Design and synthesis of novel isoxazole-based HDAC inhibitors

Article abstract of DOI:10.1016/j.ejmech.2010.06.035

A series of isoxazole-based histone deacetylase (HDAC) inhibitors structurally related to SAHA were designed and synthesized. The isoxazole moiety was inserted in the vicinity of the Zn²⁺-binding group in order to check its participation in the coordinating process. New isoxazole-based histone deacetylase inhibitors structurally related to SAHA were designed and synthesized to check the participation of the isoxazole moiety in the Zn ²⁺coordination.

Full text of DOI:10.1016/j.ejmech.2010.06.035

European Journal of Medicinal Chemistry 45 (2010) 4331e4338
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of novel isoxazole-based HDAC inhibitors
,
a
a
a
b
c
Paola Conti a , Lucia Tamborini , Andrea Pinto , Laura Sola , Roberta Ettari , Ciro Mercurio ,
*
Carlo De Micheli a
a Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy
b Dipartimento Farmaco-Chimico, Università degli Studi di Messina, Viale Annunziata, 98168 Messina, Italy
c DAC Srl, Via Adamello 16, 20139 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of isoxazole-based histone deacetylase (HDAC) inhibitors structurally related to SAHA were
designed and synthesized. The isoxazole moiety was inserted in the vicinity of the Zn2þ-binding group in
order to check its participation in the coordinating process.
Received 2 February 2010
Received in revised form
11 June 2010
Accepted 25 June 2010
Available online 30 June 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Histone deacetylase
Isoxazole
Zinc binding group
1,3-Dipolar cycloaddition
Enzyme inhibition
1. Introduction
hydrolases [9,10], with a conserved catalytic core but differing in
size, domain structure and tissue expression pattern, while class III
Altered epigenetic regulations of gene expression play an
important role in cancer onset and progression. Epigenetic alter-
ations refer mainly to DNA methylation [1] or to post-translational
modifications of histone proteins [2,3]. Histones are nuclear core
proteins accountable for the regulation of transcription and cell
cycle progression. These activities are dependent on the level of
HDACs are sirtuin related proteins and require the cofactor NADþ
for their deacetylase function [11]. The vast majority of HDAC
inhibitors, currently undergoing development as anticancer drugs,
target the zinc-dependent isozymes (classes I, II, and IV). Natural
and synthetic HDAC inhibitors entered in clinical studies as
promising anticancer drug candidates belonging to different
structural classes including hydroxamates, cyclic peptides, aliphatic
acid and ortho-amino benzamides, e.g. depsipeptide FK228, val-
proic acid, MS-275, and NVP-LAQ824 (Fig. 1) [12]; the majority of
them possess the hydroxamate moiety as the functionality capable
of chelating the zinc ion in the active site. In 2006, the US FDA
approved suberoylanilide hydroxamic acid (SAHA; ZolinzaÔ, Fig. 1)
as first-in-class HDAC inhibitor for once-daily oral treatment of
a rare cancer, cutaneous T-cell lymphoma (CTCL) [13,14]. Never-
theless, hydroxamate-based HDAC inhibitors possess some liabili-
ties, e.g. a poor oral absorption and unfavorable pharmacokinetic
parameters due to hydrolysis or rapid glucuronidation. Further-
more, the presence in the molecule of such a strong metal chelating
group causes the lack of selectivity among the different HDAC
isoforms and can also result in inhibition of other metallo-enzymes
or sequestration of metal ions giving rise to a number of side effects.
Thus, there is a considerable interest in developing non-hydrox-
amate HDAC inhibitors characterized by a selectivity for individual
isoforms or, at least, a small subset of them [5,15].
acetylation/deacetylation of specific lysine 3-amino groups of the
protein backbones. Histone acetylation/deacetylation is a reversible
phenomenon and is tightly regulated by two competing enzymes
known as histone acetyltransferases (HATs) [2,3] and histone
deacetylases (HDACs) [4,5]. Inhibition of HDACs causes hyper-
acetylation of histones leading to differentiation, growth arrest and
apoptosis of malignant cells. For such a reason such enzymes have
recently gained prominence as an emerging target of anticancer
agents [6e8]. Several biological investigations have indicated that
HDAC enzymes are present in the human genome as an heteroge-
neous cluster composed by 18 isozymes which have been catego-
rized into four classes (I, II, III and IV) based on their function and on
the sequence similarity to their yeast orthologues [9e11]. HDAC
enzymes belonging to class I, II and IV are zinc-containing amide
* Corresponding author. Tel.: þ39 02 50319329; fax: þ39 02 50319326.
E-mail address: paola.conti@unimi.it (P. Conti).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.06.035

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P. Conti et al. / European Journal of Medicinal Chemistry 45 (2010) 4331e4338
H
N
binding group (ZBG), which chelates the Zn2þ ion at the bottom of
the active site.
O
O
O
N
S
S
To assess which HDAC isozyme should be targeted for anticancer
activity, great efforts are devoted to the discovery of isoform
selective HDAC inhibitors. A recent report by Kozikowski et al. [20].
showed that the insertion of substituted aryl-isoxazoles as the CAP
group gave HDAC inhibitors potent and selective at HDAC-6 and
HDAC-3. Even if the mode of interaction of the isoxazole moiety
with the surface residues was not clarified, the same research
group, in a subsequent paper, reported the results obtained by
shifting this moiety to the linker region, adjacent to the hydrox-
amate [21]. Some of the novel compounds are provided with an
inhibitory activity in the nanomolar range, but with a marginal
isozyme selectivity.
The above reported results prompted us to disclose the results
of a related investigation carried out by us. The present paper
deals with the synthesis of derivatives 1e4, in which the CAP
region was kept identical to that of SAHA and an isoxazole or
a 4,5-dihydro-isoxazole nucleus was inserted in the linker region
adjacent to the ZBG, represented by an hydroxamic acid, to eval-
uate a possible participation of the heterocyclic nitrogen in the
coordination of the zinc ion. In addition, we envisaged that such
NH
NH
HN
H
N
O
NH2
O
O
O
O
NH
O
FK228
MS-275
OH
N
O
H
N
HO
HO
O
NVP-LAQ824 HN
Valproic acid
O
H
N
HO
N
H
O
SAHA
Fig. 1. Structures of HDAC inhibitors.
The X-ray structures of some human and bacterial HDAC
enzymes co-crystallized with SAHA and trichostatin A [16e19]
revealed the mode of interaction of the inhibitors with the amino
acid residues of the enzymes binding pocket, allowing the delin-
eation of the pharmacophore features and the design of a number
of inhibitors. Despite the variety of structural motifs, the HDAC
inhibitors can be described by a common pharmacophore, which
can be segmented into three parts: i) a surface recognition zone
(CAP group), which interacts with the amino acid residues on the
rim of the active site, ii) a linker domain, usually hydrophobic,
which occupies the narrow tube-like channel, and iii) the zinc
a
nitrogen could mimic the carbonyl oxygen of a classical
hydroxamate and we have thus designed derivatives 5e13, in
which the length of the spacer is kept to 4 carbon atoms and the
3-substituted isoxazole is intended as the ZBG. In fact, a bidentate
interaction with the metal ion could be possible, that involves the
heterocyclic nitrogen, mimicking the carbonyl oxygen, and an
appropriate substituent appended at the 3 position, i.e. eNH2,
eCH2OH, eCH2SH, eCOOH, eCH]NOH, eCONHNH2, tetrazo-
lylmethyl-, o-phenol, o-aniline (Fig. 2).
O
O
HOHN
HOHN
H
N
O
N
Ph
Ph
N
Ph
Ph
N
H
O
O
O
O
O
1
2
O
N
O
N
HOHN
HOHN
H
N
O
N
H
O
O
3
4
O
N
O
N
H2N
HO
HO
H2NHN
H
N
H
N
H
N
N
Ph
Ph
Ph
Ph
O
O
O
O
O
O
O
5
6
7
HO
N
HS
H
N
H
H
N
N
N
N
Ph
N
O
Ph
O
O
O
9
10
8
N
HN
H
N
N
N
H
N
H
N
HO
H2N
N
O
Ph
N
N
Ph
Ph
O
O
O
O
O
11
12
Fig. 2. Compounds synthesized and tested in this study.
13

P. Conti et al. / European Journal of Medicinal Chemistry 45 (2010) 4331e4338
4333
O
EtO
O
O
a
O
b
b
c
c
1: n = 3
2: n = 4
N
()n OH
()n NHPh
()n
NHPh
NHPh
O
22: n = 3
23: n = 4
14: n = 3
15: n = 4
18: n = 3
19: n = 4
O
N
EtO
O
O
a
O
3: n = 3
4: n = 4
()n OH
()n NHPh
()n
O
24: n = 3
25: n = 4
16: n = 3
17: n = 4
20: n = 3
21: n = 4
Scheme 1. a) DCC, DMAP, aniline, CH2Cl2; b) Ethyl 2-chloro-2-(hydroxyimino)acetate, NaHCO3, AcOEt, MW, 100 ꢀC; c) NH2OH$HCl, KOH, MeOH.
2. Results and discussion
An ethanolic solution of ester 23 was also reacted with sodium
borohydride supported on alumina to give primary alcohol 8
(Scheme 3). The latter was used as the starting material for the
synthesis of target derivatives 9, 10 and 11 (Scheme 3). Alcohol 8
was mildly oxidized to the corresponding aldehyde, with
DesseMartin’s reagent, and immediately condensed with hydrox-
ylamine to afford oxime 9 (Scheme 3). The synthesis of thiol
derivative 10 was achieved through the transformation of alcohol 8
into the corresponding mesylate, followed by treatment with
potassium thioacetate, to give a S-acetyl derivative, easily con-
verted into the desired compound 10 by alkaline hydrolysis
(Scheme 3). Finally, mesylate 26 turned out to be a useful inter-
mediate also for the preparation of tetrazolyl-derivative 11. This
transformation was accomplished in two steps: i) the nucleophilic
displacement of the mesylate group by the cyanide anion and ii)
treatment of the nitrile intermediate with sodium azide and
ammonium chloride in DMF at 120 ꢀC (Scheme 3).
For the synthesis of the last two designed derivatives 12 and 13,
it was necessary to prepare suitable 1,3-dipoles to be used in the
cycloaddition reaction with alkyne 19. Chloroximes 29 [26] and 30
were prepared from commercially available salicylaldehyde and 2-
N-tert-butoxycarbonylamino benzaldehyde 28 [27], respectively,
following the procedure reported in the literature [26]. Treatment
of the freshly prepared chloroximes with excess sodium bicar-
bonate in the presence of dipole 19, under microwave irradiation,
produced cycloadduct 12 and the N-Boc derivative 31, that, upon
treatment with a 30% solution of trifluoroacetic acid in CH2Cl2
yielded final derivative 13 (Scheme 4).
The synthesis of compounds 1e13 (Fig. 2) was accomplished by
1,3-dipolar cycloaddition of suitably substituted formonitrile
oxides to alkynes 18 and 19, or alkenes 20 and 21, to yield the
desired isoxazoles or 4,5-dihydro-isoxazoles intermediates,
respectively.
The selected dipolarophiles contain i) the cap region, repre-
sented by the aromatic ring, ii) the linker, which is an aliphatic
chain of 3e4 carbon atoms connected to the cap through an amide
bond, and iii) a terminal unsaturation needed to build up the
designed isoxazole or isoxazoline ring. Alkynes 18, 19 and alkenes
20, 21 were obtained by reacting commercially available carboxylic
acids 14e17 with aniline under standard reaction conditions (DCC
and catalytic DMAP) (Scheme 1).
In order to obtain hydroxamates 1e4, dipolarophiles 18e21
were reacted with ethoxycarbonylformonitrile oxide, generated in
situ from its stable precursor ethyl 2-chloro-2-(hydroxyimino)
acetate [22] by adding solid NaHCO3. The pericyclic reaction was
carried out under microwave irradiation to speed up the process
and afforded the desired cycloadducts 22e25, which were then
converted into final products 1e4 by treatment with hydroxyl-
amine hydrochloride in
hydroxide (Scheme 1) [23].
a methanolic solution of potassium
3-Ethoxycarbonyl-isoxazole 23 represented also the key
intermediate in the preparation of target derivatives 5e11. In
detail, ester derivative 23 was subjected to alkaline hydrolysis to
afford the corresponding carboxylic acid 5 (Scheme 2). Alterna-
tively, the same derivative 23 was reacted with hydrazine in
ethanol to give hydrazide 6 [24], which, upon treatment with
a solution of sodium nitrite in a 1:1 mixture of 10% hydrochloric
acid/acetone, followed by heating in a 1:1 mixture of watere-
acetic acid, was converted into the 3-amino-substituted isoxazole
7 (Scheme 2) [25].
HO
N
HO
O
O
a
b, c
23
N
N
()4
()4
NHPh
O
NHPh
NHPh
O
O
8
9
O
HS
HO
O
e,f
a
23
MsO
N
N
()4
NHPh
()4
NHPh
O
O
O
d
8
5
N
()4
O
26
g,h
N
N
O
H2N
O
HN
N
H2NHN
c, d
b
O
O
23
N
()4
NHPh
N
O
11
N
()4
NHPh
()4
NHPh
O
O
7
6
Scheme 3. a) NaBH4 10% on alumina, EtOH; b) DesseMartin periodinane, CH2Cl2; c)
NH2OH$HCl, TEA, MeOH; d) MsCl, TEA, CH2Cl2; e) KSCOCH3, DMF, 90 ꢀC; f) 1 M NaOH,
MeOH; g) KCN, DMF; h) NaN3, NH4Cl, DMF, 120 ꢀC.
Scheme 2. a) NaOH, EtOH; b) NH2NH2$H2O, EtOH; c) NaNO2, 10% HCl, acetone, H2O; d)
H2O/CH3COOH 1:1,
D.

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P. Conti et al. / European Journal of Medicinal Chemistry 45 (2010) 4331e4338
Table 2
R
H
R
a, b
In vitro inhibitory activity of compounds 2 and 4 at different HDAC isoforms.
O
NOH
Compds
HDAC-1
IC50 M)
4.95
17.7
0.17
HDAC-2
HDAC-3
HDAC-6
HDAC-10
IC50 M)
9.05
23.9
0.21
Cl
(
m
IC50
(
mM)
IC50
(
mM)
IC50
(
mM)
(m
R = OH: 27
R = NHBoc: 28
R = OH: 29
R = NHBoc: 30
2
4
18.9
36.2
0.39
26.7
28.1
0.20
0.43
0.76
0.0093
SAHA
c
19 + 29
O
HO
demonstrate that none of the 3-substituted isoxazole rings can effi-
ciently vicariate the hydroxamate moiety except for the 3-mercap-
tomethyl-isoxazole, which probably own its efficacy to the powerful
zinc-binding properties typical of the thiol group.
N
()4
12
NHPh
O
c
d
19
+
30
O
O
H2N
BocHN
N
N
()4
NHPh
()4
31
NHPh
O
O
3. Conclusion
13
In conclusion, we have tried to identify new effective iso-
xazole-based zinc binding groups that could be used instead of
the hydroxamate in the design of structurally diverse HDAC
inhibitors.
Scheme 4. a) NH2OH$HCl, NaOAc, EtOH, H2O; b) NCS, Py, CH2Cl2; 40 ꢀC; c) NaHCO3,
AcOEt, MW, 100 ꢀC; d) 30% TFA, CH2Cl2.
The new compounds 1e13 were submitted to biological evalu-
ation to identify the molecules endowed with HDAC inhibitory
activity. The assays were carried out using a commercially available
HDAC assay kit, with HeLa cell nuclear extracts as enzyme source
and a fluorogenic acetylated histone peptide fragment (Fluor de
Lys) as a substrate. As shown in Table 1 the hydroxamates
compounds 1e4 demonstrated to have a dose dependent moderate
biochemical activity (percentage of inhibition in a range between 9
However, though the electronic properties of the 3-
substituted isoxazole proposed could have justified an isosterism
with the hydroxamate group, the biological results clearly show
that, in this case, bioisosterism did not occur, with the exception
of the 3-mercaptomethyl-isoxazole. Moreover, the insertion of an
isoxazole or isoxazoline ring into the SAHA skeleton did not
significantly modify the selectivity profile towards different
HDAC isoforms. Structure optimization of compound 10,
including modification of the cap as well as the spacer region,
could lead to the development of a new type of non-hydroxamate
HDAC inhibitors.
and 23 at 1
mM and between 56 and 76.5% at 10 mM). However,
the inhibitory activity was lower than that measured for the
reference compound SAHA, especially when measured at low
concentrations. Therefore
a participation of the heterocyclic
nitrogen in the coordination of the zinc ion, in the presence of
a hydroxamate moiety, has to be ruled out. We then decided to
evaluate whether the insertion of the heterocycle into the SAHA
skeleton could play a role in the selectivity profile. To this aim we
chose compounds 2 and 4, having an isoxazole and an isoxazoline
moiety, respectively, and tested them for inhibition activity at
different HDAC isoforms. The results reported in Table 2 show that
both derivatives display 10e70 times lower IC50s at HDAC-6 than at
HDAC 1, 2, 3 and 10. This type of selectivity profile parallels that
reported for SAHA, therefore a beneficial effect of the heterocycle
was not observed.
4. Experimental
4.1. Materials and methods
Ethyl 2-chloro-2-(hydroxyimino)acetate [22], tert-butyl 2-for-
mylphenylcarbamate 28 [27] and N,2-dihydroxybenzimidoyl chlo-
ride 29 [26] were prepared following literature procedures.
1H NMRand 13C NMR spectrawere recorded with a Varian Mercury
300 (300 MHz) spectrometer. Chemical shifts (d) are expressed in ppm
and coupling constants (J) in hertz. TLC analyses were performed on
commercial silica gel 60 F254 aluminium sheets; spots were further
evidenced by spraying with a dilute alkaline potassium permanganate
solution. Melting points were determined on a model B 540 Büchi
apparatus and are uncorrected. Microanalyses (C, H, N) of new
compounds agreed with the theoretical value within ꢁ0.4%.
Concerning the non-hydroxamate derivatives, the only one
provided with a moderate inhibitoryactivity was the thiol compound
10 (22% inhibition at 1
mM and 80% inhibition at 10 mM), whereas
all the other derivatives turned out to be inactive. These outcomes
Table 1
In vitro HDAC inhibitory activity.
4.2. General procedure for the amide bond formation
Compds
Inhibition 0.1
m
M (%)
Inhibition 1
m
M (%)
Inhibition 10 mM (%)
1
2
3
4
5
6
7
8
4.5
1.5
3.5
4
0
0
4.5
0
0
4.5
0
0
23.75
26
9.75
16.75
0
0
0
0
0.75
22
0
0
0
78.5
78.5
56
77
0.5
8
0
0
7.5
80
5
A solution of DCC (0.41 g, 2.0 mmol) in CH2Cl2 (5.0 ml) was added
dropwise to a cooled (0 ꢀC) solution of carboxylic acid 14e17
(2.0 mmol), aniline (182 ml, 2.0 mmol) and DMAP (24 mg, 0.2 mmol) in
CH2Cl2 (5.0 ml). The mixture was allowed to warm at room tempera-
ture and stirred for 4 h. The progress of the reaction was monitored by
TLC (dichloromethane/methanol 9:1). After disappearance of the
starting material, the solid was filtered off and the mixture was washed
with 1 N HCl (10 ml), 5% solution of NaHCO3 (10 ml) and brine. The
organic layer was dried over anhydrous sodium sulfate and concen-
trated to give the crude material, which was purified by flash chro-
matography (petroleum ether/ethyl acetate 8:2) to yield compound 18
[28], 19 [28], 20 [29], and 21 [30] (about 80% yield).
9
10
11
12
13
SAHA
0
0.5
95
0
47
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P. Conti et al. / European Journal of Medicinal Chemistry 45 (2010) 4331e4338
4335
4.3. General procedure for the cycloaddition reaction
the cycloadduct 22e25 (0.5 mmol) in EtOH (0.5 ml) was added. The
reaction was stirred overnight at room temperature and then was
diluted with water (5 ml), made neutral with 2 N HCl and filtered
under vacuum. The solid was collected and recrystallized.
Ethyl 2-chloro-2-(hydroxyimino)acetate (0.30 g, 2.0 mmol) and
an excess of solid NaHCO3 (0.67 g, 8.0 mmol) were added to
a solution of compound 18e21 (1.0 mmol) in AcOEt (10 ml). The
mixture was irradiated with
m
W for 90 min at 100 ꢀC. The progress
4.4.1. N-Hydroxy-5-[4-oxo-4-(phenylamino)butyl]isoxazole-3-
carboxamide (1)
of the reaction was monitored by TLC. Water was added to the
reaction mixture and the organic layer was separated and dried
over anhydrous sodium sulfate. The solvent was evaporated and the
product was purified by crystallization.
Crystallized from 2-propanol as white prisms; mp 175e176 ꢀC;
yield 54%; Rf ¼ 0.20 (dichloromethane/methanol 95:5 þ1%
CH3COOH). 1H NMR (DMSO-d6): 1.95 (ddd, J ¼ 7.7, 7.7, 14.9, 2H);
2.37 (t, J ¼ 7.7, 2H); 2.84 (t, J ¼ 7.7, 2H); 6.58 (s, 1H), 7.00 (t, J ¼ 8.5,
1H); 7.25 (t, J ¼ 8.5, 2H); 7.56 (d, J ¼ 8.5, 2H), 9.35 (s, 1H), 9.90 (s,
1H), 11.45 (s, 1H). 13C NMR (DMSO-d6): 23.46, 26.06, 35.87, 101.43,
119.75, 123.63, 129.36, 139.90, 156.99, 158.17, 171.06, 174.81. Anal.
Calcd for C14H15N3O4 (289.11): C, 58.13; H, 5.23; N, 14.53. Found: C,
58.10; H, 5.28; N, 14.60.
4.3.1. Ethyl 5-[4-oxo-4-(phenylamino)butyl]isoxazole-3-
carboxylate (22)
Crystallized from diisopropyl ether/2-propanol 95/5 as white
prisms; mp 81e82 ꢀC; yield 80%; Rf ¼ 0.26 (cyclohexane/ethyl acetate
6:4). 1H NMR (CDCl3): 1.40 (t, J ¼ 7.1, 3H); 2.16 (m, 2H); 2.43 (t, J ¼ 7.2,
2H); 2.87 (t, J ¼ 7.2, 2H); 4.41 (q, J ¼ 7.1, 2H); 6.45 (s,1H); 7.10 (t, J ¼ 7.4,
1H); 7.24 (bs, 1H); 7.32 (t, J ¼ 7.4, 2H); 7.50 (d, J ¼ 7.4, 2H). 13C NMR
(CDCl3): 14.34, 23.35, 26.15, 36.13, 62.40,102.17,120.20,124.57,129.19,
138.10, 156.67, 160.30, 170.52, 174.98. Anal. Calcd for C16H18N2O4
(302.13): C, 63.56; H, 6.00; N, 9.27. Found: C, 63.89; H, 6.28; N, 9.39.
4.4.2. N-Hydroxy-5-[5-oxo-5-(phenylamino)pentyl]isoxazole-3-
carboxamide (2)
Crystallized from 2-propanol as white prisms; mp 175e176 ꢀC;
yield 36%; Rf ¼ 0.20 (dichloromethane/methanol 95:5 þ1%
CH3COOH). 1H NMR (DMSO-d6): 1.55e1.75 (m, 4H); 2.32 (m, 2H);
2.84 (m, 2H); 6.54 (s,1H), 7.00 (t, J ¼ 8.2,1H); 7.26 (t, J ¼ 8.2, 2H); 7.56
(d, J ¼ 8.2, 2H), 9.35(s,1H), 9.87 (s,1H),11.45 (s,1H).13C NMR (DMSO-
d6): 25.10, 26.28, 27.19, 36.52, 101.25, 119.73, 123.63, 129.35, 139.95,
157.02,158.14,171.60,175.14. Anal. Calcd for C15H17N3O4 (303.12): C,
59.40; H, 5.65; N, 13.85. Found: C, 59.30; H, 5.58; N, 13.90.
4.3.2. Ethyl 5-[5-oxo-5-(phenylamino)pentyl]isoxazole-3-
carboxylate (23)
Crystallized from diisopropyl ether/2-propanol 95/5 as white
prisms; mp 97e99 ꢀC; yield 86%; Rf ¼ 0.19 (cyclohexane/ethyl
acetate 7:3). 1H NMR (CDCl3): 1.40 (t, J ¼ 7.0, 3H); 1.75e1.85 (m, 4H);
2.35e2.45 (m, 2H); 2.80e2.93 (m, 2H); 4.42 (q, J ¼ 7.0, 2H); 6.42 (s,
1H); 7.10 (t, J ¼ 7.9, 1H); 7.24 (bs, 1H); 7.31 (t, J ¼ 7.9, 2H); 7.52 (d,
J ¼ 7.9, 2H). 13C NMR (CDCl3): 14.35, 24.95, 26.72, 27.12, 37.09, 62.32,
101.92, 120.08, 124.49, 129.19, 138.12, 156.63, 160.36, 170.92, 175.25.
Anal. Calcd for C17H20N2O4 (316.14): C, 64.54; H, 6.37; N, 8.86.
Found: C, 64.26; H, 6.60; N, 9.00.
4.4.3. N-Hydroxy-5-[4-oxo-4-(phenylamino)butyl]-4,5-
dihydroisoxazole-3-carboxamide (3)
Crystallized from 2-propanol as pink prisms;mp 144e146 ꢀC;yield
46%; Rf ¼ 0.19 (dichloromethane/methanol 95:5 þ1% CH3COOH). 1H
NMR (DMSO-d6): 1.50e1.75 (m, 4H); 2.30 (m, 2H); 2.81 (dd, J ¼ 8.2,
17.6, 1H); 3.22 (dd, J ¼ 10.8, 17.6, 1H); 4.65 (m, 1H); 7.00 (t, J ¼ 7.5, 1H);
7.25 (t, J ¼ 7.5, 2H); 7.56 (d, J ¼ 7.5, 2H), 9.15 (s,1H), 9.85 (s,1H),11.20 (s,
1H). 13C NMR (DMSO-d6): 21.60, 34.58, 36.57, 82.11, 119.70, 123.64,
129.35, 139.96, 153.41, 157.83, 171.55. Anal. Calcd for C14H17N3O4
(291.12): C, 57.72; H, 5.88; N, 14.42. Found: C, 57.90; H, 6.00; N, 14.57.
4.3.3. Ethyl 5-[4-oxo-4-(phenylamino)butyl]-4,5-dihydroisoxazole-
3-carboxylate (24)
Crystallized from diisopropyl ether/2-propanol 95/5 as white
prisms; mp 66e67 ꢀC; yield 98%; Rf ¼ 0.18 (petroleum ether/ethyl
acetate 7:3). 1H NMR (CDCl3): 1.36 (t, J ¼ 7.1, 3H); 1.70e1.95 (m, 4H);
2.35e2.45 (m, 2H); 2.88 (dd, J ¼ 8.2, 17.6, 1H); 3.29 (dd, J ¼ 11.0, 17.6,
1H); 4.34 (q, J ¼ 7.1, 2H); 4.85 (m, 1H); 7.10 (t, J ¼ 7.4, 1H); 7.26 (bs,
1H); 7.31 (t, J ¼ 7.4, 2H); 7.52 (d, J ¼ 7.4, 2H). 13C NMR (CDCl3): 14.39,
21.51, 34.50, 37.06, 38.82, 62.32, 84.18, 120.01, 124.55, 129.26,
138.00, 151.83, 160.93, 170.76. Anal. Calcd for C16H20N2O4 (304.14):
C, 63.14; H, 6.62; N, 9.20. Found: C, 62.89; H, 6.58; N, 9.10.
4.4.4. N-Hydroxy-5-[5-oxo-5-(phenylamino)pentyl]-4,5-
dihydroisoxazole-3-carboxamide (4)
Crystallized from 2-propanol as pink prisms; mp 166e168 ꢀC;
yield 40%; Rf ¼ 0.19 (dichloromethane/methanol 95:5 þ1%
CH3COOH). 1H NMR (DMSO-d6): 1.20e1.45 (m, 2H); 1.50e1.65 (m,
4H); 2.30 (t, J ¼ 7.1, 2H); 2.79 (dd, J ¼ 8.2, 17.6, 1H); 3.23 (dd, J ¼ 10.7,
17.6, 1H); 4.65 (m, 1H); 7.00 (t, J ¼ 7.7, 1H); 7.25 (t, J ¼ 7.7, 2H); 7.56
(d, J ¼ 7.7, 2H), 9.15 (s, 1H), 9.85 (s, 1H), 11.20 (s, 1H). 13C NMR
(DMSO-d6): 25.14, 25.55, 34.83, 36.92, 82.25, 119.70, 123.63, 129.35,
140.00, 153.40, 157.86, 171.78. Anal. Calcd for C15H19N3O4 (305.14):
C, 59.01; H, 6.27; N, 13.76. Found: C, 58.90; H, 6.00; N, 13.49.
4.3.4. Ethyl 5-[5-oxo-5-(phenylamino)pentyl]-4,5-
dihydroisoxazole-3-carboxylate (25)
Crystallized from diisopropyl ether/2-propanol 95/5 as white
prisms; mp 62e63 ꢀC; yield 98%; Rf ¼ 0.22 (cyclohexane/ethyl
acetate 6:4). 1H NMR (CDCl3): 1.27 (t, J ¼ 7.1, 3H); 1.58e1.74 (m, 6H);
2.30 (t, J ¼ 7.4, 2H); 2.74 (dd, J ¼ 8.5, 17.6, 1H); 3.16 (dd, J ¼ 11.0, 17.6,
1H); 4.25 (q, J ¼ 7.1, 2H); 4.70 (m, 1H); 7.00 (t, J ¼ 7.7, 1H); 7.20 (t,
J ¼ 7.7, 2H); 7.48 (d, J ¼ 7.7, 2H); 8.35 (bs, 1H). 13C NMR (CDCl3):
14.33, 25.11, 25.39, 35.03, 37.37, 38.66, 62.28, 84.11, 120.21, 124.36,
129.11, 138.34, 151.77, 160.99, 172.67. Anal. Calcd for C17H22N2O4
(318.16): C, 64.13; H, 6.97; N, 8.80. Found: C, 64.00; H, 6.84; N, 9.00.
4.4.5. Synthesis of 5-[4-(phenylcarbamoyl)butyl]isoxazole-3-
carboxylic acid (5)
Compound 23 (158 mg, 0.5 mmol) was dissolved in EtOH
(1.0 ml) and 1 N NaOH (0.5 ml) was added. The mixture was stirred
for 1 h at room temperature. The organic solvent was evaporated
and the aqueous phase was extracted with Et2O (1 ꢂ2 ml). The
aqueous layer was made acidic with 2 N HCl and newly extracted
with AcOEt (4 ꢂ 2 ml). The organic extracts were dried over anhy-
drous sodium sulfate. The solvent was removed under vacuum to
give 129 mg (90% yield) of compound 5.
Compound 5: crystallized from 2-propanol as white prisms;
mp > 148 ꢀC dec; Rf ¼ 0.18 (dichloromethane/methanol 95:5 þ1%
CH3COOH). 1H NMR (DMSO-d6): 1.55e1.78 (m, 4H); 2.28e2.39 (m,
2H); 2.78e2.85 (m, 2H); 6.60 (s, 1H); 7.00 (t, J ¼ 8.2, 1H); 7.25 (t,
4.4. General procedure for the synthesis of hydroxamic acids
A solution of NH2OH$HCl (70 mg,1.0 mmol) in EtOH (0.5 ml) was
prepared and refluxed. In a separate flask, a solution of KOH (84 mg,
1.5 mmol) in EtOH (0.5 ml) was refluxed. After cooling at T < 40 ꢀC,
the solution of KOH was added, in one portion, to the solution of
NH2OH$HCl. The mixturewas stirred for 5 min and then a solution of

4336
P. Conti et al. / European Journal of Medicinal Chemistry 45 (2010) 4331e4338
J ¼ 8.2, 2H); 7.56 (d, J ¼ 8.2, 2H); 9.30 (s, 1H). 13C NMR (DMSO-d6):
25.08, 26.32, 27.13, 36.51, 102.45, 119.72, 123.65, 129.33, 139.95,
157.70, 161.75,171.59,175.87. Anal. Calcd for C15H16N2O4 (288.11): C,
62.49; H, 5.59; N, 9.72. Found: C, 62.31; H, 5.41; N, 9.75.
temperature and stirred for 2 h. The mixture was poured into
a solution of 10% NaHCO3 (10 ml) containing 1% Na2S2O3, the
phases were separated and the organic layer was washed with
brine (10 ml) and dried over anhydrous sodium sulfate. The solvent
was evaporated to obtain the crude aldehyde that was used in the
next step without any additional purification.
b) The aldehyde obtained from the previous step (190 mg,
0.70 mmol) was dissolved in MeOH (2.0 ml). In a separate flask a solu-
tion of NH2OH$HCl(48 mg,0.70 mmol)andTEA(97 ml, 0.70 mmol) was
4.4.6. Synthesis of 5-[3-(hydrazinecarbonyl)isoxazol-5-yl]-N-
phenylpentanamide (6)
To a stirred solution of 23 (474 mg, 1.5 mmol) in EtOH (5.0 ml) at
0 ꢀC was added NH2NH2$H2O (218
ml, 4.5 mmol). The solution was
warmed to room temperature and stirred for 30 min. The solid was
filtered off and the solvent was evaporated under reduced pressure
to obtain a yellow solid, which was triturated with water and
recrystallized from ethanol (263 mg, yield 58%).
Compound 6: crystallized from ethanol as pale yellow prisms;
mp > 148 ꢀC dec; Rf ¼ 0.35 (dichloromethane/methanol 95/5). 1H
NMR (DMSO-d6): 1.56e1.74 (m, 4H); 2.32 (t, J ¼ 7.4, 2H); 2.81 (t,
J ¼ 7.4, 2H); 4.45e4.79 (bs, 2H); 6.55 (s, 1H); 7.00 (t, J ¼ 7.4,1H); 7.25
(t, J ¼ 7.4, 2H); 7.56 (d, J ¼ 7.4, 2H), 9.90 (s, 1H). 13C NMR (DMSO-d6):
25.11, 26.29, 27.19, 36.52, 101.11, 119.71, 123,65, 129.33, 139.95,
158.62, 158.75, 171.59, 175.07. Anal. Calcd for C15H18N4O3 (302.14):
C, 59.59; H, 6.00; N, 18.53. Found: C, 59.67; H, 6.11; N, 18.78.
prepared in MeOH (1.0 ml) and added to the solution of aldehyde. The
reaction mixture was stirred for 2 h at room temperature. The solvent
was evaporated, the residue was dissolved in AcOEt (5 ml) and washed
with 1 N HCl (5 ml). The organic layer was dried over anhydrous
sodium sulfate and the solvent was evaporated. The crude material was
purified by column chromatography (petroleum ether/ethyl acetate
6:4) to obtain compound 9 (116 mg, 58% yield).
Compound 9: crystallized from methanol as white prisms; mp
164e167 ꢀC; Rf ¼ 0.27 (petroleum ether/ethyl acetate 1:1). 1H NMR
(DMSO-d6): 1.55e1.75 (m, 4H); 2.30 (t, J ¼ 6.9, 2H); 2.80 (t, J ¼ 6.9,
2H); 6.50 (s, 1H); 7.00 (t, J ¼ 8.5, 1H); 7.25 (t, J ¼ 8.5, 2H); 7.56 (d,
J ¼ 8.5, 2H); 8.15 (s,1H), 9.90 (bs,1H); 12.00 (s,1H).13C NMR (DMSO-
d6): 25.14, 26.23, 27.17, 36.54, 98.83, 119.72, 123.64, 129.33, 139.90,
139.96, 159.04, 171.61, 174.33. Anal. Calcd for C15H17N3O3 (287.31):
C, 62.71; H, 5.96; N, 14.63. Found: C, 62.89; H, 6.18; N, 14.89.
4.4.7. Synthesis of 5-(3-aminoisoxazol-5-yl)-N-phenylpentanamide
(7)
a) The hydrazide 6 (151 mg, 0.50 mmol) was dissolved in a 1:1
mixture of acetone/10% HCl (2.0 ml). The solution was cooled in an
ice bath and a solution of NaNO2 (52 mg, 0.75 mmol) in water
(0.5 ml) was added dropwise. The mixture was stirred for 30 min.
An additional 1.0 ml of water was added and the product was
collected by filtration as white plates (137 mg, 88% yield).
b) The acyl azide (137 mg, 0.44 mmol) was suspended in a 1:1
mixture of water/acetic acid (7.0 ml) and the mixture was brought
to reflux. After disappearance of the starting material (1 h), the
solvents were removed under vacuum and the solid residue was
crystallized from ethanol to give compound 7 (88 mg, 77% yield).
Compound 7: crystallized from ethanol as white prisms;
mp > 150 ꢀC dec; Rf ¼ 0.20 (cyclohexane/ethyl acetate). 1H NMR
(DMSO-d6): 1.50e1.70 (m, 4H); 2.32 (m, 2H); 2.55 (m, 2H); 5.40 (bs, 2H);
5.56 (s, 1H); 7.00 (t, J ¼ 7.4, 1H); 7.26 (t, J ¼ 7.4, 2H); 7.56 (d, J ¼ 7.4, 2H);
9.85 (s,1H).13CNMR(DMSO-d6): 25.22, 26.54, 27.23, 36.61, 94.34,119.71,
123.64, 129.33, 139.97, 164.57, 171.66, 175.80. Anal. Calcd for C14H17N3O2
(259.13): C, 64.85; H, 6.61; N, 16.20. Found: C, 64.98; H, 6.76; N, 16.35.
4.4.10. Synthesis of (5-(4-(phenylcarbamoyl)butyl)isoxazol-3-yl)
methyl methanesulfonate (26)
TEA (208
compound 8 (274 mg,1.0 mmol) in CH2Cl2 (10 ml). The mixture was
cooled at 0 ꢀC and methanesulfonyl chloride (116
l, 1.5 mmol) was
ml, 1.5 mmol) was added to a stirred solution of
m
added dropwise. The reaction was stirred at room temperature for
4 h. The organic phase was washed with 1 N HCl (2 ꢂ 5 ml), 5%
solution of NaHCO3 (2 ꢂ 5 ml), brine (5 ml) and dried over anhy-
drous sodium sulfate. The solvent was evaporated and the product
was purified by crystallization with 2-propanol to obtain
compound 26 (274 mg, 79% yield).
Compound 26: crystallized from 2-propanol as white prisms;
mp > 78 ꢀC dec; Rf ¼ 0.32 (cyclohexane/ethyl acetate 4:6). 1H NMR
(CDCl3): 1.70e1.82 (m, 4H); 2.35e2.42 (m, 2H); 2.78e2.85 (m, 2H);
3.03 (s, 3H); 5.25 (s, 2H); 6.18 (s, 1H); 7.05 (t, J ¼ 7.9, 1H); 7.32 (t,
J ¼ 7.9, 2H); 7.38 (bs, 1H); 7.50 (d, J ¼ 7.9, 2H). 13C NMR (CDCl3):
24.98, 26.70, 27.08, 37.08, 38.36, 62.30, 100.95, 120.11, 124.53,
129.21, 138.14, 158.27, 171.09, 174.88. Anal. Calcd for C16H20N2O5S
(352.11): C, 54.53; H, 5.72; N, 7.95. Found: C, 54.78; H, 5.90; N,
7.90.
4.4.8. Synthesis of 5-(3-(hydroxymethyl)isoxazol-5-yl)-N-
phenylpentanamide (8)
To a stirred solution of compound 23 (316 mg, 1.0 mmol) in
ethanol (10 ml) NaBH4 on alumina (NaBH4 10%, 1.3 mmol, 390 mg)
was added. The mixture was stirred at room temperature for 24 h,
until disappearance of the starting material. The solid was filtered off
and the solvent was evaporated, obtaining compound 8 as a white
solid, which was purified by crystallization (260 mg, 95% yield).
Compound 8: crystallized from 2-propanol white prisms; mp
113e116 ꢀC; Rf ¼ 0.26 (dichloromethane/methanol 95:5). 1H NMR
(CD3OD): 1.70e1.82 (m, 4H); 2.38e2.44 (m, 2H); 2.78e2.85 (m,
2H); 4.58 (s, 2H); 6.20 (s,1H); 7.07 (t, J ¼ 7.9,1H); 7.28 (t, J ¼ 7.9, 2H);
7.53 (d, J ¼ 7.9, 2H); 9.75 (bs, 1H). 13C NMR (CD3OD): 25.02, 25.99,
27.01, 36.21, 55.63, 99.91, 120.06, 123.97, 128.59, 138.66, 164.33,
172.91, 173.78. Anal. Calcd for C15H18N2O3 (274.13): C, 65.68; H,
6.61; N, 10.21. Found: C, 65.40; H, 6.50; N, 10.45.
4.4.11. Synthesis of 5-(3-(mercaptomethyl)isoxazol-5-yl)-N-
phenylpentanamide (10)
a) Compound 26 (138 mg, 0.39 mmol) was dissolved in DMF
(2.0 ml) and CH3COSK (53 mg, 0.47 mmol) was added. The mixture
was stirred overnight at 90 ꢀC. After disappearance of the starting
material, water (3 ml) was added and the aqueous phase was
extracted with AcOEt (3 ꢂ 5 ml). The organic layer was dried over
anhydrous sodium sulfate and the solvent was evaporated under
reduced pressure. The crude material was purified by column
chromatography (cyclohexane/ethyl acetate 7/3) to give the
product as a yellow oil (65 mg, 50% yield).
b) The S-acetyl intermediate obtained from the previous step
(65 mg, 0.19 mmol) was dissolved in MeOH (0.20 ml) and 1 N NaOH
was added (0.19 ml). The mixture was stirred 1 h at room tempera-
ture. Water (2 ml) was added and the aqueous phase was extracted
with Et2O (1 ꢂ 2 ml). The aqueous layer was made acidic with 2 N
HCl and newly extracted with AcOEt (4 ꢂ 2 ml). The organic extracts
were dried over anhydrous sodium sulfate. The solvent was removed
under vacuum to give 48 mg (88% yield) of compound 10.
4.4.9. Synthesis of (E)-5-(3-((hydroxyimino)methyl)isoxazol-5-yl)-
N-phenylpentanamide (9)
a) To a cooled (0 ꢀC) solution of 8 (192 mg, 0.70 mmol) in dry
CH2Cl2 (10 ml) was added DesseMartin periodinane (326 mg,
0.77 mmol). The reaction mixture was warmed to room

P. Conti et al. / European Journal of Medicinal Chemistry 45 (2010) 4331e4338
4337
Compound 10: crystallized from 2-propanol as white prisms;
mp > 135 ꢀC dec; Rf ¼ 0.16 (cyclohexane/ethyl acetate 1:1). 1H NMR
(CDCl3): 1.55 (bs, 1H); 1.70e1.85 (m, 4H); 2.30e2.42 (m, 2H);
2.70e2.82 (m, 2H); 3.85 (s, 2H); 6.08 (s, 1H); 7.10 (m, 1H); 7.30 (m,
2H); 7.50 (m, 2H). 13C NMR (CDCl3): 25.12, 25.15, 27.48, 32.16, 34.34,
101.05, 120.51, 124.93, 129.61, 139.04, 158.27, 171.09, 174.88. Anal.
Calcd for C15H18N2O2S (290.11): C, 62.04; H, 6.25; N, 9.65. Found: C,
62.24; H, 6.35; N, 9.78.
added. The temperature was raised to 40 ꢀC and NCS (266 mg,
2.0 mmol) was added to the resulting solution. The reaction
mixture was stirred at this temperature for 4 h and then was
diluted with CH2Cl2 (10 ml). The organic layer was washed with
water (2 ꢂ 10 ml) and brine, dried over anhydrous sodium sulfate,
and concentrated under reduced pressure to afford compound 30,
which was purified by crystallization from EtOH (403 mg, 83%
yield).
Compound 30: crystallized from 2-propanol as pale yellow
prisms; Rf ¼ 0.24 (cyclohexane/ethyl acetate 95/5). 1H NMR (CDCl3):
1.55 (s, 9H); 7.02 (t, J ¼ 7.7, 2H); 7.20 (d, J ¼ 7.7, 1H); 7.35 (t, J ¼ 7.7,
1H); 8.38 (bd, J ¼ 8.3, 1H); 9.70 (bs, 1H). 13C NMR (CDCl3): 28.60,
80.69, 117.76, 118.74, 121.96, 131.01, 132.33, 138.73, 145.20, 153.57.
Anal. Calcd for C12H15ClN2O3 (270.08): C, 53.24; H, 5.58; N, 10.35.
Found: C, 53.56; H, 5.76; N, 10.59.
4.4.12. Synthesis of 5-(3-((2H-tetrazol-5-yl)methyl)isoxazol-5-yl)-
N-phenylpentanamide (11)
a) Compound 26 (138 mg, 0.39 mmol) was dissolved in DMSO
(2.0 ml) and KCN was added (38 mg, 0.58 mmol). The solution was
stirred at room temperature for 45 min. After disappearance of the
starting material, water (3 ml) was added and the aqueous phase
was extracted with AcOEt (3 ꢂ 5 ml). The organic layer was dried
over anhydrous sodium sulfate and the solvent was evaporated
under reduced pressure. The crude material was purified by column
chromatography (petroleum ether/ethyl acetate 3/7) to give the
product as a white solid (73 mg, 66% yield).
b) The intermediate obtained from the previous step (73 mg,
0.26 mmol) was dissolved in DMF (1.0 ml); NaN3 (270 mg,
4.16 mmol) and NH4Cl (220 mg, 4.16 mmol) were added and the
resulting solution was stirred at 120 ꢀC for 24 h. The mixture was
poured into ice cold water (2 ml) and acidified with 1 N HCl. The
aqueous layer was extracted with AcOEt (3 ꢂ 3 ml); the organic
layer was washed with brine, dried over anhydrous sodium sulfate
and concentrated to give the crude product which was purified by
flash chromatography to yield compound 11 as a white solid
(63 mg, 75% yield).
4.4.15. Synthesis of tert-butyl 2-(5-(3-(2-phenylacetamido)propyl)
isoxazol-3-yl)phenylcarbamate (31)
Prepared following the general procedure for the cycloaddition
reaction using alkyne 19 (0.7 mmol) and chloroxime 30.
Compound 31: crystallized from 2-propanol as pale yellow
prisms; mp 145e146 ꢀC; yield 65%; Rf ¼ 0.22 (cyclohexane/ethyl
acetate 7/3). 1H NMR (CDCl3): 1.51 (s, 9H); 1.60e1.80 (m, 4H);
2.35e2.45 (m, 2H); 2.65e2.80 (m, 2H); 6.38 (s, 1H); 7.03e7.15 (m,
2H); 7.23 (bs, 1H); 7.28e7.43 (m, 3H); 7.47e7.55 (m, 3H); 8.42 (d,
J ¼ 8.5, 1H); 9.68 (bs, 1H). 13C NMR (CDCl3): 25.05, 26.60, 27.22,
28.60, 37.30, 80.58, 100.50, 116.01, 119.78, 119.98, 122.20, 124.56,
129.09, 129.27, 130.86, 137.99, 138.14, 153.45, 162.65, 170.70, 172.80.
Anal. Calcd for C25H29N3O4 (435.22): C, 68.95; H, 6.71; N, 9.65.
Found: C, 69.18; H, 6.89; N, 9.78.
Compound 11: crystallized from 2-propanol as white prisms;
mp > 142 ꢀC dec; Rf ¼ 0.25 (cyclohexane/ethyl acetate 7:3). 1H NMR
(CDCl3): 1.75e1.85 (m, 4H); 2.35e2.45 (m, 2H); 2.75e2.85 (m, 2H);
4.38 (s, 2H); 6.06 (s, 1H); 7.02 (t, J ¼ 7.7, 1H); 7.18 (bs, 1H); 7.32 (t,
J ¼ 7.7, 2H); 7.51 (d, J ¼ 7.7, 2H). 13C NMR (CDCl3): 24.98, 26.79, 27.15,
37.27, 45.96, 100.46, 119.97, 124.57, 129.27, 137.99, 159.38, 163.80,
170.72, 174.44. Anal. Calcd for C16H18N6O2 (326.35): C, 58.88; H,
5.56; N, 25.75. Found: C, 59.00; H, 5.66; N, 25.80.
4.4.16. Synthesis of 5-(3-(2-aminophenyl)isoxazol-5-yl)-N-
phenylpentanamide (13)
Compound 31 (196 mg, 0.45 mmol) was treated with a 30%
CH2Cl2 solution (0.8 ml) of trifluoroacetic acid at 0 ꢀC. The solu-
tion was stirred at room temperature for 2 h until disappearance
of the starting material. The volatiles were removed under
vacuum and the residue was purified by flash chromatography
(cyclohexane/ethyl acetate 7/3) to give compound 13 (136 mg,
90% yield).
Compound 13: crystallized from 2-propanol as white prisms;
mp 112e113 ꢀC; Rf ¼ 0.19 (cyclohexane/ethyl acetate 7/3). 1H NMR
(CDCl3): 1.80e1.95 (m, 4H); 2.35e2.45 (m, 2H); 2.80e2.93 (m, 2H);
5.42 (bs, 2H); 6.37 (s, 1H); 6.70e6.80 (m, 2H); 7.10 (t, J ¼ 7.4, 1H);
7.15 (bs, 1H); 7.16e7.21 (m, 2H); 7.28e7.36 (t, J ¼ 8.2, 2H); 7.42 (t,
J ¼ 7.7,1H); 7.52 (d, J ¼ 7.7,1H). 13C NMR (CDCl3): 25.09, 26.58, 27.28,
37.37, 99.85, 112.06, 116.65, 117.23, 119.98, 124.54, 129.26, 129.40,
130.73, 137.99, 146.21, 163.27, 170.80, 171.92. Anal. Calcd for
C20H21N3O2 (335.16): C, 71.62; H, 6.31; N, 12.53. Found: C, 71.78; H,
6.56; N, 12.68.
4.4.13. Synthesis of 5-(3-(2-hydroxyphenyl)isoxazol-5-yl)-N-
phenylpentanamide (12)
Prepared following the general procedure for the cycloaddition
reaction, using alkyne 19 (1.0 mmol) and chloroxime 29.
Compound 12: crystallized from 2-propanol as pale yellow
prisms; mp 124e127 ꢀC; yield 56%; Rf ¼ 0.24 (cyclohexane/ethyl
acetate 7:3). 1H NMR (CDCl3): 1.80e1.95 (m, 4H); 2.35e2.45 (m,
2H); 2.83e2.95 (m, 2H); 6.43 (s, 1H); 6.95 (t, J ¼ 7.7, 1H); 7.08 (t,
J ¼ 7.7, 2H); 7.14 (bs, 1H); 7.38e7.48 (m, 4H); 7.44e7.55 (m, 2H);
9.55 (s, 1H). 13C NMR (CDCl3): 25.01, 26.58, 27.20, 37.27, 99.03,
113.62, 117.66, 119.91, 119.98, 124.59, 128.12, 129.27, 131.70, 137.95,
156.82, 162.78, 170.66, 172.79. Anal. Calcd for C20H20N2O3 (336.15):
C, 71.41; H, 5.99; N, 8.33. Found: C, 71.56; H, 6.08; N, 8.47.
4.5. Histone deacetylase assay
4.4.14. Synthesis of tert-butyl 2-[chloro(hydroxyimino)methyl]
phenylcarbamate (30)
The in vitro activity of HDAC inhibitors was assayed using
a BIOMOL Kit AK-500, according to the instructions from the
manufacturer (Biomolecular Research Laboratories). The assay is
automated and performed by a Tecan Freedom EVOÒ work station.
a) A solution of NaOAc (328 mg, 4.0 mmol in 2.0 ml of H2O) was
added to a stirred solution of tert-butyl 2-formylphenylcarbamate
28 (442 mg, 2.0 mmol), prepared following a literature procedure
[27], and NH2OH$HCl (278 mg, 4.0 mmol) in 80% aqueous EtOH
(5.0 ml). After refluxing for 3 h, the reaction mixture was diluted
with water (2 ml) and then cooled to 0 ꢀC. The solid obtained was
filtered off, washed with H2O, and recrystallized from EtOH to yield
the oxime (429 mg, 91% yield).
On detail, 15
l), was diluted to 50
inhibitor and the substrate (lysine with acetylated amino group on
the side chain) at a concentration of 100 M.
m
l of 30 ꢂ diluted nuclear fraction of HeLa cells (9
mg/
m
m
l with the assay buffer containing the HDAC
m
The samples were incubated for 15 min at room temperature (RT)
and then exposed to a developer (10 min at RT). In this last step
afluorophorewasproduced,whosefluorescencewasmeasuredusing
an excitation wavelength of 355 nm and an emission at 460 nm.
b) To a solution of the oxime (429 mg, 1.8 mmol), obtained from
the previous step, in CH2Cl2 (2.0 ml) pyridine (16 ml, 0.2 mmol) was

4338
P. Conti et al. / European Journal of Medicinal Chemistry 45 (2010) 4331e4338
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4.6. Determination of IC50 at HDAC 1, 2, 3, 6 and 10
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Compounds 2 and 4 were tested by Reaction Biology Corpora-
tion, One Great Valley Parkway, Suite 8, Malvern, PA 19355 (www.
reactionbiology.com).
Compounds were tested in 10-dose IC50 mode in duplicate with
3-fold serial dilution starting at 100
p53 residues 379e382 (RHKKAc) was used as general substrate.
m
M. Fluorogenic peptide from
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