Design, synthesis, and biological evaluation of potent and selective class IIa histone deacetylase (HDAC) inhibitors as a potential therapy for huntington's disease

Article abstract of DOI:10.1021/jm4011884

Inhibition of class IIa histone deacetylase (HDAC) enzymes have been suggested as a therapeutic strategy for a number of diseases, including Huntington's disease. Catalytic-site small molecule inhibitors of the class IIa HDAC4, -5, -7, and -9 were developed. These trisubstituted diarylcyclopropanehydroxamic acids were designed to exploit a lower pocket that is characteristic for the class IIa HDACs, not present in other HDAC classes. Selected inhibitors were cocrystallized with the catalytic domain of human HDAC4. We describe the first HDAC4 catalytic domain crystal structure in a "closed-loop" form, which in our view represents the biologically relevant conformation. We have demonstrated that these molecules can differentiate class IIa HDACs from class I and class IIb subtypes. They exhibited pharmacokinetic properties that should enable the assessment of their therapeutic benefit in both peripheral and CNS disorders. These selective inhibitors provide a means for evaluating potential efficacy in preclinical models in vivo.

Full text of DOI:10.1021/jm4011884

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Potent and Selective
Class IIa Histone Deacetylase (HDAC) Inhibitors as a Potential
Therapy for Huntington’s Disease
Roland W. Burli,† Christopher A. Luckhurst,† Omar Aziz,† Kim L. Matthews,† Dawn Yates,†
̈
Kathy. A. Lyons,∥ Maria Beconi,§ George McAllister,† Perla Breccia,† Andrew J. Stott,†
Stephen D. Penrose,† Michael Wall,† Marieke Lamers,† Philip Leonard,† Ilka Muller,†
̈
Christine M. Richardson,† Rebecca Jarvis,† Liz Stones,† Samantha Hughes,† Grant Wishart,†
Alan F. Haughan,† Catherine O’Connell,† Tania Mead,† Hannah McNeil,† Julie Vann,† John Mangette,‡
Michel Maillard,§ Vahri Beaumont,§ Ignacio Munoz-Sanjuan,§ and Celia Dominguez*,§
†BioFocus, Chesterford Research Park, Saffron Walden, Essex, CB10 1XL, United Kingdom
‡AMRI, Inc., 26 Corporate Circle, Albany, New York 12212, United States
§CHDI Management/CHDI Foundation Inc., 6080 Center Drive, Suite 100, Los Angeles, California 90045, United States
S
* Supporting Information
ABSTRACT: Inhibition of class IIa histone deacetylase (HDAC) enzymes have been suggested as a therapeutic strategy for a
number of diseases, including Huntington’s disease. Catalytic-site small molecule inhibitors of the class IIa HDAC4, -5, -7, and -9
were developed. These trisubstituted diarylcyclopropanehydroxamic acids were designed to exploit a lower pocket that is
characteristic for the class IIa HDACs, not present in other HDAC classes. Selected inhibitors were cocrystallized with the
catalytic domain of human HDAC4. We describe the first HDAC4 catalytic domain crystal structure in a “closed-loop” form,
which in our view represents the biologically relevant conformation. We have demonstrated that these molecules can differentiate
class IIa HDACs from class I and class IIb subtypes. They exhibited pharmacokinetic properties that should enable the
assessment of their therapeutic benefit in both peripheral and CNS disorders. These selective inhibitors provide a means for
evaluating potential efficacy in preclinical models in vivo.
The “classical” HDAC family consists of 11 zinc-dependent
HDACs that are grouped into three classes (classes I, II, and
IV). Class I members (HDACs 1−3, 8) as well as the class IV
enzyme, HDAC11, contain an N-terminal catalytic domain that
constitutes most of their ∼440 amino acid length, contain
nuclear localization motifs (HDACs 1, 3 and 8), or lack nuclear
export signals (HDACs 1 and 2), allowing them to reside either
preferentially or exclusively in the nucleus (except HDAC3,
which contains both nuclear import and nuclear export
sequences). The class I enzymes are the “prototypical enzymes”
INTRODUCTION
■
The histone deacetylase (HDAC) superfamily consists of 18
members (HDACs 1−11 and the class III sirtuins 1−7)
originating from two different evolutionary starting points,
which exhibit a common deacetylase activity of acetylated ε-
amino groups of lysine side chains. In addition to deacetylation
of histones, these enzymes act on numerous proteins, the so-
called lysine ‘”acetylome”,1 and they have been implicated in a
diverse array of biological pathways governing transcriptional
and epigenetic control, cell differentiation and cell death,
cardiac function, inflammation, protein proteostatis, and
neuroplasticity, often as part of complexes with other proteins
(extensively reviewed).2−5
Received: August 2, 2013
Published: November 21, 2013
© 2013 American Chemical Society
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Figure 1. Structures of selected literature compounds.
in that they display very efficient histone deacetylation activity;
however, HDAC8 is atypical and resembles a class IIa enzyme
in substrate recognition.6
Class II HDACs are subdivided into class IIa (HDACs 4, 5,
7, 9) and class IIb (HDACs 6, 10) enzymes. The class IIb
enzymes are catalytically avid, similar to class I enzymes, but are
characterized by two catalytic domains and a cytoplasmic
location; however, only in HDAC6 are the two domains
functional.7
such as the pathophysiology of colitis (HDAC9)25 and liver
disease.26
A particular interest of our group is in the role of HDAC4 in
Huntington’s disease.27 When HDAC4 heterozygous knockout
animals were crossed with the R6/2 mouse model of
Huntington’s disease (HD), it led to significant improvement
in motor coordination and neurological phenotypes, improved
medium spiny neuron properties and corticostriatal synaptic
function, and increased R6/2 lifespan.28 Furthermore, HDAC4
reduction delayed cytoplasmic aggregate formation in both R6/
2 and the full length mHtt Q150 knock-in mouse model.28
Class IIa HDACs have also been implicated in the etiology of
other CNS disorders, such as Alzheimers disease29 and mood
disorders30−34 and perhaps most prominently in their potential
as treatment for muscle wasting disorders and motor neurone
diseases. HDAC4 in particular has been strongly implicated in
controlling muscle differentiation and neuromuscular homeo-
stasis through a nuclear MEF2-dependent process that relies
predominantly on CamKII regulation of subcellular enzyme
location.35,36 The expression of HDAC4 appears to be highly
controlled in muscle by specific microRNAs,37,38 contributing
to the tight regulation of muscle differentiation. HDAC4 is
robustly induced in either acutely denervated muscles or those
affected by motor neuron diseases such as amyotrophic lateral
sclerosis (ALS). Muscle atrophy represents a rare instance
where the published biology supports a specific and essential
role for the catalytic domain of HDAC4.39
In most of the biological studies cited here, the suggestion for
the potential therapeutic benefit from class IIa HDAC
inhibition has arisen either from genetic suppression studies
of various class IIa isoforms or from a combination of genetic
evidence indicating class IIa enzyme involvement with
additional efficacy achieved with broad spectrum HDAC
inhibitors. Because of the unique nature of the class IIa
enzymes, namely, the regulatory role of the extended N-
terminal domain interactions and the weak deacetylase activity,
a critical issue is whether or not occupancy of the class IIa
catalytic domain with small molecules will be capable of
replicating, either fully or in part, the beneficial effects of class
IIa HDAC knockdown proven to be efficacious in various
preclinical paradigms. The work described here on the
development of novel selective class IIa inhibitors with potent
cellular activity and improved pharmacokinetic properties
sufficient to evaluate their therapeutic benefit in vitro and in
vivo will help greatly to address these questions. Given our
particular interest in HDAC4 inhibition (for the treatment of
The class IIa members are about 1000 amino acids in length
and are exemplified by the presence of a well-conserved
extended N-terminus containing multiple transcription factor
binding sites that are important for the regulatory function of
these proteins.8 These enzymes reside in both the cytoplasm
and nucleus, and cellular trafficking of these HDACs is
regulated by intrinsic nuclear import and export signals as
well as binding sites for 14-3-3 proteins. Binding to the 14-3-3
proteins stimulates the cytoplasmic retention or nuclear export
of the class IIa HDACs in a phosphorylation-dependent
manner of which multiple kinase signaling pathways have
been implicated. This regulation of subcellular location in turn
regulates the transcriptional activity of the class IIa enzymes by
limiting accessibility to their nuclear transcription partners,
including members of the MEF2 family.8,9 Compared to the
class I HDACs, the catalytic deacetylation activity of all class IIa
enzymes is intrinsically much weaker (∼1000-fold lower)
because of a Tyr to His switch in the active site. No natural
substrate of class IIa enzymes has been conclusively identified
that can be ascribed purely to the catalytic activity of the
enzyme rather than its class I/III HDAC binding partners.6,10,11
This questions the class IIa HDACs’ bona fide deacetylase
activity; the “catalytic domain” may serve instead as a
recognition domain for binding to Nε-acetyllysine residues of
proteins to orchestrate protein−protein interactions and
macromolecular protein signaling.12,13
Class IIa HDACs have been implicated as potential targets
for the treatment of a number of diseases. The role and
potential benefit of HDACs 4, 5, 7, and 9 “inhibition” in a
number of cancers are comprehensively reviewed elsewhere.9
Class IIa enzymes have also been suggested as viable drug
targets for various metabolic disorders, including type II
diabetes, owing to their regulation of glucose homeostasis
and muscle glucose metabolism.14,15 Class IIa enzyme
inhibition has also been proposed for the treatment of
pathological cardiac hypertrophy,3,16−22 acute ischemic injury
(HDAC4),23 and inflammatory or autoimmune conditions,24
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Table 1. Biochemical and Cell-Based IC50 Values for Reported Trifluoromethyl Ketones, Hydroxamic Acids, and SAHA
cell activity
HDAC4
(μM)
HDAC5
(μM)
HDAC7
(μM)
HDAC9
(μM)
HDAC3
(μM)
HDAC6
(μM)
HDAC8
(μM)
Lys-TFA
(μM)
Lys-Ac
(μM)
tPSA
(Å2)
AlogP
Mw
1
2
3
4
5
0.01
0.25
0.31
41
0.01
0.16
0.15
4.6
0.07
0.65
0.07
41
0.02
0.51
0.12
49
0.38
>50
39
0.28
11
0.22
45
0.26
2.9
0.85
>50
>50
0.04
3.5
2.4
2.1
2.0
4.3
104
49
422
227
225
264
514
4.9
15
0.97
>10
49
0.25
>43
0.03
2.3
0.64
3.6
78
a
0.13
0.08
0.04
0.02
90
a
Literature data.
Figure 2. Schematic presentation of the deacetylase mechanism of class I HDACs. Key residues of class I HDAC protein are in green, and
numbering of residues is according to HDAC8. The position of Tyr306 in the class I HDAC subtypes overlaps with His976 (HDAC4 numbering) in
the class IIa subtypes. The His residue can rotate away from the Zn2+ ion, opening up the lower pocket.
Huntington’s disease), we have focused our medicinal
chemistry strategy on this enzyme as representative of the
class IIa isoforms.40
IIa/HDAC8-specific” cell-based assays, in which the conversion
of either a Boc-Lys-Ac (Ac) or a Boc-Lys-TFA (TFA) substrate
was monitored.12,41 When compared to SAHA, all four
reference compounds (1−3) demonstrated a very different
selectivity profile and were more potent inhibitors for the class
IIa isoforms. The hydroxamic acids (2 and 3), although less
potent HDAC4 inhibitors, generally showed a lower shift in
potency from biochemical to cellular activity (TFA) and did
not show significant activity up to 50 μM under the “class I/IIb-
specific” assay conditions.
RESULTS AND DISCUSSION
■
In order to assess the efficacy of compounds against HDAC
activity, a selection of biochemical assays were performed as
described previously.41 Compound potency against the
following HDACs was interrogated either as full length
proteins (HDAC1, HDAC2, HDAC3-NCoR2, HDAC6,
HDAC8) or as catalytic site truncated proteins expressed
recombinantly (HDAC4, HDAC5, HDAC7, and HDAC9).
Cellular assays employed artificial substrates Boc-Lys(Ac)-
AMC42,43 and Boc-Lys(TFA)-AMC10,44 in Jurkat E6.1 cells
using endogenous HDAC activities, as described previously.41
Utilizing gene selective shRNAs, class IIa HDACs were
knocked-down individually and collectively in Jurkat E6.1
cells to demonstrate that the majority of class IIa HDAC
activity in this cell line is derived from HDAC4 (Supporting
Information).
Trifluoromethyl ketones are intrinsically electrophilic,19
whereas hydroxamic acids have successfully been progressed
to proof-of-concept studies preclinically and in humans for
other indications.53 We reasoned that optimization of HDAC4
inhibitors bearing a hydroxamic acid metal binding group
would provide a rapid avenue toward a molecule suitable for a
proof-of-concept study.
To understand the binding mode of these ligands, publicly
available structural data across the HDAC family, along with
mutation data and SAR for HDAC4, were analyzed.45−47 Most
relevant to this work, Bottomley et al. published two structures
of the catalytic domain complexed with a trifluoromethyl
ketone and an analogous hydroxamic acid.45,54 A comparison of
these structures with the previously reported HDAC8
substrate−mimetic complex revealed the existence of a lower
pocket in HDAC4.55 This is formed by replacement of a
tyrosine residue by a histidine (His976 in HDAC4), which
rotates away from the binding site. This Tyr to His switch is
highly conserved and characteristic for the class IIa enzymes
and accounts for their decreased deacetylation activity.45 The
HDAC4 lower pocket therefore appears to be specific for the
class IIa HDAC family and was envisaged to provide an
opportunity to pursue selectivity. Furthermore the HDAC4
lower pocket differs from the previously described HDAC2 foot
pocket.56 For HDAC4 the backbone path between residues
At the outset of this project, two structural types of selective
class IIa HDAC inhibitors had been reported: trifluoromethyl
ketones and hydroxamic acids (selected examples are shown in
Figure 1).45−51 In order to understand which chemotype may
serve as a suitable starting point for the development of a tool
molecule, we evaluated key representatives of each series and
compared them to SAHA (4, Table 1). During the preparation
of this manuscript structures of trifluoromethyloxadiazole class
IIa inhibitors were disclosed (5, Table 1).52 SAHA was found to
be a potent inhibitor of class I isoforms (HDAC1, 0.03 μM;
HDAC2, 0.92 μM; HDAC3, 0.25 μM; HDAC8, 0.64 μM) and
class IIb (HDAC6, 0.03 μM) in biochemical assays, with
relatively weak inhibitory activity for the class IIa isoforms. This
observation was further confirmed by the difference in activity
in the Lys-Ac “class I/IIb-specific” versus the Lys-TFA “class
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Figure 3. Modeled representation of the HDAC4 active site: (A) HDAC4 active site illustrating the zinc binding region, the lower selectivity pocket,
and a channel bounded by Phe871 and Phe812; (B) docked compound 2 showing occupation of the lower pocket, the linker region, and zinc
interactions; (C) docked compound (2R,3R)-15 showing occupation of the lower pocket, the linker region, and zinc interactions.
a
Table 2. Activity of the First Di- and Trisubstituted Cyclopropanes
a
HDAC4 biochemical and cell data (Lys-TFA and Lys-Ac) are shown. Compound syntheses are described in the Supporting Information.
Gly797 and Gly801 deviates from that of HDAC2, resulting in
the placement of HDAC4 residue Pro800 into the previously
described foot pocket region. Therefore, the HDAC2 foot
pocket is not present in the disclosed structures of HDAC4.
Figure 2 illustrates the proposed deacetylase mechanism of
class I, IIb, and IV HDAC families, in which a Tyr participates
in the catalytic event.
The known HDAC7/SAHA and trichostatin A (TSA) bound
structures also indicated the presence of a similar lower
pocket.57 We hypothesized that this pocket could be exploited
to obtain selectivity over the class I and class IIb HDAC
isoforms.
Sequence analysis revealed that the class IIa HDAC isotypes
are highly homologous around the catalytic deacetylation site
(described in Supporting Information). It is therefore not
surprising that the HDAC4 inhibitors (1−4) displayed very
similar activity across all class IIa isotypes (Table 1). On the
basis of this high degree of similarity within the catalytic site, we
anticipated difficulty in rationally designing HDAC4 inhibitors
with a pharmacologically relevant degree of selectivity over the
other class IIa isoforms.
Contrary to most HDAC structures, the loop regions
between residues Glu24 and Arg37 as well as Thr81 and
Ala126 (numbering based on published data)45 of the reported
HDAC4 (catalytic domain) structure adopted an open
conformation. Mutation data suggest that a loop-closed form
is necessary for enzymatic activity.45,58,18,26 We developed an
HDAC4 model in which this loop resides in a closed
conformation. On the basis of the docking result with
compound 2 (Figure 3), we envisaged that a trisubstituted
cyclopropane core may provide a scaffold with suitable vectors
to occupy the catalytic site. Initial docking of N-hydroxy-2,3-
diphenylcyclopropanecarboxamide showed that the trans-
(2R,3R)-cyclopropane isomer (single enantiomer of compound
15 in Table 2) positioned the hydroxamic acid function next to
the zinc(II) cation and one phenyl in the lower pocket while
directing the second phenyl group toward a hydrophobic
channel or linker region (Figure 3). It was reasoned that the
second phenyl group may be further substituted with
functionalities capable of projecting toward the solvent space
(capping region). Conversely, docking of the diastereomer cis-
(1r,2S,3R)-16 (Table 2) predicted that the configuration of this
compound precluded occupancy of both the lower pocket and
linker regions by the phenyl groups.
Chemistry. Synthetic access to these cyclopropane scaffolds
proved to be challenging. Our initial attempts to cyclo-
propanate styrenes with a metallocarbenoid were at best very
low yielding. However, we identified suitable conditions using a
sulfur ylide-mediated cyclization to facilitate the key synthetic
step in accessing a trisubstituted cyclopropane scaffold.59−61 To
the best of our knowledge, this is the first reported example of a
semistabilized sulfur ylide reacting with a cinnamic ester leading
to a 1,2,3-substituted cyclopropane core. Treatment of the
esters 6a−c with the corresponding sulfur ylide gave rise to the
cyclopropane esters 7a−c as mixtures of diastereoisomers.
Notably, addition of 12-crown-4 ether improved the diaster-
eoselectivity in favor of the desired trans isomer (1R*,2R*,3R*)
over the cis form (1R*,2S*,3R*) (Scheme 1), providing a 1:1
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a
Scheme 1. Synthesis of Phenyl-Linked Cyclopropanes
a
Reagents and conditions: (a) 8 (OTf or Br salt), LiHMDS, 12-crown-4, −20 °C, 2 h, 25%. (b) For 9a−c: amine, Pd2(dba)3, xantphos, Cs2CO3,
dioxane, 90 °C, 16 h, 59−82%. For 9e and 9k: boronic acid, Pd(PPh3)4, CsF, MeOH−DME (1:5), 120 °C microwave, 1 h, 93%. For 9d and 9f−h:
(Bpin)2, Pd(dppf)Cl2, KOAc, 100 °C, 2 h, then R-X, Pd(PPh3)4, Na2CO3, 100 °C, 2 h, 46−100%. For 9i, 9j, 9l: stannane, Pd(PPh3)4, dioxane, 150
°C microwave, 1 h, 73−84%. For 9m−n: oxazole, Pd(OAc)2, tetramethyl-di-tBuXPhos, K2CO3, pivalic acid, DMA, 110 °C for 16 h, 65−100%. (c)
NH2OH, KOH, MeOH−THF (1:1), rt, 1 h, 65−100%.
trans/cis ratio instead of the 1:2.5 ratio obtained without
additive. Palladium-catalyzed coupling reactions under Heck,
Suzuki, or Stille conditions installed the capping group and
provided ester intermediates 9a−n, which were converted to
their corresponding hydroxamic acids.
Similarly, final compounds 32−60 were obtained from their
corresponding α,β-unsaturated esters (Scheme 2). The starting
of the trans diasteromer 18 points toward the hydrophobic
channel (later confirmed by X-ray of close analogues; vide
infra). These observations highlighted the large gain in affinity
upon engagement of the lower pocket, and initial cell potency
data confirmed the predicted selectivity over the class I
isoforms.
The capping group was investigated with the objective of
improving potency while achieving balanced physicochemical
properties suitable to achieve CNS exposure. Accordingly, we
aimed for molecules with AlogP values ranging from 2 to 4, a
polar surface area below 80 Å2, and a limit of two hydrogen
bond donor functions. Simple heterocycloalkyl capping groups
showed no improvement in potency as illustrated by the
activity of racemic, cyclic anilines 19, 20, and 21, respectively
(Table 3). An improvement in both biochemical and cell-based
potency was observed following para-substitution of the
phenylene linker with heteroaryl capping groups; for instance,
pyridazine 22 and pyrimidines 23 and 24 (with AlogP values
between 2 and 3) displayed HDAC4 inhibitory activity below
100 nM.
Scheme 2. Synthesis of Bicyclic and Heterocyclic
a
Compounds (32−60)
a
Reagents and conditions: (a) (EtO)2P(O)CH2CO2Et, NaH, THF, 0
The high ligand efficiency (LE = 0.4)62 and ligand
lipophilicity efficiency (LLE = 4.9)63 observed for 24 warranted
further exploration of the general phenyl linker-capping
heterocycle architecture (Table 3). The 4-fluoro-substituted
analogue (R,R,R)-25 demonstrated a cellular potency of
roughly 300 nM in the “Lys-TFA” assay, while no significant
inhibition was detected under “Lys-Ac” conditions (up to 50
μM). Compound 25 was among the analogues with higher
passive permeability in Madin−Darby canine kidney (MDCK)
cells (PappA→B = 294 nm/s) and was not a substrate of P-gp in
vitro as determined by an effective efflux ratio (EER) of 1.4 in
MDCK-MDR1 cell monolayers. Hydroxamic acids are known
to be glucuronidated and metabolized by aldehyde oxi-
dase;64−66 the fractional contribution of these processes to
the in vivo clearance is difficult to predict from in vitro systems.
Hydroxamic acids can also undergo CYP metabolism; thus, our
first objective was to minimize the potential for oxidative
metabolism, and hence, we evaluated the stability of the
compounds in liver microsomes. Compound 25 had relatively
poor metabolic stability in MLM (Clint = 688 mL min−1 kg−1)
but acceptable stability in HLM (Clint = 42 mL min−1 kg−1).
We reasoned that while increasing the number of heteroatoms
may reduce metabolic liability, escalating polar surface area
would lead to reduced distribution to brain tissue.
°C to rt, 1 h, 44−99%; (b) RCl or RBr, Pd(OAc)2, P(o-tol)3, NEt3,
MeCN, 80 °C, 3−18 h, 17−93%; (c) RBr, Pd(PPh3)4, K2CO3,
dioxane, 100 °C, 17 h, 68%; (d) 8 (OTf or Br salt), LiHMDS, 12-
crown-4, −20 °C, 2 h, 16−100% (dr > 98:2 to 5:4); (e) NH2OH,
KOH, MeOH−THF (1:1), rt, 1 h, 5−86%.
enoates (13) were synthesized either via Heck, Wittig, or
Suzuki reactions from the corresponding aldehydes or
bromides. The identity and purity of all final molecules were
1
determined by H NMR, mass spectrometry, and analytical
HPLC. Purities were in excess of 95%. Separation of
enantiomers was achieved for selected compounds using
HPLC and a chiral stationary phase (Supporting Information).
The enantiomeric excess values for the compounds described
herein all exceeded 95%.
Structure−Activity Relationships. The trisubstituted
cyclopropane 15, in which the two phenyl groups are in
trans-configuration relative to each other, displayed submicro-
molar inhibitory activity for HDAC4 in the biochemical assay
and low micromolar activity in the cellular context (Table 2). In
contrast, its diastereomer 16 showed no significant HDAC4
inhibition and validated our modeling hypothesis described
earlier. The biochemical activity of the two disubstituted
diastereomers (17, 18) differed by about an order of
magnitude. Modeling suggested that the phenyl group of the
cis fragment 17 fills the lower pocket, whereas the phenyl unit
Given the encouraging potency and properties observed with
the pyrimidin-2-yl capping group, additional polar azole
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Table 3. Biological, in Vitro ADME Data and Selected Calculated Properties for Phenylene Linker Scaffold with Varying
a
Capping Group Substituents
a
Generic structure shown in Scheme 1. Intrinsic clearance (Clint) values of >257 mL min−1 kg−1 in mouse liver microsomes and >52 mL min−1 kg−1
for human liver microsomes indicate a rapid rate of oxidative metabolism. Under the assay conditions used Clint values of <65 mL min−1 kg−1 in
MLM and <36 mL min−1 kg−1 in HLM indicate low rate of metabolism by CYP450 enzymes. An effective efflux ratio (EER) value of >4 predicts that
a compound is a substrate for P-gp, whereas values of <2 suggest the compound is not a P-gp substrate.
substitution patterns were explored (Table 3). N-Methylimi-
dazole 27 displayed a loss in potency against HDAC4 (0.29
μM), indicating that ortho-substitution on the capping group
was deleterious, likely because of a twist of the capping group
and loss of biaryl planarity. Transfer of the methyl group to the
adjacent carbon heterocyclic position (28) improved bio-
chemical and cellular activity by 3- to 5-fold. This NH-
imidazole was relatively stable in MLM (Clint = 74 mL min−1
kg−1); however, introduction of the additional H-bond donor
function reduced its passive permeability (PappA→B = 10 nm/s)
and is therefore likely to have reduced oral exposure and CNS
access compared to other compounds in the series. In an
attempt to mitigate this, a fluorine atom was incorporated on
the adjacent phenyl linker of compound 29. It was anticipated
that this modification would shield the NH function of the
imidazole element. Indeed, the fluoro analogue 29 demon-
strated a 10-fold increase in passive permeability relative to 28
but showed high P-gp-mediated efflux (EER = 8.2) and was
metabolically unstable (MLM Clint = 476 mL min−1 kg−1).
Oxazole capping groups were investigated with the objective
of exploiting a pharmacophore similar to the imidazoles
described above, with removal of the extra hydrogen bond
donor (Table 3). The 2-substituted oxazole (rac)-30 was
potent under biochemical and cellular conditions but displayed
high intrinsic clearance in mouse liver microsomes, likely
because of the exposure of the oxazole C(4)−H and C(5)−H.
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Figure 4. HDAC4 cocrystal X-ray structures.
However, the 5-substituted regioisomer 31 was relatively stable
in liver microsomes and had the highest passive permeability of
all the analogues evaluated. It also exhibited slightly improved
potency (HDAC4, 25 nM; cell, 220 nM), excellent ligand
efficiency (LE = 0.43; LLE = 5.4), and effective efflux ratio
(EER) of 2.4. It was progressed to in vivo pharmacokinetic
studies (PK discussed in pharmacokinetic studies section).
Compound 31 was found to be a potent inhibitor of human
hepatic CYP450 isoforms CYP2C9 (IC50 ≈ 0.4 μM) and
CYP2C19 (IC50 ≈ 0.06 μM) and a moderate inhibitor of
CYP2D6 (IC50 ≈ 2.7 μM), which was attributed to the C(2)
unsubstituted oxazole moiety. While further work with oxazole
capping groups to address the CYP450 inhibition was
undertaken (discussed later), this was not a concern for
moving compounds forward to nonclinical PoC.
In order to determine the configuration of active cyclo-
propane inhibitors and their key interactions with HDAC4, we
obtained a cocrystal structure of 5-fluoropyrimidine 25
complexed to the catalytic domain of wild type human
HDAC4 (Figure 4A). This structure crystallized in the space
group P32 with three molecules per asymmetric unit (data not
shown). In contrast to the previously reported HDAC4
structures,45 the protein adopts a closed-loop conformation,
which is consistent with the previously documented structures
for HDAC7 and HDAC8.57,58 Inspection of the crystal packing
in the complex between HDAC4 and 25 showed that the ligand
binding site forms part of a packing interface in which residue
Leu728 of a neighboring HDAC4 chain is positioned close to
the ligand and has the potential to affect the bound compound
conformation. In addition, it was observed that amino acids 729
to 760 were not visible in the electron density maps for any of
the three molecules in the asymmetric unit and were omitted
from the model because of disorder. As a result, we could not
study the interaction between the ligand and key residue
Asp759, a mutation to alanine that has previously resulted in a
significant loss of deacetylase activity.67
In order to improve crystal packing and avoid the potential
interference of Leu728 as described above, we mutated this
residue to a sterically less bulky alanine. Indeed, HDAC4(cd)
(L728A) yielded cocrystal structures with several ligands,
including lead molecule 31 (Figure 4B) in a novel crystal form.
The complex with 31 has been solved at 2.72 Å resolution and
contains four protein molecules per asymmetric unit.
Importantly, no crystal packing contacts were observed near
the ligand binding site, and the loop between residues 727 and
761, which forms the rim of the binding site, was resolved in
this structure. Activity of the L728A mutant of the HDAC4
catalytic domain was assessed in an identical manner as that of
the wt HDAC4 catalytic domain, with activity compared at
equal amounts of protein for both enzymes (0.2 μg/mL) and
using the same substrate (Boc-Lys(TFA)-AMC) (data not
shown). Higher concentrations of the L728A mutant clearly
demonstrate that the activity scales well with increasing
concentration and that this activity was inhibited to the same
extent using a reference compound, when compared to wt
HDAC4. These data demonstrate not only that the L728A
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mutant HDAC4 catalytic domain protein is active but also that
the extent of the activity was identical to that of the wt HDAC4
catalytic domain protein using the assays detailed.
Table 4. Structure−Activity Data for Cyclopentane and
a
Heteroaromatic Linkers with Small Substituents
The cocrystal structure of the 5-fluoropyrimidine 25 with
HDAC4(cd) (WT) allowed the unambiguous assignment of
the absolute configuration of this ligand and confirmed the
binding pose as anticipated from docking studies (Figure 4A).
A detailed analysis of the crystal structure showed that the
hydroxamic acid moiety forms a bidentate interaction with the
zinc(II) ion, with the carbonyl and the hydroxy oxygen atoms
located 2.00 and 2.36 Å apart from the metal ion. This
chelation completes the coordination sphere for the zinc ion,
which includes a carboxylate oxygen atom of Asp840, one
interaction from a His842 ring nitrogen, and one contact from
Asp934 (Figure 4A). The hydroxamate OH group of 25 also
serves as a hydrogen bond donor to His802 with a distance of
2.36 Å between heavy atoms. While the distance (2.97 Å) and
angle are not ideal, an interaction may be postulated between
His803 and the hydroxamic acid NH. The lower pocket, as
anticipated, is formed by His976 oriented away from the metal
ion and is occupied by the ligand’s phenyl group, which
undergoes favorable edge-to-face π-stacking interactions with
Arg681 and Phe812. In addition, a number of lipophilic
interactions were observed, for example, to Pro676, Pro800,
and Leu943. The ligand linker region occupies a hydrophobic
channel, bordered by Gly811, Phe812, His842, Pro942,
Leu943, and Phe871. Phe871 forms a face−face π-stacking
interaction with the linker phenyl ring, while the terminal
pyrimidine cap extends toward solvent. Apart from a van der
Waals contact with Pro942 (4.36 Å), no interactions with the
capping group were observed.
The crystal structure of oxazole 31 complexed with the
HDAC4(cd) (L728A) (Figure 4B) is consistent with the 25
HDAC4(cd) (WT) structure (Figure 4A); a minor change in
the His803 conformation appeared to improve the hydrogen
bonding between this residue and the NH of the hydroxamic
acid functionality. Interestingly, the loop region bearing Asp759
is more ordered in this structure and no direct interaction
between Asp759 and the ligand was observed.
a
Generic structure shown in Scheme 2.
ophilicity efficiencies as exemplified by 35 (LE = 0.5; LLE =
5.7).
To confirm the importance of an aromatic linker π-stacking
interaction with Phe871 in the HDAC4 binding site, the phenyl
ring was replaced by a cyclopentane in 32 (Table 4). This led
to a 25-fold drop in HDAC4 biochemical potency, likely due to
the absence of this interaction.
From the X-ray structural information of compounds 25 and
31, it appeared that there were no direct interactions between
the capping group heterocycles and the protein residues. This
encouraged the design and synthesis of lower molecular weight
molecules bearing small aliphatic substituents. It was envisaged
that the desirable physicochemical properties that had been
conferred by the polar capping groups with the phenyl linker
could be attained with a heterocyclic linker in its own right.
Accordingly, a series of heteroaromatic linker scaffolds were
evaluated (Table 4).
Compounds comprising azole linkers were synthesized and
profiled. The N-methylpyrazole 33 and thiazole 34 displayed
only moderate HDAC4 activity. The 2-methyl-5-thiazole 35
was roughly 5-fold more potent in the biochemical assay,
although all three compounds showed cellular activity in the
low micromolar range. We therefore concluded that a
heteroatom in proximity to the cyclopropane core may be
detrimental for HDAC4 activity. Nevertheless, these com-
pounds displayed high ligand efficiencies and ligand-lip-
With a clear positional preference of the heteroatom, we
evaluated six-membered heterocyclic linkers with heteroatoms
located in meta and para positions (Table 4). The 5-pyrimidine
36 and 4-pyridazine 37, each without substituents, were both
relatively weak HDAC4 inhibitors, whereas addition of a 2-
cyclopropyl substituent to the pyrimidine cap (38) improved
biochemical and cell potency. Modeling suggested that
introduction of this cyclopropane substituent in the meta-
position relative to the biaryl junction may be preferred. The
pyridazine 39 and in particular the cyclopropylpyridine 40 both
demonstrated a significant improvement in potency (for 40:
HDAC4, 20 nM; cell (Lys-TFA), 670 nM). However, this
compound showed poor metabolic stability in MLM (Clint
=
367 mL min−1 kg−1) and was a potential substrate for P-gp
(EER = 9.2; Table 5). Replacing the cyclopropyl by a CF3
group (41) led to a pronounced drop in HDAC4 potency as
illustrated by the CF3-substituted pyridines 42 and 43.
Although this subseries of heterocyclic linkers exhibited good
HDAC4 potency, most of the compounds displayed poor
MLM intrinsic clearance and high P-gp efflux.
Evidence from our X-ray structures suggested that occupancy
of the predominantly hydrophobic channel of the HDAC4
protein could be optimized by inclusion of a bicyclic linker
group. Guided by docking studies, a number of bicyclic
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Table 5. In Vitro ADME Data for Selected Heteroaromatic Linker Compounds Shown in Table 4
a
Table 6. Structure, Activity, and in Vitro ADME Data for Fused Heteroaromatic Linkers
a
Generic structure shown in Scheme 2.
heteroaromatic groups were investigated (Table 6). Here, we
found that a five-membered heterocyclic ring proximal to the
cyclopropane core was deleterious for potency as illustrated by
44, 45, and 46. The weak biochemical activity of imidazopyr-
idine 44 confirmed our previous observation that heteroatoms
placed close to the core were not tolerated. In contrast, fusion
of heterocycles to the phenylene linker led to potent inhibitors
as exemplified by the activity of benzothiazole 47 and
quinoxaline 49. Most of these compounds were relatively
stable in human liver microsomes but not in mouse liver
microsomes. While introduction of a CF3 or a cyclopropyl
substituent (in 50 and 52) improved metabolic stability and
retained biological activity, most of the compounds in this
series were potential P-gp substrates.
the fused phenyl moiety increased potency, as expected from
modeling studies, but introduced efflux by P-gp. Compounds
53 and 54 were unstable in mouse liver microsomes, with
metabolism likely to occur at the ethylene bridge. The geminal
dimethyl-substituted benzodioxane 55 was designed to block
this potential metabolic soft spot and indeed demonstrated
improved stability in MLM without a marked reduction in
HDAC4 inhibitory activity. Modifying the ring heteroatom of
the potent tetrahydroquinoline 56 resulted in a compound with
high passive permeability in MDCK cells that was not a
substrate of P-gp in vitro (EER = 1.7), despite the addition of a
further hydrogen bond donor.
HDAC Selectivity. In order to understand the selectivity of
these cyclopropane-based HDAC inhibitors, we investigated
the inhibitory activity of selected compounds representing the
different subchemotypes against a panel of other HDAC
isoforms. Specifically, we compared HDAC4 activity with the
other class IIa isoforms and class I HDACs 1, 2, 3, and 8, along
In order to investigate the impact of a fused heterocycloalkyl
ring system on potency and other properties, a number of
analogues were synthesized and profiled as shown in Table 7.
For benzodioxanes 53 and 54, addition of a chlorine atom on
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a
Table 7. Structure, activity, and in Vitro ADME Data for Partially Saturated Bicyclic Linkers
a
Generic structure shown in Scheme 2.
Table 8. Selectivity Data for Representative Lead Molecules
cat. HDAC4
cat. HDAC5
cat. HDAC7
cat. HDAC9
HDAC1
(μM)
HDAC2
(μM)
HDAC3
(μM)
HDAC6
(μM)
HDAC8
(μM)
(μM)
(μM)
(μM)
(μM)
(rac)-15
25
0.34
0.05
0.09
0.02
0.54
0.02
0.02
0.02
0.03
0.34
1.1
1.8
33
9.4
26
12
25
18
8.9
4.7
16
9.7
1.9
5.3
4.3
0.03
0.11
0.22
0.03
0.46
0.12
0.01
0.02
0.06
0.19
0.37
0.04
0.79
0.26
0.03
0.05
0.08
21
1.4
(rac)-26
31
0.05
5.3
0.004
0.15
32
43
0.36
6.7
33
>50
>50
40
0.03
5.6
4.8
49
0.007
0.009
0.009
22
17
24
>50
31
0.87
0.83
1.5
54
57
>50
Figure 5. Concentrations of 57 in plasma, brain, and muscle over time following a single iv administration at 5 mg/kg (left) and po administration at
10 mg/kg (right) to fed male C57Bl/6 mice.
with HDAC6 as a representative of class IIb (Table 8). As
anticipated by the high homology within the catalytic site of the
class IIa subfamily, only minor differences in activity were
observed in most cases. Perhaps the most notable discrepancy
was detected for the truncated pyridine 40, whereby the
compound was roughly 15-fold more active for HDAC4
compared to HDAC9. We have demonstrated that these
cyclopropane molecules can differentiate class IIa HDACs from
class I and class IIb subtypes, which confirmed our design
principles of exploiting the lower pocket. Compounds
demonstrated excellent biochemical HDAC4 versus class I
HDAC selectivities, in particular over HDACs 1, 2, and 3, with
selectivity, albeit less pronounced, over HDAC8, a class I
HDAC that resembles a class IIa enzyme in substrate
recognition. For example, compound 31 displayed 2000-fold
selectivity over HDAC2 (class I) and less than 20-fold
selectivity versus HDAC8, whereas compound 40 showed
200-fold selectivity against the latter. Compounds demon-
strated selectivity over HDAC6 (class IIb) in excess of 100-fold.
Pharmacokinetic in Vivo Studies. The benzopyrrolidi-
none 57 displayed good biochemical and cell-based potency
and adequate metabolic stability in MLM and HLM. Its
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a
Table 9. Phenyl Linker Scaffold with Oxazole Capping Groups: Regiochemistry and Substitution
a
Generic structure shown in Schemes 1 and 2.
Table 10. Pharmacokinetic Parameters of 57, 31, and 59 after Administration to Fed Male C57Bl/6 mice
po (10 mg/kg)
iv (5 mg/kg)
compd
compartment
F (%)
AUCN (nM h kg/mg)
Cmax (nM kg/mg)
Tmax (h)
tissue/plasma
Clp (L h−1 kg−1
1.4
)
Vdss (L/kg)
4.6
T1/2 (h)
57
plasma
brain
62
1100
17
280
5.7
0.5
0.5
1
3.2
0.01−0.04
0.5−1.5
muscle
plasma
brain
1000
1300
700
160
1200
670
730
810
200
420
31
59
84
73
0.5
0.5
0.5
0.5
1.0
1
2.0
1.4
0.74
1.6
0.4
1.7
0.2−1
0.2−1.3
muscle
plasma
brain
810
1500
390
0.3−0.6
0.4−1.6
muscle
1000
Figure 6. Concentrations of 31 (left) and 59 (right) in plasma and brain over time following iv administration (6.3 and 7.1 mg/kg, respectively) to
fed male C57Bl/6 mice.
moderate-to-high effective efflux ratio of 4 and relatively low
passive permeability (PappA→B = 130 nm/s) indicated that CNS
exposure may be limited. With the aim of assessing the
relationship between in vitro and in vivo pharmacokinetic data,
the compound was evaluated in vivo in mice.
Following iv administration (5 mg/kg) of 57 to mice,
moderate plasma clearance was observed (1.4 L h−1 kg−1) with
a moderate-to-large volume of distribution at steady state (4.6
L/kg) and a half-life of 3.2 h (Table 10). Following po
administration (10 mg/kg), absorption was rapid with the time
of maximal concentration (Tmax) occurring between 0.5 and 1 h
for plasma, brain, and muscle (Figure 5). The compound
displayed acceptable oral bioavailability (62%).
Brain concentrations were low relative to the plasma
exposure (brain-to-plasma ratios of 0.01−0.04), and it was
not possible to distinguish between compound in parenchyma
versus contamination from residual blood in the brain
homogenate. The low brain to plasma ratios observed for
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Figure 7. Concentrations of 31 (left) and 59 (right) in plasma, brain, and muscle over time following po administration (12 and 16 mg/kg,
respectively) to fed male C57Bl/6 mice.
compound 57 are likely the consequence of both lower
permeability and efflux by P-gp, suggesting that in our in vitro
systems, an effective efflux ratio of ∼4 combined with relatively
low permeability (PappA→B 130 nm/s) will translate to poor
CNS exposure relative to plasma. Compound 57 was
distributed to muscle tissue with muscle-to-plasma ratios
ranging from 0.5 to 1.5 (AUCN = 1000 nM·h·kg/mg following
oral administration) and thus may serve as a suitable tool
molecule to interrogate peripheral HDAC4 (or class IIa
isoforms) inhibition.
Both compounds were distributed from plasma into brain
tissue, with compound 31 displaying slightly higher distribution
than 59 (brain to plasma ratios of 0.2−1.0 vs 0.3−0.6,
respectively). This is consistent with the in vitro data that
showed compound 59 had lower cell permeability and greater
potential for efflux by P-gp than 31. Both compounds had
similar distribution to muscle tissue with muscle-to-plasma
ratios ranging from 0.2 to 1.3 for compound 31 and from 0.4 to
1.6 for 59 (AUCN of 810 and 1000 nM·h·kg/mg following oral
administration, respectively).
Optimization for potency, selectivity, metabolic stability, and
minimal efflux by P-gp in a single molecule was challenging.
The oxazole 31 fulfilled this criterion, but it was an inhibitor of
human hepatic CYP450. Several close analogues were evaluated
to address CYP450 inhibition. C(2) methylation of the oxazolyl
element maintained HDAC4 potency (58) but resulted in
much higher intrinsic clearance in MLM than 31 (Table 9).
Blocking the oxazole 2-position diminished its potential for
human CYP450 inhibition in vitro (IC50 values for 58:
CYP2C9, >100 μM; CYP2C19, >100 μM; CYP2D6, >39
μM). Other alkyl analogues were also found to be unstable in
MLM. The cyclopropyl analogue 59, however, retained good
potency, was not a potent CYP inhibitor (IC50 values: CYP2C9,
23 μM; CYP2C19, >100 μM; CYP2D6, >45 μM), and was
relatively stable in mouse liver microsomes (Clint = 71 mL
min−1 kg−1). However, its passive permeability was among the
lowest for these analogues (PappA→B = 255 nm/s), and the EER
value was 4, higher than that of the unsubstituted oxazole 31
and similar to that of compound 57 which translated to poor
brain exposure in mice.
The pharmacokinetic behavior of the unsubstituted oxazole
31 and its cyclopropyl analogue 59 was investigated. Following
iv administration to mice (Table 10, Figure 6), both
compounds displayed moderate plasma clearance and a low
to moderate volume of distribution at steady state. Compound
59 exhibited an apparent biphasic elimination with a terminal
half-life of 1.7 h measured between 2 and 8 h postdose. The
pharmacokinetic profile of 31 appeared to have a single
elimination phase with a half-life value of 0.4 h measured
between 0.25 and 6 h postdose. However, the presence of a
longer terminal phase for 31 at concentrations below the
analytical limit of quantification cannot be discounted. The
compounds were rapidly absorbed following an oral dose
(Figure 7), with Tmax in plasma at 0.5 h and oral bioavailability
values of 84% and 73%. Metabolites related to epimerization at
the core cyclopropane ring of 31 were not detected in
circulation in the plasma.
Total concentrations in brain tissue, determined from tissue
homogenates, do not directly relate to target engagement. The
unbound fraction of these two compounds in the presence of
brain homogenate, as determined in vitro by equilibrium
dialysis, was 0.026 for 31 and 0.0037 for 59. Hence, when
dosed at 10 mg/kg po, we estimated free concentrations of 31
to reach its cell-based IC50 value, whereas 59 requires
administration at higher dosage to achieve free concentations
at or above its IC50. However, estimation of free concentrations
based on in vitro equilibrium dialysis data can be difficult to
interpret and may not be a valid approach to determine the
brain concentration necessary for target engagement. HDAC4
is an intracellular protein shuttling between nucleus and
cytosol; therefore, steady-state drug concentrations in the
HDAC4 environment may not be identical to that of the
estimated extracellular levels in brain. Understanding the on
and off rates of bound inhibitors is also important for
estimating target coverage and more important than a free
fraction determined in equilibrium against a buffer. Finally, in
the absence of a pharmacodynamics marker, it is difficult to
take into account whether concentrations corresponding to an
in vitro determined IC20, IC50, or IC90 value will be required to
exert a biological effect. These questions are currently being
addressed and will be reported in due course.
CONCLUSIONS
■
Class IIa HDACs are large proteins with multiple functions
including transcription factor binding, N-acetyllysine recog-
nition, and perhaps weak deacetylation activity. Thus, a critical
issue is whether or not occupancy of the class IIa HDAC
catalytic domain with small molecules will be capable of
replicating, either fully or in part, the beneficial effects of the
class IIa HDAC genetic suppression studies proven to be
efficacious in the various preclinical paradigms and so would
provide a rationale for small molecule therapy. Given our
interest in the development of HDAC4 inhibitors as a potential
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trans-N-Hydroxy-2,3-diphenylcyclopropanecarboxamide
(15). To a stirred solution of 2,3-diphenylcyclopropanecarboxylic
acid68 (62 mg, 0.26 mmol), BOP (128 mg, 0.29 mmol), and
triethylamine (0.10 mL, 0.78 mmol) in pyridine (1 mL) was added
hydroxylamine hydrochloride (20 mg, 0.29 mmol). The reaction
mixture was stirred at room temperature for 2 h. The reaction mixture
was diluted with water (10 mL) and extracted into EtOAc (3 × 20
mL). The combined organic layers were washed with water (2 × 20
mL), dried (MgSO4), and concentrated. The crude material was
purified by preparative HPLC and PEAX cartridge (DCM−MeOH,
1:1). The solvent was removed in vacuo to afford the title compound
(25 mg, 38%) as a white solid. LCMS (ES+) 254 (M + H)+, (ES−) 252
therapeutic strategy for Huntington’s disease, we developed
molecules with optimized selectivity for class IIa over class I
and class IIb isotypes and good brain and muscle exposure
following oral administration. This optimization process was
guided by crystallography and structure-based design. The
introduction of a well-designed single amino acid substitution
that maintains the molecular environment within the catalytic
site has enabled provision of important structural information,
i.e., with compound 31. The data sets for compounds 25 and
31 confirmed the existence of a lower selectivity pocket and
formed the basis for detailed docking studies. Selectivity over
class I and class IIb has clearly been achieved within this
compound series, whereas pharmacologically relevant selectiv-
ity levels over the other class IIa isoforms have been difficult, as
predicted by their high similarity. Nevertheless, these molecules
exhibit a very different profile from those of SAHA and other
previously studied HDAC inhibitors and in particular do not
interact with the class I isoforms which are very actively
involved in chromatin remodeling and transcriptional regu-
lation. Optimization of the ADME properties for CNS
permeability constituted a challenge; however, balancing the
predicted physicochemical properties led to molecules with
suitable microsomal stability, and limited P-gp mediated efflux.
This culminated in the identification of tool molecules with
distribution to brain and muscle, the oxazoles 31 and 59, for
the evaluation of compound efficacy and proof of concept
studies in disease models where deregulation of class II HDAC
biology has been implicated. In addition, we identified a
molecule (57) that is well distributed to muscle but does not
partition into brain, for indications in which CNS activity may,
in fact, be undesirable. Further in vitro and in vivo proof-of-
concept experiments with these and other class IIa inhibitors
will be reported in due course.
1
(M − H)−, tR = 2.97 min (analytical method 1). H NMR δ (ppm)
(DMSO-d6): 10.55 (1 H, s), 8.69 (1 H, s), 7.36−7.16 (10 H, m), 3.09
(1 H, dd, J = 6.8, 5.4 Hz), 2.83 (1 H, dd, J = 9.6, 6.8 Hz), 2.20 (1 H,
dd, J = 9.6, 5.4 Hz).
General Procedure A for the Synthesis of 16−61
(Hydroxamic Acid Formation). To a stirred solution of ester
(0.30 mmol) in THF/MeOH (1:1, 3 mL) were added hydroxylamine
(0.2 mL, 50% aqueous solution, 3.00 mmol) and potassium hydroxide
(0.60 mmol). The mixture was stirred at room temperature for 2 h,
neutralized with 1 M HCl(aq), and extracted with DCM. The combined
organic layers were washed with brine (10 mL), passed through a
phase separator, and concentrated.
(2R,3S)-N-Hydroxy-2,3-diphenylcyclopropanecarboxamide
(16). The synthesis involved following procedure A from (2R,3S)-
ethyl 2,3-diphenylcyclopropanecarboxylate69(180 mg, 0.68 mmol).
The residual gum obtained after workup was dissolved in DCM (10
mL) and the resulting white precipitate was collected by vacuum
filtration and washed with DCM (2 × 5 mL) to give the title
compound (55 mg, 32%). LCMS (ES+) 254 (M + H)+, (ES−) 252 (M
− H)−, tR = 3.00 min (analytical method 1). 1H NMR δ (ppm)
(DMSO-d6): 10.76 (1 H, s), 8.94 (1 H, s), 7.17−7.04 (6 H, m), 7.02−
6.94 (4 H, m), 2.83 (2 H, d, J = 5.4 Hz), 2.52 (1 H obscured by
DMSO).
(1S,2R)-N-Hydroxy-2-phenylcyclopropanecarboxamide (17).
The synthesis involved following procedure A from (1S,2R)-ethyl 2-
phenylcyclopropanecarboxylate70 (550 mg, 2.89 mmol). The residual
gum obtained after workup was dissolved in DCM (10 mL), and the
resulting white precipitate was collected by vacuum filtration and
washed with DCM (2 × 5 mL). Purification by preparative HPLC gave
the title compound (105 mg, 20%). LCMS (ES+) 178 (M + H)+, tR =
2.66 min (analytical method 1). 1H NMR δ (ppm) (DMSO-d6): 10.43
(1 H, s), 8.55 (1 H, s), 7.21 (4 H, d, J = 4.3 Hz), 7.16−7.09 (1 H, m),
2.36 (1 H, dd, J = 16.6, 8.3 Hz), 1.87−1.80 (1 H, m), 1.54−1.48 (1 H,
m), 1.24−1.16 (1 H, m). HRMS (ESI) calcd for C21H24N2O2 [M +
H]+ 178.0868, found 178.0871.
(1S,2S)-N-Hydroxy-2-phenylcyclopropanecarboxamide (18).
The synthesis involved following procedure A from (1S,2S)-ethyl 2-
phenylcyclopropanecarboxylate70 (17) (1.05 g, 5.53 mmol). The
residual gum obtained after workup was dissolved in DCM (10 mL),
and the resulting white precipitate was collected by vacuum filtration
and washed with DCM (2 × 5 mL) to give the title compound (723
mg, 74%). LCMS (ES+) 178 (M + H)+, tR = 2.22 min (analytical
method 2). 1H NMR δ (ppm) (DMSO-d6): 10.60 (1 H, s), 8.81 (1 H,
s), 7.34−7.25 (2 H, m), 7.24−7.11 (3 H, m), 2.29−2.22 (1 H, m),
1.73−1.66 (1 H, m), 1.41−1.33 (1 H, m), 1.28−1.21 (1 H, m).
General Procedure B for the Synthesis of 6a, 6c, and 13a−l.
To a stirred solution of triethyl phosphonoacetate (24.4 mmol) in
THF (30 mL) at 0 °C was added sodium hydride (24.4 mmol)
portionwise. The mixture was stirred for 1 h before addition of
aldehyde (12.2 mmol). The reaction mixture was allowed to warm to
room temperature, stirred for 17 h, quenched with water (50 mL), and
extracted into EtOAc (2 × 50 mL). The organic layers were combined
and washed with water (2 × 50 mL), dried (MgSO4), filtered, and
concentrated.
EXPERIMENTAL SECTION
■
General Comments on Experimental Data. All chemicals
purchased from commercial suppliers were used as received. Flash
chromatography was carried out with prepacked SiO2 SNAP cartridges
(KP-SIL) from Biotage using a Biotage Isolera Four system using
gradient elution. Analytical thin-layer chromatography (TLC) was
performed on silica using PolygramSIL G/UV254 with fluorescent
indicator (200 μm thickness) and visualized under UV light. NMR
spectra were recorded on a Bruker AV 400 (1H, 400.13 MHz)
instrument spectrometer and referenced in CDCl3 to tetramethylsilane
(0.00 ppm) and in DMSO-d6 referenced to DMSO-d5 (2.50 ppm).
The following abbreviations are used: br = broad signal, s = singlet, br
s = broad singlet, d = doublet, dd = doublet of doublets, t = triplet, q =
quartet, m = multiplet. Preparative HPLC was performed on a Waters
Sunfire OBD C18 10 μm column (150 mm × 19 mm), Phenomenex
Luna phenylhexyl 10 μm column (150 mm × 21.2 mm), or a Waters
Xbridge phenyl 5 μm column (100 mm × 19 mm), eluting with
mixtures of water−acetonitrile or water−methanol, optionally
containing a modifier (0.1% v/v formic acid or 10 mM ammonium
bicarbonate). Low-resolution mass spectra were recorded on a Waters
ZQ single quadrapole LCMS instrument in ESCi+, ESCi− mode or on
a Quattro Micro LC−MS−MS instrument in ESCi+, ESCi− mode. All
final compounds were purified to >95% chemical purity as assayed by
HPLC/MS (detailed analytical methods in Supporting Information).
HRMS experiments were performed on a Waters Acquity UPLC
system and Waters Xevo G2 TOF mass spectrometer. Compounds
were named with the aid of the Cambridgesoft Chemistry Cartridge
(version 9.0.0.182) software. All reactions involving air- or moisture-
sensitive reagents were performed under a nitrogen atmosphere using
dried solvents and glassware. Racemic mixtures of the cyclopropyl core
are denoted using asterisks, e.g., (1R*,2R*,3R*). Chirally pure
compounds are denoted without asterisks, e.g., (1R,2R,3R).
General Procedure C for the Synthesis of 7a−c, 14a−k, and
14a′−q′ (Cyclopropanation Reaction). A mixture of sulfonium salt
(8.92 mmol), enoate (5.96 mmol), and 12-crown-4 (8.92 mmol) in
DCM (20 mL) was cooled to −20 °C. LiHMDS (8.92 mL) was then
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added dropwise. After complete addition, the mixture was warmed to
room temperature, stirred for 2 h, and quenched with water (30 mL).
The biphasic mixture was separated, and the organic layers were
washed with brine (2 × 30 mL), separated, dried (MgSO4), filtered,
and concentrated.
General Procedure D for the Synthesis of 9a−c (Buchwald
Reaction). To a stirred solution of aryl bromide (0.76 mmol) and
amine (0.86 mmol) in dioxane (4 mL) were added xantphos (0.048
mmol), cesium carbonate (1.66 mmol), and Pd2(dba)3 (0.024 mmol).
The mixture was stirred for 16 h at 90 °C, diluted with water, and
extracted into DCM (20 mL). The organic layers were passed through
a phase separator and concentrated, and the crude mixture was
purified by flash silica column chromatography.
General Procedure E for the Synthesis of 9d and 9f−g
(Suzuki Coupling). To a stirred solution of 7a or 7b (3.02 mmol) in
dioxane (5 mL) was added bis-pinacolatodiboron (3.32 mmol),
Pd(dppf)Cl2 (0.30 mmol), and potassium acetate (15.1 mmol). The
mixture was degassed with nitrogen and then heated to 100 °C for 2 h.
The reaction mixture was diluted with water (20 mL) and extracted
into DCM (2 × 20 mL). The organic layers were passed through a
phase separator and concentrated. The crude residue was dissolved in
dioxane, and an aliquot (0.66 mmol) was added to a reaction tube. To
this were added heterocyclic halide (0.69 mmol), Pd(PPh3)4 (0.066
mmol), and aqueous Na2CO3 (5 mL, 1 M solution). The mixture was
heated at 100 °C for 2 h. The mixture was diluted with water (10 mL)
and extracted into DCM (20 mL). The organic layers were passed
through a phase separator and concentrated.
0.76 mmol) and 2-oxa-6-azaspiro[3.3]heptane formate salt (160 mg,
0.86 mmol). Purification by flash silica column chromatography
(gradient elution i-hex/EtOAc 0−100%) gave (1R*,2R*,3R*)-methyl-
2-(4-(2-oxa-6-azaspiro[3. 3] heptan-6-yl)phenyl)-3-
phenylcyclopropanecarboxylate (9b) as a clear oil [210 mg, 59%,
LCMS (ES+) 364 (M + H)+], which was progressed into the next step.
The synthesis involved following procedure A from 9b (214 mg, 0.61
mmol). Purification by preparative HPLC gave the title compound as a
white solid (23 mg, 14%). LCMS (ES+) 351 (M + H)+, tR = 8.07 min
(analytical method 3). 1H NMR δ (ppm) (DMSO-d6): 10.50 (1 H, s),
8.64 (1 H, d, J = 1.8 Hz), 7.31 (2 H, d, J = 7.6 Hz), 7.28−7.21 (2 H,
m), 7.19−7.08 (3 H, m), 6.60 (2 H, d, J = 8.2 Hz), 3.70−3.61 (4 H,
m), 3.48−3.39 (4 H, m), 2.99 (1 H, dd, J = 6.9, 5.4 Hz), 2.70 (1 H, dd,
J = 9.5, 6.9 Hz), 2.08 (1 H, dd, J = 9.5, 5.4 Hz). HRMS (ESI) calcd for
C21H22N2O3 [M + H]+ 351.1708, found 351.1706.
(1R*,2R*,3R*)-N-Hydroxy-2-(4-(3,3-difluoropyrrolidin-1-yl)-
phenyl)-3-phenylcyclopropanecarboxamide (21). The synthesis
involved following procedure D from 7b (250 mg, 0.76 mmol) and
3,3-difluoropyrrolidine (124 mg, 0.86 mmol). Purification by flash
silica column chromatography (gradient elution i-hex/EtOAc 0−
100%) gave (1R*,2R*,3R*)-methyl-2-(4-(3,3-difluoropyrrolidin-1-yl)-
phenyl)-3-phenylcyclopropanecarboxylate (9c) as a clear oil (210 mg,
59%) which was progressed into the next step. The synthesis involved
following procedure A from 9c (205 mg, 0.57 mmol). Purification by
preparative HPLC gave the title compound as a white solid (35 mg,
17%). LCMS (ES+) 359 (M + H)+, tR = 9.27 min (analytical method
1
5). H NMR δ (ppm) (DMSO-d6): 10.50 (1 H, s), 8.64 (1 H, d, J =
General Procedure F for the Synthesis of 9i, 9j, and 9l (Stille
Coupling). A mixture of aryl bromide (0.67 mmol), stannane (0.81
mmol), and Pd(PPh3)4 (0.034 mmol) in 1,4-dioxane (4 mL) was
heated in the microwave at 150 °C for 1 h. The mixture was
concentrated and purified by flash silica column chromatography.
General Procedure G for the Synthesis of 9m and 9n
(Oxazole Coupling). A stirred solution of 7b or 7a (1.46 mmol),
oxazole (2.42 mmol), Pd(Oac)2 (0.07 mmol), di-tert-butyl(2′,4′,6′-
triisopropyl-3,4,5,6-tetramethylbiphenyl-2-yl)phosphine (0.146
mmol), K2CO3 (4.38 mmol), and pivalic acid (0.58 mmol) in DMA
(7.5 mL) was degassed with nitrogen for 15 min before heating at 110
°C for 16 h. The mixture was cooled and diluted with DCM (20 mL)
and washed with water (3 × 30 mL). The organic layers were passed
through a phase separator and concentrated.
General Procedure H for the Synthesis of 13a′, 13b′, 13d′−
k′, 13m′, 13n′, 13p′, 13q′ (Heck Reaction). A stirred mixture of
aryl bromide (10.0 mmol), ethyl acrylate (15.0 mmol), palladium
acetate (1.00 mmol), P(o-tol)3 (2.00 mmol), and triethylamine (20.0
mmol) in MeCN (50 mL) was degassed with nitrogen for 15 min and
heated to 80 °C for 3−18 h. The reaction mixture was cooled and the
MeCN removed in vacuo. The residue was partitioned between DCM
and water and the organic layers were passed through a phase
separator and concentrated.
1.8 Hz), 7.31 (2 H, d, J = 7.6 Hz), 7.25 (2 H, t, J = 7.4 Hz), 7.19−7.08
(3 H, m), 6.60 (2 H, d, J = 8.2 Hz), 3.66 (2 H, t, J = 13.4 Hz), 3.44 (2
H, m), 2.99 (1 H, m), 2.70−2.65 (2 H, m), 2.33 (1 H, m), 2.08 (1 H,
dd, J = 9.5, 5.4 Hz). HRMS (ESI) calcd for C20H20F2N2O2 [M + H]+
359.1571, found 359.1565.
(1R*,2R*,3R*)-N-Hydroxy-2-phenyl-3-(4-(pyridazin-3-yl)-
phenyl)cyclopropanecarboxamide (22). The synthesis involved
following procedure A from 9d (68 mg, 0.21 mmol). Purification using
an Isolute anion exchange SPE (elution DCM−MeOH, 1:1) gave the
title compound as a white solid (49 mg, 70%). LCMS (ES+) 332 (M +
H)+, tR = 2.95 min (analytical method 1). 1H NMR δ (ppm) (DMSO-
d6): 10.59 (1 H, s), 9.20 (1 H, dd, J = 4.8, 1.5 Hz), 8.71 (1 H, s), 8.23
(1 H, dd, J = 8.6, 1.5 Hz), 8.14 (2 H, d, J = 8.1 Hz), 7.78 (1 H, dd, J =
8.6, 4.9 Hz), 7.48 (2 H, d, J = 8.1 Hz), 7.37 (2 H, d, J = 7.5 Hz), 7.32−
7.24 (2 H, m), 7.22−7.16 (1 H, m), 3.19 (1 H, dd, J = 6.8, 5.4 Hz),
2.93 (1 H, dd, J = 9.6, 6.8 Hz), 2.31 (1 H, dd, J = 9.6, 5.4 Hz). HRMS
(ESI) calcd for C20H17N3O2 [M + H]+ 332.1399, found 332.1393.
(1R*,2R*,3R*)-N-Hydroxy-2-phenyl-3-(4-(pyrimidin-5-yl)-
phenyl)cyclopropanecarboxamide (23). To a stirred solution of
7b (130 mg, 0.39 mmol) in MeOH/DME (1:5, 5 mL) were added
Pd(PPh3)4 (45 mg, 0.039 mmol), cesium fluoride (119 mg, 0.78
mmol), and 5-pyrimidineboronic acid (58 mg, 0.47 mmol). The
mixture was degassed with nitrogen for 15 min before heating in the
microwave at 120 °C for 1 h. The reaction mixture was diluted with
H2O (10 mL) and extracted into DCM (50 mL). The organic layers
were dried over MgSO4, filtered, and concentrated. Purification by
column chromatography (gradient elution i-hex to 5% EtOAc in i-hex)
gave (1R*,2R*,3R*)-methyl-2-phenyl-3-(4-(pyrimidin-5-yl)phenyl)-
cyclopropane carboxylate (9e) as a colorless oil [120 mg, 93%,
LCMS (ES+) 345 (M + H)+] which was progressed to the next step.
The synthesis involved following procedure A from 9e (120 mg, 0.36
mmol). Purification by flash silica column chromatography (gradient
elution DCM to 5% MeOH in DCM) and Isolute anion exchange SPE
(elution DCM−MeOH, 1:1) gave the title compound as a white solid
(21 mg, 17%). LCMS (ES+) 332 (M + H)+, tR = 3.07 min (analytical
method 1). 1H NMR δ (ppm) (DMSO-d6): 10.60 (1 H, s), 9.18 (1 H,
s), 9.15 (2 H, s), 8.72 (1 H, s), 7.79 (2 H, d, J = 8.1 Hz), 7.45 (2 H, d,
J = 8.1 Hz), 7.35 (2 H, d, J = 7.6 Hz), 7.27 (2 H, t, J = 7.6 Hz), 7.19 (1
H, t, J = 7.2 Hz), 3.18 (1 H, dd, J = 6.8, 5.4 Hz), 2.92 (1 H, dd, J = 9.6,
6.8 Hz), 2.27 (1 H, dd, J = 9.6, 5.4 Hz). HRMS (ESI) calcd for
C20H17N3O2 [M + H]+ 332.1399, found 332.1392.
(1R*,2R*,3R*)-2-(4-(3,3-Dimethylazetidin-1-yl)phenyl)-N-hy-
droxy-3-phenylcyclopropanecarboxamide (19). The synthesis
involved following procedure D from 7b (250 mg, 0.76 mmol) and
3,3-dimethylazetidine (105 mg, 0.86 mmol). Purification by flash silica
column chromatography (gradient elution i-hex/EtOAc 0−100%)
gave (1R*,2R*,3R*)-methyl-2-(4-(3,3-dimethylazetidin-1-yl)phenyl)-
3-phenylcyclopropanecarboxylate (9a) as a clear oil (210 mg, 59%)
which was progressed into the next step. The synthesis involved
following procedure A from 9a (200 mg, 0.59 mmol). Purification by
preparative HPLC gave the title compound as a white solid (23 mg,
14%). LCMS (ES+) 337 (M + H)+, tR = 3.42 min (analytical method
1
1). H NMR δ (ppm) (DMSO-d6): 10.49 (1 H, s), 8.63 (1 H, s),
7.33−7.20 (4 H, m), 7.19−7.12 (1 H, m), 7.05 (2 H, d, J = 8.2 Hz),
6.37 (2 H, d, J = 8.2 Hz), 3.48 (4 H, s), 2.97 (1 H, dd, J = 6.9, 5.4 Hz),
2.68 (1 H, dd, J = 9.5, 6.9 Hz), 2.06 (1 H, dd, J = 9.5, 5.4 Hz), 1.27 (6
H, s). HRMS (ESI) calcd for C21H24N2O2 [M + H]+ 337.1916, found
337.1899.
(1R*,2R*,3R*)-2-(4-(2-Oxa-6-azaspiro[3.3]heptan-6-yl)-
phenyl)-N-hydroxy-3-phenylcyclopropanecarboxamide (20).
The synthesis involved following procedure D from 7b (250 mg,
(1R*,2R*,3R*)-N-Hydroxy-2-phenyl-3-(4-(pyrimidin-2-yl)-
phenyl)cyclopropanecarboxamide (24). The synthesis involved
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following procedure E from the crude boronate derived from 7b (274
mg), and 2-chloropyrimidine (87 mg, 0.76 mmol). Purification by flash
silica column chromatography (gradient elution i-hex to 25% EtOAc in
i-hex) gave (1R*,2R*,3R*)-methyl 2-phenyl-3-(4-(pyrimidin-2-yl)-
phenyl)cyclopropanecarboxylate (9f) as a pale yellow oil [110 mg,
46%, LCMS (ES+) 345 (M + H)+] which was progressed to the next
step. The synthesis involved following procedure A from 9f (110 mg,
0.33 mmol). Purification by column chromatography (gradient elution
DCM to 5% MeOH in DCM) gave the title compound as a white
solid (75 mg, 67%). LCMS (ES+) 332 (M + H)+, (ES−) 330 (M +
H)−, tR = 2.78 min (analytical method 1). 1H NMR δ (ppm) (DMSO-
d6): 10.59 (1 H, s), 8.90 (2 H, d, J = 4.8 Hz), 8.72 (1 H, s), 8.36 (2 H,
d, J = 8.2 Hz), 7.48−7.40 (3 H, m), 7.36 (2 H, d, J = 7.6 Hz), 7.27 (2
H, t, J = 7.6 Hz), 7.19 (1 H, t, J = 7.3 Hz), 3.18 (1 H, dd, J = 6.8, 5.4
Hz), 2.93 (1 H, dd, J = 9.6, 6.8 Hz), 2.30 (1 H, dd, J = 9.6, 5.4 Hz).
HRMS (ESI) calcd for C20H17N3O2 [M + H]+ 332.1399, found
332.1390.
H, s), 3.19−3.13 (1 H, dd, J = 6.8, 5.4 Hz), 2.90 (1 H, dd, J = 9.6, 6.8
Hz), 2.26 (1 H, dd, J = 9.6, 5.4 Hz). HRMS (ESI) calcd for
C20H19N3O2 [M + H]+ 334.1555, found 334.1543.
(1R*,2R*,3R*)-N-Hydroxy-2-(4-(5-methyl-1H-imidazol-2-yl)-
phenyl)-3-phenylcyclopropanecarboxamide (28). The synthesis
involved following procedure F from crude boronate derived from 7b
(1.5 mmol) and 2-bromo-4-methylimidazole (242 mg, 1.5 mmol).
Purification by flash silica column chromatography (gradient elution
DCM−MeOH 0−10%) gave (1R*,2R*,3R*)-methyl 2-(4-(5-methyl-
1H-imidazol-2-yl)phenyl)-3-phenylcyclopropanecarboxylate (9j) as a
yellow oil (285 mg). The crude material was used in the next step [
LCMS (ES+) 335 (M + H)+]. The synthesis involved following
procedure A from 9j (280 mg, 0.84 mmol). Purification by preparative
HPLC gave the title compound as a white solid (4.8 mg, 1% yield over
three steps). LCMS (ES+) 334 (M + H)+, tR = 7.93 min (analytical
method 3). 1H NMR δ (ppm) (DMSO-d6): 10.62 (1 H, s), 8.42 (1 H,
s), 7.88 (2 H, d, J = 8.0 Hz), 7.41−7.28 (6 H, m), 7.25−7.20 (1 H, m),
6.84 (1 H, s), 3.16 (1 H, dd, J = 6.9, 5.5 Hz), 2.92 (1 H, dd, J = 9.4, 6.7
Hz), 2.31−2.20 (4 H, m), OH not observed. HRMS (ESI) calcd for
C20H19N3O2 [M + H]+ 334.1555, found 334.1545.
(1R*,2R*,3R*)-2-(4-(5-Fluoropyrimidin-2-yl)phenyl)-N-hy-
droxy-3-phenylcyclopropanecarboxamide (25). The synthesis
involved following procedure E from the crude boronate derived from
7b (250 mg) and 2-chloro-5-fluoropyrimidine (91 mg, 0.69 mmol).
Purification by flash silica column chromatography (gradient elution i-
hex to 10% EtOAc in i-hex) gave (1R*,2R*,3R*)-methyl 2-(4-(5-
fluoropyrimidin-2-yl)phenyl)-3-phenylcyclopropanecarboxylate (9g)
as a colorless oil [240 mg, 100%, LCMS (ES+) 363 (M + H)+],
which was progressed to the next step. The synthesis involved
following procedure A from 9g (220 mg, 0.63 mmol). Purification by
flash silica column chromatography (gradient elution DCM to 5%
MeOH in DCM) gave the title compound as a white solid (81 mg,
37%). LCMS (ES+) 350 (M + H)+, tR = 3.13 min (analytical method
(1R,2R,3R)-2-(3-Fluoro-4-(5-methyl-1H-imidazol-2-yl)-
phenyl)-N-hydroxy-3-phenylcyclopropanecarboxamide (29).
To the crude boronate derived from 7c (1.5 mmol) in DME/
MeOH (4:1, 10 mL) were added 2-bromo-4-methyl-1H-imidazole
(353 mg, 2.20 mmol), cesium fluoride (333 mg, 2.20 mmol), and
Pd(PPh3)4 (84 mg, 0.073 mmol). This mixture was heated under
microwave irradiation at 110 °C for 1 h. The reaction mixture was
cooled, diluted with water, and extracted with DCM. The organic
layers were combined, washed with water, dried (MgSO4), filtered, and
concentrated to give a brown gum. Purification by flash silica column
chromatography (gradient elution i-hex to 100% EtOAc in i-hex) gave
(1R*,2R*,3R*)-ethyl 2-(3-fluoro-4-(5-methyl-1H-imidazol-2-yl)-
phenyl)-3-phenylcyclopropanecarboxylate (9k) as an off white solid
[134 mg, 25%, 7:3 trans/cis, LCMS (ES+) 365 (M + H)+], which was
progressed to the next step. The synthesis involved following
procedure A from 9k (154 mg, 0.42 mmol) to yield an off white
gum (200 mg). Purification by preparative HPLC gave the racemic
product as a pale yellow solid (16 mg, 10%). Preparative chiral
purification gave the title compound as a formate salt (3 mg, 2%,
Chiralpak IC 40/60 IPA/MeOH (50/50/0.1% formic acid)/heptane
5.0 mL/min). LCMS (ES+) 352 (M + H)+, 2.29 min (analytical
1
1). H NMR δ (ppm) (DMSO-d6): 10.58 (1 H, s), 8.97 (2 H, d, J =
0.8 Hz), 8.70 (1 H, s), 8.30 (2 H, d, J = 8.2 Hz), 7.44 (2 H, d, J = 8.2
Hz), 7.36 (2 H, d, J = 7.6 Hz), 7.27 (2 H, t, J = 7.5 Hz), 7.20 (1 H, d, J
= 7.2 Hz), 3.18 (1 H, dd, J = 6.7, 5.6 Hz), 2.93 (1 H, dd, J = 9.6, 6.7
Hz), 2.30 (1 H, dd, J = 9.6, 5.4 Hz). HRMS (ESI) calcd for
C20H16FN3O2 [M + H]+ 350.1304, found 350.1298.
(1R*,2R*,3R*)-2-(3-(5-Fluoropyrimidin-2-yl)phenyl)-N-hy-
droxy-3-phenylcyclopropanecarboxamide (26). The synthesis
involved following procedure E from the crude boronate derived from
7a (300 mg) and 2-chloro-5-fluoropyrimidine (107 mg, 0.81 mmol).
Purification by flash silica column chromatography (gradient elution i-
hex to 10% EtOAc in i-hex) gave (1R*,2R*,3R*)-ethyl 2-(3-(5-
fluoropyrimidin-2-yl)phenyl)-3-phenylcyclopropanecarboxylate (9h)
as a colorless oil [140 mg, 50%, LCMS (ES+) 363 (M + H)+],
which was progressed to the next step. The synthesis involved
following procedure A from 9h (140 mg, 0.39 mmol). Purification by
flash silica column chromatography (gradient elution DCM to 7%
MeOH in DCM) and preparative HPLC gave the title compound as a
racemic mixture (12 mg, 9%). LCMS (ES+) 350 (M + H)+, tR = 3.61
1
method 1). H NMR δ (ppm) (DMSO-d6): 11.88 (0.5 H, s), 11.80
(0.5 H, s), 10.56 (1 H, s), 8.70 (1 H, s), 8.52 (1 H, s), 7.90 (1 H, q, J =
8.6 Hz), 7.34 (2 H, d, J = 7.6 Hz), 7.29−7.24 (3 H, m), 7.19 (2 H, s),
6.93 (0.5 H, s), 6.74 (0.5 H, s), 3.15 (1 H, dd, J = 6.8, 5.2 Hz), 2.93 (1
H, dd, J = 9.7, 6.8 Hz), 2.26 (1 H, dd, J = 9.7, 5.2 Hz), 2.24 (1.5 H, s),
2.17 (1.5 H, s). HRMS (ESI) calcd for C20H18FN3O2 [M + H]+
352.1461, found 352.1464.
(1R*,2R*,3R*)-N-Hydroxy-2-(4-(oxazol-2-yl)phenyl)-3-
phenylcyclopropanecarboxamide (30). The synthesis involved
following procedure F from 7b (250 mg, 0.75 mmol) and 2-(tri-n-
butylstannyl)oxazole (0.230 mL, 1.1 mmol). Purification by flash silica
column chromatography (gradient elution DCM to 10% MeOH in
DCM) afforded (1R*,2R*,3R*)-methyl-2-(4-(oxazol-2-yl)phenyl)-3-
phenylcyclopropanecarboxamide (9l) as a white solid [175 mg, 73%,
LCMS (ES+) 334 (M + H)+] which was progressed to the next step.
The synthesis involved following procedure A from 9l (160 mg, 0.50
mmol). Crystallization from MeOH gave the title compound as a
white solid (71 mg, 45%). LCMS (ES+) 321 (M + H)+, tR = 2.80 min
(analytical method 1). 1H NMR δ (ppm) (DMSO-d6): 10.58 (1 H, s),
8.70 (1 H, s), 8.21 (1 H, d, J = 0.8 Hz), 7.94 (2 H, d, J = 8.1 Hz), 7.45
(2 H, d, J = 8.1 Hz), 7.39−7.33 (3 H, m), 7.31−7.23 (2 H, m), 7.22−
7.15 (1 H, m), 3.20−3.13 (1 H, m), 2.91 (1 H, dd, J = 9.6, 6.8 Hz),
2.28 (1 H, dd, J = 9.6, 5.3 Hz). HRMS (ESI) calcd for C19H16N2O3 [M
+ H]+ 321.1239, found 321.1229.
1
min (analytical method 1). H NMR δ (ppm) (DMSO-d6): 10.59 (1
H, s), 9.01 (2 H, s), 8.71 (1 H, s), 8.20 (2 H, t, J = 4.0 Hz), 7.51−7.48
(2 H, m), 7.37 (2 H, d, J = 7.6 Hz), 7.29−7.24 (2 H, m), 7.20 (1 H, d,
J = 7.3 Hz), 3.22 (1 H, dd, J = 6.8, 5.5 Hz), 2.85 (1 H, dd, J = 9.6, 6.8
Hz), 2.32 (1 H, dd, J = 9.6, 5.4 Hz). HRMS (ESI) calcd for
C20H16FN3O2 [M + H]+ 350.1304, found 350.1307.
(1R*,2R*,3R*)-N-Hydroxy-2-(4-(1-methyl-1H-imidazol-2-yl)-
phenyl)-3-phenylcyclopropanecarboxamide (27). The synthesis
involved following procedure F from 7b (222 mg, 0.67 mmol) and 1-
methyl-2-(tributylstannyl)imidazole (300 mg, 0.81 mmol). Purification
by flash silica column chromatography (gradient elution DCM to 10%
MeOH in DCM) gave (1R*,2R*,3R*)-methyl-2-(4-(1-methyl-1H-
imidazol-2-yl)phenyl)-3-phenylcyclopropanecarboxamide (9i) as a
yellow solid [187 mg, 84%, LCMS (ES+) 347 (M + H)+] which was
progressed to the next step. The synthesis involved following
procedure A from 9i (187 mg, 0.56 mmol). Crystallization from
DCM and trituration with MeOH gave the title compound as a white
solid (98 mg, 53%). LCMS (ES+) 334 (M + H)+, tR = 9.74 min
(analytical method 3). 1H NMR δ (ppm) (DMSO-d6): 10.58 (1 H, s),
8.70 (1 H, s), 7.65 (2 H, d, J = 8.1 Hz), 7.44−7.31 (4 H, m), 7.30−
7.24 (3 H, m), 7.23−7.15 (1 H, m), 6.99 (1 H, d, J = 1.1 Hz), 3.75 (3
(1R,2R,3R)-N-Hydroxy-2-(4-(oxazol-5-yl)phenyl)-3-phenyl-
cyclopropanecarboxamide (31). The synthesis involved following
procedure A from 9m. Purification by flash silica column
chromatography (gradient elution DCM to 5% MeOH in DCM)
gave the racemic mixture as a white solid (88 mg, 29%). Preparative
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chiral HPLC gave the title compound (Chiralpak IC 20/80 EtOH (0.1
H, dd, J = 9.6, 6.9 Hz), 2.37 (1 H, dd, J = 9.7, 5.5 Hz). HRMS (ESI)
calcd for C14H13N3O2 [M + H]+ 256.1086, found 256.1083.
(1R*,2R*,3R*)-N-Hydroxy-2-phenyl-3-(pyridazin-4-yl)-
cyclopropanecarboxamide (37). The synthesis involved following
procedure A from 14a′ (45 mg, 0.17 mmol). Purification by flash silica
column chromatography (gradient elution DCM to 4% MeOH in
DCM) and reversed phase HPLC gave the title compound as a white
solid (3 mg, 21%). LCMS (ES+) 256 (M + H)+, tR = 7.02 min
(analytical method 3). 1H NMR δ (ppm) (DMSO-d6): 10.69 (1 H, s),
9.34 (1 H, s), 9.15 (1 H, d, J = 5.4 Hz), 8.81 (1 H, s), 7.63−7.61 (1 H,
m), 7.40−7.30 (4 H, m), 7.27−7.24 (1 H, m), 3.22 (1 H, dd, J = 5.2,
6.4 Hz), 3.13 (1 H, dd, J = 6.4, 9.5 Hz), 2.45 (1 H, dd, J = 9.5, 5.3 Hz).
HRMS (ESI) calcd for C14H13N3O2 [M + H]+ 256.1086, found
256.1081.
formic acid)/heptane, 1.0 mL/min, tR = 18.7 min). LCMS (ES+) 321
1
(M + H)+, tR = 2.77 min (analytical method 1). H NMR δ (ppm)
(DMSO-d6): 10.63 (1 H, s), 8.76 (1 H, s), 8.49 (1 H, s), 7.76−7.72 (3
H, m), 7.47−7.36 (4 H, m), 7.35−7.29 (2 H, m), 7.25−7.20 (1 H, m),
3.18 (1 H, dd, J = 6.8, 5.4 Hz), 2.94 (1 H, dd, J = 9.6, 6.8 Hz), 2.30 (1
H, dd, J = 9.6, 5.4 Hz). HRMS (ESI) calcd for C19H16N2O3 [M + H]+
321.1239, found 321.1235.
(1S*,2R*,3R*)-2-Cyclopentyl-N-hydroxy-3-phenyl-
cyclopropanecarboxamide (32). The synthesis involved following
procedure A from 14a (1.05 g, 4.06 mmol). Purification by flash silica
column chromatography (gradient elution DCM to 4% MeOH in
DCM) and preparative HPLC gave the title compound as a white solid
(23 mg, 9%). LCMS (ES+) 246 (M + H)+, tR = 3.18 min (analytical
method 1). 1H NMR δ (ppm)(DMSO-d6): 10.57 (1 H, s), 8.76 (1 H,
s), 7.35−7.27 (2 H, m), 7.24−7.18 (3 H, m), 1.88 (1 H, t, J = 5.0 Hz),
1.71−1.60 (1 H, m), 1.58−1.47 (2 H, m), 1.50−1.16 (8 H, m). HRMS
(ESI) calcd for C15H19NO2 [M + H]+ 246.1494, found 246.1491.
(1R,2R,3R)-N-Hydroxy-2-(1-methyl-1H-pyrazol-4-yl)-3-
phenylcyclopropanecarboxamide (33). The synthesis involved
following procedure A from 14b (493 mg, 1.83 mmol). Purification by
flash silica column chromatography (gradient elution DCM to 5%
MeOH in DCM) and then preparative HPLC gave the racemic
mixture as a white solid (235 mg, 50%). Preparative chiral HPLC gave
the title compound (Chiralpak IC 30/70 IPA/MeOH (50/50/0.1%
formic acid)/heptane, 1.0 mL/min, tR = 10.3 min). LCMS (ES+) 258
(M + H)+, (ES−) 256 (M + H)−, tR = 2.65 min (analytical method 1).
1H NMR δ (ppm) (DMSO-d6): 10.50 (1 H, s), 8.63 (1 H, s), 7.61 (1
(1R,2R,3R)-2-(2-Cyclopropylpyrimidin-5-yl)-N-hydroxy-3-
phenylcyclopropanecarboxamide (38). The synthesis involved
following procedure A from 14b′ (310 mg). Purification by preparative
HPLC gave the racemic compound (30 mg, 10%). Preparative chiral
purification gave the title compound (Chiralpak IC 40/60 IPA/
MetOH (0.1% formic acid)/heptane 1.0 mL/min). LCMS (ES+) 296
1
(M + H)+, tR = 2.93 min (analytical method 1). H NMR δ (ppm)
(DMSO-d6): 10.48 (1 H, s), 8.59 (1 H, s), 8.51−8.48 (6 H, m), 7.25
(3 H, d, J = 7.6 Hz), 7.21−7.16 (3 H, m), 7.12 (1 H, d, J = 7.23 Hz),
2.99 (1 H, dd, J = 6.7, 5.4 Hz), 2.86 (1 H, dd, J = 9.5, 6.8 Hz), 2.21 (1
H, dd, J = 9.6, 5.5 Hz), 2.12−2.08 (1 H, m), 0.96−0.91 (2 H, m),
0.91−0.87 (2 H, m). HRMS (ESI) calcd for C17H17N3O2 [M + H]+
296.1399, found 296.1399.
(1R,2R,3R)-2-(6-Cyclopropylpyridazin-4-yl)-N-hydroxy-3-
phenylcyclopropanecarboxamide (39). The synthesis involved
following procedure A from 14o′ (0.1 g, 0.3 mmol). Preparative chiral
purification gave the title compound (Chiralpak IC 30/70 [IPA/
MeOH (50/50/0.1% formic acid)]/heptane 5.0 mL/min, tR = 28.95
min). LCMS (ES+) 296 (M + H)+, tR = 2.82 min (analytical method
H, s), 7.34 (1 H, s), 7.28−7.13 (5 H, m), 3.78 (3 H, s), 2.86 (1 H, dd,
J = 6.9, 5.3 Hz), 2.63 (1 H, dd, J = 9.4, 6.9 Hz), 1.97 (1 H, dd, J = 9.4,
5.3 Hz). HRMS (ESI) calcd for C14H15N3O2 [M + H]+ 258.1242,
found 258.1237.
(1R,2R,3S)-N-Hydroxy-2-(5-methylthiazol-2-yl)-3-phenyl-
cyclopropanecarboxamide (34). The synthesis involved following
procedure A from 14c (92 mg). Purification by flash silica column
chromatography (gradient elution DCM to 4% MeOH in DCM) and
preparative HPLC gave the racemic compound (38 mg, 86% based on
purity of SM). Preparative chiral purification gave the title compound
(Chiralpak IA 15/85 MeOH (50/50/0.1 formic acid)/heptane 1.0
mL/min, tR = 12.3 min). LCMS (ES+) 275 (M + H)+, tR = 3.07 min
(analytical method 1). 1H NMR δ (ppm) (DMSO-d6): 10.55 (1 H, s),
8.63 (1 H, s), 7.29 (1 H, d, J = 1.4 Hz), 7.26−7.22 (3 H, m), 7.21−
7.16 (2 H, m), 7.15−7.09 (1 H, m), 3.31 (1 H, dd, J = 6.34, 5.2 Hz),
2.87 (1 H, dd, J = 9.7, 6.4 Hz), 2.40 (1 H, dd, J = 9.7, 5.2 Hz), 2.34 (3
H, d, J = 1.2 Hz). HRMS (ESI) calcd for C14H14N2O2S [M + H]+
275.0854, found 275.0853.
(1R,2R,3S)-N-Hydroxy-2-(5-methylthiazol-2-yl)-3-phenyl-
cyclopropanecarboxamide (35). The synthesis involved following
procedure A from 14i (92 mg). Purification by flash silica column
chromatography (gradient elution DCM to 4% MeOH in DCM) and
preparative HPLC gave the racemic compound (38 mg, 86% based on
purity of SM). Preparative chiral purification gave the title compound
(Chiralpak IA 15/85 MeOH (50/50/0.1 formic acid)/heptane 1.0
mL/min, tR = 12.3 min). LCMS (ES+) 275 (M + H)+, tR = 3.07 min
(analytical method 1). 1H NMR δ (ppm) (DMSO-d6): 10.55 (1 H, s),
8.63 (1 H, s), 7.29 (1 H, d, J = 1.4 Hz), 7.26−7.22 (3 H, m), 7.21−
7.16 (2 H, m), 7.15−7.09 (1 H, m), 3.31 (1 H, dd, J = 6.34, 5.2 Hz),
2.87 (1 H, dd, J = 9.7, 6.4 Hz), 2.40 (1 H, dd, J = 9.7, 5.2 Hz), 2.34 (3
H, d, J = 1.2 Hz). HRMS (ESI) calcd for C14H14N2O2S [M + H]+
275.0854, found 275.0850.
1
1). H NMR δ (ppm) (DMSO-d6): 10.63 (1 H, s), 9.07 (1 H, d, J =
2.1 Hz), 8.76 (1 H, s), 7.42−7.35 (3 H, m), 7.32 (2 H, t, J = 7.4 Hz),
7.25 (1 H, t, J = 7.1 Hz), 3.17−3.08 (2 H, m), 2.45 (1 H, dd, J = 9.5,
5.6 Hz), 2.28−2.21 (1 H, m), 1.15−1.11 (4 H, m). HRMS (ESI) calcd
for C17H17N3O2 [M + H]+ 296.1399, found 296.1406.
(1R,2R,3R)-2-(2-Cyclopropylpyridin-4-yl)-N-hydroxy-3-
phenylcyclopropanecarboxamide (40). The synthesis involved
following procedure A from 14f′ (330 mg, 1.07 mmol). Purification by
flash silica column chromatography (gradient elution DCM to 5%
MeOH in DCM) gave the racemic mixture as a white solid (60 mg,
19%). Preparative chiral HPLC gave the title compound (Chiralpak IC
30/70 IPA/MeOH (50/50/0.1 formic acid)/heptane, 1.0 mL/min, tR
= 9.6 min). LCMS (ES+) 295 (M + H)+, (ES−) 293 (M − H)−, tR =
2.12 min (analytical method 1). 1H NMR δ (ppm) (DMSO-d6): 10.65
(1 H, s), 8.78 (1 H, s), 8.32 (1 H, d, J = 5.1 Hz), 7.37 (2 H, d, J = 7.6
Hz), 7.34−7.28 (2 H, m), 7.25 (2 H, d, J = 8.2 Hz), 7.07 (1 H, dd, J =
5.2, 1.7 Hz), 3.10 (1 H, dd, J = 6.7, 5.6 Hz), 2.99 (1 H, dd, J = 9.7, 6.7
Hz), 2.34 (1 H, dd, J = 9.7, 5.6 Hz), 2.12−2.07 (1 H, m), 0.98−0.93 (4
H, m). HRMS (ESI) calcd for C18H18N2O2 [M + H]+ 295.1446, found
295.1450.
(1R,2R,3R)-N-Hydroxy-2-phenyl-3-(2-(trifluoromethyl)-
pyridin-4-yl)cyclopropanecarboxamide (41). The synthesis in-
volved following procedure A from 14h′ (130 mg, 0.41 mmol).
Purification by flash silica column chromatography (gradient elution
DCM to 5% MeOH in DCM) followed by preparative HPLC gave the
racemic mixture as a white solid (90 mg, 68%). Preparative chiral
HPLC gave the title compound (Chiralpak IC 20/80 IPA/MeOH
(50/50/0.1 formic acid)/heptane, 1.0 mL/min, tR = 10.3 min). LCMS
(ES+) 323 (M + H)+, tR = 3.50 min (analytical method 1). 1H NMR δ
(ppm) (DMSO-d6): 10.66 (1 H, s), 8.81 (1 H, s), 8.71 (1 H, d, J = 5.0
Hz), 7.92 (1 H, s), 7.70 (1 H, d, J = 5.1 Hz), 7.40 (2 H, d, J = 7.5 Hz),
7.32 (2 H, t, J = 7.5 Hz), 7.27−7.22 (1 H, m), 3.35 (1H, dd, J = 6.6,
5.4 Hz), 3.16 (1 H, dd, J = 9.5, 6.6 Hz), 2.48 (1 H, dd, J = 9.5, 5.4 Hz).
HRMS (ESI) calcd for C16H13F3N2O2 [M + H]+ 323.1007, found
323.1006.
(1R*,2R*,3R*)-N-Hydroxy-2-phenyl-3-(pyrimidin-5-yl)-
cyclopropanecarboxamide (36). The synthesis involved following
procedure A from 14d (270 mg, 1.08 mmol). Purification by flash
silica chromatography (gradient elution DCM to 7% MeOH in DCM)
and reversed phase HPLC gave the title compound as a yellow solid (9
mg, 5%). LCMS (ES+) 256 (M + H)+, tR = 2.52 min (analytical
method 1). 1H NMR δ (ppm) (DMSO-d6): 10.61 (1 H, s), 9.06 (1 H,
s), 8.79 (2 H, s), 8.72 (1 H, s), 7.34 (2 H, d, J = 7.6 Hz), 7.27 (2 H, t, J
= 7.5 Hz), 7.22−7.17 (1 H, m), 3.15 (1 H, dd, J = 6.8, 5.5 Hz), 3.04 (1
(1R,2R,3R)-N-Hydroxy-2-phenyl-3-(6-(trifluoromethyl)-
pyridin-3-yl)cyclopropanecarboxamide (42). The synthesis in-
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volved following procedure A from compound 14j′ (720 mg, 2.24
mmol). Purification by flash silica column chromatography (gradient
elution DCM to 5% MeOH in DCM) followed by preparative HPLC
gave the racemic mixture as a white solid (83 mg, 11%). Preparative
chiral HPLC gave the title compound (Chiralpak IC 20/80 IPA/
MeOH (50/50/0.1 formic acid)/heptane, 1.0 mL/min, tR = 14.5 min).
white solid (450 mg, 69%). An amount of 200 mg sent to preparative
chiral purification gave the title compound as an off-white solid (95
mg, 47%) (Chiralpak IC 40/60 IPA/MeOH (50/50/0.1% formic
acid)/heptane 5.0 mL/min). LCMS (ES+) 325 (M + H)+, 3.37 min
(analytical method 1). 1H NMR δ (ppm) (DMSO-d6): 10.56 (1 H, s),
8.69 (1 H, s), 7.96 (1 H, d, J = 1.8 Hz), 7.85 (1 H, d, J = 8.4 Hz),
7.41−7.33 (3 H, m), 7.31−7.23 (2 H, m), 7.19 (1 H, dd, J = 7.2, 7.2
Hz), 3.23 (1 H, dd, J = 6.8, 5.4 Hz), 2.92 (1 H, dd, J = 9.6, 6.8 Hz),
2.79 (3 H, s), 2.27 (1 H, dd, J = 9.6, 5.4 Hz). HRMS (ESI) calcd for
C18H16N2O2S [M + H]+ 325.1010, found 325.1018.
(1R,2R,3R)-2-(2-cyclopropylbenzo[d]oxazol-6-yl)-N-hydroxy-
3-phenylcyclopropanecarboxamide (48). The synthesis involved
following procedure A from 14l (240 mg, 0.69 mmol) to yield an
orange glass (265 mg). Purification by preparative HPLC gave the
racemic product as an off white solid (11 mg, 9%). Preparative chiral
purification gave the title compound as a off white solid (Chiralpak IC
40/60 IPA/MeOH (50/50/0.1% formic acid)/heptane 5.0 mL/min).
LCMS (ES+) 335 (M + H)+, 3.39 min (analytical method 1). 1H NMR
δ (ppm) (DMSO-d6):10.55 (1 H, s), 8.68 (1 H, s), 7.56−7.52 (2 H,
m), 7.36−7.31 (2 H, m), 7.30−7.22 (3 H, m), 7.21−7.15 (1 H, m),
3.22 (1 H, dd, J = 6.9, 5.36 Hz), 2.90 (1 H, dd, J = 9.5, 6.9 Hz), 2.29−
2.21 (2 H, m), 1.20−1.09 (4 H, m).
1
LCMS (ES+) 323 (M + H)+, tR = 8.60 min (analytical method 3). H
NMR δ (ppm) (DMSO-d6): 10.70 (1 H, s), 8.84 (1 H, s), 8.82 (1H,
s), 8.02−7.97 (1 H, m), 7.91 (1 H, d, J = 8.2 Hz), 7.40 (2 H, d, J = 7.6
Hz), 7.33 (2 H, t, J = 7.5 Hz), 7.28−7.22 (1 H, m), 3.32 (1 H, dd, J =
6.7 and 5.6 Hz), 3.09 (1 H, dd, J = 9.7, 6.8 Hz), 2.43 (1 H, dd, J =9.7
and 5.6 Hz). HRMS (ESI) calcd for C16H13F3N2O2 [M + H]+
323.1007, found 323.1013.
(1R,2R,3R)-N-Hydroxy-2-phenyl-3-(5-(trifluoromethyl)-
pyridin-3-yl)cyclopropanecarboxamide (43). The synthesis in-
volved following procedure A from 14l′ (250 mg, 0.78 mmol).
Purification by flash silica column chromatography (gradient elution
DCM to 5% MeOH in DCM) followed by preparative HPLC gave the
racemic mixture as a white solid (83 mg, 11%). Preparative chiral
HPLC gave the title compound (Chiralpak IC 20/80 EtOH (0.1%
formic acid)/heptane, 1.0 mL/min, tR = 11.3 min). LCMS (ES+) 323
1
(M + H)+, tR = 3.30 min (analytical method 1). H NMR δ (ppm)
(DMSO-d6): 10.64 (1 H, s), 8.96 (1 H, s), 8.88 (1 H, s), 8.79 (1 H, s),
8.15 (1 H, s), 7.41 (2 H, d, J = 7.6 Hz), 7.32 (2 H, t, J = 7.4 Hz), 7.24
(1 H, t, J = 7.2 Hz), 3.41 (1 H, obscured by water), 3.15 (1 H, dd, J =
9.8, 6.9 Hz), 2.44 (1 H, dd, J = 9.8, 5.4 Hz). HRMS (ESI) calcd for
C16H13F3N2O2 [M + H]+ 323.1007, found 323.1012.
(1R,2R,3R)-N-Hydroxy-2-phenyl-3-(quinoxalin-6-yl)-
cyclopropanecarboxamide (49). The synthesis involved following
procedure A from 14g (490 mg, 1.54 mmol). Purification by
preparative HPLC gave the racemic compound (110 mg, 36%).
Preparative chiral purification gave the title compound [Chiralpak IC
40/60 IPA/MeOH (50/50/0.1% formic acid)/heptane 1.0 mL/min].
LCMS (ES+) 306 (M + H)+, tR = 2.96 min (analytical method 1). 1H
NMR δ (ppm) (DMSO-d6): 10.64 (1 H, s), 8.99 (1 H, d, J = 1.8 Hz),
8.95 (1 H, d, J = 1.8 Hz), 8.75 (1 H, s), 8.12 (1 H, d, J = 8.6 Hz), 8.03
(1 H, s), 7.87 (1 H, dd, J = 8.7, 2.0 Hz), 7.45 (2 H, d, J = 7.5 Hz), 7.33
(2 H, t, J = 7.5 Hz), 7.27−7.22 (1 H, m), 3.43 (1 H, dd, J = 6.8, 5.4
Hz), 3.10 (1 H, dd, J = 9.7, 6.8 Hz), 2.49 (1 H, dd, J = 9.7, 5.4 Hz).
(1R,2R,3R)-N-Hydroxy-2-(imidazo[1,2-a]pyridin-2-yl)-3-
phenylcyclopropanecarboxamide (44). The synthesis involved
following procedure A from 14a (430 mg, 1.4 mmol) to yield a yellow
gum. Purification by preparative HPLC gave the racemic product as a
white solid (118 mg, 29%). Preparative chiral purification gave the title
compound (Chiralpak IA 20/80 IPA/MeOH(50/50/0.1% formic
acid)/heptane 5.0 mL/min). LCMS (ES+) 294 (M + H)+, 2.07 min
(analytical method 1). 1H NMR δ (ppm) (DMSO-d6): 10.60 (1 H, s),
8.68 (1 H, s), 8.52 (1 H, d, J = 6.7 Hz), 7.99 (1 H, s), 7.50 (1 H, d, J =
9.1 Hz), 7.39 (2 H, d, J = 7.6 Hz), 7.33−7.18 (4 H, m), 6.89 (1 H, m,),
3.25 (1 H, dd, J = 6.5, 5.3 Hz), 2.96 (1 H, dd, J = 9.5, 6.5 Hz), 2.48 (1
H, dd, J = 9.6, 5.2 Hz). HRMS (ESI) calcd for C17H15N3O2 [M + H]+
294.1242, found 294.1245.
(1R,2R,3R)-N-Hydroxy-2-phenyl-3-(pyrazolo[1,5-a]pyridin-2-
yl)cyclopropanecarboxamide (45). The synthesis involved follow-
ing procedure A from 14f (624 mg, 2.04 mmol) to yield a yellow gum
(650 mg). Purification by preparative HPLC gave the racemic product
as a pale yellow solid (378 mg, 63%). Preparative chiral purification
gave the title compound (Chiralpak IC 50/50 IPA/MeOH (50/50/
0.1% formic acid)/heptane 5.0 mL/min). LCMS (ES+) 294 (M + H)+,
3.37 min (analytical method 1). 1H NMR δ (ppm) (DMSO-d6): 10.64
(1 H, s), 8.78 (1 H, s), 8.10 (1 H, d, J = 2.4 Hz), 7.66 (1 H, d, J = 8.8
Hz), 7.46 (2 H, d, J = 7.5 Hz), 7.30 (2 H, t, J = 7.4 Hz), 7.21 (2 H, t, J
= 7.6 Hz), 6.80 (1 H, d, J = 6.8 Hz), 6.71 (1 H, d, J = 2.3 Hz), 3.77 (1
H, dd, J = 6.9, 5.6 Hz), 3.05 (1 H, dd, J = 9.5, 6.9 Hz), 2.52−2.48 (1H,
obscured by DMSO). HRMS (ESI) calcd for C17H15N3O2 [M + H]+
294.1242, found 294.1238.
HRMS (ESI) calcd for C18H15N3O [M + H]+ 306.1242, found
2
306.1241.
(1R,2R,3R)-N-Hydroxy-2-phenyl-3-(3-(trifluoromethyl)-
quinoxalin-6-yl)cyclopropanecarboxamide (50). The synthesis
involved following procedure A from 14p′ (178 mg, 2.16 mmol). The
target compound was obtained after preparative HPLC purification
followed by chiral HPLC purification (13.7 mg) (Chiralpak IC 20/80
[IPA/MeOH (50/50/0.1% formic acid)]/heptane 1.0 mL/min, tR
=
9.2 min). LCMS (ES+) 374 (M + H)+, tR = 3.68 min (analytical
method 1). 1H NMR δ (ppm) (DMSO-d6): 10.53 (1 H, s), 9.30 (1 H,
s), 8.64 (1 H, s), 8.16 (1 H, d, J = 8.7 Hz), 8.09 (1 H, s), 7.98 (1 H, d,
J = 8.8 Hz), 7.33 (2 H, d, J = 7.58 Hz), 7.21 (2 H, t, J = 7.50 Hz),
7.15−7.12 (1 H, m), 3.37 (1 H, dd, J = 6.7, 5.4 Hz), 3.06 (1 H, dd, J =
9.7, 6.7 Hz), 2.44−2.41 (1 H, dd, obscured by DMSO). HRMS (ESI)
calcd for C19H14F3N3O2 [M + H]+ 374.1116, found 374.1119.
(1R,2R,3R)-2-(3-cyclopropylquinoxalin-6-yl)-3-phenyl-
cyclopropanecarbohydroxamic Acid (51). The synthesis involved
following procedure A from 14m′ (384g, 1.07 mmol). Purification by
flash silica column chromatography (gradient elution DCM to 10%
MeOH in DCM) and preparative HPLC gave the racemic compound
(172 mg, 46%). Preparative chiral purification gave the title compound
as an off white solid (Chiralpak IC 30/70 EtOH (0.1% formic acid)/
heptane 5.0 mL/min, tR = 9.67 min). LCMS (ES+) 346 (M + H)+, tR =
3.47 min (analytical method 1). 1H NMR δ (ppm) (DMSO-d6): 10.60
(1 H, s), 8.97−8.84 (1 H, m), 8.72 (1 H, s), 7.95−7.85 (2 H, m), 7.72
(1 H, dd, J = 8.7, 2.1 Hz), 7.39 (2 H, d, J = 7.6 Hz), 7.28 (2 H, dd, J =
7.6, 7.3 Hz), 7.20 (1 H, dd, J = 7.3, 7.3 Hz), 3.35 (1 H, dd, partly
obscured by water peak), 3.03 (1 H, dd, J = 9.6, 6.8 Hz), 2.44−2.35 (2
H, m), 1.20−1.12 (4 H, m). HRMS (ESI) calcd for C21H19N3O2 [M +
H]+ 346.1556, found 346.1558.
(1R,2R,3R)-N-Hydroxy-2-phenyl-3-(6-(trifluoromethyl)-
imidazo[2,1-b]thiazol-2-yl)cyclopropanecarboxamide (46). The
synthesis involved following procedure A from 14c′ (158 mg, 0.42
mmol). Purification by preparative HPLC gave the racemic compound
(55 mg, 36%). Preparative chiral purification gave the title compound
(Chiralpak IC 30/70 [IPA/MeOH (50/50/0.1% formic acid)]/
heptane 1.0 mL/min, tR = 7.8 min). LCMS (ES+) 368 (M + H)+, tR
1
= 3.46 min (analytical method 1). H NMR δ (ppm) (DMSO-d6):
10.57 (1 H, s), 8.66 (1 H, s), 8.25 (1 H, s), 7.92 (1 H, s), 7.26−7.09 (5
H, m), 3.21 (1 H, dd, obscured by water peak), 2.85 (1 H, dd, J = 9.8,
6.7 Hz), 2.23 (1 H, dd, J = 9.8, 5.3 Hz). HRMS (ESI) calcd for
C16H12F3N3O2S [M + H]+ 368.0680, found 368.0687.
(1R,2R,3R)-N-hydroxy-2-(2-methylbenzo[d]thiazol-5-yl)-3-
phenylcyclopropanecarboxamide (47). Following procedure A
from 14d′ (680 mg, 2.02 mmol) gave a yellow glass (846 mg).
Purification by preparative HPLC gave the racemic product as an off
(1R,2R,3R)-2-(2-Cyclopropylquinoxalin-6-yl)-3-phenyl-
cyclopropanecarbohydroxamic Acid (52). The synthesis involved
following procedure A from 14n′ (129 mg, 0.37 mmol). Purification
by flash silica column chromatography (gradient elution DCM to 10%
MeOH in DCM) and preparative HPLC gave the racemic compound
(129 mg, 48%). Preparative chiral purification gave the title compound
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as an off white solid (Chiralpak IC 40/60 EtOH (50/50/0.1% formic
acid)/heptane 1.0 mL/min, tR = 9.38 min). LCMS (ES+) 346 (M +
H)+, tR = 3.52 min (analytical method 1). 1H NMR δ (ppm) (DMSO-
d6): 10.58 (1 H, s), 8.88 (1 H, s), 8.72 (1 H, s), 7.99 (1 H, d, J = 8.6
Hz), 7.77 (1 H, s), 7.69 (1 H, dd, J = 8.6, 2.1 Hz), 7.39 (2 H, d, J = 7.6
Hz), 7.28 (2 H, dd, J = 7.6, 7.3 Hz), 7.20 (1 H, d, J = 7.3 Hz), 3.02 (1
H, dd, J = 9.7, 6.9 Hz), 1.21−1.13 (4 H, m). Peaks were not seen in
the region 2.65−2.35 (3H, m, obscured by DMSO). HRMS (ESI)
calcd for C21H19N3O2 [M + H]+ 346.1556, found 346.1559.
(1R,2R,3R)-2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-N-hy-
droxy-3-phenylcyclopropanecarboxamide (53). The synthesis
involved following procedure A from compound 14k (652 mg, 2.01
mmol). Purification by flash silica column chromatography (gradient
elution DCM to 5% MeOH in DCM) gave the racemic mixture as a
white solid (420 mg, 67%). Preparative chiral HPLC gave the title
compound (Chiralpak IC 30/70 IPA/MeOH (50/50/0.1% formic
acid)/heptane, 1.0 mL/min, tR = 8.1 min). LCMS (ES+) 312 (M +
H)+, tR = 8.59 min (analytical method 4). 1H NMR δ (ppm) (DMSO-
d6): 10.57 (1 H, s), 8.73 (1 H, s), 7.35 (2 H, d, J = 7.59 Hz), 7.29 (2
H, t, J = 7.5 Hz), 7.24−7.17 (1 H, m), 6.85 (1 H, d, J = 8.5 Hz), 6.80−
6.74 (2 H, m), 4.26 (4 H, s), 3.02 (1 H, dd, J = 6.8, 5.4 Hz), 2.78 (1 H,
dd, J = 9.6, 6.8 Hz), 2.14 (1 H, dd, J = 9.6, 5.4 Hz). HRMS (ESI) calcd
for C18H17NO4 [M + H]+ 312.1236, found 312.1243.
(1R,2R,3R)-2-(8-Chloro-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
N-hydroxy-3-phenylcyclopropanecarboxamide (54). The syn-
thesis involved following procedure A from 14h (484 mg, 1.35 mmol).
Purification by flash silica column chromatography (gradient elution
DCM to 5% MeOH in DCM) and then preparative HPLC gave the
racemic product as a white solid (174 mg, 37%). Preparative chiral
HPLC gave the title compound (Chiralpak IC 20/80 IPA/MeOH
(50/50/0.1% formic acid)/heptane, 1.0 mL/min, tR = 10.4 min).
LCMS (ES+) 346, 348 (M + H)+, tR = 3.57 min (analytical method 1).
1H NMR δ (ppm) (DMSO-d6): 10.50 (1 H, s), 8.68 (1 H, s), 7.31 (2
H, d, J = 7.6 Hz), 7.25 (2 H, t, J = 7.4 Hz), 7.21−7.13 (1 H, m), 6.93
(1 H, d, J = 2.1 Hz), 6.77 (1 H, d, J = 2.1 Hz), 4.34−4.26 (4 H, m),
3.00 (1 H, dd, J = 6.8, 5.4 Hz), 2.79 (1 H, dd, J = 9.6, 6.8 Hz), 2.14 (1
H, dd, J = 9.8, 5.4 Hz). HRMS (ESI) calcd for C18H16ClNO4 [M +
H]+ 346.0846, found 346.0853.
(analytical method 1). 1H NMR δ (ppm) (DMSO-d6): 10.61 (1 H, s),
8.72 (1 H, s), 7.70 (1 H, d, J = 7.9 Hz), 7.56 (1 H, s), 7.45 (1 H, d, J =
8.0 Hz), 7.35 (2 H, d, J = 7.6 Hz), 7.27 (2 H, t, J = 7.5 Hz), 7.19 (1 H,
t, J = 7.2 Hz), 4.60 (2 H, s), 4.39 (2 H, q, J = 9.7 Hz), 3.24 (1 H, dd, J
= 6.8, 5.4 Hz), 2.93 (1 H, dd, J = 9.7, 6.8 Hz), 2.30 (1 H, dd, J = 9.7,
5.4 Hz). HRMS (ESI) calcd for C20H17F3N2O3 [M + H]+ 391.1269,
found 391.1267.
(1R,2R,3R)-N-Hydroxy-2-(4-(2-methyloxazol-5-yl)phenyl)-3-
phenylcyclopropanecarboxamide (58). The synthesis involved
following procedure A from 14i′ (460 mg). Purification by flash silica
column chromatography (gradient elution DCM to 5% MeOH in
DCM) and then preparative HPLC gave the racemic mixture as a
white solid (35 mg, 59%). Preparative chiral HPLC gave the title
compound (Chiralpak IC 40/60 EtOH (0.1% formic acid)/heptane,
1.0 mL/min, tR = 12.0 min). LCMS (ES+) 335 (M + H)+. tR = 3.28
1
min (analytical method 1). H NMR δ (ppm) (DMSO-d6): 10.50 (1
H, s), 8.63 (1 H, s), 7.55 (2 H, d, J = 8.1 Hz), 7.43 (1 H, s), 7.34−7.23
(4 H, m), 7.23−7.15 (2 H, m), 7.14−7.07 (1 H, m), 3.05 (1 H, dd, J =
6.8, 5.4 Hz), 2.81 (1 H, dd, J = 9.6, 6.8 Hz), 2.40 (3 H, s), 2.16 (1 H,
dd, J = 9.6, 5.4 Hz). HRMS (ESI) calcd for C20H18N2O3 [M + H]+
335.1396, found 335.1391.
(1R,2R,3R)-2-(4-(2-Cyclopropyloxazol-5-yl)phenyl)-N-hy-
droxy-3-phenylcyclopropanecarboxamide (59). The synthesis
involved following procedure A from compound 14k′ (300 mg, 0.80
mmol). Purification by flash silica column chromatography (gradient
elution DCM to 3% MeOH in DCM) gave the racemic mixture as a
white solid (300 mg, 78%). Preparative chiral HPLC gave the title
compound (Chiralpak IC 30/70 EtOH (0.1 formic acid)/heptane, 1.0
mL/min, tR = 19.8 min). LCMS (ES+) 361 (M + H)+, (ES−) 359 (M
− H)−, tR = 3.67 min (analytical method 1). 1H NMR δ (ppm)
(DMSO-d6): 10.62 (1 H, s), 8.76 (1 H, s), 7.65 (2 H, d, J = 8.2 Hz),
7.52 (1 H, s), 7.42−7.35 (4 H, m), 7.31 (2 H, t, J = 7.5 Hz), 7.23 (1 H,
t, J = 7.2 Hz), 3.17 (1 H, dd, J = 6.9, 5.4 Hz), 2.92 (1 H, dd, J = 9.7, 6.9
Hz), 2.28 (1 H, dd, J = 9.6, 5.4 Hz), 2.23−2.17 (1 H, m), 1.15−1.08 (2
H, m), 1.09−1.03 (2 H, m). HRMS (ESI) calcd for C22H20N2O3 [M +
H]+ 361.1552, found 361.1550.
(1R,2R,3R)-N-Hydroxy-2-(4-(oxazol-4-yl)phenyl)-3-phenyl-
cyclopropanecarboxamide (60). The synthesis involved following
procedure A from 14q′ (0.12 g, 0.37 mmol). Preparative chiral
purification gave the title compound (Chiralpak IC 30/70 [IPA/
MeOH (50/50/0.1% formic acid)]/heptane 5.0 mL/min, tR = 11.47
min). LCMS (ES+) 321 (M + H)+, tR = 9.76 min (analytical method
(1R,2R,3R)-2-(8-Chloro-3,3-dimethyl-2,3-dihydrobenzo[b]-
[1,4]dioxin-6-yl)-N-hydroxy-3-phenylcyclopropane-
carboxamide (55). The synthesis involved following procedure A
from 14j (0.2 g, 0.54 mmol). Preparative chiral purification gave the
title compound (Chiralpak IC 30/70 [IPA/MeOH (50/50/0.1%
formic acid)]/heptane 5.0 mL/min, tR = 15.6 min). LCMS (ES+) 374
1
1
(M + H)+, tR = 7.50 min (analytical method 1). H NMR δ (ppm)
1). H NMR δ (ppm) (DMSO-d6): 10.56 (1 H, s), 8.69 (1 H, d, J =
1.8 Hz), 8.62 (1 H, s), 8.46 (1 H, d, J = 0.9 Hz), 7.76 (2 H, d, J = 8.1
Hz), 7.35 (4 H, d, J = 8.1 Hz), 7.30−7.24 (2 H, m), 7.20−7.15 (1 H,
m), 3.12 (1 H, dd, J = 6.8, 5.4 Hz), 2.87 (1 H, dd, J = 9.5, 6.8 Hz), 2.24
(1 H, dd, J = 9.5, 5.4 Hz). HRMS (ESI) calcd for C22H20N2O3 [M +
H]+ 321.1239, found 321.1241.
(DMSO-d6): 10.48 (1 H, s), 8.67 (1 H, s), 7.30 (2 H, d, J = 7.4 Hz),
7.24 (2 H, t, J = 7.4 Hz), 7.17 (1 H, d, J = 7.4 Hz), 6.91 (1 H, d, J = 2.1
Hz), 6.73 (1 H, d, J = 2.1 Hz), 4.02 (2 H, s), 2.99 (1 H, dd, J = 6.8, 5.0
Hz), 2.79 (2 H, dd, J = 9.4, 6.8 Hz), 2.13 (1 H, dd, J = 9.4, 5.3 Hz),
1.29 (6 H, s). HRMS (ESI) calcd for C20H20ClNO4 [M + H]+
374.1159, found 374.1151.
(1R,2R,3R)-N-Hydroxy-2-(3-(oxazol-5-yl)phenyl)-3-phenyl-
cyclopropanecarboxamide (61). The synthesis involved following
procedure G from 7b (500 mg, 1.45 mmol). Purification by flash silica
column chromatography (gradient elution i-hex to 25% EtOAc in i-
hex) gave (1R*,2R*,3R*)-ethyl 2-(3-(oxazol-5-yl)phenyl)-3-phenyl-
cyclopropanecarboxylate (9n) as a colorless oil [494 mg, 100%, LCMS
(ES+) 334 (M + H)+], which was progressed to the next step. The
synthesis involved following procedure A from 9n (482 mg, 1.45
mmol). Purification by flash silica column chromatography (gradient
elution DCM to 5% MeOH in DCM) gave the racemic mixture as a
white solid (233 mg, 50%). Preparative chiral HPLC gave the title
compound (Chiralpak IC 40/60 EtOH (0.1 formic acid)/heptane, 1.0
mL/min, tR = 7.7 min). LCMS (ES+) 321 (M + H)+, tR = 2.82 min
(analytical method 1). 1H NMR δ (ppm) (DMSO-d6): 10.62 (1 H, s),
8.76 (1 H, s), 8.52 (1 H, s), 7.80 (1 H, s), 7.70 (1 H, s), 7.64 (1 H, d, J
= 7.8 Hz), 7.49 (1 H, t, J = 7.7 Hz), 7.42−7.28 (5 H, m), 7.24 (1 H, t, J
= 7.2 Hz), 3.22 (1 H, dd, J = 6.9, 5.4 Hz), 2.98 (1 H, dd, J = 9.6, 6.9
Hz), 2.32 (1 H, dd, J = 9.6, 5.4 Hz). HRMS (ESI) calcd for
C19H16N2O3 [M + H]+ 321.1239, found 321.1241.
(1R,2R,3R)-2-(8-Chloro-1,2,3,4-tetrahydroquinolin-6-yl)-N-
hydroxy-3-phenylcyclopropanecarboxamide (56). Following
procedure A from 14e′ (136 mg, 0.38 mmol) yielded a yellow glass
(200 mg). Purification by preparative HPLC gave the racemic product
as an off white glass (44 mg, 33%). Preparative chiral purification gave
the title compound (Chiralpak IC 20/80 IPA/MeOH (50/50/0.1%
1
formic acid)/heptane 5.0 mL/min). LCMS (ES+) 343 (M + H)+. H
NMR δ (ppm) (DMSO-d6): 10.46 (1 H, s), 8.64 (1 H, s), 7.32−7.19
(4 H, m), 7.19−7.12 (1 H, m), 6.94 (1 H, s), 6.76 (1 H, s), 5.42 (1 H,
s), 3.25 (2 H, s), 2.91 (1 H, dd, J = 6.8, 5.4 Hz), 2.72−2.64 (3 H, m),
2.05 (1 H, dd, J = 9.6, 5.4 Hz), 1.81−1.75 (2 H, m). HRMS (ESI)
calcd for C19H19ClN2O2 [M + H]+ 343.1213, found 343.1224.
(1R,2R,3R)-N-Hydroxy-2-(1-oxo-2-(2,2,2-trifluoroethyl)-
isoindolin-5-yl)-3-phenylcyclopropanecarboxamide (57). The
synthesis involved following procedure A from 14g′ (gradient elution
DCM to 6% MeOH in DCM), and then preparative HPLC gave the
racemic mixture as a white solid. Preparative chiral HPLC gave the title
compound (Chiralpak IC 30/70 EtOH (0.1 formic acid)/heptane, 1.0
mL/min, tR = 12.1 min). LCMS (ES+) 391 (M + H)+, tR = 3.47 min
9951
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Journal of Medicinal Chemistry
Article
(6) Fischle, W.; Dequiedt, F.; Hendzel, M. J.; Guenther, M. G.; Lazar,
M. A.; Voelter, W.; Verdin, E. Enzymatic activity associated with class
II HDACs is dependent on a multiprotein complex containing
HDAC3 and SMRT/N-CoR. Mol. Cell 2002, 9, 45−57.
(7) Yang, X. J.; Gregoire, S. Class II histone deacetylases: from
sequence to function, regulation, and clinical implication. Mol. Cell.
Biol. 2005, 25, 2873−2884.
(8) Verdin, E.; Dequiedt, F.; Kasler, H. G. Class II histone
deacetylases: versatile regulators. Trends Genet. 2003, 19, 286−293.
(9) Clocchiatti, A.; Florean, C.; Brancolini, C. Class IIa HDACs:
from important roles in differentiation to possible implications in
tumourigenesis. J. Cell. Mol. Med. 2011, 15, 1833−1846.
(10) Lahm, A.; Paolini, C.; Pallaoro, M.; Nardi, M. C.; Jones, P.;
Neddermann, P.; Sambucini, S.; Bottomley, M. J.; Lo Surdo, P.; Carfi,
A.; Koch, U.; De Francesco, R.; Steinkuhler, C.; Gallinari, P.
Unraveling the hidden catalytic activity of vertebrate class IIa histone
deacetylases. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 17335−17340.
(11) Jones, P.; Altamura, S.; De Francesco, R.; Gallinari, P.; Lahm, A.;
Neddermann, P.; Rowley, M.; Serafini, S.; Steinkuhler, C. Probing the
elusive catalytic activity of vertebrate class IIa histone deacetylases.
Bioorg. Med. Chem. Lett. 2008, 18, 1814−1819.
(12) Bradner, J. E.; West, N.; Grachan, M. L.; Greenberg, E. F.;
Haggarty, S. J.; Warnow, T.; Mazitschek, R. Chemical phylogenetics of
histone deacetylases. Nat. Chem. Biol. 2010, 6, 238−243.
(13) Fischle, W.; Kiermer, V.; Dequiedt, F.; Verdin, E. The emerging
role of class II histone deacetylases. Biochem. Cell Biol. 2001, 79, 337−
348.
(14) Mihaylova, M. M.; Vasquez, D. S.; Ravnskjaer, K.; Denechaud,
P. D.; Yu, R. T.; Alvarez, J. G.; Downes, M.; Evans, R. M.; Montminy,
M.; Shaw, R. J. Class IIa histone deacetylases are hormone-activated
regulators of FOXO and mammalian glucose homeostasis. Cell 2011,
145, 607−621.
ASSOCIATED CONTENT
* Supporting Information
Modeling, crystallographic method, data collection and refine-
ment statistics, synthesis of intermediates, biological activity
methods, procedures used for the in vitro ADME and in vivo
studies as well as metabolism and PK studies. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: +1 310-342-5503. E-mail: Celia.Dominguez@
CHDIFoundation.org.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
■
Notes
The authors declare no competing financial interest.
∥K.A.L.: Consultant to CHDI.
ACKNOWLEDGMENTS
■
The authors express their gratitude to Simon Yau, Mark Sandle,
and Paul Bond for analytical support, Saretius for conducting
the in-life phase of the PK studies, and the ADME team at
BioFocus for conducting the in vitro ADME studies and
bioanalysis.
ABBREVIATIONS USED
■
(15) Raichur, S.; Teh, S. H.; Ohwaki, K.; Gaur, V.; Long, Y. C.;
Hargreaves, M.; McGee, S. L.; Kusunoki, J. Histone deacetylase 5
regulates glucose uptake and insulin action in muscle cells. J. Mol.
Endocrinol. 2012, 49, 203−211.
ee, enantiomeric excess; iv, intravenous; po, per os (oral); rt,
room temperature; AUCN, area under the curve normalized for
dose level; SAR, structure−activity relationship; BOP, benzo-
triazole-1-yloxytris(dimethylamino)phosphonium hexafluoro-
phosphate; DCM, dichloromethane; DIPEA, diisopropylethyl-
amine; DMA, dimethylacetamide; DME, dimethoxyethane;
DMF, dimethylformamide; DMSO, dimethylsulfoxide; ES+,
electrospray positive ionization; ES−, electrospray negative
ionization; Et2O, diethyl ether; EtOAc, ethyl acetate; h, hour;
HPLC, high performance liquid chromatography; i-hex,
isohexane; LCMS, liquid chromatography mass spectrometry;
LiHMDS, lithium bis(trimethylsilyl)amide; M, mass; MeCN,
acetonitrile; MeOH, methanol; Pd2(dba)3 , tris-
(dibenzylideneacetone)dipalladium(0); Pd(dppf)Cl2, [1,1′-bis-
(diphenylphosphino)ferrocene]dichloropalladium(II); o-tol,
ortho tolyl; tR, retention time; SPE, solid phase extraction;
THF, tetrahydrofuran; xantphos, 4,5-bis(diphenylphosphino)-
9,9-dimethylxanthene
(16) Ago, T.; Liu, T.; Zhai, P.; Chen, W.; Li, H.; Molkentin, J. D.;
Vatner, S. F.; Sadoshima, J. A redox-dependent pathway for regulating
class II HDACs and cardiac hypertrophy. Cell 2008, 133, 978−993.
(17) Ling, S.; Sun, Q.; Li, Y.; Zhang, L.; Zhang, P.; Wang, X.; Tian,
C.; Li, Q.; Song, J.; Liu, H.; Kan, G.; Cao, H.; Huang, Z.; Nie, J.; Bai,
Y.; Chen, S.; Li, Y.; He, F.; Zhang, L.; Li, Y. CKIP-1 inhibits cardiac
hypertrophy by regulating class II histone deacetylase phosphorylation
through recruiting PP2A. Circulation 2012, 126, 3028−3040.
(18) McKinsey, T. A.; Kuwahara, K.; Bezprozvannaya, S.; Olson, E.
N. Class II histone deacetylases confer signal responsiveness to the
ankyrin-repeat proteins ANKRA2 and RFXANK. Mol. Biol. Cell 2006,
17, 438−447.
(19) McKinsey, T. A.; Olson, E. N. Dual roles of histone deacetylases
in the control of cardiac growth. Novartis Found. Symp. 2004, 259,
132−141 (discussion 141−145, 163−169).
(20) Kee, H. J.; Kook, H. Roles and targets of class I and IIa histone
deacetylases in cardiac hypertrophy. J. Biomed. Biotechnol. 2011, 2011,
928326.
(21) Kong, Y.; Tannous, P.; Lu, G.; Berenji, K.; Rothermel, B. A.;
Olson, E. N.; Hill, J. A. Suppression of class I and II histone
deacetylases blunts pressure-overload cardiac hypertrophy. Circulation
2006, 113, 2579−2588.
(22) Matsushima, S.; Kuroda, J.; Ago, T.; Zhai, P.; Park, J. Y.; Xie, L.
H.; Tian, B.; Sadoshima, J. Increased oxidative stress in the nucleus
caused by Nox4 mediates oxidation of HDAC4 and cardiac
hypertrophy. Circ. Res. 2013, 112, 651−663.
(23) Granger, A.; Abdullah, I.; Huebner, F.; Stout, A.; Wang, T.;
Huebner, T.; Epstein, J. A.; Gruber, P. J. Histone deacetylase inhibition
reduces myocardial ischemia−reperfusion injury in mice. FASEB J.
2008, 22, 3549−3560.
(24) Hancock, W. W.; Akimova, T.; Beier, U. H.; Liu, Y.; Wang, L.
HDAC inhibitor therapy in autoimmunity and transplantation. Ann.
Rheum. Dis. 2012, 71 (Suppl. 2), i46−i54.
REFERENCES
■
(1) Choudhary, C.; Kumar, C.; Gnad, F.; Nielsen, M. L.; Rehman,
M.; Walther, T. C.; Olsen, J. V.; Mann, M. Lysine acetylation targets
protein complexes and co-regulates major cellular functions. Science
2009, 325, 834−840.
(2) Aka, J. A.; Kim, G. W.; Yang, X. J. K-acetylation and its enzymes:
overview and new developments. Handb. Exp. Pharmacol. 2011, 206,
1−12.
(3) McKinsey, T. A. The biology and therapeutic implications of
HDACs in the heart. Handb. Exp. Pharmacol. 2011, 206, 57−78.
(4) Grabiec, A. M.; Tak, P. P.; Reedquist, K. A. Function of histone
deacetylase inhibitors in inflammation. Crit. Rev. Immunol. 2011, 31,
233−263.
(5) Haggarty, S. J.; Tsai, L. H. Probing the role of HDACs and
mechanisms of chromatin-mediated neuroplasticity. Neurobiol. Learn.
Mem. 2011, 96, 41−52.
9952
dx.doi.org/10.1021/jm4011884 | J. Med. Chem. 2013, 56, 9934−9954

Journal of Medicinal Chemistry
Article
(25) de Zoeten, E. F.; Wang, L.; Sai, H.; Dillmann, W. H.; Hancock,
W. W. Inhibition of HDAC9 increases T regulatory cell function and
prevents colitis in mice. Gastroenterology 2010, 138, 583−594.
(26) Mannaerts, I.; Eysackers, N.; Onyema, O. O.; Van Beneden, K.;
Valente, S.; Mai, A.; Odenthal, M.; van Grunsven, L. A. Class II HDAC
inhibition hampers hepatic stellate cell activation by induction of
MicroRNA-29. PLoS One 2013, 8, e55786.
McAllister, G.; Dominguez, C.; Munoz-Sanjuan, I.; Beaumont, V. Oral
administration of the pimelic diphenylamide HDAC inhibitor HDACi
4b is unsuitable for chronic inhibition of HDAC activity in the CNS in
vivo. PLoS One 2012, 7, e44498.
(42) Hoffmann, K.; Brosch, G.; Loidl, P.; Jung, M. A non-isotopic
assay for histone deacetylase activity. Nucleic Acids Res. 1999, 27,
2057−2058.
(27) Mielcarek, M.; Benn, C. L.; Franklin, S. A.; Smith, D. L.;
Woodman, B.; Marks, P. A.; Bates, G. P. SAHA decreases HDAC 2
and 4 levels in vivo and improves molecular phenotypes in the R6/2
mouse model of Huntington’s disease. PLoS One 2011, 6, e27746.
(28) Mielcarek, M.; Landles, C.; Weiss, A.; Bradaia, A.; Seredenina,
T.; Inuabasi, L.; Osborne, G. F.; Wadel, K.; Touller, C.; Butler, R.;
Robertson, J.; Franklin, S. A.; Smith, D. L.; Park, L.; Marks, P. A.;
Wanker, E. E.; Olson, E. N.; Luthi-Carter, R.; van der Putten, H.;
Beaumont, V.; Bates, G. P. HDAC4 reduction: a novel therapeutic
strategy to target cytoplasmic huntingtin and ameliorate neuro-
degeneration. PLoS Biol., submitted.
(29) Sung, Y. M.; Lee, T.; Yoon, H.; DiBattista, A. M.; Song, J. M.;
Sohn, Y.; Moffat, E. I.; Turner, R. S.; Jung, M.; Kim, J.; Hoe, H. S.
Mercaptoacetamide-based class II HDAC inhibitor lowers Abeta levels
and improves learning and memory in a mouse model of Alzheimer’s
disease. Exp. Neurol. 2013, 239, 192−201.
(30) Hobara, T.; Uchida, S.; Otsuki, K.; Matsubara, T.; Funato, H.;
Matsuo, K.; Suetsugi, M.; Watanabe, Y. Altered gene expression of
histone deacetylases in mood disorder patients. J. Psychiatr. Res. 2010,
44, 263−270.
(31) Iga, J.; Ueno, S.; Yamauchi, K.; Numata, S.; Kinouchi, S.;
Tayoshi-Shibuya, S.; Song, H.; Ohmori, T. Altered HDAC5 and CREB
mRNA expressions in the peripheral leukocytes of major depression.
Prog. Neuropsychopharmacol. Biol. Psychiatry 2007, 31, 628−632.
(32) Renthal, W.; Maze, I.; Krishnan, V.; Covington, H. E., 3rd; Xiao,
G.; Kumar, A.; Russo, S. J.; Graham, A.; Tsankova, N.; Kippin, T. E.;
Kerstetter, K. A.; Neve, R. L.; Haggarty, S. J.; McKinsey, T. A.; Bassel-
Duby, R.; Olson, E. N.; Nestler, E. J. Histone deacetylase 5
epigenetically controls behavioral adaptations to chronic emotional
stimuli. Neuron 2007, 56, 517−529.
(33) Tsankova, N.; Renthal, W.; Kumar, A.; Nestler, E. J. Epigenetic
regulation in psychiatric disorders. Nat. Rev. Neurosci. 2007, 8, 355−
367.
(34) Tsankova, N. M.; Berton, O.; Renthal, W.; Kumar, A.; Neve, R.
L.; Nestler, E. J. Sustained hippocampal chromatin regulation in a
mouse model of depression and antidepressant action. Nat. Neurosci.
2006, 9, 519−525.
(35) Cohen, T. J.; Barrientos, T.; Hartman, Z. C.; Garvey, S. M.; Cox,
G. A.; Yao, T. P. The deacetylase HDAC4 controls myocyte enhancing
factor-2-dependent structural gene expression in response to neural
activity. FASEB J. 2009, 23, 99−106.
(36) Simmons, B. J.; Cohen, T. J.; Bedlack, R.; Yao, T. P. HDACs in
skeletal muscle remodeling and neuromuscular disease. Handb. Exp.
Pharmacol. 2011, 206, 79−101.
(37) Chen, J. F.; Mandel, E. M.; Thomson, J. M.; Wu, Q.; Callis, T.
E.; Hammond, S. M.; Conlon, F. L.; Wang, D. Z. The role of
microRNA-1 and microRNA-133 in skeletal muscle proliferation and
differentiation. Nat. Genet. 2006, 38, 228−233.
(38) Williams, A. H.; Valdez, G.; Moresi, V.; Qi, X.; McAnally, J.;
Elliott, J. L.; Bassel-Duby, R.; Sanes, J. R.; Olson, E. N. MicroRNA-206
delays ALS progression and promotes regeneration of neuromuscular
synapses in mice. Science 2009, 326, 1549−1554.
(39) Choi, M. C.; Cohen, T. J.; Barrientos, T.; Wang, B.; Li, M.;
Simmons, B. J.; Yang, J. S.; Cox, G. A.; Zhao, Y.; Yao, T. P. A direct
HDAC4-MAP kinase crosstalk activates muscle atrophy program. Mol.
Cell 2012, 47, 122−132.
(43) Ciossek, T.; Julius, H.; Wieland, H.; Maier, T.; Beckers, T. A
homogeneous cellular histone deacetylase assay suitable for compound
profiling and robotic screening. Anal. Biochem. 2008, 372, 72−81.
(44) Riester, D.; Wegener, D.; Hildmann, C.; Schwienhorst, A.
Members of the histone deacetylase superfamily differ in substrate
specificity towards small synthetic substrates. Biochem. Biophys. Res.
Commun. 2004, 324, 1116−1123.
(45) Bottomley, M. J.; Lo Surdo, P.; Di Giovine, P.; Cirillo, A.;
Scarpelli, R.; Ferrigno, F.; Jones, P.; Neddermann, P.; De Francesco,
R.; Steinkuhler, C.; Gallinari, P.; Carfi, A. Structural and functional
analysis of the human HDAC4 catalytic domain reveals a regulatory
structural zinc-binding domain. J. Biol. Chem. 2008, 283, 26694−
26704.
(46) Jones, P.; Bottomley, M. J.; Carfi, A.; Cecchetti, O.; Ferrigno, F.;
Lo Surdo, P.; Ontoria, J. M.; Rowley, M.; Scarpelli, R.; Schultz-
Fademrecht, C.; Steinkuhler, C. 2-Trifluoroacetylthiophenes, a novel
series of potent and selective class II histone deacetylase inhibitors.
Bioorg. Med. Chem. Lett. 2008, 18 (141−145), 163−169.
(47) Muraglia, E.; Altamura, S.; Branca, D.; Cecchetti, O.; Ferrigno,
F.; Orsale, M. V.; Palumbi, M. C.; Rowley, M.; Scarpelli, R.;
Steinkuhler, C.; Jones, P. 2-Trifluoroacetylthiophene oxadiazoles as
potent and selective class II human histone deacetylase inhibitors.
Bioorg. Med. Chem. Lett. 2008, 18, 6083−6087.
(48) Tessier, P.; Smil, D. V.; Wahhab, A.; Leit, S.; Rahil, J.; Li, Z.;
Deziel, R.; Besterman, J. M. Diphenylmethylene hydroxamic acids as
selective class IIa histone deacetylase inhibitors. Bioorg. Med. Chem.
Lett. 2009, 19, 5684−5688.
(49) Ontoria, J. M.; Altamura, S.; Di Marco, A.; Ferrigno, F.; Laufer,
R.; Muraglia, E.; Palumbi, M. C.; Rowley, M.; Scarpelli, R.; Schultz-
Fademrecht, C.; Serafini, S.; Steinkuhler, C.; Jones, P. Identification of
novel, selective, and stable inhibitors of class II histone deacetylases.
Validation studies of the inhibition of the enzymatic activity of
HDAC4 by small molecules as a novel approach for cancer therapy. J.
Med. Chem. 2009, 52, 6782−6789.
(50) Mai, A.; Massa, S.; Pezzi, R.; Simeoni, S.; Rotili, D.; Nebbioso,
A.; Scognamiglio, A.; Altucci, L.; Loidl, P.; Brosch, G. Class II (IIa)-
selective histone deacetylase inhibitors. 1. Synthesis and biological
evaluation of novel (aryloxopropenyl)pyrrolyl hydroxyamides. J. Med.
Chem. 2005, 48, 3344−3353.
(51) Mai, A.; Massa, S.; Pezzi, R.; Rotili, D.; Loidl, P.; Brosch, G.
Discovery of (aryloxopropenyl)pyrrolyl hydroxyamides as selective
inhibitors of class IIa histone deacetylase homologue HD1-A. J. Med.
Chem. 2003, 46, 4826−4829.
(52) Lobera, M.; Madauss, K. P.; Pohlhaus, D. T.; Wright, Q. G.;
Trocha, M.; Schmidt, D. R.; Baloglu, E.; Trump, R. P.; Head, M. S.;
Hofmann, G. A.; Murray-Thompson, M.; Schwartz, B.; Chakravorty,
S.; Wu, Z.; Mander, P. K.; Kruidenier, L.; Reid, R. A.; Burkhart, W.;
Turunen, B. J.; Rong, J. X.; Wagner, C.; Moyer, M. B.; Wells, C.;
Hong, X.; Moore, J. T.; Williams, J. D.; Soler, D.; Ghosh, S.; Nolan, M.
A. Selective class IIa histone deacetylase inhibition via a nonchelating
zinc-binding group. Nat. Chem. Biol. 2013, 9, 319−325.
(53) Verma, R. P. Hydroxamic acids as matrix metalloproteinase
inhibitors. EXS 2012, 103, 137−176.
(54) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.
N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data
Bank. Nucleic Acids Res. 2000, 28, 235−242.
(40) Hebach, C.; Kallen, J.; Nozulak, J.; Tintelnot-Blomley, M.;
Widler, L. Novel Trifluoromethyl-oxadiazole Derivatives and Their
Use in the Treatment of Disease. WO 2013/080120, June 6, 2013.
(41) Beconi, M.; Aziz, O.; Matthews, K.; Moumne, L.; O’Connell, C.;
Yates, D.; Clifton, S.; Pett, H.; Vann, J.; Crowley, L.; Haughan, A. F.;
Smith, D. L.; Woodman, B.; Bates, G. P.; Brookfield, F.; Burli, R. W.;
(55) Vannini, A.; Volpari, C.; Gallinari, P.; Jones, P.; Mattu, M.; Carfi,
A.; De Francesco, R.; Steinkuhler, C.; Di Marco, S. Substrate binding
to histone deacetylases as shown by the crystal structure of the
HDAC8-substrate complex. EMBO Rep. 2007, 8, 879−884.
(56) Bressi, J. C.; Jennings, A. J.; Skene, R.; Wu, Y.; Melkus, R.; De
Jong, R.; O’Connell, S.; Grimshaw, C. E.; Navre, M.; Gangloff, A. R.
9953
dx.doi.org/10.1021/jm4011884 | J. Med. Chem. 2013, 56, 9934−9954

Journal of Medicinal Chemistry
Article
Exploration of the HDAC2 foot pocket: synthesis and SAR of
substituted N-(2-aminophenyl)benzamides. Bioorg. Med. Chem. Lett.
2010, 20, 3142−3145.
(57) Schuetz, A.; Min, J.; Allali-Hassani, A.; Schapira, M.; Shuen, M.;
Loppnau, P.; Mazitschek, R.; Kwiatkowski, N. P.; Lewis, T. A.;
Maglathin, R. L.; McLean, T. H.; Bochkarev, A.; Plotnikov, A. N.;
Vedadi, M.; Arrowsmith, C. H. Human HDAC7 harbors a class IIa
histone deacetylase-specific zinc binding motif and cryptic deacetylase
activity. J. Biol. Chem. 2008, 283, 11355−11363.
(58) Dowling, D. P.; Gantt, S. L.; Gattis, S. G.; Fierke, C. A.;
Christianson, D. W. Structural studies of human histone deacetylase 8
and its site-specific variants complexed with substrate and inhibitors.
Biochemistry 2008, 47, 13554−13563.
(59) Corey, E. J.; CHaykovsky, M. Dimethylsulfoxonium methylide.
J. Am. Chem. Soc. 1962, 84, 867−868.
(60) Corey, E. J.; Chaykovsky, M. Dimethyloxosulfonium methylide
((CH3)2SOCH2) and dimethylsulfonium methylide ((CH3)2SCH2).
Formation and application to organic synthesis. J. Am. Chem. Soc.
1965, 87, 1353−1364.
(61) Corey, E. J.; Jautelat, M. Construction of ring systems
containing the gemdimethylcyclopropane unit using diphenylsulfo-
nium isopropylide. J. Am. Chem. Soc. 1967, 89, 3912−3914.
(62) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a
useful metric for lead selection. Drug Discovery Today 2004, 9, 430−
431.
(63) Leeson, P. D.; Springthorpe, B. The influence of drug-like
concepts on decision-making in medicinal chemistry. Nat. Rev. Drug
Discovery 2007, 6, 881−890.
(64) Wu, H.; Du, L. M. Spectrophotometric determination of anilines
based on charge-transfer reaction. Spectrochim. Acta, Part A 2007, 67,
976−979.
(65) Mulder, G. J.; Meerman, J. H. Sulfation and glucuronidation as
competing pathways in the metabolism of hydroxamic acids: the role
of N,O-sulfonation in chemical carcinogenesis of aromatic amines.
Environ. Health Perspect. 1983, 49, 27−32.
(66) Obach, R. S. Potent inhibition of human liver aldehyde oxidase
by raloxifene. Drug Metab. Dispos. 2004, 32, 89−97.
(67) Tang, H.; Macpherson, P.; Marvin, M.; Meadows, E.; Klein, W.
H.; Yang, X. J.; Goldman, D. A histone deacetylase 4/myogenin
positive feedback loop coordinates denervation-dependent gene
induction and suppression. Mol. Biol. Cell 2009, 20, 1120−1131.
(68) Sivaguru, J.; Sunoj, R. B.; Wada, T.; Origane, Y.; Inoue, Y.;
Ramamurthy, V. Enhanced diastereoselectivity via confinement:
diastereoselective photoisomerization of 2,3-diphenyl-1-benzoylcyclo-
propane derivatives within zeolites. J. Org. Chem. 2004, 69, 5528−
5536.
(69) Martin, S. F.; Austin, R. E.; Oalmann, C. J.; Baker, W. R.;
Condon, S. L.; deLara, E.; Rosenberg, S. H.; Spina, K. P.; Stein, H. H.;
Cohen, J.; et al. 1,2,3-Trisubstituted cyclopropanes as conformationally
restricted peptide isosteres: application to the design and synthesis of
novel renin inhibitors. J. Med. Chem. 1992, 35, 1710−1721.
(70) Doyle, M. P.; Loh, K.-L.; DeVries, K. M.; Chinn, M. S.
Enhancement of stereoselectivity in catalytic cyclopropanation
reactions. Tetrahedron Lett. 1987, 28, 833−836.
NOTE ADDED AFTER ASAP PUBLICATION
■
This paper was published ASAP on December 5, 2013, with
incorrect structures for compounds 56 and 57 in Table 7. The
corrected version was reposted on December 12, 2013.
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dx.doi.org/10.1021/jm4011884 | J. Med. Chem. 2013, 56, 9934−9954