrogen and indole
NH may have rendered this analogue less polar than first
anticipated. The 3-pyridyl 5-methoxy moiety 11m was designed
to force the pyridine ring out of planarity with the indole ring and
thus prevent the formation of a similar internal hydrogen bond.
The iCSI of this analogue is improved relative to 11k and 11l, and
we therefore decided to further explore this tactic of utilizing
amphiphilicity as a wedge between hERG and HDAC activity.
We next examined 5-membered heterocycles at the indole
2-position, and one of the most potent hERG blockers within this
We next explored discrete structural modifications aimed at
exploiting structural differences between the HDAC and hERG
proteins and utilized a previously disclosed hERG homology
model.67 11a was docked into this model (Figure 5), and several
key interactions appeared to correlate with the hERG SAR in this
series. In the hERG homology model, the amine sits in a
hydrophilic pocket (gray), while the indole and phenyl rings
bind within a hydrophobic belt (brown) in the hERG channel.
The HDAC homology model was in agreement with data that
the linker amine and the phenyl ring of 11 make key interactions
with Asp99 and Phe205, respectively (Figure 3). The indole
moiety lies on the surface of the HDAC enzyme but in a
hydrophobic pocket when docked into the hERG homology
model. We hypothesized that these differences could be used in
4757
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
series was 11n (IC50 = 480 nM). Low yielding Suzuki reactions
hampered a more thorough exploration at this position, however
11o demonstrated that bulky, polar substituents at this position
could significantly decrease hERG activity. The loss of HDAC
activity of 11o was surprising given the range of steric bulk that
had previously been tolerated at this position (e.g., 11m). 11p has
an improved iCSI relative to 11a. We wondered if replacing a
methyl group of 11p with a hydroxyl group might provide the best
balance of steric bulk while simultaneously introducing some
lipophilicity to disfavor binding to the hERG channel. While the
direct hydroxy analogue of 11p was not synthesized, 11r was
tested in a manual patch clamp assay and the IC50 in this assay was
determined to be >30 μM (36% inhibition at 30 μM) while being
nearly 5-fold more potent in the HCT116 cell growth inhibition
assay than 3, resulting in an iCSI greater than 7500. By way of a
comparison of 11q and 11a, we conclude that the reduction in
hERG binding was not due to the incorporation of the N-
isopropyl moiety. Given this evidence that tertiary amines in-
crease hERG channelblockade withinthisseries, 11r validated the
hypothesis of using targeted amphiphilicity to improve the iCSI.
The PK parameters of 11r in HCT116 tumor bearing nude
in vivo PK is dominated by a high volume of distribution, rapid
extra hepatic clearance, and a short half-life which limits the
overall exposure when dosed orally or intravenously. The in vivo
PK of 11r was similar to other compounds in this class that we
have examined (data not shown), however the high in vivo
clearance was greater than predicted by the stability observed in
mouse liver microsomes. To investigate this apparent in vitro/
in vivo disconnect further, we examined the plasma stability of
11r. In mouse plasma, only 54% of the compound remained after
three hours of incubation indicating that plasma instability was
likely contributing to the rapid clearance. Compound 3, like
other analogues based on scaffold 11, has low rodent plasma
stability but is stable in human plasma, suggesting a species-
dependent effect that could overestimate the rate of human
clearance. Internal studies demonstrated hydroxamates in this
series are stable in thermally denatured mouse plasma. Taken
together with recent publications point to enzymatic processing
of esters and hydroxamates in rodent plasma, rat PK may be an
extremely poor predictor of human PK with this class of
compounds.68 For these reasons, rodent PK was a less decisive
factor in compound progression with this class of compounds.
Because of the low oral exposure in mice, the in vivo efficacy of
11r was evaluated in the HCT-116 human colon xenograft model
using iv administration (Table 3). Following a determination of
€
mice and naive SpragueꢀDawley rats were determined. The
Figure 2. Calculated species distribution plots of tertiary amines
11bꢀe. The predicted amount of the protonated amine species is
plotted as a function of pH.
Figure 4. Experimentally determined pKa versus in vitro activity.
Triangles represent activity in the HDAC-1 biochemical assay. Squares
represent activity in the HCT116 cellular proliferation assay.
Figure 3. HDAC-1 Homology model with the docked structure of 11a. Proposed interactions of 25i and HDAC-1 enzyme based on homology model.
4758
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
Table 2. Inhibitory Activity and iCSI of Compounds against HDAC-1, HCT116, and hERG
a Radioligand binding assay IC50. b Manual patch clamp assay (% inhibition at 30 μM).
the maximum tolerated dose (MTD: defined as the dose that
resulted in no deaths and less than 20% body weight loss
following 5 days of dosing), animals were treated once daily
with 11r at 10 mg/kg, iv, for a total of 7 days. Results are reported
as %T/C, determined by the ratio of the change in tumor volume
of treated animals to the change in tumor volume of the control
animals, or % regression, determined by the ratio of the change in
tumor volumes of the treated animals to the initial volume of the
treated group. At this dose, the maximum efficacy observed was
22% tumor regression and mean body weight change of ꢀ17.7%
(Figure 6). Dosing was discontinued on day 7 due to body weight
loss greater than 20% in a subset of animals. The positive control,
5-fluorouracil, administered at 75 mg/kg, iv, resulted in T/C =
43%. Tumor regressions were also observed in the HH human
cutaneous T-cell lymphoma (CTCL) xenograft model following
once daily treatment with 11r at 5 mg/kg (data not shown).
Building on the success of amphiphilic modifications, we
focused on improving the iCSI by replacing the indole moiety
with a more polar heterocycle. A systematic exploration of
azaindole analogues of 11a was initiated where single and double
nitrogen replacements were synthesized. While analogues with
nitrogen replacements of C4ꢀC7, either singly or in combina-
tion (25aꢀe), resulted in no appreciable hERG inhibition up to
30 μM (Table 4), the HCT116 antiproliferative activity in this
series was also diminished to varying degrees. Of these analogues,
the 7-azaindole analogue 25e retained the most HCT116 anti-
proliferative activity and was explored further. Introduction of a
tert-butyl group at the 2-position of 7-azaindole ring resulted in
25f with an iCSI at least 16-fold better than 3.
For C8 and C9 azaindoles, the imidazo[1,2-a]pyridine mod-
ification 25g was not well tolerated but more encouraging results
were obtained with the pyrazolo[1,5-a]pyrine analogue 25h.
This modification was the only azaindole that increased the
HDAC-1 enzymatic and cellular antiproliferative activity with a
concomitant reduction of affinity for the hERG ion channel
relative to 3. The SAR was expanded at the 2-position of the
pyrazolo[1,5-a]pyrine and on the amine nitrogen of the linker.
Introduction of a 2-methyl group (25i) resulted in an additional
4-fold improvement in cellular activity without an apparent effect
on hERG activity, resulting in an iCSI of greater than 6667-fold.
4759
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
Figure 5. hERG homology models of 11a with transparent binding
surface (top) and binding preferences (bottom). Orange volumes
represent areas in which hydrophobic groups are favored, while the
light-blue volumes represent regions in which hydrophilic groups are
favored.
Figure 6. Tumor volumes and body weight changes in the HCT-116
human colon xenograft model in athymic female nude mice following
treatment with vehicle, 11r, or 5-fluorouracil. Treatments were initiated
on day 13 post implantation when mean tumor volume reached
approximately 180 mm3. 11r was administered in tartaric acid, iv, qd
for 8 total doses. 5-Fluorouracil was administered in 1ꢁ phosphate
buffered saline as a single dose. Data is expressed as mean ( standard
error of the mean (SEM). p < 0.05 compared to vehicle controls using a
one-way ANOVA.
Table 3. PK Parameters of 11r in SpragueꢀDawley Rats and
HCT116 Tumor Bearing Nude Mice
mean PK parameters
rat
mouse
route
iv
po
iv
po
above the HCT116 LD50 value (4 nM) for 24 h while plasma and
tumor concentrations are well below the hERG IC50. Despite
high in vivo clearance and low plasma stability, plasma levels of
25i remained above the cellular LD50 and well below the hERG
IC50 (31% inhibition at 30 μM) for 8 h, suggesting the potential
for achieving a suitable CSI following oral administration in
higher species. Although the plasma exposure of 25f is slightly
higher than 25i in mice when dosed orally, 25f could not be
quantified in the tumor homogenate. On the basis of excellent
solubility (>1 g/L at pH 6.8) and higher tumor exposure, 25i was
selected for in vivo efficacy studies.
The in vivo efficacy of 11r and 25i were compared against
previous studies of 1 and 3. Because of poor solubility, we were
unable to formulate and dose 1 greater than 150 mg/kg iv.
Although others have reported modest efficacy at these doses, we
did not observe significant in vivo tumor growth inhibition in our
efficacy models at these doses (data not shown). Because 1 had
demonstrated clinical efficacy, we chose to compare 11r and 25i
with the published HCT116 xenograft data from the FDA
submission of 1, where 57% growth inhibition was observed
(Figure 7). At 100 mg/kg, iv, 3 resulted in statistically significant
tumor growth inhibition with a T/C = 20%. Although 11r
was not tolerated when dosed on a daily schedule for more than
one week, significant tumor regressions on a truncated dosing
dose (mg/kg)
5
10
5
20
AUC (μM h)
1.307
66
7
0.336
0.463
176
5.9
1.0
0.0653
3
CL (mL/min/kg)
Vss (L/kg)
T1/2 (h)
1.3
Cmax (μM)
F %
0.204
5
0.056
4
Larger hydrophobic groups such as ethyl (25j) and phenyl (25k)
had little effect on cell growth, but in the case of 25k, apparently
re-established the hERG pharmacophore (hERG IC50 = 10.2
μM). Introduction of the isopropyl group on the linker nitrogen
resulted in increased affinity for the hERG ion channel, confirm-
ing the difficulty of avoiding hERG activity with tertiary amines
on this scaffold. It is noteworthy that in each and every case the
azaindoles abrogated hERG activity and even facilitated the
reintroduction of additional hydrophobic groups with a minimal
increase in hERG blockade.
Compounds 25f and 25i were selected for more extensive in
vitro and in vivo characterization. Compound 25i has higher
intravenous exposure, lower clearance, and a lower volume of
distribution than 11r or 25f (Table 5). After a single 5 mg/kg
intravenous dose, tumor concentrations of 25i are maintained
4760
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
Table 4. Inhibitory Activity and iCSI of Compounds against HDAC-1, HCT116, and hERGa of Azaindole Analogues
a Radioligand binding assay (% inhibition at 30 μM). b hERG patch clamp assay.
schedule were observed, suggesting that greater in vitro potency
in the cellular antiproliferation assays may translate into greater
in vivo efficacy with a better tolerated compound.
On a daily intravenous dosing schedule, compound 25i dosed
at 25 or 50 mg/kg resulted in antitumor activity and tumor
regressions, respectively, following 13 days of treatment in the
HCT-116 human colon xenograft model (Figure 8). The anti-
tumor activity of 25i corresponded to approximately 15% mean
body weight loss at both dose levels. Common dose limiting
toxicities of HDAC inhibitors include anorexia, fatigue, body
4761
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
Table 5. PK Parameters of 25i and 25f in SpragueꢀDawley
Rats and HCT116 Tumor Bearing Nude Mice
25i
25f
mean PK parameters
rat
mouse
po
mouse
route
iv
po
iv
iv
po
dose (mg/kg)
2
10z
5
20
5
20
AUC (μM h)
2.56
36.5
1.99
0.915
5.10
46.4
1.46
1.44
0.362
2.44
95.0
4.5
3.3
0.474
3
CL (mL/min/kg)
Vss (L/kg)
T1/2 (h)
Figure 8. Tumor volumes and body weight changes in the HCT-116
human colon xenograft model in athymic female nude mice following
treatment with vehicle or 25i at 25 or 50 mg/kg. Treatments were
initiated on day 12 post implantation when mean tumor volume reached
approximately 193 mm3. 25i was administered in tartaric acid, iv, qd for
13 total doses. Data is expressed as mean ( standard error of the mean
(SEM). p < 0.05 compared to vehicle controls using a one-way ANOVA.
Cmax (μM)
F %
0.061
nd
0.202
2
0.849
4
Table 6. Comparison of the in Vivo Efficacy of 11r and 25i
with 1 and 3 in the HCT116 Xenograft Model
body wt
dosea
duration
%
change
survive
total
compd (mg/kg)
(days) % T/C regressions (% yield)
vehicle
100
43
ꢀ1.8
NR
8/8
NR
8/8
8/8
8/8
8/8
8/8
1
150 ipb
100
10
21
14
8
3
20
1.4
11r
22
52
>15
10
8
>15
25i
25
13
13
10
ꢀ14.2
ꢀ14.8
50
9
a Dosed iv on a QD schedule. b Dosed ip on a QD schedule (data from
NDA no. 21991 study report PD005, FDA submission). NR: not
reported.
Figure 7. Plasma and tumor pharmacokinetics of 25i in HCT116 tumor
bearing mice.
weight loss, thrombocytopenia, lethargy, and gastrointestinal
effects. In the current studies, the more pronounced efficacy
and body weight loss of 11r and 25i may be reflective of the
greater HDAC potency of these compounds rather than any
generalized toxicity of the compounds per se, however more
definitive toxicology studies are warranted to determine if these
speculations are accurate (Table 6).
Table 7. Comparison of Cellular Activity and iCSI of 11r, 25f,
and 25i with Select Clinical HDAC Inhibitors
IC50 (nM)
hERG inhibition
compd
HCT116
H1299
hERGa
at 30 μM (%)
iCSId
The iCSI of several HDAC inhibitors that are undergoing
clinical trials were determined and compared with several
compounds in this series. Whereas 3 is the only clinical com-
pound in this set that exhibits activity in the hERG patch clamp
assay at concentrations less than 30 μM, the remaining com-
pounds are significantly less potent in the HCT116 proliferation
assay, creating a challenge in the assessment of cardiac safety.
Because cardiac safety is typically predicted based on ratios of
concentrations needed to affect the hERG channel function and
those needed for target inhibition, low patch clamp activity does
not provide for a complete safety assessment and may lead to
unexpected results in more advanced and costly animal models,
especially with compounds of relatively low on-target activity or
with the potential for high Cmax levels. Many organizations
choose to use hERG IC20 instead of hERG IC50 for this very
reason, and it is worth noting that in our assays 1 achieves 18%
inhibition at 30 μM in the automated QPatch assay. For the
clinical compounds in Table 7, the iCSI ranges from greater than
1
810
160
15
8200
460
100
1440
>4000
26
>30,000
>30,000
10,300b
>30,000
>30,000
>30,000b
>30,000
>30,000
18
18
57
30
38
5c
>37
>188
542
2
3
5
310
670
4
>97
6
>45
11r
25f
25i
>7500
>9000
>7000
3.4
21
34
4.5
29
31
a AutomatedQ-patchassay. b Manualpatchclamp.c Manualpatchclamp%
inhibition at 10 μM. d iCSI calculated from HCT116 cellular data.
37-fold for 1 to greater than 542-fold for 3. Compounds 11r, 25f,
and 25i have at least another order of magnitude improvement in
their CSI relative to 3. Combined with the enhanced potency of
the compounds in this series, the iCSI of at least 4 orders of
magnitude suggests that with a suitable ADME profile, these
compounds might be able to effectively inhibit HDAC-1 without
4762
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
significant hERG blockade in vivo. By employing iCSI as a
decision making tool during lead optimization, compounds were
selected for in vivo studies that ultimately allowed toxicology
studies to understand which cardiac observations may be off-
target and which may be directly attributable to HDAC inhibi-
tion in vivo.
Compounds 6, 11a, 11b, 11f, 11k, 11l, 11n, 11p, and 11q were
prepared as previously described. The purity of all compounds was
determined using high resolution HPLC/MS characterization and
found to be >95% pure except as noted. High resolution LC/ESI-MS
data were recorded using an Agilent 6220 mass spectrometer with
electrospray ionization source and an Agilent 1200 liquid chromato-
graph with a gradient from 5% to 95% acetonitrile in water on a C18
reverse phase column with a diode array detection. The resolution of the
MS system was approximately 11000 (fwhm definition). HPLC separa-
tion was performed using one of the methods (denoted in the header
text) listed at end of report. Purine and hexakis(1H,1H,3H-tetrafluor-
opropoxy)phosphazine (protonated molecules m/z 121.05087 and
922.00979, respectively) were used as a reference. The mass accuracy
of the system has been found to be <2 ppm.
’ CONCLUSIONS
In conclusion, utilizing previously published approaches to
mitigate hERG affinity in a series of hydroxamate based HDAC
inhibitors, it was discovered that the pharmacophore for these
two targets share a high degree of similarity and were not readily
differentiated. A new approach toward reducing hERG activity
was required and a more targeted approach utilizing homology
models was employed. Compounds 11r and 25i were discovered
through a novel approach of seeking divergence between the
hERG and HDAC homology models based on the targeted
introduction of amphiphilicity. This tactic may have broader
application to other scaffolds that are recalcitrant to hERG
optimizations, but the reliability of this approach needs to be
evaluated more extensively before any general conclusions can be
made. Analogues with an improved iCSI were generated and
found to be efficacious in mouse tumor xenograft models. When
compared to several HDAC inhibitors in clinical trials, 11r and
25i are more potent, efficacious, and provide a greater in vitro
cardiac safety margin with which the QT effects observed in
clinical trials could been investigated in preclinical safety models
with the aim of translating these findings into a clinical setting.
Further work in this area is ongoing, and the results of these
studies will be published in due course.
Method A. Typical procedure for reductive amination using
NaBH(OAc)3:
To a solution of the tryptamine analogue (1 mmol) and (E)-3-(4-
formyl-phenyl)-acrylic acid methyl ester (0.96 mmol) in THF (4 mL)
was added solid NaHCO3 (2.5 mmol), and the mixture was stirred for 48
h at ambient temperature. NaBH(OAc)3 (1.4 mmol) was then added
and the mixture was stirred until judged complete by LCMS analysis,
typically 5ꢀ16 h. It was quenched with aqueous NH4Cl and washed with
excess EtOAc (2ꢁ). The combined organic layers were dried (MgSO4),
filtered, concentrated, and purified by silica gel chromatography.
Method B. Typical procedure for the conversion of esters to
hydroxamic acids:
To a stirred solution of a methyl ester (4 mmol) in methanol (5 mL)
at 0 ꢀC was added sodium methoxide (5 equiv, 20 mmol of a 25%
solution in methanol) and 10 equiv of hydroxylamine (50% solution in
water). The reaction is monitored by LCMS, and upon completion the
reaction mixture was brought to pH 7ꢀ8 with 1 N hydrochloric acid
whereupon a precipitate formed. The solid is filtered, washed with water,
and dried in vacuo to yield the desired hydroxamic acid.
Method C. Typical procedure for the formation of 3-dimethylami-
nomethyl azaindoles:
’ EXPERIMENTAL SECTION
A mixture of azaindole (22 mmol), dimethylamine hydrochloride
(2.0 g, 25 mmol), and paraformaldehyde (0.75 g, 2.5 mmol) in 1-butanol
(60 mL) were stirred at reflux for 3 h. The resulting solution was
evaporated in vacuo and purified via flash column chromatography.
Method D. Typical procedure for the formation of 3-azaindole
acetonitriles from 3-dimethylaminomethyl indoles:
Sulfuric acid dimethyl ester (2.1 g, 16 mmol) was added to a solution
of dimethyl 3-azindoylmethyl-amine (15 mmol) in THF (12 mL). The
mixture was stirred at reflux for 30 min. The solution was then placed in
an ice bath, and most of the THF is decanted out to give a gummy
intermediate which was washed several times with ether. Water (6 mL)
and sodium cyanide (950 mg, 19 mmol) were added, and the solution
was then stirred at reflux for 1 h. Upon completion of the reaction, the
solution was cooled and extracted with EtOH:DCM (1:4) several times.
The combined organic layers were washed with brine and dried over
Na2SO4. The organic solvent was removed to provide crude product,
which was purified via flash column chromatography (MeOH:DCM,
1:99 to 20:90).
Method E. (2-Methyl-1H-indol-3-yl)-acetaldehyde. Starting with
(2-methyl-1H-indol-3-yl)-acetic acid ethyl ester (4.0 g, 18 mmol) the
title compound (2.3 g, 74% yield) was prepared according to the
previously described procedure.69 1H NMR (400 MHz, CDCl3) δ
9.69 (t, J = 2.9 Hz, 1 H), 7.99 (br, 1 H), 7.48 (d, J = 7.5 Hz, 1 H),
7.33 (d, J = 8.0 Hz, 1 H), 7.16 (ddt, J = 19.6, 10.4, 3.7 Hz, 2 H), 3.74 (d,
J = 3.0 Hz, 2 H), 2.42 (s, 3 H). MS m/z 174.1 (MH+).
(E)-3-{4-[(2-Fluoro-ethylamino)-methyl]-phenyl}-acrylicAcidMethyl
Ester (8c). 2-Fluoro-ethylamine hydrochloride (110 mg, 1.1 mmol) was
added to a solution of (E)-3-(4-formyl-phenyl)-acrylic acid methyl ester
(7) (200 mg, 1.1 mmol) and acetic acid (0.5 mL) in DMF (2 mL) at
Materials and Methods. Manual Patch Clamp Electrophysiol-
ogy. Cell culture: HEK293 cells stably transfected with the R-subunits of
the hERG (University of Wisconsin, USA) were continuously main-
tained and passaged using standard cell culture media (Gibco-BRL,
Switzerland) For experiments, the cells were plated onto sterile glass
coverslips in 35 mm2 dishes at a density of 1.1ꢀ1.5 ꢁ 105 cells per dish.
The dishes were stored in a humidified and gassed (5% CO2) incubator
at 37 ꢀC until use.
Test system: The effect on hERG currents was assessed by means of
the patch clamp technique in the whole-cell configuration at 35 ( 2 ꢀC.
The corresponding vehicle for all superfusion concentrations was 0.1%
DMSO. The vehicle effect was investigated in 5 cells. The effect of the
positive controls 100 nM E-4031 (Calbiochem, Switzerland) was
investigated in 2 cells. Cells were exposed to the test item for
approximately 10 min of hERG tail currents were elicited by voltage
jumps from ꢀ75 mV to +10 mV (500 ms) and then to ꢀ40 mV (500
ms) at 0.1 Hz. The composition of the extracellular solutions was [mM]:
NaCl 137; KCl 4; CaCl2 1.8; MgCl2 1.0; D-glucose 10; N-2-hydro-
xyethylpiperazine-N0-2-ethanesulfonic acid (HEPES) 10; pH 7.4
(adjusted with 5 M NaOH). The composition of the pipet solutions
was (mM): KCl 130; MgCl2 1.0; ethylene glycol-bis(ss-aminoethyl
ether)-N,N,N0,N0-tetraacetic acid (EGTA) 5; Mg-ATP 5; HEPES 10;
pH 7.2 (adjusted with 1 M KOH). The compound effect on the currents
was corrected by the mean vehicle rundown which was observed (n = 5)
in the two cell lines. Data capturing and analysis was performed by using
Pulse (Heka Electronics, Germany) and Excel.
Enzymatic, cellular, and in vivo efficacy studies were performed as
previously described.52
4763
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
room temperature. The mixture was stirred at room temperature for
10 min. SiliaBond cyanoborohydride (1.1 g, 1.1 mmol, 1 g/mmol) was
added and stirred at room temperature for another 10 min. The mixture
was then heated via microwave irradiation at 150 ꢀC for 5 min, filtered,
and concentrated under vacuum. The crude product was purified via
flash column chromatography (EtOAc:hexanes, 10:90 to 100:0; MeOH:
DCM, 1:99 to 10:90) to provide product (100 mg, 38% yield). 1H NMR
(400 MHz, CDCl3) δ 7.70 (d, J = 15.9 Hz, 1 H), 7.50 (d, J = 8.1 Hz, 2 H),
7.42 (d, J = 8.2 Hz, 2 H), 6.44 (d, J = 16.0 Hz, 1 H), 4.55 (dt, J = 47.7,
5.0 Hz, 2 H), 3.83 (s, 3 H), 3.72 (s, 2 H), 2.82 (dt, J = 26.9, 5.1 Hz, 2 H).
MS m/z 238.2 (MH+).
8g (460 mg, 1.7 mmol) and (2-methyl-1H-indol-3-yl)-acetaldehyde
(200 mg, 1.2 mmol). H NMR (400 MHz, CDCl3) δ 7.90 (br, 1 H),
1
7.74 (d, J = 16.0 Hz, 1 H), 7.51 (d, J = 8.4 Hz, 2 H), 7.40 (d, J = 8.0 Hz,
2 H), 7.32 (d, J = 7.8Hz, 1 H), 7.24 (dt, J = 8.0, 0.9Hz, 1 H), 7.10 (ddd, J =
8.1, 7.0, 1.2 Hz, 1 H), 7.03 (ddd, J = 7.8, 7.0, 1.0 Hz, 1 H), 6.48 (d, J = 15.9
Hz, 1 H), 3.87 (s, 2 H), 3.85 (s, 3 H), 2.96 (t, J = 13.2 Hz, 2 H), 2.89ꢀ2.79
(m, 4 H), 2.29 (s, 3 H), 1.65 (t, J = 18.8 Hz, 3 H). MS m/z 427.2 (MH+).
(E)-3-(4-{[[2-(1H-Indol-3-yl)-ethyl]-(3,3,3-trifluoro-propyl)-amino]-
methyl}-phenyl)-acrylic Acid Methyl Ester (9h). Following method A,
the title compound (160 mg, 41% yield) was prepared from 8h (300 mg,
0.90 mmol) and 3,3,3-trifluoro-propionaldehyde (105 mg, 0.94 mmol).
1H NMR (400 MHz, CDCl3) δ 7.97 (s, 1 H), 7.71 (d, J = 16.1 Hz, 1 H),
7.49ꢀ7.46 (m, 3 H), 7.39ꢀ7.35 (m, 2 H), 7.20 (ddd, J = 8.1, 6.9, 1.3 Hz,
1 H), 7.09 (ddd, J = 8.0, 7.2, 1.0 Hz, 1 H), 7.00 (d, J = 2.9 Hz, 1 H), 6.46
(d, J = 16.3 Hz, 1 H), 3.84 (s, 3 H), 3.73 (s, 2 H), 2.99ꢀ2.93 (m, 2 H),
2.90ꢀ2.81 (m, 4 H), 2.36 (m, 2 H). MS m/z 431.2 (MH+).
(E)-3-{4-[(2,2-Difluoro-ethylamino)-methyl]-phenyl}-acrylic Acid
Methyl Ester (8d). Starting from 2,2-difluoro-ethylamine (250 mg,
3.1 mmol) the title compound was prepared following the procedure
for 8c (550 mg, 70% yield). 1H NMR (400 MHz, CD3OD) δ 7.69 (d,
J = 16.3 Hz, 1 H), 7.58 (d, J = 8.3 Hz, 2 H), 7.40 (d, J = 8.3 Hz, 2 H), 6.52
(d, J = 15.6 Hz, 1 H), 5.90 (tt, J = 56.3, 4.3 Hz, 1 H), 3.84 (s, 2 H), 3.78
(s, 3 H), 2.90 (td, J = 15.6, 4.6 Hz, 2 H). MS m/z 256.1 (MH+).
(E)-3-{4-[(3,3-Difluoro-propylamino)-methyl]-phenyl}-acrylic Acid
Methyl Ester (8g). Starting from 3,3-difluoro-propylamine hydrochlor-
ide (250 mg, 2.1 mmol) the title compound was prepared following the
procedure for 8c (460 mg, 81% yield). MS m/z 270.1 (MH+).
(E)-3-[4-({(2-Fluoro-ethyl)-[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino}-
methyl)-phenyl]-acrylic Acid Methyl Ester (9c). Following method A,
the title compound (100 mg, 60% yield) was prepared from 8c (100 mg,
0.42 mmol) and (2-methyl-1H-indol-3-yl)-acetaldehyde (80 mg,
(E)-3-[4-({(2-Fluoro-ethyl)-[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino}-
methyl)-phenyl]-N-hydroxy-acrylamide (11c). Following method B, the
title compound (8.9 mg, 9.3% yield) was prepared from 9c (100 mg,
0.24 mmol). 1H NMR (400 MHz, CD3OD) δ 7.56 (d, J = 15.6 Hz, 1 H),
7.54 (d, J = 7.6 Hz, 1 H), 7.41 (d, J = 7.9 Hz, 2 H), 7.29 (d, J = 8.1 Hz, 2 H),
7.25 (d, J = 8.3 Hz, 1 H), 7.02 (ddd, J = 8.1, 7.0, 1.1 Hz, 1 H), 6.93 (ddd, J =
8.1, 7.0, 1.1 Hz, 1 H), 6.56 (d, J = 16.0 Hz, 1 H), 4.63 (dt, J = 47.4, 5.4 Hz,
2 H), 3.86(s, 2H), 3.03(dt, J= 26.8, 5.2 Hz, 2 H), 2.95ꢀ2.78 (m, 4 H), 2.32
(s, 3 H). MS m/z 396.2 (MH+). HRMS calcd for C23H26N3O2F (MH+)
396.2087, found 396.2092.
(E)-3-[4-({(2,2-Difluoro-ethyl)-[2-(2-methyl-1H-indol-3-yl)-ethyl]-
amino}-methyl)-phenyl]-N-hydroxy-acrylamide (11d). Following
method B, the title compound (180 mg, 46% yield) was prepared
from 9d (400 mg, 0.92 mmol). 1H NMR (400 MHz, CD3OD) δ 7.58
(d, J = 15.5 Hz, 1 H), 7.43 (d, J = 7.8 Hz, 2 H), 7.27 (d, J = 7.8 Hz, 2 H),
7.22 (d, J = 5.3 Hz, 1 H), 7.20 (d, J = 4.7 Hz, 1 H), 6.97 (t, J = 7.3 Hz,
1 H), 6.88 (t, J = 7.5 Hz, 1 H), 6.48 (d, J = 15.8 Hz, 1 H), 5.78 (tt, J =
56.2, 4.4 Hz, 1 H), 3.67 (s, 2 H), 2.88 (td, J = 14.9, 4.2 Hz, 2 H),
2.79ꢀ2.64 (m, 4 H), 2.23 (s, 3 H). MS m/z 414.2 (M + 1). 13C NMR
(400 MHz, CD3OD) δ 166.50, 142.87, 141.54, 137.11, 135.14, 132.62,
130.52, 129.89, 128.79, 121.22, 119.3, 118.35, 117.98, 111.24, 109.32,
60.26, 57.37 (t), 56.29, 49.46, 23.23, 11.35. HRMS calcd for
C23H25N3O3F2 (Mꢀ) 412.1837, found 412.1854.
(E)-N-Hydroxy-3-(4-{[[2-(1H-indol-3-yl)-ethyl]-(2,2,2-trifluoro-ethyl)-
amino]-methyl}-phenyl)-acrylamide (11e). Following method B, the
title compound (26 mg, 19% yield) was prepared from 9e (140 mg, 0.39
mmol). 1H NMR (400 MHz, CD3OD) δ 7.58 (d, J = 15.8 Hz, 1 H), 7.54
(d, J = 8.2 Hz, 2 H), 7.50 (d, J = 8.2 Hz, 1 H), 7.45 (d, J = 7.6 Hz, 2 H), 7.34
(d, J = 8.2 Hz, 1 H), 7.13 (t, J = 8.2 H2, 1 H), 7.03 (t, J = 7.6 Hz, 1 H), 6.47
(d, J = 15.8 Hz, 1 H), 4.28 (q, J = 7.6 Hz, 1 H), 4.00 (d, J = 13.9 Hz, 1 H),
3.86(d, J= 13.9 Hz, 1 H), 3.43 (m, 1 H), 3.15 (d, J= 13.2, 5.0 Hz, 1 H), 3.01
(m, 1H), 2.61 (dd, J= 17.0, 3.2 Hz, 1 H). MS m/z(M +1). 13CNMR(400
MHz, CD3OD) δ 142.09, 141.40, 138.40, 135.56, 130.56, 128.88, 127.78,
125.07, 123.23, 119.98, 119.19, 118.25, 112.20, 111.90, 79.04, 59.01, 46.65,
17.72. HRMS calcd for C22H22N3O2F3 (MH)+ 418.1742, found 416.1611.
(E)-3-[4-({(3,3-Difluoro-propyl)-[2-(2-methyl-1H-indol-3-yl)-ethyl]-
amino}-methyl)-phenyl]-N-hydroxy-acrylamide (11g). Following
method B, the title compound (200 mg, 53% yield) was prepared
from 9g (380 mg, 0.85 mmol). 1H NMR (400 MHz, CD3OD) δ 7.59
(d, J = 15.4 Hz, 1 H), 7.51 (d, J = 7.9 Hz, 2 H), 7.38 (d, J = 8.2 Hz, 2 H),
7.21 (d, J = 3.4 Hz, 1 H), 7.19 (d, J = 3.7 Hz, 1 H), 6.96 (td, J = 7.5, 1.2
Hz, 1 H), 6.86 (td, J = 7.5, 1.3 Hz, 1 H), 6.48 (d, J = 15.3 Hz, 1 H), 3.81
(s, 2 H), 2.93 (t, J = 13.2 Hz, 2 H), 2.86ꢀ2.82 (m, 2 H), 2.75ꢀ2.71 (m,
2 H), 2.26 (s, 3 H), 1.58 (t, J = 19.2 Hz, 3 H). MS m/z 428.3 (MH+).
HRMS calcd for C24H27N3O2F2 (MH+) 428.2150, found 428.2148.
(E)-N-Hydroxy-3-(4-{[[2-(1H-indol-3-yl)-ethyl]-(3,3,3-trifluoro-propyl)-
amino]-methyl}-phenyl)-acrylamide (11h). Following method B, the
title compound (33 mg, 21% yield) was prepared from 9h (160 mg,
1
0.46 mmol). H NMR (400 MHz, CDCl3) δ 7.80 (s, 1 H), 7.74 (d,
J = 15.9 Hz, 1 H), 7.49 (d, J = 8.9 Hz, 2 H), 7.40 (d, J = 8.0 Hz, 2 H), 7.38
(d, J = 7.3 Hz, 1 H), 7.26 (d, J = 8.1 Hz, 1 H), 7.13 (t, J = 7.5 Hz, 1 H),
7.06 (t, J = 7.5 Hz, 1 H), 6.48 (d, J = 15.7 Hz, 1 H), 4.58 (dt, J = 47.6, 5.3
Hz, 2 H), 3.86 (s, 3 H), 3.83 (s, 2 H), 3.01ꢀ2.78 (m, 6 H), 2.32 (s, 3 H).
MS m/z 395.2 (MH+).
(E)-3-[4-({(2,2-Difluoro-ethyl)-[2-(2-methyl-1H-indol-3-yl)-ethyl]-
amino}-methyl)-phenyl]-acrylic Acid Methyl Ester (9d). Following
method A, the title compound (400 mg, 81% yield) was prepared from
8d (550 mg, 2.1 mmol) and (2-methyl-1H-indol-3-yl)-acetaldehyde
(200 mg, 1.2 mmol). 1H NMR (400 MHz, CDCl3) δ 7.75 (br, 1 H), 7.72
(d, J = 15.8 Hz, 1 H), 7.49 (d, J = 7.9 Hz, 2 H), 7.41ꢀ7.37 (m, 2 H), 7.34
(d, J = 7.8 Hz, 1 H), 7.27 (d, J = 8.2 Hz, 1 H), 7.11 (td, J = 7.5, 1.3 Hz,
1 H), 7.04 (td, J = 7.5, 0.9 Hz, 1 H), 6.46 (d, J = 15.6 Hz, 1 H), 5.81 (t, J =
56.2 Hz, 1 H), 3.85 (s, 2 H), 3.84 (s, 3 H), 3.00 (td, J = 14.4, 3.8 Hz, 2 H),
2.89ꢀ2.83 (m, 4 H), 2.32 (s, 3 H). MS m/z 413.2 (MH+).
(E)-3-(4-{[[2-(1H-Indol-3-yl)-ethyl]-(2,2,2-trifluoro-ethyl)-amino]-
methyl}-phenyl)-acrylic Acid Methyl Ester (9e). 2,2,2-Trifluoro-ethane-
1,1-diol (100 mg, 0.90 mmol) and p-toluenesulfonic acid (6 mg,
0.03 mmol) were added to a solution of (E)-3-(4-{[2-(1H-indol-3-yl)-
ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (200 mg,
0.60 mmol) in toluene (2 mL). The mixture was stirred at reflux
temperature under DeanꢀStark trap. Upon the completion of reaction,
THF (2 mL) and sodium triacetoxyborohydride (150 mg, 0.72 mmol)
were added. The resulting mixture was stirred at room temperature, and
then sodium bicarbonate solution was added. The aqueous layer was
extracted several times with EtOAc. The combined organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated. The
resulting crude product was purified via flash column chromatography to
1
afford a white powder (215 mg, 86% yield). H NMR (CDCl3) δ
ppm 8.0 (br s, 1H), 7.72 (d, J = 16.0 Hz, 1 H), 7.61 (d, J = 8.6 Hz, 1 H),
7.54 (m, 4 H), 7.41 (d, J = 8.2 Hz, 1 H), 7.29 (m, 2 H), 7.20 (d, J = 7.7 Hz,
1 H), 6.48 (d, J = 16.0 Hz, 1 H), 4.29 (br s, 1 H), 4.14 (m, 5 H), 3.91 (s,
1 H), 3.84 (s, 4 H), 3.56 (m, 1 H), 3.35 (m, 1 H), 3.07 (br s, 1 H), 2.78
(m, 1 H), 2.07 (m, 2 H), 1.8 (m, 1 H).
(E)-3-[4-({(3,3-Difluoro-propyl)-[2-(2-methyl-1H-indol-3-yl)-ethyl]-
amino}-methyl)-phenyl]-acrylic Acid Methyl Ester (9g). Following
method A, the title compound (380 mg, 74% yield) was prepared from
4764
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
1
0.37 mmol). H NMR (400 MHz, CD3OD) δ 7.53ꢀ7.46 (m, 3 H),
[2-(3,5-Dimethyl-isoxazol-4-yl)-1H-indol-3-yl]-acetonitrile (12o).
To a solution of (2-bromo-1H-indol-3-yl)-acetonitrile (2.0 g, 8.51
mmol) in toluene (15 mL) in a dry pressure tube under N2 were added
3,5-dimethyl-isoxazol-4-boronic acid (1.32 g, 9.36 mmol), trisdibenzy-
lidinedipalladium (0.78 g, 0.85 mmol), tri-tert-butylphosphonium tetra-
fluoroborate (0.49 g, 1.70 mmol), and potassium fluoride (1.48 g, 25.5
mmol), and the suspension was bubbled with N2. The tube was sealed
and the mixture was heated to 50 ꢀC for 16 h. The crude material was
diluted with EtOAc and filtered through a Celite plug. The filtrate was
concentrated and purified by column (0ꢀ40% EtOAc in heptane) to
give the title compound (1.35 g, 5.37 mmol, 63% yield) as a yellow solid.
1H NMR (CDCl3) δ 8.21 (br s, 1H), 7.74 (d, J = 7.7 Hz, 1H), 7.44 (d, J =
8.0 Hz, 1H), 7.30 (m, 2H), 3.66 (s, 2H), 2.41 (s, 3H), 2.23 (s, 3H). m/z
251.9 (MH+).
2-[2-(4-Methoxy-pyridin-3-yl)-1H-indol-3-yl]-ethylamine (13m). In
a dry Parr bottle was dissolved 12m (592 mg, 2.25 mmol) in EtOH
(15 mL). Then aqueous NH4OH (7.5 mL) followed by rhodium on
alumina (400 mg) were added to the mixture and stirred at room
temperature for 40 h under 45 psi of H2 pressure when LCMS showed
complete product formation. The reaction mixture was filtered through
a Celite pad, and the pad was washed with excess MeOH. The combined
filtrates were concentrated to give the title compound (590 mg, 2.21
mmol, 98% yield) as a crude yellowꢀbrown solid that was used in the
next step directly. 1H NMR (CD3OD) δ 8.40 (m, 1H), 7.94 (dd, J = 2.3,
8.5 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.42 (d, J = 7.9 Hz, 1H), 7.19 (m,
1H), 7.11 (m, 1H), 6.98 (d, J = 8.7 Hz, 1H), 4.01 (s, 3H), 3.23 (m, 4H).
m/z 267.9 (MH+).
7.37ꢀ7.31 (m, 4 H), 7.06 (ddd, J = 8.1, 7.1, 1.0 Hz, 1 H), 7.01 (s, 1 H),
6.94 (ddd, J = 8.0, 7.0, 1.1 Hz, 1 H), 6.49 (d, J = 15.7 Hz, 1 H), 3.68 (s,
2 H), 2.94ꢀ2.90 (m, 2 H), 2.85ꢀ2.76 (m, 4 H), 2.39ꢀ2.30 (m, 2 H).
13C NMR (400 MHz, CD3OD) δ 166.5, 142.7, 141.52, 138.15, 135.12,
130.6, 128.79, 127.08, 123.22, 122.2, 119.44, 119.23, 118.0, 114.02,
112.2, 59.11, 55.59, 49.38 (q) 47.60 (q), 32.45 (q), 24.20. MS m/z 432.2
(MH+). HRMS calcd for C23H24N3O2F3 (MH+) 432.1899, found
432.1901.
3-(2-Amino-ethyl)-1H-indole-2-carboxylic Acid Ethyl Ester Hydro-
chloride (14). Starting from 2-(3-chloro-propyl)-malonic acid diethyl
ester (11.8 g, 49.9 mmol) the title compound (4.4 g, 33% yield) was
prepared according to the procedure as previously described.70 1H NMR
(400 MHz, DMSO-d6) δ 8.15 (br, 2 H), 7.79 (d, J = 8.3 Hz, 1 H), 7.45
(d, J = 8.7 Hz, 1 H), 7.29 (t, J = 7.6 Hz, 1 H), 7.11 (t, J = 7.5 Hz, 1 H),
4.38 (q, J = 7.1 Hz, 2 H), 3.38 (m, 2 H), 1.42ꢀ1.34 (m, 5 H). MS m/z
233 (MH+).
3-{2-[4-((E)-2-Methoxycarbonyl-vinyl)-bezylamino]-ethyl}-1H-in-
dole-2-carboxylic Acid Ethyl Ester (9j). Following method A, the title
compound was prepared from 14. 1H NMR (400 MHz, CDCl3) δ 8.67
(s, 1 H), 7.64 (dd, J = 8.1, 1.1 Hz, 1 H), 7.57 (d, J = 16.2 Hz, 1 H), 7.37 (d,
J = 8.0 Hz, 2 H), 7.29 (ddd, J = 9.2, 8.1, 1.2 Hz, 1 H), 7.24 (d, J = 8.0 Hz, 2
H), 7.07 (ddd, J = 8.1, 6.7, 1.2 Hz, 1 H), 6.31 (d, J = 16.0 Hz, 1 H), 4.32 (q,
J = 7.2 Hz, 2 H), 3.79 (s, 2 H), 3.73 (s, 3 H), 3.28 (t, J = 7.4 Hz, 2 H), 2.91
(t, J = 7.6 Hz, 2 H), 1.32 (t, J = 7.5 Hz, 3 H). MS m/z 313.2 (M + 1)
3-{2-[4-((E)-2-Hydroxycarbamoyl-vinyl)-benzylamino]-ethyl}-1H-
indole-2-carboxylic Acid (11j). Powdered sodium hydroxide (100 mg,
2.5 mmol) was added to a solution of 9j (190 mg, 0.47 mmol) and
hydroxylamine (0.16 mL, 5 mmol, 50% in water) in ethanol (3 mL).
The mixture was stirred at room temperature overnight and purified
via preparative HPLC to give both the title compound (13 mg,
7% yield) and (E)-N-hydroxy-3-[4-(1-oxo-1,3,4,9-tetrahydro-β-carbo-
lin-2-ylmethyl)-phenyl]-acrylamide (9.5 mg, 5% yield). 1H NMR (400
MHz, CD3OD) δ 7.59 (dt, J = 8.0, 1.0 Hz, 1 H), 7.54 (d, J = 15.9 Hz,
1 H), 7.49 (d, J = 7.9 Hz, 2 H), 7.41 (dt, J = 8.2, 1.0 Hz, 1 H), 7.32 (d, J =
8.4 Hz, 2 H), 7.23 (ddd, J = 8.3, 7.0, 1.3 Hz, 1 H), 7.08 (ddd, J = 8.0, 6.9,
1.2 Hz, 1 H), 6.46 (d, J = 15.8 Hz, 1 H), 3.80 (s, 2 H), 3.24 (t, J = 6.5 Hz,
2 H), 3.07 (t, J = 6.1 Hz, 2 H). MS m/z 380.1 (M + 1)
(2-Bromo-1H-indol-3-yl)-acetonitrile. To a solution of (1H-indol-3-
yl)-acetonitrile (5.0 g, 32.0 mmol) in DCM (300 mL) under N2
atmosphere was slowly added N-bromosuccinimide (5.98 g, 33.6
mmol), and the mixture was stirred for 30 min at ambient temperature.
The reaction mixture was added with hexane, and the solvents were
evaporated in vacuo (with no heat) and more hexane was added to make
a slurry. The suspension was transferred to the top of a large SiO2 plug
and filtered with 0ꢀ30% EtOAc in hexanes to give the title compound
(5.03 g, 21.4 mmol, 67% yield) as a brown oil. 1H NMR (CDCl3) δ 8.23
(br s, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.32 (m, 1H), 7.22 (m, 2H), 3.79 (s,
2H); m/z 236.8 (MH+).
2-[2-(3,5-Dimethyl-isoxazol-4-yl)-1H-indol-3-yl]-ethylamine (13o).
To a solution of 12o (830 mg, 3.3 mmol) in THF (20 mL) was added
BH3 THF (9.9 mL, 1M, 9.9 mmol), and the mixture was stirred for 5 h
3
at ambient temperature. It was then cooled in ice bath and quenched
with MeOH. The crude material was concentrated to an oil, combined
with 5 mL of aqueous 6N NaOH solution and stirred for 30 min. Then
EtOAc was added and the layers were separated. The aqueous layer was
washed twice with EtOAc, and the combined organic layers were dried
with MgSO4, concentrated, and purified by flash column (100% EtOAc,
then 10% MeOH in DCM with a few drops of NH4OH) to give the title
compound (388 mg, 1.52 mmol, 48% yield, 90% pure) as a crude brown
solid. 1H NMR (CD2Cl2) δ 8.31 (br s, 1H), 7.67 (d, J = 8.6 Hz, 1H), 7.41
(d, J = 7.9 Hz, 1H), 7.24 (m, 1H), 7.15 (m, 1H), 3.44 (s, 1H), 2.89 (m,
2H), 2.77 (m, 2H), 2.36 (s, 3H), 2.20 (s, 3H). m/z 255.9 (MH+).
(E)-3-[4-({2-[2-(4-Methoxy-pyridin-3-yl)-1H-indol-3-yl]-ethylamino}-
methyl)-phenyl]-acrylic Acid Methyl Ester (8m). Following method A,
20m (200 mg, 0.75 mmol) was converted to the title compound
(120 mg, 0.27 mmol, 36% yield) as a yellow oil. 1H NMR (CDCl3) δ
8.41 (m, 1H), 8.08 (br s, 1H), 7.83 (m, 1H), 7.63 (m, 2H), 7.40 (m, 3H),
7.30 (m, 2H), 7.24 (m, 1H), 7.16 (m, 1H), 6.86 (d, J = 8.5 Hz, 1H), 6.37
(d, J = 15.8 Hz, 1H), 4.02 (s, 3H), 3.83 (m, 5H), 3.05 (m, 4H). m/z
441.9 (MH+).
[2-(4-Methoxy-pyridin-3-yl)-1H-indol-3-yl]-acetonitrile (12m). In
an oven-dried pressure tube under N2 atmosphere were taken (2-
bromo-1H-indol-3-yl)-acetonitrile (100 mg, 0.43 mmol), 2-methoxy-
5-pyridine boronic acid (130 mg, 0.85 mmol), and K2CO3 (88 mg, 0.64
mmol) in toluene (4 mL), and N2 was bubbled through the suspension
for 10 min. Then tetrakis(triphenylphosphine)palladium (49 mg, 0.04
mmol) was added to it, and the tube was sealed. The mixture was heated
to 90 ꢀC for 16 h, filtered through a Celite plug, and the plug was washed
further with more DCM. All the filtrates were combined together and
concentrated to a crude oil that was purified by flash column (0ꢀ30%
EtOAc in heptane) to give the title compound (59 mg, 0.22 mmol, 53%
yield) as a light-yellow oil. 1H NMR (CDCl3) δ 8.37 (d, J = 2.3 Hz, 1H),
8.26 (br s, 1H), 7.77 (dd, J = 2.5, 8.5 Hz, 1H), 7.73 (d, J = 7.7 Hz, 1H),
7.45 (d, J = 7.9 Hz, 1H), 7.29 (m, 2H), 6.93 (d, J = 8.5 Hz, 1H), 4.04 (s,
3H), 3.87 (s, 2H). m/z 263.9 (MH+).
(E)-3-[4-({2-[2-(3,5-Dimethyl-isoxazol-4-yl)-1H-indol-3-yl]-ethylamino}-
methyl)-phenyl]-acrylic Acid Methyl Ester (8o). Following method A,
20o (279 mg, 1.09 mmol) was converted to the title compound (142 mg,
1
0.33 mmol, 36% yield) as a yellow oil. H NMR (CDCl3) δ 7.65 (m,
2H), 7.42 (d, J = 7.7 Hz, 2H), 7.37 (d, J = 7.7 Hz, 1H), 7.23 (m, 3H), 7.14
(m, 1H), 6.40 (d, J = 16.5 Hz, 1H), 3.79 (s, 3H), 3.73 (s, 2H), 2.84 (br s,
2H), 2.25 (s, 3H), 2.10 (s, 3H). m/z 430.1 (MH+).
(E)-N-Hydroxy-3-[4-({2-[2-(4-methoxy-pyridin-3-yl)-1H-indol-3-yl]-
ethylamino}-methyl)-phenyl]-acrylamide (11m). Following method B,
8m (120 mg, 0.27 mmol) was converted to the title compound (46 mg,
1
0.10 mmol, 38% yield) as a yellow solid after HPLC purification. H
NMR (CD3OD) δ 8.35 (s, 1H), 7.86 (d, J = 8.9 Hz, 1H), 7.54 (d, J = 7.6
Hz, 1H), 7.46 (m, 3H), 7.37 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 7.8 Hz,1H),
7.13 (m, 1H), 7.02 (m, 1H), 6.88 (d, J = 8.9 Hz,1H), 6.48 (d, J = 15.6
Hz,1H), 3.97 (s, 3H), 3.73 (s, 2H), 3.09 (m, 2H), 2.85 (m, 2H).
4765
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
13C NMR (CD3OD) δ 166.13, 164.86, 146.95, 141.95, 140.06, 139.11,
137.96, 135.68, 132.97, 130.17, 129.94, 128.65, 124.37, 122.97, 120.15,
119.63, 119.53, 112.08, 111.72, 110.75, 54.27, 53.69, 50.18, 25.41. m/z
443.1 (MH+). HRMS (MH+) calcd for C26H26N4O3, 443.2083, found
443.2074.
2-(3-{2-[(4-Bromo-benzyl)-isopropyl-amino]-ethyl}-1H-indol-2-yl)-
propan-2-ol (5.0 g, 12 mmol) was added to a suspension of tris-
(dibenzylideneacetone)dipalladium(0) (100 mg, 0.12 mmol) and tribu-
tylphosphine tetrafluoroborate (140mg, 0.47 mmol) indioxane (10 mL).
The suspension was degassed and filled with nitrogen several times, and
then dicyclohexyl-methylamine (3 mL, 14 mmol) was added under
nitrogen. The mixture was stirred at room temperature for 10 min and
then acrylic acid methyl ester (2.1 mL, 23 mmol) and water (0.21 mL,
12 mmol) was added. It was then heated via microwave at 100 ꢀC for
12 min. The crude product mixture was purified via flash column
chromatography directly (EtOAc:hexanes, 10:90 to 100:0) to provide
yellow syrup product (4.2 g, 83% yield). 1H NMR(400MHz, CD3OD) δ
7.47 (d, J = 15.7 Hz, 1 H), 7.32 (d, J = 7.8 Hz, 2 H), 7.26ꢀ7.18 (m, 4 H),
7.03 (t, J = 7.3 Hz, 1 H), 6.93 (t, J = 7.3 Hz, 1 H), 6.45 (d, J = 15.7 Hz,
1 H), 4.02 (s, 2 H), 3.37 (s, 3 H), 3.34ꢀ3.32 (m, 2 H), 3.18 (t, J = 6.3 Hz,
2 H), 1.66 (s, 6 H), 1.40 (d, J = 6.3 Hz, 6 H). MS m/z 436.2 (MH+).
(E)-N-Hydroxy-3-{4-[({2-[2-(1-hydroxy-1-methyl-ethyl)-1H-indol-
3-yl]-ethyl}-isopropyl-amino)-methyl]-phenyl}-acrylamide (11r).
Following method A, the title compound (45 mg, 32% yield) was
prepared from 16 (140 mg, 0.32 mmol). 1H NMR (400 MHz, DMSO-
d6) δ10.74 (s, 1 H), 10.36 (s, 1 H), 9.03 (br, 1 H), 7.54ꢀ7.42 (m, 5 H),
7.26 (d, J = 8.1 Hz, 1 H), 7.10 (d, J = 7.8 Hz, 1 H), 6.93 (t, J = 7.4 Hz,
1 H), 6.81 (t, J = 7.3 Hz, 1 H), 5.24 (s, 1 H), 3.65 (s, 2 H), 3.34ꢀ3.31
(m, 2 H), 3.04ꢀ3.00 (m, 1 H), 2.87ꢀ2.83 (m, 2 H), 1.45 (s, 6 H), 1.04
(d, J = 6.0 Hz, 6 H). 13C NMR (400 MHz, CD3OD) δ166.44, 142.7,
142.36, 141.25, 136.21, 135.05, 130.91, 130.42, 128.66, 121.95,
119.56, 118.78, 118.24, 112.06, 108.12, 71.18, 58.41, 55.53, 52.41,
52.12, 31.44, 25.78, 18.49, 18.43. MS m/z 436 (MH+). HRMS calcd
for C26H34N3O3 (MH+) 436.2600, found 436.2607.
(E)-3-[4-({2-[2-(3,5-Dimethyl-isoxazol-4-yl)-1H-indol-3-yl]-ethylamino}-
methyl)-phenyl]-N-hydroxy-acrylamide (11o). Following method B,
8o (180 mg, 0.42 mmol) was converted to the title compound (145 mg,
0.337 mmol, 80% yield) TFA salt as a yellow solid after RP-HPLC
1
purification. H NMR (CD3OD) δ 7.61 (m, 4H), 7.46 (d, J = 8.1
Hz, 2H), 7.38 (d, J = 7.7 Hz, 1H), 7.19 (m, 1H), 7.10 (m, 1H), 6.53 (d,
J = 15.3 Hz, 1H), 4.23 (s, 2H), 3.18 (m, 2H), 3.04 (m, 2H), 2.34 (s,
3H), 2.17 (s, 3H). 13C NMR (CD3OD) δ 169.4, 165.9, 161.2, 140.4,
138.4, 137.5, 134.1, 131.5, 131.4, 129.7, 129.5, 128.6, 125.3, 123.6, 120.6,
120.0, 119.2, 112.5, 110.3, 109.9, 51.7, 23.0, 11.6, 10.5. m/z 430.9
(MH+). HRMS (MH+) calcd for C25H26N4O3, 431.2083, found
431.2085.
2-(3-{2-[(4-Bromo-benzyl)-isopropyl-amino]-ethyl}-1H-indol-2-yl)-
propan-2-ol (15). 3-[2-(4-Bromo-benzylamino)-ethyl]-1H-indole-2-
carboxylic acid ethyl ester: 3-(2-Amino-ethyl)-1H-indole-2-carboxylic
acid ethyl ester hydrochloride (8.5 g, 27 mmol) was added to a solution
of 4-bromo-benzaldehyde (4.7 g, 26 mmol) and acetic acid (2.3 mL, 41
mmol) in THF (120 mL). The mixture was stirred at room temperature
for 1 h. To this mixture, sodium triacetoxyborohydride (8.4 g, 38 mol)
was added, and the reaction mixture was stirred at room temperature
overnight. Satd NaHCO3 solution was added, and the aqueous layer was
extracted with EtOAc several times. The organic layer was combined,
dried over Na2SO4, filtered, and evaporated off. The crude product was
purified via flash column chromatography (EtOAc:hexanes, 10:90 to
60:40) to provide product (9 g, 83%yield). 1H NMR(400 MHz, CDCl3)
δ 8.77 (br, 1 H), 7.72 (d, J = 8.2 Hz, 1 H), 7.54ꢀ7.09 (m, 7 H), 4.39 (q,
J = 7.0 Hz, 2 H), 3.88 (s, 2 H), 3.42 (t, J = 7.2 Hz, 2 H), 3.06 (t, J = 7.0 Hz,
2 H), 1.40 (t, J = 7.2 Hz, 3 H). MS m/z 402 (MH+).
2-tert-Butyl-1H-pyrrolo[2,3-b]pyridine (17f). To an ice-cold solution
of 3-methyl-pyridin-2-ylamine (25 g, 0.23 mol) in toluene (500 mL)
under N2 atmosphere was added potassium carbonate (32.0 g, 0.23
mol). The mixture was stirred at room temperature for 30 min, followed
by dropwise addition of trimethylacetyl chloride (31.3 mL, 0.25 mol).
The mixture was stirred at 25 ꢀC for 16 h and filtered. The precipitate
was washed EtOAc. The combined filtrates were washed with aqueous
NH4Cl and brine. The organic layer was dried (MgSO4) and concen-
trated to give 2,2-dimethyl-N-(3-methyl-pyridin-2-yl)-propionamide
(21.3 g, 0.11 mol, 48% yield) as an off-white solid. To a cold suspension
(ꢀ20 ꢀC) of this solid (21.3 g, 0.11 mol) in THF (300 mL) under N2
was slowly added butyllithium (10 M in hexane, 33 mL, 0.33 mol), and
the flask was allowed to warm up to room temperature and stand for 16
h. The mixture was then cooled below 0 ꢀC and quenched with 25 mL of
aqueous 1N HCl, diluted with ether, and the layers were separated. The
organic layer was washed with aqueous NaHCO3. The aqueous wash-
ings were extracted with additional ether, and the combined organic
layers were washed with brine, dried (MgSO4), and concentrated to give
the title compound (22.7 g) as a crude white solid that was used directly
3-{2-[(4-Bromo-benzyl)-isopropyl-amino]-ethyl}-1H-indole-2-car-
boxylic acid ethyl ester: 3-[2-(4-Bromo-benzylamino)-ethyl]-1H-in-
dole-2-carboxylic acid ethyl ester (9.0 g, 222 mmol) was added to a
solution of 2-iodo-propane (76 g, 448 mmol) and triethylamine (62 mL,
448 mmol) in CH3CN (200 mL). The mixture was refluxed overnight.
The solvent was then removed under vacuum. EtOAc and water were
added. The aqueous layer was extracted several times with EtOAc. The
combined organic layer was washed with brine, dried over Na2SO4,
filtered, and concentrated in vacuo. The crude product was purified via
flash column chromatography (EtOAc:hexanes, 10:90 to 90:10) to
1
provide slightly yellow solid (6.5 g, 65% yield). H NMR (400 MHz,
CDCl3) δ 8.64 (br, 1 H), 7.45 (d, J = 8.3 Hz, 1 H), 6.87ꢀ7.27 (m, 4 H),
7.15 (d, J = 8.1 Hz, 2 H), 7.09 (t, J = 7.5 Hz, 1 H), 4.35 (q, J = 7.1 Hz, 2
H), 3.59 (s, 2 H), 3.18 (t, J = 7.9 Hz, 2 H), 2.09ꢀ2.00 (m, 1 H), 2.69 (t,
J = 7.5 Hz, 2 H), 1.37 (t, J = 7.3 Hz, 3 H), 1.02 (d, J = 6.9 Hz, 6 H). MS
m/z 444 (MH+).
1
in the next step without additional purification. H NMR (CDCl3) δ
Methyl lithium (18 mL, 44 mmol, 2.5 M in diethoxymethane) was
added to a solution of 3-{2-[(4-bromo-benzyl)-isopropyl-amino]-
ethyl}-1H-indole-2-carboxylic acid ethyl ester (6.5 g, 15 mmol) in
THF (65 mL) at ꢀ65 ꢀC. The mixture was stirred at same temperature
for 2 h. Ice water was added slowly. The aqueous layer was then extracted
several times with EtOAc. The combined organic layer was washed with
brine, dried over Na2SO4, filtered, and evaporated off. The crude
product was purified via flash column chromatography (EtOAc:hexanes,
10:90 to 100:0) to provide yellow oil (5.5 g, 87% yield). 1H NMR (400
MHz, CDCl3) δ 8.05 (s, 1 H), 7.32 (d, J = 7.9 Hz, 1 H), 6.78ꢀ6.73 (m, 3
H), 6.64ꢀ6.60 (m, 3 H), 6.54 (t, J = 7.3 Hz, 1 H), 3.44 (s, 2 H),
3.01ꢀ2.94 (m, 1 H), 2.89 (t, J = 7.0 Hz, 2 H), 2.68 (t, J = 6.9 Hz, 2 H),
1.58 (s, 6 H), 1.01 (d, J = 6.7 Hz, 6 H). MS m/z 430 (MH+).
(E)-3-{4-[({2-[2-(1-Hydroxy-1-methyl-ethyl)-1H-indol-3-yl]-ethyl}-
isopropyl-amino)-methyl]-phenyl}-acrylic Acid Methyl Ester (16).
8.25 (d, J = 4.4 Hz, 1H), 7.87 (br s, 1H), 7.56 (d, J = 6.9 Hz, 1H), 7.11
(dd, J = 4.8, 7.2 Hz, 1H), 2.23 (s, 3H), 1.35 (s, 9H). m/z 193.06 (MH+).
Dimethyl-(1H-pyrrolo[3,2-b]pyridin-3-ylmethyl)-amine (18a). The
title compound (1.1 g, >99% yield) was prepared according to method C
1
from 1H-pyrrolo[3,2-b]pyridine (750 mg, 6.4 mmol). H NMR (400
MHz, CD3OD) δ 8.45 (d, J = 4.4 Hz, 1 H), 7.93 (dd, J = 8.4, 1.6 Hz,
1 H), 7.85 (s, 1 H), 7.29 (dd, J = 8.2, 4.8 Hz, 1 H), 4.46 (s, 2 H), 2.81 (s,
6 H). MS m/z 176.1 (MH+).
Dimethyl-(1H-pyrrolo[3,2-c]pyridin-3-ylmethyl)-amine (18d). The
title compound (2.6 g, 68% yield) was prepared according to method C
from 1H-pyrrolo[3,2-c]pyridine (2.6 g, 22 mmol). 1H NMR (400 MHz,
CD3OD) δ 9.13 (s, 1 H), 8.23 (d, J = 6.3 Hz, 1 H), 7.77 (s, 1 H), 7.56 (d,
J = 6.4 Hz, 1 H), 4.50 (s, 2 H), 2.82 (s, 6 H). MS m/z 176.2 (MH+).
(2-tert-Butyl-1H-pyrrolo[2,3-b]pyridin-3-ylmethyl)-dimethyl-amine
(18f). In a dry vial was taken paraformaldehyde (517 mg, 17.2 mmol) in
4766
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
dimethylacetamide:n-butanol (29:1 v/v, 3 mL) and subjected to micro-
wave irradiation (100 ꢀC for 13 min). Dimethylamine hydrochloride
(1.4 g, 17.2 mmol) was added, and the mixture was heated and sonicated.
To the resulting clear solution was added 17f (2.0 g, 11.5 mmol), and the
mixture was subjected to microwave irradiation (100 ꢀC for 20 min).
The mixture was diluted with MeOH and concentrated in vacuo. The
residue was dissolved in DCM and washed with a small volume of
aqueous Na2CO3. The aqueous washings were rewashed (2ꢁ) with
DCM, and the combined organic layers were dried (MgSO4), concen-
trated, and dried to give the title compound (2.31 g, 9.99 mmol, 87%
yield) as a yellow solid. 1H NMR (CDCl3) δ 9.4 (br s, 1H), 8.21 (dd, J =
1.4, 4.8 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.02 (dd, J = 4.8, 7.9 Hz, 1H),
3.63 (s, 2H), 2.23 (s, 6H), 1.53 (s, 9H). m/z 232.0 (MH+).
Imidazo[1,2-a]pyridin-3-ylmethyl-dimethyl-amine (18g). The title
compound (900 mg, 68% yield) was prepared according to method C
from imidazo[1,2-a]pyridine (880 mg, 7.5 mmol). 1H NMR (400 MHz,
CDCl3) δ 8.19 (d, J = 6.8 Hz, 1 H), 7.60 (d, J = 9.4 Hz, 1 H), 7.53 (s, 1
H), 7.22 (ddd, J = 9.2, 6.7, 1.3 Hz, 1 H), 6.84 (t, J = 6.7 Hz, 1 H), 4.78 (s,
2 H), 3.60 (s, 6 H). MS m/z 176.1 (MH+).
2-Imidazo[1,2-a]pyridin-3-yl-ethylamine (20g). The title com-
pound (140 mg, 79% yield) was prepared according to same procedure
as 20d from 20g (180 mg, 1.1 mmol). 1H NMR (400 MHz, CD3OD) δ
8.32 (d, J = 6.9 Hz, 1 H), 7.56 (d, J = 8.9 Hz, 1 H), 7.44 (s, 1 H), 7.32
(ddd, J = 9.1, 6.8, 1.2 Hz, 1 H), 6.99 (t, J = 6.8 Hz, 1 H), 3.12 (t, J = 6.8
Hz, 2 H), 3.05 (t, J = 6.8 Hz, 2 H). MS m/z 162.1 (MH+).
Typical Procedure for the Formation of 2-Ylpyrazolo[1,5-
a]pyridine-3-carboxylic Acid Ethyl Esters. To a solution of amino-
pyridinium iodide (22 g, 99 mmol) and anhydrous potassium carbonate
(17.8 g, 128.7 mmol) in dimethylformamide (225 mL) was added an
alkyl- or aryl-2-pentynoate (198 mmol). The solution was stirred at
room temperature for 24 h. The reaction mixture was poured into 1.2 L
of ice water. The resulting light-brown precipitate was collected by
vacuum filtration and air-dried overnight to yield 11.9 g (55% yield) of
crude product, which was used without further purification.
2-Methyl-pyrazolo[1,5-a]pyridine-3-carboxylic Acid Ethyl Ester
(21i). The title compound (6.2 g, 52% yield) was prepared according
to the above procedure from amino-pyridinium iodide (13.0 g, 58.5
mmol) and methyl-2-butynoate (13.6 mL, 117 mmol). 1H NMR (400
MHz, CDCl3) δ 8.42ꢀ8.39 (m, 1 H), 8.09ꢀ8.06 (m, 1 H), 7.37ꢀ7.33
(m, 1 H), 6.89ꢀ6.86 (m, 1 H), 4.39 (q, J = 7.9 Hz, 2 H), 2.68 (s, 3 H),
1.43 (t, J = 9.0 Hz, 3 H). MS m/z 205 (MH+).
(1H-Pyrrolo[3,2-b]pyridin-3-yl)-acetonitrile (19a). The title com-
pound (340 mg, 34% yield) was prepared according to method D from
18a (1.1 g, 6.3 mmol). MS m/z 158.1 (MH+).
2-Ethyl-pyrazolo[1,5-a]pyridine-3-carboxylic Acid Ethyl Ester (21j).
The title compound (11 g, 55% yield) was prepared according to the
above procedure from amino-pyridinium iodide (22 g, 99 mmol) and
ethyl-2-butynoate (24.98 mL, 198 mmol). 1H NMR (400 MHz, CDCl3)
δ 8.43 (ddd, J = 6.9, 1.1, 1.1 Hz, 1 H), 8.10 (ddd, J = 8.8, 1.2, 1.3 Hz, 1 H),
7.35 (ddd, J = 9.0, 6.8, 1.2 Hz, 1 H), 6.88 (ddd, J = 7.7, 6.1, 0.7 Hz, 1 H),
4.39 (q, J = 7.1 Hz, 2 H), 3.13 (q, J = 7.5 Hz, 2 H), 1.43 (t, J = 7.0 Hz, 3
H), 1.36 (t, J = 7.8 Hz, 3 H). MS m/z 219 (MH+).
2-Phenyl-pyrazolo[1,5-a]pyridine-3-carboxylic Acid Ethyl Ester
(21k). The title compound (20 g, 74% yield) was prepared according
to the above procedure from amino-pyridinium iodide (13.0 g, 58.5
mmol) and phenyl-propynoic acid ethyl ester (19.3 mL, 117 mmol). 1H
NMR (400 MHz, CDCl3) δ 8.52 (ddd, J = 7.0, 1.2, 1.2 Hz, 1 H), 8.21
(ddd, J = 8.9, 1.2, 1.2 Hz, 1 H), 7.80ꢀ7.77 (m, 2 H), 7.48ꢀ7.38 (m, 3 H),
6.95 (ddd, J = 7.7, 6.2, 0.8 Hz, 1 H), 4.32 (q, J = 7.2 Hz, 2 H), 1.31 (t, J =
7.1 Hz, 3 H). MS m/z 267 (MH+).
Typical Procedure for the Formation of 2-Ylpyrazolo[1,5-a]-
pyridine-3-carbaldehydes. A solution of pyrazolo[1,5-a]pyridine-3-
carboxylic acid ethyl ester (68.7 mmol) in THF (300 mL) was cooled
in an ice bath, and lithium aluminum hydride (2.6 g, 68.7 mmol) was
added. The reaction mixture was stirred at room temperature overnight.
Water was added until bubbling stopped. Silica gel was added, and the
solvent was evaporated in vacuo. The resulting residue was purified via
silica gel chromatography eluted with 20% EtOAc:hexanes to 100% to
yield 9.67 g (80% yield) of product as a yellowꢀbrown oil.
1H-Pyrrolo[3,2-c]pyridin-3-yl)-acetonitrile (19d). The title com-
pound (600 mg, 26% yield) was prepared according to method D from
18d (2.6 g, 15 mmol).
(2-tert-Butyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-acetonitrile (19f). To a
solution of 18f (2.0 g, 8.66 mmol) in THF (40 mL) was slowly added
dimethyl sulfate (900 μL, 9.51 mmol), and the mixture was refluxed for
30 min. The solvent was removed in vacuo, and the residue was
suspended in 30 mL water to which NaCN (0.57 g, 11.2 mmol) was
added. After refluxing for 1 h, the aqueous suspension was washed with
excess EtOAc (3ꢁ), and the combined organic layers were dried
(MgSO4) and concentrated to give a residue that was purified by flash
column (0ꢀ40% EtOAc in Hex) to give the title compound (0.33 g,1.55
mmol, 18% yield) as a white solid. 1H NMR (CDCl3) δ 9.87 (br s, 1H),
8.31 (dd, J = 1.4, 4.9 Hz, 1H), 7.92 (d, J = 7.4 Hz, 1H), 7.12 (dd, J = 4.9,
8.1 Hz, 1H), 3.95 (s, 2H), 1.56 (s, 9H). m/z 214.1 (MH+).
Imidazo[1,2-a]pyridin-3-yl-acetonitrile (19g). The title compound
(110 mg, 5.1% yield) was prepared according to method D from 18g
(2.6 g, 15 mmol). MS m/z 158.0 (MH+).
2-(1H-Pyrrolo[3,2-b]pyridin-3-yl)-ethylamine (20a). The title com-
pound (340 mg, 98% yield) was prepared according to same procedure
as 13m from 19a (340 mg, 2.2 mmol). MS m/z 162.1 (MH+).
2-(6-Methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-ethylamine (20b).
The title compound (140 mg, 72% yield) was prepared according to
same procedure as 13m from (6-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)-acetonitrile (200 mg, 1.1 mmol). 1H NMR (400 MHz, DMSO-d6) δ
8.81 (s, 1 H), 8.60 (s, 1 H), 2.77ꢀ2.75 (m, 4 H), 2.30 (s, 3 H). MS m/z
177.1 (MH+).
2-(6-Methyl-5H-pyrrolo[2,3-b]pyrazin-7-yl)-ethylamine (20c). The
title compound (200 mg, 98% yield) was prepared according to same
procedure as 13m from (6-methyl-5H-pyrrolo[2,3-b]pyrazin-7-yl)-acet-
onitrile (200 mg, 1.1 mmol). MS m/z 177.1 (MH+).
2-(1H-Pyrrolo[3,2-c]pyridin-3-yl)-ethylamine (20d). Rhodium on
alumina (200 mg, 0.1 mmol) was added to a solution of 19d (200 mg,
1.3 mmol) in ammonium hydroxide (6 mL) and ethanol (12 mL). The
mixture was stirred under hydrogen atmosphere (50 psi) in a Parr shaker
overnight. Upon completion of the reaction, the solution was filtered
and evaporated off. The resulting product (200 mg, 98% yield) was used
directly in the next step.
To a solution of (pyrazolo[1,5-a]pyridin-3-yl)-methanol (54.9
mmol) in THF (500 mL) was added MnO2 (23.88 g, 274.5 mmol,
Aldrich cat. no. 217646, 85%, dried overnight in oven at 120 ꢀC) and
stirred at reflux for 1.5 h. The reaction mixture was filtered through
Celite and concentrated under reduced pressure to give 9.4 g (98%
yield) of product, which was used without further purification.
2-Methyl-pyrazolo[1,5-a]pyridine-3-carbaldehyde (22i). (2-Meth-
yl-pyrazolo[1,5-a]pyridin-3-yl)-methanol (3.9 g, 80% yield) was pre-
pared according to same procedure as above from 21i (6.1 g, 30 mmol).
1H NMR (400 MHz, CDCl3) δ 8.28 (dt, J = 7.0, 1.0 Hz, 1 H),
7.47 (dt, J = 9.0, 1.2 Hz, 1 H), 7.06 (ddd, J = 8.9, 6.8, 1.1 Hz, 1 H),
6.65 (td, J = 6.9, 1.6 Hz, 1 H), 4.78 (s, 2 H), 2.43 (s, 3 H). MS m/z 163.1
(MH+).
2-(2-tert-Butyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-ethylamine (20f). The
title compound (524 mg, 2.03 mmol, 86% yield, 84% purity) was
prepared according to same procedure as 13m from 19f (500 mg,
2.34 mmol) and was directly used in the next step. m/z 218.2 (MH+).
The title compound (3.8 g, 97% yield) was prepared according to
same procedure as above from (2-methyl-pyrazolo[1,5-a]pyridin-3-yl)-
methanol (3.9 g, 24 mmol). 1H NMR (400 MHz, CDCl3) δ 10.02 (s,
1 H), 8.40 (dt, J = 6.8, 1.0 Hz, 1 H), 8.14 (dt, J = 8.7, 1.3 Hz, 1 H), 7.42
4767
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
(ddd, J = 8.7, 7.0, 1.1 Hz, 1 H), 6.93 (ddd, J = 7.7, 6.2, 0.8 Hz, 1 H), 2.62
(s, 3 H). MS m/z 161.1 (MH+).
solution. The organic layer was washed with brine. The brine layer was
further extracted with CH2Cl2:EtOH (4:1) several times until no
product was detected by TLC in brine layer. The organic layers were
combined, concentrated under vacuum, and purified on a short silica gel
column, eluting with CH2Cl2 to 25% MeOH/CH2Cl2 with 1% NH4OH,
(82.5ꢀ95% yield).
2-Ethyl-pyrazolo[1,5-a]pyridine-3-carbaldehyde (22j). (2-Ethyl-
pyrazolo[1,5-a]pyridin-3-yl)-methanol (9.67 g 80% yield) was pre-
pared according to same procedure as above from 22i (15 g,
1
68.7 mmol) H NMR (400 MHz, CDCl3) δ 8.35 (dt, J = 8.3, 1.3
Hz, 1 H), 7.51 (dt, J = 8.9, 1.3 Hz, 1 H), 7.09 (ddd, J = 9.1, 6.5, 1.2 Hz,
1 H), 6.68 (ddd, J = 7.5, 6.2, 0.6 Hz, 1 H), 4.83 (s, 2 H), 2.79 (q, J = 7.5
Hz, 2 H), 1.28 (t, J = 7.6 Hz, 3 H). MS m/z 177.1 (MH+).
2-(2-Methyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamine (24i). The title
compound (820 mg, 95% yield) was prepared according to same procedure
as above from 23i (1.0 g, 4.9 mmol). 1H NMR (400 MHz, CD3OD) δ8.69
(dt, J = 7.0, 0.9 Hz, 1 H), 8.00 (dt, J = 9.1, 1.2 Hz, 1 H), 7.76 (ddd, J = 9.0,
7.1, 1.0 Hz, 1 H), 7.40 (ddd, J = 7.7, 6.3, 0.6 Hz, 1 H), 3.36ꢀ3.31 (m, 2 H),
3.23ꢀ3.18 (m, 2 H), 2.61 (s, 3 H). MS m/z 176.1 (MH+).
The title compound (9.4 g 98% yield) was prepared according to
same procedure as above from (2-ethyl-pyrazolo[1,5-a]pyridin-3-yl)-
methanol (9.67 g, 54.9 mmol). 1H NMR (400 MHz, CDCl3) δ 10.04 (s,
1 H), 8.41 (dt, J = 6.8, 1.3 Hz, 1 H), 8.15 (dt, J = 8.9, 1.5 Hz, 1 H), 7.41
(ddd, J = 8.8, 6.8, 1.1 Hz, 1 H), 6.93 (ddd, J = 7.8, 6.1, 0.8 Hz, 1 H), 3.03
(q, J = 7.6 Hz, 2 H), 1.35 (t, J = 7.5 Hz, 3 H). MS m/z 175.1 (MH+).
2-Phenyl-pyrazolo[1,5-a]pyridine-3-carbaldehyde (22k). (2-Phen-
yl-pyrazolo[1,5-a]pyridin-3-yl)-methanol (3.6 g, 43% yield) was pre-
pared according to same procedure as above from 21k (10 g, 38 mmol).
1H NMR (400 MHz, CDCl3) δ 8.49 (dt, J = 7.0, 1.1 Hz, 1 H), 7.91 (dt,
J = 6.7, 1.6 Hz, 2 H), 7.65 (dt, J = 8.9, 1.1 Hz, 1 H), 7.54ꢀ7.49 (m, 2 H),
7.45 (dt, J = 7.2, 1.9 Hz, 1 H), 7.19 (ddd, J = 8.9, 6.7, 1.1 Hz, 1 H), 6.81
(ddd, J = 7.5, 6.2, 0.8 Hz, 1 H), 4.96 (s, 2 H). MS m/z 225.0 (MH+).
The title compound (870 mg, 98% yield) was prepared according to
same procedure as above from (2-phenyl-pyrazolo[1,5-a]pyridin-3-yl)-
methanol (900 mg, 4.0 mmol). 1H NMR (400 MHz, CDCl3) δ 10.04 (s,
1 H), 8.53 (dt, J = 6.9, 1.0 Hz, 1 H), 8.36 (dt, J = 8.8, 1.2 Hz, 1 H),
7.73ꢀ7.70 (m, 2 H), 7.50ꢀ7.44 (m, 4 H), 7.03 (ddd, J = 7.6, 6.3, 0.6 Hz,
1 H). MS m/z 223.0 (MH+).
2-(2-Ethyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamine (24j). The title
compound (5.75 g, 85% pure, 82.5% yield) was prepared according to
same procedure as above from 23i (6.8 g, 31 mmol) and used in the next
step without further purification. 1H NMR (400 MHz, CD3OD) δ 8.43
(dt, J = 7.1, 1.1 Hz, 1 H), 7.60 (dt, J = 8.8, 1.3 Hz, 1 H), 7.24 (ddd, J = 8.9,
6.8, 1.0 Hz, 1 H), 6.86 (ddd, J = 7.6, 6.0, 0.7 Hz, 1 H), 3.35ꢀ3.31 (m, 2
H), 3.14ꢀ3.09 (m, 2 H), 2.86 (q, J = 7.4 Hz, 2 H), 1.36 (t, J = 7.8 Hz, 3
H). MS m/z 190.1 (MH+).
2-(2-Phenyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamine (24k). The ti-
tle compound (700 mg, 78% yield) was prepared according to same
procedure as above from 23k (1.0 g, 3.8 mmol). 1H NMR (400 MHz,
CD3OD) δ 8.55 (dt, J = 7.1, 1.1 Hz, 1 H), 7.76ꢀ7.70 (m, 3 H),
7.57ꢀ7.53 (m, 2 H), 7.49 (tt, J = 7.3, 1.8 Hz, 1 H), 7.33 (ddd, J = 9.1, 6.8,
1.1 Hz, 1 H), 6.97 (ddd, J = 7.5, 6.2, 0.7 Hz, 1 H), 3.30 (t, J = 8.7 Hz, 2 H),
3.09 (t, J = 8.0 Hz, 2 H). MS m/z 238.0 (MH+).
(E)-N-Hydroxy-3-(4-{[2-(1H-pyrrolo[3,2-b]pyridin-3-yl)-ethylamino]-
methyl}-phenyl)-acrylamide (25a). Following method A, (E)-3-(4-
{[2-(1H-pyrrolo[3,2-b]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-acrylic
acid methyl ester (200 mg, 48% yield) was prepared from 20a (200 mg,
1.2 mmol). 1H NMR (400 MHz, CD3OD) δ 8.27 (dd, J = 4.7, 1.4 Hz, 1
H), 7.86 (dd, J = 8.2, 1.4 Hz, 1 H), 7.75 (s, 1 H), 7.72ꢀ7.69 (m, 2 H), 7.56
(d, J = 8.3 Hz, 2 H), 7.51 (s, 1 H), 7.22 (dd, J = 8.1, 4.7 Hz, 1 H), 6.61 (d,
J = 15.9Hz, 1 H), 4.24 (s, 2 H), 3.81 (s, 3 H), 3.33 (t, J = 6.1 Hz, 2 H), 3.22
(t, J = 6.5 Hz, 2 H). MS m/z 336.2 (MH+).
The title compound (10 mg, 5.5% yield) was prepared according
to method B from (E)-3-(4-{[2-(1H-pyrrolo[3,2-b]pyridin-3-yl)-
ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (190 mg, 0.54
mmol). 1H NMR (400 MHz, CD3OD) δ 8.28 (dd, J = 4.7, 1.4 Hz, 1 H),
7.80 (dd, J = 8.3, 1.4 Hz, 1 H), 7.55 (d, J = 16.0 Hz, 1 H), 7.51 (d, J = 8.3
Hz, 2 H), 7.43 (s, 1 H), 7.35 (d, J = 8.6 Hz, 2 H), 7.17 (dd, J = 8.3, 4.9 Hz,
1 H), 6.46 (d, J = 16.3 Hz, 1 H), 3.87 (s, 2 H), 3.10 (t, J = 6.8 Hz, 2 H),
3.01 (t, J = 6.7 Hz, 2 H). MS m/z 336.9 (MH+). HRMS calcd for
C19H20N4O2 (Mꢀ) 335.1508, found 335.1518.
(E)-N-Hydroxy-3-(4-{[2-(6-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-
ethylamino]-methyl}-phenyl)-acrylamide (25b). Following method A,
(E)-3-(4-{[2-(6-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-ethylamino]-
methyl}-phenyl)-acrylic acid methyl ester (60 mg, 21% yield) was
prepared from 20b (140 mg, 0.80 mmol). 1H NMR (400 MHz, CD3OD)
δ 8.76 (s, 1 H), 8.61(s, 1 H), 7.68 (d, J = 16.2Hz, 1 H), 7.55 (d, J = 8.3 Hz,
2 H), 7.35 (d, J = 8.4 Hz, 2 H), 6.52 (d, J = 15.8 Hz, 1 H), 3.82 (s, 2 H),
3.80 (s, 3 H), 2.96 (t, J = 7.6 Hz, 2 H), 2.85 (t, J = 7.5 Hz, 2 H), 2.43 (s, 3
H). MS m/z 351.1 (MH+).
The title compound (4 mg, 7% yield) was prepared according to
method B from (E)-3-(4-{[2-(6-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (60 mg, 0.17
mmol). 1H NMR (400 MHz, DMSO-d6) δ 8.81 (s, 1 H), 8.60 (s, 1 H),
7.48 (d, J = 8.2 Hz, 2 H), 7.42 (d, J = 15.9 Hz, 1 H), 7.33 (d, J = 7.8 Hz, 2
H), 6.43 (d, J = 16.1 Hz, 1 H), 3.75 (s, 2 H), 2.83 (t, J = 6.6 Hz, 2 H), 2.73
(t, J = 7.0 Hz, 2 H), 2.34 (s, 3 H). MS m/z 351.8 (MH+). HRMS calcd for
C19H21N5O2 (MH+) 352.1774, found 352.1784.
Typical Procedure for the Formation of 2-Yl-3-((E)-2-nitro-vinyl)-
pyrazolo[1,5-a]pyridines. To a solution of 2-yl-pyrazolo[1,5-a]pyridine-
3-carbaldehyde (48 mmol) in nitromethane (29 g, 480 mmol) was added
ammonium acetate (18 g, 240 mmol) and heated at 100 ꢀC for 1 h. The
reaction mixture was concentrated under vacuum, and the resulting
residue was chromatographed (silica gel, 10% EtOAc/hexane to 100%
EtOAc/hexane) to obtain the title compound (62ꢀ83% yields).
2-Methyl-3-((E)-2-nitro-vinyl)-pyrazolo[1,5-a]pyridine (23i). The ti-
tle compound (1 g, 83% yield) was prepared according to same
1
procedure as above from 22i (950 mg, 5.9 mmol). H NMR (400
MHz, CDCl3) δ 8.50 (dt, J = 6.9, 1.1 Hz, 1 H), 8.31 (d, J = 13.6 Hz, 1 H),
7.70 (dt, J = 8.8, 1.0 Hz, 1 H), 7.58 (d, J = 13.7 Hz, 1 H), 7.49 (ddd, J =
8.8, 6.9, 1.2 Hz, 1 H), 7.00 (ddd, J = 7.6, 6.1, 0.6 Hz, 1 H), 2.62 (s, 3 H).
MS m/z 204.0 (MH+).
2-Ethyl-3-((E)-2-nitro-vinyl)-pyrazolo[1,5-a]pyridine (23j). The title
compound (6.3 g, 62% yield) was prepared according to same procedure
as above from 22j (8.3 g, 48 mmol). 1H NMR (400 MHz, CDCl3) δ 8.52
(dt, J = 6.9, 1.0 Hz, 1 H), 8.31 (d, J = 13.6 Hz, 1 H), 7.70 (dt, J = 8.9, 1.2
Hz, 1 H), 7.58 (d, J = 13.5 Hz, 1 H), 7.49 (ddd, J = 8.7, 6.9, 1.1 Hz, 1 H),
7.01 (ddd, J = 7.6, 6.2, 0.7 Hz, 1 H), 2.99 (q, J = 7.4 Hz, 2 H), 1.42 (t, J =
7.6 Hz, 3 H). MS m/z 218.0 (MH+).
3-((E)-2-Nitro-vinyl)-2-phenyl-pyrazolo[1,5-a]pyridine (23k). The
title compound (1 g, 99% yield) was prepared according to same
1
procedure as above from 22k (850 mg, 3.8 mmol) H NMR (400
MHz, CDCl3) δ 8.55 (dt, J = 6.9, 1.1 Hz, 1 H), 8.26 (d, J = 13.5 Hz, 1 H),
7.72 (dt, J = 8.8, 1.2 Hz, 1 H), 7.61ꢀ7.58 (m, 2 H), 7.53 (d, J = 13.7 Hz,
1 H), 7.50ꢀ7.44 (m, 4 H), 7.01 (ddd, J = 7.6, 6.3, 0.7 Hz, 1 H). MS m/z
265.9 (MH+).
Typical Procedure for the Formation of 2-Yl-pyrazolo[1,5-a]pyridin-
3yl Ethylamines. To a solution of 2-yl-3-((E)-2-nitro-vinyl)-pyrazolo-
[1,5-a]pyridine (31 mmol) in THF (300 mL) was added BF3 (93 mL,
1 M in THF) and BH3 (188 mL, 1 M in THF) and stirred at 65 ꢀC
overnight. The reaction was quenched with MeOH until bubbling
ceased. The solution was concentrated in vacuo, and aqueous NaOH
(1N) was added followed by extraction with CH2Cl2:EtOH (4:1)
several times until no product was detected by TLC in the aqueous
(E)-N-Hydroxy-3-(4-{[2-(6-methyl-5H-pyrrolo[2,3-b]pyrazin-7-yl)-
ethylamino]-methyl}-phenyl)-acrylamide (25c). Following method A,
4768
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
(E)-3-(4-{[2-(6-methyl-5H-pyrrolo[2,3-b]pyrazin-7-yl)-ethylamino]-
methyl}-phenyl)-acrylic acid methyl ester (130 mg, 41% yield) was
prepared from 20c (160 mg, 0.91 mmol). 1H NMR (400 MHz,
CD3OD) δ 8.76 (s, 1 H), 8.61 (s, 1 H), 7.68 (d, J = 16.2 Hz, 1 H),
7.54 (d, J = 7.9 Hz, 2 H), 7.34 (d, J = 8.1 Hz, 2 H), 6.51 (d, J = 16.0 Hz,
1 H), 3.82 (s, 2 H), 3.80 (s, 3 H), 2.96 (t, J = 7.3 Hz, 2 H), 2.85 (t, J = 7.1
Hz, 2 H), 2.42 (s, 3 H). MS m/z 350.8 (MH+).
3-yl-ethylamino)-methyl]-phenyl}-acrylic acid methyl ester (50 mg,
0.15 mmol). H NMR (400 MHz, CD3OD) δ 8.24 (d, J = 7.7 Hz,
1
1 H), 7.57ꢀ7.49 (m, 4 H), 7.39 (d, J = 14.4 Hz, 1 H), 7.36 (d, J = 8.0 Hz,
2 H), 7.30 (t, J = 7.8 Hz, 1 H), 6.96ꢀ6.92 (m, 1 H), 6.46 (d, J = 15.5 Hz,
1 H), 3.87 (s, 2 H), 3.17 (t, J = 7.7 Hz, 2 H), 3.01 (t, J = 7.4 Hz, 2 H). 13
C
NMR (400 MHz, CD3OD) δ 166.30, 146.67, 141.44, 141.19, 135.53,
131.21, 130.31, 129.03, 126.09, 125.17, 123.60, 118.47, 117.70, 113.85,
53.61, 46.96, 24.27. MS m/z 336.7 (MH+). HRMS calcd for
C19H20N4O2 (MH+) 337.1665, found 337.1652.
The title compound (33 mg, 25% yield) was prepared according to
method B from (E)-3-(4-{[2-(6-methyl-5H-pyrrolo[2,3-b]pyrazin-7-
yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (130 mg,
(E)-N-Hydroxy-3-(4-{[2-(2-methyl-pyrazolo[1,5-a]pyridin-3-yl)-
ethylamino]-methyl}-phenyl)-acrylamide (25i). (E)-3-(4-{[2-(2-
Methyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-
acrylic acid methyl ester (510 mg, 31% yield) was prepared according to
1
0.37 mmol). H NMR (400 MHz, DMSO-d6) δ 8.80 (s, 1 H), 8.60
(s, 1 H), 7.47 (d, J = 8.1 Hz, 2 H), 7.42 (d, J = 15.2 Hz, 1 H), 7.33 (d, J =
8.7 Hz, 2 H), 6.42 (d, J = 15.5 Hz, 1 H), 3.74 (s, 2 H), 2.82 (t, J = 6.7 Hz,
2 H), 2.71 (t, J = 7.2 Hz, 2 H), 2.33 (s, 3 H). 13C NMR (400 MHz,
CD3OD) δ 166.35, 152.32, 150.57, 146.53, 141.23, 136.83, 135.35,
130.18, 128.96, 128.28, 120.93, 118.36, 108.64, 53.75, 50.03, 24.51,
11.30. MS m/z 351.7 (MH+). HRMS calcd for C19H21N5O2 (MH+)
352.1774, found 352.1775.
(E)-N-Hydroxy-3-(4-{[2-(1H-pyrrolo[3,2-c]pyridin-3-yl)-ethylamino]-
methyl}-phenyl)-acrylamide (25d). Following method A, (E)-3-(4-
{[2-(1H-pyrrolo[3,2-c]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-acrylic
acid methyl ester (270 mg, 65% yield) was prepared from 20d (200 mg,
1.2 mmol). 1H NMR (400 MHz, CD3OD) δ 8.80 (s, 1 H), 8.14 (d, J =
6.2 Hz, 1 H), 7.68 (d, J = 16.1 Hz, 1 H), 7.58 (d, J = 7.9 Hz, 2 H),
7.43ꢀ7.39 (m, 3 H), 7.27 (s, 1 H), 6.53 (d, J = 16.2Hz, 1 H), 3.96(s, 2 H),
3.80 (s, 3 H), 3.33ꢀ3.32 (m, 2 H), 2.13ꢀ2.06 (m, 2 H). MS m/z 336.1
(MH+).
1
general procedure method A from 24i (820 mg, 4.7 mmol). H NMR
(400 MHz, CDCl3) δ 8.28 (d, J = 6.7 Hz, 1 H), 7.53 (d, J = 16.6 Hz, 1 H),
7.44 (d, J = 7.2 Hz, 2 H), 7.40ꢀ7.37 (m, 3 H), 7.02 (dd, J = 8.9, 6.8 Hz,
1 H), 6.63 (t, J = 7.0 Hz, 1 H), 6.32 (d, J = 16.3 Hz, 1 H), 3.81 (s, 3 H),
3.70 (s, 2 H), 3.00ꢀ2.98 (m, 4 H), 2.39 (s, 3 H). MS m/z 349.9 (MH+).
The title compound (50 mg, 25% yield) was prepared according to
method B from (E)-3-(4-{[2-(2-methyl-pyrazolo[1,5-a]pyridin-3-yl)-
ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (200 mg, 0.57
mmol). 1H NMR (400 MHz, CD3OD) δ 8.38 (d, J = 7.1, 1 H), 7.63 (d,
J = 7.6 Hz, 2 H), 7.61 (d, J = 9.6, 1 H), 7.54 (dd, J = 13.1, 3.5 Hz, 1 H),
7.50 (d, J = 7.6, 2 H), 7.20 (dd, J = 7.6, 7.1 Hz, 1 H), 6.81 (t, J = 7.1 Hz,
1 H), 6.52 (d, J = 15.6 Hz, 1 H), 4.18 (s, 2 H), 3.12 (m, 4 H), 2.42 (s,
3 H). 13C NMR (400 MHz, CD3OD) δ 166.2, 150.89, 141.00, 140.75,
140.05, 135.87, 130.52, 129.09, 128.83, 124.65, 118.72, 117.11, 112.69,
106.03, 53.31, 49.71, 22.90, 11.81. MS m/z 351.0 (MH+). HRMS calcd
for C12H25N4O2 (MH+) 351.1821, found 351.1831.
The title compound (20 mg, 7.8% yield) was prepared according
to method
B from (E)-3-(4-{[2-(1H-pyrrolo[3,2-c]pyridin-3-yl)-
(E)-3-(4-{[2-(2-Ethyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamino]-
methyl}-phenyl)-N-hydroxy-acrylamide (25j). (E)-3-(4-{[2-(2-Ethyl-
pyrazolo[1,5-a]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-acrylic acid
methyl ester (1.0 g, 99% yield) was prepared according to general
ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (270 mg, 0.77
mmol). 1H NMR (400 MHz, DMSO-d6) δ 8.79 (s, 1 H), 8.12 (d, J = 5.8
Hz, 1 H), 7.48 (d, J = 7.8 Hz, 2 H), 7.43 (d, J = 16.0 Hz, 1 H), 7.36 (d, J =
8.2 Hz, 2 H), 7.30 (dd, J = 5.6, 1.2 Hz, 1 H), 7.21 (s, 1 H), 6.43 (d, J =
17.2 Hz, 1 H), 3.77 (s, 2 H), 2.91 (t, J = 7.3 Hz, 2 H), 2.82 (t, J = 7.0 Hz, 2
H). 13C NMR (400 MHz, CD3OD) δ 166.33, 142.06, 141.99, 141.91,
141.19, 140.12, 135.33, 130.18, 128.95, 125.76, 125.63, 118.34, 114.21,
108.25, 53.72, 50.19, 25.68. MS m/z 337 (MH+). HRMS calcd for
C19H20N4O2 (MH+) 337.1665, found 337.1657.
1
procedure method A from 24j (530 mg, 2.8 mmol). H NMR (400
MHz, CDCl3) δ 8.33 (dt, J = 7.0, 1.2 Hz, 1 H), 7.51 (d, J = 15.9 Hz, 1 H),
7.48ꢀ7.39 (m, 5 H), 7.07ꢀ7.03 (m, 1 H), 6.69ꢀ6.64 (m, 1 H), 6.31 (d,
J = 15.9 Hz, 1 H), 3.82 (s, 3 H), 3.71 (s, 2 H), 3.45 (t, J = 6.4 Hz, 2 H),
2.92 (t, J = 7.0 Hz, 2 H), 2.75 (q, J = 7.7 Hz, 2 H), 1.28 (t, J = 7.7 Hz, 3 H).
MS m/z 363.9 (MH+).
(E)-3-(4-{[2-(2-tert-Butyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-ethylamino]-
methyl}-phenyl)-N-hydroxy-acrylamide (25f). Following method A,
20f (524 mg, 2.03 mmol, approximately 84% pure) was converted into
(E)-3-(4-{[2-(2-tert-butyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-ethylamino]-
methyl}-phenyl)-acrylic acid methyl ester (392 mg, 1.0 mmol, 49%
yield). 1H NMR (DMSO-d6) δ 10.99 (br s, 1H), 8.09 (dd, J = 1.5, 5.0 Hz,
1H), 7.77 (d, J = 7.8 Hz, 1H) 7.66 (m, 3H), 7.40 (d, J = 7.9 Hz, 2H), 6.95
(dd, J = 4.8, 7.8 Hz, 1H), 6.61 (d, J = 15.6 Hz, 1H), 3.83 (s, 2H), 3.73 (s,
3H), 2.99 (m, 2H), 2.73 (m, 2H), 1.40 (s, 9H). m/z 392.0 (MH+).
Following method B, (E)-3-(4-{[2-(2-tert-butyl-1H-pyrrolo[2,3-
b]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl ester
(400 mg, 1.02 mmol) was converted to 25f (52 mg, 0.13 mmol, 13%
yield) after HPLC purification. 1H NMR (CD3OD) δ 8.07 (d, J = 4.7
Hz, 1H) 7.82 (d, J = 8.0 Hz, 1H), 7.51 (m, 3H), 7.35 (d, J = 8.8 Hz, 2H),
6.99 (dd, J = 5.0, 8.0 Hz, 1H), 6.47 (d, J = 15.9 Hz, 1H), 3.82 (s, 2H),
3.09 (m, 2H), 2.80 (m, 2H), 1.46 (s, 9H). 13C NMR (CD3OD) δ 166.0,
148.3, 146.1, 142.7, 140.7, 136.9, 131.1, 129.4, 127.2, 123.5, 119.5, 116.4,
104.6, 52.5, 34.7, 31.1, 24.1. m/z 393.2 (MH+). HRMS (MH+) calcd for
C23H28N4O2, 393.2291, found 393.2291.
The title compound (47 mg, 47% yield) was prepared according to
general procedure method B from (E)-3-(4-{[2-(2-ethyl-pyrazolo[1,5-
a]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl ester
(100 mg, 0.27 mmol). 1H NMR (400 MHz, CD3OD) δ 8.43 (d, J = 7.0,
1 H), 7.68 (d, J = 8.1 Hz, 2 H), 7.63ꢀ7.57 (m, 4 H), 7.23 (dd, J = 8.7, 6.8
Hz, 1 H), 6.85 (td, J = 6.8, 1.0 Hz, 1 H), 6.56 (d, J = 16.0 Hz, 1 H), 4.31
(s, 2 H), 3.25ꢀ3.16 (m, 4 H), 2.85 (q, J = 7.7 Hz, 2 H), 1.34 (t, J = 7.6 Hz,
3 H). 13C NMR (400 MHz, CD3OD) δ 165.94, 156.47, 140.82, 140.48,
134.16, 131.7, 129.5, 129.12, 125.11, 119.94, 117.07, 113.08, 103.05,
51.97, 49.75, 20.88, 20.63, 14.57. HRMS C12H25N4O2 (MH+). calcd
365.1978, found 365.1985.
(E)-N-Hydroxy-3-(4-{[2-(2-phenyl-pyrazolo[1,5-a]pyridin-3-yl)-
ethylamino]-methyl}-phenyl)-acrylamide (25k). (E)-3-(4-{[2-(2-
Phenyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-
acrylic acid methyl ester (640 mg, 53% yield) was prepared according
1
to general procedure method A from 24k (700 mg, 3.0 mmol). H
NMR (400 MHz, CDCl3) δ 8.44 (d, J = 6.9 Hz, 1 H), 7.74ꢀ7.70 (m,
3 H), 7.52ꢀ7.41 (m, 4 H), 7.38 (d, J = 7.6 Hz, 2 H), 7.29 (d, J = 7.9
Hz, 2 H), 7.11 (dd, J = 8.8, 6.8 Hz, 1 H), 6.76 (ddd, J = 7.5, 6.2, 0.7 Hz,
1 H), 6.28 (d, J = 16.1 Hz, 1 H), 3.86 (s, 3 H), 3.83 (s, 2 H), 3.09 (t, J =
7.9 Hz, 2 H), 2.94 (t, J = 7.5 Hz, 2 H). MS m/z 412.1 (MH+).
The title compound (35 mg, 35% yield) was prepared according to
general procedure method B from (E)-3-(4-{[2-(2-phenyl-pyrazolo-
[1,5-a]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl
(E)-N-Hydroxy-3-{4-[(2-imidazo[1,2-a]pyridin-3-yl-ethylamino)-
methyl]-phenyl}-acrylamide (25g). (E)-3-{4-[(2-Imidazo[1,2-a]-
pyridin-3-yl-ethylamino)-methyl]-phenyl}-acrylic acid methyl ester
(50 mg, 17% yield) was prepared according to general method A from
20g (140 mg, 0.87 mmol). MS m/z 335.7 (MH+).
The title compound (13 mg, 26% yield) was prepared according to
general procedure method B from (E)-3-{4-[(2-imidazo[1,2-a]pyridin-
1
ester (100 mg, 0.24 mmol). H NMR (400 MHz, CD3OD) δ 8.48
4769
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
(dt, J = 7.0, 1.1 Hz, 1 H), 7.70ꢀ7.67 (m, 2 H), 7.62 (dt, J = 9.2, 1.2 Hz,
1 H), 7.54ꢀ7.43 (m, 6 H), 7.27 (d, J = 8.0 Hz, 2 H), 7.21 (ddd, J = 9.0,
6.6, 1.2 Hz, 1 H), 6.89 (dt, J = 9.7, 3.5 Hz, 1 H), 6.47 (d, J = 15.8 Hz, 1 H),
3.74 (s, 2 H), 3.13 (t, J = 7.8 Hz, 2 H), 2.78 (t, J = 7.8 Hz, 2 H). 13C NMR
(400 MHz, CD3OD) δ 166.35, 153.31, 142.23, 141.45, 141.14, 135.23,
134.58, 131.05, 130.07, 129.78, 129.47, 129.07, 128.92, 124.66, 118.35,
118.03, 113.71, 106.78, 53.54, 49.86, 24.03. MS m/z 412.9 (MH+).
HRMS calcd for C25H24N4O2 (Mꢀ) 411.1821, found 411.1830.
(E)-N-Hydroxy-3-[4-({isopropyl-[2-(2-methyl-pyrazolo[1,5-a]pyridin-
3-yl)-ethyl]-amino}-methyl)-phenyl]-acrylamide (25l). (E)-3-[4-
({Isopropyl-[2-(2-methyl-pyrazolo[1,5-a]pyridin-3-yl)-ethyl]-amino}-
methyl)-phenyl]-acrylic acid methyl ester (400 mg, 70% yield) was
prepared according to same procedure as the intermediate for
25m below from (E)-3-(4-{[2-(2-methyl-pyrazolo[1,5-a]pyridin-3-
yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (900 mg,
2.48 mmol). 1H NMR(400MHz, CDCl3) δ8.27 (dt,J =7.0, 1.2Hz, 1 H),
7.68 (d, J = 16.7 Hz, 1 H), 7.40 (d, J = 7.5 Hz, 2 H), 7.30 (d, J = 8.0 Hz, 2
H), 7.09 (dt, J = 8.8, 1.2 Hz, 1 H), 6.89 (ddd, J = 8.8, 6.7, 1.1 Hz, 1 H), 6.56
(td, J = 6.9, 1.4 Hz, 1 H), 6.41 (d, J = 16.0 Hz, 1 H), 3.81 (s, 3 H), 3.62 (s,
2 H), 3.05 (sept, J = 6.5 Hz, 1 H), 2.69 (t, J = 7.6 Hz, 2 H), 2.56 (t, J = 7.9
Hz, 2 H), 2.32 (s, 3 H), 1.05 (d, J = 6.3 Hz, 6 H). MS m/z 391.9 (MH+).
The title compound (280 mg, 72% yield) was prepared according to
(E)-N-Hydroxy-3-[4-({isopropyl-[2-(2-phenyl-pyrazolo[1,5-a]pyridin-
3-yl)-ethyl]-amino}-methyl)-phenyl]-acrylamide (25n). (E)-3-[4-
({Isopropyl-[2-(2-phenyl-pyrazolo[1,5-a]pyridin-3-yl)-ethyl]-amino}-
methyl)-phenyl]-acrylic acid methyl ester (300 mg, 52% yield) was
prepared according to same procedure as above from (E)-3-(4-{[2-(2-
phenyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-ac-
1
rylic acid methyl ester (520 mg, 1.26 mmol). H NMR (400 MHz,
CDCl3) δ 8.39 (dt, J = 7.1, 1.1 Hz, 1 H), 7.69 (d, J = 14.5 Hz, 1 H),
7.66ꢀ7.64 (m, 2 H), 7.42ꢀ7.37 (m, 5 H), 7.27 (d, J = 8.6 Hz, 2 H), 7.20
(dt, J = 9.0, 1.4 Hz, 1 H), 6.96 (ddd, J = 8.9, 6.6, 1.1 Hz, 1 H), 6.67 (ddd,
J = 7.5, 6.3, 0.7 Hz, 1 H), 6.42 (d, J = 15.8 Hz, 1 H), 3.82 (s, 3 H), 3.57 (s,
2 H), 2.99 (sept, J = 5.2 Hz, 1 H), 2.92 (t, J = 7.6 Hz, 2 H), 2.62 (t, J = 7.8
Hz, 2 H), 1.00 (d, J = 6.4 Hz, 6 H). MS m/z 453.9 (MH+).
The title compound (270 mg, 92% yield) was prepared according to
the general procedure method B from (E)-3-[4-({isopropyl-[2-(2-
phenyl-pyrazolo[1,5-a]pyridin-3-yl)-ethyl]-amino}-methyl)-phenyl]-
acrylic acid methyl ester (300 mg, 0.66 mmol). 1H NMR (400 MHz,
CD3OD) δ 8.41 (dt, J = 7.0, 1.1 Hz, 1 H), 7.62 (dt, J = 6.0, 2.0 Hz, 2 H),
7.47ꢀ7.40 (m, 0 H), 7.34 (d, J = 8.0 Hz, 2 H), 7.30 (dt, J = 8.9, 1.3 Hz,
1 H), 7.15 (d, J = 8.1 Hz, 2 H), 7.08 (ddd, J = 9.0, 6.7, 1.0 Hz, 1 H), 6.82
(dt, J = 9.7, 3.5 Hz, 1 H), 6.47 (d, J = 15.8 Hz, 1 H), 3.53 (s, 2 H), 3.02
(sept, J = 6.6 Hz, 1 H), 2.93 (t, J = 7.2 Hz, 2 H), 2.60 (t, J = 7.7 Hz, 2 H),
1.00 (d, J = 6.5 Hz, 6 H). 13C NMR (400 MHz, CD3OD) δ 166.56,
153.26, 144.31, 141.67, 141.3, 134.79, 130.36, 129.79, 129.67, 129.62,
129.35, 129.30,128.94, 128.9, 128.51, 128.41, 126.34, 124.18, 118.13,
117.73, 113.46, 108.14, 54.69, 54.43, 51.47, 51.34, 23.83, 18.28.
HRMS calcd for C28H30N4O2 (MH+) 455.2447, found 455.2442.
method
B
from (E)-3-[4-({isopropyl-[2-(2-methyl-pyrazolo[1,5-
a]pyridin-3-yl)-ethyl]-amino}-methyl)-phenyl]-acrylic acid methyl es-
ter (390 mg, 0.99 mmol). 1H NMR (400 MHz, CD3OD) δ 8.29 (dt, J =
7.0, 1.1 Hz, 1 H), 7.48 (d, J = 16.1 Hz, 1 H), 7.35 (d, J = 8.3 Hz, 2 H),
7.21ꢀ7.18 (m, 3 H), 7.00 (ddd, J = 8.9, 6.7, 1.0 Hz, 1 H), 6.70 (dt, J = 9.5,
3.5 Hz, 1 H), 6.44 (d, J = 16.0 Hz, 1 H), 3.60 (s, 2 H), 3.13 (sept, J = 6.6
Hz, 1 H), 2.72 (t, J = 7.1 Hz, 2 H), 2.61 (t, J = 7.2 Hz, 2 H), 2.27 (s, 3 H),
1.11 (d, J = 6.5 Hz, 6 H). MS m/z 393.0 (MH+). HRMS calcd for
C23H28N4O2 (MH+) 393.2291, found 39.2289.
(E)-3-[4-({[2-(2-Ethyl-pyrazolo[1,5-a]pyridin-3-yl)-ethyl]-isopropyl-
amino}-methyl)-phenyl]-N-hydroxy-acrylamide (25m). (E)-3-(4-
{[2-(2-Ethyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-
acrylic acid methyl ester (900 mg, 2.48 mmol) was added to a solution
of 2-iodo-propane (4.21 g, 24.8 mmol) and triethylamine (3.44 mL,
24.8 mmol) in CH3CN(20mL). The mixture wasrefluxedovernight. The
solvent was then removed under vacuum. EtOAc and water were added.
The aqueous layer was extracted several times with EtOAc. The combined
organic layers were washed with brine, dried over Na2SO4, filtered, and
evaporated off. The crude product was purified via flash column chroma-
tography (EtOAc:hexanes, 10:90 to 100:0) to provide (E)-3-[4-({[2-(2-
ethyl-pyrazolo[1,5-a]pyridin-3-yl)-ethyl]-isopropyl-amino}-methyl)-
phenyl]-acrylic acid methyl ester as a slightly yellow oil (300 mg, 30%
yield). 1H NMR (400 MHz, CDCl3) δ 8.30 (dt, J = 7.0, 1.2 Hz, 1 H),
7.69 (d, J = 16.1 Hz, 1 H), 7.42 (d, J = 7.9 Hz, 2 H), 7.34 (d, J = 8.0 Hz,
2 H), 7.11 (dt, J = 8.8, 1.3 Hz, 1 H), 6.90 (ddd, J = 8.9, 6.6, 1.2 Hz, 1 H),
6.57 (ddd, J = 7.8, 6.0, 0.7 Hz, 1 H), 6.42 (d, J = 15.9 Hz, 1 H), 3.81 (s,
3 H), 3.64 (s, 2 H), 3.06 (sept, J = 6.6 Hz, 1 H), 2.72ꢀ2.66 (m, 4 H),
2.56 (t, J = 8.1 Hz, 2 H), 1.24 (t, J = 7.6 Hz, 3 H), 1.07 (d, J = 6.8 Hz,
6 H). MS m/z 406.6 (MH+).
’ ASSOCIATED CONTENT
S
Supporting Information. Scatter plot of clogP and hERG
b
radioligand displacement IC50. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 617-871-7551. Fax: 617-871-4081. E-mail: michael.
shultz@novartis.com.
Present Addresses
†Department of Chemistry, Massachusetts Institute of Technol-
ogy, Cambridge, Massachusetts, United States.
‡Alkermes, 852 Winter Street, Waltham, Massachusetts 02451,
United States.
§Memorial Sloan Kettering Cancer Center, New York, New York,
United States.
WuXi AppTec, Shanghai, China.
^Concert Pharmaceuticals Inc., 99 Hayden Avenue, Lexington
Massachusetts, 02421, United States.
The title compound (160 mg, 54% yield) was prepared according to
the general procedure method B from (E)-3-[4-({[2-(2-ethyl-pyrazolo-
[1,5-a]pyridin-3-yl)-ethyl]-isopropyl-amino}-methyl)-phenyl]-acrylic
acid methyl ester (300 mg, 0.73 mmol). 1H NMR (400 MHz, CD3OD)
δ 8.30 (d, J = 6.9 Hz, 1 H), 7.54 (d, J = 15.8 Hz, 1 H), 7.40 (d, J = 8.3 Hz,
2 H), 7.26 (d, J = 7.8 Hz, 2 H), 7.21 (d, J = 8.7 Hz, 1 H), 7.01 (ddd, J =
8.9, 6.7, 1.0 Hz, 1 H), 6.70 (dt, J = 9.5, 3.5 Hz, 1 H), 6.43 (d, J = 15.8 Hz,
1 H), 3.66 (s, 2 H), 3.18 (sept, J = 6.4 Hz, 1 H), 2.72 (t, J = 7.2 Hz, 2 H),
2.66ꢀ2.60 (m, 4 H), 1.19 (t, J = 7.6 Hz, 3H), 1.14 (d, J = 6.4 Hz, 6H).
13C NMR (400 MHz, CD3OD) δ 166.48, 155.99, 141.52, 140.59,
134.96, 130.65, 128.71, 128.63, 123.98, 117.96, 117.47, 112.45, 107.15,
54.77, 52.10, 51.68, 23.36, 20.64, 18.36, 14.63. MS m/z 407.0 (MH+).
HRMS C24H30N4O2 (Mꢀ) calcd 405.2291, found 405.2323
’ ABBREVIATIONS USED
CSI, cardiac safety index; fwhm, full width at half-maximum;
H1299, human nonsmall cell lung cancer cell line; HCT-116,
human colorectal carcinoma cell line; HDAC, histone deacety-
lase; hERG, human ether-a-go-go related gene product; iCSI, in
vitro cardiac safety index; PK, pharmacokinetic; SAHA, suberoy-
lanilide hydroxamic acid; SAR, structureꢀactivity relationships
’ REFERENCES
(1) Wang, H.; Holloway, M. P.; Ma, L.; Cooper, Z. A.; Riolo, M.;
Samkari, A.; Elenitoba-Johnson, K. S.; Chin, Y. E.; Altura, R. A.
4770
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
Acetylation directs survivin nuclear localization to repress STAT3
oncogenic activity. J. Biol. Chem. 2010, 46, 36129–36137.
(2) Lu, Z.; Scott, I.; Webster, B. R.; Sack, M. N. The emerging
characterization of lysine residue deacetylation on the modulation of
mitochondrial function and cardiovascular biology. Circ. Res. 2009,
105, 830–841.
(20) Colussi, C.; Illi, B.; Rosati, J.; Spallotta, F.; Farsetti, A.; Grasselli,
A.; Mai, A.; Capogrossi, M. C.; Gaetano, C. Histone deacetylase
inhibitors: keeping momentum for neuromuscular and cardiovascular
diseases treatment. Pharmacol. Res. 2010, 62, 3–10.
(21) Cho, Y. K.; Eom, G. H.; Kee, H. J.; Kim, H. S.; Choi, W. Y.;
Nam, K. I.; Ma, J. S.; Kook, H. Sodium valproate, a histone deacetylase
inhibitor, but not captopril, prevents right ventricular hypertrophy in
rats. Circ. J. 2010, 74, 760–770.
(22) Kee, H. J.; Sohn, I. S.; Nam, K. I.; Park, J. E.; Qian, Y. R.; Yin, Z.;
Ahn, Y.; Jeong, M. H.; Bang, Y. J.; Kim, N.; Kim, J. K.; Kim, K. K.;
Epstein, J. A.; Kook, H. Inhibition of histone deacetylation blocks cardiac
hypertrophy induced by angiotensin II infusion and aortic banding.
Circulation 2006, 113, 51–59.
(23) Meinke, P. T.; Liberator, P. Histone deacetylase: a target for
antiproliferative and antiprotozoal agents. Curr. Med. Chem. 2001,
8, 211–235.
(24) Kavanaugh, S. M.; White, L. A.; Kolesar, J. M. Vorinostat: a
novel therapy for the treatment of cutaneous T-cell lymphoma. Am. J.
Health-Syst. Pharm. 2010, 67, 793–797.
(25) Gimsing, P. Belinostat: a new broad acting antineoplastic
histone deacetylase inhibitor. Expert. Opin. Invest. Drugs 2009, 18, 501–
508.
(26) Atadja, P.; Hsu, M.; Kwon, P.; Trogani, N.; Bhalla, K.;
Remiszewski, S. Molecular and cellular basis for the anti-proliferative
effects of the HDAC inhibitor LAQ824. Novartis Found. Symp. 2004,
259, 249–266.
(3) Shandilya, J.; Swaminathan, V.; Gadad, S. S.; Choudhari, R.;
Kodaganur, G. S.; Kundu, T. K. Acetylated NPM1 localizes in the
nucleoplasm and regulates transcriptional activation of genes implicated
in oral cancer manifestation. Mol. Cell. Biol. 2009, 29, 5115–5127.
(4) Faus, H.; Haendler, B. Androgen receptor acetylation sites
differentially regulate gene control. J. Cell Biochem. 2008, 104, 511–524.
(5) di Bari, M. G.; Ciuffini, L.; Mingardi, M.; Testi, R.; Soddu, S.;
Barila, D. c-Abl acetylation by histone acetyltransferases regulates its
nuclear-cytoplasmic localization. EMBO Rep. 2006, 7, 727–733.
(6) Zhao, L. J.; Subramanian, T.; Zhou, Y.; Chinnadurai, G. Acetyla-
tion by p300 regulates nuclear localization and function of the transcrip-
tional corepressor CtBP2. J. Biol. Chem. 2006, 281, 4183–4189.
(7) Gay, F.; Calvo, D.; Lo, M. C.; Ceron, J.; Maduro, M.; Lin, R.; Shi,
Y. Acetylation regulates subcellular localization of the Wnt signaling
nuclear effector POP-1. Genes Dev. 2003, 17, 717–722.
(8) Du, Z.; Song, J.; Wang, Y.; Zhao, Y.; Guda, K.; Yang, S.; Kao,
H. Y.; Xu, Y.; Willis, J.; Markowitz, S. D.; Sedwick, D.; Ewing, R. M.;
Wang, Z. DNMT1 Stability Is Regulated by Proteins Coordinating
Deubiquitination and Acetylation-Driven Ubiquitination. Sci. Signaling
2010, 3 (ra80), 1–10.
(9) Kim, S.; Jho, E. H. The protein stability of Axin, a negative
regulator of Wnt signaling, is regulated by Smad Ubiquitination Reg-
ulatory Factor 2. J. Biol. Chem. 2010, 47, 36420–36426.
(10) Chen, J. Y.; Wang, M. C.; Hung, W. C. Bcr-Abl induced tyrosine
phosphorylation of emi1 to stabilize skp2 protein via inhibition of
ubiquitination in chronic myeloid leukemia cells. J. Cell Physiol. 2010,
226, 407–413.
(11) Nie, J.; Liu, L.; Wu, M.; Xing, G.; He, S.; Yin, Y.; Tian, C.; He,
F.; Zhang, L. HECT ubiquitin ligase Smurf1 targets the tumor suppres-
sor ING2 for ubiquitination and degradation. FEBS Lett. 2010,
584, 3005–3012.
(12) Patwardhan, P.; Resh, M. D. Myristoylation and membrane
binding regulate c-Src stability and kinase activity. Mol. Cell. Biol. 2010,
30, 4094–4107.
(13) Singh, B. N.; Zhang, G.; Hwa, Y. L.; Li, J.; Dowdy, S. C.; Jiang,
S. W. Nonhistone protein acetylation as cancer therapy targets. Expert.
Rev. Anticancer Ther. 2010, 10, 935–954.
(14) Mann, B. S.; Johnson, J. R.; Cohen, M. H.; Justice, R.; Pazdur, R.
FDA approval summary: vorinostat for treatment of advanced primary
cutaneous T-cell lymphoma. Oncologist 2007, 12, 1247–1252.
(15) Vandermolen, K. M.; McCulloch, W.; Pearce, C. J.; Oberlies,
N. H. Romidepsin (Istodax, NSC 630176, FR901228, FK228,
depsipeptide): a natural product recently approved for cutaneous T-cell
lymphoma. J. Antibiot. (Tokyo) 2011, (Epub ahead of print) doi:
10.1038/ja.2011.35
(16) Thomas, E. A.; Coppola, G.; Desplats, P. A.; Tang, B.; Soragni,
E.; Burnett, R.; Gao, F.; Fitzgerald, K. M.; Borok, J. F.; Herman, D.;
Geschwind, D. H.; Gottesfeld, J. M. The HDAC inhibitor 4b ameliorates
the disease phenotype and transcriptional abnormalities in Huntington’s
disease transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 15564–
15569.
(17) Gray, S. G.; Dangond, F. Rationale for the use of histone
deacetylase inhibitors as a dual therapeutic modality in multiple sclerosis.
Epigenetics 2006, 1, 67–75.
(18) Minamiyama, M.; Katsuno, M.; Adachi, H.; Waza, M.; Sang, C.;
Kobayashi, Y.; Tanaka, F.; Doyu, M.; Inukai, A.; Sobue, G. Sodium
butyrate ameliorates phenotypic expression in a transgenic mouse model
of spinal and bulbar muscular atrophy. Hum. Mol. Genet. 2004, 13, 1183–
1192.
(27) Yong, W. P.; Goh, B. C.; Soo, R. A.; Toh, H. C.; Ethirajulu, K.;
Wood, J.; Novotny-Diermayr, V.; Lee, S. C.; Yeo, W. L.; Chan, D.; Lim,
D.; Seah, E.; Lim, R.; Zhu, J. Phase I and pharmacodynamic study of an
orally administered novel inhibitor of histone deacetylases, SB939, in
patients with refractory solid malignancies. Ann. Oncol. 2011, (advance
access) doi: 10.1093/annonc/mdq784.
(28) Boumber, Y.; Younes, A.; Garcia-Manero, G. Mocetinostat
(MGCD0103): a review of an isotype-specific histone deacetylase
inhibitor. Expert. Opin. Invest. Drugs 2011, 20, 823–829.
(29) Hess-Stumpp, H.; Bracker, T. U.; Henderson, D.; Politz, O.
MS-275, a potent orally available inhibitor of histone deacetylases—the
development of an anticancer agent. Int. J. Biochem. Cell Biol. 2007,
39, 1388–1405.
(30) Piekarz, R. L.; Frye, R.; Prince, H. M.; Kirschbaum, M. H.; Zain,
J.; Allen, S. L.; Jaffe, E. S.; Ling, A.; Turner, M.; Peer, C. J.; Figg, W. D.;
Steinberg, S. M.; Smith, S.; Joske, D.; Lewis, I.; Hutchins, L.; Craig, M.;
Fojo, A. T.; Wright, J. J.; Bates, S. E. Phase II trial of romidepsin in
patients with peripheral T-cell lymphoma. Blood 2011, 117, 5827–5834.
(31) Campas-Moya, C. Romidepsin for the treatment of cutaneous
T-cell lymphoma. Drugs Today 2009, 45, 787–795.
(32) Kouraklis, G.; Theocharis, S. Histone deacetylase inhibitors and
anticancer therapy. Curr. Med. Chem. Anticancer Agents 2002, 2, 477–
484.
(33) Marks, P. A.; Richon, V. M.; Kelly, W. K.; Chiao, J. H.; Miller, T.
Histone deacetylase inhibitors: development as cancer therapy. Novartis
Found. Symp. 2004, 259, 269–281.
(34) Kouraklis, G.; Theocharis, S. Histone deacetylase inhibitors: a
novel target of anticancer therapy (review). Oncol. Rep. 2006,
15, 489–494.
(35) Mai, A.; Massa, S.; Rotili, D.; Cerbara, I.; Valente, S.; Pezzi, R.;
Simeoni, S.; Ragno, R. Histone deacetylation in epigenetics: an attractive
target for anticancer therapy. Med. Res. Rev. 2005, 25, 261–309.
(36) Federico, M.; Bagella, L. Histone deacetylase inhibitors in the
treatment of hematological malignancies and solid tumors. J. Biomed.
Biotechnol. 2011, 2011, 475641–475652.
(37) Quintas-Cardama, A.; Santos, F. P.; Garcia-Manero, G. Histone
deacetylase inhibitors for the treatment of myelodysplastic syndrome
and acute myeloid leukemia. Leukemia 2011, 25, 226–235.
(38) Carafa, V.; Nebbioso, A.; Altucci, L. Histone deacetylase
inhibitors: recent insights from basic to clinical knowledge and patenting
of anti-cancer actions. Recent Pat. Anticancer Drug Discovery 2011, 6,
131–145.
(19) Pang, M.; Zhuang, S. Histone deacetylase: a potential thera-
peutic target for fibrotic disorders. J. Pharmacol. Exp. Ther. 2010, 335,
266–272.
4771
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772

Journal of Medicinal Chemistry
ARTICLE
(39) Jain, N.; Odenike, O. Emerging role of the histone deacetylase
inhibitor romidepsin in hematologic malignancies. Expert Opin. Phar-
macother. 2010, 11, 3073–3084.
(40) Marks, P. A. The clinical development of histone deacetylase
inhibitors as targeted anticancer drugs. Expert. Opin. Invest. Drugs 2010,
19, 1049–1066.
(41) Marsoni, S.; Damia, G.; Camboni, G. A work in progress: the
clinical development of histone deacetylase inhibitors. Epigenetics 2008,
3, 164–171.
(42) Bruserud, O.; Stapnes, C.; Ersvaer, E.; Gjertsen, B. T.; Ryningen,
A. Histone deacetylase inhibitors in cancer treatment: a review of the
clinicaltoxicityandthemodulation ofgene expression incancercell. Curr.
Pharm. Biotechnol. 2007, 8, 388–400.
(43) Rambaldi, A.; Dellacasa, C. M.; Finazzi, G.; Carobbio, A.;
Ferrari, M. L.; Guglielmelli, P.; Gattoni, E.; Salmoiraghi, S.; Finazzi,
M. C.; Di, T. S.; D’Urzo, C.; Vannucchi, A. M.; Barosi, G.; Barbui, T. A
pilot study of the histone-deacetylase inhibitor Givinostat in patients
with JAK2V617F positive chronic myeloproliferative neoplasms. Br. J.
Haematol. 2010, 150, 446–455.
(44) Molife, R.; Fong, P.; Scurr, M.; Judson, I.; Kaye, S.; de Bono, J.
HDAC inhibitors and cardiac safety. Clin. Cancer Res. 2007, 13, 1068–
1069.
(55) Recanatini, M.; Poluzzi, E.; Masetti, M.; Cavalli, A.; De, P. F.
QT prolongation through hERG K(+) channel blockade: current
knowledge and strategies for the early prediction during drug develop-
ment. Med. Res. Rev. 2005, 25, 133–166.
(56) Lagrutta, A. A.; Trepakova, E. S.; Salata, J. J. The hERG channel
and risk of drug-acquired cardiac arrhythmia: an overview. Curr. Top.
Med. Chem. 2008, 8, 1102–1112.
(57) Redfern, W. S.; Carlsson, L.; Davis, A. S.; Lynch, W. G.; MacK-
enzie, I.; Palethorpe, S.; Siegl, P. K.; Strang, I.; Sullivan, A. T.; Wallis, R.;
Camm, A. J.; Hammond, T. G. Relationships between preclinical cardiac
electrophysiology, clinical QT interval prolongation and torsade de
pointes for a broad range of drugs: evidence for a provisional safety
margin in drug development. Cardiovasc. Res. 2003, 58, 32–45.
(58) Kerr, J. S.; Galloway, S.; Lagrutta, A.; Armstrong, M.; Miller, T.;
Richon, V. M.; Andrews, P. A. Nonclinical safety assessment of the
histone deacetylase inhibitor vorinostat. Int. J. Toxicol. 2010, 29, 3–19.
(59) Munster, P. N.; Rubin, E. H.; Van, B. S.; Friedman, E.;
Patterson, J. K.; Van, D. K.; Li, X.; Comisar, W.; Chodakewitz, J. A.;
Wagner, J. A.; Iwamoto, M. A single supratherapeutic dose of vorinostat
does not prolong the QTc interval in patients with advanced cancer.
Clin. Cancer Res. 2009, 15, 7077–7084.
(60) Borchard, U.; Hafner, D. Ion channels and arrhythmias.
(45) Strevel, E. L.; Ing, D. J.; Siu, L. L. Molecularly targeted oncology
therapeutics and prolongation of the QT interval. J. Clin. Oncol. 2007,
25, 3362–3371.
Z. Kardiol. 2000, 89 (Suppl 3), 6–12.
(61) Szantay, Cs.; Szabo, L.; Kalaus, G. A New Route to Trypta-
mines. Synthesis 1974, 5, 354–356.
(46) Subramanian, S.; Bates, S.; Wright, J.; Espinoza-Delgado, I.;
Piekarz, R. Clinical Toxicities of Histone Deacetylase Inhibitors. Phar-
maceuticals 2010, 3, 2751–2767.
(62) Lee, N.; Authier, S.; Pugsley, M. K.; Curtis, M. J. The continuing
evolution of torsades de pointes liability testing methods: is there an end
in sight? Toxicol. Appl. Pharmacol. 2010, 243, 146–153.
(47) Khan, N.; Jeffers, M.; Kumar, S.; Hackett, C.; Boldog, F.;
Khramtsov, N.; Qian, X.; Mills, E.; Berghs, S. C.; Carey, N.; Finn,
P. W.; Collins, L. S.; Tumber, A.; Ritchie, J. W.; Jensen, P. B.;
Lichenstein, H. S.; Sehested, M. Determination of the class and isoform
selectivity of small-molecule histone deacetylase inhibitors. Biochem. J.
2008, 409, 581–589.
(63) Pugsley, M. K.; Hancox, J. C.; Curtis, M. J. Perception of validity
of clinical and preclinical methods for assessment of torsades de pointes
liability. Pharmacol. Ther. 2008, 119, 115–117.
(64) Jones, K. A.; Garbati, N.; Zhang, H.; Large, C. H. Automated
patch clamping using the QPatch. Methods Mol. Biol. 2009, 565,
209–223.
(48) Beckers, T.; Burkhardt, C.; Wieland, H.; Gimmnich, P.; Ciossek,
T.; Maier, T.; Sanders, K. Distinct pharmacological properties of second
generation HDAC inhibitors with the benzamide or hydroxamate head
group. Int. J. Cancer 2007, 121, 1138–1148.
(49) Remiszewski, S. W. The discovery of NVP-LAQ824: from
concept to clinic. Curr. Med. Chem. 2003, 10, 2393–2402.
(50) Catley, L.; Weisberg, E.; Tai, Y. T.; Atadja, P.; Remiszewski, S.;
Hideshima, T.; Mitsiades, N.; Shringarpure, R.; LeBlanc, R.; Chauhan,
D.; Munshi, N. C.; Schlossman, R.; Richardson, P.; Griffin, J.; Anderson,
K. C. NVP-LAQ824 is a potent novel histone deacetylase inhibitor with
significant activity against multiple myeloma. Blood 2003, 102, 2615–
2622.
(51) Cho, Y. S.; Whitehead, L.; Li, J.; Chen, C. H.; Jiang, L.; Vogtle,
M.; Francotte, E.; Richert, P.; Wagner, T.; Traebert, M.; Lu, Q.; Cao, X.;
Dumotier, B.; Fejzo, J.; Rajan, S.; Wang, P.; Yan-Neale, Y.; Shao, W.;
Atadja, P.; Shultz, M. Conformational refinement of hydroxamate-based
histone deacetylase inhibitors and exploration of 3-piperidin-3-ylindole
analogues of dacinostat (LAQ824). J. Med. Chem. 2010, 53, 2952–2963.
(52) Remiszewski, S. W.; Sambucetti, L. C.; Bair, K. W.; Bontempo,
J.; Cesarz, D.; Chandramouli, N.; Chen, R.; Cheung, M.; Cornell-
Kennon, S.; Dean, K.; Diamantidis, G.; France, D.; Green, M. A.;
Howell, K. L.; Kashi, R.; Kwon, P.; Lassota, P.; Martin, M. S.; Mou,
Y.; Perez, L. B.; Sharma, S.; Smith, T.; Sorensen, E.; Taplin, F.; Trogani,
N.; Versace, R.; Walker, H.; Weltchek-Engler, S.; Wood, A.; Wu, A.;
Atadja, P. N-Hydroxy-3-phenyl-2-propenamides as novel inhibitors of
human histone deacetylase with in vivo antitumor activity: discovery
of (2E)-N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]-
amino]methyl ]phenyl]-2-propenamide (NVP-LAQ824). J. Med. Chem.
2003, 46, 4609–4624.
(65) Kutchinsky, J.; Friis, S.; Asmild, M.; Taboryski, R.; Pedersen, S.;
Vestergaard, R. K.; Jacobsen, R. B.; Krzywkowski, K.; Schroder, R. L.;
Ljungstrom, T.; Helix, N.; Sorensen, C. B.; Bech, M.; Willumsen, N. J.
Characterization of potassium channel modulators with QPatch auto-
mated patch-clamp technology: system characteristics and performance.
Assay Drug Dev. Technol. 2003, 1, 685–693.
(66) Taglialatela, M.; Castaldo, P.; Pannaccione, A.; Giorgio, G.;
Genovese, A.; Marone, G.; Annunziato, L. Cardiac ion channels and
antihistamines: possible mechanisms of cardiotoxicity. Clin. Exp. Allergy
1999, 29 (Suppl 3), 182–189.
(67) Farid, R.; Day, T.; Friesner, R. A.; Pearlstein, R. A. New insights
about HERG blockade obtained from protein modeling, potential
energy mapping, and docking studies. Bioorg. Med. Chem. 2006, 14,
3160–3173.
(68) Flipo, M.; Charton, J.; Hocine, A.; Dassonneville, S.; Deprez,
B.; Deprez-Poulain, R. Hydroxamates: relationships between structure
and plasma stability. J. Med. Chem. 2009, 52, 6790–6802.
(69) Ihara, M.; Noguchi, K.; Fukumoto, K.; Kametani, T. Conver-
sion of indoles into quinolines through the n-1-c-2 fission by singlet-
oxygen as a model experiment of biomimetic synthesis of quinine
alkaloids[, 1974 354ꢀ356]. Tetrahedron 1985, 41, 2109–2114.
(70) Szꢀantay, C.; Szabꢀo, L.; Kalaus, G. Synthesis 1974, 354ꢀ356
(53) Tamargo, J. Drug-induced torsade de pointes: from molecular
biology to bedside. Jpn. J. Pharmacol. 2000, 83, 1–19.
(54) Witchel, H. J.; Hancox, J. C. Familial and acquired long
qt syndrome and the cardiac rapid delayed rectifier potassium current.
Clin. Exp. Pharmacol. Physiol. 2000, 27, 753–766.
4772
dx.doi.org/10.1021/jm200388e |J. Med. Chem. 2011, 54, 4752–4772