Synthesis and biological characterization of spiro[2H-(1,3)-benzoxazine-2, 4′-piperidine] based histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.03.061

Histone Deacetylases (HDACs) have become important targets for the treatment of cancer and other diseases. In previous studies we described the development of novel spirocyclic HDAC inhibitors based on the combination of privileged structures with hydroxamic acid moieties as zinc binding group. Herein, we report further explorations, which resulted in the discovery of a new class of spiro[2H-(1,3)-benzoxazine-2,4′-piperidine] derivatives. Several compounds showed good potency of around 100 nM and less in the HDAC inhibition assays, submicromolar IC₅₀ values when tested against tumour cell lines and a remarkable stability in human and mouse microsomes. Two representative examples exhibited a good pharmacokinetic profile with an oral bioavailability equal or higher than 35% and one of them studied in an HCT116 murine xenograft model showing a robust tumour growth inhibition. In addition, the two benzoxazines were found to have a minor affinity for the hERG potassium channel compared to their corresponding ketone analogues.

Full text of DOI:10.1016/j.ejmech.2013.03.061

European Journal of Medicinal Chemistry 64 (2013) 273e284
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological characterization of spiro[2H-(1,3)-
benzoxazine-2,40-piperidine] based histone deacetylase inhibitors
,
b
,
b
,
,
c
,
,c,2
Florian Thaler a , Mario Varasi c, Agnese Abate b 1, Giacomo Carenzi b
,
*
Andrea Colombo d 3, Chiara Bigogno d 4, Roberto Boggio a 5, Roberto Dal Zuffo b
,
,
,
,
,c
Daniela Rapetti d, Anna Resconi d, Nickolas Regalia d, Stefania Vultaggio b
,
,
c
,
,
Giulio Dondio d 4, Stefania Gagliardi d, Saverio Minucci e f, Ciro Mercurio c
a Genextra Group, Congenia s.r.l., Via Adamello 16, 20139 Milan, Italy
b Drug Discovery Program, Department of Experimental Oncology, IEO e European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy
c Genextra Group, DAC s.r.l., Via Adamello 16, 20139 Milan, Italy
d NiKem Research s.r.l., Via Zambeletti 25, 20021 Baranzate, Italy
e IEO e European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy
f Dept. of Life Sciences, University of Milan, Via Celoria, 26, 20133 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Histone Deacetylases (HDACs) have become important targets for the treatment of cancer and other
diseases. In previous studies we described the development of novel spirocyclic HDAC inhibitors based
on the combination of privileged structures with hydroxamic acid moieties as zinc binding group. Herein,
we report further explorations, which resulted in the discovery of a new class of spiro[2H-(1,3)-ben-
zoxazine-2,40-piperidine] derivatives. Several compounds showed good potency of around 100 nM and
less in the HDAC inhibition assays, submicromolar IC50 values when tested against tumour cell lines and a
remarkable stability in human and mouse microsomes. Two representative examples exhibited a good
pharmacokinetic profile with an oral bioavailability equal or higher than 35% and one of them studied in
an HCT116 murine xenograft model showing a robust tumour growth inhibition. In addition, the two
benzoxazines were found to have a minor affinity for the hERG potassium channel compared to their
corresponding ketone analogues.
Received 30 November 2012
Received in revised form
27 March 2013
Accepted 28 March 2013
Available online 10 April 2013
Keywords:
Histone deacetylases
Privileged scaffolds
Hydroxamates
Antiproliferation
Pharmacokinetics
In vivo antitumor activity
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
histone acetyltransferases (HATs) promote the acetylation of his-
tones leading to nucleosomal relaxation and generally to gene
The histone acetylation status plays an important role in chro-
matin structure and control of gene expression [1]. Acetylation and
deacetylation are regulated by two distinct enzyme families:
transcription, histone deacetylases (HDACs) catalyze the cleavage of
the acetyl group resulting in a condensed form of the chromatin
and generally gene silencing. Recent studies showed that HDACs
are responsible for the deacetylation of a series of non-histone
proteins, among them proteins involved in the control of various
cellular functions [2,3]. To date, 18 distinct human histone deace-
tylases have been reported and are grouped into four classes. Class I
(HDAC 1e3 and HDAC 8), class II (HDAC 4e7 and HDAC 9e10) and
class IV (HDAC 11) contain a zinc atom in their catalytic site,
whereas class III HDACs or sirtuins (Sir 1e7) have an unique
enzymatic mechanism and use NADþ as cofactor [4e6]. In the past
decade, HDACs have emerged as an attractive target for the
development of new agents with anticancer or anti-inflammatory
properties or active in neurodegenerative diseases [7e12]. A se-
ries of HDAC inhibitors have entered clinical studies and the
hydroxamic acid Zolinza (known also as SAHA or vorinostat) [13]
* Corresponding author. Drug Discovery Program, Department of Experimental
Oncology, IEO e European Institute of Oncology, Via Adamello 16, 20139 Milan,
Italy. Tel.: þ39 02 94375130; fax: þ39 02 94375138.
E-mail address: florian.thaler@ieo.eu (F. Thaler).
Current address: Sbarro Health Research Organization, Bio Life Science Building,
1
Temple University, 1900 North 12th Street, Philadelphia, PA 19122-6099, USA.
2
Current address: Fondazione Istituto Insubrico di Ricerca per la Vita, Via Lepetit,
34, 21040 Gerenzano, Italy.
3
Current address: Teva Pharmaceuticals Fine Chemicals, Strada Statale Briantea,
23892 Bulciago, Italy.
4
Current address: Cyathus Exquirere Italia s.r.l., Via Roberto Lepetit 34, 21040
Gerenzano, Italy.
5
Current address: Nerviano Medical Sciences, Via Pasteur 10, 20014 Nerviano,
Italy.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.061

274
F. Thaler et al. / European Journal of Medicinal Chemistry 64 (2013) 273e284
and the cyclic depsipeptide Istodax (romidepsin or FK228) [14,15]
gained approval by the FDA for the treatment of the cutaneous T-
cell lymphoma (CTCL). Other compounds in clinical trials for the
treatment of hematological and solid tumours comprise the
hydroxamic acid derivatives panobinostat (or LBH589) [16], beli-
nostat (or PXD101) [17] and the benzamides SNDX-275 [18] and
MGCD-0103 [19,20] (Fig. 1).
We have previously identified a new series of histone deacety-
lase inhibitors composed by a spirocyclic privileged structure and a
hydroxamic acid as zinc binding moiety [21,22]. Compounds 1e3
(see Fig. 2) were found to be potent HDAC inhibitors with good
antiproliferative activity against three tumour cell lines. The in-
hibitors exhibited a good oral bioavailability in mouse, substantially
higher than other hydroxamic acid HDAC derivatives, currently in
advanced clinical studies such as SAHA, PXD101 or LBH589 [23,24],
and showed activity in an HCT116 xenograft model after oral
administration [21,22].
1: n=1, R=H
2: n=1, R=F
3: n=2, R=H
Fig. 2. Lead compounds 1e3.
directed towards unsubstituted, 3-methyl and 3-benzyl benzox-
azine derivatives with the 40-piperidine either unsubstituted or
substituted by N-methyl, benzyl, 4-fluorobenzyl, or 2-phenylethyl
(Fig. 3).
While studying the spirocyclic structures we found that the
ketone group was important for the biochemical and anti-
proliferative activity: the reduced secondary alcohol (E)-N-hy-
droxy-3-{10-benzyl-4-hydroxyspiro[chromane-2,40-piperidine]-6-
yl}acrylamide as well as the corresponding chromane, chromene
and the N-acetylamino derivatives were indeed less potent com-
pounds [21]. Herein, we wish to present the synthesis of a new
series of 4-oxospiro[2H-(1,3)-benzoxazine-2,40-piperidin]-6-yl}-N-
hydroxyacrylamides, which contain an amide instead of the
important ketone moiety, and their biological characterization. This
further exploration was also stimulated by previously published
metabolic studies of L-691,121, an antiarrhythmic agent, which
shares the 4-oxospiro[chromane-2,40-piperidin] core structure
with the lead compounds 1e3 and which had shown that one
major metabolic path was the reduction of the ketone group to the
secondary alcohol [25]. In specific, planned structures were
2. Results and discussion
(E)-3-{3,4-Dihydro-4-oxospiro[2H-(1,3)-benzoxazine-2,40-
piperidin]-6-yl}-N-hydroxyacrylamide (8) was prepared as illus-
trated in Scheme 1. Cyclization of 5-bromo salicylamide (4) and 1-
(tert-butyloxycarbonyl)piperidine-4-one (1-BOC piperidin-4-one)
in presence of pyrrolidine and under microwave irradiation gave
Fig. 3. Exploration of the spiro[2H-(1,3)-benzoxazine-2,40-piperidine] series.
Zolinza
Istodax
panobinostat
belinostat
SNDX-275
MGCD-0103
Fig. 1. HDAC inhibitors approved or in advanced clinical development.

F. Thaler et al. / European Journal of Medicinal Chemistry 64 (2013) 273e284
275
a
b
4
5
6
c
d, e
8
7
Scheme 1. Reagents and conditions: (a) N-BOC-piperidin-4-one, pyrrolidine, MW, 72 ꢀC, 1 h; (b) DMF, TEA, Pd(OAc)2, P(o-tol)3, methyl acrylate, 100 ꢀC, 9 h; (c) dioxane/H2O (1:1,
v:v), NaOH, RT, overnight; (d) CH2Cl2, EDC, HOBt, TEA, NH2OTHP, RT, overnight; (e) CH2Cl2, dioxane, HCl, RT, 3 h.
the spirocycle 5. Heck reaction with methyl acrylate in presence of
palladium acetate [Pd(OAc)2], tris(o-tolyl)phosphine [P(o-tol)3],
and triethylamine (TEA) in DMF afforded the methyl acrylic ester 6.
The methyl ester 6 was hydrolyzed with sodium hydroxide (NaOH)
in water/dioxane and the resulting carboxylic acid 7 was then
coupled with NH2OTHP (O-(tetrahydropyran-2-yl)hydroxylamine)
in the presence of EDC (N-(3-dimethylaminopropyl)-N0-ethyl-
carbodiimide hydrochloride) and HOBt (N-hydroxybenzotriazole).
The tetrahydropyran-2-yl and BOC protecting groups were finally
cleaved with hydrogen chloride (HCl) affording the requisite
hydroxamic acid 8.
The N-substituted spirocycles 12aed were prepared as outlined
in Scheme 2. Cleavage of the BOC protecting group of spirocycle 6
with HCl gave the free amine 9, which was then alkylated or
reductively alkylated using sodium cyanoborohydride [NaBH3(CN)]
as reducing agent. Hydrolysis of the methyl ester with NaOH and
subsequent introduction of the hydroxamic acid according to the
procedure described above for the compound 8 (Scheme 1) fur-
nished the desired hydroxamic acid derivatives 12aed.
The synthesis of the N-methyl and N-benzyl amide analogues 17,
18, 25aed, and 26aed was performed as described in Scheme 3.
Spirocycle 6 was first deprotonated with NaH and then reacted
with methyl iodide at room temperature or benzyl bromide at
80 ꢀC. The N-methyl and the N-benzyl spirocycle methyl esters 13
and 14 were then hydrolyzed with NaOH and the resulting car-
boxylic acids 15 and 16 were then coupled with NH2OTHP in
presence of EDC and HOBt. Acidic cleavage of the THP and of the
tert-butyl ester groups with HCl gave the desired hydroxamates 17
and 18. Removal of the BOC protecting group of 13 and 14 with HCl
gave the free amines 19 and 20, which were then alkylated or
reductively alkylated giving the N-substituted spirocycles 21ae
d and 22aed. Saponification of the methyl ester proceeded under
basic conditions, and the resulting carboxylic acids 23aed and
24aed were converted into the desired hydroxamic acid de-
rivatives first by coupling with NH2OTHP in presence of EDC and
HOBt and then cleavage of the THP protecting group with HCl.
The synthesized 4-oxospiro[2H-(1,3)-benzoxazine-2,40-piper-
idin]-6-yl}-N-hydroxyacrylamides were initially examined for their
HDAC inhibiting properties. For this purpose, the spirocycles were
tested using a commercial HDAC assay kit, containing partially
purified nuclear extracts from HeLa cells as enzyme source. As
summarized in Table 1, no major differences in biochemical activ-
ities were found among the amides 8 and 12aed: the free
spiropiperidine derivative 8 exhibited an IC50 value of 0.078
N-methyl analogue 12a 0.099 M, and the IC50 values for the
benzyl, the para fluorobenzyl and 2-phenylethyl analogues 12be
d were between 0.083 and 0.103 M, in the range of the previously
disclosed biochemical activities of the three reference 4-
oxospirochromanes 1e3 as well as of SAHA, which are included
in Table 1 for the purpose of comparison [21,22]. The HDAC inhib-
itory activity of the 3-methyl-benzoxazine derivatives 17 and 25ae
mM, the
m
m
d were between 0.136 mM (benzyl analogue 25b) and 0.527 mM
(methyl analogue 25a). When we compare the results with those of
the unsubstituted amides, the N-methylation has led to a slight
decrease in potency. Major differences in the biochemical activity
were found for the 3-benzyl benzoxazines: the free spiropiperidine
18 was with an IC50 value of 0.059
mM around three times more
potent than the N-methyl piperidine (0.204
mM) and about ten
times more potent than the bulkier benzyl or 2-phenylethyl ana-
logues 26bed.
As further characterization of the compounds, the anti-
proliferative activity of the spirocycles was assessed in three
tumour cell lines of different tissue origins: chronic myelogenous
leukaemia cell line K562, carcinomic human alveolar basal
epithelial cells A549 and human colon cancer cells HCT116. As
compiled in Table 1, all compounds with exception of the unsub-
stituted spiropiperidines 8 and 17 showed good antiproliferative
activity in the three cell lines with IC50 values ranging from sub-
micromolar to the low micromolar values. Compounds 8 and 17
were less potent with IC50 values of ꢁ5
mM in the tested cell lines,
potentially due to limited cellular membrane permeability.
Furthermore, the compounds showed in general higher anti-
proliferative potency against the K562 and HCT116 cell lines than in
the lung cancer A549 cells. In specific, the N-benzyl and 2-
phenylethyl spiropiperidines 12bed, which exhibited anti-
proliferative activities similar to the reference spirochromanes 1e3
and SAHA, had a slightly superior cellular activity than the methyl
analogue 12a. For example, spiropiperidine 12a was with an IC50 of
1.49
mM around three times less active in K562 cells than the 2-
phenylethyl analogue 12d (IC50 ¼ 0.49
m
M). Differences in activ-
ities among the spiropiperidines 12aed are even minor in the other
two cell lines. Similarly, also the antiproliferative activities were
almost identical for the N-substituted spiropiperidines within the
3-methyl-benzoxazine series. The para fluorobenzyl spiropiper-
idine 25c exhibited in K562, A549 and HCT116 cell lines IC50 values
of 0.734, 2.10, and 0.85 mM, respectively, and was less than two

276
F. Thaler et al. / European Journal of Medicinal Chemistry 64 (2013) 273e284
a
b
6
10a-d
9
c
d, e
12a-d
11a-d
Scheme 2. Reagents and conditions: (a) CH2Cl2, dioxane, HCl, RT, 4 h; (b) MeOH, R0CHO, NaBH3(CN), RT, overnight or CH2Cl2, TEA, RBr, RT, 5 h for 10b; (c) dioxane/H2O (1:1, v:v),
NaOH, RT, overnight or MeOH, NaOH, 50 ꢀC, 2 h for 11b; (d) CH2Cl2, EDC, HOBt, TEA, NH2OTHP, RT, overnight; (e) CH2Cl2, dioxane, HCl, RT, 3 h.
times more potent than the methyl derivative 25a. On the contrary,
major differences in antiproliferative potency was found within the
3-benzyl-benzoxazine series. The N-methyl analogue 26a showed
to be the most active compound within this subseries with IC50
normal electrical activity in the heart and several studies have
shown that inhibition of the hERG channel are linked to a prolon-
gation of the QT interval of the surface electrocardiogram and to the
polymorphic ventricular arrhythmia Torsades de Pointes [26e28].
The affinity of the tested compounds as well as their corresponding
spirochromanes 1 and 2 was measured in a [3H]astemizole
displacement binding assay [29]. The two spirochromane de-
rivatives exhibited hERG binding affinities with IC50 values of
values of 0.360
mM (K562), 0.912 mM (A549), and 0.326 mM
(HCT116), respectively. The selection criteria for compounds to be
further investigated was based also taking into account the activ-
ities of the three reference spirochromanes 1e3: thus, compounds,
which did not have antiproliferative activities in the submicromolar
range against at least two cellular lines as well as good biochemical
potency, were not further characterized.
3.81 ꢂ 0.61 and 2.38 ꢂ 0.73
mM for compounds 1 and 2, respec-
tively. Changing the ketone to an amide resulted in a significantly
reduced affinity for the hERG Kþ channel. At the highest tested dose
Subsequently, the metabolic stability of the spirocycles was
assessed in mouse and human microsomes to verify the initial
hypothesis that ketone substitution may lead to an increased sta-
bility. For this purpose, the inhibitors were exposed to a micro-
somal preparation for 30 min at 37 ꢀC. The reaction was stopped by
adding an aliquot of CH3CN and the amount of the unmetabolized
derivative was determined by LCeMS/MS running in MRM mode.
Table 2 comprises the experimental results together with the pre-
viously obtained data of metabolic stability of the lead compounds
1e3. The N-benzyl and 4-fluorobenzyl analogues 12b and 12c
exhibited a superior microsomal stability than the corresponding
spiroketone analogues 1 and 2. In particular, only 25% of 12b was
metabolized in mouse microsomes compared to 68% of spirocycle 1.
The 4-fluorobenzyl analogue 12c was fully recovered after 30 min
incubation in mouse microsomes, while less than 50% of the ketone
analogue 2 was found after 30 min incubation. Compounds 12b and
12c showed good stability also in human microsomes, with a re-
covery of 77% and 71% of 12b and 12c, respectively. On the other
hand, an increase of stability both in mouse and human micro-
somes was not found for the 2-phenylethyl analogue 12d compared
to the corresponding ketone 3. The two further tested spirocycles
25c and 26a showed higher clearance rates in mouse microsomes
than 12b and 12c: the recovery of unaltered compound after 30 min
of incubation was 39 and 19%, respectively.
of 30 mM, the percentages of inhibition were 54% and 70% for the
benzyl derivative 12b and the 4-fluorobenzyl 12c analogue,
respectively.
Then, the pharmacokinetic behaviour of the two spirocycles was
determined. The inhibitors were administrated to CD-1 mice in
single intravenous (iv) or oral doses of 5 or 15 mg/kg, respectively.
The compounds were dissolved in water containing 3% DMSO and
9.7% encapsin for the iv dose, or in water containing 5% DMSO and
9.5% encapsin for the oral administration. As summarized in
Table 3, both compounds showed medium clearance rates with
3.27 L/h/kg for 12b and 2.66 L/h/kg for 12c, respectively, which
were lower than the clearance of the previously reported corre-
sponding ketone analogues 1 (5.88 L/h/kg) and 2 (3.35 L/h/kg) [22].
The elimination half-lives were found to be 13.4 h for compound
12b and 6.9 h for 12c. The estimated steady-state volume of dis-
tribution Vss of 12b and 12c were 8.5 and 3.05 L/kg, respectively,
and indicate an extensive tissue distribution of the compounds. A
rapid absorption of the two spiro-benzoxazines from the gastro-
intestinal tract was observed after oral administration; and the
maximum plasma concentrations (cmax) were 2.71 (12b) and
5.05 mM (12c), respectively. The AUC0eN after oral administration
were practical identical for both compounds and the calculated oral
bioavailability was 42.8% for 12b and 35% for 12c. The date
demonstrated that both compounds showed a good PK profile with
an oral bioavailability equal or higher than 35% of the administered
dose.
Compound 12b was then chosen as representative compound to
be further characterized in further in vitro assays and finally in an
in vivo efficacy model. Firstly, its biochemical selectivity profile was
evaluated with respect to HDAC 2 and HDAC 3 as representatives of
class I HDACs and HDAC 6 as representative of class II HDACs. As
reported in Table 4, compound 12b, similar to the previously
The benzyl and the 4-fluorobenzyl spirobenzoxazines 12b and
12c emerged as the most stable compounds in the mouse and
human microsome assays. The solubility at pH 7.4 of both com-
pounds exceeded 200 mM, and we selected both compounds for
further in vitro and in vivo characterizations.
Subsequently, the affinity of the compounds the voltage-gated
potassium channel encoded by the human ether-a-go-go related
gene (hERG) was assessed. The hERG channels are fundamental for

F. Thaler et al. / European Journal of Medicinal Chemistry 64 (2013) 273e284
277
a
13 R1...Me
14 R1...Bn
6
c, d
b
13 R1...Me
14 R1...Bn
17 R1...Me
18 R1...Bn
15 R1...Me
16 R1...Bn
e
f
13 R1...Me
14 R1...Bn
19 R1...Me
20 R1...Bn
21a-d R1...Me
22a-d R1...Bn
g
h, i
25a-d R1...Me
26a-d R1...Bn
23a-d R1...Me
24a-d R1...Bn
Scheme 3. Reagents and conditions: (a) DMF, NaH, CH3I, 4 ꢀC to RT, 40 min for 13 or DMF, PhCH2Br, 80 ꢀC, 3 h for 14; (b) dioxane/water (1:1, v:v), NaOH, RT, overnight for 15 and
MeOH, NaOH, 50 ꢀC, 2 h for 16; (c) þ (h) CH2Cl2, EDC, HOBt, TEA, NH2OTHP, RT, overnight; (d) þ (i) CH2Cl2, dioxane, HCl, RT, 3 h; (e) CH2Cl2, dioxane, HCl, RT, 4 h; (f) CH2Cl2, R0CHO,
NaBH(CN)3, RT, overnight for 21aed, or CH2Cl2, R0CHO, NaBH(OAc)3, RT, 3 h for 22a, 22ced, or CH2Cl2, TEA; PhCH2Br, RT, 5 h for 22b; (g) dioxane/water (2:1, v:v), NaOH, RT, 5 h for
23aed and acetic acid, 6 M HCl, 85 ꢀC, 4 h for 24aed.
disclosed spirochromanes 1e3, did not show a preferential activity
versus the different tested HDAC isoforms.
for 5 days/week for 2 weeks. No significant body weight differences
among the groups of mice and no signs of evident toxicity were
observed during the treatment. As shown in Fig. 6, oral adminis-
tration at different dose level determined and evident and signifi-
cant dose dependent decrease in tumour volume relative to the
control. Specifically, the calculated tumour/control ratio (T/C) at the
dose of 75 mg/kg and 150 mg/kg were of 0.59 and 0.33,
respectively.
Overall, the in vivo efficacy, as evaluated by T/C ratio for 12b at
the highest tested dose appear statistically significant and similar
to SAHA, for which we had obtained a calculated T/C ratio of around
0.34 after 2 weeks of treatment administering the substance
intraperitoneally once a day (50 mg/kg) [24]. Furthermore, 12b
exhibited a tumour growth inhibition comparable to spirocycle 1,
and slightly, but not significantly inferior compared to spirocycles 2
and 3 [22]. A lower permeability behaviour in Caco-2 cells [30]
between 12b and 1 (4 nm/s versus 56 nm/s, respectively, which
possibly translates into a minor tumour penetration of 12b) could
The ability of 12b to inhibit HDACs in cells was also confirmed by
dose dependent increase in acetylation level in K562 cells as indi-
cated by flow cytometry (Fig. 4). Possible cell cycle perturbations
and cell death inductions were analyzed by flow cytometry
following the cell cycle profile of colon carcinoma cells HCT116
exposed to 12b for 24 h. As reported in Fig. 5, cells treated with
compound 12b showed an increased percentage of G0/G1 popu-
lation at lower doses and an increase of G2/M population at the
highest tested dose. Furthermore a dose dependent increase of sub-
G1 population, an indicator for cell death, could be observed.
Finally,12b was submitted to in an in vivo efficacy experiment in
an established human HCT116 xenograft model. Human cancer
cells HCT-116 (5 ꢃ 106) were injected subcutaneously in the flank of
female CD-1 nude mice. The treatment initiated once the tumours
reached a volume of 100 mm3 and the compound was tested at the
doses of 75 and 150 mg/kg. 12b was administered orally once daily

278
F. Thaler et al. / European Journal of Medicinal Chemistry 64 (2013) 273e284
Table 1
Table 3
Enzymatic and antiproliferative activity of 4-oxospiro[2H-(1,3)-benzoxazine-2,40-
piperidin]-6-yl}-N-hydroxyacrylamides 8, 12aed, 21, 22, 25aed and 26aed.a
Pharmacokinetic properties of compounds 1, 2, 3, 12b and 12c in mice.
Parameter
Cmax, iv M]
AUCiv0eN [ng$h/mL]
t1/2 [h]
Cl [L/h/kg]
Vss [L/kg]
1
2
3
12b
12c
[
m
12.5
847
8.4
11
1494
13
3.35
5.9
6.34
1042
9.1
4.79
8.6
13.5
1527
13.4
3.27
8.5
13
1875
6.9
2.66
3.1
5.88
8.9
Cmax, iv
tmax [min]
AUCos0eN [ng$h/mL]
F [%]
[
m
M]
0.823
1.83
15
1238
27.6
2.49
15
1598
51.1
2.71
15
1961
42.8
5.05
5
1977
35
15
800
31.5
R
R1
HDACb
M)
K562
M)
A549
M)
HCT-116
(mM)
(
m
(
m
(
m
SAHA
1
2
3
8
12a
12b
12c
12d
17
25a
25b
25c
25d
18
0.102
0.121
0.108
0.113
0.078
0.099
0.083
0.103
0.091
0.204
0.527
0.136
0.137
0.258
0.059
0.174
0.540
0.667
0.645
0.645
0.399
0.681
0.180
8.93
1.079
0.773
1.322
0.551
4.81
2.70
1.41
1.67
1.42
0.757
0.477
0.777
0.201
6.86
0.886
0.515
0.498
0.469
12.1
1.40
0.979
0.848
1.06
3. Conclusion
In summary, the synthesis and biological characterization of
new spiro[2H-(1,3)-benzoxazine-2,40-piperidine] hydroxamic acid
derivatives has been described. The unsubstituted 3-H-benzox-
azines exhibited good HDAC inhibitory activities comparable to that
of the previously described spirochromanes. Major differences in
the biochemical activity was found for the 3-benzyl benzoxazines:
H
Me
Bn
4-F-Bn
Phe(CH2)2e
H
Me
Bn
4-F-Bn
Phe(CH2)2e
H
Me
Bn
H
H
H
H
1.49
0.582
0.961
0.494
14.2
1.70
H
Me
Me
Me
Me
Me
Bn
Bn
Bn
Bn
Bn
17.9
3.54
1.33
2.22
2.10
2.55
3.19
0.912
2.62
3.04
1.51
the free spiropiperidine 18 was with an IC50 value of 0.059
mM
0.734
0.795
1.52
0.360
1.72
around three times more potent than the N-methyl piperidine 17
and about ten times more potent than the benzyl or 2-phenylethyl
analogues 26bed. With exception of the unsubstituted spi-
0.857
0.326
1.87
2.03
1.67
26a
26b
26c
26d
ropiperidines
8 and 17 all compounds showed good anti-
4-F-Bn
Phe(CH2)2e
1.57
1.25
proliferative activity with IC50 values in the submicromolar to the
low micromolar range in three tumour cell lines. Microsomial
stability studies of selected compounds resulted in a remarkable
stability. The in vitro ADME data were confirmed by a good in vivo
pharmacokinetic profile of two representative spirocycles 12b and
12c with an oral bioavailability equal or higher than 35%. The good
PK behaviour correlated well with a good in vivo antitumor activity
of the representative spirocycle 12b in an HCT-116 xenograft model.
These results, together with the fact that the benzyl and 4-
fluorobenzyl benzoxazines 12b and 12c showed a reduced hERG
binding affinity, confirm the spiropiperidine system as a versatile
privileged scaffold to obtain potent HDAC inhibitors with good
in vivo potency after oral administration.
a
Assays done in replicates (n ꢁ 2). Mean values are shown and the standard
deviations are <30% of the mean.
b
HeLa cell nuclear extracts.
justify that the better pharmacokinetic profile of 12b compared to
compound 1 was not confirmed by an increased in vivo potency.
In order to correlate the observed in vivo efficacy with HDAC
inhibition, tumour sections were taken at the end of treatment
from mice treated with the vehicle and from those treated with the
spirocycle 12b and analyzed by immunohistochemistry, following
the histone acetylation level [31]. As illustrated in Fig. 6B, treatment
with 12b resulted in a considerable increase of the histone acety-
lation level in the tumour section of the treated animals versus the
vehicle group in terms of positive cells and signal intensity.
4. Experimental section
4.1. Chemistry
Table 2
Stability of selected 4-oxospiro[2H-(1,3)-benzoxazine-2,40-piperidin]-6-yl}-N-
hydroxyacrylamides in mouse and human microsomes.a
All reagents and solvents were of commercially available re-
agent grade quality and were used without further purification.
Flash chromatography purifications were performed on Merck sil-
ica gel 60 (0.04e0.063 mm). Nuclear magnetic resonance spectra
(1H NMR and 13C NMR) were recorded on a Bruker spectrometer at
300 MHz and 75 MHz, respectively, with TMS as internal standard,
and, unless stated otherwise, at 300 K. The spectra are referenced in
ppm (d) and coupling constants (J) are expressed in hertz (Hz).
Standard abbreviations indicating multiplicity were used as fol-
lows: s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet
and bs ¼ broad signal. HPLCeMS experiments were performed
either on an Acquity UPLC apparatus. Method A was carried out on a
Waters 2777 Sample Manager, Waters 1525 Binary HPLC Pump,
equipped with a Waters 2996 diode array and a Micromass ZQ 2000
R
R1
Liver Microsomes [%]
Mouse
Human
1
2
3
12b
12c
12d
25c
26a
32
47
57
75
98
46
39
19
55
63
66
77
71
68
60
53
Bn
H
H
H
Me
Bn
Single Quadrupole (Waters) and using an Atlantis dC18 (3
mm,
4-F-Bn
Phe(CH2)2e
4-F-Bn
Me
100 ꢃ 2.1 mm) column. The flow rate was adjusted to 0.30 mL/min.
Method B was carried out on an Acquity UPLC apparatus, equipped
with a diode array and a Micromass ZQ single quadruple (Waters)
a
and using a BEH C18 (1.7
was adjusted to 0.60 mL/min and the splitting ratio between the
m
m, 50 ꢃ 2.1 mm) column. The flow rate
Assays done in replicates (n ꢁ 2). Mean values are shown and the standard
deviations are <30% of the mean.

F. Thaler et al. / European Journal of Medicinal Chemistry 64 (2013) 273e284
279
Table 4
HDAC selectivity profile for SAHA, 1, 2, 3, and 12b.a
Comp
IC50 (mM)
IC50 enzymaticb
HDAC2
HDAC3
HDAC6
SAHA
1
2 [22]
3 [22]
12b
0.102
0.121
0.108
0.113
0.083
0.197
0.27
0.31
0.19
0.192
0.055
0.065
0.09
0.13
0.087
0.030
0.103
0.19
0.16
0.11
a
Assays done in replicates (n ꢁ 2). Mean values are shown and the standard
deviations are ꢄ30% of the mean.
b
HeLa cell nuclear extracts.
amount submitted to the mass spectrometer and to the waste was
1:4. Mobile phase A was composed by a mixture of water and
Fig. 5. Cell cycle profiles of human cancer HCT-116 cells treated for 24 h with 12b at
different doses.
acetonitrile (95:5) containing 0.1% TFA and mobile phase
B
composed by a mixture of water and acetonitrile (5:95) containing
0.1% TFA. Purity refers to UV detection at 254 nm.
with water and brine, dried over Na2SO4 and evaporated. The res-
idue was triturated in AcOEt and petroleum ether to give the
Method A: gradient: 0.00e0.20 min (A: 95%, B: 5%), 0.20e
5.00 min (A: 0%, B: 100%), 5.00e6.00 min (A: 0%, B: 100%), 6.00e
6.10 min (A: 95%, B: 5%); 6.10e7.00 min (A: 95%, B: 5%).
Method B: gradient: 0.00e0.25 min (A: 95%, B: 5%), 0.25e
3.30 min (A: 0%, B: 100%), 3.30e4.00 min (A: 0%, B: 100%), 4.00e
4.10 min (A: 95%, B: 5%); 4.10e5.00 min (A: 95%, B: 5%).
methyl ester 6 (5.05 g, 99%). 1H NMR (300 MHz, DMSO-d6):
d
¼ 8.82
(s, 1H), 8.03 (d, J ¼ 2.05 Hz, 1H), 7.93 (dd, J ¼ 8.66, 2.20 Hz, 1H), 7.68
(d, J ¼ 16.14 Hz, 1H), 7.12 (d, J ¼ 8.51 Hz, 1H), 6.57 (d, J ¼ 16.14 Hz,
1H), 3.73e3.83 (m, 2H), 3.72 (s, 3H), 2.98e3.25 (m, 2H), 1.90e2.08
(m, 2H), 1.60e1.83 ppm (m, 2H), 1.41 (s, 9H).
Compound 6 (300 mg, 0.745 mmol) was suspended in dioxane
(5 mL) and water (5 mL). 1 M NaOH (0.93 mL) was added and the
resulting mixture was stirred overnight at room temperature. The
mixture was neutralized with 1 M citric acid and concentrated
under vacuum. The pH was brought to 4 with 1 M citric acid. The
4.1.1. (E)-3-{3,4-Dihydro-4-oxospiro[2H-(1,3)-benzoxazine-2,40-
piperidin]-6-yl}-N-hydroxyacrylamide (8)
Three batches of 5-bromo salicylamide (4, 5.00
g each,
23.1 mmol), N-Boc-piperidin-4-one (3.45 g, 17.3 mmol) and pyr-
rolidine (1.64 g, 23.1 mmol) were heated in different flasks at 72 ꢀC
under MW irradiation for 1 h. Further N-Boc-piperidin-4-one
(3.45 g, 17.3 mmol) was added and the mixtures were heated for
1 h at 72 ꢀC. The resulting precipitates were collected by filtration
and washed with MeOH to give the spirocycle 5 (17.2 g, 63%) as a
white solid. 1H NMR (300 MHz, DMSO-d6):
J ¼ 2.35 Hz, 1H), 7.69 (dd, J ¼ 8.66, 2.49 Hz, 1H), 7.06 (d, J ¼ 8.80 Hz,
1H), 3.64e3.88 (m, 2H), 3.02e3.21 (m, 2H),1.91e2.10 (m, 2H), 1.63e
1.78 (m, 2H), 1.40 ppm (s, 9H).
d
¼ 8.86 (s, 1H), 7.82 (d,
Compound 5 (5.00 g, 12.6 mmol) was dissolved in hot DMF
(15 mL) and then TEA (5.26 mL, 37.8 mmol), tris(o-tolyl)phosphine
(P(o-tol)3) (153 mg, 0.504 mmol), Pd(OAc)2 (56 mg, 0.25 mmol) and
methyl acrylate (3.25 g, 37.8 mmol) were added at room temper-
ature. The mixture was heated at 100 ꢀC for 6 h under N2, then
further P(o-tol)3 (153 mg, 0.504 mmol) and Pd(OAc)2 (56 mg,
0.25 mmol) was added after 3 h, and the reaction mixture was
partitioned between brine and AcOEt. The organic phase was rinsed
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Fig. 6. A) Antitumor activity of 12b against HCT-116 human tumour xenografts
implanted in mice, expressed as mean tumour volume (expressed as mm3) ꢂ standard
error of the mean (SEM). B) Immunohistochemical analysis of histone acetylation
staining of the section of HCT-116 xenograft tumour at the end of treatment with
compound 12b. Representative sections of the staining for the different treatment
groups are reported.
0
0.1
0.5
1
3
12b / µM
Fig. 4. Dose-dependent histone acetylation of 12b in K562 cell line. The numbers
represent the ratio of the acetylation level of treated cells over controls.

280
F. Thaler et al. / European Journal of Medicinal Chemistry 64 (2013) 273e284
resulting solid was decanted and dried to give the acrylic acid 7
(287 mg, 99%). 1H NMR (300 MHz, DMSO-d6):
8.81 (s, 1H), 7.99 (d, J ¼ 2.35 Hz, 1H), 7.89 (dd, J ¼ 8.51, 2.35 Hz, 1H),
7.60 (d, J ¼ 16.14 Hz, 1H), 7.11 (d, J ¼ 8.51 Hz, 1H), 6.45 (d,
J ¼ 16.14 Hz, 1H), 3.69e3.87 (m, 2H), 3.04e3.23 (m, 2H), 1.91e2.16
(m, 2H), 1.65e1.84 (m, 2H), 1.41 ppm (s, 9H).
The acrylic acid 7 (287 mg, 0.739 mmol) was then dissolved in
CH2Cl2 (20 mL) and TEA (0.17 mL, 1.23 mmol). EDC (169 mg,
0.88 mmol), HOBt (119 mg, 0.88 mmol) and NH2OTHP (101 mg,
0.87 mmol) were added and the mixture was stirred at room
temperature overnight. The mixture was partitioned between
CH2Cl2 and 5% aqueous NaHCO3 and the organic phase was dried
over Na2SO4 and evaporated. The crude mixture was purified by
column chromatography (eluent:CH2Cl2/MeOH 95:5). The resulting
intermediate was dissolved in CH2Cl2 and treated with 4 M HCl in
dioxane for 3 h. The precipitate was filtered off and washed with
CH2Cl2 to give the desired hydroxamic acid hydrochloride 8
(109 mg, 43% from 7) as a white solid. 1H NMR (300 MHz, DMSO-
NaHCO3, and the organic phase was dried over Na2SO4 and evap-
orated. The crude mixture was purified by column chromatography
(eluent:CH2Cl2/MeOH 95:5). The resulting intermediate was dis-
solved in CH2Cl2 and treated with 4 M HCl in dioxane for 3 h. The
precipitate was filtered off and washed with CH2Cl2 to give the
requisite hydroxamic acid 12d (hydrochloride salt, 175 mg, 64%) as
d
¼ 12.29 (bs, 1H),
a white solid. 1H NMR (300 MHz, DMSO-d6):
d
¼ 10.81 (bs, 1H),
10.71 (bs, 1H), 9.11 (s, 1H), 7.96 (d, J ¼ 2.05 Hz, 1H), 7.77 (dd, J ¼ 8.51,
1.76 Hz, 1H), 7.46 (d, J ¼ 15.55 Hz, 1H), 7.22e7.40 (m, 5H), 7.15 (d,
J ¼ 8.22 Hz, 1H), 6.46 (d, J ¼ 15.85 Hz, 1H), 3.47e3.66 (m, 2H), 3.15e
3.45 (m, 4H), 3.04e3.13 (m, 2H), 2.17e2.39 ppm (m, 4H); 13C NMR
(125 MHz, DMSO-d6):
d
¼ 30.02, 32.67 (2C), 48.21 (2C), 59.12, 84.61,
118.30, 118.35, 119.22, 126.17, 127.31, 129.16 (4C), 129.95, 134.48,
137.39, 137.50, 155.32, 160.82, 163.01 ppm; LCeMS (ESI) m/z: 408
[M þ H]þ; HPLC purity: 100%, tR ¼ 1.23 min (Method B).
4.1.3. (E)-3-{10-Methyl-3,4-dihydro-4-oxospiro[2H-(1,3)-
benzoxazine-2,40-piperidin]-6-yl}-N-hydroxyacrylamide (12a)
The methyl ester 10a was obtained starting from free amine 9
(300 mg, 0.855 mmol) and formaldehyde as described for inter-
d6):
d
¼ 9.29e9.48 (m, 1H), 9.15e9.29 (m, 1H), 9.11 (s, 1H), 7.94 (d,
J ¼ 2.05 Hz, 1H), 7.74 (dd, J ¼ 8.36, 1.91 Hz, 1H), 7.45 (d, J ¼ 15.85 Hz,
1H), 7.16 (d, J ¼ 8.51 Hz,1H), 6.46 (d, J ¼ 15.85 Hz,1H), 3.17e3.40 (m,
2H), 2.95e3.17 (m, 2H), 2.02e2.33 ppm (m, 4H); LCeMS (ESI) m/z:
304 [M þ H]þ; HPLC purity: 99%, tR ¼ 2.05 min (Method A).
mediate 10d (235 mg, 84%). 1H NMR (300 MHz, DMSO-d6):
d
¼ 8.76
(s, 1H), 8.02 (d, J ¼ 2.1 Hz, 1H), 7.90 (dd, J ¼ 8.5, 2.3 Hz, 1H), 7.67 (d,
J ¼ 15.8 Hz, 1H), 7.08 (d, J ¼ 8.5 Hz,1H), 6.56 (d, J ¼ 16.1 Hz, 1H), 3.72
(s, 3H), 2.53e2.68 (m, 2H), 2.23e2.35 (m, 2H), 2.21 (s, 3H), 1.92e
2.09 (m, 2H), 1.68e1.92 ppm (m, 2H). LCeMS (ESI) m/z: 317.2
[M þ H]þ.
4.1.2. (E)-3-{10-(2-Phenylethyl)-3,4-dihydro-4-oxospiro[2H-(1,3)-
benzoxazine-2,40-piperidin]-6-yl}-N-hydroxyacrylamide (12d)
A 4 M solution of HCl in dioxane (4 mL) was added to a solution
of 6 (1.20 g, 2.98 mmol) in CH2Cl2 (20 mL) and the mixture was
stirred at room temperature for 4 h. The precipitated solid was
filtered off and washed with CH2Cl2, then dried under vacuum and
collected to give the free piperidine 9 (694 mg, 69%) as a white solid
The methyl ester 10a was hydrolyzed according to the procedure
for 11d, and the crude acrylic acid 11a was then converted into the
requisite hydroxamic acid 12a according to synthetic procedure
described for compound 12d (126 mg, 48% from 10a, hydrochloride
salt). 1H NMR (300 MHz, DMSO-d6):
d
¼ 10.93 (bs, 1H), 9.06 (s, 1H),
(hydrochloride salt). 1H NMR (300 MHz, DMSO-d6):
d
¼ 9.20 (s, 1H),
7.95 (d, J ¼ 1.76 Hz, 1H), 7.76 (dd, J ¼ 8.36, 1.91 Hz, 1H), 7.45 (d,
J ¼ 15.85 Hz, 1H), 7.16 (d, J ¼ 8.51 Hz, 1H), 6.46 (d, J ¼ 15.85 Hz, 1H),
3.28e3.51 (m, 2H), 2.97e3.28 (m, 2H), 2.78 (d, J ¼ 4.70 Hz, 3H),
2.07e2.39 ppm (m, 4H); LCeMS (ESI) m/z: 318 [M þ H]þ; HPLC
purity: 97%, tR ¼ 2.08 min (Method A).
9.01 (bs, 1H), 8.08 (d, J ¼ 2 Hz, 1H), 8.00 (dd, J ¼ 8, 2 Hz, 1H), 7.72 (d,
J ¼ 16 Hz, 1H), 7.21 (d, J ¼ 8 Hz, 1H), 6.63 (d, J ¼ 16 Hz, 1H), 3.76 (s,
3H), 3.30 (bs, 2H), 3.16 (bs, 2H), 2.26 (bs, 2H), 2.09 ppm (bs, 2H);
LCeMS (ESI) m/z: 303 [M þ H]þ.
The amine hydrochloride salt 9 (300 mg, 0.886 mmol) was
dissolved in MeOH (30 mL). The pH was adjusted to 5 with AcOH
and 1 M NaOH in MeOH, and then phenylacetaldehyde (128 mg,
1.06 mmol) and NaCNBH3 (67 mg, 1.06 mmol) were added. The
mixture was stirred overnight at room temperature, then the sol-
vent was removed, and the residue partitioned between 5%
aqueous NaHCO3 and CH2Cl2. The organic phase was dried over
Na2SO4 and evaporated. The crude product was purified by column
chromatography (eluent:CH2Cl2/MeOH 95:5) to afford the methyl
4.1.4. (E)-3-{10-Benzyl-3,4-dihydro-4-oxospiro[2H-(1,3)-
benzoxazine-2,40-piperidin]-6-yl}-N-hydroxyacrylamide (12b)
Benzyl bromide (0.33 mL, 2.79 mmol) was added to a solution of
the free amine 9 (0.28 g, 0.83 mmol) and TEA (0.26 mL, 1.9 mmol) in
CH2Cl2 (10 mL), and the solution was stirred for 5 h at room tem-
perature. The mixture was washed with water, and the pH value
was adjusted to 5 with a 0.5 M HCl. The solution was then dried,
concentrated, and the crude residue was purified by column
chromatography (eluent:CH2Cl2/MeOH 95:5) to give 10b as a white
ester 10d (322 mg, 92%). 1H NMR (300 MHz, DMSO-d6):
d
¼ 8.77 (s,
1H), 8.02 (d, J ¼ 2.05 Hz, 1H), 7.91 (dd, J ¼ 8.51, 2.35 Hz, 1H), 7.67 (d,
J ¼ 16.14 Hz, 1H), 7.12e7.35 (m, 5H), 7.08 (d, J ¼ 8.51 Hz, 1H), 6.56 (d,
J ¼ 15.85 Hz, 1H), 3.72 (s, 3H), 2.67e2.88 (m, 4H), 2.54e2.62 (m,
2H), 2.29e2.45 (m, 2H), 1.93e2.11 (m, 2H), 1.67e1.93 ppm (m, 2H).
10d (292 mg, 0.719 mmol) was then suspended in dioxane
(5 mL) and water (5 mL). 1 M NaOH (0.93 mL) was added and the
resulting mixture was stirred overnight at room temperature. The
mixture was neutralized with 1 M HCl and concentrated under
vacuum. The pH was brought to 4 with 1 M HCl and the resulting
solid was decanted and dried to give the acrylic acid 11d (265 mg,
solid (145 mg, 45%). 1H NMR (300 MHz, CDCl3):
1H), 7.60e7.67 (m, 2H), 7.31e7.32 (m, 5H), 6.99 (d, J ¼ 8 Hz, 1H),
6.40 (d, J ¼ 16 Hz, 1H), 3.80 (s, 3H), 3.56 (s, 2H), 2.49e2.64 (m, 4H),
2.17e2.20 (m, 2H), 1.87e1.94 ppm (m, 2H).
d
¼ 8.10 (d, J ¼ 2 Hz,
The methyl ester 10b (145 mg, 0.46 mmol), dissolved in 3 mL of
MeOH, was hydrolyzed with 2 M NaOH (0.8 mL). The mixture was
stirred at 50 ꢀC for 2 h, then the solvent was evaporated and the pH
of the aqueous phase was adjusted with 1 M HCl to 5. The resulting
suspension was extracted with CH2Cl2, the combined organic
phases dried over Na2SO4 and concentrated to give the acrylic acid
11b as a white solid (110 mg, 79%). 1H NMR (300 MHz, DMSO-d6):
94%). 1H NMR (300 MHz, DMSO-d6):
d
¼ 12.30 (bs, 1H), 8.81 (bs,
1H), 7.99 (d, J ¼ 2.05 Hz, 1H), 7.88 (dd, J ¼ 8.66, 2.20 Hz, 1H), 7.60 (d,
J ¼ 15.85 Hz,1H), 7.15e7.34 (m, 5H), 7.09 (d, J ¼ 8.51 Hz,1H), 6.45 (d,
J ¼ 16.14 Hz, 1H), 2.57e3.02 (m, 6H), 1.65e2.45 ppm (m, 6H).
11d (240 mg, 0.612 mmol) was then dissolved in CH2Cl2 (20 mL)
and TEA (0.17 mL, 1.23 mmol). EDC (169 mg, 0.88 mmol), HOBt
(119 mg, 0.88 mmol), and NH2OTHP (86 mg, 0.74 mmol) were
added, and the mixture was stirred at room temperature overnight.
The mixture was partitioned between CH2Cl2 and 5% aqueous
d
¼ 11.63 (bs,1H), 9.21 (s,1H), 8.34 (m, 2H), 7.49e7.73 (m, 6H), 7.17e
7.19 (m, 1H), 6.53 (d, J ¼ 16 Hz,1H), 4.42 (bs, 2H), 3.30e3.70 (m, 4H),
2.27e2.40 ppm (m, 4H); LCeMS (ESI) m/z: 379 [M þ H]þ.
The resulting product was treated with NH2OTHP according to
the procedure described for compound 12d giving the N-(tetrahy-
dropyran-2-yloxy)acrylamide intermediate as
(50 mg, 36%). 1H NMR (300 MHz, CDCl3):
¼ 10.98 (bs, 1H), 8.24e
8.25 (m, 1H), 7.63e7.67 (m, 3H), 7.23e7.34 (m, 4H), 7.95 (d, J ¼ 8 Hz,
a yellow solid
d

F. Thaler et al. / European Journal of Medicinal Chemistry 64 (2013) 273e284
281
1H), 6.56e6.59 (m, 1H), 5.03 (bs, 1H), 4.06e4.08 (m, 1H), 3.61e3.74
(m, 1H), 3.55 (s, 2H), 2.63e2.69 (m, 2H), 2.42e2.51 (m, 2H), 1.60e
1.92 ppm (m, 10H). Finally, removal of the THP protecting group
gave the desired N-hydroxyacrylamide 12b (12 mg, 27%) as its hy-
salt starting from the acrylic acid 15 following the procedure
described for the hydroxamic acid 8 and was obtained as white
solid (334, 77%, hydrochloride salt). 1H NMR (300 MHz, DMSO-d6):
d
¼ 10.30 (s, 1H), 9.06e9.63 (m, 2H), 7.96 (d, J ¼ 2.05 Hz, 1H), 7.76
drochloride salt. 1H NMR (300 MHz, DMSO-d6):
d
¼ 11.00 (bs, 1H),
(dd, J ¼ 8.51, 2.05 Hz, 1H), 7.46 (d, J ¼ 15.85 Hz, 1H), 7.22 (d,
J ¼ 8.51 Hz, 1H), 6.48 (d, J ¼ 15.85 Hz, 1H), 3.18e3.42 (m, 2H), 3.06e
3.21 (m, 2H), 3.02 (s, 3H), 2.38e2.58 (m, 2H), 2.10e2.27 ppm (m,
2H); LCeMS (ESI) m/z: 318 [M
tR ¼ 2.17 min (Method A).
9.12 (s, 1H), 7.94 (d, J ¼ 2.05 Hz, 1H), 7.77 (dd, J ¼ 8.51, 1.47 Hz, 1H),
7.59e7.70 (m, 2H), 7.37e7.54 (m, 4H), 7.12 (d, J ¼ 8.51 Hz, 1H), 6.45
(d, J ¼ 15.55 Hz, 1H), 4.36 (d, J ¼ 4.69 Hz, 2H), 2.97e3.44 (m, 4H),
þ
H]þ; HPLC purity: 93%,
2.11e2.41 ppm (m, 4H); 13C NMR (125 MHz, DMSO-d6):
d
¼ 32.45
(2C), 47.91 (2C), 59.12, 84.71, 118.20, 118.29, 119.21, 126.15, 129.24
(2C), 129.98, 130.302, 131.89 (3C), 134.50, 137.34, 155.32, 160.82,
163.01 ppm; LCeMS (ESI) m/z: 394 [M þ H]þ; HPLC purity: 99%,
tR ¼ 1.07 min (Method B).
4.1.7. (E)-3-{10-(4-Fluorobenzyl)-3,4-dihydro-3-methyl-4-oxospiro
[2H-(1,3)-benzoxazine-2,40-piperidin]-6-yl}-N-hydroxyacrylamide
(25c)
Compound 13 (3.00 g, 7.21 mmol) was dissolved together with
4 M HCl in dioxane (5 mL) in CH2Cl2 (150 mL) and stirred at room
temperature for 4 h. The precipitate was filtered off to give the free
piperidine 19 as its hydrochloride salt (2.46 g, 97%). 1H NMR
4.1.5. (E)-3-{10-(4-Fluorobenzyl)-3,4-dihydro-4-oxospiro[2H-(1,3)-
benzoxazine-2,40-piperidin]-6-yl}-N-hydroxyacrylamide (12c)
The methyl ester 10c was obtained starting from spirocycle 9
(0.560 g, 1.65 mmol) and 4-fluorobenzaldehyde as described for
intermediate 10d (0.535 g, 79%). 1H NMR (300 MHz, DMSO-d6):
(DMSOed6)
d
(ppm): 9.21 (bs, 2H), 8.06 (d, J ¼ 2.35 Hz,1H), 7.98 (dd,
J ¼ 8.66, 2.20 Hz, 1H), 7.70 (d, J ¼ 15.85 Hz, 1H), 7.24 (d, J ¼ 8.51 Hz,
1H), 6.60 (d, J ¼ 16.14 Hz, 1H), 3.73 (s, 3H), 3.21e3.40 (m, 2H), 3.05e
3.20 (m, 2H), 3.03 (s, 3H), 2.38e2.48 (m, 2H), 2.12e2.29 (m, 2H).
Spirocycle 19 (600 mg, 1.70 mmol) was treated with 4-
fluorobenzaldehyde according to the procedure described for
compound 10d to give the methyl ester 21c (542 mg, 75%). 1H NMR
d
¼ 8.80 (s, 1H), 8.02 (d, J ¼ 2.3 Hz, 1H), 7.91 (dd, J ¼ 8.5, 2.3 Hz, 1H),
7.67 (d, J ¼ 15.8 Hz,1H), 7.34 (m, 2H), 7.14 (m, 2H), 7.07 (d, J ¼ 8.5 Hz,
1H), 6.55 (d, J ¼ 15.8 Hz, 1H), 3.72 (s, 3H), 3.51 (bs, 2H), 2.55e2.72
(m, 2H), 2.16e2.44 (m, 2H), 1.93e2.09 (m, 2H), 1.64e1.93 ppm (m,
2H).
Hydrolysis of the methyl ester group following the procedure for
(300 MHz, DMSO-d6):
d
¼ 8.03 (d, J ¼ 2.05 Hz, 1H), 7.93 (dd, J ¼ 8.51,
11d gave the acrylic acid 11c (495 mg, 95%). 1H NMR (300 MHz,
2.35 Hz, 1H), 7.68 (d, J ¼ 16.14 Hz, 1H), 7.28e7.46 (m, 2H), 7.10e7.20
(m, 2H), 7.11 (d, J ¼ 8.51 Hz, 1H), 6.56 (d, J ¼ 15.85 Hz, 1H), 3.72 (s,
3H), 3.51 (s, 2H), 3.02 (s, 3H), 2.65e2.80 (m, 2H), 2.21e2.41 (m, 2H),
2.02e2.19 (m, 2H), 1.88e2.02 ppm (m, 2H). The methyl ester 21c
(520 mg, 1.22 mmol) was suspended in dioxane (20 mL) and water
(10 mL). 1 M NaOH (1.59 mL) was added and the resulting mixture
was stirred at room temperature for 5 h. The mixture was
neutralized with 1 M HCl and the solvent was removed. The
DMSO-d6):
d
¼ 12.31 (bs, 1H), 8.79 (bs, 1H), 7.98 (d, J ¼ 1.8 Hz, 1H),
7.82e7.92 (m,1H), 7.59 (d, J ¼ 15.8 Hz,1H), 7.29e7.43 (m, 2H), 6.98e
7.26 (m, 3H), 6.44 (d, J ¼ 16.1 Hz, 1H), 3.49 (bs, 2H), 2.56e2.71 (m,
2H), 2.21e2.41 (m, 2H), 1.93e2.14 (m, 2H), 1.66e1.93 ppm (m, 2H).
The acrylic acid 11c was then converted to the corresponding
hydroxamic acid 12c (352 mg, 65%, hydrochloride salt) according to
synthetic procedure described for 12d. 1H NMR (300 MHz, DMSO-
d6):
d
¼ 11.23 (bs, 1H), 9.12 (s, 1H), 7.94 (d, J ¼ 1.76 Hz, 1H), 7.77 (dd,
resulting acrylic acid 23c (1H NMR (300 MHz, DMSO-d6):
d
¼ 7.89
J ¼ 8.80, 1.76 Hz, 1H), 7.67e7.75 (m, 2H), 7.45 (d, J ¼ 15.85 Hz, 1H),
7.20e7.38 (m, 2H), 7.12 (d, J ¼ 8.51 Hz,1H), 6.46 (d, J ¼ 15.85 Hz,1H),
4.36 (d, J ¼ 4.70 Hz, 2H), 3.08e3.41 (m, 4H), 2.15e2.43 ppm (m, 4H);
(d, J ¼ 2.3 Hz, 1H), 7.77 (dd, J ¼ 8.5, 2.3 Hz, 1H), 7.25e7.41 (m, 3H),
7.08e7.20 (m, 2H), 7.05 (d, J ¼ 8.2 Hz, 1H), 6.41 (d, J ¼ 15.8 Hz, 1H),
3.50 (s, 2H), 3.01 (s, 3H), 2.62e2.83 (m, 2H), 2.23e2.43 (m, 2H), 2.08
(td, J ¼ 12.7, 4.3 Hz, 2H), 1.90e2.01 ppm (m, 2H)) was dissolved in
CH2Cl2 (10 mL) and TEA (0.338 mL, 2.44 mmol). EDC (350 mg,
1.83 mmol), HOBt (247 mg, 1.83 mmol) and NH2OTHP (172 mg,
1.46 mmol) were added to the resulting solution. The mixture was
stirred for 6 h at room temperature and was then partitioned be-
tween 5% aqueous NaHCO3 and CH2Cl2. The organic layer was dried
over Na2SO4 and evaporated. The crude intermediate was purified
by column chromatography (eluent:CH2Cl2/MeOH 97:3) and the
resulting compound was dissolved in CH2Cl2 and treated with 4 M
HCl in dioxane for 4 h. The precipitate was filtered off to afford the
requisite N-hydroxyacrylamide hydrochloride 25c (451 mg, 80%
13C NMR (125 MHz, DMSO-d6):
d
¼ 32.42 (2C), 47.77 (2C), 58.12,
84.72, 116.12 (2C, J ¼ 21 Hz), 118.29, 118.37, 119.22, 126.30, 126.64,
129.94, 134.31 (2C, J ¼ 8 Hz), 134.50, 137.32, 155.32, 160.81, 163.00,
163.17 (J ¼ 245 Hz) ppm; LCeMS (ESI) m/z: 412 [M þ H]þ; HPLC
purity: 99%, tR ¼ 1.16 min (Method B).
4.1.6. (E)-3-{3,4-Dihydro-3-methyl-4-oxospiro[2H-(1,3)-
benzoxazine-2,40-piperidin]-6-yl}-N-hydroxyacrylamide (17)
Spirocycle 6 (4.02 g, 10.0 mmol) was dissolved in DMF (35 mL)
and added to a stirred suspension of NaH (60% oil dispersion,
480 mg, 12 mmol) in DMF (35 mL) at 4 ꢀC. After 10 min CH3I (2.13 g,
15.0 mmol) was added, the resulting mixture was stirred at room
temperature for 30 min and was then partitioned between satu-
rated an aqueous solution of NH4Cl and AcOEt. The organic phase
was washed with water, dried over Na2SO4 and evaporated. The
crude mixture was purified by trituration in AcOEt and petroleum
ether to give the 3-methyl analogue 13 (3.66 g, 88%). 1H NMR
from 21c) as a white solid. 1H NMR (300 MHz, DMSO-d6):
d
¼ 11.32
(bs, 1H), 10.72 (bs, 1H), 7.97 (d, J ¼ 1.76 Hz, 1H), 7.79 (dd, J ¼ 8.36,
1.91 Hz, 1H), 7.64e7.75 (m, 2H), 7.46 (d, J ¼ 15.85 Hz, 1H), 7.23e7.39
(m, 2H), 7.18 (d, J ¼ 8.51 Hz, 1H), 6.47 (d, J ¼ 15.85 Hz, 1H), 4.38 (d,
J ¼ 4.40 Hz, 2H), 3.10e3.48 (m, 4H), 3.01 (s, 3H), 2.58e2.85 (m, 2H),
2.11e2.34 ppm (m, 2H); 13C NMR (125 MHz, DMSO-d6):
d
¼ 27.33,
(300 MHz, DMSO-d6):
d
¼ 8.04 (d, J ¼ 2.35 Hz, 1H), 7.94 (dd, J ¼ 8.51,
29.51 (2C), 47.91 (2C), 58.08, 84.72, 116.192 (2C, J ¼ 22 Hz), 118.10,
118.22, 119.36, 126.33, 126.67, 130.26, 134.17 (2C, J ¼ 8 Hz), 134.55,
137.30,154.06,160.29,162.96,163.16 (J ¼ 246 Hz) ppm; LCeMS (ESI)
m/z: 426 [M þ H]þ; HPLC purity: 100%, tR ¼ 1.23 min (Method B).
2.35 Hz, 1H), 7.69 (d, J ¼ 15.85 Hz, 1H), 7.19 (d, J ¼ 8.51 Hz, 1H), 6.58
(d, J ¼ 15.85 Hz, 1H), 3.96 (d, J ¼ 13.20 Hz, 2H), 3.73 (s, 3H), 3.01 (s,
3H), 2.95e3.14 (m, 2H), 1.72e2.19 (m, 4H), 1.41 ppm (s, 9H). The
methyl ester 13 (507 mg, 1.60 mmol) was then hydrolyzed ac-
cording to the procedure for the acrylic acid intermediate 7
4.1.8. (E)-3-{10-Methyl-3,4-dihydro-3-methyl-4-oxospiro[2H-(1,3)-
benzoxazine-2,40-piperidin]-6-yl}-N-hydroxyacrylamide (25a)
The methyl ester 21a was obtained starting from spirocycle 19
(574 mg, 1.69 mmol) and formaldehyde, following the synthetic
procedure described for compound 10d (420 mg, 75%). 1H NMR
(480 mg, 99%). 1H NMR (300 MHz, DMSO-d6):
d
¼ 7.97 (d, J ¼ 2.1 Hz,
1H), 7.88 (dd, J ¼ 8.5, 2.3 Hz, 1H), 7.54 (d, J ¼ 16.1 Hz, 1H), 7.17 (d,
J ¼ 8.5 Hz, 1H), 6.46 (d, J ¼ 15.8 Hz, 1H), 3.85e4.12 (m, 2H), 3.04e
3.15 (m, 2H), 3.01 (s, 3H), 1.93e2.15 (m, 4H), 1.42 ppm (s, 9H). The
hydroxamic acid 17 was obtained as white solid as its hydrochloride
(300 MHz, DMSO-d6):
d
¼ 8.04 (d, J ¼ 2.3 Hz, 1H), 7.92 (dd, J ¼ 8.8,

282
F. Thaler et al. / European Journal of Medicinal Chemistry 64 (2013) 273e284
2.3 Hz, 1 H), 7.68 (d, J ¼ 16.1 Hz, 1H), 7.12 (d, J ¼ 8.2 Hz, 1H), 6.57 (d,
J ¼ 16.1 Hz, 1H), 3.72 (s, 3H), 3.01 (s, 3H), 2.59e2.75 (m, 2H), 2.21 (s,
3H), 2.19e2.31 (m, 2H), 2.04e2.18 (m, 2H), 1.85e2.03 ppm (m, 2H).
Hydrolysis of the methyl ester group according to the procedure for
compound 23c gave the acrylic acid 23a, which was used in the
next without further purification. 1H NMR (300 MHz, DMSO-d6):
2.37 ppm (m, 2H); LCeMS (ESI) m/z: 422 [M þ H]þ; HPLC purity:
100%, tR ¼ 1.30 min (Method B).
4.1.11. (E)-3-{3,4-Dihydro-3-benzyl-4-oxospiro[2H-(1,3)-
benzoxazine-2,40-piperidin]-6-yl}-N-hydroxyacrylamide (18)
Spirocycle 6 (1.8 g, 4.5 mmol) was dissolved in dry DMF (25 mL)
under N2, and NaH (268.6 mg, 6.71 mmol, 60% suspension in
mineral oil) was added. After 10 min stirring, benzyl bromide
(1.07 mL, 8.95 mmol) was added dropwise and the mixture was
heated for 3 h at 80 ꢀC. The mixture was poured into water and a
saturated NH4Cl solution was added. The aqueous phase was
extracted with CH2Cl2, and the combined organic phases were
dried and concentrated. The crude residue was purified by column
chromatography (eluent:petroleum ether/AcOEt 90:10e70:30) to
give the 3-benzyl intermediate 14 (1.3 g, 59%) as a white solid. 1H
d
¼ 8.00 (d, J ¼ 2.3 Hz, 1H), 7.89 (dd, J ¼ 8.5, 2.3 Hz, 1H), 7.60 (d,
J ¼ 15.8 Hz, 1H), 7.12 (d, J ¼ 8.5 Hz, 1H), 6.45 (d, J ¼ 16.1 Hz, 1H), 3.01
(s, 3H), 2.65e2.83 (m, 2H), 2.27e2.36 (m, 2H), 2.24 (s, 3H), 2.07e
2.21 (m, 2H), 1.84e2.03 ppm (m, 2H). The acrylic acid 23a was
converted to the requisite hydroxamic acid 25a according to the
synthetic procedure for 25c giving a white powder (250 mg, 55%
from 21c, hydrochloride salt). 1H NMR (300 MHz, DMSO-d6):
d
¼ 11.26 (bs, 1H), 10.28 (bs, 1H), 7.97 (d, J ¼ 2.05 Hz, 1H), 7.78 (dd,
J ¼ 8.36, 1.91 Hz, 1H), 7.46 (d, J ¼ 15.85 Hz, 1H), 7.22 (d, J ¼ 8.51 Hz,
1H), 6.48 (d, J ¼ 15.85 Hz, 1H), 3.32e3.49 (m, 2H), 3.15e3.32 (m,
2H), 3.02 (s, 3H), 2.79 (d, J ¼ 4.40 Hz, 3H), 2.63 (td, J ¼ 13.79,
4.40 Hz, 2H), 2.08e2.34 ppm (m, 2H); LCeMS (ESI) m/z: 332
[M þ H]þ; HPLC purity: 99%, tR ¼ 2.20 min (Method A).
NMR (300 MHz, DMSO-d6):
d
¼ 8.14 (d, J ¼ 2.05 Hz, 1H), 7.99 (dd,
J ¼ 8.51, 2.35 Hz, 1H), 7.72 (d, J ¼ 16.14 Hz, 1H), 7.12e7.46 (m, 6H),
6.61 (d, J ¼ 15.85 Hz, 1H), 4.85 (s, 2H), 3.77e3.98 (m, 2H), 3.73 (s,
3H), 2.82e3.14 (m, 2H), 1.75e2.07 (m, 4H), 1.37 ppm (s, 9H). The
methyl ester 14 (800 mg, 1.63 mmol) was hydrolyzed with NaOH
following the procedure described for the 11b to give the acrylic
acid 16 as a white solid (750 mg, 96%). 1H NMR (300 MHz, DMSO-
4.1.9. (E)-3-{10-Benzyl-3,4-dihydro-3-methyl-4-oxospiro[2H-(1,3)-
benzoxazine-2,40-piperidin]-6-yl}-N-hydroxyacrylamide (25b)
Methyl ester 21c was obtained starting from spirocycle 19
(280 mg, 0.826 mmol) and benzaldehyde, following the synthetic
procedure described for compound 10d (235 mg, 85%). 1H NMR
d6):
d
¼ 8.10 (d, J ¼ 2.05 Hz, 1H), 7.95 (dd, J ¼ 8.51, 2.05 Hz, 1H), 7.64
(d, J ¼ 15.85 Hz,1H), 7.22 (d, J ¼ 8.51 Hz,1H), 7.16e7.49 (m, 5H), 6.49
(d, J ¼ 15.85 Hz, 1H), 4.85 (s, 2H), 3.75e3.93 (m, 2H), 2.86e3.10 (m,
2H), 1.75e2.08 (m, 4H), 1.36 ppm (s, 9H). The resulting acid 16 was
treated with NH2OTHP following the procedure described for the
hydroxamic acid 8 and purified by preparative HPLC giving the
requisite hydroxamic acid 18. (208 mg, 56%) as its trifluoroacetate
(300 MHz, DMSO-d6):
d
¼ 8.03 (d, J ¼ 2.1 Hz, 1H), 7.93 (dd, J ¼ 8.7,
2.2 Hz, 1H), 7.68 (d, J ¼ 15.8 Hz, 1H), 7.18e7.41 (m, 5H), 7.11 (d,
J ¼ 8.5 Hz,1H), 6.57 (d, J ¼ 16.1 Hz,1H), 3.72 (s, 3H), 3.52 (s, 2H), 3.02
(s, 3H), 2.63e2.86 (m, 2H), 2.23e2.41 (m, 2H), 2.10 (td, J ¼ 12.6,
4.4 Hz, 2H), 1.85e2.02 ppm (m, 2H). Hydrolysis of the methyl ester
group according to compound 23c gave the acrylic acid 23b
(150 mg, 70%). LCeMS (ESI) m/z: 393 [M þ H]þ. The desired
hydroxamic acid 25b was obtained as white powder starting from
the acrylic acid 23b according to the procedure for 25c (80 mg, 49%,
salt. 1H NMR (300 MHz, DMSO-d6):
d
¼ 10.71 (bs,1H), 8.59e8.74 (m,
1H), 8.20e8.51 (m, 1H), 8.06 (d, J ¼ 2.05 Hz, 1H), 7.82 (dd, J ¼ 8.80,
1.76 Hz, 1H), 7.50 (d, J ¼ 15.85 Hz, 1H), 7.08e7.41 (m, 6H), 6.48 (d,
J ¼ 15.85 Hz,1H), 4.80 (s, 2H), 3.19e3.33 (m, 2H), 2.88e3.16 (m, 2H),
1.99e2.25 ppm (m, 4H); LCeMS (ESI) m/z: 394 [M þ H]þ; HPLC
purity: 100%, tR ¼ 1.22 min (Method B).
hydrochloride salt). 1H NMR (300 MHz, DMSO-d6):
d
¼ 11.25 (bs,
1H), 7.97 (d, J ¼ 2.05 Hz, 1H), 7.73e7.89 (m, 1H), 7.58e7.72 (m, 2H),
7.35e7.57 (m, 4H), 7.18 (d, J ¼ 8.51 Hz, 1H), 6.47 (d, J ¼ 15.85 Hz, 1H),
4.38 (d, J ¼ 4.99 Hz, 2H), 3.10e3.45 (m, 4H), 3.01 (s, 3H), 2.58e2.86
(m, 2H), 2.10e2.34 ppm (m, 2H); LCeMS (ESI) m/z: 408 [M þ H]þ;
HPLC purity: 100%, tR ¼ 1.17 min (Method B).
4.1.12. (E)-3-{10-Methyl-3,4-dihydro-3-benzyl-4-oxospiro[2H-
(1,3)-benzoxazine-2,40-piperidin]-6-yl}-N-hydroxyacrylamide (26a)
Intermediate 14 (900 mg, 1.83 mmol) was deprotected with 4 M
HCl in dioxane (3 mL). The mixture was stirred at room tempera-
ture for 4 h, then precipitated solid was filtered off and washed with
CH2Cl2, dried under vacuum and collected giving the free amine 20
(700 mg, 89%) as its hydrochloride salt. 1H NMR (300 MHz, DMSO-
4.1.10. (E)-3-{10-(2-Phenylethyl)-3,4-dihydro-3-methyl-4-oxospiro
[2H-(1,3)-benzoxazine-2,40-piperidin]-6-yl}-N-hydroxyacrylamide
(25d)
The methyl ester 21d was obtained starting from spirocycle 19
(561 mg, 1.66 mmol) and phenylacetaldehyde, following the syn-
thetic procedure described for compound 10d (411 mg, 59%). 1H
d6):
d
¼ 9.09 (bs, 2H), 8.16 (d, J ¼ 2.35 Hz, 1H), 8.03 (dd, J ¼ 8.66,
2.20 Hz, 1H), 7.73 (d, J ¼ 16.14 Hz, 1H), 7.14e7.47 (m, 6H), 6.63 (d,
J ¼ 16.14 Hz, 1H), 4.81 (s, 2H), 3.74 (s, 3H), 3.18e3.31 (m, 2H), 2.93e
3.18 (m, 2H), 2.39 (td, J ¼ 13.64, 4.70 Hz, 2H), 1.99e2.20 ppm (m,
2H). Spirocycle 20 (600 mg, 1.40 mmol) was suspended in 1 M
NMR (300 MHz, DMSO-d6):
d
¼ 8.04 (d, J ¼ 2.1 Hz, 1H), 7.93 (dd,
J ¼ 8.5, 2.3 Hz, 1H), 7.69 (d, J ¼ 16.1 Hz, 1H), 7.16e7.36 (m, 5H), 7.13
(d, J ¼ 8.5 Hz, 1H), 6.57 (d, J ¼ 15.8 Hz, 1H), 3.72 (s, 3H), 3.02 (s, 3H),
2.81e2.94 (m, 2H), 2.67e2.81 (m, 2H), 2.54e2.66 (m, 2H), 2.35 (t,
J ¼ 10.7 Hz, 2H), 2.02e2.17 (m, 2H), 1.89e2.03 ppm (m, 2H). The
acrylic acid 23d was then obtained as described for compound 23c,
which was used in the next step without further purification. 1H
K2CO3 (10 mL), and then extracted with CH2Cl2$HCHO (125 mL,
1.68 mmol, 37% aqueous solution) and sodium triacetoxyborohy-
dride (0.45 g, 2.1 mmol) were added subsequently, and the mixture
was stirred for 3 h. After washing with water, the pH was adjusted
to 8 with aqueous ammonia, the product was extracted with
CH2Cl2, and the organic phases were dried and concentrated. The
crude compound was purified by column chromatography
(eluent:CH2Cl2/MeOH 99:1 to 97:3) to give intermediate 22a as
NMR (300 MHz, DMSO-d6):
d
¼ 12.27 (s,1H), 8.00 (d, J ¼ 2.3 Hz,1H),
7.90 (dd, J ¼ 8.5, 2.1 Hz, 1H), 7.61 (d, J ¼ 15.8 Hz, 1H), 7.16e7.37 (m,
5H), 7.14 (d, J ¼ 8.5 Hz, 1H), 6.46 (d, J ¼ 16.1 Hz, 1H), 3.03 (s, 3H),
2.55e2.98 (m, 8H), 1.84e2.45 ppm (m, 4H); LCeMS (ESI) m/z: 407
[M þ H]þ. Conversion into the desired hydroxamic acid 25d was
performed as for compound 25c giving a white powder (289 mg,
65% from 21d, hydrochloride salt). 1H NMR (300 MHz, DMSO-d6):
white solid (495 mg, 87%). 1H NMR (300 MHz, DMSO-d6):
d
¼ 8.14
(d, J ¼ 2.35 Hz, 1H), 7.97 (dd, J ¼ 8.80, 2.35 Hz, 1H), 7.72 (d,
J ¼ 16.14 Hz, 1H), 7.19e7.48 (m, 5H), 7.15 (d, J ¼ 8.51 Hz, 1H), 6.61 (d,
J ¼ 16.14 Hz, 1H), 4.83 (s, 2H), 3.73 (s, 3H), 2.53e2.66 (m, 2H), 2.16e
2.27 (m, 2H), 2.14 (s, 3H), 1.77e2.06 ppm (m, 4H). The methyl ester
22a (488 mg, 1.20 mmol) was dissolved in acetic acid (10 mL). 6 M
HCl (10 mL) was added and the resulting suspension was heated at
85 ꢀC for 4 h. Then, the solvents were evaporated and the residue
was dried under vacuum giving the acrylic acid 24a hydrochloride
d
¼ 11.31 (bs, 1H), 7.98 (d, J ¼ 1.76 Hz, 1H), 7.79 (dd, J ¼ 8.51, 1.76 Hz,
1H), 7.46 (d, J ¼ 15.85 Hz,1H), 7.24e7.41 (m, 5H), 7.21 (d, J ¼ 8.51 Hz,
1H), 6.48 (d, J ¼ 15.85 Hz, 1H), 3.49e3.70 (m, 2H), 3.16e3.49 (m,
4H), 3.07e3.16 (m, 2H), 3.04 (s, 3H), 2.58e2.82 (m, 2H), 2.18e

F. Thaler et al. / European Journal of Medicinal Chemistry 64 (2013) 273e284
283
as a white solid (490 mg, 95%). 1H NMR (300 MHz, DMSO-d6):
¼ 11.05 (bs, 1H), 8.68 (bs, 1H), 8.11 (d, J ¼ 2.05 Hz, 1H), 8.02 (dd,
2.06 ppm (m, 4H). The methyl ester 22c (533 mg, 1.07 mmol) was
hydrolyzed with HCl 20% and acetic acid following the procedure
described for intermediate 24a, giving the acrylic acid hydrochlo-
ride 24c as a white solid (510 mg, 92%). 1H NMR (300 MHz, DMSO-
d
J ¼ 8.66, 2.20 Hz, 1H), 7.65 (d, J ¼ 15.85 Hz, 1H), 7.18e7.47 (m, 6H),
6.51 (d, J ¼ 15.85 Hz, 1H), 4.79 (s, 2H), 3.12e3.45 (m, 4H), 2.76 (d,
J ¼ 4.69 Hz, 3H), 2.55e2.68 (m, 2H), 2.02e2.23 ppm (m, 2H). The
resulting acid 24a (460 mg, 1.07 mmol) was treated with NH2OTHP
according to the procedure described for compound 8, giving the N-
(tetrahydropyran-2-yloxy)acrylamide intermediate as a yellow
solid (150 mg, 28%). Finally, removal of the THP protecting group
gave after purification by preparative HPLC the requisite hydroxa-
mic acid 26a as its trifluoroacetate salt (12 mg, 7.5%). 1H NMR
d6):
d
¼ 12.19 (bs, 1H), 10.73 (bs, 1H), 8.11 (d, J ¼ 2.35 Hz, 1H), 8.03
(dd, J ¼ 8.36,1.91 Hz,1H), 7.54e7.76 (m, 2H), 7.14e7.45 (m, 9H), 6.52
(d, J ¼ 16.14 Hz, 1H), 4.78 (s, 2H), 4.35 (d, J ¼ 4.11 Hz, 2H), 3.03e3.29
(m, 4H), 2.53e2.67 (m, 2H), 2.02e2.24 ppm (m, 2H). The acrylic
acid 24c (497 mg, 0.95 mmol) was treated with NH2OTHP according
to the procedure described for compound 8, giving the N-(tetra-
hydropyran-2-yloxy)acrylamide intermediate as a white solid
(343 mg, 62%). Finally, removal of the THP protecting group gave
the desired hydroxamic acid 26c (hydrochloride salt, 197 mg, 65%)
(300 MHz, DMSO-d6):
d
¼ 10.71 (bs, 1H), 9.39 (bs, 1H), 8.07 (d,
J ¼ 1.76 Hz, 1H), 7.85 (dd, J ¼ 8.36, 1.91 Hz, 1H), 7.50 (d, J ¼ 15.55 Hz,
1H), 7.12e7.44 (m, 6H), 6.48 (d, J ¼ 15.85 Hz, 1H), 4.79 (s, 2H), 3.12e
3.46 (m, 4H), 2.81 (bs 3H), 2.04e2.33 ppm (m, 4H); 13C NMR
as a white solid. 1H NMR (300 MHz, DMSO-d6):
d
¼ 11.09 (bs, 1H),
8.05 (d, J ¼ 2.05 Hz, 1H), 7.84 (dd, J ¼ 8.36, 1.61 Hz, 1H), 7.66 (m, 2H),
7.49 (d, J ¼ 15.85 Hz, 1H), 7.24e7.40 (m, 8H), 6.49 (d, J ¼ 15.85 Hz,
1H), 4.79 (s, 2H), 4.34 (d, J ¼ 4.40 Hz, 2H), 3.06e3.35 (m, 4H), 2.55e
2.77 (m, 2H), 2.00e2.23 ppm (m, 2H); LCeMS (ESI) m/z: 502
[M þ H]þ; HPLC purity: 97%, tR ¼ 1.62 min (Method B).
(125 MHz, DMSO-d6):
d
¼ 31.08 (2C), 42.57, 44.44, 50.09(2C),
88.57,118.18, 118.45, 119.46, 126.67, 127.29 (2C), 127.69, 129.15 (2C),
130.48, 134.73, 137.35, 138.45, 154.26, 161.06, 163.03 ppm; LCeMS
(ESI) m/z: 408 [M þ H]þ; HPLC purity: 97%, tR ¼ 1.21 min (Method B).
4.1.13. (E)-3-{10-Benzyl-3,4-dihydro-3-benzyl-4-oxospiro[2H-(1,3)-
benzoxazine-2,40-piperidin]-6-yl}-N-hydroxyacrylamide (26b)
Spirocycle 22b was obtained by reacting the free amine 20
(485 mg, 1.13 mmol) with benzyl bromide, according to the pro-
cedure described for compound 10b giving a white solid (544 mg,
4.1.15. (E)-3-{10-(2-Phenylethyl)-3,4-dihydro-3-benzyl-4-oxospiro
[2H-(1,3)-benzoxazine-2,40-piperidin]-6-yl}-N-hydroxyacrylamide
(26d)
The 2-phenylethyl derivative 22d was obtained by reaction be-
tween amine 20 (485 mg, 1.13 mmol) and phenylacetaldehyde
quantitative). 1H NMR (300 MHz, DMSO-d6):
d
¼ 8.13 (d, J ¼ 2.05 Hz,
(171
rocycle 22a, giving a white solid (554 mg, 99%). 1H NMR (300 MHz,
DMSO-d6):
mL, 1.53 mmol), according to the procedure described for spi-
1H), 7.97 (dd, J ¼ 8.66, 2.20 Hz, 1H), 7.71 (d, J ¼ 16.14 Hz, 1H), 7.18e
7.45 (m, 10H), 7.14 (d, J ¼ 8.51 Hz, 1H), 6.60 (d, J ¼ 16.14 Hz, 1H), 4.85
(s, 2H), 3.73 (s, 3H), 3.46 (s, 2H), 2.57e2.71 (m, 2H), 2.19e2.40 (m,
2H), 1.78e2.11 ppm (m, 4H). The methyl ester 22b (500 mg,
1.03 mmol) was hydrolyzed with HCl 20% and AcOH following the
procedure described for intermediate 24a giving the acrylic acid
24b as a white solid (500 mg, 95%). 1H NMR (300 MHz, DMSO-d6):
d¼(d, J ¼ 2.05 Hz, 1H), 7.97 (dd, J ¼ 8.51, 2.05 Hz, 1H),
7.72 (d, J ¼ 15.85 Hz, 1H), 7.13e7.46 (m, 11H), 6.61 (d, J ¼ 16.14 Hz,
1H), 4.85 (s, 2H), 3.73 (s, 3H), 2.58e2.89 (m, 4H), 2.53e2.57 (m, 2H),
2.13e2.43 (m, 2H), 1.88e2.03 ppm (m, 4H). The methyl ester 22d
(540 mg, 1.09 mmol) was hydrolyzed with HCl 20% and acetic acid
following the procedure described for intermediate 24a, giving the
acrylic acid 24d as a white solid (440 mg, 78%). 1H NMR (300 MHz,
d
¼ 11.50 (bs,1H), 8.10 (d, J ¼ 2.05 Hz,1H), 8.01 (dd, J ¼ 8.66, 2.20 Hz,
1H), 7.55e7.87 (m, 3H), 7.19e7.51 (m, 9H), 6.51 (d, J ¼ 16.14 Hz, 1H),
4.79 (s, 2H), 4.35 (d, J ¼ 4.70 Hz, 2H), 3.19e3.36 (m, 4H), 2.67e2.91
(m, 2H), 1.94e2.21 ppm (m, 2H). The acid 24b was treated with
NH2OTHP according to the procedure described for compound 8,
DMSO-d6):
d
¼ 12.19 (bs, 1H), 10.67 (bs, 1H), 8.12 (d, J ¼ 2.05 Hz, 1H),
8.03 (dd, J ¼ 8.51, 2.05 Hz, 1H), 7.66 (d, J ¼ 15.85 Hz, 1H), 7.20e7.47
(m, 11H), 6.52 (d, J ¼ 16.14 Hz, 1H), 4.82 (s, 2H), 3.45e3.66 (m, 2H),
3.32e3.43 (m, 2H), 3.11e3.27 (m, 2H), 2.88e3.11 (m, 2H), 2.54e2.66
(m, 2H), 2.09e2.32 ppm (m, 2H). The acid 24d (423 mg, 0.81 mmol)
was treated with NH2OTHP according to the procedure described in
for compound 8, giving the THP-protected intermediate as a white
giving the THP-hydroxamate intermediate as
a
yellow solid
(228 mg, 40%). 1H NMR (300 MHz, DMSO-d6):
d
¼ 11.14 (bs, 1H),
8.05 (d, J ¼ 2.05 Hz, 1H), 7.78 (dd, J ¼ 8.80, 1.17 Hz, 1H), 7.52 (d,
J ¼ 15.85 Hz, 1H), 7.18e7.43 (m, 10H), 7.14 (d, J ¼ 8.51 Hz, 1H), 6.52
(d, J ¼ 15.85 Hz, 1H), 4.92 (bs, 1H), 4.85 (s, 2H), 3.86e4.03 (m, 1H),
3.50e3.63 (m, 1H), 3.46 (s, 2H), 2.59e2.69 (m, 2H), 2.19e2.38 (m,
2H), 1.81e2.04 (m, 4H), 1.42e1.78 ppm (m, 6H). Finally, removal of
the THP protecting group (177 mg, 0.312 mmol) and purification by
preparative HPLC gave the desired N-hydroxyacrylamide 26b
(150 mg, 81%) as its trifluoroacetate salt. 1H NMR (300 MHz, DMSO-
solid (422 mg, 89%). 1H NMR (300 MHz, DMSO-d6):
d
¼ 10.85 (bs,
2H), 8.06 (d, J ¼ 2.05 Hz, 1H), 7.78 (d, J ¼ 9.68 Hz, 1H), 7.52 (d,
J ¼ 16.14 Hz, 1H), 7.09e7.40 (m, 11H), 6.52 (d, J ¼ 16.73 Hz, 1H), 4.92
(bs, 1H), 4.85 (s, 2H), 3.73e4.10 (m, 1H), 3.43e3.64 (m, 1H), 2.54e
2.87 (m, 6H), 2.14e2.42 (m, 2H), 1.85e2.03 (m, 4H), 1.27e1.81 ppm
(m, 6H). Finally, removal of the THP protecting group starting from
95 mg (0.16 mmol) of the intermediate gave the desired hydroxa-
mic acid 26d (37 mg, 37%, trifluoroacetate salt) after purification by
d6):
d
¼ 10.72 (bs, 1H), 9.51 (bs, 1H), 8.06 (d, J ¼ 1.76 Hz, 1H), 7.86 (d,
J ¼ 8.80 Hz, 1H), 7.42e7.69 (m, 6H), 7.19e7.42 (m, 6H), 6.49 (d,
J ¼ 15.85 Hz, 1H), 4.76 (s, 2H), 4.36 (bs, 2H), 3.12e3.45 (m, 4H),
2.10e2.34 ppm (m, 4H); LCeMS (ESI) m/z: 484 [M þ H]þ; HPLC
purity: 100%, tR ¼ 1.55 min (Method B).
preparative HPLC. 1H NMR (300 MHz, DMSO-d6):
d
¼ 10.71 (bs, 1H),
9.62 (bs, 1H), 8.07 (d, J ¼ 1.76 Hz, 1H), 7.85 (dd, J ¼ 8.36, 1.91 Hz, 1H),
7.50 (d, J ¼ 15.85 Hz, 1H), 7.19e7.44 (m, 11H), 6.49 (d, J ¼ 15.85 Hz,
1H), 4.82 (s, 2H), 3.47e3.63 (m, 2H), 3.31e3.47 (m, 2H), 3.10e3.31
(m, 2H), 2.85e3.04 (m, 2H), 2.00e2.44 ppm (m, 4H); LCeMS (ESI)
m/z: 498 [M þ H]þ; HPLC purity: 100%, tR ¼ 1.64 min (Method B).
4.1.14. (E)-3-{10-(4-Fluorobenzyl)-3,4-dihydro-3-benzyl-4-oxospiro
[2H-(1,3)-benzoxazine-2,40-piperidin]-6-yl}-N-hydroxyacrylamide
(26c)
Spirocycle 22c was obtained by reaction between the free
piperidine 20 (485 mg, 1.13 mmol) and 4-fluorobenzaldehyde
4.2. Biological assays
(161
rocycle 22a, giving a white solid (553 mg, 98%). 1H NMR (300 MHz,
DMSO-d6):
1H), 7.71 (d, J ¼ 16.14 Hz, 1H), 7.19e7.45 (m, 7H), 7.14 (d, J ¼ 8.51 Hz,
1H), 7.06e7.15 (m, 2H), 6.60 (d, J ¼ 16.14 Hz,1H), 4.84 (s, 2H), 3.73 (s,
3H), 3.45 (s, 2H), 2.55e2.70 (m, 2H), 2.13e2.40 (m, 2H), 1.77e
m
L, 1.53 mmol), according to the procedure described for spi-
4.2.1. HDACs inhibition assay
The HDAC inhibition assay was performed as previously
described [24].
d
¼ 8.13 (d, J ¼ 2.05 Hz, 1H), 7.97 (dd, J ¼ 8.66, 2.20 Hz,
4.2.2. HDAC 2, 3, and 6 inhibition assays
The experiments were performed as previously described [22].

284
F. Thaler et al. / European Journal of Medicinal Chemistry 64 (2013) 273e284
E.R. Gardner, S.M. Steinberg, E.S. Jaffe, M. Stetler-Stevenson, S. Lade, A.T. Fojo,
4.2.3. Cell growth assay
The cell growth assays were performed according to methods
described previously [32].
S.E. Bates, Phase II multi-institutional trial of the histone deacetylase inhibitor
romidepsin as monotherapy for patients with cutaneous T-cell lymphoma,
J. Clin. Oncol. 27 (2009) 5410e5417.
[15] S. Jain, J. Zain, Romidepsin in the treatment of cutaneous T-cell lymphoma,
J. Blood Med. 2 (2011) 37e47.
[16] P. Atadja, Development of the pan-DAC inhibitor panobinostat (LBH589):
successes and challenges, Cancer Lett. 280 (2009) 233e241.
[17] P. Gimsing, Belinostat: a new broad acting antineoplastic histone deacetylase
inhibitor, Expert Opin. Investig. Drugs 18 (2009) 501e508.
4.2.4. Metabolic stability in hepatic microsomes
Microsomal stability experiments were performed as previously
described [24].
[18] I. Gojo, A. Jiemjit, J.B. Trepel, A. Sparreboom, W.D. Figg, S. Rollins, M.L. Tidwell,
J. Greer, E.J. Chung, M.J. Lee, S.D. Gore, E.A. Sausville, J. Zwiebel, J.E. Karp, Phase
1 and pharmacologic study of MS-275, a histone deacetylase inhibitor, in
adults with refractory and relapsed acute leukemias, Blood 109 (2007) 2781e
2790.
4.2.5. [3H]Astemizole displacement binding assay
The radioligand displacement assay was performed as described
previously by Chiu et al. [29].
[19] K.A. Blum, A. Advani, L. Fernandez, R. Van Der Jagt, J. Brandwein,
S. Kambhampati, J. Kassis, M. Davis, C. Bonfils, M. Dubay, J. Dumouchel,
M. Drouin, D.M. Lucas, R.E. Martell, J.C. Byrd, Phase II study of the histone
deacetylase inhibitor MGCD0103 in patients with previously treated chronic
lymphocytic leukaemia, Br. J. Haematol. 147 (2009) 507e514.
4.2.6. HDAC whole-cell assay
The HDAC whole-cell assay was performed according to the
methods described previously [21].
[20] N. Zhou, O. Moradei, S. Raeppel, S. Leit, S. Frechette, F. Gaudette, I. Paquin,
N. Bernstein, G. Bouchain, A. Vaisburg, Z. Jin, J. Gillespie, J. Wang, M. Fournel,
P.T. Yan, M.C. Trachy-Bourget, A. Kalita, A. Lu, J. Rahil, A.R. MacLeod, Z. Li,
J.M. Besterman, D. Delorme, Discovery of N-(2-aminophenyl)-4-[(4-pyridin-3-
ylpyrimidin-2-ylamino)methyl]benzamide (MGCD0103), an orally active
histone deacetylase inhibitor, J. Med. Chem. 51 (2008) 4072e4075.
[21] M. Varasi, F. Thaler, A. Abate, C. Bigogno, R. Boggio, G. Carenzi, T. Cataudella,
R. Dal Zuffo, M.C. Fulco, M.G. Rozio, A. Mai, G. Dondio, S. Minucci, C. Mercurio,
Discovery, synthesis, and pharmacological evaluation of spiropiperidine
hydroxamic acid based derivatives as structurally novel histone deacetylase
(HDAC) inhibitors, J. Med. Chem. 54 (2011) 3051e3064.
[22] F. Thaler, M. Varasi, G. Carenzi, A. Colombo, A. Abate, C. Bigogno, R. Boggio,
S. Carrara, T. Cataudella, R. Dal Zuffo, V. Reali, S. Vultaggio, G. Dondio,
S. Gagliardi, S. Minucci, C. Mercurio, Spiro[chromane-2,40-piperidine]-based
histone deacetylase inhibitors with improved in vivo activity, ChemMedChem
7 (2012) 709e721.
4.2.7. Flow cytometric cell cycle analysis
The flow-cytometric cell cycle analysis was performed as
described previously [21]. An anti-acetylated H4 clone T52 was
used as monoclonal antibody [33].
4.2.8. In vivo drug pharmacokinetic and efficacy experiments
In vivo pharmacokinetic in mice and efficacy experiments in a
human HCT116 xenograft model in mice were performed as pre-
viously reported [24]. An anti-acetylated H4 clone T25 (1:13,000)
was used as monoclonal antibody [33,34].
Acknowledgements
[23] P. Yeo, L. Xin, E. Goh, L.S. New, P. Zeng, X. Wu, P. Venkatesh, E. Kantharaj,
Development and validation of high-performance liquid chromatography-
tandem mass spectrometry assay for 6-(3-benzoyl-ureido)-hexanoic acid
hydroxyamide, a novel HDAC inhibitor, in mouse plasma for pharmacokinetic
studies, Biomed. Chromatogr. 21 (2007) 184e189.
The authors are grateful to Raimondo Fattori for his excellent
technical support in the execution of the present work.
[24] F. Thaler, A. Colombo, A. Mai, R. Amici, C. Bigogno, R. Boggio, A. Cappa,
S. Carrara, T. Cataudella, F. Fusar, E. Gianti, S. Joppolo di Ventimiglia,
M. Moroni, D. Munari, G. Pain, N. Regalia, L. Sartori, S. Vultaggio, G. Dondio,
S. Gagliardi, S. Minucci, C. Mercurio, M. Varasi, Synthesis and biological
evaluation of N-hydroxyphenylacrylamides and N-hydroxypyridin-2-
ylacrylamides as novel histone deacetylase inhibitors, J. Med. Chem. 53
(2010) 822e829.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.03.061.
[25] S. Vickers, C.A. Duncan, P.H. Kari, C.F. Homnick, J.M. Elliott, S.M. Pitzenberger,
M. Hichens, K.P. Vyas, In vivo and in vitro metabolism studies on a class III
antiarrhythmic agent, Drug Metab. Dispos. 21 (1993) 467e473.
[26] M.C. Sanguinetti, M. Tristani-Firouzi, hERG potassium channels and cardiac
arrhythmia, Nature 440 (2006) 463e469.
[27] A.M. Aronov, Ligand structural aspects of hERG channel blockade, Curr. Top.
Med. Chem. 8 (2008) 1113e1127.
[28] A.A. Lagrutta, E.S. Trepakova, J.J. Salata, The hERG channel and risk of drug-
acquired cardiac arrhythmia: an overview, Curr. Top. Med. Chem. 8 (2008)
1102e1112.
References
[1] E.I. Campos, D. Reinberg, Histones: annotating chromatin, Annu. Rev. Genet.
(2009).
[2] S. Spange, T. Wagner, T. Heinzel, O.H. Kramer, Acetylation of non-histone
proteins modulates cellular signalling at multiple levels, Int. J. Biochem. Cell.
Biol. 41 (2009) 185e198.
[3] M. Ocker, Deacetylase inhibitors e focus on non-histone targets and effects,
World J. Biol. Chem. 1 (2010) 55e61.
[29] P.J. Chiu, K.F. Marcoe, S.E. Bounds, C.H. Lin, J.J. Feng, A. Lin, F.C. Cheng,
W.J. Crumb, R. Mitchell, Validation of a [3H]astemizole binding assay in HEK293
cells expressing HERG Kþ channels, J. Pharmacol. Sci. 95 (2004) 311e319.
[30] I. Peretto, R. Forlani, C. Fossati, G.A. Giardina, A. Giardini, M. Guala, E. La Porta,
P. Petrillo, S. Radaelli, L. Radice, L.F. Raveglia, E. Santoro, R. Scudellaro,
F. Scarpitta, C. Bigogno, P. Misiano, G.M. Dondio, A. Rizzi, E. Armani, G. Amari,
M. Civelli, G. Villetti, R. Patacchini, M. Bergamaschi, M. Delcanale, C. Salcedo,
A.G. Fernandez, B.P. Imbimbo, Discovery of diaryl imidazolidin-2-one de-
rivatives, a novel class of muscarinic M3 selective antagonists (part 1), J. Med.
Chem. 50 (2007) 1571e1583.
[31] R. Di Micco, G. Sulli, M. Dobreva, M. Liontos, O.A. Botrugno, G. Gargiulo, R. dal
Zuffo, V. Matti, G. d’Ario, E. Montani, C. Mercurio, W.C. Hahn, V. Gorgoulis,
S. Minucci, F. d’Adda di Fagagna, Interplay between oncogene-induced DNA
damage response and heterochromatin in senescence and cancer, Nat. Cell.
Biol. 13 (2011) 292e302.
[4] A.J. de Ruijter, A.H. van Gennip, H.N. Caron, S. Kemp, A.B. van Kuilenburg,
Histone deacetylases (HDACs): characterization of the classical HDAC family,
Biochem. J. 370 (2003) 737e749.
[5] M. Haberland, R.L. Montgomery, E.N. Olson, The many roles of histone
deacetylases in development and physiology: implications for disease and
therapy, Nat. Rev. Genet. 10 (2009) 32e42.
[6] K. Huber, G. Superti-Furga, Afterthe grape rush:sirtuins asepigenetic drugtargets
in neurodegenerative disorders, Bioorg. Med. Chem. 19 (2011) 3616e3624.
[7] S. Minucci, P.G. Pelicci, Histone deacetylase inhibitors and the promise of
epigenetic (and more) treatments for cancer, Nat. Rev. Cancer 6 (2006) 38e51.
[8] M. Paris, M. Porcelloni, M. Binaschi, D. Fattori, Histone deacetylase inhibitors:
from bench to clinic, J. Med. Chem. 51 (2008) 1505e1529.
[9] A.G. Kazantsev, L.M. Thompson, Therapeutic application of histone deacety-
lase inhibitors for central nervous system disorders, Nat. Rev. Drug Discov. 7
(2008) 854e868.
[32] F. Thaler, A. Colombo, A. Mai, C. Bigogno, R. Boggio, S. Carrara, S. Joppolo di
Ventimiglia, D. Munari, N. Regalia, G. Dondio, S. Gagliardi, S. Minucci,
C. Mercurio, M. Varasi, Synthesis and biological characterization of
amidopropenyl-hydroxamates as HDAC inhibitors, ChemMedChem 5 (2010)
1359e1372.
[10] R. Glauben, E. Sonnenberg, M. Zeitz, B. Siegmund, HDAC inhibitors in models
of inflammation-related tumorigenesis, Cancer Lett. 280 (2009) 154e159.
[11] F. Thaler, S. Minucci, Next generation histone deacetylase inhibitors: the
answer to the search for optimized epigenetic therapies? Expert Opin. Drug
Discov. 6 (2011) 393e404.
[33] S. Ronzoni, M. Faretta, M. Ballarini, P. Pelicci, S. Minucci, New method to
detect histone acetylation levels by flow cytometry, Cytometry, Part A 66
(2005) 52e61.
[12] M. Federico, L. Bagella, Histone deacetylase inhibitors in the treatment of
hematological malignancies and solid tumors, J. Biomed. Biotechnol. 2011
(2011) 475641.
[34] L. Marquard, L.M. Gjerdrum, I.J. Christensen, P.B. Jensen, M. Sehested,
E. Ralfkiaer, Prognostic significance of the therapeutic targets histone deace-
tylase 1, 2, 6 and acetylated histone H4 in cutaneous T-cell lymphoma, His-
topathology 53 (2008) 267e277.
[13] P.A. Marks, Discovery and development of SAHA as an anticancer agent,
Oncogene 26 (2007) 1351e1356.
[14] R.L. Piekarz, R. Frye, M. Turner, J.J. Wright, S.L. Allen, M.H. Kirschbaum, J. Zain,
H.M. Prince, J.P. Leonard, L.J. Geskin, C. Reeder, D. Joske, W.D. Figg,