Achiral Derivatives of Hydroxamate AR-42 Potently Inhibit Class i HDAC Enzymes and Cancer Cell Proliferation

Article abstract of DOI:10.1021/acs.jmedchem.0c00230

AR-42 is an orally active inhibitor of histone deacetylases (HDACs) in clinical trials for multiple myeloma, leukemia, and lymphoma. It has few hydrogen bond donors and acceptors but is a chiral 2-arylbutyrate and potentially prone to racemization. We report achiral AR-42 analogues incorporating a cycloalkyl group linked via a quaternary carbon atom, with up to 40-fold increased potency against human class I HDACs (e.g., JT86, IC50 0.7 nM, HDAC1), 25-fold increased cytotoxicity against five human cancer cell lines, and up to 70-fold less toxicity in normal human cells. JT86 was ninefold more potent than racAR-42 in promoting accumulation of acetylated histone H4 in MM96L melanoma cells. Molecular modeling and structure-activity relationships support binding to HDAC1 with tetrahydropyran acting as a hydrophobic shield from water at the enzyme surface. Such potent inhibitors of class I HDACs may show benefits in diseases (cancers, parasitic infections, inflammatory conditions) where AR-42 is active.

Full text of DOI:10.1021/acs.jmedchem.0c00230

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Article
Achiral Derivatives of Hydroxamate AR-42 Potently Inhibit
Class I HDAC Enzymes and Cancer Cell Proliferation
Jiahui Tng, Junxian Lim, Kai-Chen Wu, Andrew J Lucke, Weijun Xu, Robert C Reid, and David P. Fairlie
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00230 • Publication Date (Web): 08 May 2020
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Journal of Medicinal Chemistry
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Jiahui Tng,† Junxian Lim,† § Kai-Chen Wu,† § Andrew J. Lucke,†, Weijun Xu,† Robert C. Reid,†,
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David P. Fairlie†,
† Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, ‡Australian Research Council Centre
of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, and Centre for Inflammation and
Disease Research, Institute for Molecular Bioscience; The University of Queensland, Brisbane, QLD 4072, Australia
“S” Supporting Information
§
ABSTRACT: AR-42 is an orally active inhibitor of histone
deacetylases (HDACs) in clinical trials for multiple myeloma,
leukemia and lymphoma. It has few hydrogen bond donors and
acceptors but is a chiral 2-arylbutyrate and potentially prone to
racemization. We report achiral AR-42 analogues incorporating
a cycloalkyl group linked via a quaternary carbon atom, with up
to 40-fold increased potency against human class I HDACs (e.g.
JT86, IC50 0.7 nM, HDAC1), 25-fold increased cytotoxicity against
five human cancer cell lines and up to 70-fold less toxicity in normal human cells. JT86 was 9-fold more potent than racAR-
42 in promoting accumulation of acetylated histone H4 in MM96L melanoma cells. Molecular modelling and structure-activity
relationships support binding to HDAC1 with tetrahydropyran acting as a hydrophobic shield from water at the enzyme
surface. Such potent inhibitors of class I HDACs may show benefit in diseases (cancers, parasitic infections, inflammatory
conditions) where AR-42 is active.
INTRODUCTION
affinity-determining zinc-binding group (ZBG), a linker or
spacer that additionally forms van der Waals interactions
with residues in the elongated 14 Å hydrophobic tubular
groove, and a capping group that interacts with surface
residues at the entrance to the substrate-binding active site
that might enable some selectivity for different isozymes.36,
37 Four HDACi have been approved by the US Food and Drug
Administration (FDA) for human use, three of which are
Histone deacetylases (HDACs) have important epigenetic
properties in eukaryotes where they control gene
regulation, in part through their enzymatic removal of the
acetyl group from the lysine ε-amino nitrogen of histone
and other proteins, causing chromatin condensation and
transcriptional repression.1, 2 Human HDACs 1-11 have a
conserved catalytic zinc ion (Zn2+), while another seven
HDAC enzymes are NAD+-dependent sirtuins (SIRT1-7).3, 4
Many studies have shown that HDAC1 deacetylates lysine
residues near the N-terminal region of core histone proteins
(H2A, H2B, H3, H4). Class I HDACs (HDAC 1-3, 8) also
deacetylate many non-histone proteins, activating binding
of transcription factors that leads to inhibition of tumor
suppressors, including the DNA-binding transcription
factor p53, transcription factor NF-κB, cytoskeletal protein
hydroxamate-based,
Vorinostat
(suberoylanilide
hydroxamic acid or SAHA),38 Belinostat (FXD101),39
Panobinostat (LBH589),40 and one a naturally occurring
cyclic tetrapeptide disulfide prodrug Romidepsin (FK228)
with a masked Zn2+-binding thiolate.41 These compounds
inhibit multiple classes of HDACs and are being used mostly
in oncology applications.42-54 In 2015, a benzamide HDACi,
Chidamide (HBI-8000), was approved in China for treating
peripheral T-cell lymphoma (PTCL).55-57 There are
numerous other HDACi in different phases of experimental,
preclinical or clinical trials, most being antiproliferative and
cytotoxic against solid or haematological tumours.58, 59
α-tubulin, molecular chaperone heat shock protein (HSP)
and other transcription factors.5,
The diverse
4, 6-11
epigenetic and cellular roles of class I HDACs include
regulating cell proliferation and differentiation and
inhibitors of these enzymes have been found to inhibit
AR-42 was discovered by structure-based design using a
histone deacetylase-like protein and starting with a simple
achiral 4-phenylbutyramide (IC50 44 nM, HDAC nuclear
extracts) and culminated in an optically active α-branched
derivative.60 AR-42 ((S)-N-hydroxy-4-(3-methyl-2-phenyl-
butanamido)-benzamide) 1 is a broad spectrum HDAC
inhibitor with its (S)-enantiomer being 5-fold more potent
multifactorial
cardiovascular
diseases,
and
including
metabolic
cancers,12-16
dysfunction,17-22
inflammatory disorders,23, 17, 24-31 rheumatoid arthritis32 and
parasitic infections33-35
.
Most HDAC inhibitors (HDACi) can be characterized by a
general pharmacophore model with three components: an
1
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Scheme 1. General approach to the synthesis of quaternary carbon linked inhibitors 1, 10-69 of class I HDACs.
1
2
3
4
5
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7
8
9
Reagents and conditions: (a) NaH, THF. (b) alkyl halide or dihaloalkane. (c) NaOH, MeOH/H2O. (d) oxalyl chloride or thionyl chloride,
DCM. (e) ethyl 4-aminobenzoate, Et3N, DCM. (f) HONH2 (50% Aq), NaOH, MeOH.
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60
Scheme 2. General method for the synthesis of heterocyclic inhibitors 35-41 of class I HDACs.
Reagents and conditions: (a) Cs2CO3, 1,4-dibromobutane, DMF; (b) Br2, EtOH; (c) benzamidines (X=NH) or thioamides (X=S), Et3N,
EtOH; (d) NaOH, MeOH/H2O; (e) oxalyl chloride or thionyl chloride, DCM. (f) methyl 4-aminobenzoate, Et3N, DCM; (g) NH2OH (50%
Aq), NaOH, MeOH.
(IC50 16 nM) than the (R)-enantiomer (IC50 84 nM).60, 61
AR-42 is orally active and antiproliferative against cancer
cells in vitro and in vivo in solid tumors (hepatocellular
carcinoma (HCC),62 osteosarcoma (OS),63 vestibular
schwannoma (VS) and meningioma64, 65) and other
malignancies (myeloma,66-68 lymphoma,69, 68 pancreatic,70
prostate,71 breast,72 ovarian,73 bladder74 and leukemia75, 76).
A mouse model of human prostate cancer showed the PC-3
xenograft tumor growth suppressed by 67% when AR-42
was administered orally (50 mg/kg, every other day), while
treatment with SAHA resulted in 31% growth
suppression.77 AR-42 is reportedly in Phase Ia/b clinical
trials for treating patients with advanced or relapsed
multiple myeloma (MM), lymphomas or chronic
lymphocytic leukemia (clinicaltrials.gov). The maximum
tolerated dose for humans was 40 mg (in total)
administered orally, 3 times per week for 3 weeks of a 28-
day cycle.67 AR-42 may be synergistic when given in
combination with other drugs, suggesting some promise for
cancer therapy.74, 78, 72
RESULTS AND DISCUSSION
Chemical Synthesis. A generic 5-step synthetic route to
give AR-42 and the achiral analogues (Tables 1-4) was used
as outlined (Scheme 1). Commercially available methyl
arylacetates 2 were deprotonated with NaH and reacted
with the appropriate alkyl halides or dihaloalkanes at
elevated temperatures giving the achiral compounds with a
quaternary carbon bearing geminal dialkyl substituents or
rings such as cycloalkyl, tetrahydropyran or piperidine 3.
Subsequent ester hydrolysis gave the respective acids
which were converted to their acid chlorides using oxalyl
chloride, and the reactive acid chloride intermediates were
coupled (Schotten-Baumann) with ethyl 4-aminobenzoate
to produce the amides 4. Conversion of the ester group of 4
to hydroxamic acids (1, 10-69) was either conducted under
dry conditions using NH2OH in MeOH (generated from pre-
dried hydroxylamine hydrochloride and freshly prepared
sodium methoxide in methanol) or, more conveniently,
using NH2OH (50% aqueous, 10 equiv.) and NaOH (5 equiv.)
at room temperature. A small amount of ester hydrolysis
usually accompanied the latter procedure, but products
were purified by preparative HPLC. Substituted aryl
compounds 42-53, 57-62 were prepared via an additional
Suzuki coupling step with the 4-bromophenyl precursor
and boronic acids. The resulting esters were converted to
the hydroxamic acids in a similar manner as described
above.
An attractive feature of AR-42 is its compact size with
minimal hydrogen bond donors and acceptors. There are no
AR-42 analogues reported with more potent inhibition of
HDACs or greater attenuation of tumor growth. Although
there is established methodology to prepare the chiral
precursor (3S)-methyl-2-phenylbutanoic acid by resolution
of diastereomeric salts60 or enantioselective synthesis79, α-
branched phenylacetic acid derivatives are prone to
racemization due to their relatively acidic α-CH.80 We
hypothesized that a chiral center may not be required for
potency and that other structural changes might produce
potent inhibitors. In this study, we report achiral analogues
of racemic AR-42 that are more potent inhibitors of human
class I HDAC enzymes, and more cytotoxic against human
cancer cells as determined by MTT and Caspase-Glo3/7
assays.
Analogues 35-41, incorporating an imidazole or a thiazole
ring, were prepared using the general method (Scheme 2).
Ethyl acetoacetate 5 was alkylated with 1,4-dibromobutane
to install the cyclopentane ring in ethyl 1-
acetylcyclopentane-1-carboxylate 6, which was brominated
in situ giving ethyl 1-(2-bromoacetyl)cyclopentane-1-
carboxylate 7. Condensation with either a benzamidine or
thioamide (via a Hantzsch type imidazole synthesis or
Hantzsch reaction, respectively) gave the heterocyclic
esters 8, which were hydrolyzed to the corresponding acids.
The acid chlorides were generated and coupled to methyl 4-
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aminobenzoate followed by conversion to the hydroxamic
acids 35-41 as described above. All final compounds were
purified by preparative rp-HPLC and lyophilized as white
powders. Detailed experimental descriptions are given in
the Supporting Information.
1
2
3
4
5
6
7
8
Structure-activity relationships for AR-42. There are no
crystal structures of 1 (AR-42) bound to a HDAC enzyme,
however its rigidity and Zn2+ binding hydroxamate
permitted AR-42 to be modeled into the HDAC1 active site
with a high degree of confidence (Figure. 1a). AR-42 was
predicted to bind to Zn2+ with its hydroxamate group
chelating in a bidentate manner similar to other ligands
bound to Class I HDACs.81, 82 The model suggested that the
adjacent aromatic ring of AR-42 occupied the narrow 14 Å
hydrophobic cavity, making pi-interactions with both
Phe150 and Phe205. The amide NH made a hydrogen bond
with the side chain carboxylate of Asp99 and the terminal
phenyl ring filled a hydrophobic pocket lined by residues
Phe150, Pro29, Leu271 and His28. The isopropyl group was
positioned at the exposed hydrophilic surface of the enzyme
and did not directly contact any HDAC residue, but instead
provided a “hydrophobic cap” above the H-bonding amide
NH…OC Asp99 atoms, thereby excluding entry of solvating
water molecules that would weaken this interaction.
9
10
11
12
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17
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21
22
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This proposed binding mode for AR-42 was supported
through assaying a set of synthetic analogues that
specifically probed contributions to HDAC1 inhibition from
each component of AR-42 (Table 1). Replacing the Zn2+-
binding hydroxamate with o-aminobenzamide 9 reduced
inhibitor potency ten-fold, consistent with lower affinity for
Zn2+. N-methylation of amide 10 reduced potency 18-fold,
supporting a hydrogen bond between the amide NH and
Asp99. This was further supported by an 85-fold reduction
in potency for the inverse amide 11 that disrupts the
geometry required for the H-bond. We next tested the
simple phenylacetamide 12, lacking the isopropyl group of
AR-42, which caused a modest 3-fold loss of potency. Since
the isopropyl group did not contact any protein residue it is
unlikely to confer potency through binding interactions.
Instead it may prevent bulk water from accessing the active
site and disrupting the hydrogen bond between the amide
NH and Asp99. Further, ligand docking of 12 suggested that
the phenyl group had moved from the hydrophobic pocket
to the region formerly occupied by the isopropyl group, a
new orientation stabilized by a T-shaped edge-to-face
aromatic interaction with Phe205 (Figure 1b). In this
arrangement the methylene and phenyl groups would be
able to compensate for loss of the hydrophobic shield. The
only inhibitor able to do this was unsubstituted
phenylacetamide 12, as alkylated derivatives in this specific
conformation have steric strain between the alkyl group
and amide carbonyl oxygen. This unique shield presents
new opportunities to rationally design molecules to
optimize this effect (see ahead for tetrahydropyran).
Figure 1. a) AR-42 (green) docked in the active site of hHDAC1
homology model (built on hHDAC2 crystal structures 4LY1.pdb
and 4LXZ.pdb) shows chelation by hydroxamate to Zn2+ and
hydrogen bonds between hydroxamate carbonyl and NH to
Tyr303 and His141, respectively. The aromatic linker adjacent
to hydroxamate makes parallel displaced aromatic interactions
with Phe150 and Phe205 (cyan), and a hydrogen bond (yellow)
between amide NH and Asp99. The (S)-isopropyl group shields
this hydrogen bond from water at the exposed enzyme surface.
b) Phenylacetamide analogue 12 compensates for loss of the
isopropyl group with the terminal phenyl group moving to
make an edge-to-face aromatic interaction (cyan) with Phe205,
shielding the hydrogen bond (yellow) between amide NH and
Asp99.
the aromatic ring of the benzamide component (15 and 16),
but an isosteric pyridine ring (17) in place of the aromatic
ring was equipotent with 1 (AR-42, Table 1).
Achiral analogues of AR-42. AR-42 is a chiral derivative of
phenylbutyrate and potentially prone to racemization.
Since the chiral (S)-isopropyl group seems only to act as a
hydrophobic shield and exclude water from the
environment around the amide NH hydrogen bond donor,
we decided to remove the chiral center by inserting
symmetrical groups linked through a quaternary carbon
(Table 2).
Substituting isopropyl with phenyl gave the symmetrical
diphenylacetamide 13 without a chiral center, but this led
to
a
10-fold loss in potency. Likewise, the 2-
propylpentanamide 14 derived from valproic acid was 30-
fold less active than AR-42. Together these results support
a combination of aromatic and aliphatic substituents being
needed for potent HDAC1 inhibition in this series. Finally, a
chlorine atom was not tolerated at the 2- or 3-postions of
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1
2
Table 1. Racemic AR-42 and analogues identify key structural components for inhibition of HDAC1.
3
Entry
Structure
IC50 ± SEM (nM)
Entry Structure
IC50 ± SEM (nM)
4
5
6
1
13
25  3.7
250  137
7
8
(AR-42)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9
14
231  112
738  145
10
11
12
15
16
17
439  147
2,116  749
75  13
8,680  4,800
1,065  753
22  16
Table 2. Inhibition of HDAC1 by achiral analogues of AR-42 bearing a benzylic quaternary carbon atom.
Entry
R
IC50 ± SEM (nM)
218  42
Entry
R
IC50 ± SEM (nM)
15  4.9
18
22
19
23
93  24
28  5.2
20
21
24
25
430  92
28  4.7
12  2
132  84
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The simple dimethyl analogue (18) was only a weak HDAC1
inhibitor, modeling suggesting that it binds in a similar way
1
2
3
4
5
6
7
8
to AR-42 rather than as described above for unsubstituted
phenylacetamide (12). The methyl groups were too small to
provide an effective hydrophobic shield, as was the
cyclopropane group (19). The larger but more flexible
diethyl derivative (20) was more potent but did not map
onto the same space as the isopropyl group of AR-42.
Instead of introducing longer and more flexible alkyl chains
with an associated entropy penalty for binding,83 we
examined ring substituents.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
32
33
34
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40
41
42
43
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50
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57
58
59
60
A cyclopentane ring (21) conferred the same potency as the
isopropyl group of AR-42, but importantly replaced both
the chiral center and the oxidizable benzylic position with a
stable quaternary carbon. Ligand docking into HDAC1,
suggested that a cyclopentane (21) or larger cyclohexane
(22) ring could efficiently shield the ligand-protein amide
NH‧‧‧OC Asp99 hydrogen bond, that would otherwise be
exposed to water at the enzyme boundary. These rings
introduce some conformational rigidity to the molecule.
The preferred conformation for 22 based on modeling
placed the phenyl ring at the equatorial position and the
amide axial in the cyclohexane chair conformation, which
was predicted by DFT calculations to be slightly lower in
energy than the alternative ring-flipped conformation by
0.4 kcal/mol. This was reflected by 21 being equipotent
with AR-42 as an inhibitor of HDAC1, and 22 showed 2-fold
greater inhibitor potency. Since the 4-position of the
cyclohexane ring was directed towards the solvated surface
of the enzyme, it appeared rational to introduce a polar O or
N atom that might also improve solubility and reduce log P.
The tetrahydropyran 23 and piperidine 24 were equipotent
with carbocyclic analogues and, in more advanced
analogues, conferred considerable potency gains (see
ahead). Unlike compound 17, a pyridine ring in place of the
benzene ring caused compound 25 to be 5-fold less potent
when compared to the parent compound 21.
Figure 2. a) Docked pose of 42 (green) in hHDAC1 homology
model (derived from HDAC2 crystal structures 4LY1.pdb and
4LXZ.pdb) shows similar interactions as AR-42. The additional
pyrazole ring makes a H-bond with the carbonyl of Gly27 and a
T-shaped interaction with His28. These interactions may be
optimal for HDAC1 inhibition (IC50 3.0  0.6 nM). b) Docked
pose of 68 (JT86, IC50 0.72  0.29 nM, green) in HDAC1 with
similar interactions as AR-42, plus an additional T-shaped
interaction between the naphthyl ring and His28. The chair
conformation adopted by the tetrahydropyran ring presents
the polar O atom to interact with water solvent at the surface
and the other five carbocyclic atoms provide a hydrophobic
shield over the hydrogen bond, excluding water to make it the
most potent HDAC1 inhibitor in the series.
Optimizing
capping
groups
for
surface
complementarity. Installing a cyclopentane ring in a panel
of analogues 26-53, we explored the effects of hydrophobic
capping groups (Table 3). A 4-fluoro substituent 26 (IC50 41
nM) decreased activity 2-fold, whereas
a 4-chloro
substituent 27 (IC50 12 nM) increased activity by 2-fold and
a para-bromo derivative 28 (IC50 7 nM) was 4-fold more
potent than AR-42, suggesting that bulkier halogens fill
space better at the surface. Therefore, analogues with
bulkier capping groups were investigated. Incorporating
both 3-fluoro and 4-trifluoromethyl substituents together
was detrimental 29 (IC50 227 nM). Inserting a 4-
dimethylamino group on the phenyl capping group
improved activity by ~8-fold 30 (IC50 4 nM) compared to
the unsubstituted 21.
improving potency in a way that the topology of the
biphenyls 31 and 32 did not allow (see Figure 2b for similar
binding in the tetrahydropyran series). We then postulated
that a 3-indole analogue with an additional H-bond
donating NH near Asp99 could result in stronger HDAC1
inhibition but 34 (IC50 23 nM) only gave comparable IC50
relative to AR-42. Replacing the phenyl ring next to the
quaternary carbon with a 2-phenylthiazole, 35 (IC50 12 nM)
and the 2-(3-methoxyphenyl)thiazole derivative 36 (IC50 15
nM) inhibited human HDAC1 with 2-fold greater potency
than AR-42. The more acute angles of the 5-membered
heterocyclic ring in compounds 35-41 directs the phenyl
capping group differently when compared to biphenyl 31,
with some presentations giving more optimal aromatic-
Extending the phenyl capping group by installing the larger
para biphenyl group 31 (IC50 16 nM) had similar activity to
the smaller 4-chloro derivative. Similarly, the 2-fluoro-
biphenyl derivative 32 (IC50 42 nM) was 3-fold less potent
than biphenyl 31, the electron-withdrawing fluorine being
undesirable. The 2-naphthyl derivative 33 (IC50 11 nM) was
a potent analogue. We anticipated through computer-
assisted modelling (Figure 2) that changing the phenyl
capping group to 2-naphthyl may give rise to an additional
pi-interaction between the naphthalene ring and His28 thus
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Table 3. HDAC1 inhibitors with a quaternary carbon linked cyclopentane ring and diverse capping groups.
1
2
3
4
5
6
7
8
9
Entry
R
IC50 ± SEM (nM)
41  7.2
Entry
R
IC50 ± SEM (nM)
10  1.6
26
40
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
27
28
41
42
12  7.3
7  2.6
6  0.98
3  0.64
29
30
43
44
227  142
4  1.1
4  0.75
5  1.2
31
32
33
45
46
47
16  5.3
42  16
11  5.0
8  1.9
43  25
0.94  0.56
34
35
36
37
48
49
50
51
23  12
4  2.1
12  4.5
15  7.6
431  134
5  2.2
5  2.0
240  134
38
39
>10,000
52
53
8  3.3
2  1.5
3  0.92
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Table 4. HDAC1 inhibitors with quaternary carbon linked six-membered rings and diverse capping groups.
2
3
4
5
6
7
8
9
Entry
R
IC50 ± SEM (nM)
29  19
Entry
R
IC50 ± SEM (nM)
2  0.96
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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64
42  31
31  19
2  0.54
63  23
57
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59
65
66
67
10  5.0
9  5.8
3  1.6
9  1.1
22  3.7
6  4.1
68
60
61
10  6.4
2  1.1
0.72  0.29
43  21
(JT86)
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aromatic interactions between the terminal phenyl group the catalytic surface. Similarly, the N-(5-amino-1,1'-
1
2
3
4
5
6
7
8
and His28. When a phenoxy group was attached at the 2-
position (37, IC50 431 nM) potency decreased 60-fold
compared to unsubstituted 35. The 3-phenoxy analogue
was inactive (38, IC50 >10 μM). Clashes between the
phenoxy moiety and side chains at the surface may have
reduced inhibitor activity. Next, we replaced the thiazole
ring with an imidazole, where its ring NH and amide NH
could both potentially form H-bond interactions with Asp99
biphenyl-2-yl)acetamide 60, (IC50 10 nM) was also a potent
inhibitor. The 4-pyrazolylphenyl derivatives with
tetrahydropyran ring 61 (IC50 2 nM) and cyclohexane ring
62 (IC50 2 nM) had equal activity. The 4-bromophenyl
tetrahydropyran 63 (IC50 2 nM) was 37-fold more potent
than the analogous cyclohexane 64 (IC50 63 nM) in
surprising contrast to the 4-pyrazolylphenyl pair 61 and 62.
When compared to the cyclopentane series, 63 was a 4x
more potent HDAC1 inhibitor than 28. Substituting the
para-position with a nitro group 65 (IC50 9 nM) decreased
potency by 5-fold. The 4-amino analogue 66 (IC50 22 nM)
in a bidentate fashion. The imidazole derivatives (39, IC50
3
9
nM; 40, IC50 10 nM; 41, IC50 6 nM) were 3- to 10-fold more
potent than AR-42. Compounds 42-53 were prepared via
Suzuki coupling with the ester intermediate of 4-
bromophenyl compound 28 and various heteroaryl boronic
acids with the intention of improving activity by making
new H-bond interactions with the amide carbonyl of Gly27
at the surface of the catalytic pocket. The 4-pyrazole 42 (IC50
3 nM) and 3-pyrazole 43 (IC50 4 nM) were equipotent,
possibly because each can present H-bond acceptors and
donors in a similar range of permutations through
tautomerization and rotation of the biaryl bond (Figure 2a).
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was 13-fold less potent. The dichloro analogue 67 (IC50
6
nM) was a potent inhibitor, possibly due to space filling
chlorine atoms. Occupying similar space with a naphthalene
ring,
the
inhibitory
potency
of
the
4-(2-
naphthyl)tetrahydropyran derivative 68 (JT86, IC50 0.72
nM) was the best in the series.
This result again supports the role of the fused naphthalene
ring in forming a T-shaped interaction with His28 (Figure
2a) and as described previously for the analogous
cyclopentane compound 33. This tetrahydropyran
compound 68 was 40-fold more active than its cyclohexane
analogue 69 (IC50 43 nM) and 15-fold more potent than the
cyclopentane compound 33. Both the 4-bromophenyl and
2-naphthyl substituted tetrahydropyran compounds
demonstrate that the electronegative oxygen atom of the
tetrahydropyran ring improves activity greatly compared
to the cycloalkane analogues. The oxygen atom also
increased water solubility almost 3-fold (e.g. 68 JT86, 0.045
mg/mL vs 69, 0.016 mg/mL) and reduced lipophilicity (e.g.
68 JT86, experimental log P 2.5; calculated log P 3.2 vs 69,
experimental log P 3.8; calculated log P 4.9 ChemDraw Pro
version 19).
However, the potency of the 5-methyl and 3,5-dimethyl-4-
pyrazole compounds 44 and 45 slightly decreased,
suggesting that adding substituents to the ring may not be
beneficial for surface complementarity. The 2-acetyl-3-
methylthiophene oxime containing compound 46 was ~15-
fold less potent than pyrazoles 42 and 43, suggesting that
the oxime OH was too far away to interact with Gly27. The
2-acetanilide compound 47 had sub-nanomolar potency
(IC50 0.94 nM) suggesting it makes an effective H-bond with
Gly27, in addition to other complementary interactions. In
contrast 48-50 have a HBD/HBA at the meta-position of the
ring and were 5-fold less active (IC50 ~5 nM). The benzyl
ether 51 (IC50 240 nM) was 48-fold less potent than
unsubstituted 50, consistent with the large O-benzyl group
not being optimal for the catalytic pocket. Incorporating a
HBD at the para-position was less favorable than a HBD at
the ortho- or meta-position, as shown for the indole-
containing compound 52 (IC50 8 nM). Interestingly, the
benzoic acid compound 53 (IC50 2 nM) was a potent
inhibitor and this could be rationalised by a H-bond
interaction between the carboxylate oxygen and the NH of
Gly27.
Class I isozyme selectivity. Selectivity of 1 (AR-42), 68
(JT86), 30, 39, 53, 62 and 63 was tested against all four
class I HDAC enzymes (recombinant human HDAC1, 2, 3 and
8) and HDAC7 (class IIb) (Table 5). Isozyme selectivity data
is reported for the first time for AR-42, which was a pan-
inhibitor of class I HDACs with very little selectivity. AR-42
was a 2-fold more potent inhibitor of HDAC3 (IC50 12 nM)
than HDAC1 (IC50 25 nM), but was 2-fold less potent against
HDAC2 (IC50 44 nM) and 4-fold less active against HDAC8
(IC50 87 nM). In comparison, the achiral compounds showed
some isozyme selectivity. All inhibited HDAC1 more
potently than the other class I HDAC enzymes, but HDAC3
inhibition was only 2-3-fold weaker. Inhibitor selectivity
was 5-12 fold greater than for HDAC2 and 14-61 fold
greater than for HDAC8 . 68 (JT86) was the most potent
inhibitor of HDAC1 (IC50 0.7 nM), showing approximately
35-fold increased potency over AR-42. It was also the most
selective HDAC1 inhibitor with 61 fold selectivity over
HDAC8 (IC50 44 nM) and 11 fold over HDAC2 (IC50 8 nM)
(Table 5). All compounds were much less active against the
class IIa enzyme HDAC7. Selectivity for HDAC1 was 140-
600 fold greater than for HDAC7 (Table 5), whereas AR-42
showed only 30 fold higher selectivity.
Effects of the electronegative oxygen. A series of six-
membered
ring
analogues,
cyclohexanes
and
tetrahydropyrans, 54-69, were potent inhibitors for HDAC1
(Table 4). The 3,4-dimethoxyphenyl compound 53 (IC50 29
nM) was equipotent with AR-42. Ring closing the methoxy
groups did not improve IC50 value of the 3,4-
methylenedioxyphenyl analogue 55 (IC50 42 nM). Replacing
the 3-methoxy group with bromine gave the 3-bromo-4-
methoxyphenyl derivative 56 (IC50 31 nM) with potency
improved by 1.5-fold. Increasing substituent size from
bromine to 3-aminophenyl improved potency 3-fold for 57
(IC50 10 nM). The 2-acetamido-2’-methoxybiphenyl
compound 58 had similar activity (IC50 9 nM). Replacing the
acetanilide fragment with a 4-pyrazole further improved
potency by 3-fold for 59 (IC50 3 nM). The results showed
that substitutions at the meta- and para-positions of the
second phenyl ring from the quaternary carbon center were
tolerable. The low nanomolar potency may result from
molecular interactions between the NHs and side chains at
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Table 5 Selective inhibition of rhHDAC enzymes. demonstrated improved cytoselectivity (2- to 70-fold)
1
2
3
4
5
6
7
8
compared with AR-42 in killing cancer cells versus normal
human NFF. 62 was 13-fold more potent in inducing
caspase 3/7 release than AR-42, with significant
improvement in cytoselectivity in MM96L (170-fold) and
MDA-MB-231 (60-fold) cell lines. Compound 63 was 11-fold
more cytotoxic than AR-42 in A549 and 54-fold more
cytoselective for MM96L cells. Compounds 23 and 33 were
comparable to AR-42 in cytotoxicity and cytoselectivity for
the cancer cell lines and NFF while compound 53 showed
no activity (< 100 µM). Despite differences in potencies
observed in MTT and Caspase3/7-Glo viability assays, the
rank order and potency of these HDAC1 inhibitors were
comparable to AR-42. From the cell viability data (Figures
3-4 and Supplementary Tables 1-2; Supporting
Information), JT86 consistently exhibited higher
cytotoxicity while maintaining significant cytoselectivity for
cancer cells versus NFF compared with AR-42.
IC50 ± SEM (nM)a
cpd
HDAC1
HDAC2 HDAC3 HDAC8
HDAC7
1
25 ± 3.7
44 ± 6
12 ± 2
87 ± 24 770 ± 180
0.72
±0.3
68
8 ± 0.9 2 ± 0.3 44 ± 11 210 ± 35
30
39
4 ± 1.1
3 ± 0.9
49 ± 8
21 ± 3
9 ± 2
89 ± 22 2500±770
41 ± 11 470 ± 110
10 ± 1
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118
53
2 ± 1.5
13 ± 2
7 ± 0.7
920 ± 160
±26
62
63
2 ± 0.96
2 ± 0.54
10 ± 2
17 ± 2
2 ± 0.2 69 ± 14 570 ± 120
4 ± 0.5 59 ± 17 270 ± 42
aInhibitor potency (IC50) as mean ± SEM (n>3 experiments).
Developing isozyme-specific inhibitors for class I HDACs
has been a challenge due to the highly conserved side
chains4 that line their active sites so, the modest selectivity
over HDAC2 here was surprizing. As expected, these
compounds were more selective over HDAC8 because of
structural differences in the HDAC8 active site that make it
more flexible compared to HDACs 1, 2 and 3, particularly in
loop 1 S30-K36 and loop2 P91-T105.84,85 This new series of
compounds has more favourable properties than chiral AR-
42 in being achiral and hence not subject to racemization,
more potent inhibitors of HDAC1, more cytotoxic against
human cancer cells, and more selective inhibitors of HDAC1,
providing a possible platform for next generation HDAC1
inhibitors with higher selectivity over other class I HDAC
isozymes.
Histone H4 acetylation. Acetylation of histone H4 has been
implicated in the modulation of melanoma,86 therefore we
extended activity profiles for the most potent HDAC1
inhibitors (JT86 and 63) in MM96L cells. Treatment of
AR-42, JT86 or 63 for 24 h induced the accumulation of
acetylated Lys5, Lys8, Lys12 and Lys16 of histone H4 as
determined by western blot (Figure 5). AR-42 (Figure 5a),
68 (JT86, Figure 5b) and 63 (Figure 5c) resulted in a
concentration-dependent accumulation of acetylated
histone H4 in MM96L melanoma cells indicating inhibition
of endogenous HDAC1. Consistent with the improved
potency observed in the HDAC1 enzyme and cell viability
assays, JT86 (EC50 0.2 µM) and 63 (EC50 0.6 µM) were 9- and
3-fold more potent respectively in hyperacetylation of
histone H4 than AR-42 (EC50 1.8 µM).
Cytotoxicity of 68 (JT86). On the basis of the HDAC1
enzyme data, potent inhibitors 1 (AR-42), 68 (JT86), 30, 39,
53, 62, and 63 were investigated for cytotoxicity in five
different human cancer cell lines (A549, HT-29, HeLa, MDA-
MB-231 and MM96L) using MTT and Caspase-Glo 3/7
viability assays. Their cytoselectivity was also compared
with normal human neonatal foreskin fibroblasts (NFF)
(Figure 3, Figure 4 and Supplementary Tables 1 and 2). JT86
was the most cytotoxic (IC50 0.02 – 0.1 µM) across the five
cancer cell lines with 15- to 23-fold increased potency over
AR-42. JT86 also exhibited increased cytoselectivity (4- to
20-fold) in killing cancer cells versus normal human NFF,
whereas AR-42 showed little or no cytoselectivity.
Compounds 62 and 63 were 2- to 7-fold more cytotoxic
than AR-42 in the five cancer cell lines. 62 demonstrated up
to 8-fold increased cytoselectivity, but 63 showed little or
no cytoselectivity over NFF. Compounds 30 and 39 were
comparable to AR-42 in cytotoxicity and cytoselectivity in
these cell lines, while 53 was completely inactive (> 100
µM).
CONCLUSION
The chiral isopropyl group and the tertiary benzylic
position of AR-42 have been replaced by achiral five or six-
membered rings linked via a quaternary carbon atom to
circumvent racemization. We propose that the potency was
driven in part by an important hydrogen bond between the
amide NH of the ligand and one of the carboxylate oxygens
of Asp99 and that an aliphatic group is a hydrophobic shield
from bulk water at the enzyme surface. Low nanomolar
concentrations of new achiral analogues of AR-42 were able
to inhibit human class 1 HDAC enzymes. The most potent
inhibitor 68 (JT86, IC50 0.7 nM, HDAC1) was also the most
cytotoxic against five human cancer cell lines, with up to
25 fold increased potency over AR-42 in MTT assays and
up to 70 fold less toxicity in normal human cells (NFF)
versus cancer cells in Caspase-Glo 3/7 assays. Further, JT86
demonstrated a 9-fold increase in potency over AR-42 in the
accumulation of acetylated histone H4 in malignant
melanoma cells (MM96L). JT86 and other potent inhibitors
described here for class 1 HDACs may be more promising
than AR-42 in inhibiting a range of cancers, parasitic
infections, and inflammatory conditions where AR-42 has
shown some benefit.
In a Caspase-Glo 3/7 assay, the release of intracellular
caspase 3/7 is proportional to cell death, indicative of
cytotoxicity of the compounds. HDAC1 inhibitors were
incubated with the cells for 72 h and activity of caspase3/7
(EC50) was calculated from the concentration-response
curves compared with AR-42 (Figure. 4 and Supplementary
Table 2; Supporting Information). Similar to MTT assay,
JT86 was the most potent (EC50 0.06 - 2.6 µM) at inducing
caspase3/7 release, a 6- to 15-fold improvement in potency
over AR-42 in the five cancer cell lines. JT86 also
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1
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Figure 3. MTT cytotoxicity concentration-response curves of
Figure 4. Concentration-dependent caspase 3/7 activity
HDAC1 inhibitors in different cell lines. Cell viability of
respective cell line was determined after 72 h incubation with
HDAC1 inhibitor at the indicated concentrations. NFF (normal
neonatal foreskin fibroblasts IC50: AR-42 0.5 µM; JT86 0.4 µM),
A549 (lung carcinoma, IC50: AR-42 1.5 µM; JT86 0.1 µM), HT-29
(colon carcinoma, IC50: AR-42 0.6 µM; JT86 0.04 µM), HeLa
(cervical carcinoma, IC50: AR-42 0.7 µM; JT86 0.03 µM), MDA-
MB-231 (breast carcinoma, IC50: AR-42 1.5 µM; JT86 0.1 µM)
and MM96L (melanoma carcinoma, IC50: AR-42 0.3 µM; JT86
0.02 µM). Each data point represents mean ± SEM (n > 3).
induced by HDAC1 inhibitors in different cell lines. Caspase 3/7
activity of respective cell line was determined after 72 h
incubation with HDAC1 inhibitor at the indicated
concentrations. NFF (normal neonatal foreskin fibroblasts,
EC50: AR-42 24.3 µM; JT86 4.3 µM), A549 (lung carcinoma, EC50:
AR-42 15.6 µM; JT86 2.6 µM), HT-29 (colon carcinoma, EC50:
AR-42 1.7 µM; JT86 0.2 µM), HeLa (cervical carcinoma, EC50:
AR-42 8.1 µM; JT86 0.7 µM), MDA-MB-231 (breast carcinoma,
EC50: AR-42 2.4 µM; JT86 0.3 µM) and MM96L (melanoma
carcinoma, EC50: AR-42 0.9 µM; JT86 0.06 µM). Each data point
represents mean ± SEM (n > 3).
EXPERIMENTAL SECTION
1H NMR spectra in DMSO-d6 were referenced to the residual
1H signal at 2.50 ppm, CDCl3 solutions were referenced to
internal TMS. 13C NMR spectra were referenced to the
solvent peak DMSO-d6 39.51 ppm. The exact concentration
of the compounds was determined by quantitative NMR
integration ‘PULCON’ experiments. Analytical UPLC-MS was
performed on a Shimadzu Nexera equipped with a 2020
ESI-MS and diode array detectors. Column was Zorbax
Eclipse plus C18, 1.8 m, 95 Å, 2.1 × 100 mm monitored at
three different wavelengths (λ 214, 230 and 254 nm).
Standard conditions were used for all compounds, linear
gradient 100% A – 100% B over 5 min followed by a further
1 min at 100% B, flow rate 0.5 mL/min. Solvent A was water
+ 0.1% formic acid and B was 90% MeCN 10% water + 0.1%
formic acid. Preparative rpHPLC was conducted on a
Phenomenex Luna column C18, 21.5 x 250 mm at flow rate
20 mL/min using same solvents as analytical except TFA 0.1
% was used in place of formic acid.
General chemistry. All reagents were purchased from
Sigma-Aldrich, Combi-Blocks Inc. or Chem-Impex
International Inc. Microwave-driven reactions were
performed using Biotage® Robot Eight 2.3 build 6250.
Preparative rpHPLC separations were performed on a
Phenomenex Luna C18 10 m, 250 × 21.2 mm column.
Standard conditions were used for elution of all compounds
100% A to 100% B linear gradient over 15 min followed by
a further 10 min at 100% B where solvent A was H2O + 0.1%
TFA and solvent B was 90% MeCN, 10% H2O + 0.1% TFA at
a flow rate of 20 mL/min. Detection was by UV and pure
fractions were lyophilized.
All final compounds (see Supporting Information) were
analyzed by 1H and 13C NMR, UPLC-MS and HRMS and purity
was determined to be >95%. 1H and 13C NMR spectra were
recorded on a Bruker Avance III HD 600 spectrometer at
298 K in the deuterated solvents indicated.
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High-resolution mass spectra (ESI-HRMS) measurements
1
2
3
4
5
6
7
8
were obtained on a Bruker microTOF mass spectrometer
equipped with a Dionex LC system (Chromeleon) in positive
ion mode by direct infusion in MeCN at 100 μL/h using
sodium formate clusters as an internal calibrant. Data was
processed using Bruker Daltonics DataAnalysis 3.4
software. Mass accuracy was consistently better than 1 ppm
error. All final compounds were checked for PAINS and
aggregators at http://zinc15.docking.org/patterns/home
and no alerts were found. Full experimental details and
spectroscopic data including intermediates are reported in
Supporting Information.
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General method A: Installation of a cycloalkyl ring at the α-
benzylic carbon of methyl 2-arylacetates.
Methyl
4-(naphthalen-2-yl)tetrahydro-2H-pyran-4-
carboxylate (70). To a solution of methyl 2-(naphthalen-2-
yl)acetate (3.10 g, 15.5 mmol) in dry THF (50 mL) at 0 °C,
was added NaH (60% dispersion in mineral oil, 1.78 g, 46.5
mmol, 3 equiv.) in three portions at 10-min intervals. The
mixture was stirred at 0 °C until no further effervescence
was observed then a solution of di-2-iodoethyl ether (4.20
g, 12.9 mmol, 0.8 equiv.) in dry THF (10 mL) was added
dropwise at 0 °C. The mixture was stirred at 0 °C for 10 min
and then, slowly warmed to RT. Stirring was continued at
50 °C for 18 h. The mixture was diluted with EtOAc and H2O
and the aqueous layer was extracted with EtOAc. The
combined extracts were washed with brine, dried over
MgSO4, and concentrated in vacuo. The residue was purified
by flash chromatography eluting with 10-30% EtOAc /
petroleum to give the ester 70 as a white solid (2.11 g, 61%,
Rf 0.39 petrol/EtOAc 2:1). 1H NMR (400 MHz, CDCl3): 훿 7.79-
7.84 (m, 4H), 7.45-7.54 (m, 3H), 4.12 (q, J = 7.2 Hz, 2H), 3.96-
4.01 (m, 2H), 3.59-3.66 (m, 2H), 2.62-2.66 (m, 2H), 2.07-
2.15 (m, 2H), 1.26 (t, J = 7.2 Hz, 3H).
General method B: Ester hydrolysis - synthesis of
quaternary carboxylic acids.
4-(Naphthalen-2-yl)tetrahydro-2H-pyran-4-carboxylic
acid (71). A suspension of the ester 70 (2.11 g, 7.8 mmol)
in a 1:1 mixture of MeOH/H2O (40 mL) was treated with
NaOH (0.94 g, 23.5 mmol, 3 equiv.). The mixture was stirred
and refluxed for 3 h. The mixture was then diluted with H2O
and the MeOH was mostly removed in vacuo. The H2O
solution was washed with ether, acidified to pH 1 with 2 M
HCl and extracted with EtOAc. The combined EtOAc extracts
were washed with brine, dried over MgSO4, and
concentrated in vacuo to give the acid 71 a white solid (1.52
g, 76%). ESI-MS: m/z 257 [M+H]+.
General method C: Preparation of amides of ethyl 4-
aminobenzoate using acid chlorides.
Ethyl 4-(4-(naphthalen-2-yl)tetrahydro-2H-pyran-4-
carboxamido)benzoate (72). To a suspension of the acid
71 (0.5 g, 2.0 mmol, 1 equiv.) in DCM (20 mL) at 0 °C, was
added oxalyl chloride (0.5 mL, 5.9 mmol, 3 equiv.). The
mixture was stirred at 0 °C until a clear solution was formed.
Excess oxalyl chloride was removed under a stream of N2.
The residue was dissolved in DCM (20 mL) and treated with
ethyl 4-aminobenzoate (0.32 g, 2.0 mmol, 1.1 equiv.) and
DIPEA (1 mL, 5.9 mmol, 3 equiv.). The mixture was stirred
at 0 °C for 10 min then slowly warmed to RT. Stirring was
continued at RT for 1 h. The mixture was diluted with DCM
and washed with H2O. The aqueous layer was further
Figure 5. AR-42, JT86 and 63 induced accumulation of
50
51
52
53
54
55
56
57
58
59
60
acetylated histone H4 in MM96L cells. MM96L cells were
treated with indicated concentrations of HDAC inhibitor (a)
AR-42, (b) JT86 and (c) 63 for 24 h and accumulation of
acetylated histone H4 (Ac-H4) was determined by western
blot. Blots are representative of 3 independent experiments
and GAPDH was used as the loading control. Densitometric
analysis of bands was quantified using ImageJ and expressed as
arbitrary unit (A.U) against log concentration of HDAC
inhibitor. Each data point represents mean ± SEM (n=3).
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extracted with DCM. The combined organic layers were
1H NMR (600 MHz, DMSO-d6): 훿 11.07 (br, s, 1H), 9.44 (s,
1
2
3
4
5
6
7
8
dried over MgSO4 and concentrated in vacuo. The yellow
oily residue was purified by flash column chromatography
with 50% EtOAc / petroleum to give the ester 72 as a white
solid (0.55 g, 70%, Rf 0.25 petrol / EtOAc 2:1). 1H NMR (600
MHz, CDCl3): 훿 7.97-7.98 (m, 2H), 7.46-7.51 (m, 4H), 6.94 (s,
1H), 4.35 (q, J = 6.0, 7.2 Hz, 2H), 3.80-3.87 (m, 4H), 2.43-2.45
(m, 2H), 2.11-2.15 (m, 2H), 1.38 (t, J = 7.2 Hz, 3H).
1H), 8.17 (s, 1H), 7.90-7.92 (m, 2H), 7.66-7.70 (m, 5H), 7.59-
7.64 (m, 3H), 7.51-7.57 (m, 3H), 2.61-2.68 (m, 2H), 1.93-
2.00 (m, 2H), 1.65-1.72 (m, 4H). 13C NMR (150 MHz, CDCl3):
훿 173.7, 167.2, 163.8, 143.5, 141.9, 140.0, 137.4, 131.5,
131.0, 129.3, 128.8, 128.2, 127.4, 127.24, 127.16, 126.7,
-
119.3, 60.1, 35.8, 23.3. HRMS m/z calculated for C26H23N2O5
443.1607, found 443.1609. UPLC tR = 3.2 min.
General method D: Conversion of methyl/ethyl esters to
hydroxamic acids.
4-(1-(4-(1H-pyrazol-4-yl)phenyl)cyclohexane-1-
carboxamido)-N-hydroxybenzamide (62). Prepared
from ethyl 4-(1-(4-(1H-pyrazol-4-yl)phenyl)cyclohexane-
1-carboxamido)benzoate using general method D. 1H NMR
(600 MHz, DMSO-d6): 훿 11.08 (br, s, 1H), 9.28 (s, 1H), 8.02
(s, 2H), 7.67 (s, 4H), 7.56-7.57 (m, 2H), 7.38-7.39 (m, 2H),
2.53-2.5 (m, 2H), 1.74-1.78 (m, 2H), 1.57-1.64 (m, 3H), 1.46-
1.52 (m, 2H), 1.27-1.32 (m, 2H). 13C NMR (150 MHz, DMSO-
d6): 훿 173.4, 163.9, 142.1, 141.9, 131.2, 127.33, 127.30,
126.2, 125.2, 120.8, 119.6, 51.1, 34.2, 25.1, 23.2. HRMS m/z
9
N-(4-(hydroxycarbamoyl)phenyl)-4-(naphthalen-2-
yl)tetrahydro-2H-pyran-4-carboxamide (68 JT86).
NaOH (0.2 g, 5 mmol) was dissolved in 50% aqueous NH2OH
(0.67 mL, 10 mmol, Aldrich) then a 0.5 mL aliquot of this
solution was added to a solution of ethyl 4-(4-(naphthalen-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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51
52
53
54
55
56
57
58
59
60
2-yl)tetrahydro-2H-pyran-4-carboxamido)benzoate
72
(0.91 g, 2.3 mmol) in MeOH (10 mL). The mixture was
stirred at RT for 45 min and the reaction was followed by
UPLC-MS. The mixture was diluted with H2O. Solvent
(MeOH) was removed in vacuo and the water phase was
acidified to pH 1 with 2 M HCl. The aqueous layer was then
extracted with EtOAc. The combined organic layers were
washed with 10% NaHCO3, brine, dried over MgSO4, and
evaporated to give the crude product as a white solid (0.85
g). Further purification by preparative reverse phase HPLC
and lyophilization gave the pure hydroxamic acid 68 as a
white powder (0.75 g, 85%). 1H NMR (600 MHz, DMSO-d6):
훿 11.10 (br, s,1H), 9.50 (s, 1H), 7.67-7.69 (m, 2H), 7.60-7.65
(m, 4H), 7.39 (dd, J = 2.4, 8.4 Hz, 1H), 3.78-3.80 (m, 2H),
-
calculated for C23H23N4O3 403.1770, found 403.1774. UPLC
tR = 3.0 min.
4-(4-Bromophenyl)-N-(4-(hydroxycarbamoyl)phenyl)-
tetrahydro-2H-pyran-4-carboxamide (63). Prepared
from ethyl 4-(4-(4-bromophenyl)tetrahydro-2H-pyran-4-
1
carboxamido)benzoate using general method D. H NMR
(600 MHz, DMSO-d6): 훿 11.10 (br, s, 1H), 9.45 (s, 1H), 7.67-
7.69 (m, 2H), 7.62-7.64 (m, 2H), 7.57-7.58 (m, 2H), 7.37-
7.39 (m, 2H), 3.78-3.80 (m, 2H), 3.50-3.54 (m, 2H), 2.54-
2.56 (m, 2H), 1.32-1.35 (m, 2H). 13C NMR (150 MHz, DMSO-
d6): 훿 172.0, 163.8, 142,8, 141.3, 131.5, 128.2, 127.7, 127.4,
120.2, 119.8, 64.5, 49.0, 34.0. HRMS m/z calculated for
C19H20N2O4+ 419.0601, found 419.0604. UPLC tR = 2.8 min.
3.50-3.54 (m, 2H), 2.55-2.57 (m, 2H), 1.95-2.00 (m, 2H). 13
C
NMR (150 MHz, DMSO-d6): 훿 171.6, 163.8, 144.3, 141.2,
131.3, 130.8, 129.8, 128.1, 127.8, 127.4, 126.5, 120.0, 64.4,
Ethyl 1- acetylcyclopentane-1-carboxylate (6). To a
stirred solution of ethyl acetoacetate 5 (4.9 mL, 38 mmol) in
MeCN (20 mL), was added Cs2CO3 (25 g, 77 mmol, 2 equiv.)
in three portions at 10-min intervals. The mixture was
stirred at 0 °C until no further effervescence was observed
then 1,4-dibromobutane (5.5 mL, 46 mmol, 1.2 equiv.) was
added dropwise. The mixture was stirred at 0 °C for 10 min
then slowly warmed to RT. Stirring was continued at 50 °C
for 19 h. The reaction mixture was quenched with H2O and
extracted with EtOAc. The combined organic layers were
washed with brine, dried over MgSO4 and concentrated in
vacuo. The residue was purified by flash column
chromatography (petrol/EtOAc 20:1) to give the product 6
as a clear colourless oil (4.89 g, 70%, Rf 0.52 petrol/EtOAc
10:1). 1H NMR (600 MHz, CDCl3): 훿 4.21 (q, J = 7.2 Hz, 2H),
2.17 (s, 3H), 2.11-2.13 (m, 4H), 1.61-1.72 (m, 4H), 1.27 (t, J
= 7.1 Hz, 3H).
+
48.9, 33.8. HRMS m/z calculated for C19H19Cl2N2O4
409.0716 found 409.0715. UPLC tR = 3.0 min.
4-(1-(4-(Dimethylamino)phenyl)cyclopentane-1-
carboxamido)-N-hydroxybenzamide (30). Prepared
from ethyl 4-(1-(4-(dimethylamino)phenyl)cyclopentane-
1-carboxamido)benzoate using general method D. 1H NMR
(600 MHz, DMSO-d6): 훿 11.1 (s, 1H), 9.25 (s, 1H), 7.58-7.66
(m, 4H), 7.27-7.28 (m, 2H), 6.85 (s. 2H), 2.90 (s, 6H), 2.55-
2.58 (m, 2H), 1.88-1.92 (m, 2H), 1.58-1.68 (m, 4H). 13C NMR
(150 MHz, DMSO-d6): 훿 174.2, 163.8, 159.8, 158.0, 142.0,
127.3, 127.1, 127.0, 125.7, 119.6, 119.2, 59.4, 40.0. 35.7,
23.1. HRMS m/z calculated for C18H21N2O3+ 368.1969, found
368.1970. UPLC tR = 2.3 min.
N-Hydroxy-4-(1-(2-phenyl-1H-imidazol-4-
yl)cyclopentane-1-carboxamido)benzamide
(39).
Prepared from ethyl 4-(1-(2-phenyl-1H-imidazol-4-
yl)cyclopentane-1-carboxamido)benzoate was prepared
using general method D. 1H NMR (600 MHz, DMSO-d6): 훿
11.10 (s, 1H), 9.57 (br, s, 1H), 8.94 (br, s, 1H), 8.01-8.02 (m,
2H), 7.61-7.82 (m, 8H), 2.43-2.49 (m, 2H), 2.24-2.25 (m,
2H), 1.77-.82 (m, 2H), 1.65-1.72 (m, 2H). 13C NMR (150 MHz,
DMSO-d6): 훿 164.2, 158.4, 145.2, 141.8, 130.1, 128.2, 127.9,
127.2, 120.4, 54.3, 36.3, 24.1, tautomerization broadened
General method E - Preparation of heterocycles.
1-(2-Phenyl-1H-imidazol-4-yl)cyclopentane-1-
carboxylic
acid
(73).
Ethyl
1-(2-
bromoacetyl)cyclopentane-1-carboxylate 7 was generated
in situ by treating a solution of ethyl 1-acetylcyclopentane-
1-carboxylate 6 (1 g, 5.4 mmol) in EtOH (5 mL) at 0 C with
Br2 (0.3 mL, 6.0 mmol, 1.1 equiv.), added dropwise and the
mixture was stirred at 0 C for 30 min. [1H NMR (600 MHz,
CDCl3): 훿 4.21 (q, J = 7.2 Hz, 2H), 4.09 (s, 2H), 2.13-2.23 (m,
4H), 1.71-1.76 (m, 2H). 1.64- 1.69 (m, 2H), 1.28 (t, J = 7.1 Hz,
3H)]. Benzamidine hydrochloride hydrate (1.02 g, 6.5
mmol, 1.2 equiv.) and Et3N (2.3 mL, 16.5 mmol, 3 equiv.)
were added and the resulting mixture was stirred and
refluxed for 5 h. Solvent was removed in vacuo and the
+
signals 3C not seen. HRMS m/z calculated for C22H23N4O3
391.1765, found 391.1766. UPLC tR = 2.2 min.
4'-(1-((4-(hydroxycarbamoyl)phenyl)carbamoyl)-
cyclopentyl)-[1,1'-biphenyl]-3-carboxylic acid (53).
Prepared
from
4'-(1-((4-
(ethoxycarbonyl)phenyl)carbamoyl)-cyclopentyl)-[1,1'-
biphenyl]-3-carboxylic acid using general method D.
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Journal of Medicinal Chemistry
residue was partitioned between H2O and EtOAc. The
into HDAC1 was performed using Gold.95 In GOLD, the
1
2
3
4
5
6
7
8
organic layer was separated while the H2O layer was further
extracted with EtOAc. The combined organic layers were
washed with brine, dried over MgSO4 and concentrated in
vacuo. The residue was purified by flash column
chromatography (petrol/EtOAc 1:1) to give the imidazole
product 8 as a yellow oil (0.36 g, 23%, Rf 0.58 petrol/EtOAc
1:1). This ester was hydrolyzed to its acid 73 as a white solid
docking site was defined by a search radius of 15 Å about
the HDAC1 ligand Vorinostat. Docked ligands had a scaffold
constraint of hydroxamic acid heavy atoms CONO applied
and also included protein H-bond constraints to Tyr303-
OH, His140-N, His141-N and Asp99 sidechain O atoms.
HDAC1 Asp99 sidechain was allowed to rotate freely during
docking. Ligands were submitted to 50 GA runs at 200%
search efficiency using PLP fitness score to rank poses.
1
(80 mg, 98%) using the general method B. H NMR (400
MHz, DMSO-d6): 8.24-8.27 (m, 2H), 7.71 (br, s, 1H), 7.61-
7.62 (m, 3H), 2.23-2.34 (m, 4H), 1.72-1.81 (m, 2H), 1.62-
1.68 (m, 2H).
DFT calculations. Two conformers of compound 22 were
constructed with phenyl and amide groups appended to the
cyclohexane ring, in the chair conformation, at either axial
or equatorial positions. Structures were energy minimized
and frequencies calculated using DFT, M06-2X 6-311g
(2d,2p) with empirical dispersion correction (GD3) in
vacuum with Gaussian 16. There were no negative
frequencies, indicating that true minima were obtained. The
most stable conformer had the lowest energy −1110.42602
versus −1110.42539 hartree respectively, corresponding to
a difference of 0.4 kcal/mol, and showed the phenyl group
in the equatorial position and amide group axial.
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60
General method F: Preparation of hetero-biaryls via Suzuki
coupling.
4-(1-(4-(1H-Pyrazol-4-yl)phenyl)cyclopentane-1-
carboxamido)-N-hydroxybenzamide (42). Ethyl 4-(1-(4-
bromophenyl)cyclopentane-1-carboxamido)benzoate (83
mg, 0.20 mmol), (1H-pyrazol-4-yl)boronic acid (45 mg, 0.40
mmol, 2 equiv.), triphenylphosphine (42 mg, 0.16 mmol, 0.8
equiv.), and K2CO3 (84 mg, 0.60 mmol, 3 equiv.) in 2:1
mixture of DME/H2O (6 mL) were degassed with argon.
Pd(OAc)2 (2 mg, 0.04 mmol, 0.2 equiv.) was added and the
mixture was reacted under microwave conditions (110°C)
for 2 h. The mixture was diluted with H2O and the aqueous
layer was extracted with EtOAc. The combined organic
layers were washed with brine, dried over MgSO4, and
concentrated in vacuo. The brown oil was purified by flash
column chromatography to give the ester as a white solid
(90 mg, 89%). 1H NMR (600 MHz, CDCl3): 훿 10.39 (br, s, 3H),
8.04 (br, s, 2H), 7.93 (d, J = 8.8 Hz, 2H), 7.43-7.55 (m, 6H),
7.01 (br, s, 1H), 4.33 (q, J = 7.1 Hz, 2H), 2.57-2.61 (m, 2H),
2.11-2.15 (m, 2H), 1.90-1.92 (m, 2H), 1.75-1.77 (m, 2H),
1.36 (t, J = 7.1 Hz, 3H). This ester was then converted to the
hydroxamic acid 42 using general method D giving a white
solid. 1H NMR (600 MHz, DMSO-d6): 훿 11.08 (br, s, 1H), 9.35
(s, 1H), 8.03 (s, 2H), 7.65-7.68 (m, 4H), 7.56-7.57 (m, 2H),
7.37-7.38 (m, 2H), 2.61-2.64 (m, 2H), 1.94-1.98 (m, 2H),
1.63-1.73 (m, 4H). 13C NMR (150 MHz, DMSO-d6): 훿 174.4,
164.3, 142.4, 141.7, 131.7, 127.8, 127.6, 127.3, 126.6 (from
2D-HSQC), 125.6, 121.31, 119.8, 60.5, 36.2, 23.7. HRMS m/z
Human recombinant HDAC enzymes assays. The
inhibition of human recombinant HDAC enzymes was
assessed in vitro using a black 384-well plate (Corning Inc.)
and the volume of each reagent used is stated in
Supplementary Information (Table S3). All reagents and
compounds were dissolved in DMSO as stock solutions and
further diluted with assay buffer (25 mM Tris-HCl, 137 mM
NaCl, 2.7 mM KCl, 1 mM MgCl2 and 0.1 mg/mL bovine serum
albumin; pH 8) to the final indicated concentration. Various
concentrations of inhibitor were pre-incubated with human
HDAC1 (BPS Bioscience), 50 ng/mL; human HDAC2 (BPS
Bioscience), 50 ng/mL; human HDAC3 (Reaction Biology
Corp), 100 ng/mL; or human HDAC8 (Reaction Biology
Corp), 2000 ng/mL enzyme for 30 min at room temperature
on a plate shaker. HDAC1/2/3/8 substrate Ac-Leu-Gly-
Lys(Ac)-AMC (12 µM) was then added and incubated for 90
min at 37°C in the dark. The enzymatic reaction was
stopped with the developer solution containing trypsin (1
mg/mL) and SAHA (25 μM) and co-incubated for 15 min at
room temperature. For HDAC7 enzyme assay, various
concentrations of inhibitor were pre-incubated with human
HDAC7 (Reaction Biology Corp), 5 ng/mL enzyme for 15
min at room temperature on a plate shaker. HDAC7
substrate Ac-Leu-Gly-Lys(trifluroAc)-AMC (20 µM) was
then added and incubated for 60 min at 37°C in the dark.
The enzymatic reaction was stopped with the developer
solution containing trypsin (1 mg/mL) and SAHA (10 μM)
and co-incubated for 15 min at room temperature.
Fluorescence intensity was measured using a PHERAstar
plate reader (BMG Labtech) at 350 nm excitation and 460
nm emission wavelengths.
-
calculated for C22H21N4O3 389.1619, found 389.1614.
Ligand docking. A homology model of HDAC1 was
generated by aligning the HDAC1 sequence (UniProtKB -
Q13547) to the sequences of HDAC2 in crystal structures
4LY1.pdb and 4LXZ.pdb (resolution 1.56 and 1.85 Å
respectively).87 A single Prime88 energy-based protein
homology model was constructed including the active site
Zn2+ atom and inhibitor Vorinostat. The HDAC1 model was
then submitted to the Protein Preparation Wizard89 where
atom and bond types were corrected and protonation states
of ionizable species adjusted to pH 7.4  1.0 by Epik.90 N-
and C-terminal residues were capped with neutral acetyl
and N-methyl amide groups respectively. Potassium ions
were added to the HDAC1 model in positions that replicated
the sodium and calcium ions in 4LY1.pdb. The HDAC1
homology model was minimized using the OPLS3e91 force
field to a converged heavy atom RMSD  0.3 Å. HDAC1
model quality was assessed with a Molprobity92 score of
1.12 and QMEAN93 score of -0.09, both indicators of a
quality model. Ligands were prepared using LigPrep94 with
protonation states set at pH 7.4 1.0 by Epik and structures
minimized using the OPLS3e force field. Docking of ligands
Cell culture. A549, HeLa and HT-29 cell lines were
purchased from ATCC. NFF, MM96L and MDA-MB-231 cells
were obtained from the Queensland Institute of Medical
Research. All cell culture media and reagents were
purchased from Invitrogen unless otherwise stated. A549
cells were cultured in F-12K supplemented with 10% fetal
bovine
serum
(FBS),
GlutaMAX
and
penicillin/streptomycin. NFF, HeLa, HT-29, MM96L and
HeLa cells were cultured in DMEM supplemented with 10%
FBS, GlutaMAX and penicillin/streptomycin. MDA-MB-231
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Page 14 of 19
Junxian Lim: 0000-0002-8309-0704
cells were cultured in L-15 supplemented with 10% FBS,
GlutaMAX and penicillin/streptomycin.
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7
8
Kai-Chen Wu: 0000-0003-0622-1165
Andrew J. Lucke: 0000-0002-3985-7429
Robert C. Reid: 0000-0002-0829-239X
David P. Fairlie: 0000-0002-7856-8566
MTT viability assays. Cells (5000/well) were added to a
96-well plate and allow to adhere overnight. Media were
then removed and cells were treated with 10% FBS media
supplemented with varying concentrations of the HDAC1
inhibitor for 72 h. MTT (1 mg/mL, 50 µL/well) was
incubated with the cells for 90 min followed by addition of
isopropanol to solubilize the formazan product. Absorbance
was measured at 570 nm using a PHERAstar plate reader.
Cytoselectivity was defined as IC50 determined by MTT of
normal human NFF divided by IC50 of cancer cell line.
JT and RCR synthesised the compounds; KCW and JL performed
all enzyme and cellular assays; WX and AJL performed docking
and molecular simulations; JT, RR and DF designed all
compounds, syntheses and pharmacological profiles and wrote
the manuscript. All authors approved the final submission.
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Caspase-Glo 3/7 viability assay. Cells (5000/well) were
added to a 96-well plate and allow to adhere overnight.
Media were then removed and cells were treated with 10%
FBS media supplemented with varying concentrations of a
HDAC1 inhibitor for 72 h. Caspase 3/7 activity was
measured using Caspase-Glo 3/7 (Promega) according to
manufacturer’s protocol. An equal volume of cell culture
media was mixed with Caspase-Glo 3/7 Reagent in a white
384-well plate and allow to incubate for 60 min at room
temperature. Luminescence values were measured using
PHERAstar plate reader. Cytoselectivity is defined as the
EC50 as determined by Caspase-Glo 3/7 assay of normal
human NFF divided by the EC50 of cancer cell line.
Authors declare no competing interests.
We thank the National Health and Medical Research Council of
Australia (NHMRC) for a Senior Principal Research Fellowship
to D.F. (1117017) and a grant (1093378) and the ARC
(CE140100011) for general lab support to D.F.
We thank Dr. Ping Chen (visiting researcher) and Mr Darren Do
for contributing to early syntheses of some compounds. We
thank Alun Jones (Institute for Molecular Bioscience, UQ.
Australia) for assistance with mass spectrometry.
Western blot analysis of histone H4 acetylation. MM96L
cells (12,500/well) were seeded into 96-well plate and
allow to adhere overnight. Cells were treated with 10% FBS
DMEM supplemented with varying concentrations of
AR-42, JT86 or 63 for 24 h. After treatment, cells were lysed
on ice with Cell Lysis Buffer supplemented with
Protease/Phosphatase Inhibitor Cocktail (Cell Signaling
Technology). Cell lysates were immediately separated on 4-
12% Bis-Tris gels and transferred using iBlot 2 Dry Blotting
System. Membranes were then blocked with 5% skim milk
for 1 h at room temperature and incubated with primary
antibodies against acetylated-histone H4 (Lys5, Lys8, Lys12
and Lys16, 1:20,000, ab177790, Abcam) or GAPDH
(1:30,000, G9545, Sigma-Aldrich) for 1 h at room
temperature. Image acquisition was performed using
ChemiDoc MP (Bio-Rad Laboratories) and densitometric
analysis of bands was quantified using ImageJ.
Akt, a serine/threonine-specific protein kinase also known as
protein kinase B (PKB); Asp, L-aspartate; ATL, adult T-cell
lymphoma; A549, lung carcinoma; CDCl3, deuterated
chloroform; CLL, chronic lymphocytic leukemia; CTCL,
cutaneous T-cell lymphoma; DMSO-d6, deuterated dimethyl
sulfoxide; EC50, half maximal effective concentration; ESI,
electrospray ionisation; FBS, foetal bovine serum, Gly, glycine;
HDACs, histone deacetylases; HDACi, histone deacetylase
inhibitor; HeLa, cervical carcinoma, His, histidine; HPLC, high-
performance liquid chromatography; HRMS, high resolution
mass spectrometry; HSP90, heat shock protein 90; HT-29,
colon carcinoma, IC50, half maximal inhibitory concentration;
LCMS, low resolution mass spectrometry; MDA-MB-231, breast
carcinoma; MS, mass spectrometry; MM, multiple myeloma;
MM96L, malignant melanoma human carcinoma; NFF,
neonatal foreskin human fibroblast; NF-κB, nuclear factor
kappa B; NMR, nuclear magnetic resonance; Phe, L-
phenylalanine; PTCL, peripheral T-cell lymphoma; rp-HPLC,
reversed-phase high-performance liquid chromatography; RT,
room temperature; SAHA, suberoylanilide hydroxamic acid;
SAR, structure-activity relationship; TFA, trifluoroacetic acid;
TMS, tetramethylsilane; Tyr, tyrosine; UPLC, ultra-
performance liquid chromatography; ZBG, zinc binding group.
Data analysis. Triplicate measurements were made for
each data point and error bars represent mean ± SEM of
three independent experiments. All data were plotted and
analyzed using four parameters variable slope model in
Prism 8 for Mac OS X (GraphPad Software).
Supporting Information. Additional experimental proce-
dures, figures and tables illustrating synthesis, compound char-
acterization, and cell viability, SMILES strings of all compounds
(CSV) and atomic coordinates of the homology model used for
ligand docking are available free of charge on the ACS Publica-
tions website.
1.
Gupta, P.; Reid, R. C.; Iyer, A.; Sweet, M. J.; Fairlie, D. P.
Towards isozyme-selective HDAC inhibitors for interrogating
disease. Curr. Top. Med. Chem. 2012, 12, 1479-1499.
2.
Iyer, A.; Fairlie, D. P.; Brown, L. Lysine acetylation in
obesity, diabetes and metabolic disease. Immunol. Cell Biol. 2012,
90, 39-46.
3.
Trojer, P.; Brandtner, E. M.; Brosch, G.; Loidl, P.; Galehr, J.;
Linzmaier, R.; Haas, H.; Mair, K.; Tribus, M.; Graessle, S. Histone
deacetylases in fungi: novel members, new facts. Nucleic Acids Res.
2003, 31, 3971-3981.
* Professor David P. Fairlie (E-mail: d.fairlie@imb.uq.edu.au) or
* Dr Robert C. Reid (E-mail: r.reid@imb.uq.edu.au).
ORCID:
Jiahui Tng: 0000-0002-3507-8831
14
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4.
Seto, E.; Yoshida, M. Erasers of histone acetylation: the
26.
Shakespear, M. R.; Halili, M. A.; Irvine, K. M.; Fairlie, D. P.;
histone deacetylase enzymes. Cold Spring Harb. Perspect. Biol.
Sweet, M. J. Histone deacetylases as regulators of inflammation and
1
2
3
4
5
6
7
8
2014, 6, a018713.
immunity. Trends Immunol. 2011, 32, 335-343.
5.
and deacetylation of non-histone proteins. Gene 2005, 363, 15-23.
6. Verdone, L.; Agricola, E.; Caserta, M.; Di Mauro, E. Histone
Glozak, M. A.; Sengupta, N.; Zhang, X.; Seto, E. Acetylation
27.
Choi, S. W.; Gatza, E.; Hou, G.; Sun, Y.; Whitfield, J.; Song,
Y.; Oravecz-Wilson, K.; Tawara, I.; Dinarello, C. A.; Reddy, P. Histone
deacetylase inhibition regulates inflammation and enhances Tregs
after allogeneic hematopoietic cell transplantation in humans.
Blood 2015, 125, 815-819.
acetylation in gene regulation. Brief Funct Genomic Proteomic
2006, 5, 209-221.
7.
Spange, S.; Wagner, T.; Heinzel, T.; Kramer, O. H.
28.
Halili, M. A.; Andrews, M. R.; Labzin, L. I.; Schroder, K.;
Acetylation of non-histone proteins modulates cellular signalling
at multiple levels. Int. J. Biochem. Cell Biol. 2009, 41, 185-198.
Matthias, G.; Cao, C.; Lovelace, E.; Reid, R. C.; Le, G. T.; Hume, D. A.;
Irvine, K. M.; Matthias, P.; Fairlie, D. P.; Sweet, M. J. Differential
effects of selective HDAC inhibitors on macrophage inflammatory
responses to the Toll-like receptor 4 agonist LPS. J. Leukoc. Biol.
2010, 87, 1103-1114.
9
8.
Singh, B. N.; Zhang, G. H.; Hwa, Y. L.; Li, J. P.; Dowdy, S. C.;
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Jiang, S. W. Nonhistone protein acetylation as cancer therapy
targets. Expert Rev. Anticancer Ther. 2010, 10, 935-954.
9.
Luo, Y.; Jian, W.; Stavreva, D.; Fu, X. Q.; Hager, G.; Bungert,
29.
Chen, X.; Barozzi, I.; Termanini, A.; Prosperini, E.;
J.; Huang, S. M.; Qiu, Y. Trans-regulation of histone deacetylase
activities through acetylation. J. Biol. Chem. 2009, 284, 34901-
34910.
Recchiuti, A.; Dalli, J.; Mietton, F.; Matteoli, G.; Hiebert, S.; Natoli, G.
Requirement for the histone deacetylase HDAC3 for the
inflammatory gene expression program in macrophages. Proc.
Natl. Acad. Sci. U. S. A. 2012, 109, E2865-E2874.
10.
Yao, Y.-L.; Yang, W.-M. Beyond histone and deacetylase:
an overview of cytoplasmic histone deacetylases and their
nonhistone substrates. Biomed. Res. Int. 2010, 2011.
11.
30.
Barnes, P. J.; Adcock, I. M.; Ito, K. Histone acetylation and
deacetylation: importance in inflammatory lung diseases. Eur.
Respir. J. 2005, 25, 552-563.
31.
Groot, D.; Sanders, M. E.; Prakken, B. J.; Maslinski, W.; Eldering, E.;
Tak, P. P.; Reedquist, K. A. Histone deacetylase inhibitors suppress
inflammatory activation of rheumatoid arthritis patient synovial
macrophages and tissue. J. Immunol. 2010, 184, 2718-2728.
Peng, L.; Seto, E. Deacetylation of nonhistone proteins by
HDACs and the implications in cancer. In Histone Deacetylases: The
Biology and Clinical Implication, Springer: Berlin, 2011; pp 39-56.
12.
development and therapy. Cold Spring Harb. Perspect. Med. 2016,
6, a026831.
13.
(HDACs) in human cancer. Mol. Oncol. 2007, 1, 19-25.
14. Glozak, M. A.; Seto, E. Histone deacetylases and cancer.
Oncogene 2007, 26, 5420-5432.
Grabiec, A. M.; Krausz, S.; de Jager, W.; Burakowski, T.;
Li, Y.; Seto, E. HDACs and HDAC inhibitors in cancer
Ropero, S.; Esteller, M. The role of histone deacetylases
32.
Göschl, L.; Preglej, T.; Boucheron, N.; Saferding, V.;
Müller, L.; Platzer, A.; Hirahara, K.; Shih, H.-Y.; Backlund, J.;
Matthias, P.; Niederreiter, B.; Hladik, A.; Kugler, M.; Gualdoni, G. A.;
Scheinecker, C.; Knapp, S.; Seiser, C.; Holmdahl, R.; Tillmann, K.;
Plasenzotti, R.; Podesser, B.; Aletaha, D.; Smolen, J. S.; Karonitsch,
T.; Steiner, G.; Ellmeier, W.; Bonelli, M. Histone deacetylase 1
(HDAC1): A key player of T cell-mediated arthritis. J. Autoimmun.
2019, 102379.
33.
parasitic diseases. Immunol. Cell Biol. 2012, 90, 66-77.
34. Andrews, K. T.; Gupta, A. P.; Tran, T. N.; Fairlie, D. P.;
15.
West, A. C.; Johnstone, R. W. New and emerging HDAC
inhibitors for cancer treatment. J. Clin. Invest. 2014, 124, 30-39.
16.
deacetylase inhibitors in cancer cells: mechanisms and potential
clinical implications. Clin. Cancer Res. 2009, 15, 3947-3957.
17.
with histone deacetylase inhibitors. Mol. Med. 2011, 17, 434-441.
18. McKinsey, T. A. Therapeutic potential for HDAC
inhibitors in the heart. Annu. Rev. Pharmacol. Toxicol. 2012, 52,
303-319.
Schrump, D. S. Cytotoxicity mediated by histone
Andrews, K. T.; Haque, A.; Jones, M. K. HDAC inhibitors in
McKinsey, T. A. Targeting inflammation in heart failure
Gobert, G. N.; Bozdech, Z. Comparative gene expression profiling of
P. falciparum malaria parasites exposed to three different histone
deacetylase inhibitors. PLoS One 2012, 7, e31847.
19.
Cao, D. J.; Wang, Z. V.; Battiprolu, P. K.; Jiang, N.; Morales,
35.
Chua, M. J.; Arnold, M. S.; Xu, W.; Lancelot, J.; Lamotte, S.;
C. R.; Kong, Y.; Rothermel, B. A.; Gillette, T. G.; Hill, J. A. Histone
deacetylase (HDAC) inhibitors attenuate cardiac hypertrophy by
suppressing autophagy. Proc. Natl. Acad. Sci. U. S. A. 2011, 108,
4123-4128.
Spath, G. F.; Prina, E.; Pierce, R. J.; Fairlie, D. P.; Skinner-Adams, T.
S.; Andrews, K. T. Effect of clinically approved HDAC inhibitors on
Plasmodium, Leishmania and Schistosoma parasite growth. Int. J.
Parasitol. Drugs Drug Resist. 2017, 7, 42-50.
20.
Montgomery, R. L.; Davis, C. A.; Potthoff, M. J.; Haberland,
36.
Finnin, M. S.; Donigian, J. R.; Cohen, A.; Richon, V. M.;
M.; Fielitz, J.; Qi, X.; Hill, J. A.; Richardson, J. A.; Olson, E. N. Histone
deacetylases 1 and 2 redundantly regulate cardiac morphogenesis,
growth, and contractility. Genes Dev. 2007, 21, 1790-1802.
Rifkind, R. A.; Marks, P. A.; Breslow, R.; Pavletich, N. P. Structures of
a histone deacetylase homologue bound to the TSA and SAHA
inhibitors. Nature 1999, 401, 188-193.
21.
Kook, H.; Lepore, J. J.; Gitler, A. D.; Lu, M. M.; Yung, W. W.-
37.
Wang, D.-F.; Wiest, O.; Helquist, P.; Lan-Hargest, H.-Y.;
M.; Mackay, J.; Zhou, R.; Ferrari, V.; Gruber, P.; Epstein, J. A. Cardiac
hypertrophy and histone deacetylase–dependent transcriptional
repression mediated by the atypical homeodomain protein Hop. J.
Clin. Invest. 2003, 112, 863-871.
Wiech, N. L. On the function of the 14 Å long internal cavity of
histone deacetylase-like protein: implications for the design of
histone deacetylase inhibitors. J. Med. Chem. 2004, 47, 3409-3417.
38.
Mann, B. S.; Johnson, J. R.; He, K.; Sridhara, R.; Abraham,
22.
Bogaard, H. J.; Mizuno, S.; Hussaini, A. A.; Toldo, S.;
S.; Booth, B. P.; Verbois, L.; Morse, D. E.; Jee, J. M.; Pope, S.;
Harapanhalli, R. S.; Dagher, R.; Farrell, A.; Justice, R.; Pazdur, R.
Vorinostat for treatment of cutaneous manifestations of advanced
primary cutaneous T-cell lymphoma. Clin. Cancer Res. 2007, 13,
2318-2322.
Abbate, A.; Kraskauskas, D.; Kasper, M.; Natarajan, R.; Voelkel, N. F.
Suppression of histone deacetylases worsens right ventricular
dysfunction after pulmonary artery banding in rats. Am. J. Respir.
Crit. Care Med. 2011, 183, 1402-1410.
23.
Haberland, M.; Montgomery, R. L.; Olson, E. N. The many
39.
Poole, R. M. Belinostat: first global approval. Drugs 2014,
roles of histone deacetylases in development and physiology:
implications for disease and therapy. Nat. Rev. Genet. 2009, 10, 32-
74, 1543-1554.
40.
Oki, Y.; Buglio, D.; Fanale, M.; Fayad, L.; Copeland, A.;
42.
Romaguera, J.; Kwak, L. W.; Pro, B.; de Castro Faria, S.; Neelapu, S.;
Fowler, N.; Hagemeister, F.; Zhang, J.; Zhou, S.; Feng, L.; Younes, A.
Phase I study of panobinostat plus everolimus in patients with
relapsed or refractory lymphoma. Clin. Cancer Res. 2013, 19, 6882-
6890.
24.
agents. Br. J. Pharmacol. 2007, 150, 829-831.
25. Halili, M. A.; Andrews, M. R.; Sweet, M. J.; Fairlie, D. P.
Adcock, I. M. HDAC inhibitors as anti-inflammatory
Histone deacetylase inhibitors in inflammatory disease. Curr. Top.
Med. Chem. 2009, 9, 309-319.
41.
Whittaker, S. J.; Demierre, M. F.; Kim, E. J.; Rook, A. H.;
Lerner, A.; Duvic, M.; Scarisbrick, J.; Reddy, S.; Robak, T.; Becker, J.
C.; Samtsov, A.; McCulloch, W.; Kim, Y. H. Final results from a
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 19
multicenter, international, pivotal study of romidepsin in
refractory cutaneous T-cell lymphoma. J. Clin. Oncol. 2010, 28,
55.
Lu, X.; Ning, Z.; Li, Z.; Cao, H.; Wang, X. Development of
chidamide for peripheral T-cell lymphoma, the first orphan drug
1
2
3
4
5
6
7
8
4485-4491.
approved in China. Intractable Rare Dis Res. 2016, 5, 185-191.
42.
Yeo, W.; Chung, H. C.; Chan, S. L.; Wang, L. Z.; Lim, R.;
56.
Shi, Y.; Dong, M.; Hong, X.; Zhang, W.; Feng, J.; Zhu, J.; Yu,
Picus, J.; Boyer, M.; Mo, F. K. F.; Koh, J.; Rha, S. Y.; Hui, E. P.; Jeung, H.
C.; Roh, J. K.; Yu, S. C. H.; To, K. F.; Tao, Q.; Ma, B. B.; Chan, A. W. H.;
Tong, J. H. M.; Erlichman, C.; Chan, A. T. C.; Goh, B. C. Epigenetic
therapy using belinostat for patients with unresectable
hepatocellular carcinoma: A multicenter phase I/II study with
biomarker and pharmacokinetic analysis of tumors from patients
in the Mayo phase II consortium and the cancer therapeutics
research group. J. Clin. Oncol. 2012, 30, 3361-3367.
L.; Ke, X.; Huang, H.; Shen, Z.; Fan, Y.; Li, W.; Zhao, X.; Qi, J.; Huang,
H.; Zhou, D.; Ning, Z.; Lu, X. Results from a multicenter, open-label,
pivotal phase II study of chidamide in relapsed or refractory
peripheral T-cell lymphoma. Ann. Oncol. 2015, 26, 1766-1771.
57.
Ning, Z. Q.; Li, Z. B.; Newman, M. J.; Shan, S.; Wang, X. H.;
Pan, D. S.; Zhang, J.; Dong, M.; Du, X.; Lu, X. P. Chidamide
(CS055/HBI-8000): a new histone deacetylase inhibitor of the
benzamide class with antitumor activity and the ability to enhance
immune cell-mediated tumor cell cytotoxicity. Cancer Chemother.
Pharmacol. 2012, 69, 901-909.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
43.
Lee, H. Z.; Kwitkowski, V. E.; Del Valle, P. L.; Ricci, M. S.;
Saber, H.; Habtemariam, B. A.; Bullock, J.; Bloomquist, E.; Li Shen,
Y.; Chen, X. H.; Brown, J.; Mehrotra, N.; Dorff, S.; Charlab, R.; Kane,
R. C.; Kaminskas, E.; Justice, R.; Farrell, A. T.; Pazdur, R. FDA
approval: Belinostat for the treatment of patients with relapsed or
refractory peripheral T-cell lymphoma. Clin. Cancer Res. 2015, 21,
2666-2670.
58.
Mottamal, M.; Zheng, S. L.; Huang, T. L.; Wang, G. D.
Histone deacetylase inhibitors in clinical studies as templates for
new anticancer agents. Molecules 2015, 20, 3898-3941.
59.
in cancer therapy. J. Clin. Oncol. 2009, 27, 5459-5468.
60. Lu, Q.; Wang, D. S.; Chen, C. S.; Hu, Y. D.; Chen, C. S.
Lane, A. A.; Chabner, B. A. Histone deacetylase inhibitors
44.
Lachenmayer, A.; Toffanin, S.; Cabellos, L.; Alsinet, C.;
Hoshida, Y.; Villanueva, A.; Minguez, B.; Tsai, H. W.; Ward, S. C.;
Thung, S.; Friedman, S. L.; Llovet, J. M. Combination therapy for
hepatocellular carcinoma: additive preclinical efficacy of the HDAC
inhibitor panobinostat with sorafenib. J. Hepatol. 2012, 56, 1343-
1350.
Structure-based optimization of phenylbutyrate-derived histone
deacetylase inhibitors. J. Med. Chem. 2005, 48, 5530-5535.
61.
Lu, Q.; Yang, Y. T.; Chen, C. S.; Davis, M.; Byrd, J. C.;
Etherton, M. R.; Umar, A.; Chen, C. S. Zn2+-chelating motif-tethered
short-chain fatty acids as a novel class of histone deacetylase
inhibitors. J. Med. Chem. 2004, 47, 467-474.
45.
Laubach, J. P.; Moreau, P.; San-Miguel, J. F.; Richardson, P.
G. Panobinostat for the Treatment of Multiple Myeloma. Clin.
Cancer Res. 2015, 21, 4767-4773.
62.
Zhang, J. G.; Shi, Y.; Hong, D. F.; Song, M.; Huang, D.; Wang,
C. Y.; Zhao, G. MiR-148b suppresses cell proliferation and invasion
in hepatocellular carcinoma by targeting WNT1/beta-catenin
pathway. Sci. Rep. 2015, 5, 8087.
46.
Thurn, K. T.; Thomas, S.; Moore, A.; Munster, P. N.
Rational therapeutic combinations with histone deacetylase
inhibitors for the treatment of cancer. Future Oncol. 2011, 7, 263-
283.
63.
Murahari, S.; Jalkanen, A. L.; Kulp, S. K.; Chen, C. S.;
Modiano, J. F.; London, C. A.; Kisseberth, W. C. Sensitivity of
osteosarcoma cells to HDAC inhibitor AR-42 mediated apoptosis.
BMC Cancer 2017, 17, 67.
47.
Tate, C. R.; Rhodes, L. V.; Segar, H. C.; Driver, J. L.;
Pounder, F. N.; Burow, M. E.; Collins-Burow, B. M. Targeting triple-
negative breast cancer cells with the histone deacetylase inhibitor
panobinostat. Breast Cancer Res. 2012, 14, R79.
64.
Bush, M. L.; Oblinger, J.; Brendel, V.; Santarelli, G.; Huang,
J.; Akhmametyeva, E. M.; Burns, S. S.; Wheeler, J.; Davis, J.; Yates, C.
W.; Chaudhury, A. R.; Kulp, S.; Chen, C. S.; Chang, L. S.; Welling, D. B.;
Jacob, A. AR42, a novel histone deacetylase inhibitor, as a potential
therapy for vestibular schwannomas and meningiomas. Neuro
Oncol. 2011, 13, 983-999.
48.
Rathkopf, D.; Wong, B. Y.; Ross, R. W.; Anand, A.; Tanaka,
E.; Woo, M. M.; Hu, J.; Dzik-Jurasz, A.; Yang, W.; Scher, H. I. A phase
I study of oral panobinostat alone and in combination with
docetaxel in patients with castration-resistant prostate cancer.
Cancer Chemother. Pharmacol. 2010, 66, 181-189.
65.
Jacob, A.; Oblinger, J.; Bush, M. L.; Brendel, V.; Santarelli,
49.
Coiffier, B.; Pro, B.; Prince, H. M.; Foss, F.; Sokol, L.;
G.; Chaudhury, A. R.; Kulp, S.; La Perle, K. M.; Chen, C. S.; Chang, L.
S.; Welling, D. B. Preclinical validation of AR42, a novel histone
deacetylase inhibitor, as treatment for vestibular schwannomas.
Laryngoscope 2012, 122, 174-189.
Greenwood, M.; Caballero, D.; Morschhauser, F.; Wilhelm, M.;
Pinter-Brown, L.; Padmanabhan Iyer, S.; Shustov, A.; Nielsen, T.;
Nichols, J.; Wolfson, J.; Balser, B.; Horwitz, S. Romidepsin for the
treatment of relapsed/refractory peripheral T-cell lymphoma:
pivotal study update demonstrates durable responses. J. Hematol.
Oncol. 2014, 7, 11.
66.
Zhang, S.; Suvannasankha, A.; Crean, C. D.; White, V. L.;
Chen, C. S.; Farag, S. S. The novel histone deacetylase inhibitor, AR-
42, inhibits gp130/Stat3 pathway and induces apoptosis and cell
cycle arrest in multiple myeloma cells. Int. J. Cancer 2011, 129, 204-
213.
50.
cell lymphoma. Clin. Cancer Res. 2012, 18, 3509-3515.
51. Piekarz, R. L.; Frye, R.; Prince, H. M.; Kirschbaum, M. H.;
Prince, H. M.; Dickinson, M. Romidepsin for cutaneous T-
67.
Sborov, D. W.; Canella, A.; Hade, E. M.; Mo, X.; Khountham,
Zain, J.; Allen, S. L.; Jaffe, E. S.; Ling, A.; Turner, M.; Peer, C. J.; Figg,
W. D.; Steinberg, S. M.; Smith, S.; Joske, D.; Lewis, I.; Hutchins, L.;
Craig, M.; Fojo, A. T.; Wright, J. J.; Bates, S. E. Phase 2 trial of
romidepsin in patients with peripheral T-cell lymphoma. Blood
2011, 117, 5827-5834.
S.; Wang, J.; Ni, W.; Poi, M.; Coss, C.; Liu, Z.; Phelps, M. A.; Mortazavi,
A.; Andritsos, L.; Baiocchi, R. A.; Christian, B. A.; Benson, D. M.;
Flynn, J.; Porcu, P.; Byrd, J. C.; Pichiorri, F.; Hofmeister, C. C. A phase
1 trial of the HDAC inhibitor AR-42 in patients with multiple
myeloma and T- and B-cell lymphomas. Leuk Lymphoma 2017, 58,
2310-2318.
52.
Butler, L. M.; Zhou, X.; Xu, W. S.; Scher, H. I.; Rifkind, R. A.;
Marks, P. A.; Richon, V. M. The histone deacetylase inhibitor SAHA
arrests cancer cell growth, up-regulates thioredoxin-binding
protein-2, and down-regulates thioredoxin. Proc. Natl. Acad. Sci. U.
S. A. 2002, 99, 11700-11705.
68.
Zimmerman, B.; Sargeant, A.; Landes, K.; Fernandez, S. A.;
Chen, C. S.; Lairmore, M. D. Efficacy of novel histone deacetylase
inhibitor, AR42, in a mouse model of, human T-lymphotropic virus
type 1 adult T cell lymphoma. Leuk. Res. 2011, 35, 1491-1497.
53.
Min, A.; Im, S. A.; Kim, D. K.; Song, S. H.; Kim, H. J.; Lee, K.
69.
Lucas, D. M.; Alinari, L.; West, D. A.; Davis, M. E.; Edwards,
H.; Kim, T. Y.; Han, S. W.; Oh, D. Y.; Kim, T. Y.; O'Connor, M. J.; Bang,
Y. J. Histone deacetylase inhibitor, suberoylanilide hydroxamic acid
(SAHA), enhances anti-tumor effects of the poly (ADP-ribose)
polymerase (PARP) inhibitor olaparib in triple-negative breast
cancer cells. Breast Cancer Res. 2015, 17, 33.
R. B.; Johnson, A. J.; Blum, K. A.; Hofmeister, C. C.; Freitas, M. A.;
Parthun, M. R.; Wang, D.; Lehman, A.; Zhang, X.; Jarjoura, D.; Kulp,
S. K.; Croce, C. M.; Grever, M. R.; Chen, C. S.; Baiocchi, R. A.; Byrd, J.
C. The novel deacetylase inhibitor AR-42 demonstrates pre-clinical
activity in B-cell malignancies in vitro and in vivo. PLoS One 2010,
5, e10941.
54.
Chiao, M. T.; Cheng, W. Y.; Yang, Y. C.; Shen, C. C.; Ko, J. L.
Suberoylanilide hydroxamic acid (SAHA) causes tumor growth
slowdown and triggers autophagy in glioblastoma stem cells.
Autophagy 2013, 9, 1509-1526.
70.
Chen, Y. J.; Wang, W. H.; Wu, W. Y.; Hsu, C. C.; Wei, L. R.;
Wang, S. F.; Hsu, Y. W.; Liaw, C. C.; Tsai, W. C. Novel histone
deacetylase inhibitor AR-42 exhibits antitumor activity in
16
ACS Paragon Plus Environment

Page 17 of 19
Journal of Medicinal Chemistry
pancreatic cancer cells by affecting multiple biochemical pathways.
PLoS One 2017, 12, e0183368.
and inhibition of proliferation of melanoma cells. Cancer Lett.
2001, 163, 103-107.
1
2
3
4
5
6
7
8
71.
Elshafae, S. M.; Kohart, N. A.; Altstadt, L. A.; Dirksen, W.
87.
Lauffer, B. E.; Mintzer, R.; Fong, R.; Mukund, S.; Tam, C.;
P.; Rosol, T. J. The effect of a histone deacetylase inhibitor (AR-42)
on canine prostate cancer growth and metastasis. Prostate 2017,
77, 776-793.
Zilberleyb, I.; Flicke, B.; Ritscher, A.; Fedorowicz, G.; Vallero, R.
Histone deacetylase (HDAC) inhibitor kinetic rate constants
correlate with cellular histone acetylation but not transcription
and cell viability. J. Biol. Chem. 2013, 288, 26926-26943.
72.
Zhou, R.; Wu, J.; Tang, X.; Wei, X.; Ju, C.; Zhang, F.; Sun, J.;
Shuai, D.; Zhang, Z.; Liu, Q.; Lv, X. B. Histone deacetylase inhibitor
AR-42 inhibits breast cancer cell growth and demonstrates a
synergistic effect in combination with 5-FU. Oncol. Lett. 2018, 16,
1967-1974.
88.
Jacobson, M. P.; Pincus, D. L.; Rapp, C. S.; Day, T. J.; Honig,
B.; Shaw, D. E.; Friesner, R. A. A hierarchical approach to all-atom
protein loop prediction. Proteins 2004, 55, 351-367.
89.
Sastry, G. M.; Adzhigirey, M.; Day, T.; Annabhimoju, R.;
9
73.
Yang, Y. T.; Balch, C.; Kulp, S. K.; Mand, M. R.; Nephew, K.
Sherman, W. Protein and ligand preparation: parameters,
protocols, and influence on virtual screening enrichments. J.
Comput. Aided Mol. Des. 2013, 27, 221-234.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
P.; Chen, C. S. A rationally designed histone deacetylase inhibitor
with distinct antitumor activity against ovarian cancer. Neoplasia
2009, 11, 552-563.
90.
Shelley, J. C.; Cholleti, A.; Frye, L. L.; Greenwood, J. R.;
74.
Li, D. R.; Zhang, H.; Peek, E.; Wang, S.; Du, L.; Li, G.; Chin,
Timlin, M. R.; Uchimaya, M. Epik: a software program for pK( a )
prediction and protonation state generation for drug-like
molecules. J. Comput. Aided Mol. Des. 2007, 21, 681-691.
A. I. Synergy of histone-deacetylase inhibitor AR-42 with cisplatin
in bladder cancer. J. Urol. 2015, 194, 547-555.
75.
Guzman, M. L.; Yang, N.; Sharma, K. K.; Balys, M.; Corbett,
91.
Roos, K.; Wu, C.; Damm, W.; Reboul, M.; Stevenson, J. M.;
C. A.; Jordan, C. T.; Becker, M. W.; Steidl, U.; Abdel-Wahab, O.;
Levine, R. L.; Marcucci, G.; Roboz, G. J.; Hassane, D. C. Selective
activity of the histone deacetylase inhibitor AR-42 against
Lu, C.; Dahlgren, M. K.; Mondal, S.; Chen, W.; Wang, L.; Abel, R.;
Friesner, R. A.; Harder, E. D. OPLS3e: Extending force field coverage
for drug-like small molecules. J. Chem. Theory Comput. 2019, 15,
1863-1874.
leukemia stem cells:
myelogenous leukemia. Mol. Cancer Ther. 2014, 13, 1979-1990.
76. Mims, A.; Walker, A. R.; Huang, X.; Sun, J.; Wang, H.;
a novel potential strategy in acute
92.
Chen, V. B.; Arendall, W. B., 3rd; Headd, J. J.; Keedy, D. A.;
Immormino, R. M.; Kapral, G. J.; Murray, L. W.; Richardson, J. S.;
Richardson, D. C. MolProbity: all-atom structure validation for
Santhanam, R.; Dorrance, A. M.; Walker, C.; Hoellerbauer, P.;
Tarighat, S. S.; Chan, K. K.; Klisovic, R. B.; Perrotti, D.; Caligiuri, M.
A.; Byrd, J. C.; Chen, C. S.; James Lee, L.; Jacob, S.; Mrozek, K.;
Bloomfield, C. D.; Blum, W.; Garzon, R.; Schwind, S.; Marcucci, G.
Increased anti-leukemic activity of decitabine via AR-42-induced
upregulation of miR-29b: a novel epigenetic-targeting approach in
acute myeloid leukemia. Leukemia 2013, 27, 871-878.
macromolecular crystallography. Acta Crystallogr.
Crystallogr. 2010, 66, 12-21.
D
Biol.
93.
Benkert, P.; Biasini, M.; Schwede, T. Toward the
estimation of the absolute quality of individual protein structure
models. Bioinformatics 2011, 27, 343-350.
94.
LigPrep, Schrödinger Release 2019-2, Schrödinger, LLC,
77.
Kulp, S. K.; Chen, C. S.; Wang, D. S.; Chen, C. Y.; Chen, C. S.
New York, NY., 2019.
Antitumor effects of
a
novel phenylbutyrate-based histone
95.
Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
deacetylase inhibitor, (S)-HDAC-42, in prostate cancer. Clin. Cancer
Res. 2006, 12, 5199-5206.
Development and validation of a genetic algorithm for flexible
docking. J. Mol. Biol. 1997, 267, 727-748.
78.
Kong, Y.; Barisone, G. A.; Abuhay, M.; O'Donnell, R. T.;
Buksh, Z.; Yousefian, F.; Tuscano, J. M. Histone deacetylase
inhibition enhances the lymphomacidal activity of the anti-CD22
monoclonal antibody HB22.7. Leuk. Res. 2014, 38, 1320-1326.
79.
Stivala, C. E.; Zakarian, A. Highly enantioselective direct
alkylation of arylacetic acids with chiral lithium amides as traceless
auxiliaries. J. Am. Chem. Soc. 2011, 133, 11936-11939.
80.
Wsol, V.; Skalova, L.; Szotakova, B. Chiral inversion of
drugs: coincidence or principle? Curr. Drug Metab. 2004, 5, 517-
533.
81.
Watson, P. J.; Millard, C. J.; Riley, A. M.; Robertson, N. S.;
Wright, L. C.; Godage, H. Y.; Cowley, S. M.; Jamieson, A. G.; Potter, B.
V.; Schwabe, J. W. Insights into the activation mechanism of class I
HDAC complexes by inositol phosphates. Nat. Commun. 2016, 7,
11262.
82.
Gottesfeld, J. M. Milestones in transcription and
chromatin published in the journal of biological chemistry. J. Biol.
Chem. 2019, 294, 1652-1660.
83.
Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.;
Ward, K. W.; Kopple, K. D. Molecular properties that influence the
oral bioavailability of drug candidates. J. Med. Chem. 2002, 45,
2615-2623.
84.
Chakrabarti, A.; Oehme, I.; Witt, O.; Oliveira, G.; Sippl, W.;
Romier, C.; Pierce, R. J.; Jung, M. HDAC8: a multifaceted target for
therapeutic interventions. Trends Pharmacol. Sci. 2015, 36, 481-
492.
85.
Somoza, J. R.; Skene, R. J.; Katz, B. A.; Mol, C.; Ho, J. D.;
Jennings, A. J.; Luong, C.; Arvai, A.; Buggy, J. J.; Chi, E.; Tang, J.; Sang,
B.-C.; Verner, E.; Wynands, R.; Leahy, E. M.; Dougan, D. R.; Snell, G.;
Navre, M.; Knuth, M. W.; Swanson, R. V.; McRee, D. E.; Tari, L. W.
Structural snapshots of human HDAC8 provide insights into the
class I histone deacetylases. Structure 2004, 12, 1325-1334.
86.
Demary, K.; Wong, L.; Spanjaard, R. A. Effects of retinoic
acid and sodium butyrate on gene expression, histone acetylation
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