Discovery, synthesis, and pharmacological evaluation of spiropiperidine hydroxamic acid based derivatives as structurally novel histone deacetylase (HDAC) inhibitors

Article abstract of DOI:10.1021/jm200146u

New spiro[chromane-2,4′-piperidine] and spiro[benzofuran-2,4′- piperidine] hydroxamic acid derivatives as HDAC inhibitors have been identified by combining privileged structures with a hydroxamic acid moiety as zinc binding group. The compounds were evaluated for their ability to inhibit nuclear extract HDACs and for their in vitro antiproliferative activity on different tumor cell lines. This work resulted in the discovery of spirocycle 30d that shows good oral bioavailability and tumor growth inhibition in an HCT-116 murine xenograft model.

Full text of DOI:10.1021/jm200146u

ARTICLE
pubs.acs.org/jmc
Discovery, Synthesis, and Pharmacological Evaluation of
Spiropiperidine Hydroxamic Acid Based Derivatives as Structurally
Novel Histone Deacetylase (HDAC) Inhibitors
Mario Varasi,†,‡ Florian Thaler,*,§,‡ Agnese Abate,†,‡ Chiara Bigogno,|| Roberto Boggio,§ Giacomo Carenzi,†,‡
Tiziana Cataudella,† Roberto Dal Zuffo,§ Maria Carmela Fulco,†,‡ Marco Giulio Rozio,|| Antonello Mai,^
Giulio Dondio,|| Saverio Minucci,‡,# and Ciro Mercurio†
†Genextra Group, DAC SRL, Via Adamello 16, 20139 Milan, Italy
‡European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy
§Genextra Group, Congenia SRL, Via Adamello 16, 20139 Milan, Italy
NiKem Research SRL, Via Zambeletti 25, 20021 Baranzate (MI), Italy
^Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti,
Universitꢀa degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy
#Department of Biomolecular Sciences and Biotechnologies, University of Milan, Via Celoria, 26, 20133 Milan, Italy
S Supporting Information
b
ABSTRACT: New spiro[chromane-2,40-piperidine] and spiro[benzofuran-2,40-piperidine]
hydroxamic acid derivatives as HDAC inhibitors have been identified by combining privileged
structures with a hydroxamic acid moiety as zinc binding group. The compounds were
evaluated for their ability to inhibit nuclear extract HDACs and for their in vitro anti-
proliferative activity on different tumor cell lines. This work resulted in the discovery of
spirocycle 30d that shows good oral bioavailability and tumor growth inhibition in an HCT-
116 murine xenograft model.
’ INTRODUCTION
SAHA 112,13 (also known as vorinostat) and the cyclic depsipep-
tide romidepsin or FK228 214 are the first two compounds to
gain market authorization: both HDAC inhibitors have been
approved by the FDA for the treatment of cutaneous T-cell
lymphoma (CTCL). Other compounds in clinical trials for the
treatment of hematological and solid tumors include the hydro-
xamic acids panobinostat or LBH589 3 (Novartis, phase III),15
PXD101 or belinostat 4 (Topotarget, phase III),16 SB-939 5
(S*BIO, phase II),17 the benzamides SNDX-275 or MS-275 6
(Syndax, phase II),18 and MGCD-0103 7 (methylgene, phase
II)19(Figure 1).
We have recently described the synthesis and the biological
activity of phenyloxopropenyl and amidopropenyl hydroxa-
mic acid derivatives20,21 as potent inhibitors of HDACs. The
compounds, comparable to the most advanced hydroxamic
acid HDAC inihibitors in clinical studies, such as 1, 3, and 4,
exhibited an oral bioavailability of less than 20% in mice.
Therefore, we explored new chemical entities with a (potential)
superior oral bioavailability. Privileged structures with their
inherent affinity for diverse biological targets represent an ideal
source of core scaffolds for the design of molecules able to
target various receptors.22ꢀ24 Conformationally constrained
Chemical modifications of DNA and histones play an essential
role for modulating transcription processes in eukaryotic cells.
Histone acetylation leads to the relaxation of chromatin and thus
generally to gene activation. Conversely, hypoacetylation causes
chromatin condensation resulting predominantly in transcrip-
tional repression.1,2 The acetylation status is mediated by two
enzyme families, histone acetyl transferases (HATs) and histone
deacetylases (HDACs). The HDAC family can be classified into
two groups: the “classical” HDACs, which are zinc-dependent
amidohydrolases (classes I, II, and IV), and the NADþ depen-
dent sirtuins (class III). Class I HDACs comprise HDACs 1ꢀ3
and HDAC 8. They are predominantly nuclear enzymes and are
ubiquitously expressed, whereas class II HDACs (HDACs 4ꢀ7,
9, and 10) are found in the nucleus and in the cytosol.3,4 HDAC
11 is the sole member of class IV HDACs. Perturbations of the
acetylation level are associated with aberrant transcriptional
activity, and HDACs have been identified as attractive targets
for cancer therapy. Indeed, even though the exact mechanism of
HDAC inhibitors as anticancer agents has not been fully eluci-
dated, several compounds have demonstrated potent antitumoral
activity in preclinical and clinical studies by increasing the acetyl-
ation status of histones and non-histones and inducing transcrip-
tional events involved in growth arrest, differentiation, and
apoptotic cell death.5ꢀ11 The hydroxamic acid derivative
Received: February 9, 2011
Published: March 21, 2011
r
2011 American Chemical Society
3051
dx.doi.org/10.1021/jm200146u J. Med. Chem. 2011, 54, 3051–3064
|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. HDAC inhibitors in clinical development with compounds 1 and 2 having marketing approval for the treatment of cutaneous T-cell
lymphoma (CTCL).
Scheme 1a
a Reagents and conditions: (a) MeOH, N-BOC-piperidin-4-one, pyrrolidine, reflux; (b) HCl, dioxane, room temp; (c) CH2Cl2, TEA; R-Br or R-Cl,
room temp; (d) MeOH, N-Me-piperidin-4-one, pyrrolidine, reflux; (e) MeOH/H2O, NaOH, 50 ꢀC; (f) CH2Cl2, EDC, HOBT, NH2OTHP, room
temp; (g) CH2Cl2, Et2O, HCl, room temp.
4-oxospiro[chroman-2,40-piperidine] ring systems are privileged
structures in the terminology of Evans et al.:25 the reduced
molecular flexibility, the small size, and polarity present favorable
physical properties for oral bioavailability. The spirochromane
moiety is present in structures acting as antiarrhythmic agents,26
R1a-receptor antagonists,27 inhibitors of histamine release,28
3052
dx.doi.org/10.1021/jm200146u |J. Med. Chem. 2011, 54, 3051–3064

Journal of Medicinal Chemistry
ARTICLE
Scheme 2a
a Reagents and conditions: (a) MeOH, N-BOC-piperidin-4-one, pyrrolidine, reflux; (b) DMF, TEA, Pd(OAc)2, PPh3, methyl acrylate, 100 ꢀC; (c)
MeOH/H2O, NaOH, 50 ꢀC; (d) CH2Cl2, EDC, HOBT, NH2OTHP, room temp; (e) CH2Cl2, dioxane, HCl, room temp.
growth hormone secretagogues,29 dementia alleviating agents,30
δ-opioid receptor ligands,31,32 stearoyl-CoA desaturase 1
inhibitors,33,34 acetyl-CoA carboxylase inhibitors,35 or antiproli-
ferative agents.36 In the present study, we describe the synthesis
and biological characterization of spiro[chromane-2,40-
piperidine] and spiro[benzofuran-2,40-piperidine] combined
with a hydroxamic acid moiety as zinc binding group to give
derivatives with HDAC inhibitory activity.
described above for compounds 13aꢀd (Scheme 1) furnished the
N-hydroxyspiro[chromane-2,40-piperidine]acrylamides 22 and 23.
N-Substituted spiro[chromane-2,40-piperidine]acrylhydroxamates
30aꢀe and 31aꢀe were prepared as exemplified in Scheme 3.
The BOC protection groups of the acrylic acid methyl esters 18
and 19 were cleaved in acidic conditions, giving the free amines
24 and 25, which were then functionalized through alkylation,
reductive amination, or acylation to afford 26b, 26d, 26e, and
27aꢀe. The tert-butyl esters 26a and 26c were obtained by
spirocyclization of 14 with 1-methylpiperidin-4-one or 1-ethy-
loxycarbonylpiperidin-4-one and subsequent Heck reaction with
tert-butyl acrylate. Cleavage of methyl ester with NaOH and the
tert-butyl ester with trifluoracetic acid (TFA) gave the carboxylic
acids, which were then coupled with NH2OTHP utilizing EDC
and HOBt. Acidic deprotection of tetrahydropyranyl moiety
afforded the desired hydroxamic acids 30aꢀe and 31aꢀe.
(E)-N-Hydroxy-3-{3-oxospiro[benzofuran-2(3 H),40-piperidin]-
5-yl}acrylamide (35) was synthesized as described in Scheme 4.
The spirobenzofuran 3239 was coupled with methyl acrylate
following the procedures outlined in Scheme 2 for the acrylic
esters 18 and 19. Basic hydrolysis of the methyl ester afforded the
acrylic acid 34, which was then subsequently converted into the
desired hydroxamic acid according to Scheme 1.
’ CHEMISTRY
The N-hydroxy-spiro[chromane-2,40-piperidine]-6-yl}carboxa-
mides 13aꢀd were prepared starting from 3-acetyl-4-hydroxy-
benzoic acid methyl ester (8),37 as illustrated in Scheme 1.
Reaction of 8 with 1-(tert-butyloxycarbonyl)piperidine-4-one
(N-BOC-piperidin-4-one) or 1-methylpiperidin-4-one in the
presence of pyrrolidine gave the spirocyclic methyl ester 9 or
11a. Treatment of 9 with hydrogen chloride (HCl) in dioxane led
to the free amine 10, which was then further functionalized either
by alkylation with benzyl bromide or by acylation with acetyl or
benzoyl chloride, giving intermediates 11bꢀd. Saponification of
the methyl esters 11aꢀd with sodium hydroxide (NaOH) gave
the carboxylic acids 12aꢀd, which were coupled with NH2OTHP
(O-(tetrahydropyran-2-yl)hydroxylamine) in the presence of
EDC (N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydro-
chloride) and HOBt (N-hydroxybenzotriazole). Cleavage of
the protecting group with hydrogen chloride (HCl) in diethyl
ether afforded the hydroxamates 13aꢀd.
N-Substituted spirobenzofuran acrylhydroxamates 39aꢀe
were prepared starting from the BOC protected spiro[benzofuran-
2(3H)-40-piperidin]acrylic acid methyl ester 33 (Scheme 4).
Deprotection of the BOC group gave the free amine 36, which
was then either alkylated with benzyl bromide or acylated
with acetyl chloride, benzoyl chloride, or ethyl chloroformate
in presence of TEA. Reductive amination with formaldehyde
in the presence of sodium triacetoxyborohydride [NaBH-
(OAc)3] as reducing agent gave the N-methyl intermediate
37a. Hydrolysis of the methyl esters followed by EDC cou-
pling with NH2OTHP and final cleavage of the tetrahydropyr-
anyl protecting group furnished the hydroxamic acid deriva-
tives 39aꢀe.
(E)-N-Hydroxy-3-{4-oxospiro[chromane-2,40-piperidine]-6-yl}-
acrylamide (22) and the spiro[chromane-2,40-piperidine]-7-yl deri-
vative 23 were prepared as shown in Scheme 2. Reaction of the
commercially available 1-(5-bromo-2-hydroxyphenyl)ethanone
(14) and 1-(4-bromo-2-hydroxyphenyl)ethanone (15)38 with N-
BOC piperidin-4-one was carried out according to the procedure
described above in Scheme 1. The obtained spiropiperidines 16 and
17 were then submitted to the Heck reaction with methyl acrylate in
the presence of palladium acetate [Pd(OAc)2], PPh3, and triethyl-
amine (TEA) in DMF, affording the spirocyclic methyl esters 18
and 19. Hydrolysis of the methyl ester with NaOH and sub-
sequent conversion to the hydroxamic acid following the procedure
(E)-N-Hydroxy-3-{10-benzyl-4-hydroxyspiro[chromane-2,
40-piperidine]-6-yl}acrylamide (40) was prepared by reducing the
corresponding oxospiropiperidine derivative 30d with sodium
borohydride (NaBH4) as outlined in Scheme 5.
3053
dx.doi.org/10.1021/jm200146u |J. Med. Chem. 2011, 54, 3051–3064

Journal of Medicinal Chemistry
ARTICLE
Scheme 3a
a Reagents and conditions: (a) CH2Cl2, dioxane, HCl, room temp; (b) CH2Cl2, TEA, R-Br or R-Cl, room temp or for 27a CH2Cl2, HCHO, NaBH-
(Ac)3, room temp; (c) MeOH, N-Me-piperdin-4-one or N-ethyloxycarbonyl-piperidin-4-one, pyrrolidine, reflux; (d) DMF, TEA, Pd(OAc)2, tert-butyl
acrylate, 100 ꢀC; (e) MeOH/H2O, NaOH, 50 ꢀC or for 27a dioxane/water, NaOH, room temp or for 26a and 26c CH2Cl2, TFA, room temp; (f)
CH2Cl2, EDC, HOBT, NH2OTHP, room temp; (g) CH2Cl2, dioxane or Et2O, HCl, room temp.
Scheme 4a
a Reagents and conditions: (a) DMF, TEA, Pd(OAc)2, PPh3, methyl acrylate, 100 ꢀC; (b, g) MeOH/H2O, NaOH, 50 ꢀC; (c, h) CH2Cl2, EDC, HOBT,
NH2OTHP, room temp; (d, i) CH2Cl2, Et2O, HCl, room temp; (e) HCl, dioxane, room temp; (f) CH2Cl2, TEA, R-Br or R-Cl, room temp or for 37a
CH2Cl2, HCHO, NaBH(Ac)3, room temp.
3054
dx.doi.org/10.1021/jm200146u |J. Med. Chem. 2011, 54, 3051–3064

Journal of Medicinal Chemistry
ARTICLE
Scheme 5a
a Reagents and conditions: (a) MeOH, NaBH4, room temp.
Scheme 6a
a Reagents and conditions: (a)MeOH, N-Bn-piperidin-4-one, pyrrolidine, reflux; (b) MeOH, NaBH4, room temp; (c, i, n) DMF, TEA, Pd(OAc)2, PPh3,
methyl acrylate, 100 ꢀC; (d) THF, p-TsOH, reflux; (e, j, o) MeOH/H2O, NaOH, 50 ꢀC; (f, k, p) CH2Cl2, EDC, HOBT, NH2OTHP, room temp; (g, l, q)
CH2Cl2, dioxane, HCl, room temp; (h) EtOH, Zn, HCl, room temp; (m) CH3CN, H2SO4, ꢀ10 to 0 ꢀC, then room temp.
The benzylspiropiperidine compounds 44, 47, and 50 were
prepared as illustrated in Scheme 6. Condensation of 1-(5-bromo-
2-hydroxyphenyl)ethanone (14) with N-benzylpiperidin-4-one in
the presence of pyrrolidine gave the spiropiperidine 41. Reduction
of the carbonyl group with NaBH4 furnished intermediate 42. Heck
reaction with methyl acrylate in the presence of Pd(OAc)2, PPh3,
and TEA and subsequent elimination of the alcohol gave the
spirochromene methyl ester 43. Conversion of the methyl ester
into the hydroxamic acid 44 was achieved following the procedures
described in Scheme 2. Reduction of the oxospiropiperidine 41
with zinc in HCl furnished the spirochromane 45. Heck reaction
with methyl acrylate and subsequent saponification of the ester
gave the acrylic acid 46. EDC coupling with NH2OTHP and
final cleavage of the tetrahydropyranyl protecting group with HCl
in dioxane afforded the requisite hydroxamic acid 47. Ritter
reaction of 42 with acetonitrile and sulfuric acid led to acetylami-
nospiropiperidine 48, which was then converted into the desired
N-hydroxyacrylamide 50 as described for compound 47.
3055
dx.doi.org/10.1021/jm200146u |J. Med. Chem. 2011, 54, 3051–3064

Journal of Medicinal Chemistry
ARTICLE
’ BIOLOGY
HDAC assay kit, with partially purified HeLa nuclear extracts as
the source for HDACs and a fluorogenic acetylated histone
peptide fragment (Fluor de Lys) as the substrate. In addition to
these biochemical experiments, we also investigated the ability of
these compounds to inhibit cancer cell growth using the human
chronic myelogenous leukemia cell line K562, carcinomic human
alveolar basal epithelial cells A549, and human colon cancer cells
HCT-116. HDAC inhibitory data and cellular activity of the
compounds are compiled in Tables 1ꢀ3. As shown in Table 1,
the methyl and the acetyl derivatives 13a and 13b were virtually
inactive in the enzymatic assay, whereas the benzyl and the
benzoyl analogues 13c and 13d exhibited IC50 of 24 and 29 μM,
respectively. Compound 13c showed inhibitory activities with
IC50 of around 20 μM for all three cell lines in the antiprolifera-
tive assay, whereas the three other compounds had IC50 exceed-
ing 50 μM.
The HDAC inhibitory activity of the synthesized spiropiper-
idine derivatives was measured using a commercially available
Table 1. HDAC Enzyme and Antiproliferative Activity Data
for Spiro[chromane-2,40-piperidine]-6-yl}-N-hydroxybenza-
mides 13aꢀda
activity (μM)
Recently we reported about hydroxamic acid derivatives
having minimum structural requirements for HDAC inhibition.20
We had found that N-hydroxybenzamide showed a modest
HDAC inhibitory activity. Introduction of an acrylic group
resulted in a substantial increase of activity: N-hydroxycinnamide
inhibited 55% of the HDAC enzyme activity at 1 μM.20 These
results stimulated the synthesis of new spiropiperidine acrylhy-
droxamate derivatives with the goal to obtain compounds with
an increased biochemical and cellular activity. The first series
R
HDAC
K562
A549
HCT-116
13a
13b
13c
13d
CH3
>50
>50
24
>50
>50
16
>50
>50
22
>50
>50
15
acetyl
benzyl
benzoyl
29
>50
>50
>50
a Assays done in replicates (n g 2). Mean values are shown, and the
standard deviations are <30% of the mean.
Table 2. HDAC Enzyme and Antiproliferative Activity Data for Spiro[chromane-2,40-piperidine] and Spiro[benzofuran-2(3H)-
40-piperidin]-N-hydroxyacrylamides 22, 23, 30aꢀe, 31aꢀe, 35, and 39aꢀea
activity (μM)
R
HDAC
K562
A549
HCT-116
22
H
0.082
0.288
0.641
0.140
0.121
0.316
1.41
5.77
0.806
9.37
2.08
0.399
1.65
>50
2.72
1.04
26.2
5.27
0.773
5.52
24.8
3.76
11.3
2.78
12.2
8.78
1.09
1.07
>50
20.2
5.94
22.1
1.59
0.266
5.66
2.14
0.477
2.20
35.5
5.72
36.0
3.03
8.41
4.92
0.832
0.337
>50
30a
30b
30c
30d
30e
23
CH3
acetyl
ethyloxycarbonyl
benzyl
benzoyl
H
31a
31b
31c
31d
31e
35
CH3
5.84
4.50
6.08
2.19
8.94
3.19
1.69
0.397
>50
acetyl
1.04
ethyloxycarbonyl
benzyl
0.408
2.173
0.366
0.221
0.538
>50
benzoyl
H
39a
39b
39c
39d
39e
CH3
acetyl
ethyloxycarbonyl
benzyl
2.99
2.61
4.11
7.43
11.8
3.10
12.3
0.932
benzoyl
2.55
a Assays done in replicates (n g 2). Mean values are shown, and the standard deviations are <30% of the mean.
3056
dx.doi.org/10.1021/jm200146u |J. Med. Chem. 2011, 54, 3051–3064

Journal of Medicinal Chemistry
Table 3. HDAC Enzyme and Antiproliferative Activity Data for Spiropiperidine-N-hydroxyacrylamides 40, 44, 47, and 50a
ARTICLE
a Assays done in replicates (n g 2). Mean values are shown, and the standard deviations are <30% of the mean.
comprised N-hydroxy-{4-oxospiro[chromane-2,40-piperidine]-
6-yl}acrylamides with various N-piperidine substitutents (22
and 30aꢀe). As summarized in Table 2, a remarkable potency
increase was observed: the benzyl and the benzoyl analogues
30d and 30e with IC50 of 0.121 and 0.316 μM, respectively,
were around 100 times more active than the corresponding
N-hydroxy-spiro[chromane-2,40-piperidine]-6-yl}carboxamides.
A similar increase was found also for the methyl and the acetyl
derivatives 30a and 30b, which showed potencies in the sub-
micromolar range. Within the series no major differences in
activities among the compounds were observed: compound 22
(R = H) with an IC50 of 0.082 μM was around 8-fold more active
than the least potent one within this series, the acetyl analogue
30b. With the exception of 30b, all compounds of this subseries
also showed good antiproliferative activity in the three cell lines
in the micromolar or submicromolar range. The benzyl derivative
30d emerged as the most potent agent in two cell lines, with IC50
of 0.399 μM in K562 cells and 0.773 μM in the A549 cell line.
The methyl analogue 30a was slightly more active in the HCT-
116 cell line (IC50 = 0.266 μM) than the benzyl derivative
(IC50 = 0.477 μM). Furthermore, the antiproliferative activity
was in general slightly superior in the K562 and HCT-116 cell
lines than in A549 cells.
compared to the spiro[chromane-2,40-piperidine]-6-yl analo-
gues. In addition, 23 (R = H), 31a (R = Me), and 31d (R =
Bn) were about 10 times less potent in the three cell lines than
the related analogues 22, 30a, and 30d. On the other hand, no
major differences in the antiproliferative potencies were observed
between the 7-spiropiperidines 31b, 31c, and 31e (IC50 in the
low micromolar range) and the corresponding spiro[chromane-
2,40-piperidine]-6-yl analogues 30b, 30c, and 30e.
Subsequently, our attention was directed to N-substituted
spirobenzofuran acrylhydroxamates. The enzymatic activity of
the prepared analogues (Table 2, compounds 35 and 39aꢀe)
was overall lower than the corresponding spiro[chromane-2,
40-piperidine]-6-yl derivatives. 35 with R = H (IC50 = 0.221 μM)
was around 3 times less potent than the corresponding spiro-
chromane analogue 22, and the methyl analogue 39a (IC50
=
0.538 μM) was 2 times less potent than the corresponding 30a.
The decrease in the enzymatic activity was more evident in the
cases of 39b, with R = acetyl (g100-fold), and 39c, with R =
ethyloxycarbonyl (∼20-fold). With the exception of 35 and 39a,
all other spirobenzofuran representatives were less potent than
the corresponding spirochromanes in the cellular assays. Com-
pounds 35 (R = H) and 39a (R = Me), with IC50 values in the
submicromolar/low micromolar range in the three cell lines, had
activity comparable to that of the corresponding spirochromane
derivative 22 and 30a.
On the basis of the enzymatic and antiproliferative activities,
the spiro[chromane-2,40-piperidine]-6-yl series was selected
for further exploration. Since the introduction of different N-
subsitutents was well tolerated, our attention was directed toward
the preparation of further different spiropiperidines, in particular
to assess the importance of the ketone moiety. In specific, ana-
logues of the benzylspiropiperidine analogue 30d were prepared
The significant increase of activity of N-hydroxyacrylamide
series compared to the N-hydroxybenzamide analogues
prompted us to investigate further modifications of the spiropi-
peridine scaffold: the first step was the synthesis of N-hydroxy-
spiro[chromane-2,40-piperidine]acrylamides, wherein the acry-
lamide moiety was shifted to position 7. As shown in Table 2, the
benzoyl derivative 31e (IC50 = 0.366 μM) was essentially
equipotent to 30e in the enzymatic assay, whereas the other
four compounds of this series were 2- to 20-fold less active
3057
dx.doi.org/10.1021/jm200146u |J. Med. Chem. 2011, 54, 3051–3064

Journal of Medicinal Chemistry
ARTICLE
Figure 2. (A) Dose-dependent inhibition of whole-cell HDAC activity in cultured HCT-116 human colon cancer cells by 30d. Cells were incubated
with 30d for 2, 4, or 8 h. (B) Whole-cell HDAC inhibitory activity of 30d in HCT-116 cells in a washout experiment. Cells were treated with 30d at
various doses for 4 h. The inhibitor was then washed out with PBS, and fresh drug-free medium was added. The whole-cell HDAC activity was measured
at the reported times. (C) Dose and time dependent induction of histone acetylation in HCT-116 human colon cancer cells after treatment with 30d.
Cells were incubated with 30d for the reported times and concentrations before histone acetylation of the whole-cell lysates was analyzed by cytoblot.
Table 4. Pharmacokinetic Properties of 30d in Mice
parameter
30d
C
max,iv [μM]
12.5
847
8.4
AUCiv,0ꢀ¥ [ng h/mL]
t1/2 [h]
Cl [(L/h)/kg]
Vss [L/kg]
5.88
8.9
Cmax,os [μM]
tmax [h]
0.823
0.25
800
31.5
AUCos,0ꢀ¥ [ng h/mL]
F [%]
In summary, the benzyl derivative 30d exhibited a good
enzymatic and cellular potency, and thus, we selected it as a
representative compound for a more extensive characterization.
First, 30d was submitted to Amphora Corp. (Research Triangle
Park, NC, U.S.) to evaluate its inhibitory activity against HDACs
1, 3, 4, 6, and 8 subtypes. 30d exhibited IC50 of 93.8, 9.4, 127,
25.7, and 1760 nM against HDACs 1, 3, 4, 6 and 8, respectively.
This biological profile is typical for pan inhibitors, such as 1,
trichostatin A, and the previously published amidopropenyl
hydroxamic acid derivatives.21,40,41
On the basis of the biochemical inhibition data (using both
human recombinant proteins and HeLa nuclear extracts as
enzymatic sources), we investigated the enzymatic inhibitory
activity of 30d in intact human HCT-116 cancer cells in order to
measure its whole-cell HDAC inhibitory activity.42 As shown in
Figure 2A, 30d exhibited a dose dependent inhibitory activity
with an IC50 of around 0.1 μM. These results are in good
Figure 3. Cell cycle profiles of human cancer HCT-116 cells treated for
24 h with 30d at different doses.
and tested; the hydroxy derivative 40 and the chromene 44 were
around 10-fold less active than 30d in the enzymatic assay.
Furthermore, both compounds showed a lower antiproliferative
activity in K562, A549, and HCT-116 cells. Also, the chromane
47 (IC50 = 9.0 μM) and the N-acetyl derivatives 50 (IC50
=
5.15 μM) showed a significantly reduced HDAC inhibitory
activity and a substantially lower antiproliferative potency. These
data demonstrate that the carbonyl group is critical for the
biochemical and cellular potency of this series.
3058
dx.doi.org/10.1021/jm200146u |J. Med. Chem. 2011, 54, 3051–3064

Journal of Medicinal Chemistry
ARTICLE
Figure 4. (A) Antitumor activity of 30d against HCT-116 human tumor xenografts implanted in mice, expressed as mean tumor volume (expressed as
mm3) ( standard error of the mean (SEM). (B) Analysis by immunohistochemistry of histone acetylation staining of the section of HCT-116 xenograft
tumor at the end of treatment. Tumor sections were fixed in formalin, paraffin-embedded, and stained with an antibody recognizing acetylated histone
H4. Representative sections of the staining for the different treatment groups are reported.
agreement with the biochemical data obtained using HeLa
nuclear extract as the enzymatic source. Interestingly, the HDAC
inhibitory activity was already maximal after 2 h of incubation.
Moreover, 30d completely inhibited the measurable enzymatic
activity in HCT-116 cells, reaching the maximal plateau at 100 μM.
The possible persistence of the inhibitory activity in whole
cells was then evaluated in a drug washout experiment (Figure 2B).
To this end, HCT-116 cells were exposed for 4 h at different
concentrations of 30d until complete inhibition of the HDAC
enzymatic activity. Cells were then subsequently washed with
drug-free media. As shown the Figure 2B, the inhibitory activity
of 30d was partially reversed and almost completly reversed after
2 and 4 h of drug washout.
Subsequently, a cytoblot assay dedicated to evaluate histone
acetylation was carried out as further confirmation of the enzy-
matic inhibition and to define a temporal correlation between
inhibition and its possible biological readout on well-known
targets of HDAC inhibition (Figure 2C).43 The experiments,
which were performed at the same experimental conditions used
for the whole cell HDAC inhibitory assay, underscored a dose
and time dependent hyperacetylation of histone H3. The time
dependency of histone hyperacetylation clearly indicated the
presence of a lag time between the enzymatic inhibition, already
maximal after 2 h of compound exposure, and biological readout,
which was more evident after 4 h of exposure. Moreover, while
the enzymatic inhibitory activity appeared to be already maximal
after 2 h of exposure with no further increase after that time, the
induced hyperacetylation of histones appeared to increase over
the time of enzymatic inhibition.
population at both 1 and 3 μM. A dose dependent increase of
the subG1 population, an indication of cell death, was also noted.
Given the promising in vitro properties of spirocycle 30d, we
evaluated its in vivo behavior. Pharmacokinetic studies were
carried out in male CD-1 mice, and the compound was adminis-
tered in a single intravenous (iv) dose of 5 mg/kg or an oral dose
of 15 mg/kg.20 The main pharmacokinetic parameters obtained
are reported in Table 4. The compound showed a systemic
plasma clearance slightly greater than the hepatic blood flow of
5.4 (mL/h)/kg in mice44 but lower than the previously pub-
lished clearance rates of the hydroxamic acid derivatives 1 (6.73
(mL/h)/kg),45,20 3 (18.3 (mL/h)/kg),45 and 4 (11.6 (mL/h)/
kg).45 The estimated elimination half-life was 8.4 h, substantially
higher than that of 1 (0.38 h), 3 (1.37 h), and 4 (1.21 h).45,20 The
calculated steady state volume of distribution (8.9 L/kg) suggests
an extensive tissue distribution. Moreover, the compound was
quickly absorbed after oral administration with a tmax of 15 min
and showed an oral bioavailability (F = 31.5%) substantially
higher than that of the three reference compounds 1, 3, and 4
(less than 10%45).
The in vivo antitumor activity of spirocycle 30d was then
studied in an established human xenograft model obtained by
implanted human colon carcinoma cells HCT-116 in athymic
mice. Human cancer cells (HCT-116, 5 ꢁ 106) were injected
subcutaneously in the flank of female CD-1 nude mice, and the
treatment was initiated once the tumors reached a volume of
around 100 mm3. The compound, dissolved in water containing
5% DMSO and 9.5% encapsin, was administrated orally at
35ꢀ150 mg/kg for 5 days/week for 17 days. As shown in
Figure 4A, the compound was able to reduce significantly the
tumor volume in a dose dependent manner with respect to the
control mice. Specifically, the calculated T/C ratio at the highest
tested dose of 150 mg/kg was around 0.3, which was highly
statistically significant (p < 0.0072). No significant body weight
Next, the cell cycle profile of the human cancer cell line HCT-
116 treated with 30d was analyzed by flow cytometry. As
reported in the Figure 3, 30d induced a strong increment of
G0/G1 phase population, an associated decrease of S and G2/M
population at the lowest dose, and an increase of G2M
3059
dx.doi.org/10.1021/jm200146u |J. Med. Chem. 2011, 54, 3051–3064

Journal of Medicinal Chemistry
ARTICLE
differences among the groups of mice and no signs of overt
toxicity were observed during the treatment. As previously des-
cribed,20 we had found that compound 1 at doses of 50 mg/kg
was able to reduce, similarly to 30d, the tumor growth in HCT-
116 in athymic mice with a calculated T/C ratio of around 0.34
after 2 weeks of treatment. However, the compound was
administered intraperitoneally once a day, which corresponds,
to our best knowledge, to the optimal route of administration for
compound 1 in these animal models.
Finally, tumor sections taken from mice treated with the
vehicle or 30d were analyzed by immunohistochemistry in order
to establish a relationship between the antitumor activity ob-
served in vivo in the xenograft model and HDAC inhibition. The
histone acetylation level was followed as readout for HDAC
inhibition.
array and a Bruker ion trap Esquire 3000þ. Purity was monitored at
254 nm, and the purities of the compounds used for biological tests were
found to be at least 95% with the exception of 13c (92%) and 22 (83%)
(see Supporting Information).
N-Hydroxy-[10-benzyl-4-oxospiro(chromane-2,40-piperidine)-
6-yl]carboxamide (13c). A mixture of 3-acetyl-4-hydroxybenzoic
acid methyl ester (8, 2.4 g, 12 mmol), N-BOC-piperidine-4-one (2.4 g,
12 mmol), and pyrrolidine (0.51 mL, 6.1 mmol) in MeOH (80 mL) was
heated for 4 h at reflux. The solution was concentrated under vacuum
and then poured into water. Then 1 M HCl was added until a neutral pH
was obtained, and the product was extracted with AcOEt. The organic
phase was dried over Na2SO4, filtered, and evaporated. The crude
mixture was purified by silica gel chromatography (CH2Cl2/MeOH
97:3) to give the spirocycle 9 (1.45 g, 32%). 1H NMR (CDCl3)
δ (ppm): 8.55 (d, J = 2.02 Hz, 1 H), 8.16 (dd, J = 8.74, 2.14 Hz,
1 H), 7.03 (d, J = 8.74 Hz, 1 H), 3.90 (s, 3 H), 3.25ꢀ3.19 (m, 2 H), 2.75
(s, 2 H), 2.46ꢀ2.43 (m, 2 H), 2.05ꢀ2.00 (m, 2 H), 1.67ꢀ1.59 (m, 2 H),
1.46 (s, 9 H). MS (ESI) m/z: 276 [M þ H ꢀ 100]þ.
As shown in the Figure 4B, the observed tumor response
correlated well with a significant increase of histone acetylation
level in the tumor section of groups treated with 30d versus the
vehicle group, in terms of both positive cells and signal intensity.
A 4 M solution of HCl in dioxane (10 mL) was added to a solution of
compound 9 (1.4 g, 3.7 mmol) in CH2Cl2 (10 mL), and the mixture was
stirred at room temperature for 4 h. The product was filtered off, washed
with CH2Cl2, dried, and collected, giving the free amine 10 as its
hydrochloride salt (1.03 g, 89%) as a white solid. 1H NMR (DMSO-d6)
δ (ppm): 9.35 (bs, 1 H), 8.35 (d, J = 2.25 Hz, 1 H), 8.18 (dd, J = 8.71,
2.25 Hz, 1 H), 7.31 (d, J=8.71 Hz, 1 H), 3.89 (s, 3 H), 3.25ꢀ3.15(m, 4H),
3.04 (s, 2 H), 2.14ꢀ2.18 (m, 2 H), 1.94ꢀ2.00 (m, 2 H). MS (ESI) m/z:
276 [M þ H]þ.
’ CONCLUSIONS
New spiro[chromane-2,40-piperidine] and spiro[benzofuran-
2,40-piperidin] hydroxamic acid derivatives as HDAC inhibitors
have been developed integrating a privileged structure with a
hydroxamic acid moiety as zinc binding group. Four examples of
N-hydroxy-spiro[chromane-2,40-piperidine]-6-yl}carboxamides
exhibited only a modest in vitro HDAC and cell growth
inhibitory activity, but introduction of a N-hydroxyacrylamide
group as zinc binding moiety led to a substantial increase in
activity. The compounds exhibited IC50 in the submicromolar
range against HDACs and in the submicromolar to low micro-
molar range in the antiproliferative assay against three tumor cell
lines. The synthesis of N-hydroxy[spiro(chromane-2,40-piperidine)-
7-yl]acrylamide analogues and the spirobenzofuran derivatives
led to structures that were in general less active in the enzy-
matic and cellular assays. Furthermore, reduction, elimination, or
general replacement of the carbonyl group of the spirochromane
led to compounds with reduced potency. On the basis of the in
vitro biochemical and antiproliferative activity, the spirocycle
30d was selected for further in vitro and in vivo pharmacokinetic
and activity experiments. Whole-cell HDAC inhibitory activity of
30d was assessed in HCT-116 cells, and the cell cycle profiles of
human cancer HCT-116 cells showed that the compound
induced a strong increment of G0/G1 phase population at the
lowest dose and an increase of G2M population at 1 and 3 μM.
The compound, based on the good oral bioavailability
(F = 31.5%) and its ability to inhibit tumor growth in a HCT-
116 murine xenograft model in a dose dependent manner, was
selected for further investigation.
A suspension of the spirocycle 10 (250 mg, 0.80 mmol) in CH2Cl2
(10 mL) was treated with DIPEA (0.28 mL, 1.6 mmol) and benzyl
bromide (95 μL, 0.80 mmol). After being stirred at room temperature
for 2 h, the mixture was concentrated, and the crude residue was puri-
fied by column chromatography (CH2Cl2/MeOH 95:5) to give 11c
(275 mg, 94%) as a light yellow solid. 1H NMR (CDCl3) δ (ppm): 8.51
(d, J=2.03 Hz,1 H), 8.12 (dd, J=8.76, 2.03 Hz,1 H), 7.36ꢀ7.25 (m, 5 H),
7.02 (d, J = 8.76 Hz, 1 H), 4.56 (s, 2 H), 3.86 (s, 3 H), 2.73 (s, 2 H),
2.53ꢀ2.72 (m, 4 H), 2.04ꢀ2.06 (m, 2 H), 1.80ꢀ2.00 (m, 2 H). MS (ESI)
m/z: 366 [M þ H]þ.
NaOH (60 mg, 1.5 mmol) in H2O (4 mL) was added to a suspension
of methyl ester 11c (275 mg, 0.753 mmol) in MeOH (10 mL). The
mixture was stirred at reflux for 1 h, then concentrated. The pH of the
aqueous phase was adjusted with 1 M HCl to 5. The resulting suspension
was extracted with CH2Cl2, the organic phase dried over Na2SO4 and
concentrated to give the carboxylic acid 12c (250 mg, 95%) as a white
1
solid. H NMR (DMSO-d6) δ (ppm): 8.31 (bs, 1 H), 8.16ꢀ8.18 (m,
1 H), 7.60ꢀ7.70 (m, 2 H), 7.40ꢀ7.50 (m, 3 H), 7.21ꢀ7.23 (m, 1 H),
4.40 (s, 2 H), 3.17ꢀ3.22 (m, 4 H), 2.93 (s, 2 H), 2.09ꢀ2.22 (m, 4 H)).
MS (ESI) m/z: 352 [M þ H]þ.
A solution of compound 12c (250 mg, 0.712 mmol) in CH2Cl2
(20 mL) was cooled to 0 ꢀC. EDC (203 mg, 1.06 mmol) and HOBt (143
mg, 1.06 mmol) were added, and the mixture was stirred at room
temperature for 3 h. NH2OTHP (100 mg, 0.85 mmol) in CH2Cl2
(1 mL) was added dropwise, and the solution was stirred at room
temperature overnight. The reaction mixture was then washed with
saturated NaHCO3 and brine, dried over Na2SO4, and concentrated.
The crude residue was purified by silica gel chromatography (CH2Cl2/
MeOH 95:5) to give [10-benzyl-4-oxospiro(chromane-2,40-piperidine)-
6-yl}-N-(tetrahydro-pyran-2-yloxy)amide (85 mg, 26%). 1H NMR
(CDCl3) δ (ppm): 8.18 (d, J = 2.30 Hz, 1 H), 8.01 (dd, J = 8.82,
2.49 Hz, 1 H), 7.26ꢀ7.31 (m, 5 H), 7.04 (d, J = 8.82 Hz, 1 H), 5.07
(s, 1 H), 3.97ꢀ4.02 (m, 1 H), 3.62ꢀ3.64 (m, 1 H), 3.54 (s, 2 H), 2.72
(s, 2 H), 2.61ꢀ2.63 (m, 2 H), 2.41ꢀ2.46 (m, 2 H), 1.81ꢀ2.00 (m, 10 H).
MS (ESI) m/z: 451 [M þ H]þ. Then 1 M HCl in Et2O (5 mL) was
added dropwise to a solution of the THP hydroxamate (79.8 mg,
0.177 mmol) in CH2Cl2 (5 mL). The precipitating solid was filtered
’ EXPERIMENTAL SECTION
Chemistry. Reagents and solvents used, unless stated otherwise,
were of commercially available reagent grade quality and were used
without further purification. Flash chromatography purifications were
performed on Merck silica gel 60 (0.04ꢀ0.063 mm). Nuclear magnetic
resonance spectra (1H NMR) were recorded on a Bruker 400 MHz
spectrometer at 300 K and are referenced in ppm (δ) relative to TMS.
Coupling constants (J) are expressed in hertz (Hz). HPLCꢀMS
experiments were performed either on an Acquity UPLC apparatus,
equipped with a diode array and a Micromass SQD single quadruple
(Waters) spectrometer or on an Agilent 1100, equipped with a diode
3060
dx.doi.org/10.1021/jm200146u |J. Med. Chem. 2011, 54, 3051–3064

Journal of Medicinal Chemistry
ARTICLE
off, washed with CH2Cl2, and dried giving the requisite hydroxamic acid
13c as a light yellow solid (55 mg, 80%). 1H NMR (DMSO-d6)
δ (ppm): 10.52 (bs, 1 H), 9.05 (bs, 1 H), 8.23 (s, 1 H), 8.06ꢀ8.08
(m, 1 H), 7.63ꢀ7.64 (m, 2 H), 7.51 (s, 3 H), 7.20ꢀ7.21 (m, 1 H), 4.41
(s, 2 H), 3.23ꢀ3.36 (m, 4 H), 2.96 (s, 2 H), 2.08ꢀ2.24 (m, 4 H). MS
(ESI) m/z: 367 [M þ H]þ.
1 H), 7.19 (d, J = 8.4 Hz, 1 H), 6.45 (d, J = 16.0 Hz, 1 H), 3.06ꢀ3.19
(m, 4 H), 2.95 (s, 2 H), 2.10ꢀ2.14 (m, 2 H), 1.89ꢀ1.93 (m, 2 H). MS
(ESI) m/z: 627 [2MNa]þ.
(E)-N-Hydroxy-3-{10-benzyl-4-oxospiro[chromane-2,40-
piperidine]-6-yl}acrylamide (30d). A 4 M solution of HCl in
dioxane (4 mL) was added to compound 18 (1.18 g, 2.94 mmol)
in CH2Cl2 (20 mL), and the mixture was stirred at room temperature for
4 h. The precipitated solid was filtered off, washed with CH2Cl2, then
dried under vacuum to give 24 as a white solid (748 mg, 77%,
(E)-N-Hydroxy-3-{4-oxospiro[chromane-2,40-piperidine]-
6-yl}acrylamide (22). A mixture of 2-hydroxy-5-bromoacetophe-
none (14, 10.7 g, 50.0 mmol), N-BOC-piperidin-4-one (9.96 g, 50.0 mmol),
and pyrrolidine (2.1 mL, 25 mmol) in MeOH (80 mL) was heated to
reflux for 11 h. The solvent was then removed, and the crude mixture was
purified by column chromatography (hexane/AcOEt 9:1 to 8:2) to give
the spirocycle 16 as a yellow solid (18.55 g, 94%). 1H NMR (CDCl3)
δ (ppm): 7.96 (s, 1 H), 7.55 (d, J = 8.8 Hz, 1 H), 6.82 (d, J = 7.6 Hz, 1 H),
3.83ꢀ3.86 (m, 2 H), 3.18 (t, J = 11.6 Hz, 2 H), 2.70 (s, 2 H), 1.98ꢀ2.01
(m, 2 H), 1.58ꢀ1.62 (m, 2 H), 1.44 (s, 9 H). MS (ESI) m/z: 420
[M þ Na]þ.
1
hydrochloride salt). H NMR (DMSO-d6) δ (ppm): 8.95 (bs, 2 H),
8.01ꢀ8.06 (m, 2 H), 7.69 (d, J = 16.0 Hz, 1 H), 7.20 (d, J = 7.2 Hz, 1 H),
6.60 (d, J = 16.0 Hz, 1 H), 3.72 (s, 3 H), 3.17ꢀ3.21 (m, 2 H), 3.07ꢀ3.13
(m, 2 H), 2.99 (s, 2 H), 2.08ꢀ2.13 (m, 2 H), 1.88ꢀ1.95 (m, 2 H). MS
(ESI) m/z: 302 [M þ H]þ.
A suspension of compound 24 (451 mg, 1.34 mmol) in CH2Cl2
(12 mL) was treated with TEA (0.42 mL, 3.0 mmol) and benzyl bromide
(0.54 mL, 4.5 mmol), and the mixture was stirred at room temperature
for 5 h. The mixture was washed with water, and the pH value was
adjusted to 5 with 0.5 M HCl. The organic phase was dried, concen-
trated, and the crude residue was purified by silica gel chromatography
(CH2Cl2/MeOH 95:5) to give the spirocycle 26d as a light yellow solid
(440 mg, 84%). 1H NMR (CDCl3) δ (ppm): 8.02 (d, J = 2.0 Hz, 1 H),
7.61ꢀ7.67 (m, 2 H), 7.51ꢀ7.57 (m, 2 H), 7.47ꢀ7.50 (m, 3 H), 7.01 (d,
J = 9.2 Hz, 1 H), 6.38 (d, J = 16.0 Hz, 1 H), 3.80 (s, 3 H), 3.51ꢀ3.58 (m,
2 H), 2.74ꢀ2.77 (m, 2 H), 2.60ꢀ2.64 (m, 2 H), 2.45ꢀ2.47 (m, 2 H),
2.03ꢀ2.05 (m, 2 H), 1.73ꢀ1.80 (m, 2 H), 1.52ꢀ1.59 (m, 2 H). MS
(ESI) m/z: 805.5 [2MNa]þ.
A mixture of compound 16 (1.04 g, 2.62 mmol), Pd(OAc)2 (59 mg,
0.26 mmol), PPh3 (137 mg, 0.522 mmol), TEA (0.73 mL, 5.2 mmol),
methyl acrylate (0.47 mL, 5.2 mmol) in dry DMF (10 mL) was heated at
100 ꢀC for 8 h. The mixture was cooled to room temperature, filtered on
a Celite pad, and washed with AcOEt (100 mL). The filtrate was washed
with NH4Cl, saturated NaHCO3, and brine. The organic phase was
dried over Na2SO4 and evaporated under vacuum. The crude residue
was purified by column chromatography (hexane/AcOEt 9:1 to 7:3) to
give the acrylic acid methyl ester 18 as a light yellow solid (594 mg, 56%).
1H NMR (CDCl3) δ (ppm): 8.00 (bs, 1 H), 7.60ꢀ7.65 (m, 2 H), 7.00
(d, J = 8.4 Hz, 1 H), 6.31 (d, J = 16.0 Hz, 1 H), 3.85 (bs, 2 H), 3.78
(s, 3 H), 3.20 (t, J = 12.0 Hz, 2 H), 2.73 (s, 2 H), 2.00 (d, J = 15.2 Hz,
2 H), 1.53ꢀ1.70 (m, 2 H), 1.44 (s, 9 H). MS (ESI) m/z: 424
[M þ Na]þ.
Hydrolysis of the methyl ester 26d (414 mg, 1.06 mmol) was
performed as described for 20, giving the carboxylic acid 28d as a white
solid (380 mg, 96%). 1H NMR (CDCl3) δ (ppm): 8.02 (d, J = 2.0 Hz,
1 H), 7.64ꢀ7.68 (m, 2 H), 7.44ꢀ7.49 (m, 5 H), 7.01 (d, J = 9.2 Hz, 1 H),
6.38 (d, J = 16.0 Hz, 1 H), 3.62ꢀ3.70 (m, 2 H), 3.15ꢀ3.22 (m, 2 H),
2.68ꢀ2.77 (m, 4 H), 2.35ꢀ2.44 (m, 2 H), 1.99ꢀ2.08 (m, 2 H). MS
(ESI) m/z: 378 [M þ H]þ.
NaOH (160 mg, 4.00 mmol) in H2O (2 mL) was added to a sus-
pension of methyl ester 18 (462 mg, 1.15 mmol) in MeOH (6 mL), and
the mixture was stirred at 50 ꢀC. After 2 h, MeOH was evaporated and
the pH of the aqueous phase was adjusted to 5 with 1 M HCl. The
resulting suspension was extracted with CH2Cl2, the organic phase dried
over Na2SO4 and concentrated to give the carboxylic acid 20 as a white
solid (410 mg, 92%). 1H NMR (CDCl3) δ (ppm): 8.05 (d, J = 2.0 Hz,
1 H), 7.68ꢀ7.74 (m, 2 H), 7.04 (d, J = 8.8 Hz, 1 H), 6.39 (d, J = 16.0 Hz,
1 H), 3.85ꢀ3.94 (m, 2 H), 3.19ꢀ3.25 (m, 2 H), 2.75 (s, 2 H), 1.59ꢀ1.68
(m, 4 H), 1.50 (s, 9 H). MS (ESI) m/z: 410 [M þ Na]þ.
Reaction of the acrylic acid 28d (365 mg, 0.967 mmol) with
NH2OTHP was carried out following the procedure for compound
22. The crude THP intermediate was purified by column chromatog-
raphy (CH2Cl2/MeOH 97:3) (328 mg, 69%). 1H NMR (CDCl3)
δ (ppm): 8.31 (bs, 1 H), 8.05 (d, J = 2.0 Hz, 1 H), 7.54ꢀ7.68 (m,
2 H), 7.32ꢀ7.40 (m, 4 H), 6.96ꢀ6.98 (m, 1 H), 6.36 (bs, 1 H), 4.99 (bs,
1 H), 3.93ꢀ3.97 (m, 1 H), 3.68ꢀ3.66 (m, 1 H), 3.45ꢀ3.54 (m, 1 H),
2.76 (s, 2 H), 2.63ꢀ2.66 (m, 2 H), 2.40ꢀ2.50 (m, 2 H), 2.04ꢀ2.12 (m,
2 H), 1.81ꢀ1.84 (m, 4 H), 1.51ꢀ1.60 (m, 6 H). MS (ESI) m/z: 477
[M þ H]þ. Deprotection of tetrahydropyranyl group gave the requisite
hydroxamic acid 30d as a light yellow solid (230 mg, 78%, hydrochloride
salt). 1H NMR (DMSO-d6) δ (ppm): 10.69 (bs, 1 H), 7.91 (d, J = 2.0 Hz
1 H), 7.84 (dd, J = 8.6,1.9 Hz 1 H), 7.55ꢀ7.69 (m, 2 H), 7.36ꢀ7.54
(m, 4 H), 7.15 (d, J = 8.5 Hz, 1 H), 6.45 (d, J = 15.8 Hz, 1 H), 4.37 (d, J =
5.2 Hz, 2 H), 3.06ꢀ3.34 (m, 4 H), 2.88 (s, 2 H), 1.97ꢀ2.25 (m, 4 H).
MS (ESI) m/z: 393 [M þ H]þ.
A solution of the acrylic acid 20 (387 mg, 1.00 mmol) in CH2Cl2
(15 mL) was cooled to 0 ꢀC. EDC (383 mg, 2.00 mmol) and HOBt
(135 mg, 1.00 mmol) were added, and the mixture was stirred at room
temperature for 1 h. NH2OTHP (146 mg, 1.25 mmol) in CH2Cl2
(1 mL) was added dropwise, and the mixture was stirred at room
temperature for 4 h. Then the solution was washed with saturated
NaHCO3 and brine, dried over Na2SO4, and concentrated. The crude
residue was purified by silica gel chromatography (CH2Cl2/MeOH
98:2) to give (E)-3-{10-tert-butoxycarbonyl-4-oxospiro[chromane-2,
40-piperidine]-6-yl}-N-(tetrahydropyran-2-yloxy)acrylamide as a light
yellow oil (464 mg, 95%). 1H NMR (DMSO-d6) δ (ppm): 7.91 (s, 1 H),
7.80 (d, J = 7.6 Hz, 1 H), 7.48 (d, J = 16.0 Hz, 1 H), 7.14 (d, J = 8.4 Hz,
1 H), 6.46 (d, J = 16.0 Hz, 1 H), 4.90 (bs, 1 H), 3.84ꢀ3.90 (m, 1 H),
3.70ꢀ3.77 (m, 2 H), 3.52ꢀ3.55 (m, 1 H), 3.10ꢀ3.16 (m, 2 H), 2.88
(s, 2 H), 1.81ꢀ1.90 (m, 4 H), 1.53ꢀ1.70 (m, 6 H), 1.40 (s, 9 H). MS
(ESI) m/z: 995.7 [2MNa]þ. Then 4 M HCl in dioxane (2 mL) was
added dropwise to a solution of the N-(tetrahydropyran-2-yloxy)
acrylamide intermediate (434 mg, 0.892 mmol) in CH2Cl2 (10 mL) and
the mixture was stirred at room temperature for 2 h. The precipitate was
filtered off, washed with CH2Cl2, and dried to give the desired hydro-
xamic acid 22 as a white solid (260 mg, 86%, hydrochloride salt).
1H NMR (DMSO-d6) δ (ppm): 10.27 (bs, 1 H), 8.98 (bs, 1 H), 7.90
(d, J = 2.0 Hz, 1 H), 7.81 (dd, J = 8.8, 2.4 Hz, 1 H), 7.44 (d, J = 16.0 Hz,
(E)-N-Hydroxy-3-{10-benzyl-4-hydroxyspiro[chromane-2,
40-piperidine]-6-yl}acrylamide (40). A mixture of the hydroxamic
acid 30d (60 mg, 0.14 mmol) and NaBH4 (18 mg, 0.48 mmol) in MeOH
(5 mL) was stirred at room temperature. After 3 h the solution was
evaporated and the crude residue was purified by preparative HPLC to
give the hydroxy derivative 40 (45 mg, 58%, trifluoroacetate). 1H NMR
(DMSO-d6) δ (ppm): 10.64 (bs, 1 H), 9.60 (bs, 1 H), 7.36ꢀ7.63
(m, 8 H), 6.89 (d, J = 8.52 Hz, 1 H), 6.30 (d, J = 16.0 Hz, 1 H), 4.71 (bs,
1 H), 4.39 (s, 2 H), 3.25ꢀ3.29 (m, 6 H), 2.18ꢀ1.81 (m, 4 H). MS (ESI)
m/z: 395 [M þ H]þ.
Biological Assays. Pan-HDAC Inhibition Assay. Pan-HDAC in-
hibition assays were performed as previously described.20
HDACs 1, 3, 4, 6, and 8 Inhibition Assay. The inhibition assays for
HDACs 1, 3, 4, 6, and 8 were carried out by Amphora Discovery Corp.
3061
dx.doi.org/10.1021/jm200146u |J. Med. Chem. 2011, 54, 3051–3064

Journal of Medicinal Chemistry
ARTICLE
(Research Triangle Park, NC, U.S.) as previously described.41,40 Purified
HDACs were incubated with the test compounds and a carboxyfluor-
escein-labeled peptide (1 μM) as substrate for 17 h at 25 ꢀC in a buffer
consisting of 100 mM HEPES (pH 7.5), 1 mg/mL BSA, 0.01% Triton
X-100, 1% DMSO, and 25 mM KCl. The reaction was stopped by
addition of 45 μL of 100 mM HEPES (pH 7.5) and 0.08% sodium
dodecyl sulfate, and the substrate and the product were then separated
electrophoretically using a LabChip 3000 system (Caliper Life Sciences,
Hopkinton, MA) with blue laser excitation and green fluorescence
detection. All experiments were performed in duplicate. IC50 values
were determined from 12-point concentrationꢀresponse curves using a
nonlinear regression analysis.
HDAC Whole-Cell Assay. HCT-116 cells were seeded into 96-well
plates (Corning Inc., Costar) at a density of 2 ꢁ 104 cells/well in a
volume of 50 μL of the appropriate tissue culture medium. Compounds
were added with different concentrations for the indicated incubation
time at 37 ꢀC with 5% CO2. KI-104 Fluor de Lys substrate was then
added to initiate the reaction (final concentration of 500 μM). The
reaction was stopped at the reported times, and fluorescence was
developed by adding 50 μL of a freshly prepared stop mix, which is
composed of the Fluor-de-Lys developer diluted at 1:60 (BIOMOL),
1 μM TSA (BIOMOL), and 1% NP40. Substrate fluorescence was
measured using an excitation at 355 nm and an emission at 460 nm after
10 min of reaction.
Cell Growth Assay. The antiproliferative effect of the HDAC in-
hibitors on cell proliferation was evaluated against K562 (chronic
myeloid carcinoma), A549 (non-small-cell lung cancer), and HCT-
116 (human colon cancer) tumor cell lines using the CellTiter-Glo
luminescent cell viability assay (Promega, Madison, WI) according to
the manufacturer’s instructions. K562, A549, and HCT-116 cells, in
exponential growth, were incubated for 72 h at different concentrations
of the inhibitors. Then an equivalent of the CellTiter-Glo reagent was
added, the solution was mixed for 2 min in order to induce cell lysis, and
the luminescence was recorded after a further 10 min. The IC50 was
calculated using GraphPad software.
Flow Cytometric Cell Cycle Analysis. Cells were treated with 30d for
24 h, then harvested and fixed with 70% ethanol at ꢀ20 ꢀC. Nucleic acids
from fixed cells were treated with RNase type IIIA (1 mg/mL) and
stained with propidium iodide (50 mg/mL). DNA content was mea-
sured by using a fluorescence-activated cell cytometer (FACScan,
Becton Dickinson, Franklin Lakes, NJ) and analyzed using CellQuest
Pro software.
deparaffinized, rehydrated, unmasked using TEG buffer (10 mM Tris þ
0.5 mM EGTA, pH 9), and treated for 5 min with 3% H2O2. Slides were
then incubated overnight at 4 ꢀC with an antibody against acetylated
histone H4,46 revealed using the EnVision Plus/HRP detection system
(Dako), and counterstained with hematoxylin.
’ ASSOCIATED CONTENT
S
Supporting Information. Synthesis and characterization
b
of compounds 13a,b,d, 23, 30aꢀc,e, 31aꢀe, 35, 39aꢀe, 44, 47,
and 50; purity of key compounds as determined by HPLC.
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ390294375130. Fax: þ390294375138. E-mail:
florian.thaler@ifom-ieo-campus.it.
’ ACKNOWLEDGMENT
The authors are grateful to Dr. Raffaella Amici, Dr. Stefania
Gagliardi, Dr. Andrea Colombo, and Dr. Simon Plyte for their
helpful discussions and the critical review of the manuscript.
’ ABBREVIATIONS USED
HDAC, histone deacetylases; HAT, histone acetyltransferases;
K562, chronic myelogenous leukemia cell line; A549, carcinomic
human alveolar basal epithelial cells; HCT-116, human color-
ectal carcinoma cell line; BSA, bovine serum albumin; IHC,
immunohistochemistry; PK, pharmacokinetics; TBST, Tris-buf-
fered saline and Triton X-100
’ REFERENCES
(1) Hong, L.; Schroth, G. P.; Matthews, H. R.; Yau, P.; Bradbury,
E. M. Studies of the DNA binding properties of histone H4 amino
terminus. Thermal denaturation studies reveal that acetylation markedly
reduces the binding constant of the H4 “tail” to DNA. J. Biol. Chem.
1993, 268, 305–314.
(2) Selvi, R. B.; Kundu, T. K. Reversible acetylation of chromatin:
implication in regulation of gene expression, disease and therapeutics.
Biotechnol. J. 2009, 4, 375–390.
(3) de Ruijter, A. J.; van Gennip, A. H.; Caron, H. N.; Kemp, S.; van
Kuilenburg, A. B. Histone deacetylases (HDACs): characterization of
the classical HDAC family. Biochem. J. 2003, 370, 737–749.
(4) Haberland, M.; Montgomery, R. L.; Olson, E. N. The many roles
of histone deacetylases in development and physiology: implications for
disease and therapy. Nat. Rev. Genet. 2009, 10, 32–42.
(5) Minucci, S.; Pelicci, P. G. Histone deacetylase inhibitors and the
promise of epigenetic (and more) treatments for cancer. Nat. Rev.
Cancer 2006, 6, 38–51.
(6) Glozak, M. A.; Seto, E. Histone deacetylases and cancer.
Oncogene 2007, 26, 5420–5432.
(7) Spange, S.; Wagner, T.; Heinzel, T.; Kramer, O. H. Acetylation of
non-histone proteins modulates cellular signalling at multiple levels. Int.
J. Biochem. Cell Biol. 2009, 41, 185–198.
(8) Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. Histone
deacetylase inhibitors: from bench to clinic. J. Med. Chem. 2008,
51, 1505–1529.
(9) Carey, N.; La Thangue, N. B. Histone deacetylase inhibitors:
gathering pace. Curr. Opin. Pharmacol. 2006, 6, 369–375.
Cytoblot Assay. HCT-116 cells were seeded at the density of 2 ꢁ 104
per well and allowed to attach overnight at 37 ꢀC with 5% CO2. 30d was
added at the reported doses and for the indicated times. Then the cells
were fixed with 100% methanol at ꢀ20 ꢀC for 10 min, washed twice in
Tris-buffered saline buffer containing 0.1% Triton X-100 (TBST), and
finally blocked with 5% milk in TBST buffer. The hyperacetylation was
determined using an antibody recognizing acetylated histone H3
(Upstate 06599), diluted 1:200 in 5% milk in TBST, after incubation
overnight at 4 ꢀC.
In Vivo Drug Pharmacokinetic and Efficacy Experiments. In vivo
pharmacokinetic in mice and efficacy experiments in a human HCT-116
xenograft model in mice were performed as previously described.20 The
T/C ratio corresponds to the mean relative tumor volume of the treated
tumors/mean relative volume of control group. Analysis was performed
using t test statistics (two tailed). The iv formulation for the pharma-
cokinetic experiments comprised 2% DMSO and 9.8% encapsin in
water. For oral dosing the inhibitor was dissolved in water containing 5%
DMSO and 9.5% encapsin.
Immunohistochemistry (IHC) of Tumor Xenografts. Tumor xeno-
grafts were recovered 2 h after the last compound administration and
fixed in formalin. Formalin-fixed and paraffin-embedded tumors were
stained for histone H4 acetylation: the xenograft tissue sections were
3062
dx.doi.org/10.1021/jm200146u |J. Med. Chem. 2011, 54, 3051–3064

Journal of Medicinal Chemistry
ARTICLE
(10) Rasheed, W.; Bishton, M.; Johnstone, R. W.; Prince, H. M.
Histone deacetylase inhibitors in lymphoma and solid malignancies.
Expert Rev. Anticancer Ther. 2008, 8, 413–432.
(11) Khan, O.; La Thangue, N. B. Drug insight: histone deacetylase
inhibitor-based therapies for cutaneous T-cell lymphomas. Nat. Clin.
Pract. Oncol. 2008, 5, 714–726.
Phillips, B. T.; et al. 4-Oxospiro[benzopyran-2,40-piperidines] as class III
antiarrhythmic agents. Pharmacological studies on 3,4-dihydro-
10-[2-(benzofurazan-5-yl)-ethyl]-6-methanesulfonamidospiro[(2H)-1-
benzopyran-2,40-piperidin]-4-on e (L-691,121). J. Med. Chem. 1992,
35, 3973–3976.
(27) Nerenberg, J. B.; Erb, J. M.; Bergman, J. M.; O’Malley, S.;
Chang, R. S.; Scott, A. L.; Broten, T. P.; Bock, M. G. 4-Oxospiro-
[benzopyran-2,40-piperidines] as selective alpha 1a-adrenergic receptor
antagonists. Bioorg. Med. Chem. Lett. 1999, 9, 291–294.
(28) Yamato, M.; Hashigaki, K.; Tsutsumi, A.; Tasaka, K. Synthesis
and structureꢀactivity relationship of spiro[isochroman-piperidine]
analogs for inhibition of histamine release. II. Chem. Pharm. Bull.
(Tokyo) 1981, 29, 3494–3498.
(29) Yang, L.; Morriello, G.; Prendergast, K.; Cheng, K.; Jacks, T.;
Chan, W. W.; Schleim, K. D.; Smith, R. G.; Patchett, A. A. Potent
3-spiropiperidine growth hormone secretagogues. Bioorg. Med. Chem.
Lett. 1998, 8, 107–112.
(30) Harsanyi, K.; Szabadkai, I.; Borza, I.; Karpati, E.; Kiss, B.; Pellionisz,
M.; Farkas, S.; Horvath, C.; Csomor, K.; Lapis, E.; Laszlovszky, I.; Szabo,
S.; Kis-Varga, A.; Laszy, J.; Gere, A. Novel Spiro [2H-1-Benzopyran-2,
40-piperidine]-4(3H)-one Derivatives, and Addition Salts Thereof and
Pharmaceutical Compositions Containing Them. WO97/37630, 1997.
(31) Le Bourdonnec, B.; Windh, R. T.; Ajello, C. W.; Leister, L. K.;
Gu, M.; Chu, G. H.; Tuthill, P. A.; Barker, W. M.; Koblish, M.; Wiant,
D. D.; Graczyk, T. M.; Belanger, S.; Cassel, J. A.; Feschenko, M. S.;
Brogdon, B. L.; Smith, S. A.; Christ, D. D.; Derelanko, M. J.; Kutz, S.;
Little, P. J.; DeHaven, R. N.; DeHaven-Hudkins, D. L.; Dolle, R. E.
Potent, orally bioavailable delta opioid receptor agonists for the treat-
ment of pain: discovery of N,N-diethyl-4-(5-hydroxyspiro[chromene-
2,40-piperidine]-4-yl)benzamide (ADL5859). J. Med. Chem. 2008,
51, 5893–5896.
(32) Le Bourdonnec, B.; Windh, R. T.; Leister, L. K.; Zhou, Q. J.;
Ajello, C. W.; Gu, M.; Chu, G. H.; Tuthill, P. A.; Barker, W. M.; Koblish,
M.; Wiant, D. D.; Graczyk, T. M.; Belanger, S.; Cassel, J. A.; Feschenko,
M. S.; Brogdon, B. L.; Smith, S. A.; Derelanko, M. J.; Kutz, S.; Little, P. J.;
DeHaven, R. N.; DeHaven-Hudkins, D. L.; Dolle, R. E. Spirocyclic delta
opioid receptor agonists for the treatment of pain: discovery of N,
N-diethyl-3-hydroxy-4-(spiro[chromene-2,40-piperidine]-4-yl) benza-
mide (ADL5747). J. Med. Chem. 2009, 52, 5685–5702.
(33) Uto, Y.; Ueno, Y.; Kiyotsuka, Y.; Miyazawa, Y.; Kurata, H.; Ogata,
T.; Yamada, M.; Deguchi, T.; Konishi, M.; Takagi, T.; Wakimoto, S.;
Ohsumi, J. Synthesis and evaluation of novel stearoyl-CoA desaturase 1
inhibitors: 10-{6-[5-(pyridin-3-ylmethyl)-1,3,4-oxadiazol-2-yl]pyridazin-
3-yl}-3,4-dihydrospiro[chromene-2,40-piperidine] analogs. Eur. J. Med.
Chem. 2010, 45, 4788–4796.
(34) Uto, Y.; Kiyotsuka, Y.; Ueno, Y.; Miyazawa, Y.; Kurata, H.;
Ogata, T.; Deguchi, T.; Yamada, M.; Watanabe, N.; Konishi, M.;
Kurikawa, N.; Takagi, T.; Wakimoto, S.; Kono, K.; Ohsumi, J. Novel
spiropiperidine-based stearoyl-CoA desaturase-1 inhibitors: identifica-
tion of 10-{6-[5-(pyridin-3-ylmethyl)-1,3,4-oxadiazol-2-yl]pyridazin-3-
yl}-5-(trifluoromethyl)-3,4-dihydrospiro[chromene-2,40-piperidine].
Bioorg. Med. Chem. Lett. 2010, 20, 746–754.
(12) Marks, P. A. Discovery and development of SAHA as an
anticancer agent. Oncogene 2007, 26, 1351–1356.
(13) Marks, P. A.; Breslow, R. Dimethyl sulfoxide to vorinostat:
development of this histone deacetylase inhibitor as an anticancer drug.
Nat. Biotechnol. 2007, 25, 84–90.
(14) Bates, S. E.; Zhan, Z.; Steadman, K.; Obrzut, T.; Luchenko, V.;
Frye, R.; Robey, R. W.; Turner, M.; Gardner, E. R.; Figg, W. D.;
Steinberg, S. M.; Ling, A.; Fojo, T.; To, K. W.; Piekarz, R. L. Laboratory
correlates for a phase II trial of romidepsin in cutaneous and peripheral
T-cell lymphoma. Br. J. Haematol. 2010, 148, 256–267.
(15) Atadja, P. Development of the pan-DAC inhibitor panobinostat
(LBH589): successes and challenges. Cancer Lett. 2009, 280, 233–41.
(16) Steele, N. L.; Plumb, J. A.; Vidal, L.; Tjornelund, J.; Knoblauch,
P.; Rasmussen, A.; Ooi, C. E.; Buhl-Jensen, P.; Brown, R.; Evans, T. R.;
DeBono, J. S. A phase 1 pharmacokinetic and pharmacodynamic study
of the histone deacetylase inhibitor belinostat in patients with advanced
solid tumors. Clin. Cancer Res. 2008, 14, 804–810.
(17) Novotny-Diermayr, V.; Sangthongpitag, K.; Hu, C. Y.; Wu, X.;
Sausgruber, N.; Yeo, P.; Greicius, G.; Pettersson, S.; Liang, A. L.; Loh,
Y. K.; Bonday, Z.; Goh, K. C.; Hentze, H.; Hart, S.; Wang, H.; Ethirajulu,
K.; Wood, J. M. SB939, a novel potent and orally active histone
deacetylase inhibitor with high tumor exposure and efficacy in mouse
models of colorectal cancer. Mol. Cancer Ther. 2010, 9, 642–652.
(18) Gojo, I.; Jiemjit, A.; Trepel, J. B.; Sparreboom, A.; Figg, W. D.;
Rollins, S.; Tidwell, M. L.; Greer, J.; Chung, E. J.; Lee, M. J.; Gore, S. D.;
Sausville, E. A.; Zwiebel, J.; Karp, J. E. Phase 1 and pharmacologic study
of MS-275, a histone deacetylase inhibitor, in adults with refractory and
relapsed acute leukemias. Blood 2007, 109, 2781–2790.
(19) Zhou, N.; Moradei, O.; Raeppel, S.; Leit, S.; Frechette, S.;
Gaudette, F.; Paquin, I.; Bernstein, N.; Bouchain, G.; Vaisburg, A.; Jin,
Z.; Gillespie, J.; Wang, J.; Fournel, M.; Yan, P. T.; Trachy-Bourget, M. C.;
Kalita, A.; Lu, A.; Rahil, J.; MacLeod, A. R.; Li, Z.; Besterman, J. M.;
Delorme, D. Discovery of N-(2-aminophenyl)-4-[(4-pyridin-3-ylpyri-
midin-2-ylamino)methyl]benzamide (MGCD0103), an orally active
histone deacetylase inhibitor. J. Med. Chem. 2008, 51, 4072–4075.
(20) Thaler, F.; Colombo, A.; Mai, A.; Amici, R.; Bigogno, C.;
Boggio, R.; Cappa, A.; Carrara, S.; Cataudella, T.; Fusar, F.; Gianti, E.;
Joppolo di Ventimiglia, S.; Moroni, M.; Munari, D.; Pain, G.; Regalia, N.;
Sartori, L.; Vultaggio, S.; Dondio, G.; Gagliardi, S.; Minucci, S.;
Mercurio, C.; Varasi, M. Synthesis and biological evaluation of
N-hydroxyphenyl-acrylamides and N-hydroxypyridin-2-yl-acrylamides
as novel histone deacetylase inhibitors. J. Med. Chem. 2010, 53, 822–829.
(21) Thaler, F.; Colombo, A.; Mai, A.; Bigogno, C.; Boggio, R.;
Carrara, S.; Joppolo di Ventimiglia, S.; Munari, D.; Regalia, N.; Dondio,
G.; Gagliardi, S.; Minucci, S.; Mercurio, C.; Varasi, M. Synthesis and
biological characterization of amidopropenyl-hydroxamates as HDAC
inhibitors. ChemMedChem 2010, 5, 1359–1372.
(35) Shinde, P.; Srivastava, S. K.; Odedara, R.; Tuli, D.; Munshi, S.;
Patel, J.; Zambad, S. P.; Sonawane, R.; Gupta, R. C.; Chauthaiwale, V.; Dutt,
C. Synthesis of spiro[chroman-2,40-piperidin]-4-one derivatives as acetyl-
CoA carboxylase inhibitors. Bioorg. Med. Chem. Lett. 2009, 19, 949–953.
(36) Reiser, U.; Kraemer, O.; Sennhenn, P.; Spevak, W. Spiro-(thio)
Benzopyran-2,40-piperidine- and Cyclohexane Derivatives as Inhibitors
of Specific Cell Cycle Enzyme. WO2007/128782, 2007.
(37) Cochran, J. E.; Wu, T.; Padwa, A. Synthesis of polysubstituted
anilines using the DielsꢀAlder reaction of methyl 5-aminofuroate.
Tetrahedron Lett. 1996, 37, 2903–2906.
(38) Klymchenko, A. S.; Mꢁely, Y. 7-(2-Methoxycarbonylvinyl)-3-
hydroxychromones: new dyes with red shifted dual emission. Tetrahe-
dron Lett. 2004, 45, 8391–8394.
(39) Wilson, R. A.; Chan, L.; Wood, R.; Brown, R. C. Synthesis and
derivatisation of a novel spiro[1-benzofuran-2,40-piperidin]-3-one scaf-
fold. Org. Biomol. Chem. 2005, 3, 3228–3235.
(22) DeSimone, R. W.; Currie, K. S.; Mitchell, S. A.; Darrow, J. W.;
Pippin, D. A. Privileged structures: applications in drug discovery. Comb.
Chem. High Throughput Screening 2004, 7, 473–494.
(23) Costantino, L.; Barlocco, D. Privileged structures as leads in
medicinal chemistry. Curr. Med. Chem. 2006, 13, 65–85.
(24) Beghyn, T.; Deprez-Poulain, R.; Willand, N.; Folleas, B.;
Deprez, B. Natural compounds: leads or ideas? Bioinspired molecules
for drug discovery. Chem. Biol. Drug Des. 2008, 72, 3–15.
(25) Evans, B. E.; Rittle, K. E.; Bock, M. G.; DiPardo, R. M.;
Freidinger, R. M.; Whitter, W. L.; Lundell, G. F.; Veber, D. F.; Anderson,
P. S.; Chang, R. S.; et al. Methods for drug discovery: development of
potent, selective, orally effective cholecystokinin antagonists. J. Med.
Chem. 1988, 31, 2235–2246.
(26) Elliott, J. M.; Selnick, H. G.; Claremon, D. A.; Baldwin, J. J.;
Buhrow, S. A.; Butcher, J. W.; Habecker, C. N.; King, S. W.; Lynch, J. J., Jr.;
3063
dx.doi.org/10.1021/jm200146u |J. Med. Chem. 2011, 54, 3051–3064

Journal of Medicinal Chemistry
ARTICLE
(40) Blackwell, L.; Norris, J.; Suto, C. M.; Janzen, W. P. The use of
diversity profiling to characterize chemical modulators of the histone
deacetylases. Life Sci. 2008, 82, 1050–1058.
(41) Kozikowski, A. P.; Chen, Y.; Gaysin, A.; Chen, B.; D’Annibale,
M. A.; Suto, C. M.; Langley, B. C. Functional differences in epigenetic
modulators—superiority of mercaptoacetamide-based histone deacety-
lase inhibitors relative to hydroxamates in cortical neuron neuroprotec-
tion studies. J. Med. Chem. 2007, 50, 3054–3061.
(42) Fournel, M.; Bonfils, C.; Hou, Y.; Yan, P. T.; Trachy-Bourget,
M. C.; Kalita, A.; Liu, J.; Lu, A. H.; Zhou, N. Z.; Robert, M. F.; Gillespie,
J.; Wang, J. J.; Ste-Croix, H.; Rahil, J.; Lefebvre, S.; Moradei, O.;
Delorme, D.; Macleod, A. R.; Besterman, J. M.; Li, Z. MGCD0103, a
novel isotype-selective histone deacetylase inhibitor, has broad spectrum
antitumor activity in vitro and in vivo. Mol. Cancer Ther. 2008, 7
759–768.
(43) Haggarty, S. J.; Koeller, K. M.; Wong, J. C.; Grozinger, C. M.;
Schreiber, S. L. Domain-selective small-molecule inhibitor of histone
deacetylase 6 (HDAC6)-mediated tubulin deacetylation. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 4389–4394.
(44) Davies, B.; Morris, T. Physiological parameters in laboratory
animals and humans. Pharm. Res. 1993, 10, 1093–1095.
(45) Yeo, P.; Xin, L.; Goh, E.; New, L. S.; Zeng, P.; Wu, X.;
Venkatesh, P.; Kantharaj, E. Development and validation of high-
performance liquid chromatographyꢀtandem mass spectrometry assay
for 6-(3-benzoyl-ureido)-hexanoic acid hydroxyamide, a novel HDAC
inhibitor, in mouse plasma for pharmacokinetic studies. Biomed. Chro-
matogr. 2007, 21, 184–189.
(46) Ronzoni, S.; Faretta, M.; Ballarini, M.; Pelicci, P.; Minucci, S.
New method to detect histone acetylation levels by flow cytometry.
Cytometry, Part A 2005, 66, 52–61.
3064
dx.doi.org/10.1021/jm200146u |J. Med. Chem. 2011, 54, 3051–3064