4,5,6,7-Tetrahydro-isoxazolo-[4,5-c]-pyridines as a new class of cytotoxic Hsp90 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.01.056

Hsp90 is considered an interesting therapeutic target for anticancer drug development. Here we describe a new class of 4,5,6,7-tetrahydro-isoxazolo-[4,5- c]-pyridine compounds. A small library of derivatives has been synthesized and investigated. Some reported compounds show interesting properties combining both notable binding to Hsp90 and potent cell growth inhibitory activity. N-5 substitution with a 2,4 resorcinol carboxamide appears crucial for activity. Moreover, a derivative bearing a hydroxamic acid residue bound to C-3 amide portion was found to inhibit both Hsp90 and HDAC6.

Full text of DOI:10.1016/j.ejmech.2014.01.056

European Journal of Medicinal Chemistry 76 (2014) 53e60
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
4,5,6,7-Tetrahydro-isoxazolo-[4,5-c]-pyridines as a new class of
cytotoxic Hsp90 inhibitors
,
a
a
Riccardo Baruchello a, Daniele Simoni a , Paolo Marchetti , Riccardo Rondanin ,
*
Stefania Mangiola a, Cristiana Costantini a, Maria Meli b, Giuseppe Giannini c
Loredana Vesci c, Valeria Carollo c, Tiziana Brunetti c, Gianfranco Battistuzzi c,
,
,
**
,
1
Manlio Tolomeo d, Walter Cabri c
a Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Ferrara, I-44121 Ferrara, Italy
b Dipartimento di Scienze per la Promozione della Salute e Materno Infantile, Sezione di Farmacologia, Università di Palermo, Italy
c R&D Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., I-00040 Pomezia, Roma, Italy
d Centro Interdipartimentale di Ricerca in Oncologia Clinica e Dipartimento Biomedico di Medicina Interna e Specialistica, Sezione di Malattie Infettive,
Università di Palermo, Palermo, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Hsp90 is considered an interesting therapeutic target for anticancer drug development. Here we describe
a new class of 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine compounds. A small library of derivatives
has been synthesized and investigated. Some reported compounds show interesting properties
combining both notable binding to Hsp90 and potent cell growth inhibitory activity. N-5 substitution
Received 5 July 2013
Received in revised form
24 January 2014
Accepted 28 January 2014
Available online 30 January 2014
with
a 2,4 resorcinol carboxamide appears crucial for activity. Moreover, a derivative bearing
a hydroxamic acid residue bound to C-3 amide portion was found to inhibit both Hsp90 and HDAC6.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Heat shock protein 90
Anti-cancer drugs
4,5,6,7-Tetrahydro-isoxazolo-[4,5-c]-
pyridines
1. Introduction
Hsp90 is normally expressed in healthy cells representing the
1e2% of the total intracellular proteins, from which about 3% is
Heat shock protein 90 (Hsp90) is a molecular chaperone protein
that plays a key role in the conformational maturation, stability and
function of so-called “client” proteins [1,2]. Many Hsp90 client
proteins are over-expressed in cancer, often in mutated forms, and
are responsible for unrestricted cell proliferation and survival. In-
hibition of the ATPase activity of Hsp90 disrupts an ongoing
“folding” cycle, involving multiple co-chaperone proteins and, in
turn, leads to destabilization, ubiquitination, and ultimately
proteasome-mediated degradation of client proteins, causing loss
of function and inhibition of cell growth [3,4].
found in the nucleus with effects on the regulation of several nu-
clear events. In case of tumor tissues, however, its levels increase up
to 4e6% of the whole proteomic load of the cell [5,6]. Interestingly,
Hsp90 derived from tumor cells has particularly strong ATPase
activity with higher binding affinity to inhibitors than the latent
form in normal cells, allowing a possible selective targeting to tu-
mor tissues [7].
Hsp90 has thus emerged as an important target in several dis-
eases [8e11]. In particular, Hsp90 is considered an intriguing
therapeutic target for anticancer drug development because its
single inhibition represents an attack on multiple hallmark traits of
cancer.
A number of highly specific Hsp90 inhibitors have been identi-
fied [12]. They redirect Hsp90 chaperoning activity and decrease
cellular levels of its numerous cancer-related client proteins [6].
Such inhibitors exhibit promising antitumor activity as single
agents or in combination with other cytotoxic agents, and some of
them are in clinical development [8,13,14].
* Corresponding author. Dip. di Scienze Chimiche e Farmaceutiche, Università di
Ferrara, Via Fossato di Mortara 17, I-44121 Ferrara, Italy.
** Corresponding author. R&D Sigma-Tau S.p.A., Via Pontina, Km 30.400, I-00040
Pomezia, Roma, Italy.
E-mail addresses: smd@unife.it (D. Simoni), giuseppe.giannini@sigma-tau.it
(G. Giannini).
1
Present address: Indena, S.p.A., Viale Ortles 12, I-20139 Milano, Italy.
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.056

54
R. Baruchello et al. / European Journal of Medicinal Chemistry 76 (2014) 53e60
Table 1
The first-in-class Hsp90 inhibitor to enter clinical trials was the
Binding on Hsp90 (FP Assay) and cytotoxicity on NCI-H460 non-small cell lung
carcinoma cells.
17-allylamino analogue of geldanamycin (17-AAG, 1, Fig. 1) [15].
However, despite a high in vitro activity, its interest was shadowed
by poor water solubility, coupled to hepatotoxic side effects. Some
of these problems have been partially solved by the discovery of 17-
Compound
NCI-H460 (IC50
;
m
M)
Hsp90 (FP) (IC50; mM)
5
0.0024
>1
>20
>20
0.87
9.7
>20
0.45
>1
0.061
>100
0.16
3.9
0.1
0.11
1.2
0.029
>100
>100
1.4
11
13
14
15
17
18
19
20
21
22
23
25
26
27
29
dimethylaminoethylamino-17-demethoxygeldanamycin
(17-
DMAG, 2) derivative. Radicicol (3), a natural macrocyclic anti-
fungal antibiotic, was found to inhibit Hsp90 protein by interact-
ing with the same site of action of geldanamycin [16]. However, due
to its intrinsic chemical instability, it is devoid of in vivo activity.
Some synthetic compounds containing the isoxazole nucleus
(4e6) have shown potent and selective inhibition of Hsp90 [17e
20]. The presence of the heterocyclic nucleus seems to exert a key
role in the docking of these compounds to the ATP-binding site of
Hsp90 [21].
In this context, the 4,5-diarylisoxazole scaffold demonstrated of
remarkable importance, recently leading to 5, currently in Phase II
clinical trials [17]. Additionally, the benzoisoxazole derivative 4,
having a resorcinol moiety in position 3, has also been disclosed as
potent Hsp90 inhibitor [18].
However, to date, no Hsp90 inhibitors in clinical trials fully
satisfy requisites of safety and stability. Therefore, finding further
potent and selective Hsp90 inhibitors still remains an interesting
and promising goal [22].
Recently, a structural investigation on the isoxazole scaffold led
us to the discovery of 3,4-isoxazolediamide compounds, with a
nitrogen atom directly attached to the C-4 heterocycle ring,
endowed with potent Hsp90 inhibitory properties. Several de-
rivatives from this series combine potent binding and cell growth
inhibitory activities. In vivo studies proved also important anti-
tumor effect of the morpholine-bearing derivative 6 against human
epidermoid carcinoma A431 [20].
>1
>20
0.23
0.19
0.9
0.2
3.9
e
0.054
0.068
0.034
0.033
tetrahydroisoquinoline), have been thus considered in order to
build a novel similar series of potential Hsp90 inhibitors.
So, herein we will describe the synthesis and Hsp90 inhibitor
activity of a new class of 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyr-
idine compounds 7. A small library of different N-5 amide de-
rivatives were synthesized and investigated. The resorcinol portion
was firstly considered of importance by virtue of its role in the
interaction with the Hsp90 protein either in radicicol and other
synthetic series of compounds [20,23e26]. However, structural
alterations and substitution of the resorcinol group was also
investigated, as well as different N-substitutions at the C-3 amide
portion.
Some of the synthesized compounds showed interesting activ-
ity combining both notable binding properties and potent cell
growth inhibitory activity.
In the meantime, considering the efforts and the interest
recently devoted by some of us into HDAC inhibitors field [27e29],
we envisaged a strategy taking into consideration the key role of
We envisaged that isoxazole structure could constitute a portion
able to contribute to a high activity in various scaffolds, also taking
into consideration other derivatives from previous literature.
Structures described in recent papers [23e25], containing
condensed
bicyclic
groups
(as
isoindoline
or
O
O
R
O
H
O
O
O
O
N
H
N
H
O
N
HO
N
O
H
O
OH
Cl
HO
O
OH
Cl
OCONH2
R = NHCH2CH=CH2
OH
1 17-AAG
3 Radicicol
4
2 17-DMAG R = NHCH2CH2NMe2
O
O
NHEt
NHEt
N
N
O
O
N
O
O
CO-Y
NH
O
N
N
O
HO
HO
N
Z
N
O
OH
OH
6
7
5 NVP-AUY922
Fig. 1. Known Hsp90 inhibitors (1e6) and the new scaffold 7. Distinct Y and Z refer to compounds detailed in Scheme 1.

R. Baruchello et al. / European Journal of Medicinal Chemistry 76 (2014) 53e60
55
Hsp90 deacetylation performed by HDAC6 as a crucial step to
ensure its chaperon activity [30e32]. In the present series, various
C-3 amide N-substitutions demonstrated to maintain appreciable
binding and growth inhibition activity, so we tried to add in this
position a long chain hydroxamic acid residue (derivative 29) in
order to possibly obtain a Hsp90/HDAC dual inhibitor.
the latter with the opportune carboxylic acid in the presence of 1-
hydroxybenzotriazole (HOBt) and N0-(3-dimethylaminopropyl)-N-
ethyl-carbodiimide hydrochloride (EDC) in DMF gave easily the
desired 13e15. Alternatively, the amine 12 was reacted with eth-
ylamine in methanol at reflux to give in good yield the ethyl amide
16. The desired amides 17e19 were obtained by condensation of 16
with the proper carboxylic acid in the same manner as described
previously for compounds 13e15. Otherwise, compound 16 was
reacted with opportune (commercial or synthesized from aryl-
amine precursor) isocyanate to give ureas 20 and 21. The methylene
derivative 22 was obtained by the selective reduction of 17a (the
protected 2,4 dibenzylated analog of 17) using LiAlH4 in THF;
whereas 23 was simply obtained by reacting 19 with acetic anhy-
dride in DCM solution.
2. Chemistry
The synthetic route used for the preparation of the esters 13e15
and amides 17e21 (Table 1) is shown in Scheme 1. The 1,3-dipolar
cycloaddition of nitriloxide, generated in situ by treating the methyl
chlorooximineacetate with triethylamine in dichloromethane
(DCM), to the pyrrolidine-enamine derivative 9, is a known reaction
that has been exploited for the synthesis of isoxazole-fused piper-
idine derivatives [33]. The enamine 9 was obtained from the N-
acetyl-piperidone 8 and pyrrolidine in toluene at reflux.
Finally, derivatives 25e28 were prepared by reaction of the
carboxylic acid derivative 24, in turn obtained from the piperidine
12
by
condensation
with
the
bis-2,4-benzyloxyi-5-
isopropylbenzoic acid, with appropriate amine using benzo-
triazolyl-1-oxy-tripyrrolidino-phosphonium hexafluorophosphate
(PyBOP) as condensing agent and diisopropylethylamine (DIPEA).
Hydroxamic derivative 29 was quantitatively prepared starting
The isoxazoline intermediate 10 was treated with trifluoroacetic
acid (TFA) in DCM at reflux for 8 h affording the 4,5,6,7-tetrahydro-
isoxazole-[4,5-c]-pyridine derivative 11, in turn hydrolyzed with
32% hydrochloric acid to give the amine ethyl ester 12. Reaction of
N
N
O
O
N
N
N
9
O
N
O
COOMe
COOMe
b
a
c
d
N
N
O
O
O
11
8
10
N
N
O
O
N
O
COOH
OBn
CONH-R
OH
COOEt
e,k
m,n
N
N
N
x HCl
H
O
O
12
BnO
HO
24
25: R = (CH2)2-4-morpholine
26: R = cyclopentyl
e
f
27: R = (CH2)2-Cl
28: R = (CH2)6-COOMe
29: R = (CH2)6-CONHOH
o
N
N
N
O
O
O
COOEt
CONHEt
CONHEt
e
h,n
i
or g
(comp. 20 and 21)
N
N
N
X
22
23
17a
19
H
R1
R1
O
16
HO
OH
R2O
OR2
17: X = CO, R1 = Cl, R2 = H; 17a: R2 = Benzyl
18: X = CO, R1 =H, R2 = H
13: R1 = Cl
14: R1 = H
19: X = CO, R1 = CH(CH3)2, R2 = H
20: X = CONH, R1 = Cl, R2 = H
15: R1 = CH(CH3)2
21: X = CONH, R1 = CH(CH3)2, R2 = H
Scheme 1. Reagents and conditions. a) pyrrolidine, p-TosOH, rfx; b) 2-chlorohydroxyimino acetic acid methyl ester, TEA, DCM; c) TFA, DCM, rfx; d) HCl 32%, EtOH, rfx; e) HOBt, WSC,
opportune acid, DMF; f) EtNH2 in MeOH, rfx; g) Step 1: 5-chloro-2,4-dimethoxyphenyl isocyanate, 1,4 dioxan; step 2: BBr3 1 M in DCM, for comp. 20; step 1: 2,4-bis-(benzyloxy)-5-
isopropylaniline, trichloromethyl chloroformate, 1,4 dioxan, then 16; step 2: BCl3 1 M in DCM for comp. 21; h) LiAlH4, THF; i) Ac2O, DMF; k) LiOH, THF/H2O; m) PyBOP, DIPEA,
opportune amine; n) BCl3 1 M in DCM; o) NH2OH, MeOH, NaOHaq.

56
R. Baruchello et al. / European Journal of Medicinal Chemistry 76 (2014) 53e60
from the corresponding methyl ester intermediate 28 by reaction
with hydroxylamine in MeOH in the presence of aq. NaOH [28].
Removal of benzyl or methyl protecting groups at phenolic
functions were routinely conducted with good yields using boron
trichloride or boron tribromide, respectively, in DCM.
3. Biological results and discussion
Measurements of binding by a fluorescence polarization (FP)
assay and inhibition of cell growth proliferation of NCI-H460 tumor
cell line were determined for all the synthesized compounds. Flow
cytometric analysis of cell cycle was also determined for two
representative compounds (17 and 19). Among the group of esters
13e15, the isopropyl resorcinol derivative 15 was the most potent
compound in terms of antiproliferative activity and affinity to
HSP90a (Table 1); whereas, compounds 13 and 14, despite retain-
ing some binding ability, resulted devoid of cellular activity. Among
the group of the ethylamides 17e23, compound 19, along with an
important binding property, showed a notable antiproliferative
activity. Otherwise, the chlororesorcinol derivative 17 and com-
pound 18 resulted devoid of cytotoxicity. Quite surprisingly, com-
pounds 20 and 21, where the N-5 amide portion was elaborated
into an ureidic moiety resulted with no activity. Detrimental for
activity resulted also the reduction of the N-5 amide moiety in
compound 22.
Compound 25e27,29 modified at the C-3 amide portion were
synthesized and screened to better substantiate our structuree
activity investigation. Interestingly, the morpholine water solubi-
lizing group in compound 25 was well tolerated retaining good
binding and cell growth inhibiting properties. The cyclopentyl and
chloroethyl derivatives 26 and 27 as well as the hydroxamic de-
rivative 29 also retained binding and cell growth activity. Of in-
terest, compounds 25 and 27 showed stronger antiproliferative
activity than the starting ethyl amide derivative 19 indicating that
the C-3 amide region could be of some importance in modulating
the activity of this class of compounds. Of some interest resulted
also the ester derivative 23, a diacetyl prodrug of 19, whose cellular
activity result increased as compared to 19 itself notwithstanding
the drop of binding capability. Additionally, cell viability, apoptosis
and flow cytometric analysis of cell cycle was determined for
compounds 17 and 19 in comparison with 17-AAG. 17 and 19 eli-
cited a concentration-dependent growth inhibitory effect in K562
Fig. 2. Cell cycle distribution of K562 cells exposed to the Hsp90 inhibitors. The cells
were exposed to the drugs for 24 h and cell cycle was evaluated by flow cytometry. (a)
control untreated cells, (b) 17-AAG 1
mM, (c) 17 75 mM, (d) 19 10 mM. The data are
representative of three separate experiments.
Finally, the compound 29 was investigated for the HDAC activity
by measuring its potency in inhibiting 10 isolated human HDAC
isozymes in the presence of a fluorogenic peptide bound to the
RHKK(Ac), fragment of p53 (residues 379e392), and RHK(Ac)K(Ac)
for HDAC8, as the substrate, and using SAHA as the reference
compound (Table 3) [35].
In addition to being very active against Hsp90, in the HDAC
profile 29 also showed a high selectivity towards the isoform
HDAC6. This compound represents an interesting hit compound for
further studies in the dual targeting HDAC/Hsp90 inhibition
approach.
4. Conclusions
In summary, a novel class of 4,5,6,7-tetrahydro-isoxazolo-[4,5-
c]-pyridine derivatives has been synthesized and tested for their
affinity to Hsp90 and for their antiproliferative activity.
A group of compounds 13, 15, 17, 19, 22, 25e27, 29 showed
binding affinity at micromolar/nanomolar level. Compounds 15, 19,
23, 26e27, 29 showed also cytotoxic activity in sub-micromolar
range (200e900 nM). The flow cytometric analysis showed that
both 17 and 19 determined an increase in the proportion of cells in
the G1 phase of the cell cycle with a strong reduction of the S phase
fraction (Fig. 2). The effect was in agreement with that previously
reported for Hsp90 inhibitors. Attempts to investigate structural
alterations of the resorcinol portion, as well as of the N-5 amide
moiety had only deleterious effect on activity. Conversely, struc-
tural alteration of the C-3 amide seems of importance in modu-
lating the activity of the compounds.
cells with an IC50 of 32.6 and 0.72
exposure (Table 2).
mM respectively after 72 h drug
Furthermore, the flow cytometric analysis showed that both 17
and 19 determined an increase in the proportion of cells in the G1
phase of the cell cycle with a strong reduction of the S phase
fraction (Fig. 2). This effect was similar to that observed in the cells
exposed to reference compound 17-AAG (1) 1
mM and was in
agreement with previous reports that demonstrate that although
Hsp90 inhibitors can affect cell cycle progression at multiple levels,
a G1 arrest is seen in the majority of cell lines, provided that an
intact Rb pathway is present [34].
Table 3
Table 2
In vitro inhibitory activity (IC50; nM) of 29 vs. SAHA against isoforms HDAC1e8, 10e
Cytotoxic and apoptotic effects of the Hsp90 inhibitors 17 and 19 in K562 cells.
11.a
IC50
(
m
M)
AC50
(
m
M)
HDAC isoform
1
2
3
4
5
6
7
8*
10 11
17
19
17-AAG
32.6
0.72
0.15
>100
5.4
3.1
29
SAHA
2030 9490 4230 75900 1580 4.1 2510 1760 886 521
258 921 350 493 378 28.9 344 243 456 362
a
Compounds were tested in 10-dose IC50 mode, in triplicate with 3-fold serial
M solutions. Isolated human HDAC isozymes were used in
The cells were exposed to the drugs for 72 h. Cytotoxicity was evaluated by the
Trypan blue dye exclusion test, while apoptosis was measured by fluorescence
microscopy IC50: growth inhibitory concentration inducing 50% apoptotic cell death.
AC50: concentration inducing 50% apoptotic cell death.
dilution starting from 10
m
presence of a fluorogenic peptide bound to the RHKK(Ac) fragment, except for
HDAC8*: where was used the diacetyl RHK(Ac)K(Ac), as substrate. Values are the
means of three experiments and are given in nanomolar.

R. Baruchello et al. / European Journal of Medicinal Chemistry 76 (2014) 53e60
57
5. Experimental part
(400 MHz, CDCl3)
3.05 (t, J ¼ 5.6 Hz, 2H), 3.16 (sept, 1H), 3.93e3.95 (m, 2H), 4.39e4.44
(m, 2H), 4.80 (s, 2H), 6.44 (s, 1H), 7.12 (s, 1H). 13C NMR (CDCl3)
d:
d
1.21 (d, J ¼ 6.8 Hz, 6H), 1.38 (t, J ¼ 7.2 Hz, 3H),
5.1. Chemistry
14.2, 22.8, 23.7, 26.4, 62.4, 104.2, 108.4, 110.3, 111.9, 126.7, 152.4,
Reagents were purchased from commercial suppliers and used
without further purification. 1H NMR spectra were recorded, unless
otherwise indicated, in DMSO solution at 200 MHz on a Bruker AC-
200, 300 MHz on a Varian Gemini-300, 400 MHz on a Varian
Mercury Plus 400 and 500 MHz on a Varian Gemini-500 spec-
trometer, and peak positions are given in parts per million down-
field from tetramethylsilane as the internal standard. J values are
expressed in hertz. Electrospray mass spectra were recorded on a
Waters Micromass ZQ-2000 instrument or a double-focusing Fin-
nigan MAT 95 instrument with BE geometry. Thin layer chroma-
tography (TLC) was carried out using Merck precoated silica gel F-
254 plates. Flash chromatography was done using Merck silica gel
60 (0.063e0.200 mm). Solvents were dried according to standard
procedures, and reactions requiring anhydrous conditions were
performed under argon. Solutions containing the final products
were dried with Na2SO4, filtered, and concentrated under reduced
pressure using a rotatory evaporator.
157.9, 159.9, 168.8, 173.3. MS (ESI): m/z 375.3 [MþH]þ.
5.1.2.4. 5-(5-Chloro-2,4-dihydroxybenzoyl)-N-ethyl-4,5,6,7-
tetrahydroisoxazole[4,5-c]pyridine-3-carboxamide (17). From 16
and 5-chloro-2,4-dihydroxybenzoic acid. (Yield: 61%) 1H NMR
(200 MHz, DMSO, 120 ꢁC)
d
1.14 (t, J ¼ 7.2 Hz, 3H), 2.89 (t, J ¼ 5.8 Hz,
2H), 3.26e3.36 (m, 2H), 3.76 (t, J ¼ 5.8 Hz, 2H), 4.55 (s, 2H), 6.51 (s,
1H), 7.09 (s, 1H), 8.19 (br, 1H). MS (ESI): m/z 365.9 [MþH]þ.
5 .1. 2 . 5 . 5 - ( 2 , 4 - D i h y d ro x y - b e n z o yl ) - N - e t hyl - 4 , 5 , 6 , 7 -
tetrahydroisoxazole[4,5-c]pyridine-3-carboxamide (18). From 16
and 2,4-dihydroxybenzoic acid. (Yield: 68%) 1H NMR (400 MHz,
CDCl3)
d
1.23 (t, J ¼ 7.2 Hz, 3H), 3.01e3.04 (m, 2H), 3.43e3.47 (m,
2H), 3.93e3.96 (m, 2H), 4.92 (s, 2H), 6.35 (dd, J ¼ 8.4 Hz, J ¼ 2.4 Hz,
1H), 6.44 (d, J ¼ 2.4 Hz, 1H), 7.26 (d, J ¼ 8.4 Hz, 1H). 13C NMR (CDCl3)
d
: 14.5, 23.3, 34.6, 40.8, 44.7, 104.2, 107.1, 108.5, 111.4, 130.2, 153.6,
159.4, 161.1, 162.1, 169.0, 173.1. MS (ESI): m/z 331.7 [MþH]þ.
5.1.1. N-Ethyl-4,5,6,7-tetrahydroisoxazole[4,5-c]pyridine-3-
carboxamide (16)
Compound 12 (4.3 mmol, 1 g) was suspended in ethylamine 2 M
in MeOH solution (51.7 mmol) and stirred for 12 h. The solution was
concentrated in vacuo to give a residue which was purified by flash
chromatography (DCM/MeOH 9.5/0.5). Yield: 65%. 1H NMR
5.1.2.6. 5-(2,4-Dihydroxy-5-isopropylbenzoyl)-N-ethyl-4,5,6,7-
tetrahydroisoxazole[4,5-c]pyridine-3-carboxamide (19). From 16
and 5-isopropyl-2,4-dihydroxybenzoic acid. (Yield: 68%) 1H NMR
(400 MHz, DMSO)
3.09 (m, 1H), 3.23 (t, J ¼ 5.6 Hz, 2H), 3.6 (br, 2H), 4.53 (s, 2H), 6.38 (s,
d
1.06e1.10 (m, 9H), 2.88 (t, J ¼ 5.6 Hz, 2H), 3.03e
1H), 6.88 (s, 1H), 8.77 (br, 1H), 9.55 (s, 1H), 9.57 (s, 1H). 13C NMR
(400 MHz, CDCl3)
3.19 (m, 2H), 3.42e3.49 (m, 2H), 4.06 (s, 2H), 6.75 (br, 1H). MS (ESI):
m/z 196.1 [MþH]þ.
d
1.23 (t, J ¼ 7.2 Hz, 3H), 2.80e2.83 (m, 2H), 3.16e
(DMSO) d: 14.8, 23.0, 23.1, 24.5, 27.6, 35.2, 103.3, 112.5, 114.1, 127.4,
128.2, 154.9, 155.6, 159.0, 161.2, 169.9, 172.8. MS (ESI): m/z 374.1
[MþH]þ.
5.1.2. General procedure for the N-acylation of 4,5,6,7-
5.1.3. N5-(5-Chloro-2,4-dihydroxyphenyl)-N3-ethyl-6,7-
tetrahydroisoxazole[4,5-c]pyridines derivatives 12 and 16
dihydroisoxazolo[4,5-c]pyridine-3,5(4H)-dicarboxamide (20)
A mixture of the amine 12 or 16 (0.56 mmol), EDC ꢀ HCl
(0.67 mmol, 128 mg), HOBt (0.67 mmol, 90,5 mg), TEA (1.7 mmol,
171 mg, 0.24 mL) and the opportune acid (0.56 mmol) in 10 mL of
DMF was stirred at room temperature overnight and then the
solvent was removed in vacuo. The residue was partitioned be-
tween AcOEt (15 mL) and 5% aq. citric acid (10 mL), the organic
layer was separated and washed successively with saturated so-
dium bicarbonate solution (10 mL), brine (5 mL), dried over Na2SO4,
filtered and evaporated in vacuo. The crude material was purified
by flash chromatography (DCM/MeOH 9.5:0.5) to give the desired
compound (13e15, 17e19).
A
solution of 5-chloro-2,4-dimethoxyphenyl isocyanate
(0.28 mmol, 55 mg) and amine 16 (0.28 mmol, 60 mg) in dry
dioxane (10 mL) was heated at 60 ꢁC overnight. The reaction was
cooled to room temperature, the solvent was removed in vacuo and
the obtained residue was purified by flash chromatography on silica
gel
dimethoxyphenyl)-N3-ethyl-6,7-dihydroisoxazolo[4,5-c]pyridine-
3,5(4H)-dicarboxamide. Yield 73%. 1H NMR (CDCl3, 400 MHz)
1.25
(t, J ¼ 7.2 Hz, 3H), 2.92e2.95 (m, 2H), 3.45e3.50 (m, 2H), 3.85e3.89
(m, 8H), 4.65 (s, 2H), 6.50 (s, 1H), 6.85 (br, 2H), 8.02 (s 1H). MS (ESI):
m/z 408.9e410.1 [MþH]þ.
(hexane/AcOEt
3/7)
to
give
N5-(5-chloro-2,4-
d
To a solution of this compound (0.12 mmol, 47 mg) in dry DCM
(10 mL), cooled to ꢂ78 ꢁC under argon, was added BBr3 (1 M in
DCM, 0.6 mmol, 0.6 mL) and stirring was continued for 30 min at
the same temperature. The reaction was allowed to warm to rt and
stirred for another 2 h, then saturated NaHCO3 (5 mL) aqueous
solution was added and DCM was removed in vacuo. The aqueous
residue was extracted with AcOEt (3 ꢀ 10 mL), and the combined
organic phases were washed with brine and dried. The crude res-
idue obtained after evaporation was purified by flash chromatog-
raphy on silica gel (DCM/MeOH 97/3). Yield 49%. 1H NMR (DMSO,
5.1.2.1. Ethyl 5-(5-chloro-2,4-dihydroxybenzoyl)-4,5,6,7-
tetrahydroisoxazole[4,5-c]pyridine-3-carboxylate (13). From 12 and
5-chloro-2,4-dihydroxybenzoic acid. (Yield: 72%) 1H NMR
(200 MHz, DMSO, 120 ꢁC)
d
1.31 (t, J ¼ 7 Hz, 3H), 2.89e2.95 (m, 2H),
3.77 (t, J ¼ 5.6 Hz, 2H), 4.37 (q, J ¼ 7 Hz, 2H), 4.57 (s, 2H), 6.57 (s,1H),
7.11 (s, 1H). MS (ESI): m/z 366.9 [MþH]þ.
5.1.2.2. Ethyl 5-(2,4-dihydroxybenzoyl)-4,5,6,7-tetrahydroisoxazole
[4,5-c]pyridine-3-carboxylate
dihydroxybenzoic acid. (Yield: 40%) 1H NMR (200 MHz, DMSO,
120 ꢁC)
(14). From
12
and
2,4-
400 MHz)
2H), 3.75e3.77 (m, 2H), 4.53 (s, 2H), 6.45 (s, 1H), 7.21 (s, 1H), 8.05 (s
1H), 8.80 (br, 1H), 9.61 (s, 1H), 9.75 (s, 1H). 13C NMR (DMSO)
: 14.5,
d
1.11 (t, J ¼ 7.2 Hz, 3H), 2.88e2.91 (m, 2H), 3.25e3.28 (m,
d
: 1.30 (t, J ¼ 7 Hz, 3H), 2.74 (s, 2H), 3.77 (t, J ¼ 5.6 Hz, 2H),
4.35 (q, J ¼ 7 Hz, 2H), 4.57 (s, 2H), 6.28e6.34 (m, 2H), 7.01 (d,
d
J ¼ 8.2 Hz, 1H), 7.95 (br, 1H), 9.22 (br, 1H), 9.37 (br, 1H); 13C NMR
23.1, 33.6, 104.1, 108.6, 111.3, 119.4, 125.7, 149.8, 150.1, 154.5, 155.9,
(DMSO)
d
: 13.8, 23.3, 61.8, 102.2, 106.8, 111.9, 114.2, 129.4, 129.8,
158.6, 168.6. MS (ESI): m/z 380.5e382.2 [MþH]þ.
152.2, 155.2, 159.4, 159.6, 168.6, 169.3. MS (ESI): m/z 332.7 [MþH]þ.
5.1.4. N5-(2,4-Dihydroxy-5-isopropylphenyl)-N3-ethyl-6,7-
dihydroisoxazolo[4,5-c]pyridine-3,5(4H)-dicarboxamide (21)
To a solution of 2,4-bis-(benzyloxy)-5-isopropylaniline [36]
(0.58 mmol, 200 mg) in dry dioxane (15 mL) was added
5.1.2.3. Ethyl 5-(2,4-dihydroxy-5-isopropylbenzoyl)-4,5,6,7-
tetrahydroisoxazole[4,5-c]pyridine-3-carboxylate (15). From 12 and
5-isopropyl-2,4-dihydroxybenzoic acid. (Yield: 70%) 1H NMR

58
R. Baruchello et al. / European Journal of Medicinal Chemistry 76 (2014) 53e60
trichloromethyl chloroformate (0.29 mmol, 57 mg, 0.036 mL),
DIPEA (1.2 mmol, 154 mg, 0.208 mL) and the mixture was stirred at
60 ꢁC for 4 h. The reaction was cooled to room temperature, 16
(0.58 mmol, 112 mg) was added and then heated at 60 ꢁC overnight.
The dioxane was evaporated and the crude residue was purified by
flash chromatography on silica gel (hexane/ethyl acetate 9/1) to
5.1.7. 5-(2,4-Bis-(benzyloxy)-5-isopropylbenzoyl)-4,5,6,7-
tetrahydroisoxazolo[4,5-c]pyridine-3-carboxylic acid (24)
Compound 12 (0.99 mmol, 373 mg), EDC (0.99 mmol, 175
mL),
HOBt (0.99 mmol, 134 mg) and DIPEA (1.98 mmol, 344
mL) were
added to a solution of 2,4-bis-(benzyloxy)-5- isopropylbenzoic acid
[37] (0.82 mmol, 191 mg) in 5 mL of DMF. The reaction was stirred
overnight at room temperature; then it was poured into water and
extracted with AcOEt. The organic phase was washed with water
and brine, then dried and evaporated. The crude product was pu-
rified by flash chromatography (light petroleum/AcOEt 7/3) to give
the intermediate ethyl 5-(2,4-bis(benzyloxy)-5-isopropylbenzoyl)-
give
dihydroisoxazolo[4,5-c]pyridine-3,5(4H)-dicarboxamide.
52%. 1H NMR (CDCl3, 400 MHz)
1.21e1.28 (m, 9H), 2.86e2.89 (m,
N5-(2,4-bis-(benzyloxy)-5-isopropylphenyl)-N3-ethyl-6,7-
Yield
d
2H), 3.34 (ept, J ¼ 6.8 Hz,1H), 3.44e3.51 (m, 2H), 3.81e3.84 (m, 2H),
4.63 (s, 2H), 4.99 (s, 2H), 5.06 (s, 2H), 6.56 (s, 1H), 6.79 (br, 1H), 6.92
(s 1H), 7.30e7.42 (m, 10H), 7.87 (s, 1H). MS (ESI): m/z 569.1 [MþH]þ.
To a solution of this compound (0.18 mmol, 100 mg) in dry DCM
(10 mL) at ꢂ78 ꢁC was added BCl3 (1 M in DCM, 0.88 mmol,
0.88 mL) and stirring was continued for 30 min at the same tem-
perature. The reaction was allowed to warm to rt and stirred
another hour, saturated NaHCO3 (5 mL) aqueous solution was
added and DCM was removed in vacuo. The aqueous residue was
extracted with AcOEt (3 ꢀ 10 mL), and the combined organic phases
were washed with brine (5 mL) and dried. The crude residue ob-
tained after evaporation was purified by flash chromatography on
silica gel (hexane/AcOEt 1:1). Yield 64%. 1H NMR (DMSO, 400 MHz)
4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-carboxylate.
Yield
1.17 (d, J ¼ 7.0 Hz, 6H),
85%. 1H NMR (200 MHz, DMSO, 120 ꢁC):
d
1.28 (t, J ¼ 7.0 Hz, 3H), 2.79e2.85 (m, 2H), 3.26 (ept, J ¼ 7.0 Hz, 1H),
3.72e3.77 (m, 2H), 4.35 (q, J ¼ 7.0 Hz, 2H), 4.49e4.53 (m, 2H), 5.09
(s, 2H), 5.18 (s, 2H), 6.89 (s, 1H), 7.04 (s, 1H), 7.28e7.48 (m, 10H). MS
(ESI): m/z 555.3 [MþH]þ.
The obtained ester (0.7 mmol, 386 mg) was dissolved in a so-
lution of LiOH$H2O (0.58 mmol, 38 mg) in 15 mL of THF/H2O 3:2.
The reaction mixture was stirred until complete conversion of the
starting material to the corresponding carboxylic salt derivative.
THF was evaporated and the aqueous phase was acidified with 1 N
HCl and extracted with AcOEt (20 mL). The organic phase was dried
and evaporated to give the desired acid, which was used without
further purification.
d
1.08e1.11 (m, 9H), 2.90e2.92 (m, 2H), 3.06 (ept, J ¼ 6.8 Hz, 1H),
3.24e3.29 (m, 2H), 3.76e3.80 (m, 2H), 4.55 (s, 2H), 6.31 (s, 1H), 6.88
(s, 1H), 8.16 (s 1H), 8.79 (br, 1H), 8.95 (s, 1H), 9.07 (s, 1H). MS (ESI):
m/z 388.6 [MþH]þ.
5.1.7.1. 5-(2,4-Dihydroxy-5-isopropyl-benzoyl)-4,5,6,7-tetrahydro-
isoxazolo[4,5-c]pyridine-3-carboxylic acid (2-morpholin-4-yl-ethyl)-
amide (25). To a solution of acid 24 (0.17 mmol, 90 mg) in 5 mL of
5.1.5. 5-(5-Chloro-2,4-dihydroxybenzyl)-N-ethyl-4,5,6,7-
tetrahydroisoxazolo[4,5-c]pyridine-3-carboxamide (22)
DCM, 4-(2-aminoethyl)-morpholine (0.25 mmol, 32
mL), DIPEA
To a suspension of LiAlH4 (0.63 mmol, 100 mg) in dry THF (5 mL)
was added a solution of 5-(5-chloro-2,4-dibenzyloxybenzoyl)-N-
ethyl-4,5,6,7-tetrahydroisoxazole[4,5-c]pyridine-3-carboxamide
(17a) (0.36 mmol, 200 mg) in dry THF (10 mL) over 10 min. The
resulting mixture was stirred for 2 h, cooled in an ice-bath and the
excess of hydride was decomposed by the dropwise addition of
water followed by 15% sodium hydroxide and more water. After
vigorous stirring for another 20 min the mixture was filtered with
suction, the granular precipitate was washed with ethyl acetate.
The filtrate was evaporated and the residue diluted water and
extracted with AcOEt (2 ꢀ 15 mL). The organic phases were com-
bined, dried, concentrated under vacuo and the residue was puri-
fied by flash chromatography (eluent: light petroleum/AcOEt 7/3).
Yield 72%. MS (ESI): m/z 532,4e533,6 [MþH]þ.
(0.5 mmol, 86 L), and PyBOP (0.2 mmol, 106 mg) were added. The
m
reaction was stirred overnight at room temperature; the DCM was
removed in vacuo and water was added. The mixture was then
extracted with AcOEt (3 ꢀ 15 mL). The organic phase was dried over
Na2SO4 to obtain the protected product as white solid. MS (ESI): m/z
661.3 [MþNa]þ.
The desired final compound was obtained by deprotection with
BCl3 1 M in DCM as previously described for compound 21. The
crude product was purified by flash chromatography (DCM/meth-
anol from 98/2 to 96/4) to give 47 mg of the title compound. Yield
62%. 1H NMR (300 MHz, DMSO)
d
1.08 (d, J ¼ 7.0 Hz, 6H), 2.44e2.35
(m, 6H), 2.87 (m, 2H), 3.05 (m, 1H), 3.30 (m, 2H), 3.53 (t, J ¼ 4.5 Hz,
4H), 3.71 (bs, 2H), 4.52 (bs, 2H), 6.36 (s, 1H), 6.87 (s, 1H), 8.58 (t,
J ¼ 5.6 Hz, 1H), 9.55 (s, 2H). MS (ESI): m/z 459.4 [MþH]þ.
70 mg of dibenzyl intermediate were deprotected with BCl3 in
the same way as described for compound 21. Purification by flash
chromatography (DCM/MeOH 95/5). Yield 45%, 1H NMR (400 MHz,
5.1.7.2. 5-(2,4-Dihydroxy-5-isopropyl-benzoyl)-4,5,6,7-tetrahydro-
isoxazolo[4,5-c]pyridine-3-carboxylic acid cyclopentylamide (26).
To a solution of acid 24 (0.17 mmol, 90 mg) in 5 mL of DCM,
CD3OD):
2H), 3.35 (t, J ¼ 7.2 Hz, 2H), 3.69 (s, 2H), 3.81 (s, 2H), 6.38 (s, 1H),
7.05 (s, 1H). 13C NMR (DMSO)
: 14.8, 23.7, 35.2, 57.9, 104.9, 111.6,
d
1.19 (t, J ¼ 7.2 Hz, 3H), 2.88e2.91 (m, 2H), 2.93e2.96 (m,
cyclopentylamine (0.25 mmol, 25 mL), DIPEA (0.25 mmol, 43 mL),
d
and PyBOP (0.2 mmol, 106 mg) were added. The reaction was
stirred for 6 h at room temperature; then it was poured into water
and extracted with AcOEt (3 ꢀ 15 mL). The organic phase was dried
over Na2SO4 and evaporated. The crude product was purified by
flash chromatography (hexane/AcOEt 70/30) to give 84 mg of the
protected title compound. Yield 82%. MS (ESI): m/z 616.2 [MþNa]þ.
The desired final compound 26 was obtained by deprotection
with BCl3 1 M in DCM as previously described for compound 21.
111.8,112.8,115.8,131.3,154.6,155.8,158.0,161.4,169.8. MS (ESI): m/
z 352e353.9 [MþH]þ.
5.1.6. 5-(2,4-Diacetoxy-5-isopropylbenzoyl)-N-ethyl-4,5,6,7-
tetrahydroisoxazole[4,5-c]pyridine-3-carboxamide (23)
To a solution of 19 (50 mg, 0.13 mmol) in DCM (10 mL) was
added Ac2O (54 mg, 50 mL, 0.53 mmol) and catalytic amount of
pyridine. The reaction mixture was stirred for 4 h at room tem-
perature, water (10 mL) and DCM (10 mL) were then added. The
organic phase was separated, dried and the solvent evaporated
under reduced pressure to give a residue which was purified by
flash chromatography (DCM/MeOH 98/2) Yield 73%. 1H NMR
Yield 67%. 1H NMR (300 MHz, DMSO)
d
1.08 (d, J ¼ 6.9 Hz, 6H), 1.9e
1.4 (m, 8H), 2.87 (m, 2H), 3.05 (m, 1H), 3.71 (bs, 2H), 4.15 (m, 1H),
4.51 (bs, 2H), 6.36 (s,1H), 6.87 (s,1H), 8.65 (d, J ¼ 7.5 Hz,1H), 9.54 (s,
2H). MS (ESI): m/z 414.3 [MþH]þ.
(400 MHz, CD3OD):
d
1.10e1.21 (m, 9H), 2.10 (s, 3H), 2.32 (s, 3H),
5.1.7.3. 5-(2,4-Dihydroxy-5-isopropyl-benzoyl)-4,5,6,7-tetrahydro-
isoxazolo[4,5-c]pyridine-3-carboxylic acid (2-chloro-ethyl)-amide
(27). 2-Chloroethylamine hydrochloride (0.25 mmol, 29 mg),
2.90e3.08 (m, 5H), 3.24e3.35 (m, 2H), 3.75 (s, 2H), 4.54 (s, 2H), 7.03
(s, 1H), 7.33 (s, 1H), 8.23 (br, 1H). MS (ESI): m/z 458.1 [MþH]þ.

R. Baruchello et al. / European Journal of Medicinal Chemistry 76 (2014) 53e60
59
DIPEA (0.5 mmol, 86
m
L), and PyBOP (0.2 mmol, 106 mg) were
5.2. Biology
added to a solution of acid 24 (0.17 mmol, 90 mg) in 5 mL of
DCM. The reaction was stirred overnight at room temperature;
the DCM was removed in vacuo and water was added (10 mL).
The mixture was then extracted with AcOEt (3 ꢀ 15 mL). The
organic phase was dried over Na2SO4 and evaporated. The crude
product was purified by flash chromatography (hexane/AcOEt 7/
3) to give 90 mg of protected product as white solid. Yield 88%.
ESI-MS m/z ¼ 610.3/612.3 [MþNa]þ. The desired compound was
obtained by deprotection with BCl3 1 M in DCM as previously
described for compound 21. The crude product was purified by
flash chromatography (DCM/methanol 98/2) to give 40 mg of
5.2.1. Cellular sensitivity to drugs
In non-small-cell lung carcinoma cells (NCI-H460) cellular
sensitivity to drugs was evaluated by growth-inhibition assay after
72 h of drug exposure. Cells in the logarithmic phase of growth
were seeded into 96-well plates, and 24 h after seeding, the drug
was added to the medium. Cell survival was evaluated after 72 h of
drug exposure by the sulforhodamine B test. IC50 was calculated by
the ALLFIT program and was defined as the drug concentration
causing a 50% reduction of cell number compared to that of un-
treated control cells.
the title compound. Yield 65%. 1H NMR (300 MHz, DMSO)
d 1.08
(d, J ¼ 6.9 Hz, 6H), 2.88 (m, 2H), 3.05 (m, 1H), 3.54 (m, 2H), 3.69
(bm, 4H), 4.53 (bs, 2H), 6.36 (s, 1H), 6.87 (s, 1H), 8.92 (t,
J ¼ 5.5 Hz, 1H), 9.54 (s, 2H). MS (ESI): m/z 408.32/410.34
[MþH]þ.
5.2.2. Binding on Hsp90 by a fluorescence polarization assay [38]
GM-FITC, supplied by Invivogen (catalog no. 06C23-MT, CA, U.S.)
was previously dissolved in DMSO to obtain 10 mM stock solutions
and kept at ꢂ20 ꢁC until use. Hsp90, purchased from Stressgen
(catalog no. SPP-776, Victoria, BC, Canada), was previously dis-
solved in assay buffer (HFB) to form 2.2 mM stock solutions and kept
at ꢂ80 ꢁC until use. The compounds were previously dissolved in
DMSO to obtain stock solutions and kept at ꢂ20 ꢁC. On the day of
the experiment, various concentration solutions were prepared by
serial dilutions in assay buffer (HFB) containing 20 mM HEPES (K),
pH 7.3, 50 mM KCl, 5 mM MgCl2, 20 mM Na2MoO4, and 0.01% NP40.
5.1.7.4. Methyl 7-((5-(2,4-dihydroxy-5-isopropyl-benzoyl)-6,7-
dihydro-4H-isoxazolo[4,5-c]pyridine-3-carboxamido)-heptanoate
(28). Methyl 7-aminoheptanoate HCl (0.20 mmol, 39 mg), DIPEA
(0.40 mmol, 70 mL) and PyBOP (0.20 mmol,106 mg) were added to a
solution of acid 24 (0.17 mmol, 92 mg) in 5 mL of DCM. The reaction
mixture was stirred at room temperature until complete conver-
sion of starting material to amide (4 h, TLC check with eluent DCM/
MeOH 96/4) then diluted with DCM (15 mL) and washed with H2O
(10 mL). The organic phase was dried over Na2SO4 and concen-
trated at reduced pressure. The crude product was purified by flash
chromatography (DCM/MeOH ¼ 98/2) to give methyl 7-((5-(2,4-
dibenzyloxy-5-isopropyl-benzoyl)-6,7-dihydro-4H-isoxazolo[4,5-
c]pyridine-3-carboxamido)-heptanoate (98 mg, Yield 88%).1H NMR
Before each use, 0.1 mg/mL bovine
g globulin and 2 mM DTT were
freshly added. Fluorescence polarization (FP) was performed in
Opti-Plate-96F well plates (PerkineElmer, Zaventem, Belgium) us-
ing a plate reader (Wallac Envision 2101 multilabel reader, Perkine
Elmer, Zaventem, Belgium). To evaluate the binding affinity of the
molecules, an amount of 50
mL of the GM-FTC solution (5 nM) was
added to 30 nM Hsp90 in the presence of 5
mL of the test com-
pounds at increasing concentrations. The plates were shaken at 4 ꢁC
for 4 h, and the FP values in mP (millipolarization units) were
recorded. The IC50 values were calculated as the inhibitor concen-
tration that displaced 50% of the tracer, each data point being the
result of the average of triplicate wells, and were determined from a
plot using nonlinear least-squares analysis. Curve fitting was per-
formed using Prism GraphPad software program (GraphPad Soft-
ware, Inc., San Diego, CA, U.S.)
(300 MHz, DMSO, at 80 ꢁC)
d
1.16 (d, J ¼ 6.9 Hz, 6H), 1.32 (m, 4H),
1.55 (m, 4H), 2.27 (t, J ¼ 7.3 Hz, 2H), 2.78 (m, 2H), 3.24 (m, 3H), 3.58
(s, 3H), 3.72 (bs, 2H), 4.51 (bs, 2H), 5.07 (s, 2H), 5.16 (s, 2H), 6.87 (s,
1H), 7.03 (s, 1H), 7.28 (s, 5H), 7.42 (m, 5H), 8.11 (bs, 1H). MS (ESI): m/
z 690.2 [MþNa]þ.
Compound 28 was obtained by deprotection with BCl3 1 M in
DCM as previously described for compound 21. The crude product
was purified by flash chromatography (DCM/MeOH 97/3) to give
40 mg of the desired product (Yield 58%). 1H NMR (300 MHz,
5.2.3. Growth inhibition and apoptosis in K562 cells
DMSO)
d
1.08 (d, J ¼ 6.9 Hz, 6H), 1.24 (m, 4H), 1.47 (m, 4H), 2.26 (t,
5.2.3.1. Cytotoxicity assay. To evaluate the number of live and dead
cells, cells were stained with trypan blue and counted on a he-
mocytometer. Cells which showed trypan blue uptake were inter-
preted as nonviable. To determine the growth inhibitory activity of
the drugs tested, 2 ꢀ 105 cells were plated into 25-mm wells
(Costar, Cambridge, U.K.) in 1 mL of complete medium and treated
with different concentrations of each drug. After 48 h of incubation,
the number of viable cells was determined and expressed as
percent of control proliferation.
J ¼ 7.4 Hz, 2H), 2.87 (m, 2H), 3.04 (m, 1H), 3.17 (m, 2H), 3.55 (s, 3H),
3.71 (bs, 2H), 4.51 (bs, 2H), 6.36 (s, 1H), 6.86 (s, 1H), 8.72 (t,
J ¼ 5.8 Hz, 1H), 9.57 (bs, 2H). MS (ESI): m/z 488.1 [M þ H]þ, 510.1
[M þ Na]þ, 486.1 [MꢂH]ꢂ.
5.1.7.5. 5-(2,4-Dihydroxy-5-isopropyl-benzoyl)-N-[7-(hydrox-
yamino)-7-oxo-heptyl]-6,7-dihydro-4H-isoxazolo[4,5-c]pyridine-3-
carboxamide (29). Hydroxylamine (50% aqueous solution,
0.61 mmol, 41
m
L) and NaOH 1 M (0.41 mmol, 410
m
L) were
L,
added to a stirred solution of 28 (20 mg) in MeOH (410
m
5.2.3.2. Flow cytometric analysis per cell cycle and apoptosis.
The cells were washed once in ice-cold PBS and resuspended at
1 ꢀ 106 cells/mL in a hypotonic fluorochrome solution containing
propidium iodide (Sigma), 50 g/mL in 0.1% sodium citrate plus
0.03% (v/v) Nonidet P-40 (Sigma). After 30 min of incubation in this
solution, the samples were filtered through nylon cloth, and their
fluorescence was analyzed as single-parameter frequency histo-
grams using a FACSort (Becton Dickinson, Mountain View, CA). The
distribution of cells in the cell cycle was determined using the
ModFit LT program (Verity Software House, Inc.). Apoptosis was
determined by evaluating the percentage of hypoploid nuclei
accumulated in the sub-G0eG1 peak after labeling with propidium
iodide.
0.1 M) at 0 ꢁC. The reaction mixture was warmed up to room
temperature and after 30 min TLC analysis showed complete
conversion of starting material. The solution was cooled to 0 ꢁC,
neutralized with HCl 1 M (310
mL) and then extracted with
AcOEt. The organic phase was washed with water and with
brine, dried over Na2SO4 and concentrated under reduced
pressure to give 29 (20 mg, Yield 99%). 1H NMR (300 MHz,
DMSO)
d
1.08 (d, J ¼ 7.0 Hz, 6H), 1.22 (m, 4H) 1.45 (m, 4H), 1.91
(t, J ¼ 7.3 Hz, 2H), 2.87 (m, 2H), 3.04 (m, 1H), 3.17 (m, 2H), 3.71
(bs, 2H), 4.51 (bs, 2H), 6.36 (s, 1H), 6.86 (s, 1H), 8.70 (bs, 1H),
8.72 (t, J ¼ 5.6 Hz, 1H), 9.55 (bs, 2H), 10.35 (bs, 1H). MS (ESI): m/
z 489.1 [MþH]þ, 511.2 [MþNa]þ, 487.1 [MꢂH]ꢂ.

60
R. Baruchello et al. / European Journal of Medicinal Chemistry 76 (2014) 53e60
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hibitors of chaperone heat shock protein 90, Journal of Medicinal Chemistry
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5.2.3.3. Histone deacetylase profiling. HDAC profiling was per-
formed by Reaction Biology Corp. (Malvern, PA) against 10 isolated
isoforms of human HDAC (HDAC1e8, 10e11) in the presence of the
fluorogenic tetrapeptide RHKKAc (p53 residues 379e382) as the
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substrate (10 mM). Isolated human HDACs were obtained by stan-
dard purification, with the exception of HDAC3, which was isolated
in complex with NCOR2 and used as such. SAHA was used as
reference compounds. Each compound was dissolved in DMSO
(10 mM), and sequentially diluted solutions were used for testing.
IC50 values were calculated from the resulting sigmoidal dosee
response inhibition slopes.
Acknowledgments
[21] T. Taldone, W. Sun, G. Chiosis, Discovery and development of heat shock
protein 90 inhibitors, Bioorganic & Medicinal Chemistry 17 (2009) 2225e
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The authors would like to thank Dr. Alessandro Noseda, General
Manager & CEO sigma-tau Research Switzerland S.A. (STRCH), for
his valuable financing to this project of research. Moreover, we
thank Dr. M. Castorina and Dr. M.L. Cervoni for their support to
Hsp90 binding assay.
Appendix A. Supplementary data
[24] C.W. Murray, M.G. Carr, O. Callaghan, G. Chessari, M. Congreve, S. Cowan,
J.E. Coyle, R. Downham, E. Figueroa, M. Frederickson, B. Graham,
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.056.
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