Synthesis and biological characterization of amidopropenyl hydroxamates as HDAC inhibitors

Article abstract of DOI:10.1002/cmdc.201000166

A series of amidopropenyl hydroxamic acid derivatives were prepared as novel inhibitors of human histone deacetylases (HDACs). Several compounds showed potency at <100 nm in the HDAC inhibition assays, sub-micromolar IC ₅₀ values in tests again

Full text of DOI:10.1002/cmdc.201000166

DOI: 10.1002/cmdc.201000166
Synthesis and Biological Characterization of
Amidopropenyl Hydroxamates as HDAC Inhibitors
Florian Thaler,*[a, f] Mario Varasi,[b, f] Andrea Colombo,[c] Roberto Boggio,[a] Davide Munari,[b]
Nickolas Regalia,[c] Marco G. Rozio,[c] Veronica Reali,[c] Anna E. Resconi,[c] Antonello Mai,[d]
Stefania Gagliardi,[c] Giulio Dondio,[c] Saverio Minucci,[e] and Ciro Mercurio[b]
A series of amidopropenyl hydroxamic acid derivatives were
prepared as novel inhibitors of human histone deacetylases
(HDACs). Several compounds showed potency at <100 nm in
the HDAC inhibition assays, sub-micromolar IC50 values in tests
against three tumor cell lines, and remarkable stability in
human and mouse microsomes was observed. Three represen-
tative compounds were selected for further characterization
and submitted to a selectivity profile against a series of class I
and class II HDACs as well as to preliminary in vivo pharmaco-
kinetic (PK) experiments. Despite their high microsomal stabili-
ty, the compounds showed medium-to-high clearance rates in
in vivo PK studies as well as in rat and human hepatocytes, in-
dicating that a major metabolic pathway is catalyzed by non-
microsomal enzymes.
Introduction
Reversible post-translational histone modifications such as ace-
tylation and deacetylation play an essential role in modifying
chromatin structure and regulating gene transcription in eu-
karyotic cells. The acetylation status is tightly controlled by
counteracting enzyme families: histone acetyltransferases
(HATs), enzymes that catalyze histone acetylation, which is gen-
erally associated with transcriptional activation, and histone
deacetylases (HDACs), enzymes responsible for deacetylation
of lysine residues located at the N termini of the proteins; this
is normally correlated with transcriptional repression.[1,2]
HDACs can be grouped into two distinct families: Zn2+-depen-
dent enzymes, also known as “classical HDACs”, and the NAD+
-dependent sirtuins. Based on phylogenetic conservation, his-
tone deacetylases can be further grouped into four classes:
class I (HDACs 1–3 and 8), II (HDACs 4–7, 9, and 10), and IV
(HDAC 11) belong to the classical HDACs, whereas sirtuins are
represented by class III enzymes.[3,4] Aberrant regulation of his-
tone acetylation as well as acetylation of non-histone proteins
has been shown to be linked not only to the pathogenesis of
cancer, but also to several other diseases.[5–12] Over the past
few years, there has been a growing interest in the develop-
ment of HDAC inhibitors, and a common pharmacophore
based on the first compounds has been presented by Miller
et al.[13] This model pharmacophore is composed of a metal
binding group, such as a hydroxamic acid, a linking group con-
sisting of linear or cyclic structures, and a surface recognition
domain, which is generally a hydrophobic group. Many studies
have demonstrated that HDAC inhibition leads to antitumor ef-
fects, and several compounds are currently in clinical studies
for solid and hematological tumor therapy, Huntington’s dis-
ease, amyotrophic lateral sclerosis, and fungal infections.[14–16]
Currently, two HDAC inhibitors have gained approval by the
US Food and Drug Administration (FDA) for the treatment of
cutaneous T-cell lymphoma: vorinostat (1; Zolinza, also known
as SAHA or suberoylanilide hydroxamic acid),[17–19] which was
approved in October 2006, and the cyclic depsipeptide rhomi-
depsin (2; FK228, Gloucester),[20] which was approved three
years later in November 2009. Further compounds in clinical
studies include hydroxamic acids such as panobinostat (3;
LBH589, Novartis, phase III),[21] belinostat (4; PXD101, Topotar-
get, phase III),[22] and givinostat (5; ITF-2357, Italfarmaco, pha-
se II),[23] as well benzamides, such as entinostat (6; SNDX-275,
Syndax, phase II)[24] and mocetinostat (7; MGCD-0103, Methyl-
gene, phase II).[25] A further class comprises short fatty acids,
such as sodium phenylbutyrate (8; Tikvah Therapeutics, pha-
se II).[26,27]
We recently described a novel HDAC inhibitor scaffold,
namely aryloxypropenylphenyl- and aryloxypropenylpyridinyl
[a] Dr. F. Thaler, Dr. R. Boggio
Genextra Group, Congenia s.r.l., Via Adamello 16, 20139 Milan (Italy)
Fax: (+39)02-94375138
E-mail: florian.thaler@ifom-ieo-campus.it
[b] Dr. M. Varasi, Dr. D. Munari, Dr. C. Mercurio
Genextra Group, DAC s.r.l., Via Adamello 16, 20139 Milan (Italy)
[c] Dr. A. Colombo, Dr. N. Regalia, Dr. M. G. Rozio, Dr. V. Reali, Dr. A. E. Resconi,
Dr. S. Gagliardi, Dr. G. Dondio
NiKem Research s.r.l., Via Zambeletti 25, 20021 Baranzate (Italy)
[d] Prof. Dr. A. Mai
Istituto Pasteur – Fondazione Cenci Bolognetti, Dipartimento di Studi
Farmaceutici, Universitꢀ degli Studi di Roma “La Sapienza”
P.le A. Moro 5, 00185 Roma (Italy)
[e] Prof. Dr. S. Minucci
IEO – European Institute of Oncology, Via Adamello 16, 20139 Milan (Italy)
and
Department of Biomolecular Sciences and Biotechnologies
University of Milan, Via Celoria 26, 20133 Milan (Italy)
[f] Dr. F. Thaler, Dr. M. Varasi
Current address: Drug Discovery Program, Department of Experimental
Oncology, IEO – European Institute of Oncology
Via Adamello 16, 20139 Milan (Italy)
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F. Thaler et al.
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hydroxamates.[28] The study was
designed to obtain potent inhib-
itors with improved pharmacoki-
netic (PK) properties. As outlined
in the work, unsubstituted aryl-
oxypropenyl derivatives have
poor solubility and were rapidly
metabolized in mouse micro-
somes, whereas the introduction
of a solubilizing 4-methylpipera-
zin-1-yl moiety led to more solu-
ble structures with enhanced mi-
crosomal stability and a better
PK profile. In a further experi-
ment, metabolic profiling of (E)-
N-hydroxy-3-[4-((E)-3-oxo-3-
phenyl-1-propen-1-yl)phenyl]ac-
rylamide performed in rat hepa-
Scheme 1. Reagents and conditions: a) THF, NaH, triethyl phosphonoacetate, RT; b) CH2Cl2, TFA, RT; c) CH2Cl2, EDC,
tocytes showed that reduction HOBt, TEA, NH2OTHP, RT; d) 1m LiOH, THF, RT; e) DMF, EDC, HOBt, TEA, 4-R-piperazine; f) CH2Cl2, Et2O, HCl, RT.
of the a,b-unsaturated ketone
was one of the major route of metabolism for the molecule.[29]
Herein we describe the synthesis and biological activity of
amidopropenyl hydroxamic acid derivatives, which are struc-
turally related to the series described above and are devoid of
the potentially metabolically unstable a,b-unsaturated ketone
moiety.
benzotriazole (HOBt) as coupling agents, leading to the tetra-
hydropyran-2-yl-protected hydroxamic acids 13 and 14. Hy-
drolysis of the ethyl ester with lithium hydroxide in tetrahydro-
furan, amide construction via standard amide coupling, and
final deprotection of the tetrahydropyranyl moiety with hydro-
chloric acid in diethyl ether provided the desired hydroxamic
acids 17a,b and 18a,b.
Heck reaction of the commercially available bromo deriva-
tive 19 with ethyl acrylate in the presence of palladium ace-
tate, triphenylphosphine, triethylamine, and sodium bicarbon-
ate furnished (E)-3-(3-formylphenyl)acrylic acid ethyl ester (20,
see Scheme 2), which was then converted into the diester 21
under Horner–Emmons conditions using tert-butyldiethyl phos-
phonoacetate. Subsequent removal of the tert-butyl ester
group with TFA, coupling of the carboxylic acid with NH2OTHP
using the water-soluble carbodiimide reagent EDC and HOBt
gave the tetrahydropyran-2-yl-protected hydroxamic acid 22.
Deprotection of the ethyl ester, EDC coupling of the resulting
acid with the corresponding cyclic amines, and final deprotec-
tion of the THP group gave the desired amidopropenyl hy-
droxamic acid derivatives 24a,b.
Results and Discussion
Chemistry
The syntheses of the hydroxamic acid derivatives 17a,b and
18a,b were carried out by starting from (E)-tert-butyl-3-(4-for-
mylphenyl)acrylate (9)[30] and tert-butyl-(E)-3-(5-formylpyridin-2-
yl)acrylate (10).[28] As illustrated in Scheme 1, the tert-butylacry-
lates 9 and 10 were treated with triethyl phosphonoacetate ac-
cording to Horner–Emmons conditions, giving the diesters 11
and 12. Thus, the tert-butyl group was cleaved with trifluoro-
acetic acid (TFA) in dichloromethane, and the obtained acrylic
acid was then treated with O-(2-tetrahydropyranyl)hydroxyl-
amine (NH2OTHP) in the presence of N-(3-dimethylaminoprop-
yl)-N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy-
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Amidopropenyl Hydroxamates as HDAC Inhibitors
tested. For this purpose, 4-
methyl- and 4-phenylpiperazinyl
analogues of the following four
linkers were prepared: meta-
and para-substituted phenylacry-
late, and pyridin-2-ylacrylate sub-
stituted at positions 5 or 6. As
summarized in Table 1, the meta-
substituted phenylacrylate deriv-
atives 24a and 24b exhibited
IC50 values of 0.16 and 0.35 mm,
respectively, and were about 10-
fold more potent than the corre-
sponding para-substituted com-
pounds 17a and 17b. In the
pyridinyl series, the differences
between 2,5- and 2,6-disubstitut-
ed pyridin-2-ylacrylates was less
evident: 29b was about fourfold
Scheme 2. Reagents and conditions: a) DMF, TEA, NaHCO3, PPh3, Pd(OAc)2, 1008C; b) NaH, THF, tert-butyldiethyl
phosphonoacetate, 08C, then RT; c) CH2Cl2, TFA, RT; d) CH2Cl2, EDC, HOBt, TEA, NH2OTHP, RT; e) 1m LiOH, THF, RT;
f) CH2Cl2, DMF, EDC, HOBt, TEA, 4-R-piperazine, RT; g) CH2Cl2, Et2O, HCl, RT.
The diester 26 was prepared by Horner–Emmons reaction of
(E)-3-(6-formylpyridin-2-yl)acrylic acid tert-butyl ester (25)[28]
and triethyl phosphonoacetate (Scheme 3). Deprotection of
the tert-butyl group and reaction with NH2OTHP gave the tet-
rahydropyranyl-protected hydroxamic acid 27. Subsequent
cleavage of ethyl ester in 4n sodium hydroxide in THF led to
the pyridine acrylic acid 28. Amide formation and deprotection
of the hydroxamic acid following the conditions described in
Schemes 1 and 2 afforded the desired N-hydroxyacrylamides
29a–v.
more potent than 18b, whereas the two N-methylpiperazine
derivatives 29a and 18a were almost equipotent. Comparison
of the compounds containing a central phenyl ring with those
with a pyridinyl ring showed that the latter inhibitors were
generally more potent in the HDAC assay. Similar observations
had also been found for the previously described aryloxypro-
penylphenyl- and aryloxypropenylpyridinyl hydroxamates.[28]
Antiproliferative activities were overall superior for those
compounds containing the phenylpiperazine group; the meth-
ylpiperazine analogues generally exhibited IC50 values >10 mm
in the cellular assays. Limited permeability for these polar hy-
droxamic acids could explain the absence of antiproliferative
activity observed at the doses studied. Although 29b was ~5–
6-fold more potent than 18b and 24b in the HDAC assay, the
three compounds exhibited similar antiproliferative activities in
K562 and HCT116 cells. Slightly higher IC50 values in the A549
cell line were observed for the three compounds. Linking the
activity observed in a biochemical assay with cellular potency
on the basis of the physicochemical properties of a molecule is
often difficult. Several variables are known to play a role, such
as cellular membrane permeability, subcellular compartmentali-
zation, and cellular mechanisms for efficiently extruding xeno-
Biology
The compounds were profiled using HeLa cell nuclear extracts
as a source of class I, II, and IV HDAC enzymatic activity. The
absence of NAD+ under the experimental conditions allowed
exclusion of the contribution of class III sirtuins to the enzy-
matic activity. Subsequently, their antiproliferative potency was
assessed using the chronic myelogenous leukemia cell line
K562, human alveolar basal epithelial lung cancer A549 cells,
and human colon cancer HCT116 cells. Initially, a series of pi-
perazine oxypropenyl analogues with various linkers were
Scheme 3. Reagents and conditions: a) NaH, THF, triethyl phosphonoacetate, RT; b) CH2Cl2, TFA, RT; c) CH2Cl2, EDC, HOBt, TEA, NH2OTHP, RT; d) 4m NaOH, THF,
RT; e) CH2Cl2, DMF, EDC, HOBt, TEA; f) CH2Cl2, Et2O, HCl, RT.
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Table 1. SAR of 4-methyl- and 4-phenylpiperazinyl analogues 17a,b, 18a,b, 24a,b, and 29a,b.[a]
IC50 [mm]
Microsomes [%]
Solubility [mm]
Compd
X
Position
R
HDAC
K562
A549
HCT116
mouse
human
17a
17b
18a
18b
24a
24b
29a
29b
CH
CH
N
5
5
5
5
6
6
6
6
CH3
Ph
CH3
Ph
CH3
Ph
CH3
Ph
1.25
5.60
14.5
3.37
8.84
1.41
19.1
0.980
21.7
1.13
26.8
7.45
5.77
4.37
1.33
12.6
0.780
16.0
1.07
58
27
88
91
82
72
72
74
56
51
87
82
100
60
384
5
430
10
477
76
434
214
7.32
22.9
3.68
32.1
1.38
>50
2.43
0.438
0.299
0.164
0.354
0.345
0.069
N
CH
CH
N
65
77
N
[a] Assays were performed in replicate (nꢀ2); values are shown are the mean, and standard deviations are ꢁ25% of the mean.
biotics. Compound 17b exhibited, in line with the limited en-
zymatic inhibition, a lower antiproliferative activity in all three
cell lines relative to the three inhibitors 18b, 24b, and 29b.
The inhibitors were then submitted to a microsomal stability
assay, and their solubility was assessed at pH 7.4. Compounds
were exposed to mouse and human microsomes at 378C for
30 min, and the percentage of the unmetabolized derivative
was determined by LC–MS–MS. With the exception of 17b, all
compounds were metabolized by <50% under the test condi-
tions used both in mouse and human microsomes. In compari-
son, previously disclosed unsubstituted aryloxypropenyl deriva-
tives[28] were shown to be rapidly metabolized in mouse and
human microsomes. The compounds described herein (which
contain an amidic moiety instead of the ketone group) demon-
strated significantly higher stability in human and mouse mi-
crosome preparations. This is in agreement with the identifica-
tion of the a,b-unsaturated ketone moiety as a major metabol-
ic pathway of the previously described N-hydroxy-3-[4-((E)-3-
oxo-3-phenyl-1-propen-1-yl)phenyl]acrylamide.[29]
proliferative activities in A549 cells ranged between 0.61 mm
(29e) and 6 mm (29g), whereas differences in cellular activities
of these inhibitors were minor in the other two cell lines. Com-
pound 29e (IC50 =0.44 mm) was about fourfold more potent
than 29 f in K562 cells and about fivefold more active than
29g in HCT116 cells (IC50 =0.29 mm).
All compounds (with the exception of 29i with 29% remain-
ing in mouse microsomes) exhibited good stability in mouse
and human microsomes, with <50% of the inhibitors metabo-
lized after 30 min incubation. Furthermore, aqueous solubility
of all compounds exceeded 100 mm at pH 7.4.
The good potency of the compounds, together with the fa-
vorable ADME profile, prompted us to explore further modifi-
cations of the surface recognition domain. Compound 29e
was selected as a starting point for further exploration based
on its good biochemical and antiproliferative activity, and a
series of substituted 3-phenylpiperidine analogues were syn-
thesized. As shown in Table 3, the R and S isomers of com-
pound 29e were equipotent in the enzymatic and cell-based
assays. Introduction of a fluoro, methyl, or methoxy substituent
at the ortho, meta, and para positions of the phenyl ring led to
compounds that did not show substantial differences in the
enzymatic assay, whereas the two derivatives 29u and 29v,
with a naphthyl cap group in place of the phenyl ring, were
10-fold less potent than 29e. All compounds displayed good
antiproliferative activity in all three cell lines tested, particularly
K562 cells, with IC50 values <1 mm. Compound 29l, the 2-fluo-
rophenyl analogue of 29e, emerged as the most potent agent,
with an IC50 value of 0.068 mm. Compound 29l was also found
to be the most active compound in the other two cell lines,
with IC50 values of 0.057 mm (HCT116) and 0.196 mm (A549).
This sub-series also showed good microsomal stability in
general. A remarkable decrease in stability was observed be-
tween the 4-methylphenyl and 4-methoxyphenyl analogues
29q and 29t, respectively, and the unsubstituted compound
29e in human microsomes. Specifically, the methyl derivative
29q was almost completely metabolized after 30 min, and just
25% of the methoxy analogue 29q remained unaltered,
whereas <20% of 29e was degraded under the experimental
conditions. This trend, however, was not observed in mouse
All four 4-methylpiperazinyl analogues 17a, 18a, 24a, and
29a exhibited good aqueous solubility at neutral pH. Major
differences were found among the 4-phenylpiperazines: the
two para-substituted phenyl- and pyridin-2-ylacrylates 17b
and 18b had a much lower aqueous solubility (5 and 10 mm,
respectively) than the corresponding meta analogues 24b and
29b (76 and 214 mm, respectively). The hydroxamic acid 29b
was selected as a starting point for further exploration based
on the biochemical and cellular potency and the in vitro ADME
properties. The inhibitor was the most potent compound
within this group in the enzymatic assay (IC50 =69 nm); further-
more, it was found to be more soluble than 24b. Because hy-
drophobic aromatic groups represent a common surface rec-
ognition domain in several HDAC inhibitors,[13] a series of cyclic
amine derivatives substituted by phenyl were synthesized. The
results of the biological tests are reported in Table 2. HDAC IC50
values varied from 0.025 mm (compound 29e) to 0.294 mm
(compound 29c). The limited differences in activities (except
for the outlier 29e) can be ascribed to the fact that the surface
region of the HDAC enzyme is highly flexible and is able to ac-
commodate inhibitors with various capping groups.[31,32] Anti-
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Amidopropenyl Hydroxamates as HDAC Inhibitors
Table 2. SAR of phenylcycloamine analogues 29b–i.[a]
IC50 [mm]
A549
Microsomes [%]
Solubility [mm]
Compd
R
HDAC
0.294
K562
1.220
HCT116
1.300
mouse
human
29c
4.670
66
52
66
307
29d
0.269
0.616
1.230
0.432
90
218
29e
29 f
0.025
0.146
0.445
1.730
0.610
2.788
0.294
1.470
74
75
86
98
286
167
29g
29h
0.100
0.173
1.550
0.464
6.350
1.510
1.660
0.390
82
78
88
85
345
365
29b
29i
0.069
0.286
1.126
0.568
2.430
1.890
1.070
0.581
74
29
77
46
214
117
[a] Assays were performed in replicate (nꢀ2); values are shown are the mean, and standard deviations are <30% of the mean.
microsomes. On the other hand, the 4-fluorophenyl derivative
29n was fully recovered after 30 min incubation in mouse and
human microsome preparations. Table 3 also includes the solu-
bility data for this sub-series; the two naphthyl analogues 29u
and 29v were virtually insoluble at pH 7.4, whereas the aque-
ous solubility of 29l and 29m exceeded 400 mm.
The arylhydroxamate 24b and the two heteroaryl hydroxa-
mates 29b and 29e, which had been chosen as starting points
for further explorations, were selected for a more extensive
characterization. In an initial experiment the selectivity profile
against HDAC subtypes 1, 3, 4, 6, and 8 was evaluated. The re-
sults are summarized in Table 4 together with the data ob-
tained for SAHA (1), which are in agreement with recently pub-
lished data.[33] The potency of the three tested compounds as
well of 1 was in the nanomolar range against the class I and II
HDACs, with the exception of HDAC8, for which the IC50 values
were ~1 mm. The three structures can be classified as typical
pan-HDAC inhibitors upon comparison of the biological profile
with the data of 1 and other previously reported hydroxamic
acid derivatives.[5,34]
A further experiment was designed to study changes in
basal acetylation levels in K562 leukemia cells in the presence
of the inhibitors at a final concentration of 0.5 mm. The results
are included in Table 4; consistent with the ability to inhibit
cellular HDACs, all compounds were able to induce an increase
in basal acetylation levels greater than threefold relative to un-
treated K562 cells.
The three selected compounds 29b, 29e, and 24b were
then submitted to PK studies in mice. The compounds were
administrated in single intravenous (i.v.) or oral doses of 5 or
15 mgkgꢂ1, respectively. The derivatives were dissolved in
water containing 3% DMSO and 10% hydroxypropyl-b-cyclo-
dextrin (EncapsinTM) for the i.v. dose, or in water (29b) or water
containing 5% DMSO and 9.5% Encapsin (24b, 29e) for the
oral dose. The main plasma PK parameters are presented in
Table 5. For comparison, Table 5 also lists previously published
PK results for SAHA (1) obtained in house.[35] Following i.v. ad-
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Table 3. SAR of 3-phenylpiperidine analogues 29e and 29j–v.[a]
IC50 [mm]
A549
Microsomes [%]
Solubility [mm]
Compd
R
HDAC
0.025
K562
0.445
HCT116
0.294
mouse
human
29e
0.610
0.570
0.533
0.196
0.757
74
57
70
48
59
86
286
174
187
424
427
29j
29k
29l
0.025
0.031
0.020
0.033
0.214
0.229
0.068
0.249
0.196
0.169
0.057
0.240
77
75
66
69
29m
29n
0.026
0.462
1.050
0.291
100
100
113
29o
29p
29q
29r
29s
0.020
0.097
0.092
0.089
0.021
0.309
0.519
0.655
0.640
0.186
0.598
0.898
1.127
1.780
0.688
1.990
0.516
0.405
0.796
0.242
54
59
62
39
58
89
30
4
102
93
52
61
20
90
142
29t
0.068
0.299
0.948
0.383
70
25
125
29u
29v
0.307
0.215
0.764
0.390
1.560
0.763
0.601
0.284
37
92
52
15
1
100
[a] Assays were performed in replicate (nꢀ2); values are shown are the mean, and standard deviations are ꢁ30% of the mean.
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Amidopropenyl Hydroxamates as HDAC Inhibitors
LC–MS–MS. All three test compounds proved to be stable
under the conditions used, and the recovery of the products
exceeded 85%.
Table 4. HDAC selectivity profile and histone acetylation assay for 1,
24b, 29b, and 29e.[a]
Compd
IC50 [mm]
AI[b]
Once the stability of the three compounds in plasma was
confirmed, the next step was to explore their clearance behav-
ior in greater detail. For this purpose the inhibitors were incu-
bated in rat and human hepatocytes in order: 1) to explore the
metabolism in an additional species (rat), and 2) to determine
the metabolic behavior of the derivatives in hepatocytes rela-
tive to microsomes. Considering previously described clearance
classifications,[37,38] the three amidopropenyl hydroxamates un-
derwent a rapid biotransformation in rat and human hepato-
cytes (Table 6). The clearance rates were overall higher in rat
HDAC1
HDAC3
HDAC8
HDAC4
HDAC6
1
0.0453
0.203
0.098
0.025
0.0149
0.028
0.030
0.005
1.65
1.88
1.56
0.86
0.0377
0.303
0.150
0.024
0.0115
0.038
0.025
0.016
3.61
5.0
4.5
24b
29b
29e
4.2
[a] Assays were performed in replicate (nꢀ2); values are shown are the
mean, and standard deviations are <30% of the mean. [b] Acetylation in-
crement at an inhibitor concentration of 0.5 mm.
Table 5. Pharmacokinetic profiles for 1, 24b, 29b, and 29e.
Table 6. Clearance rates in rat and human hepatocytes for 1, 24b, 29b,
and 29e.
Compd
Parameter[a]
1
24b
440
1.22
11.37
5.2
29b
29e
1191
AUCi.v. 0ꢂ1 [nghmLꢂ1
]
657
0.5
7.62
1.2
12.1
590
Compd
CL [mLminꢂ1 (106 cells)ꢂ1
]
t1/2 [h]
0.27
8.47
0.8
0.67
Rat
Human
CL [Lhꢂ1 kgꢂ1
]
4.19
0.55
3.1
V
ss [Lkgꢂ1
F [%]
]
1
57.1
114.0
23.3
4.60
14.5
11.5
24b
29b
29e
10
2.9
[a] AUC: area under the plasma concentration–time curve of the drug;
t1/2: half-life; CL: volume of plasma cleared of the drug per unit time; Vss:
volume of distribution at the steady state defined by the amount of drug
in the body over the concentration of the drug in the plasma at the
steady state; F: percentage of the dose reaching blood circulation after
oral administration.
46.3
21.9
than in human hepatocytes, particularly for 24b. A high clear-
ance rate was also observed for reference compound 1 in rat
hepatocytes (57.1 mLminꢂ1 per 106 cells). On the other hand,
compound 1 showed a higher stability in human hepatocytes
than the three amidopropenyl hydroxamic acid derivatives.
These results reflected the observed in vivo PK data obtained
in mice, and showed that the metabolic stability observed in
microsomes was not found in hepatocytes.
A similar outcome has been described by Elaut et al.,[39] who
studied the in vitro biotransformation of trichostatin A (TSA).
TSA was rapidly and completely metabolized by rat hepato-
cytes, whereas the compound was metabolized slowly and in-
completely in rat and human microsome preparations. Two
major metabolites found in the hepatocytes were TSA amide,
which was obtained by reduction of the hydroxamic acid
group to amide, and N-monodemethylated TSA. The demethy-
lated metabolite was also detected in the microsomal prepara-
tion, but not the TSA amide.
ministration, the elimination half-life was found to be between
0.27 h (29b) and 1.22 h (24b). Furthermore, the three com-
pounds as well as the reference compound 1 exhibited high
clearance rates. In particular, inhibitors 24b and 29b (as well
as reference compound 1) showed clearance rates that exceed-
ed the hepatic blood flow of 5.4 Lhꢂ1 kgꢂ1 in mice.[35] The
steady-state volume of distribution Vss for 29b and 29e was in
the range of the total body volume, indicating a moderate
tissue distribution of these compounds. On the other hand,
24b exhibited a high Vss value of 5.2 Lkgꢂ1. Similarly to refer-
ence compound 1, the three amidopropenyl hydroxamates dis-
played a low oral bioavailability in mice with Fꢁ10%.
Overall, despite good in vitro ADME data (particularly the ex-
cellent stability of the compounds in human and mouse micro-
somes), the three selected derivatives showed surprisingly low
oral bioavailability in the PK studies in mice. However, limited
oral bioavailability and high clearance rates in rodent PK ex-
periments were also observed for several other HDAC inhibi-
tors containing a hydroxamic acid group.[36,14]
The rat and human hepatocyte preparations of compounds
24b, 29b, and 29e were submitted, together with reference
compound 1, to LC–MS–MS analysis and indeed, amidic me-
tabolites were detected in all samples. Thus, as further charac-
terization, a biotransformation experiment of 29b and 29e
was carried out. The compounds were incubated at 10 mm in
106 cellsmLꢂ1 at 378C. After 90 min the reaction was stopped
with acetonitrile, and LC–MS–MS analysis showed that 29b
and 29e were metabolized 77 and 86%, respectively, and that
the amidic analogues were the major metabolites in both
cases. A further hydroxylated metabolite of 29e was found,
while no relevant metabolites were detected for 29b. These re-
sults are an additional confirmation that a major metabolic
pathway of hydroxamic acid derivatives may be catalyzed by
non-microsomal enzymes, and that biotransformation investi-
The differences between the in vitro and in vivo properties,
particularly between the in vitro microsomal stability and the
high in vivo clearance of the three representative amidopro-
penyl derivatives, prompted us to perform a set of in vitro
studies in order to find plausible explanations. First, the three
selected inhibitors were submitted to a plasma stability assay.
The compounds were incubated at a concentration of 2.5 mm
in mouse plasma. After incubation for 1 h at 378C, a 50 mL ali-
quot of plasma was removed, quenched with acetonitrile
(140 mL), centrifuged, and the supernatant was analyzed by
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F. Thaler et al.
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7.00 min (A: 0%, B: 100%), 7.00–7.10 min (A: 98%, B: 2%); 7.10–
8.50 min (A: 98%, B: 2%).
gations in microsome preparations may not be predictive of
the in vivo clearance behavior of the studied examples.
Method 2: BEH C18 (1.7 mm, 2.1ꢁ50 mm) column; gradient: 0–
0.25 min (A: 100%, B: 0%), 0.25–3.30 min (A: 0%, B: 100%), 3.30–
4.00 min (A: 0%, B: 100%), 4.00–4.10 min (A: 100%, B: 0%); 4.10–
5.00 min (A: 100%, B: 0%).
Conclusions
In summary, the synthesis and biological characterization of
novel amidopropenyl hydroxamic acid derivatives has been de-
scribed. Several compounds showed good in vitro potency to-
gether with significant microsomal stability. Three representa-
tive compounds, 24b, 29b, and 29e, demonstrated nanomolar
potency against class I (HDACs 1, 3, and 8) and class II (4 and
6) HDACs, showing a profile similar to other previously de-
scribed pan-HDAC inhibitors. The remarkable stability of the
compounds in microsomes, however, was not confirmed by
in vivo PK experiments in mice. Additional studies indicated
that the three compounds showed high clearance rates in rat
and human hepatocytes, suggesting that a major metabolic
pathway for the hydroxamic acid derivatives may be catalyzed
by non-microsomal enzymes. Reduction of the hydroxamic
acid group to its corresponding amide was identified for the
three compounds as well as for SAHA as one relevant biotrans-
formation pathway. Similar observations have been previously
described for the HDAC inhibitor TSA.[39]
Method 3: BEH C18 (1.7 mm, 2.1ꢁ50 mm) column; gradient: 0–
0.25 min (A: 98%, B: 2%), 0.25–3.30 min (A: 0%, B: 100%), 3.30–
4.00 min (A: 0%, B: 100%), 4.00–4.10 min (A: 98%, B: 2%); 4.10–
5.00 min (A: 98%, B: 2%).
Method 4: BEH C18 (1.7 mm, 2.1ꢁ50 mm) column; gradient: 0–
0.25 min (A: 95%, B: 5%), 0.25–3.30 min (A: 0%, B: 100%), 3.30–
4.00 min (A: 0%, B: 100%), 4.00–4.10 min (A: 95%, B: 5%); 4.10–
5.00 min (A: 95%, B: 5%).
Method 5: Synergi (2.5 mm, 2.0ꢁ20 mm) column; gradient: 0–
0.25 min (A: 95%, B: 5%), 0.25–3.5 min (A: 0%, B: 100%), 3.35–
4.50 min (A: 0%, B: 100%), 4.50–4.60 min (A: 95%, B: 5%); 4.60–
6.00 min (A: 95%, B: 5%).
Method 6: BEH C18 (1.7 mm, 2.1ꢁ50 mm) column; gradient: 0–
0.50 min (A: 95%, B: 5%), 0.50–6.00 min (A: 0%, B: 100%), 6.00–
7.00 min (A: 0%, B: 100%), 7.00–7.10 min (A: 95%, B: 5%); 7.10–
8.50 min (A: 95%, B: 5%).
(E)-N-Hydroxy-3-{4-[(E)-3-(4-phenylpiperazin-1-yl)-3-oxo-1-
propen-1-yl]phenyl}acrylamide (17b): (E)-tert-Butyl-3-(4-formyl-
phenyl)acrylate[30] (9, 850 mg, 3.66 mmol) was dissolved in dry THF
(5 mL) and added dropwise to a stirred mixture of triethyl phos-
phonoacetate (0.878 mL, 4.43 mmol) and NaH (60% oil dispersion,
228 mg, 5.7 mmol) in dry THF (5 mL). The resulting solution was
stirred at room temperature for 7 h and then additional NaH (60%
oil dispersion, 100 mg, 2.5 mmol) was added. After stirring over-
night at room temperature the reaction was quenched with H2O
and the mixture was extracted with Et2O. The organic phase was
dried over Na2SO4 and evaporated in vacuo to afford ethyl (E)-3-[4-
((E)-2-tert-butoxycarbonylvinyl)phenyl]acrylate (11) (1.1 g), which
was used in the next step without further purification; LC–MS (ESI):
m/z: 303 [M+H]+.
Experimental Section
Chemistry
All reagents and solvents were of commercially available reagent
grade and were used without further purification. Flash chroma-
tography was performed on Merck silica gel 60 (0.04–0.063 mm).
1H and 13C NMR spectroscopic data were recorded on a Bruker
spectrometer at 300 and 75 MHz, respectively, with (CH3)4Si as in-
ternal standard, and, unless stated otherwise, at 300 K. Chemical
shift (d) values are given in ppm, and coupling constants (J) are ex-
pressed in Hz. Standard abbreviations indicating multiplicity are
used as follows: s=singlet, d=doublet, t=triplet, q=quartet, m=
multiplet, and bs=broad signal. HPLC–MS experiments were per-
formed with a Waters 2777 sample manager and a Waters 1525
binary HPLC pump equipped with a Waters 2996 diode array and a
Micromass ZQ 2000 single quadrupole (Waters), or on an Acquity
UPLC apparatus equipped with a diode array and a Micromass ZQ
single quadrupole (Waters). HPLC purity of the target compounds
was assessed either with a Waters 2777 sample manager and a
Waters 1525 binary HPLC pump equipped with a Waters 2996
diode array and a Micromass ZQ 2000 single quadrupole (Waters)
(Methods 1 and 5), or on an Acquity UPLC apparatus equipped
with a diode array and a Micromass ZQ single quadruple (Waters)
(all other methods). The flow rate was adjusted to 0.7 mLminꢂ1 for
Method 5 and 0.6 mLminꢂ1 for the other methods, and the split-
ting ratio between the amount submitted to the mass spectrome-
ter and to the waste was 1:4. Mobile phase A consisted of a mix-
ture of H2O and CH3CN (95:5) containing 0.1% TFA, and mobile
phase B consisted of a mixture of H2O and CH3CN (5:95) containing
0.1% TFA. Purity refers to UV detection at l 254 nm. High-resolu-
tion mass spectrometry experiments were performed on an Auto-
flex III smartbeam vertical MALDI-TOF (TOF/TOF) mass spectrome-
ter (Bruker Daltonics).
A solution of the tert-butyl ester 11 (2.75 g, 9.10 mmol) and TFA
(7 mL) in CH2Cl2 (20 mL) was stirred at room temperature for 1 h.
The solvent was removed in vacuo to give the acrylic acid as a
white solid (2.13 g, 95%). (E)-3-[4-((E)-2-Ethoxycarbonylvinyl)phenyl]-
acrylic acid (1.5 g, 6.1 mmol) was then dissolved in CH2Cl2 (80 mL)
and TEA (1.7 mL, 12 mmol). EDC (2.33 g, 12.2 mmol) and HOBt
(1.65 g, 12.2 mmol) were added to the resulting mixture. After
15 min, NH2OTHP (856 mg, 7.32 mmol) was added and the result-
ing mixture was stirred at room temperature for 5 h. The solution
was partitioned between 5% NaHCO3 and Et2O. The organic phase
was dried over Na2SO4, evaporated in vacuo, and purified by
column chromatography (petroleum ether/EtOAc 1:1) to give the
tetrahydropyran-2-yl-protected acrylamide 13 (1.94 g, 92%).
1H NMR (300 MHz, [D6]DMSO): d=11.24 (bs, 1H), 7.77 (d, J=
8.22 Hz, 2H), 7.66 (d, J=15.85 Hz, 1H), 7.57–7.65 (m, 2H), 7.50 (d,
J=16.14 Hz, 1H), 6.68 (d, J=16.14 Hz, 1H), 6.58 (d, J=16.43 Hz,
1H), 4.92 (bs, 1H), 4.20 (q, J=7.04 Hz, 2H), 3.96 (bs, 1H), 3.43–3.66
(m, 1H), 1.38–1.96 (m, 6H), 1.27 ppm (t, J=7.04 Hz, 3H).
LiOH (1m, 3 mL) was added to a stirred solution of 13 (520 mg,
1.5 mmol) in THF (10 mL) and the resulting mixture was stirred at
room temperature overnight. Further 1m LiOH (1.5 mL) was added,
and, after stirring at room temperature overnight, the solution was
partitioned between H2O and EtOAc. The aqueous phase was
Method 1: Atlantis dC18 (3 mm, 2.1ꢁ100 mm) column; gradient: 0–
0.50 min (A: 98%, B: 2%), 0.50–6.00 min (A: 0%, B: 100%), 6.00–
1366
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ChemMedChem 2010, 5, 1359 – 1372

Amidopropenyl Hydroxamates as HDAC Inhibitors
brought to acidic pH by adding citric acid (5% aqueous solution)
at 08C and extracted twice with EtOAc. The organic layers were
washed with brine, dried over Na2SO4, and evaporated in vacuo to
perature for 4 h. Further NH2OTHP (40 mg, 0.34 mmol) was added
and the mixture was stirred overnight. The solvent was then
evaporated and the residue was partitioned between H2O and
EtOAc. The organic layer was dried over Na2SO4 and evaporated to
dryness. The crude mixture was purified by column chromatogra-
phy (petroleum ether/EtOAc 4:6) to give the THP-protected hy-
droxamic acid 14 as a yellow powder (237 mg, 69%). 1H NMR
(300 MHz, [D6]DMSO): d=11.39 (bs, 1H), 8.92 (d, J=2.05 Hz, 1H),
8.22 (dd, J=8.07, 2.20 Hz, 1H), 7.27–7.88 (m, 3H), 7.02 (d, J=
15.26 Hz, 1H), 6.82 (d, J=16.14 Hz, 1H), 4.94 (bs, 1H), 4.21 (q, J=
7.24 Hz, 2H), 3.84–4.09 (m, 1H), 3.46–3.63 (m, 1H), 1.41–1.86 (m,
6H), 1.27 ppm (t, J=7.04 Hz, 3H).
1
give the acrylic acid 15 as a white powder (445 mg, 94%). H NMR
(300 MHz, [D6]DMSO): d=12.35 (bs, 1H), 11.23 (bs, 1H), 7.69–7.84
(m, 2H), 7.58–7.67 (m, 2H), 7.59 (d, J=16.14 Hz, 1H), 7.51 (d, J=
15.85 Hz, 1H), 6.57 (d, J=16.14 Hz, 2H), 4.92 (bs, 1H), 3.97 (bs, 1H),
3.43–3.71 (m, 1H), 1.31–1.95 ppm (m, 6H).
A mixture of 15 (200 mg, 0.63 mmol), EDC (240 mg, 1.26 mmol),
HOBt (170 mg, 1.26 mmol), TEA (0.354 mL, 2.52 mmol), and 1-phe-
nylpiperazine (192 mL, 1.26 mmol) in CH2Cl2 (6 mL) and DMF (1 mL)
was stirred overnight at room temperature. The mixture was then
partitioned between H2O and CH2Cl2. The aqueous phase was ex-
tracted with CH2Cl2 and the collected organic layers were dried
over Na2SO4 and evaporated to dryness. The crude mixture was pu-
rified by column chromatography (CH2Cl2/MeOH/NH4OH 90:10:0.1).
The resulting product was dissolved in CH2Cl2 (10 mL) and treated
with HCl/Et2O for 1 h. The precipitate was filtered off and triturated
with CH2Cl2 and Et2O to give the desired hydroxamic acid 17b
(166 mg, 63% from 15) as its HCl salt. 1H NMR (300 MHz,
[D6]DMSO): d=7.78 (m, 2H), 7.60 (m, 2H), 7.54 (d, J=15.26 Hz,
1H), 7.46 (d, J=15.85 Hz, 1H), 7.37 (d, J=15.26 Hz, 1H), 7.27–7.34
(m, 2H), 7.10–7.23 (m, 2H), 6.87–7.05 (m, 1H), 6.57 (d, J=15.85 Hz,
1H), 3.85 (bs, 4H), 3.27 ppm (bs, 4H); 13C NMR (75 MHz, [D6]DMSO):
d=164.97, 162.98, 141.74, 137.74, 136.56, 136.46, 129.98 (2C),
129.12 (2C), 128.28 (2C), 120.70, 119.35 (2C), 119.02, 52.03 (2C),
44.01, 40.58 ppm; HRMS-TOF (ESI): m/z [M+H]+ calcd for
C22H24N3O3: 378.1817, found: 378.1803; HPLC purity: 100%, tR =
1.56 min (Method 2).
LiOH (1m, 1.68 mL) was added to a stirred solution of 14 (292 mg,
0.843 mmol) in THF (8 mL) and the resulting mixture was stirred at
room temperature. Further 1m LiOH (1.26 mL) was added over
20 h. The solution was then brought to pH 6 by slow addition of a
4m HCl solution and then brought to basic pH value by adding
NH4OH. The solvent was concentrated and the residue was freeze-
dried. The crude acrylic acid 16 was dissolved in CH2Cl2 (8 mL),
DMF (8 mL), and TEA (0.234 mL, 1.68 mmol). EDC (322 mg,
1.68 mmol), HOBt (252 mg, 1.68 mmol), and 1-phenylpiperazine
(164 mg, 1.01 mmol) were added and the resulting slurry was
stirred at room temperature overnight. Further 1-phenylpiperazine
(136 mg, 0.838 mmol) was added and the mixture was heated until
dissolution of the slurry. After stirring for additional 6 h at room
temperature the solution was partitioned between H2O and EtOAc.
The resulting precipitate was filtered off and washed with EtOAc
and CH2Cl2. The obtained solid was suspended in CH2Cl2 and treat-
ed with HCl/Et2O for 4 h. The resulting precipitate was filtered off
and washed with CH2Cl2 to give the desired hydroxamic acid 18b
as its HCl salt (102 mg, 30% from 14). 1H NMR (300 MHz,
[D6]DMSO): d=8.96 (d, J=1.76 Hz, 1H), 8.34 (dd, J=8.07, 1.91 Hz,
1H), 7.73 (d, J=8.22 Hz, 1H), 7.44–7.67 (m, 3H), 7.27–7.41 (m, 2H),
7.10–7.25 (m, 2H), 7.01 (d, J=15.55 Hz, 1H), 6.92–7.02 (m, 1H),
3.67–4.15 (m, 4H), 3.30 ppm (bs, 4H); 13C NMR (75 MHz, [D6]DMSO):
d=164.46, 161.97, 154.02, 152.54, 148.41, 137.84, 137.70, 135.28,
132.10, 129.90 (2C), 125.70, 124.71, 121.66, 118.75 (2C), 51.45 (2C),
44.47, 40.88 ppm; HRMS-TOF (ESI): m/z [M+H]+ calcd for
C21H23N4O3: 379.1770, found: 379.1759; HPLC purity: 99%, tR =
1.54 min (Method 5).
(E)-N-Hydroxy-3-{4-[(E)-3-(4-methylpiperazin-1-yl)-3-oxo-1-
propen-1-yl]phenyl}acrylamide (17a): Compound 17a (35 mg,
yield 18%) was obtained as its HCl salt starting from the THP-pro-
tected hydroxamic acid 15 and 1-methylpiperazine following the
synthetic procedure of the hydroxamic acid 17b. 1H NMR
(300 MHz, [D6]DMSO): d=10.83 (bs, 1H), 7.77 (d, J=8.22 Hz, 2H),
7.60 (d, J=8.22 Hz, 2H), 7.55 (d, J=15.55 Hz, 1H), 7.46 (d, J=
15.85 Hz, 1H), 7.32 (d, J=15.55 Hz, 1H), 6.54 (d, J=15.85 Hz, 1H),
4.53 (d, J=13.50 Hz, 2H), 3.23–3.56 (m, 4H), 2.88–3.13 (m, 2H),
2.78 ppm (d, J=3.81 Hz, 3H); LC–MS (ESI): m/z: 316 [M+H]+; HPLC
purity: 99%, tR =2.31 min (Method 1).
(E)-N-Hydroxy-3-{5-[(E)-3-(4-methylpiperazin-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide (18a): Compound 18a
(52 mg, yield 19%) was obtained as its HCl salt by starting from
the THP-protected hydroxamic acid 16 and 1-methylpiperazine fol-
lowing the synthetic procedure of the hydroxamic acid 18b.
1H NMR (300 MHz, [D6]DMSO): d=10.86 (bs, 1H), 8.92 (d, J=
1.76 Hz, 1H), 8.25 (dd, J=8.22, 2.35 Hz, 1H), 7.67 (d, J=7.92 Hz,
1H), 7.60 (d, J=15.55 Hz, 1H), 7.51 (d, J=15.26 Hz, 1H), 7.46 (d,
J=15.26 Hz, 1H), 6.99 (d, J=15.26 Hz, 1H), 4.40–4.64 (m, 2H),
3.34–3.61 (m, 3H), 2.93–3.28 (m, 3H), 2.79 ppm (d, J=4.40 Hz, 3H);
LC–MS (ESI): m/z: 317 [M+H]+; HPLC purity: 95%, tR =0.55 min
(Method 2).
(E)-N-Hydroxy-3-{5-[(E)-3-(4-phenylpiperazin-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide (18b): A solution of triethyl
phosphonoacetate (373 mg, 1.66 mmol) and NaH (60% oil disper-
sion, 71.7 mg, 1.79 mmol) in dry THF (5 mL) was added dropwise
to a stirred solution of tert-butyl-(E)-3-(5-formylpyridin-2-yl)acry-
late[28] (10, 323 mg, 1.38 mmol) in dry THF (5 mL) under N2 atmos-
phere. The resulting mixture was stirred at room temperature for
4 h and was then partitioned between H2O and Et2O. The aqueous
layer was washed with Et2O, the collected organic phases were
dried over Na2SO4, and evaporated in vacuo to give the diester 12
1
(420 mg, quant). H NMR (300 MHz, CDCl3): d=8.77 (d, J=2.05 Hz,
1H), 7.86 (dd, J=8.22, 2.35 Hz, 1H), 7.68 (d, J=16.14 Hz, 1H), 7.60
(d, J=15.55 Hz, 1H), 7.45 (d, J=7.92 Hz, 1H), 6.89 (d, J=15.55 Hz,
1H), 6.54 (d, J=16.14 Hz, 1H), 4.31 (q, J=7.14 Hz, 2H), 1.56 (s, 9H),
1.37 ppm (t, J=7.19 Hz, 3H).
(E)-N-Hydroxy-3-{3-[(E)-3-(4-phenylpiperazin-1-yl)-3-oxo-1-
propen-1-yl]phenyl}acrylamide (24b): A mixture of 3-bromobenz-
aldehyde (19, 500 mg, 2.70 mmol), TEA (1.62 mL, 11.6 mmol), ethyl
acrylate (270 mg, 2.70 mmol), NaHCO3 (454 mg, 5.4 mmol), and
PPh3 (35 mg, 0.13 mmol) in DMF (13 mL) was degassed with N2
and then Pd(OAc)2 (12 mg, 0.054 mmol) was added. The resulting
slurry was heated at 1008C under N2 for 6 h and was then parti-
tioned between H2O and Et2O. The organic phase was washed
with H2O, dried over Na2SO4, and evaporated to dryness. The crude
reaction mixture was purified by column chromatography (petro-
A mixture of 12 (300 mg, 0.99 mmol) and TFA (4 mL) in CH2Cl2
(10 mL) was stirred at room temperature for 3 h. The solvent was
removed to dryness and the residue (380 mg) was dissolved in
CH2Cl2 (20 mL) and TEA (1.17 mL, 8.39 mmol). EDC (401 mg,
2.09 mmol), HOBt (283 mg, 2.10 mmol), and NH2OTHP (184 mg,
1.57 mmol) were added and the mixture was stirred at room tem-
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leum ether/EtOAc 95:5) to give the ethyl acrylate 20 (206 mg,
37%). H NMR (300 MHz, [D6]DMSO): d=10.04 (s, 1H), 8.26 (t, J=
1.61 Hz, 1H), 8.06 (dt, J=7.70, 1.43 Hz, 1H), 7.94 (dt, J=7.63,
1.32 Hz, 1H), 7.75 (d, J=16.14 Hz, 1H), 7.65 (t, J=7.63 Hz, 1H), 6.76
(d, J=16.14 Hz, 1H), 4.22 (q, J=7.04 Hz, 2H), 1.28 ppm (t, J=
7.04 Hz, 3H).
product was purified by column chromatography (CH2Cl2/MeOH/
NH4OH from 99:1:0.1 to 98:2:0.2), and the resulting product was
dissolved in CH2Cl2 (5 mL) and treated with HCl/Et2O for 2 h. The
precipitate was filtered off and washed with CH2Cl2 to give the de-
1
1
sired hydroxamic acid 24b as its HCl salt (238 mg, 73%). H NMR
(300 MHz, [D6]DMSO, 353 K): d=7.87 (t, J=1.61 Hz, 1H), 7.62–7.70
(m, 1H), 7.39–7.60 (m, 4H), 7.26–7.36 (m, 2H), 7.27 (t, J=9.10 Hz,
1H), 7.06–7.18 (m, 2H), 6.86–6.98 (m, 1H), 6.64 (d, J=15.85 Hz,
1H), 3.75–3.97 (m, 4H), 3.19–3.36 ppm (m, 4H); 13C NMR (75 MHz,
[D6]DMSO): d=164.94, 163.02, 142.01, 138.15, 136.17, 135.98,
130.08 (s, 2C), 129.80, 129.55, 128.45, 127.91, 120.55, 119.76 (2C),
119.03, 52.39 (2C), 43.92, 40.85 ppm; HRMS-TOF (ESI): m/z [M+H]+
calcd for C22H24N3O3: 378.1817, found: 378.1775; HPLC purity: 96%,
tR =1.60 min (Method 4).
A solution of 20 (206 mg, 1.01 mmol) in dry THF (5 mL) was added
dropwise under N2 atmosphere to a stirred mixture of tert-butyldi-
ethylphosphonoacetate (277 mg, 1.10 mmol) and NaH (60% oil dis-
persion, 52 mg, 1.3 mmol) in dry THF (10 mL), cooled to 08C. The
resulting solution was stirred at room temperature for 45 min, then
quenched with H2O, and the mixture was extracted with EtOAc.
The organic phase was dried, the solvent was removed in vacuo,
and the crude mixture was purified by column chromatography
(petroleum ether/EtOAc 95:5) to give the diester 21 (260 mg,
(E)-N-Hydroxy-3-{3-[(E)-3-(4-methylpiperazin-1-yl)-3-oxo-1-
propen-1-yl]phenyl}acrylamide (24a): Compound 24a (55 mg,
yield 65%) was obtained as its HCl salt starting from THP protected
hydroxamic acid 23 and 1-methylpiperazine following the synthetic
procedure of the hydroxamic acid 24b. 1H NMR (300 MHz,
[D6]DMSO+TFA): d=10.30 (bs, 1H), 7.94 (s, 1H), 7.72 (d, J=
7.63 Hz, 1H), 7.41–7.63 (m, 4H), 7.34 (d, J=15.26 Hz, 1H), 6.54 (d,
J=15.85 Hz, 1H), 4.55 (d, J=13.20 Hz, 2H), 3.48 (d, J=10.27 Hz,
3H), 2.94–3.22 (m, 3H), 2.82 ppm (s, 3H); LC–MS (ESI): m/z: 316
[M+H]+; HPLC purity: 97%, tR =0.95 min (Method 3).
1
86%). H NMR (300 MHz, [D6]DMSO): d=8.13 (s, 1H), 7.69–7.77 (m,
2H), 7.66 (d, J=16.14 Hz, 1H), 7.56 (d, J=16.14 Hz, 1H), 7.45 (t, J=
7.63 Hz, 1H), 6.78 (d, J=15.85 Hz, 1H), 6.67 (d, J=15.85 Hz, 1H),
4.21 (q, J=7.04 Hz, 2H), 1.50 (s, 9H), 1.27 ppm (t, J=7.19 Hz, 3H).
A mixture of the tert-butyl ester 21 (2.72 g, 9.00 mmol) and TFA
(13.9 mL) in CH2Cl2 (28 mL) was stirred at room temperature for
1 h. The solvent was removed in vacuo to give (E)-3-[3-((E)-2-ethoxy-
carbonylvinyl)phenyl]acrylic acid (2.13 g, 96%). 1H NMR (300 MHz,
[D6]DMSO): d=12.41 (bs, 1H), 8.11 (s, 1H), 7.67–7.78 (m, 2H), 7.67
(d, J=16.14 Hz, 1H), 7.60 (d, J=16.14 Hz, 1H), 7.46 (t, J=7.63 Hz,
1H), 6.77 (d, J=16.14 Hz, 1H), 6.67 (d, J=16.14 Hz, 1H), 4.21 (q,
J=7.04 Hz, 2H), 1.27 ppm (t, J=7.04 Hz, 3H). The acrylic acid
(2.13 g, 8.66 mmol) was then dissolved in a mixture of CH2Cl2
(80 mL) and TEA (2.4 mL, 17 mmol). EDC (3.31 g, 17.3 mmol) and
HOBt (2.34 g, 17.3 mmol) were then added to the resulting solu-
tion. After 15 min NH2OTHP (2.03 g, 17.3 mmol) was added and the
resulting mixture was stirred at room temperature overnight. The
solvent was evaporated and the residue was partitioned between
5% NaHCO3 and Et2O. The organic phase was dried over Na2SO4
and evaporated in vacuo. The crude mixture was purified by
column chromatography (petroleum ether/EtOAc 1:1) to give the
THP-protected hydroxamic acid 22 (2.62 g, 87%). 1H NMR
(300 MHz, [D6]DMSO): d=11.22 (bs, 1H), 7.95 (s, 1H), 7.66 (d, J=
15.85 Hz, 1H), 7.29–7.82 (m, 4H), 6.70 (d, J=15.85 Hz, 1H), 6.60 (d,
J=15.85 Hz, 1H), 4.93 (bs, 1H), 4.20 (q, J=7.24 Hz, 2H), 3.98 (d, J=
6.75 Hz, 1H), 3.54 (d, J=11.44 Hz, 1H), 1.35–1.89 (m, 6H), 1.27 ppm
(t, J=7.04 Hz, 3H).
(ꢃ)-(E)-N-Hydroxy-3-{6-[(E)-3-(3-phenylpiperidin-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide hydrochloride (29e): A solu-
tion of triethyl phosphonoacetate (3.34 g, 14.9 mmol) in dry THF
(10 mL) was added dropwise under N2 atmosphere to a stirred sus-
pension of NaH (60% oil dispersion, 705 mg, 17.6 mmol) in dry
THF (5 mL). The resulting mixture was stirred at room temperature
for 15 min and then added dropwise, using a syringe equipped
with a filtering septum, to a stirred solution of tert-butyl-(E)-3-(6-
formylpyridin-2-yl)acrylate[28] (25, 3.16 g, 13.6 mmol) in dry THF
(40 mL), keeping the temperature <258C. The mixture was stirred
at room temperature for 1.5 h, then diluted with EtOAc and
washed with a saturated NH4Cl solution. The aqueous phase was
washed twice with EtOAc, the collected organic layers were dried
over Na2SO4 and then evaporated to dryness. The crude product
was purified by column chromatography (petroleum ether/EtOAc
from 95:5 to 9:1) to give the diester 26 (3.92 g, 95%). 1H NMR
(300 MHz, [D6]DMSO): d=7.93 (t, J=7.63 Hz, 1H), 7.76 (d, J=
7.63 Hz, 1H), 7.75 (d, J=7.63 Hz, 1H), 7.67 (d, J=15.55 Hz, 1H),
7.58 (d, J=15.55 Hz, 1H), 6.98 (d, J=15.85 Hz, 1H), 6.89 (d, J=
15.85 Hz, 1H), 4.23 (q, J=7.04 Hz, 2H), 1.50 (s, 9H), 1.28 ppm (t, J=
7.04 Hz, 3H). A mixture of 26 (3.92 g, 12.9 mmol) and TFA (3.99 mL)
in CH2Cl2 (50 mL) was stirred at room temperature for 24 h. Further
TFA (2 mL) was added and the reaction mixture was stirred for ad-
ditional 5 h at 308C. Then the solvent was removed in vacuo, the
residue was triturated with Et2O and decanted to give (E)-3-[6-((E)-
2-ethoxycarbonylvinyl)pyridin-2-yl]acrylic acid as its trifluoroacetate
LiOH (1m, 15.2 mL) was added to a stirred solution of 22 (2.62 g,
7.59 mmol) in THF (50 mL) and the mixture was stirred at room
temperature overnight. Further 1m LiOH (10 mL) was added and
the reaction was carried out overnight. The solution was then par-
titioned between H2O and EtOAc. The aqueous phase was brought
to acidic pH by adding citric acid (5% aqueous solution) at 08C
and was extracted twice with EtOAc. The organic layer was washed
with brine, dried over Na2SO4, and evaporated in vacuo to give the
1
salt (4.6 g, 98%). H NMR (300 MHz, [D6]DMSO): d=12.58 (bs, 1H),
1
acrylic acid 23 as a white powder (2.01 g, 83%). H NMR (300 MHz,
7.93 (t, J=7.63 Hz, 1H), 7.76 (d, J=7.04 Hz, 1H), 7.74 (d, J=
7.63 Hz, 1H), 7.68 (d, J=15.85 Hz, 1H), 7.61 (d, J=15.85 Hz, 1H),
6.98 (d, J=15.55 Hz, 1H), 6.92 (d, J=15.55 Hz, 1H), 4.23 (q, J=
7.24 Hz, 2H), 1.28 ppm (t, J=7.19 Hz, 3H).
[D6]DMSO): d=11.22 (bs, 1H), 7.90 (s, 1H), 7.65–7.76 (m, 1H), 7.60
(d, J=15.85 Hz, 1H), 7.32–7.65 (m, 3H), 6.60 (d, J=16.14 Hz, 2H),
4.92 (bs, 1H), 3.80–4.15 (m, 1H), 3.46–3.69 (m, 1H), 1.31–1.95 ppm
(m, 6H). The THP-protected hydroxamic acid 23 (250 mg,
0.79 mmol) was dissolved in CH2Cl2 (10 mL) and TEA (0.110 mL,
0.789 mmol). EDC (226 mg, 1.18 mmol), HOBt (159 mg, 1.18 mmol),
and 1-phenylpiperazine (153 mg, 0.947 mmol) were added and the
mixture was stirred overnight at room temperature. The resulting
solution was partitioned between brine and CH2Cl2 and the aque-
ous phase was extracted with CH2Cl2. The collected organic layers
were dried over Na2SO4 and evaporated to dryness. The crude
The acrylic acid (8.49 g, 23.5 mmol) was then dissolved in CH2Cl2
(150 mL) and TEA (3.27 mL, 23.5 mmol). EDC (4.49 g, 23.5 mmol),
HOBt (3.18 g, 23.5 mmol), and NH2OTHP (2.75 g, 23.5 mmol) were
added and the resulting mixture was stirred at room temperature
for 5 h. Further EDC (1.35 g, 7.07 mmol), HOBt (952 mg,
7.05 mmol), and NH2OTHP (819 mg, 7.00 mmol) were added. The
solution was stirred at room temperature overnight and was then
1368
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1359 – 1372

Amidopropenyl Hydroxamates as HDAC Inhibitors
washed with H2O and brine. The organic phase was dried over
Na2SO4, evaporated to dryness, and the crude product was purified
by column chromatography (petroleum ether/EtOAc from 65:35 to
45:55) to give the tetrahydropyran-2-yl-protected acrylamide 27 as
a white powder (6.58 g, 81%). 1H NMR (300 MHz, [D6]DMSO): d=
11.39 (bs, 1H), 7.91 (t, J=7.63 Hz, 1H), 7.71 (d, J=7.34 Hz, 1H),
7.69 (d, J=15.85 Hz, 1H), 7.61 (d, J=7.63 Hz, 1H), 7.52 (d, J=
15.26 Hz, 1H), 7.10 (d, J=15.55 Hz, 1H), 7.00 (d, J=15.55 Hz, 1H),
4.94 (bs, 1H), 4.23 (q, J=7.04 Hz, 2H), 3.80–4.04 (m, 1H), 3.41–3.68
(m, 1H), 1.42–1.87 (m, 6H), 1.28 ppm (t, J=7.04 Hz, 3H).
mic acid 28 and 1-phenylpiperazine following the synthetic proce-
dure of the hydroxamic acid 29e. 1H NMR (300 MHz, [D6]DMSO):
d=7.93 (t, J=7.63 Hz, 1H), 7.80 (d, J=7.34 Hz, 1H), 7.66 (d, J=
15.26 Hz, 1H), 7.60 (d, J=8.22 Hz, 1H), 7.55 (d, J=15.55 Hz, 1H),
7.51 (d, J=15.26 Hz, 1H), 7.32 (t, J=8.07 Hz, 2H), 7.18 (d, J=
7.92 Hz, 2H), 7.04 (d, J=15.26 Hz, 1H), 6.93–7.01 (m, 1H), 3.67–4.06
(m, 4H), 3.17–3.45 ppm (m, 4H); 13C NMR (75 MHz, [D6]DMSO): d=
164.49, 162.31, 153.23, 153.04, 147.35, 140.63, 139.29, 136.71,
129.97 (2C), 124.91, 124.60 (2 C) 124.26, 123.20, 119.00 (2C), 51.51
(2C), 44.43, 40.85 ppm; HRMS-TOF (ESI): m/z [M+H]+ calcd for
C21H23N4O3: 379.1770, found: 379.1771; HPLC purity: 97%, tR =
1.81 min (Method 4).
NaOH (4n, 11.38 mL) was added dropwise to a stirred solution of
27 (6.58 g, 19.0 mmol) in THF (200 mL). The mixture was stirred at
room temperature for 2 h and was then brought to pH 5 with
citric acid (20% aqueous solution). The solution was concentrated
in vacuo, and the resulting slurry was acidified to pH 4 with citric
acid and extracted with EtOAc and CH2Cl2 several times. The col-
lected organic layers were dried over Na2SO4 and evaporated to
dryness. The crude mixture was triturated in EtOAc, THF, and petro-
leum ether to give the acrylic acid 28 as a white powder (5.7 g,
(ꢃ)-(E)-N-Hydroxy-3-{6-[(E)-3-(3-phenylpyrrolidin-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide hydrochloride (29c): Com-
pound 29c (yield 28%) was prepared by starting from the THP-
protected hydroxamic acid 28 and 3-phenylpyrrolidine according
to the procedure described for the synthesis of 29e. 1H NMR
(300 MHz, [D6]DMSO): d=7.90 (t, J=7.92 Hz, 1H), 7.76 (d, J=
7.19 Hz, 1H), 7.13–7.65 (m, 9H), 7.00 (d, J=15.55 Hz, 1H), 3.88–4.08
(m, 1H), 3.07–4.50 (m, 4H), 2.20–2.47 (m, 1H), 1.80–2.20 ppm (m,
1H); LC–MS (ESI): m/z: 364 [M+H]+; HPLC purity: 95%, tR =
1.64 min (Method 4).
1
95%). H NMR (300 MHz, [D6]DMSO): d=12.30 (bs, 1H), 11.41 (bs,
1H), 7.90 (t, J=7.63 Hz, 1H), 7.67 (d, J=7.34 Hz, 1H), 7.60 (d, J=
7.63 Hz, 1H), 7.61 (d, J=15.55 Hz, 1H), 7.54 (d, J=15.26 Hz, 1H),
7.09 (d, J=15.55 Hz, 1H), 6.93 (d, J=15.55 Hz, 1H), 4.95 (bs, 1H),
3.84–4.11 (m, 1H), 3.36–3.74 (m, 1H), 1.28–2.01 ppm (m, 6H).
(ꢃ)-(E)-N-Hydroxy-3-{6-[(E)-3-(2-phenylpiperidin-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide hydrochloride (29d): Com-
pound 29d (yield 36%) was prepared by starting from the THP-
protected hydroxamic acid 28 and 2-phenylpiperidine according to
the synthetic procedure of the hydroxamic acid 29e. 1H NMR
(300 MHz, [D6]DMSO, 353 K): d=7.84 (t, J=7.63 Hz, 1H), 7.64 (d,
J=7.63 Hz, 1H), 7.56 (d, J=15.55 Hz, 1H), 7.48–7.56 (m, 1H), 7.49
(d, J=15.55 Hz, 1H), 7.47 (d, J=15.55 Hz, 1H), 7.33–7.43 (m, 2H),
7.20–7.34 (m, 3H), 7.00 (d, J=15.55 Hz, 1H), 5.44–5.95 (m, 1H),
3.90–4.48 (m, 1H), 2.79–3.13 (m, 1H), 2.29–2.47 (m, 1H), 1.81–2.08
(m, 1H), 1.35–1.81 ppm (m, 4H); LC–MS (ESI): m/z: 378 [M+H]+;
HPLC purity: 95%, tR =1.79 min (Method 4).
3-Phenylpiperidine (42.5 mg, 0.264 mmol) was added to a stirred
solution of 28 (70 mg, 0.22 mmol), TEA (0.061 mL, 0.44 mmol), EDC
(63 mg, 0.33 mmol), and HOBt (45 mg, 0.33 mmol) in CH2Cl2 (3 mL).
The mixture was stirred at room temperature for 8 h and then
washed with 1m K2CO3. The layers were separated by a phase sep-
arator cartridge and the organic layer was shaken overnight in the
presence of PS-isocyanate (240 mg, loading: 1.58 mmolgꢂ1). The
resin was filtered off and the solvent was evaporated in vacuo. The
crude product was purified using a SiO2 cartridge (CH2Cl2/MeOH/
NH4OH 98:2:0.2) and the resulting product was dissolved in CH2Cl2
and treated with HCl/Et2O for 2 h. The precipitate was filtered off
(E)-N-Hydroxy-3-{6-[(E)-3-(4-phenylpiperidin-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide hydrochloride (29 f): Com-
pound 29 f (yield 49%) was prepared by starting from the THP-
protected hydroxamic acid 28 and 4-phenylpiperidine according to
1
to give the title compound as its HCl salt (44.4 mg, 53%). H NMR
(300 MHz, [D6]DMSO, 353 K+TFA): d=7.86 (t, J=7.63 Hz, 1H), 7.70
(d, J=7.04 Hz, 1H), 7.55 (d, J=15.85 Hz, 1H), 7.48–7.54 (m, 1H),
7.48 (d, J=15.55 Hz, 1H), 7.46 (d, J=15.26 Hz, 1H), 7.27–7.38 (m,
4H), 7.16–7.28 (m, 1H), 7.01 (d, J=15.85 Hz, 1H), 4.12–4.45 (m,
2H), 3.04 (bs, 1H), 2.62–2.82 (m, 2H), 1.92–2.13 (m, 1H), 1.69–1.92
(m, 2H), 1.40–1.69 ppm (m, 1H); 13C NMR (75 MHz, [D6]DMSO): d=
164.24, 162.13, 152.96, 143.95, 139.99, 128.93 (4C), 127.68, 127.46,
127.03, 124.50, 124.30, 52.07, 48.60, 46.24, 43.69, 42.56, 31.98,
26.76, 25.43 ppm; HRMS-TOF (ESI): m/z [M+H]+ calcd for
C22H24N3O3: 378.1817, found: 378.1850; HPLC purity: 98%, tR =
1.83 min (Method 4).
1
the methodology described for the hydroxamic acid 29e. H NMR
(300 MHz, [D6]DMSO): d=7.90 (t, J=7.78 Hz, 1H), 7.77 (d, J=
7.04 Hz, 1H), 7.63 (d, J=15.55 Hz, 1H), 7.57 (d, J=7.34 Hz, 1H),
7.51 (d, J=15.26 Hz, 1H), 7.50 (d, J=15.55 Hz, 1H), 7.14–7.38 (m,
5H), 7.01 (d, J=15.26 Hz, 1H), 4.54–4.83 (m, 1H), 4.21–4.46 (m,
1H), 3.05–3.40 (m, 1H), 2.67–2.96 (m, 2H), 1.77–2.05 (m, 2H), 1.41–
1.74 ppm (m, 2H); LC–MS (ESI): m/z: 378 [M+H]+; HPLC purity:
99%, tR =1.81 min (Method 4).
(ꢃ)-(E)-N-Hydroxy-3-{6-[(E)-3-(3-phenylpiperazin-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide hydrochloride (29g): Com-
pound 29g (yield 28%) was prepared by starting from the THP-
protected hydroxamic acid 28 according to the methodology de-
(E)-N-Hydroxy-3-{6-[(E)-3-(4-methylpiperazin-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide (29a): Compound 29a
(55 mg, yield 78%) was prepared by starting from the THP-protect-
ed hydroxamic acid 28 and 1-methylpiperazine following the syn-
thetic procedure of the hydroxamic acid 29e. 1H NMR (300 MHz,
[D6]DMSO+TFA): d=10.68 (bs, 1H), 7.91 (t, J=7.78 Hz, 1H), 7.77
(d, J=7.34 Hz, 1H), 7.60 (d, J=15.26 Hz, 1H), 7.57–7.61 (m, 1H),
7.54 (d, J=15.26 Hz, 1H), 7.50 (d, J=15.55 Hz, 1H), 7.03 (d, J=
15.55 Hz, 1H), 4.32–4.69 (m, 2H), 3.38–3.72 (m, 3H), 2.94–3.27 (m,
3H), 2.81 ppm (s, 3H); LC–MS (ESI): m/z: 317 [M+H]+; HPLC purity:
98%, tR =0.81 min (Method 6).
1
scribed for the hydroxamic acid 29e. H NMR (300 MHz, [D6]DMSO,
353 K+Na2CO3): d=10.76 (bs, 1H), 8.82 (bs, 1H), 7.86 (t, J=
7.78 Hz, 1H), 7.67–7.76 (m, 1H), 7.56–7.64 (m, 2H), 7.29–7.57 (m,
7H), 7.05 (d, 1H), 4.29–4.64 (m, 2H), 4.05 (dd, J=10.86, 2.64 Hz,
1H), 3.39 (dd, J=10.71, 4.55 Hz, 1H), 3.21–3.32 (m, 1H), 2.92–
3.03 ppm (m, 2H); LC–MS (ESI): m/z: 379 [M+H]+; HPLC purity:
98%, tR =1.01 min (Method 4).
(ꢃ)-(E)-N-Hydroxy-3-{6-[(E)-3-(4-methyl-3-phenylpiperazin-1-yl)-
(E)-N-Hydroxy-3-{6-[(E)-3-(4-phenylpiperazin-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide (29b): Compound 29b (yield
50%) was prepared by starting from the THP-protected hydroxa-
3-oxo-1-propen-1-yl]pyridin-2-yl}acrylamide
hydrochloride
(29h): Compound 29h (yield 38%) was prepared by starting from
the THP-protected hydroxamic acid 28 according to the synthetic
ChemMedChem 2010, 5, 1359 – 1372
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1369

F. Thaler et al.
MED
procedure of the hydroxamic acid 29e. 1H NMR (300 MHz,
[D6]DMSO, 353 K): d=7.87 (t, J=7.63 Hz, 1H), 7.72 (d, J=7.34 Hz,
1H), 7.67–7.83 (m, 2H), 7.38–7.62 (m, 7H), 7.04 (d, J=15.85 Hz,
1H), 4.16–4.72 (m, 3H), 3.80–4.10 (m, 1H), 3.46–3.81 (m, 2H), 3.27
(td, J=12.18, 3.81 Hz, 1H), 2.52 ppm (s, 3H); LC–MS (ESI): m/z: 393
[M-H]+; HPLC purity: 97%, tR =1.48 min (Method 6).
(ꢃ)-(E)-N-Hydroxy-3-(6-{(E)-3-[3-(3-fluorophenyl)piperidin-1-yl]-3-
oxo-1-propen-1-yl}pyridin-2-yl)acrylamide hydrochloride (29m):
Compound 29m (yield 64%) was prepared by starting from the
THP-protected hydroxamic acid 28 and 3-(3-fluorophenyl)piperi-
dine according to the procedure described for the hydroxamic acid
29e. 1H NMR (300 MHz, [D6]DMSO, 353 K): d=7.85 (t, J=7.78 Hz,
1H), 7.68 (dd, J=7.92, 0.88 Hz, 1H), 7.29–7.59 (m, 5H), 7.10–7.19
(m, 2H), 6.97–7.07 (m, 2H), 4.23–4.55 (m, 2H), 2.98–3.17 (m, 2H),
2.67–2.90 (m, 1H), 1.93–2.14 (m, 1H), 1.71–1.90 (m, 2H), 1.52–
1.66 ppm (m, 1H); LC–MS (ESI): m/z: 396 [M+H]+; HPLC purity:
98%, tR =1.84 min (Method 4).
(ꢃ)-(E)-N-Hydroxy-3-{6-[(E)-3-(4-phenylazepan-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide hydrochloride (29i): Com-
pound 29i (yield 24%) was prepared by starting from the THP-pro-
tected hydroxamic acid 28 and 4-phenylazepane according to the
methodology described for the hydroxamic acid 29e. 1H NMR
(300 MHz, [D6]DMSO): d=7.82–8.00 (m, 1H), 7.77 (d, J=7.19 Hz,
1H), 7.40–7.69 (m, 4H), 7.08–7.40 (m, 5H), 6.99 (d, J=15.41 Hz,
1H), 3.25–4.12 (m, 4H), 2.59–2.82 (m, 1H), 1.90–2.10 (m, 2H), 1.62–
1.88 ppm (m, 4H); LC–MS (ESI): m/z: 392 [M+H]+; HPLC purity:
98%, tR =1.84 min (Method 4).
(ꢃ)-(E)-N-Hydroxy-3-(6-{(E)-3-[3-(4-fluorophenyl)piperidin-1-yl]-3-
oxo-1-propen-1-yl}pyridin-2-yl)acrylamide hydrochloride (29n):
Compound 29n (yield 44%) was prepared by starting from the
THP-protected hydroxamic acid 28 and 3-(4-fluorophenyl)piperi-
dine according to the procedure described for the hydroxamic acid
29e. 1H NMR (300 MHz, [D6]DMSO): d=7.75–8.09 (m, 2H), 7.27–
7.75 (m, 6H), 6.66–7.26 (m, 3H), 4.38–4.77 (m, 1H), 3.87–4.39 (m,
1H), 3.02–3.47 (m, 1H), 2.53–3.01 (m, 2H), 1.20–2.18 ppm (m, 4H);
LC–MS (ESI): m/z: 396 [M+H]+; HPLC purity: 96%, tR =1.82 min
(Method 4).
(+)-(E)-N-Hydroxy-3-{6-[(E)-3-((R)-3-phenylpiperidin-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide hydrochloride (29j): Com-
pound 29j (yield 56%) was prepared by starting from the THP-pro-
tected hydroxamic acid 28 and (R)-3-phenylpiperidine according to
the methodology described for the hydroxamic acid 29e. [a]D =
+137.5 (c=0.5 in MeOH); 1H NMR (300 MHz, MeOD, 333 K): d=
8.25 (t, J=7.92 Hz, 1H), 8.01 (d, J=7.63 Hz, 1H), 7.94 (d, J=
8.22 Hz, 1H), 7.52–7.82 (m, 3H), 7.18–7.39 (m, 5H), 7.07 (bs, 1H),
4.66 (bs, 1H), 4.30 (bs, 1H), 3.33–3.43 (m, 1H), 2.74–2.98 (m, 2H),
2.06–2.14 (m, 1H), 1.84–2.02 (m, 2H), 1.63–1.78 ppm (m, 1H);
13C NMR (75 MHz, [D6]DMSO): d=164.24, 162.13, 152.96, 143.95,
139.99, 128.93 (4C), 127.68, 127.46, 127.03, 124.50, 124.30, 52.07,
48.60, 46.24, 43.69, 42.56, 31.98, 26.76, 25.43 ppm; HRMS-TOF (ESI):
m/z [M+H]+ calcd for C22H24N3O3: 378.1817, found: 378.1794; HPLC
purity: 99%, tR =1.78 min (Method 4).
(ꢃ)-(E)-N-Hydroxy-3-{6-[(E)-3-(3-(o-tolyl)piperidin-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide hydrochloride (29o): Com-
pound 29o (yield 67%) was prepared by starting from the THP-
protected hydroxamic acid 28 and 3-(o-tolyl)piperidine following
the synthetic procedure of the hydroxamic acid 29e. 1H NMR
(300 MHz, [D6]DMSO): d=6.56–8.27 (m, 11H), 4.41–4.74 (m, 1H),
3.96–4.41 (m, 1H), 3.01–3.36 (m, 1H), 2.56–3.03 (m, 2H), 2.32 (s,
3H), 1.69–2.06 (m, 3H), 1.34–1.69 ppm (m, 1H); LC–MS (ESI): m/z:
392 [M+H]+; HPLC purity: 94%, tR =1.90 min (Method 4).
(ꢃ)-(E)-N-Hydroxy-3-{6-[(E)-3-(3-(m-tolyl)piperidin-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide hydrochloride (29p): Com-
pound 29p (yield 40%) was prepared by starting from the THP-
protected hydroxamic acid 28 and 3-(m-tolyl)piperidine following
the synthetic procedure of the hydroxamic acid 29e. 1H NMR
(300 MHz, [D6]DMSO+Na2CO3): d=9.83 (bs, 1H), 7.27–7.93 (m,
6H), 7.22 (t, J=7.34 Hz, 1H), 6.78–7.15 (m, 4H), 4.39–4.80 (m, 1H),
4.02–4.40 (m, 1H), 2.92–3.26 (m, 1H), 2.53–2.90 (m, 2H), 2.30 (s,
3H), 1.65–2.08 (m, 3H), 1.28–1.66 ppm (m, 1H); LC–MS (ESI): m/z:
392 [M+H]+; HPLC purity: 98%, tR =1.94 min (Method 4).
(ꢂ)-(E)-N-Hydroxy-3-{6-[(E)-3-((S)-3-phenylpiperidin-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide hydrochloride (29k): Com-
pound 29k (yield 29%) was prepared by starting from the THP-
protected hydroxamic acid 28 and (S)-3-phenylpiperidine accord-
ing to the methodology described for the hydroxamic acid 29e.
[a]D =ꢂ112.9 (c=0.5 in MeOH); 1H NMR (300 MHz, [D6]DMSO,
353 K): d=7.85 (t, J=7.78 Hz, 1H), 7.68 (d, J=7.04 Hz, 1H), 7.39–
7.64 (m, 4H), 7.16–7.39 (m, 5H), 7.02 (d, J=15.26 Hz, 1H), 3.63–
4.83 (m, 1H), 2.85–3.32 (m, 2H), 2.56–2.85 (m, 2H), 1.94–2.11 (m,
1H), 1.68–1.94 (m, 2H), 1.39–1.70 ppm (m, 1H); 13C NMR (75 MHz,
[D6]DMSO): d=164.24, 162.13, 152.96, 143.95, 139.99, 128.93 (4C),
127.68, 127.46, 127.03, 124.50, 124.30, 52.07, 48.60, 46.24, 43.69,
42.56, 31.98, 26.76, 25.43 ppm; HRMS-TOF (ESI): m/z [M+H]+ calcd
for C22H24N3O3: 378.1817, found: 378.1816; HPLC purity: 95%, tR =
1.78 min (Method 4).
(ꢃ)-(E)-N-Hydroxy-3-{6-[(E)-3-(3-(p-tolyl)piperidin-1-yl)-3-oxo-1-
propen-1-yl]pyridin-2-yl}acrylamide hydrochloride (29q): Com-
pound 29q (yield 50%) was prepared by starting from the THP-
protected hydroxamic acid 28 and 3-(p-tolyl)piperidine following
the synthetic procedure of the hydroxamic acid 29e. 1H NMR
(300 MHz, [D6]DMSO): d=7.71–8.15 (m, 2H), 7.34–7.74 (m, 4H),
6.71–7.34 (m, 5H), 4.35–4.79 (m, 1H), 3.88–4.38 (m, 1H), 3.01–3.42
(m, 1H), 2.55–2.99 (m, 2H), 2.28 (s, 3H), 1.24–2.10 ppm (m, 4H);
LC–MS (ESI): m/z: 392 [M+H]+; HPLC purity: 97%, tR =1.94 min
(Method 4).
(ꢃ)-(E)-N-Hydroxy-3-(6-{(E)-3-[3-(2-fluorophenyl)piperidin-1-yl]-3-
oxo-1-propen-1-yl}pyridin-2-yl)acrylamide hydrochloride (29l):
Compound 29l (yield 53%) was prepared by starting from the
THP-protected hydroxamic acid 28 and 3-(2-fluorophenyl)piperi-
dine according to the procedure described for the hydroxamic acid
29e. 1H NMR (300 MHz, [D6]DMSO, 353 K): d=7.85 (t, J=7.78 Hz,
1H), 7.66 (d, J=7.63 Hz, 1H), 7.37–7.57 (m, 5H), 7.11–7.34 (m, 3H),
7.02 (d, J=15.85 Hz, 1H), 4.16–4.49 (m, 2H), 2.95–3.20 (m, 3H),
1.79–2.03 (m, 3H), 1.54–1.69 ppm (m, 1H); 13C NMR (75 MHz,
[D6]DMSO): d=164.34, 162.24, 162.15, 153.20, 152.71, 139.59 (2C),
136.39, 130.14, 128.94, 128.83, 128.77, 125.12, 124.23 (3C), 115.84,
47.29, 46.22, 35.76, 30.62, 26.71 ppm; HRMS-TOF (ESI): m/z [M+H]+
calcd for C22H23FN3O3: 396.1724, found: 396.1750; HPLC purity:
99%, tR =1.82 min (Method 4).
(ꢃ)-(E)-N-Hydroxy-3-(6-{(E)-3-[3-(2-methoxyphenyl)piperidin-1-
yl]-3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide
hydrochloride
(29r): Compound 29r (yield 53%) was prepared by starting from
the THP-protected hydroxamic acid 28 and 3-(2-methoxyphenyl)pi-
peridine according to the procedure described for the hydroxamic
acid 29e. 1H NMR (300 MHz, [D6]DMSO, 353 K): d=7.74–7.94 (m,
1H), 7.35–7.75 (m, 5H), 7.10–7.35 (m, 2H), 6.89–7.12 (m, 2H), 6.88
(d, J=15.85 Hz, 1H), 3.99–4.84 (m, 2H), 3.54–3.99 (m, 3H), 2.71–
3.38 (m, 3H), 1.72–2.23 (m, 3H), 1.30–1.72 ppm (m, 1H); LC–MS
(ESI): m/z: 408 [M+H]+; HPLC purity: 98%, tR =1.86 min
(Method 4).
1370
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1359 – 1372

Amidopropenyl Hydroxamates as HDAC Inhibitors
(ꢃ)-(E)-N-Hydroxy-3-(6-{(E)-3-[3-(3-methoxyphenyl)piperidin-1-
formed in duplicate. IC50 values were determined from 12-point
concentration–response curves using a nonlinear regression analy-
sis.
yl]-3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide
hydrochloride
(29s): Compound 29s (yield 47%) was prepared by starting from
the THP-protected hydroxamic acid 28 and 3-(3-methoxyphenyl)pi-
peridine according to the procedure described for the hydroxamic
acid 29e. 1H NMR (300 MHz, MeOD, 333 K): d=8.38 (dd, J=
7.92 Hz, 1H), 7.99–8.23 (m, 2H), 7.59–7.86 (m, 3H), 7.25 (dd, J=
7.92 Hz, 1H), 7.10 (d, J=16.14 Hz, 1H), 6.74–6.96 (m, 3H), 4.64 (bs,
1H), 4.29 (bs, 1H), 3.80 (s, 3H), 3.21–3.48 (m, 1H), 2.71–3.05 (m,
2H), 2.05–2.15 (m, 1H), 1.80–2.02 (m, 2H), 1.62–1.77 ppm (m, 1H);
LC–MS (ESI): m/z: 408 [M+H]+; HPLC purity: 98%, tR =1.78 min
(Method 4).
Cell growth assay: The antiproliferative effect of the HDAC inhibi-
tors on cell proliferation was evaluated against K562 (chronic mye-
loid carcinoma), A549 (non-small-cell lung cancer) and HCT-116
(human colon cancer) cell lines using the CellTiter-Glo Luminescent
Cell Viability Assay (Promega, Madison, WI, USA) according to the
manufacturer’s instructions. K562, A549, and HCT-116 cells were in-
cubated for 72 h with various inhibitor concentrations. An equiva-
lent of the CellTiter-Glo reagent was then added, the solution was
mixed for 2 min in order to induce cell lysis, and the luminescence
was recorded after a further 10 min. IC50 values were calculated
using GraphPad software.
(ꢃ)-(E)-N-Hydroxy-3-(6-{(E)-3-[3-(4-methoxyphenyl)piperidin-1-
yl]-3-oxo-1-propen-1-yl}pyridin-2-yl)acrylamide
hydrochloride
(29t): Compound 29t (yield 41%) was prepared by starting from
the THP-protected hydroxamic acid 28 and 3-(4-methoxyphenyl)pi-
peridine according to the procedure described for the hydroxamic
Metabolic stability in hepatic microsomes and hepatocytes:
Sample preparation: By adapting the protocols described by Di
et al.,[41] the compounds at 1 mm concentration were pre-incubated
for 10 min at 378C in potassium phosphate buffer (pH 7.4) togeth-
er with either 0.5 mgmLꢂ1 mouse or human hepatic microsomes
(Xenotech, Kansas City, MO, USA). The cofactor mixture comprising
NADP, G6P and G6P-DH was added, and aliquots were taken after
0 and 30 min, quenched with CH3CN, separated by centrifugation,
and analyzed by LC–MS–MS.
1
acid 29e. H NMR (300 MHz, [D6]DMSO, 353 K+TFA): d=7.85 (dd,
J=7.63 Hz, 1H), 7.60–7.73 (m, 1H), 7.36–7.60 (m, 4H), 7.22 (m, 2H),
7.02 (d, J=15.26 Hz, 1H), 6.90 (m, 2H), 4.34 (bs, 2H), 3.75 (s, 3H),
3.00 (bs, 2H), 2.59–2.80 (m, 1H), 1.92–2.07 (m, 1H), 1.78–1.92 (m,
1H), 1.66–1.78 (m, 1H), 1.48–1.66 ppm (m, 1H); LC–MS (ESI): m/z:
408 [M+H]+; HPLC purity: 99%, tR =1.76 min (Method 4).
(ꢃ)-(E)-N-Hydroxy-3-{6-[(E)-3-(3-naphthalen-1-ylpiperidin-1-yl)-3-
oxo-1-propen-1-yl]pyridin-2-yl}acrylamide hydrochloride (29u):
Compound 29u (yield 46%) was prepared by starting from the
THP-protected hydroxamic acid 28 and 3-naphthalen-1-ylpiperidine
following the synthetic procedure of the hydroxamic acid 29e.
1H NMR (300 MHz, [D6]DMSO, 353 K): d=8.04–8.41 (m, 1H), 7.28–
8.03 (m, 12H), 6.78–7.23 (m, 1H), 4.05–4.94 (m, 2H), 3.25–3.77 (m,
1H), 2.79–3.39 (m, 2H), 1.52–2.27 ppm (m, 4H); LC–MS (ESI): m/z:
428 [M+H]+; HPLC purity: 97%, tR =3.11 min (Method 6).
Rat or human cryogenically preserved hepatocytes (Xenotech,
Kansas City, MO, USA) were thawed and isolated according to the
manufacturer’s protocol using the XenoTech Hepatocyte Isolation
Kit. The inhibitors were incubated at 2.5 mm in 0.5ꢁ106 cellsmLꢂ1 at
378C. Aliquots, taken at 0, 15, 30, 60, and 90 min, were quenched
with ice-cold CH3CN, centrifuged, and the supernatant was ana-
lyzed by LC–MS–MS.
Sample analysis: LC–MS–MS analyses were performed on an Acqui-
ty UPLC, coupled with a sample organizer and interfaced with a
triple quadrupole Premiere XE (Waters, Milford, USA). Mobile
phases consisted of a phase A [0.1% formic acid in a mixture of
H2O and CH3CN (95:5 v/v)] and phase B [0.1% formic acid in a mix-
ture of H2O and CH3CN (5:95 v/v)]. LC runs were carried out at
408C on Acquity BEH C18 columns (1.7 mm, 50 mmꢁ2.1 mm at a
flow rate of 0.45 mLminꢂ1, or 1.7 mm, 50 mmꢁ1 mm at a flow rate
of 0.2 mLminꢂ1). The columns were conditioned with 2% phase B
for 0.2 min, then brought to 100% phase B within 0.01 min and
maintained at these conditions for 1.3 min. LC–MS–MS analyses
were carried out using a positive electrospray ionization (ESI(+)) in-
terface in multiple reaction monitoring (MRM) mode with verapa-
mil as internal standard. The percentage of the compound remain-
ing after 30 min incubation in microsomes was calculated accord-
ing to the following equation: [(AUC30 min)/(AUC0 min)]ꢁ100%.
(ꢃ)-(E)-N-Hydroxy-3-{6-[(E)-3-(3-naphthalen-2-ylpiperidin-1-yl)-3-
oxo-1-propen-1-yl]pyridin-2-yl}acrylamide hydrochloride (29v):
Compound 29v (yield 47%) was prepared by starting from the
THP-protected hydroxamic acid 28 and 3-naphthalen-2-ylpiperidine
following the synthetic procedure of the hydroxamic acid 29e.
1H NMR (300 MHz, [D6]DMSO, 353 K): d=7.75–7.98 (m, 5H), 7.32–
7.77 (m, 8H), 7.02 (d, J=15.85 Hz, 1H), 4.02–4.79 (m, 2H), 2.99–
3.46 (m, 2H), 2.76–3.00 (m, 1H), 2.03–2.23 (m, 1H), 1.79–2.04 (m,
2H), 1.44–1.82 ppm (m, 1H); LC–MS (ESI): m/z: 428 [M+H]+; HPLC
purity: 97%, tR =3.16 min (Method 6).
Biological assays
Pan-HDAC inhibition assay: Pan-HDAC inhibition assays were per-
formed as previously described.[28]
For the clearance studies of 24b, 29b, and 29e in rat and human
hepatocytes, MRM transitions of potentially formed amide and
acrylic acid metabolites were also monitored. The rates of intrinsic
clearance (CLi) of the compounds were calculated according to the
following equation: CLi [mLminꢂ1 (106 cells)ꢂ1]=kꢁV, for which V is
the incubation volume per 106 cells and k (minꢂ1) is the rate con-
stant derived from the exponential decay.
HDAC1, 3, 4, 6, and 8 inhibition assays: Assays for inhibition of
HDAC1, 3, 4, 6, and 8 were carried out by Amphora Discovery
Corp. (Research Triangle Park, NC, USA) as previously de-
scribed.[40,33] Purified HDACs were incubated with the test com-
pounds and a carboxyfluorescein-labeled peptide (1 mm) as sub-
strate for 17 h at 258C in a buffer consisting of 100 mm HEPES
(pH 7.5), 1 mgmLꢂ1 BSA, 0.01% Triton X-100, 1% DMSO, and
25 mm KCl. The reaction was stopped by the addition of 45 mL
0.08% sodium dodecyl sulfate in 100 mm HEPES (pH 7.5), and the
substrate and product were then separated electrophoretically
using a LabChip 3000 system (Caliper Life Sciences, Hopkinton,
MA, USA) with blue laser excitation and green fluorescence detec-
tion. The fluorescence intensity was established using the Well An-
alyzer software on the Caliper system. All experiments were per-
Histone acetylation assay: The histone acetylation assay was per-
formed as previously described.[28]
In vivo drug pharmacokinetic studies: PK experiments were per-
formed using four-week-old male nude CD-1 mice (Charles River
Laboratories, Calco, Italy). Animals were quarantined for about one
week prior to the study. They were housed under standard condi-
tions and had free access to water and standard laboratory rodent
ChemMedChem 2010, 5, 1359 – 1372
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1371

F. Thaler et al.
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Revised: May 21, 2010
Published online on June 22, 2010
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ChemMedChem 2010, 5, 1359 – 1372