Discovery of NMS-E973 as novel, selective and potent inhibitor of heat shock protein 90 (Hsp90)

Article abstract of DOI:10.1016/j.bmc.2013.09.018

Novel small molecule inhibitors of heat shock protein 90 (Hsp90) were discovered with the help of a fragment based drug discovery approach (FBDD) and subsequent optimization with a combination of structure guided design, parallel synthesis and application of medicinal chemistry principles. These efforts led to the identification of compound 18 (NMS-E973), which displayed significant efficacy in a human ovarian A2780 xenograft tumor model, with a mechanism of action confirmed in vivo by typical modulation of known Hsp90 client proteins, and with a favorable pharmacokinetic and safety profile.

Full text of DOI:10.1016/j.bmc.2013.09.018

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of NMS-E973 as novel, selective and potent inhibitor
of heat shock protein 90 (Hsp90)
⇑
Maria Gabriella Brasca , Sergio Mantegani, Nadia Amboldi, Simona Bindi, Dannica Caronni, Elena Casale,
Walter Ceccarelli, Nicoletta Colombo, Anna De Ponti, Daniele Donati, Antonella Ermoli, Gabriele Fachin,
Eduard R. Felder, Ronald D. Ferguson, Claudio Fiorelli, Marco Guanci, Antonella Isacchi, Enrico Pesenti,
Paolo Polucci, Laura Riceputi, Francesco Sola, Carlo Visco, Fabio Zuccotto, Gianpaolo Fogliatto
Nerviano Medical Sciences S.r.l., Oncology, Viale Pasteur 10, 20014 Nerviano, MI, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel small molecule inhibitors of heat shock protein 90 (Hsp90) were discovered with the help of a frag-
ment based drug discovery approach (FBDD) and subsequent optimization with a combination of struc-
ture guided design, parallel synthesis and application of medicinal chemistry principles. These efforts led
to the identification of compound 18 (NMS-E973), which displayed significant efficacy in a human ovar-
ian A2780 xenograft tumor model, with a mechanism of action confirmed in vivo by typical modulation
of known Hsp90 client proteins, and with a favorable pharmacokinetic and safety profile.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 31 May 2013
Revised 4 September 2013
Accepted 6 September 2013
Available online xxxx
Keywords:
Fragment based drug discovery
Tumor cell proliferation inhibition
Anti-cancer agents
1. Introduction
ment based drug discovery (FBDD), an approach receiving
increased recognition for its performance in delivering efficient li-
Heat shock protein 90 (Hsp90) is a molecular chaperone, which
plays a key role in the conformational maturation, stability and
function of several oncogenic proteins that are involved in signal
transduction pathways, whose dysfunctional activation have been
collectively described as constituting the hallmark traits of can-
cer.1,2 Discovery and characterization of natural inhibitors of
Hsp90 function such as geldanamycin3 and radicicol4 have at-
tracted interest on Hsp90 as a therapeutic target for anticancer
drugs. It was shown that geldanamycin inhibits Hsp90 functions
by binding to an ATP binding pocket in the N-terminal domain of
the protein. The subsequent loss of its chaperone function induces
the degradation of diverse client proteins. After the identification
of the early Hsp90 inhibitors, in particular 17-AAG5 and other
gands and focused optimization pathways.15 We screened our cor-
porate fragment library characterized by low molecular weight
compounds (<300 Da) against the N-terminal domain of human
Hsp90a using a FAXS (Fluorine chemical shift Anisotropy and eX-
change for Screening) NMR displacement assay.16 The screening
led to the identification of fragment 1 (Fig. 1), which showed a
Kd of 0.6 lM in the FAXS competitive binding assay. On the basis
of these results, fragment 1 was selected as starting point for fur-
ther explorations. To facilitate the chemical expansion we deter-
mined the crystal structure of 1 bound to the ATPase N-terminal
domain of Hsp90 (Fig. 1). The resorcinol moiety of 1 occupies the
adenosine pocket and displays a binding mode already described
for other known Hsp90 inhibitors:7,8 one of the two phenolic hy-
droxyl groups makes key interactions with Asp93 and with a con-
served water molecule, whereas the other hydroxyl substituent is
involved in a network of water mediated interactions with the
nearby Ser52 and Leu48 residues. The dichlorophenyl group is
stacked between the side chain of Leu107 and Phe138 and fills a
hydrophobic pocket adjacent to the adenosine binding site, created
by the rearrangement of residues 103–111, as previously reported
also for other Hsp90 inhibitors.11,14,17–19 Based on modeling stud-
ies, our exploration and optimization strategy was focused on
expansion of the initial hit within the ATP binding site, by adding
groups pointing to the solvent region, and on improvement of its
fitting into the induced hydrophobic pocket, through the modifica-
tion of the substitution pattern of the phenyl ring.
semi-synthetic ansamycin derivatives such as retaspimycin6
a
new group of fully synthetic second generation inhibitors was
developed with improved preclinical efficacy and ‘‘drug-likeness’’
including the intravenous drugs NVP-AUY922,7 AT-13387,8 ganet-
espib (STA-9090),9 KW-2478,10 and the oral drugs DEBIO-0932
(CUDC-305),11 NVP-HSP990,12 SNX-5422,13 and PU-H71,14 which
have progressed into clinical development.
As part of our strategy for the identification of novel chemo-
types for the development of Hsp90 inhibitors we relied on frag-
⇑
Corresponding author. Tel.: +39 0331 581533; fax: +39 0331 581347.
E-mail address: gabriella.brasca@nervianoms.com (M.G. Brasca).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.09.018
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.09.018

2
M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Comparison of 1 to crystal structures of other known Hsp90
inhibitors, in particular of VER-4900920 and NVP-AUY922,7 sug-
gested to focus initially on the 4-position of the resorcinol ring of
1, by adding a moiety like an isoxazole 3-carboxamide pointing
to the solvent region, with the aim of increasing the binding affin-
ity through additional interactions with the protein.
OH
4
2. Chemistry
Cl
Cl
Compounds 2–38 were synthesized21 according to Schemes 1–
4. Reaction of commercially available 39a–c with 1-fluoro-4-nitro-
benzene in DMSO in presence of potassium carbonate afforded
40a–c. Reduction of 40a furnished the amino derivative, which
was converted into the chloro analogue 41 via its diazonium salt.
Treatment of 40a–c and 41 with diethyl oxalate in the presence
of lithium bis(trimethylsilyl)amide in THF and final cyclization
with hydroxylamine hydrochloride in ethanol afforded isoxazole
O
HO
1
Figure 1. Compound 1 bound to the active site of Hsp90. Compound 1 is shown
with blue carbon atoms while protein is colored in grey. Ordered water molecules
are shown as red spheres and hydrogen bonds as dashed line.
O
R1
R1
Cl
NO2
b, c
a
O
41 (from 40a)
h, e
OH
O
O
R2
R2
O
O
O
40a R1 = OCH3, R2 = OCH3
40b R1 = H, R2 = OCH3
40c R1 = OCH3, R2 = H
39a R1 = OCH3, R2 = OCH3
39b R1 = H, R2 = OCH3
39c R1 = OCH3, R2 = H
d, e
d, e
(for 42)
(for 43a, 43b, 43c)
R1
R1
O
X
X
Cl
f
O
R2
O
R2
O
O
O
N
O
N
O
N
H
N
O
O
O
46
42 R1 = OCH3, R2 = OCH3, X = Cl
44 R1 = OCH3, R2 = OCH3, X = Cl
45a R1 = OCH3, R2 = OCH3, X = NO2
45b R1 = H, R2 = OCH3, X = NO2
45c R1 = OCH3, R2 = H, X = NO2
43a R1 = OCH3, R2 = OCH3, X = NO2
43b R1 = H, R2 = OCH3, X = NO2
43c R1 = OCH3, R2 = H, X = NO2
g
g
OH
R1
Cl
X
O
O
HO
R2
O
O
N
N
H
N
O
6
2 R1 = OH, R2 = OH, X = Cl
3 R1 = OH, R2 = OH, X = NO2
4 R1 = H, R2 = OH, X = NO2
5 R1 = OH, R2 = H, X = NO2
Scheme 1. Reagents and conditions: (a) 1-Fluoro-4- nitrobenzene, K2CO3, DMSO, 110 °C, 2 h; (b) 37% HCl, Pd/C, H2, MeOH/EtOAc/DCM (1:1:1), 40 psi, 8 h; (c) CuCl2, t-BuONO,
acetonitrile, 65 °C, 2 h; (d) 1 M Lithium bis(trimethylsilyl)amide in THF, ꢀ50 °C, then (COOEt)2, rt, 2 h; (e) NH4OH.HCl, EtOH, reflux, 2 h; (f) 2 N EtNH2 in THF, 100 °C, 5 h; (g)
1 M BBr3 in DCM, rt, 120 h; (h) 1 M Lithium bis(trimethylsilyl)amide in THF, ꢀ50 °C, then HCOOEt, rt, 2 h.
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M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
OH
OH
NO2
NO2
b for 7-14, 16, 18-20, 29
O
HO
O
b, c for 15
H
O
a
43a
O
N
O
N
OH
R3
O
7-16, 18-20, 29
47
Scheme 2. Reagents and conditions: (a) 1 M BBr3 in DCM, rt, 120 h; (b) primary or secondary amines, DIPEA, TBTU, DMA, rt, overnight; (c) 50% CF3COOH, DCM, rt, 1 h.
derivatives 42 and 43a–c. Conversion of the ester function to eth-
ylamides 44 and 45a–c was obtained in one step by reaction with
ethylamine in THF in a stopped flask at 100 °C. Finally, treatment
with BBr3 furnished the target compounds 2–5 (Scheme 1). Reac-
tion of 41 with ethyl formate, in the presence of lithium bis(tri-
methylsilyl)amide in THF, followed by cyclization with
hydroxylamine hydrochloride in ethanol afforded 46, which was fi-
nally converted to compound 6 by treatment with BBr3. Prepara-
tion of amides 7–16, 18–20 and 29 was accomplished according
to Scheme 2, starting from carboxylic ester 43a. BBr3 mediated re-
moval of hydroxyl protecting groups and, at the same time, sapon-
ification of the carboxylic ester afforded 47, which was reacted
with suitable amines under usual coupling condition to give 7–
14, 16, 18–20 and 29, while 15 was obtained after nitrogen depro-
tection of the piperidine ring. A better look at the previous syn-
thetic procedure led to focus our attention on hydroxyl
protecting group removal. In fact, despite the use of an excess of
BBr3, after five days the reaction was not completed. Furthermore,
recovery of pure material required extensive purification. The syn-
thetic pathway was ameliorated (Scheme 3) using a different pro-
tecting group. The in situ methoxymethylation22 of 48 afforded
49,23 which was then reacted with 1-fluoro-4-nitrobenzene at
55 °C. Condensation of 50 with diethyloxalate was carried out
using sodium tert-butoxide at ꢀ10 °C, the raw intermediate was
cyclized by treatment with hydroxylamine hydrochloride at room
temperature to isoxazole 51, which was converted to 52 by ami-
nolysis with tert-butyl 4-aminopiperidine-1-carboxylate. Removal
of the protecting groups under acidic conditions afforded 17. Alkyl-
ation of 17 with 1-bromo-4,4,4-trifluorobutane afforded 21, while
22–28 were prepared by reductive amination. Preparation of 30–
38 was accomplished according to Scheme 4. Aminolysis of 51 with
1-methyl-piperidin-4-ylamine furnished 53. Reduction of the nitro
group of 53 afforded 54, which was then converted to 30–31 and
33–34 by alkylation of the piperidine nitrogen under reductive
amination condition followed by protecting groups removal. Com-
pound 32 was prepared by reaction of 54 with 2,5-dimethoxytetra-
hydrofuran, followed by protecting groups removal. Finally,
reduction of the nitro group of 52 followed by alkylation of the
amino group with formaldehyde under reductive amination condi-
tion furnished its dimethylamino derivative 55, which was con-
verted into 35 by protecting groups removal. Alkylation of 35
with 1-bromo-4,4,4-trifluorobutane afforded 36, while 37–38 were
prepared by reductive amination.
3. Results and discussion
Table 1 summarizes the SAR of the first set of compounds 2–6.
Binding to Hsp90 was measured by fluorescence polarization (FP)
assay and inhibition of cell growth proliferation was determined
on A2780 tumor cell line. Due to interference with the fluorescence
O
O
O
OH
O
O
O
O
O
NO2
NO2
c, d
a
b
OH
O
O
OH
HO
O
O
O
O
O
O
N
O
O
51
50
48
49
O
O
OH
OH
O
O
NO2
g for 21
NO2
NO2
O
O
HO
HO
O
O
O
h for 22-28
f
e
O
N
O
N
O
N
H
H
N
N
R3
O
O
21-28
N
N
HCl
H
O
17
52
O
Scheme 3. Reagents and conditions: (a) Dimethoxymethane, ZnBr2, DCM, AcCl, then 48, DIPEA, DCM, 5 °C, 1 h, then rt, on; (b) 1-fluoro-4-nitrobenzene, K2CO3, DMSO, 55 °C,
6 h; (c) t-BuONa, (COOEt)2, THF, ꢀ10 °C, 1 h, then rt, 2 h; (d) NH2OHꢁHCl, AcONa, EtOH, rt, 2 h, then 6 N HCl, rt, 24 h; (e) t-butyl 4-aminopiperidine-1-carboxylate, 80 °C, 24 h;
(f) 4 N HCl in dioxane, EtOH, rt, 5 h; (g) 1-bromo-4,4,4-trifluorobutane, DIPEA, DMF, rt, overnight; (h) aldehydes or ketones, (CH3)4N(OAc)3BH, DMF, rt, overnight.
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M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
O
O
O
OH
O
O
O
NH2
X
NO2
c, f for 30
d, f for 32
O
O
b
O
O
H
a
O
O
e, f for 31, 33-34
51
O
O
O
N
N
H
N
H
H
N
N
N
O
O
O
53
N
54
N
N
30-34
O
O
OH
OH
N
N
N
g for 36
f
e for 37-38
O
b, c
O
O
O
HO
O
HO
52
O
N
O
N
O
N
H
H
N
N
R3
O
O
N
N
2.HCl
H
O
55
35
36-38
O
Scheme 4. Reagents and conditions: (a) 1-Methyl-piperidin-4-ylamine, 80 °C, 9 h; (b) Zn, NH4Cl, dioxane, H2O, 100 °C, 3 h; (c) 37% HCHO, (CH3)4N(OAc)3BH, AcOH, DCM,
DMF, rt, 1 h; (d) 2,5-dimethoxytetrahydrofuran, 2.5 M H2SO4, NaBH4, THF, MeOH, rt, overnight; (e) ketones, (CH3)4N(OAc)3BH, AcOH, DMF, rt, overnight; (f) 4 N HCl in
dioxane, EtOH, rt, overnight; (g) 1-bromo-4,4,4-trifluorobutane, DIPEA, DMF, rt, overnight.
signal the FP data for fragment hit 1 could not be generated, there-
fore its binding affinity was measured by the FAXS competitive
with respect to 2, while a more drastic reduction of the antiprolif-
erative activity was observed.
binding assay and the Kd resulted to be 0.6
l
M. Gratifyingly, com-
The crystal structure of 3 (Fig. 2) shows that the resorcinol moi-
ety maintains the key interactions with Asp93 and the conserved
waters. The carboxamide function, expanding towards the solvent
area, makes additional hydrogen bond interactions with the car-
bonyl backbone of Gly97 and with the nitrogen side chain of
Lys58. Interestingly, the p-nitrophenyl group of 3 does not occupy
anymore the hydrophobic pocket induced by compound 1. The aro-
matic group rotates of about 48 degrees with respect to the dichlo-
pounds 2 and 3, where an isoxazole 3-carboxamide moiety was
added on the 4-position of the resorcinol ring of 1, showed im-
proved binding affinity to Hsp90. A deeper investigation from a
biological and chemical point of view was then pursued. It was ob-
served that the replacement of chloro (2) with nitro (3) maintained
the affinity to Hsp90, while the activity in cells increased about
fourfold. Removal of one of the two hydroxyl moieties (4–5) led
to a drop in enzyme affinity and potency in cells, confirming the
crucial role of the concurrent presence of the two hydroxyl groups
in the molecule. Removal of ethylcarboxamide (6) caused approx-
imately a fourfold decrease of potency in the biochemical assay
Table 1
Preliminary exploration
R1
X
O
R2
O
N
R3
a
a
Compd R1
R2
R3
X
Hsp90 DC50
M)
A2780 IC50
M)
(
l
(l
2
3
4
5
6
OH OH CONHEt Cl
OH OH CONHEt NO2 0.048
0.052
0.300
0.080
>10
>10
>10
H
OH
OH OH
OH CONHEt NO2 1.920
CONHEt NO2 >10
Cl 0.210
Figure 2. Overlay of the crystal structures of bound compounds 3 and 1. Compound
3 is shown with yellow carbon atoms, while the protein is colored in green. Carbon
atoms of 1 and the protein residues interacting with it are shown in grey. Ordered
water molecules are shown as red spheres and hydrogen bonds as dashed line.
H
H
a
Values are means of three experiments.
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M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
Table 2
R3 expansion
OH
NO2
O
HO
O
N
R3
a
a
Compd
R3
Hsp90 DC50
(lM)
HER2 degrad IC50
(l
M)
A2780 IC50
(
lM)
Solubility pH 7 (lM)
3
7
8
9
10
11
CONHCH2CH3
CON(CH3)2
CONHCH2CH2F
CONHCH2CH2OCH3
CONHCH2CH2OH
CONH(CH2)3N(CH3)2
0.048
>10
0.018
0.021
0.016
0.021
0.114
ND
0.086
ND
ND
ND
0.080
>10
0.052
0.276
0.294
0.192
65
145
19
130
131
84
O
12
0.658
ND
ND
1.946
18
N
H
O
13
2.713
7.271
14
N
H
O
14
15
>10
ND
ND
>10
>10
<1
N
H
H
N
O
0.025
144
N
H
N
O
16
0.033
ND
7.188
125
N
H
O
17
18
0.017
0.010
ND
0.362
0.069
155
179
N
H
O
NH
N
0.110
N
H
O
19
20
0.013
0.073
ND
ND
0.396
3.404
104
ND
N
H
N
O
N
H
N
O
O
21
22
23
0.022
0.019
0.020
0.071
0.277
0.071
0.060
0.052
0.050
40
143
61
N
H
O
N
N
N
CF3
N
H
O
N
H
(continued on next page)
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M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 2 (continued)
a
a
Compd
R3
Hsp90 DC50
(lM)
HER2 degrad IC50
0.210
(l
M)
A2780 IC50
0.029
(
lM)
Solubility pH 7 (
lM)
O
24
0.054
75
N
H
O
N
N
25
26
27
0.048
0.012
0.037
0.130
ND
0.029
0.368
0.041
58
138
87
N
H
O
N
H
O
N
N
N
O
0.088
N
H
O
O
28
29
0.046
0.035
0.032
0.094
0.028
0.013
25
9
N
H
N
N
O
N
H
ND = not determined.
a
Values are means of three experiments.
rophenyl moiety of 1 and it is situated near Leu107 and Phe138
residues in a solvent exposed site. The nitro group is located in
promising compounds 3, 8 and 18, with IC50 ranging from 86 nM
for 8 to 114 nM for 3. Co-crystal structure of 18 (Fig. 3) shows that
the molecule maintains a similar binding mode to 3. The resorcinol
moiety makes the key interaction with Asp93 and the conserved
water molecules while the carboxamide group makes hydrogen
bond interactions with Gly97 and Lys58. Moreover, the basic piper-
idine nitrogen makes an additional salt interaction with the car-
boxylic residue of Asp102, thus increasing compound affinity. It
is worth noting that the p-nitrophenyl group of 18 adopts a slightly
the ADP
a-phosphate site and makes a hydrogen bond to the
Phe138 backbone nitrogen. This structural information led us to
slightly modify our chemical expansion strategy, as described later
in this paper. The initial plan to introduce bulkier groups on the
aromatic ring, which could better fit into the hydrophobic pocket,
was halted.
Based on the structural analysis of the carboxamide moiety of 3,
exposed to the solvent area, a larger series of residues linked to the
isoxazole ring was investigated.
Data concerning activity and solubility in neutral buffer of ana-
logues of 3 are summarized in Table 2. Furthermore, an assay mea-
suring Her2 client protein degradation in BT-474 breast cancer cell
line was also established to evaluate Hsp90 specific cellular effects
of interesting compounds with an IC50 <100 nM on A2780 cells.
Condensation of carboxylic acid 47 with different amines gave rise
to a series of amides 7–29, whose biochemical and cellular activity
was sharply affected by minor modifications. The secondary nature
of the amide was essential to binding, as deduced from the detri-
mental effect of a tertiary amide function on compound 7, likely
due to steric clash and loss of interaction with Gly97. Linear ali-
phatic amides bearing small hydrophilic residues (8–11) showed
better binding affinity to Hsp90 with respect to 3. Interesting po-
tency in A2780 cell proliferation assay was observed for compound
8, whereas 9–11 were less potent. Unfortunately, compound 8 dis-
played low solubility in neutral buffer. Bulkier and hydrophobic
residues such as cyclohexyl, benzyl and cyclohexylmethyl (12–
14) caused a marked decrease in solubility and in the protein affin-
ity, probably due to the lack of interactions in an area rich of polar
side chains. Improvement of solubility and affinity for Hsp90 with
respect to 3 was achieved with compounds 15–18, due to the pres-
ence of hydrophilic moieties. However, only 18 showed interesting
inhibitory potency of cell growth proliferation of A2780 tumor cell
line. Specific biological effects in BT-474 breast cancer cell line re-
lated to the inhibition of Hsp90 were determined for the most
Figure 3. X-ray structure of the N-terminal ATP binding site of Hsp90 in complex
with 18. Compound 18 is shown with yellow carbon atoms, while the protein is
colored in green. Ordered water molecules are shown as red spheres and hydrogen
bonds as dashed lines.
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M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
Table 3
X and R3 expansion
OH
X
O
HO
O
N
R3
a
Compd
X
R3
Hsp90 DC50
0.016
,
l
Ma
HER2 degrad IC50
0.033
(lM)
A2780 IC50
0.037
(l
M)
Solubility pH 7 (
lM)
O
30
N(CH3)2
161
N
H
O
N
N
N
N
31
32
0.013
0.037
0.065
0.107
0.074
0.088
158
119
N
H
N
H
O
N
N
H
O
33
34
0.061
0.104
ND
ND
0.149
1.709
ND
ND
N
H
N
H
O
O
N
H
N
N
H
O
35
36
37
38
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
0.025
0.025
0.023
0.013
ND
0.136
0.020
0.016
0.011
161
63
N
H
O
NH
N
0.025
0.020
0.028
N
H
O
CF3
79
N
H
O
N
O
O
105
N
H
N
ND = not determined.
a
Values are means of three experiments.
Table 4
Binding kinetic affinity and in vivo pharmacokinetic parameters in Balb nu/nu miceb of selected compounds
PK data (iv), dosec 10 mg/kg
a
Compd
Hsp90 KD (nM)
AUC1
(
lM h)
CL (mL/min/kg)
Vss (L/kg)
t1/2 (h)
18
30
31
38
0.346 0.001
0.120 0.060
0.220 0.069
0.057 0.017
9.26 0.36
5.92 0.26
4.80 0.14
7.60 0.69
39.91 1.70
62.29 2.81
64.80 1.97
29.35 0.26
5.83 3.18
19.73 1.61
18.37 1.59
4.94 0.42
5.55 1.07
5.55 0.01
5.44 0.53
5.46 0.41
a
b
c
Values are means of three experiments.
n = 3 Animals per study.
Dosed in 10% Tween 80 in 5% dextrose.
different conformation compared to the corresponding moiety of 3,
allowing a hydrogen bond of the nitro group with the side chain
324 reveals a rearrangement of residues 109–124 so that Lys112
points toward the binding site. However, we can not exclude that
the different conformation adopted by the p-nitrophenyl group is
due to the change in protein conformation. Actually, the flexibility
nitrogen of Lys112 and
a water mediated interaction with
Gly135 and Phe138. Comparison of the crystal structure of 18 with
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8
M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
of this protein region is known and was previously reported.25
Compound 18 became the prototype of a series of analogues. Dif-
ferent structural motifs were chosen as a replacement of the
methyl group of the piperidine ring. Modeling studies indicated
that this part of the molecule protrudes into the solvent region
allowing the accomodation of moieties of different size and
lipophilicity.
the basis of their affinity to Hsp90, antiproliferative activity, mech-
anism of action and preliminary solubility, were profiled by Surface
Plasmon Resonance analysis, with an approach of kinetic titration
as previously described,26 in order to more accurately determine
the KD (Table 4). All compounds showed a KD <1 nM. In addition,
we tested for comparison in the same assay a reference Hsp90
inhibitor, SNX-2112,13 and its measured KD was 0.726 0.015 nM.
No activity was observed when the selected compounds were
tested against the biologically related ATPase Hsc70 as well as
against a panel of 52 protein kinases (IC50 >10 nM).
In order to assess the potential of the most promising com-
pounds 18, 30, 31, and 38, a preliminary pharmacokinetic investi-
gation in healthy nude mice was performed. When administered
intravenously (iv) at 10 mg/kg, all compounds showed good expo-
sure levels and a half life >5 h (Table 4). High volume of distribu-
tion associated to a moderate systemic clearance was observed
for 18 and 38, indicating a good tissue distribution, while 30 and
31 showed an extremely high volume of distribution (Vss >18 L).
Efficacy of 18 and 38 was evaluated in vivo in the human ovar-
ian A2780 xenograft mouse model. As shown in Table 5, 18 was
administered intravenously (iv) at 30 and 60 mg/kg for 10 consec-
utive days. Both schedules resulted in significant tumor growth
inhibition (TGI = 53% and 74%, respectively), with a maximum
body weight loss (BWL) of 11.5% observed only at higher dose,
which rapidly recovered at the end of the treatment. Compound
38, when given intravenously at 30 mg/kg for 10 consecutive days,
showed higher tumor growth inhibition (TGI = 93%) with respect to
18, but lower tolerability (BWL = 23.4%).
Activity data concerning analogues of 18 (19–29) are summa-
rized in Table 2. Sterically hindered 1,2,2,6,6-pentamethylpiperi-
din-4-yl 19 showed high binding affinity but lower
antiproliferative activity on cells with respect to 18, while acyla-
tion of the piperidine nitrogen (20) led to about a sevenfold de-
crease in the biochemical assay and to a more drastic reduction
of the antiproliferative activity on the A2780 cell line. The lower
affinity of 20 could be explained by loss of saline interaction be-
tween the piperidine nitrogen and the carboxylic residue of
Asp102 of the protein, as seen for 18. Replacement of the methyl
group of the piperidine ring of 18 by linear, branched and cyclic ali-
phatic chains gave rise to compounds 21–23, which maintained
good affinity for Hsp90 and antiproliferative activity on cells.
Her2 degradation in BT-474 cell line related to the inhibition of
Hsp90 was observed for all the compounds, with an IC50 ranging
from 71 nM (21 and 23) to 277 nM (22). Solubility in neutral buffer
was moderate for 21 and 23, and good for 22. The noteworthy bio-
logical profile of 23 prompted the preparation of 24 and 25, where
the cyclohexyl moiety, located in the solvent region, was replaced
by different size rings. A slight decrease in binding affinity and an
improvement in antiproliferative activity on A2780 cells were ob-
served with respect to 23, while Her2 degradation IC50 was higher.
Moreover, compounds 24 and 25 showed moderate solubility in
neutral buffer. In order to improve solubility, the cyclohexyl moi-
ety of 23 was replaced by the 1-methylpiperidin-4-yl group in
26. However, despite its high affinity for Hsp90 and an improved
solubility, this compound showed lower antiproliferative activity
with respect to 23. Compounds 27–29 were synthesized to further
explore the solvent accessible region of Hsp90. These modifications
led to a slight decrease in the binding affinity with respect to 18, all
of them exhibiting potent antiproliferative activity on A2780.
Hsp90 dependent mechanism of action was confirmed in the
Her2 degradation assay, however the solubility in neutral buffer,
particularly for 28–29, prevented further investigation.
With the aim of improving both affinity and physico-chemical
properties we then explored the role of the nitro group of 3 by
its replacement with different amino-substituted moieties (Table 3,
30–34). Replacement of the nitro functionality with either dimeth-
ylamine or isopropylamine substituents (30–31) maintained affin-
ity to Hsp90 and solubility, with an improvement of the cellular
activity of 30 compared to 18. Introduction of the pyrrolidine
group (32) was tolerated, while cyclobutylamine and (tetrahy-
dro-pyran-4-yl)-amine (33–34) caused a decrease in binding affin-
ity and a more drastic reduction of the antiproliferative activity on
the A2780 cell line, in particular for 34. Her2 degradation assay
confirmed the mechanism of action for compounds 30–32, with
an IC50 ranging from 33 nM for 30 to 107 nM for 32.
The interesting results obtained with the dimethylamine deriv-
ative 30, as far as potency and solubility were concerned,
prompted the synthesis of carboxamides 35–38 (Table 3). The
desmethyl analogue 35 showed an interesting Hsp90 affinity, but
a lower antiproliferative effect than 30. Insertion of the most inter-
esting moieties seen in the previous expansion gave rise to com-
pounds 36–38, characterized by significant binding affinity,
potent antiproliferative activity on cells, Hsp90 specific cellular ef-
fects on Her2 client protein and moderate solubility in neutral
buffer.
A PK/PD algorithm that links the dosing regimen to the A2780
tumor growth dynamics was generated,27 suggesting
a new
administration schedule (60 mg/kg twice a day, 3 days on, 1 day
off, 3 days on), which resulted in a better efficacy profile for com-
pound 18. With this optimized schedule, treatment with either 30
or 60 mg/kg of 18 resulted in high efficacy (TGI = 68% and 91%,
respectively), associated with a moderate and reversible body
weight loss (BWL = 4.5 and 7.7%, respectively). On the contrary,
no improvement was achieved for compound 38 with this admin-
istration schedule. Treatment with either 10 or 15 mg/kg of 38
showed lower efficacy (TGI = 53% and 72%, respectively) in com-
parison to 18, with no improvement in its safety profile.
We characterized the in vitro and in vivo effects and mechanism
of action of compound 18. Incubation of A2780 tumor cells with 18
induced degradation of AKT and inhibition of the AKT and MAPK
pathways, as shown by the reduction of AKT and ERK phosphory-
lation, associated with the typical induction of Hsp70, a well-
known feedback response to Hsp90 inhibition (Fig. 4). Analogous
effects, although to a lesser extent, were observed in lysates from
A2780 xenograft tumors isolated at different times after adminis-
tration of 60 mg/kg, confirming that in vivo antitumor activity in-
duced by 18 was Hsp90 dependent (Fig. 5).
Table 5
In vivo intravenous activity of compounds 18 and 38 on A2780 ovarian carcinoma in
nude micea
Compd
Treatment
Dose mg/kg
%TGIb (day)
%BWLc (day)
18
18
38
18
18
38
38
Die 1–10 day
Die 1–10 day
Die 1–10 day
1–3 bid ꢂ 2 cycles
1–3 bid ꢂ 2 cycles
1–3 bid ꢂ 2 cycles
1–3 bid ꢂ 2 cycles
30
60
30
30
60
10
15
53 (18)
74 (18)
93 (18)
68 (14)
91 (14)
53 (14)
72 (14)
2.4 (18)
11.5 (18)
23.4 (18)
4.5 (14)
7.7 (14)
12.3 (14)
19.1 (14)
a
b
c
n = 7 Animals per study.
TGI = Tumor growth inhibition.
BWL = Body weight loss.
Due to the high potency close to the sensitivity limit of the as-
say used, all the relevant compounds 18, 30, 31, and 38, selected on
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M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
9
system with prepacked silica gel cartridges (Biotage or Varian).
Components were visualized by UV light (k: 254 nm) and by iodine
vapor. 1H NMR spectra were recorded at a constant temperature of
28 °C on
a Varian INOVA 400 spectrometer (operating at
400.5 MHz for 1H) and equipped with a 5 mm 1H{13C,15N} z axis
PFG indirect detection probe. Chemical shifts were referenced with
respect to the residual solvents signals. Data are reported as fol-
lows: chemical shift (d), multiplicity (s = singlet, d = doublet,
t = triplet, q = quartet, br s = broad signal, td = triplet of doublet,
dd = doublet of doublets, ddd = doublet of doublets of doublets,
m = multiplet), coupling constants (Hz), and number of protons.
Electrospray (ESI) mass spectra were obtained on a Finnigan LCQ
ion trap. HPLC–UV–MS analyses were carried out combining the
ion trap MS instrument with HPLC system SSP4000 (Thermo Sepa-
ration Products) equipped with an autosampler LC Pal (CTC Analyt-
ics) and UV6000LP diode array detector (UV detection 215–
400 nm). Instrument control, data acquisition and processing were
performed by using Xcalibur 1.2 software (Finnigan). HPLC chro-
matography was run at room temperature, and 1 mL/min flow rate,
Figure 4. In vitro biomarkers modulation of compound 18 in A2780 cells at two
concentrations.
using a Waters X Terra RP 18 column (4.6 ꢂ 50 mm; 3.5
lm). Mo-
bile phase A was ammonium acetate 5 mM buffer (pH 5.5 with ace-
tic acid)/acetonitrile 90:10, and mobile phase B was ammonium
acetate 5 mM buffer (pH 5.5 with acetic acid)/acetonitrile 10:90;
the gradient was from 0% to 100% B in 7 min then hold 100% B
for 2 min before requilibration. Mass data, given as m/z ratio.
ESI(+) high resolution mass spectra (HRMS), were obtained on a
Waters Q-Tof Ultima directly connected with a micro HPLC 1100
Agilent as previously described.28
5.1.1. 5-(3,4-Dichlorophenoxy)benzene-1,3-diol (1)
1H NMR (400 MHz, DMSO-d6) d 9.48 (br s, 2H), 7.57 (d,
J = 8.8 Hz, 1H), 7.23 (d, J = 2.8 Hz, 1H), 6.96 (dd, J = 2.8, 8.8 Hz,
1H), 6.00 (t, J = 2.1 Hz, 1H), 5.83 (d, J = 2.1 Hz, 2H); LC–MS (ESI):
m/z 270 [M+H]+; HRMS (ESI): m/z calcd for C12H8Cl2O3+H+
270.9923, found 270.9928.
Figure 5. Ex vivo biomarkers modulation in lysates from A2780 tumors (3 tumors/
group) isolated at different post-treatment times after single iv administration of
60 mg/kg of compound 18.
5.1.2. 1-[2,4-Dimethoxy-6-(4-nitrophenoxy)phenyl] ethanone
(40a)
A
stirred suspension of 1-(2-hydroxy-4,6-dimethoxy-
phenyl)ethanone (39a, 10.0 g, 51 mmol), potassium carbonate
(10.48 g, 76 mmol) and 1-fluoro-4-nitrobenzene (5.4 mL, 51 mmol)
in DMSO (50 mL) was heated at 110 °C for 2 h. The reaction mix-
ture was diluted with EtOAc (100 mL) and thoroughly washed with
brine (4 ꢂ 50 mL). The organic phase was dried over Na2SO4 and
then evaporated to dryness. The crude was purified by Biotage
SP1 flash chromatography (gradient elution from 3% to 20% of
EtOAc in hexane) to afford 40a (11.9 g, 63%). 1H NMR (400 MHz,
4. Conclusion
The application of a fragment based approach and subsequent
optimization led to the identification of compound 18 (NMS-
E973), which showed nanomolar affinity to Hsp90 and high selec-
tivity towards a panel of kinases, as well as against the biologically
related ATPase Hsc70. In vivo data in a mouse model of human
ovarian cancer demonstrated that the antitumor activity of com-
pound 18 is related to the inhibition of Hsp90, with a favorable
pharmacokinetic and safety profile. NMS-E973 was selected for
further characterization in additional tumor models that have been
presented in a paper submitted separately.27
DMSO-d6)
d 8.23 (d, J = 9.3 Hz, 2H), 7.09 (m, 2H), 6.62 (d,
J = 2.2 Hz, 1H), 6.36 (d, J = 2.1 Hz, 1H), 3.86 (s, 3H), 3.78 (s, 3H),
2.33 (s, 3H); LC–MS (ESI): m/z 318 [M+H]+; HRMS (ESI): m/z calcd
for C16H15NO6+H+ 318.0972, found 318.0973.
The following compounds 40b and 40c were prepared accord-
ing to the method described above.
5. Experimental
5.1. Chemistry
5.1.3. 1-[2-Methoxy-6-(4-nitrophenoxy)phenyl] ethanone (40b)
1H NMR (400 MHz, DMSO-d6) d 8.24 (d, J = 9.3 Hz, 2H), 7.49 (t,
J = 8.2 Hz, 1H), 7.10 (d, J = 9.3 Hz, 2H), 7.07 (d, J = 8.2 Hz, 1H),
6.76 (dd, J = 8.3, 0.6 Hz, 1H), 3.87 (s, 3H), 2.38 (s, 3H); LC–MS
(ESI): m/z 288 [M+H]+; HRMS (ESI): m/z calcd for C15H13NO5+H+
288.0867, found 288.0871.
All solvents and reagents, unless otherwise stated, were com-
mercially available, of the best grade and were used without fur-
ther purification. All experiments dealing with moisture-sensitive
compounds were carried out under dry nitrogen or argon atmo-
sphere. Thin-layer chromatography was performed on Merck Silica
Gel 60 F254 pre-coated plates. Column chromatography was con-
ducted either under medium pressure on silica (Merck Silica Gel
5.1.4. 1-[4-Methoxy-2-(4-nitrophenoxy)phenyl] ethanone (40c)
1H NMR (400 MHz, DMSO-d6) d 8.26 (d, J = 9.4 Hz, 2H), 7.92 (d,
J = 8.8 Hz, 1H), 7.14 (d, J = 9.4 Hz, 2H), 7.01 (dd, J = 8.8, 2.5 Hz, 1H),
6.78 (d, J = 2.4 Hz, 1H), 3.82 (s, 3H), 2.42 (s, 3H); LC–MS (ESI): m/z
40–63 lm) or performed by using a Biotage SP1 flash purification
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.09.018

10
M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
288 [M+H]+; HRMS (ESI): m/z calcd for C15H13NO5+H+ 288.0867,
found 288.0872.
5.1.9. Ethyl 5-[4-methoxy-2-(4-nitrophenoxy) phenyl]-
isoxazole-3-carboxylate (43c)
1H NMR (400 MHz, DMSO-d6) d 8.24–8.28 (m, 2H), 8.05 (d,
J = 8.8 Hz 1H), 7.25 (m, 2H), 7.11 (dd, J = 8.8, 2.5 Hz, 1H), 6.94 (s,
1H), 6.91 (d, J = 2.4 Hz, 1H), 4.35 (q, J = 7.1 Hz, 2H), 3.84 (s, 3H),
1.30 (t, J = 7.1 Hz, 3H); LC–MS (ESI): m/z 385 [M+H]+; HRMS
(ESI): m/z calcd for C19H16N2O7+H+ 385.1031, found 385.1026.
5.1.5. 1-[2-(4-Chlorophenoxy)-4,6-dimethoxy-phenyl]-
ethanone (41)
To a solution of 40a (2.2 g, 6.94 mmol) in MeOH/EtOAc/DCM
(1:1:1, 30 mL) 37% HCl (1.15 mL) and Pd/C (1.0 g) were added.
The reaction mixture was shaken at room temperature in a hydro-
gen athmosphere (40 psi) for 8 h and then filtered through a pad of
Celite that was washed with MeOH. The filtrate was concentrated,
triturated with ether and filtered to afford 1-[2-(4-amino-phen-
oxy)-4,6-dimethoxy-phenyl]-ethanone hydrochloride (1.98 g,
88%). 1H NMR (400 MHz, DMSO-d6) d 9.21 (br s, 3H), 7.19 (d,
J = 8.7 Hz, 2H), 7.00 (d, J = 8. 7 Hz, 2H), 6.49 (d, J = 2.1 Hz, 1H),
6.06 (d, J = 2.1 Hz, 1H), 3.83 (s, 3H), 3.73 (s, 3H), 2.35 (s, 3H); LC–
MS (ESI): m/z 288 [M+H]+. This intermediate (1.0 g, 3.09 mmol)
was added to a mixture of copper(II) chloride (771 mg, 4.52 mmol)
and tert-butylnitrite (0.372 mL, 3.13 mmol) in acetonitrile (40 mL)
heated at 65 °C. The reaction mixture was stirred under reflux for
2 h, then cooled to room temperature, poured into water and ex-
tracted with EtOAc (3 ꢂ 50 mL). The organic phase was dried over
Na2SO4 and then evaporated to dryness. The crude was purified by
Biotage SP1 flash chromatography (gradient elution from 3% to 20%
of EtOAc in hexane) to afford 41 (823 mg, 87%). 1H NMR (400 MHz,
DMSO-d6) d 7.37 (m, 2H), 6.99 (m, 2H), 6.27 (d, J = 2.4 Hz, 1H), 6.12
(d, J = 2.4 Hz, 1H), 3.84 (s, 3H), 3.80 (s, 3H), 2.37 (s, 3H); LC–MS
(ESI): m/z 307 [M+H]+; HRMS (ESI): m/z calcd for C16H15ClO4+H+
307.7506, found 307.7509.
5.1.10. 5-[2,4-Dimethoxy-6-(4-nitrophenoxy)phenyl] -N-ethyl-
isoxazole-3-carboxamide (45a)
A solution of 43a (828 mg, 2.0 mmol) in 2 M ethylamine in THF
solution (20 mL) in a stopped flask was heated at 100 °C for 5 h.
The solvent was removed and the residue was crystallized from a
small volume of MeOH to provide 45a (770 mg, 93%). 1H NMR
(400 MHz, DMSO-d6) d 8.69 (t, J = 5.7 Hz, 1H), 8.22 (m, 2H), 7.12
(m, 2H), 6.81 (s, 1H), 6.75 (d, J = 2.3 Hz, 1H), 6.54 (d, J = 2.2 Hz,
1H), 3.92 (s, 3H), 3.84 (s, 3H), 3.23 (m, 2H), 1.08 (t, J = 7.2 Hz,
3H); LC–MS (ESI): m/z 414 [M+H]+; HRMS (ESI): m/z calcd for C20-
H19N3O7+H+ 414.1296, found 414.1295.
The following compounds 44, 45b and 45c were prepared
according to the method described above from 42, 43b and 43c.
5.1.11. 5-[2-(4-Chlorophenoxy)-4,6-dimethoxy-phenyl]-N-
ethyl-isoxazole-3-carboxamide (44)
1H NMR (400 MHz, DMSO-d6) d 8.69 (t, J = 5.7 Hz, 1H), 7.39 (m,
2H), 6.99 (m, 2H), 6.78 (s, 1H), 6.62 (d, J = 2.2 Hz, 1H), 6.24 (d,
J = 2.2 Hz, 1H), 3.87 (s, 3H), 3.78 (s, 3H), 3.24 (m, 2H), 1.09 (t,
J = 7.2 Hz, 3H); LC–MS (ESI): m/z 403 [M+H]+; HRMS (ESI): m/z
calcd for C20H19ClN2O5+H+ 403.1056, found 403.1061.
5.1.6. Ethyl 5-[2,4-dimethoxy-6-(4-nitrophenoxy)-phenyl]-
isoxazole-3-carboxylate (43a)
5.1.12. N-Ethyl-5-[2-methoxy-6-(4-nitrophenoxy) phenyl]-
isoxazole-3-carboxamide (45b)
To a stirred solution of 40a (3.48 g, 11 mmol) in THF (50 mL) at -
50 °C was added dropwise 1 M lithium bis(trimethylsilyl)amide in
THF (12 mL, 12 mmol). After 30 min, a solution of diethyl oxalate
(1.62 mL, 12 mmol) in THF (15 mL) was slowly added and the reac-
tion mixture was stirred for 2 h at room temperature. The solution
was poured into water (50 mL) and treated with 1 M HCl solution
(13 mL), then extracted with EtOAc. The organic phase was washed
with brine, dried over Na2SO4 and the solvent removed. The crude
dissolved in EtOH (50 mL) was treated with hydroxylamine hydro-
chloride (0.74 g, 15 mmol) and refluxed for 2 h. The solution was
concentrated to a small volume and the precipitate was collected
to provide 43a (3.5 g, 78%, 2 steps). 1H NMR (400 MHz, DMSO-d6)
d 8.22 (m, 2H), 7.12 (m, 2H), 6.90 (s, 1H), 6.74 (d, J = 2.2 Hz, 1H),
6.53 (d, J = 2.3 Hz, 1H), 4.33 (q, J = 7.2 Hz, 2H), 3.91 (s, 3H), 3.84
(s, 3H), 1.29 (t, J = 7.1 Hz, 3H); LC–MS (ESI): m/z 415 [M+H]+; HRMS
(ESI): m/z calcd for C20H18N2O8+H+ 415.1136, found 415.1136.
The following compounds 42, 43b and 43c were prepared
according to the method described above from 41, 40b and 40c.
1H NMR (400 MHz, DMSO-d6) d 8.72 (t, J = 5.73 Hz, 1H), 8.22 (m,
2H), 7.46 (t, J = 8.2 Hz, 1H), 7.20 (dd, J = 8.3, 0.9 Hz, 1H), 7.11 (m,
2H), 702 (s, 1H), 6.90 (dd, J = 8.3, 0.9 Hz, 1H), 3.91 (s, 3H), 3.24
(m, 2H), 1.09 (t, J = 7.2 Hz, 3H); LC–MS (ESI): m/z 384 [M+H]+;
HRMS (ESI): m/z calcd for
C
19H17N3O6+H+ 384.1190, found
384.1193.
5.1.13. N-Ethyl-5-[4-methoxy-2-(4-nitrophenoxy) phenyl]-
isoxazole-3-carboxamide (45c)
1H NMR (400 MHz, DMSO-d6) d 8.74 (t, J = 5.7 Hz, 1H), 8.27 (d,
J = 9.4 Hz, 2H), 8.02 (d, J = 8.8 Hz, 1H), 7.22 (d, J = 9.4 Hz, 2H),
7.10 (dd, J = 8.8, 2.5 Hz, 1H), 6.90 (d, J = 2.5 Hz, 1H), 6.85 (s, 1H),
3.83 (s, 3H), 3.23 (qd, J = 7.1, 6.0 Hz, 2H), 1.08 (t, J = 7.2 Hz, 3H);
LC–MS (ESI): m/z 384 [M+H]+; HRMS (ESI): m/z calcd for C19H17N3-
O6+H+ 384.1190, found 384.1189.
5.1.14. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy) phenyl]-N-ethyl-
isoxazole-3-carboxamide (3)
5.1.7. Ethyl 5-[2-(4-chlorophenoxy)-4,6-dimethoxyphenyl]-
isoxazole-3-carboxylate (42)
To a stirred solution of 45a (670 mg, 1.62 mmol) in DCM (5 mL)
was slowly added 1 M BBr3 in DCM (4.86 mL, 4.86 mmol) at 0 °C.
After stirring for 60 h at room temperature, 1 M BBr3 in DCM
(4.86 mL, 4.86 mmol) was slowly added. After stirring for 60 h at
room temperature, the cloudy solution was concentrated and then
diluted with EtOAc and thoroughly washed with water, then with
1 M sodium hydrogen carbonate solution and dried over Na2SO4.
The solvent was removed and the residue was purified by Biotage
SP1 flash chromatography (gradient elution from 0% to 10% of
MeOH in DCM) to afford 3 (305 mg, 48%). 1H NMR (400 MHz,
DMSO-d6) d 10.71 (s, 1H), 10.30 (s, 1H), 8.65 (t, 1H), 8.21 (d,
J = 9.4 Hz, 2H), 7.10 (d, J = 9.4 Hz, 2H), 6.79 (s, 1H), 6.45 (d,
J = 2.2 Hz, 1H), 6.10 (d, J = 2.2 Hz, 1H), 3.22 (qd, J = 7.2, 5.8 Hz,
2H), 1.07 (t, J = 7.2 Hz, 3H); LC–MS (ESI): m/z 386 [M+H]+; HRMS
(ESI): m/z calcd for C18H15N3O7+H+ 386.0983, found 386.0982.
1H NMR (400 MHz, DMSO-d6) d 7.41 (m, 2H), 7.01 (m, 2H), 6.90
(s, 1H), 6.63 (d, J = 2.2 Hz, 1H), 6.24 (d, J = 2.2 Hz, 1H), 4.36 (q,
J = 7.1 Hz, 2H), 3.88 (s, 3H), 3.80 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H);
LC–MS (ESI): m/z 404 [M+H]+; HRMS (ESI): m/z calcd for C20H18-
ClNO6+H+ 404.0896, found 404.08965.
5.1.8. Ethyl 5-[2-methoxy-6-(4-nitrophenoxy) phenyl]-
isoxazole-3-carboxylate (43b)
1H NMR (400 MHz, DMSO-d6) d 8.22 (d, J = 9.4 Hz, 2H), 7.66 (t,
J = 8.4 Hz, 1H), 7.20 (dd, J = 8.5, 0.6 Hz, 1H), 7.12 (d, J = 9.4 Hz,
2H), 7.02 (s, 1H), 6.91 (dd, J = 8.2, 0.8 Hz, 1H), 4.35 (q, J = 7.1 Hz,
2H), 3.91 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H); LC–MS (ESI): m/z 385
[M+H]+; HRMS (ESI): m/z calcd for C19H16N2O7+H+ 385.1031, found
385.1035.
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.09.018

M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
11
The following compounds 2, 4 and 5 were prepared according to
the method described above from 44, 45b and 45c.
stirring for 60 h at room temperature, 1 M BBr3 in DCM (23 mL,
23 mmol) was slowly added. After stirring for other 60 h at room
temperature, the cloudy solution was diluted with DCM and
washed with 1 M sodium hydrogen carbonate solution. The aque-
ous solution was acidified with citric acid to pH 5 and thoroughly
extracted with EtOAc, and dried over Na2SO4. The solvent was re-
moved and the residue was purified twice by Biotage SP1 flash
chromatography (gradient elution from 2% to 20% of MeOH in
DCM) to afford 47 (480 mg, 29%). 1H NMR (400 MHz, DMSO-d6) d
10.72 (s, 1H), 10.31 (s, 1H), 8.22 (d, J = 9.3 Hz, 2H), 7.11 (d,
J = 9.3 Hz, 2H), 6.81 (s, 1H), 6.45 (d, J = 2.2 Hz, 1H), 6.09 (d,
J = 2.3 Hz, 1H); LC–MS (ESI): m/z 359 [M+H]+; HRMS (ESI): m/z
calcd for C16H10N2O8+H+ 359.0510, found 359.0514.
5.1.15. 5-[2-(4-Chlorophenoxy)-4,6-dihydroxy-phenyl]-N-ethyl-
isoxazole-3-carboxamide (2)
1H NMR (400 MHz, DMSO-d6) d 10.47 (s, 1H), 10.07 (s, 1H), 8.66
(t, J = 5.8 Hz, 1H), 7.41 (m, 2H), 7.01 (m, 2H), 6.77 (m, 1H), 6.32 (d,
J = 2.3 Hz, 1H), 5.87 (d, J = 2.3 Hz, 1H), 3.27 (m, 2H), 1.10 (t,
J = 7.1 Hz, 3H); LC–MS (ESI): m/z 375 [M+H]+; HRMS (ESI): m/z
calcd for C18H15ClN2O5+H+ 375.0743, found 375.0748.
5.1.16. N-Ethyl-5-[2-hydroxy-6-(4-nitrophenoxy) phenyl]-
isoxazole-3-carboxamide (4)
1H NMR (400 MHz, DMSO-d6) d 10.88 (s, 1H), 8.72 (t, J = 5.7 Hz,
1H), 8.23 (m, 2H), 7.46 (t, J = 8.2 Hz, 1H), 7.10 (m, 2H), 7.01 (dd,
J = 8.4, 0.9 Hz, 1H), 6.95 (s, 1H), 6.75 (dd, J = 8.2, 0.8 Hz, 1H), 3.24
(m, 2H), 1.09 (t, J = 7.2 Hz, 3H); LC–MS (ESI): m/z 370 [M+H]+;
5.1.21. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy) phenyl]-N-(2-
fluoroethyl)-isoxazole-3-carboxamide (8)
To a stirred solution of 47 (107 mg, 0.3 mmol) in dimethylacet-
amide (3 mL) was added N,N,N0,N0-tetramethyl-O-(benzotriazol-1-
yl)uronium tetrafluoroborate (TBTU) (115 mg, 0.36 mmol) After
stirring for 15 min at room temperature, N,N-diisopropylethyl-
amine (0.104 mL, 0.6 mmol) and 2-fluoroethylamine hydrochlo-
ride (45 mg, 0.45 mmol) were added. After stirring overnight, the
reaction mixture was poured into a saturated solution of sodium
hydrogen carbonate and thoroughly extracted with EtOAc, washed
with brine, dried over Na2SO4 and evaporated to dryness. The res-
idue was purified by Biotage SP1 flash chromatography (gradient
elution from 2% to 10% of MeOH in DCM) to afford 8 (86 mg,
71%). 1H NMR (400 MHz, DMSO-d6) d 10.73 (s, 1H), 10.31 (s, 1H),
8.83 (t, 1H), 8.22 (d, J = 9.3 Hz, 2H), 7.11 (d, J = 9.3 Hz, 2H), 6.82
(s, 1H), 6.46 (d, J = 2.2 Hz, 1H), 6.10 (d, J = 2.2 Hz, 1H), 4.49 (dt,
J(H,F) = 47.4 Hz, J = 5.1 Hz, 2H), 3.54 (q, J = 5.3 Hz, 1H), 3.48 (q,
J = 5.3 Hz, 1H); LC–MS (ESI): m/z 404 [M+H]+; HRMS (ESI): m/z
calcd for C18H14FN3O7+H+ 404.0889, found 404.0885.
HRMS (ESI): m/z calcd for
C
18H15N3O6+H+ 370.1034, found
370.1036
5.1.17. N-Ethyl-5-[4-hydroxy-2-(4-nitrophenoxy) phenyl]-
isoxazole-3-carboxamide (5)
1H NMR (400 MHz, DMSO-d6) d 10.55 (s, 1H), 8.72 (t, J = 5.5, 1H),
8.28 (d, J = 9.4 Hz, 2H), 7.90 (d, J = 8.7 Hz, 1H), 7.24 (d, J = 9.3 Hz, 2H),
6.89 (dd, J = 8.7, 2.4 Hz, 1H), 6.79 (s, 1H), 6.59 (d, J = 2.3 Hz, 1H), 3.23
(qd, J = 7.2, 5.7 Hz, 2H), 1.08 (t, 3H); LC–MS (ESI): m/z 370 [M+H]+;
HRMS (ESI): m/z calcd for C18H15N3O6+H+ 370.1034, found 370.1037.
5.1.18. 5-[2-(4-Chlorophenoxy)-4,6-dimethoxy-phenyl]-
isoxazole (46)
To a stirred solution of 41 (306 mg, 1.0 mmol) in THF (5 mL) at
ꢀ50 °C was added dropwise 1 M lithium bis(trimethylsilyl)amide
in THF (1.1 mL, 1.1 mmol). After 30 min, a solution of ethyl formate
(0.096 mL, 1.2 mmol) was slowly added and the reaction mixture
was stirred for 2 h at room temperature. The solution was poured
into water (10 mL) and treated with 1 M HCl solution (1.2 mL), ex-
tracted with EtOAc. The organic phase was washed with brine,
dried over Na2SO4 and the solvent removed. The crude dissolved
in ethanol (5 mL) was treated with hydroxylamine hydrochloride
(90.0 mg, 1.3 mmol) and refluxed for 2 h. The solution was concen-
trated to a small volume and the precipitate was collected to pro-
vide 46 (268 mg, 81%, 2 steps). 1H NMR (400 MHz, DMSO-d6) d 8.51
(d, J = 1.9 Hz, 1H), 7.38 (m, 2H), 6.95 (m, 2H), 6.61 (d, J = 2.3 Hz,
1H), 6.50 (d, J = 1.8 Hz, 1H), 6.26 (d, J = 2.3 Hz, 1H), 3.86 (s, 3H),
3.79 (s, 3H); LC–MS (ESI): m/z 332 [M+H]+; HRMS (ESI): m/z calcd
for C17H14ClNO4+H+ 332.0684, found 332.0688.
The following compounds 7, 9–16, 18–20, 29 were prepared
according to the method described above using the suitable amine.
5.1.22. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy) phenyl]-N,N-
dimethyl-isoxazole-3-carboxamide (7)
1H NMR1H NMR (400 MHz, DMSO-d6) d 10.67 (br s, 1H), 10.30
(br s, 1H), 8.20 (d, J = 9.4 Hz, 2H), 7.10 (d, J = 9.4 Hz, 2H), 6.68 (s,
1H), 6.44 (d, J = 2.2 Hz, 1H), 6.10 (d, J = 2.3 Hz, 1H), 2.96 (s, 3H),
2.95 (s, 3H); LC–MS (ESI): m/z 386 [M+H]+; HRMS (ESI): m/z calcd
for C18H15N3O7+H+ 386.0983, found 386.0991.
5.1.23. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy) phenyl]-N-(2-
methoxyethyl)-isoxazole-3-carboxamide (9)
1H NMR (400 MHz, DMSO-d6) d 10.72 (s, 1H), 10.30 (s, 1H), 8.60
(t, J = 5.4 Hz, 1H), 8.21 (m, 2H), 7.10 (m, 2H), 6.81 (s, 1H), 6.45 (d,
J = 2.2 Hz, 1H), 6.10 (d, J = 2.2 Hz, 1H), 3.40 (m, 4H), 3.23 (s, 3H);
LC–MS (ESI): m/z 416 [M+H]+; HRMS (ESI): m/z calcd for C19H17N3-
O8+H+ 416.1089, found 416.1093.
5.1.19. 5-(4-Chlorophenoxy)-4-(isoxazol-5-yl) benzene-1,3-diol
(6)
To a stirred solution of 46 (87 mg, 0.26 mmol) in DCM (2 mL) was
slowly added 1 M BBr3 in DCM (0.78 mL, 0.78 mmol) at 0 °C. After
stirring for 120 h at room temperature, the cloudy solution was di-
luted with DCM and thoroughly washed with water, then with 1 M
sodium hydrogencarbonate solutionand driedover Na2SO4. The sol-
vent was removed and the residue was purified by Biotage SP1 flash
chromatography (gradient elution from 0% to 10% of MeOH in DCM)
to afford 6 (36 mg, 45%). 1H NMR (400 MHz, DMSO-d6) d 10.29 (s,
1H), 9.97 (s, 1H), 8.45 (d, J = 1.8 Hz, 1H), 7.38 (d, J = 9.0 Hz, 2H),
6.97 (d, J = 9.0 Hz, 2H), 6.49 (d, J = 1.8 Hz, 1H), 6.30 (d, J = 2.3 Hz,
1H), 5.86 (d, J = 2.2 Hz, 1H); LC–MS (ESI): m/z 304 [M+H]+; HRMS
(ESI): m/z calcd for C15H10ClNO4+H+ 304.0371, found 304.0374.
5.1.24. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy) phenyl]-N-(2-
hydroxyethyl)-isoxazole-3-carboxamide (10)
1H NMR (400 MHz, DMSO-d6) d 10.72 (br s, 1H), 10.31 (br s, 1H),
8.50 (t, J = 5.7 Hz, 1H), 8.21 (m, 2H), 7.10 (m, 2H), 6.81 (s, 1H), 6.45
(d, J = 2.2 Hz, 1H), 6.10 (d, J = 2.2 Hz, 1H), 4.69 (t, J = 5.6 Hz, 1H),
3.46 (q, J = 6.1 Hz, 2H), 3.29 (m, 2H); LC–MS (ESI): m/z 402
[M+H]+; HRMS (ESI): m/z calcd for C18H15N3O8+H+ 402.0932, found
402.0931.
5.1.25. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy) phenyl]-N-[3-
(dimethylamino)propyl]-isoxazole-3-carboxamide (11)
1H NMR (400 MHz, DMSO-d6) d 10.75 (s, 1H), 10.34 (s, 1H), 9.19
(br s, 1H), 8.80 (t, J = 5.8 Hz, 1H), 8.22 (m, 2H), 7.10 (m, 2H), 6.81 (s,
1H), 6.46 (d, J = 2.2 Hz, 1H), 6.11 (d, J = 2.3 Hz, 1H), 3.28 (m, 2H),
5.1.20. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy)phenyl] -isoxazole-
3-carboxylic acid (47)
To a stirred solution of 43a (1.9 g, 4.6 mmol) in DCM (20 mL)
was slowly added 1 M BBr3 in DCM (23 mL, 23 mmol) at 0 °C. After
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.09.018

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M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3.03 (m, 2H), 2.75 (d, J = 4.9 Hz, 6H), 1.81 (m, 2H); LC–MS (ESI): m/z
443 [M+H]+; HRMS (ESI): m/z calcd for C21H22N4O7+H+ 443.1562,
found 443.1566.
J = 2.2 Hz, 1H), 6.11 (d, J = 2.2 Hz, 1H), 4.09–4.32 (m, 1H), 2.73 (br
s, 2H), 1.53–2.27 (m, 8H), 0.96–1.50 (m, 12H); LC–MS (ESI): m/z
511 [M+H]+; HRMS (ESI): m/z calcd for C26H30N4O7+H+ 511.2188,
found 511.2176.
5.1.26. N-Cyclohexyl-5-[2,4-dihydroxy-6-(4-
nitrophenoxy)phenyl]-isoxazole-3-carboxamide (12)
1H NMR (400 MHz, DMSO-d6) d 10.71 (s, 1H), 10.30 (s, 1H), 8.43
(d, J = 8.3 Hz, 1H), 8.22 (m, 2H), 7.10 (m, 2H), 6.79 (s, 1H), 6.45 (d,
J = 2.2 Hz, 1H), 6.10 (d, J = 2.2 Hz, 1H), 3.61–3.75 (m, 1H), 1.66–1.76
(m, 4H), 1.53–1.60 (m, 1H), 1.21–1.33 (m, 4H), 1.05–1.12 (m, 1H);
LC–MS (ESI): m/z 440 [M+H]+; HRMS (ESI): m/z calcd for C22H21N3-
O7+H+ 440.1453, found 440.1456.
5.1.33. N-(1-Acetylpiperidin-4-yl)-5-[2,4-dihydroxy-6-(4-
nitrophenoxy)phenyl]-isoxazole-3-carboxamide (20)
1H NMR (400 MHz, DMSO-d6) d 10.72 (s, 1H), 10.31 (s, 1H), 8.60
(d, J = 7.9 Hz, 1H), 8.22 (d, J = 9.3 Hz, 2H), 7.10 (d, J = 9.3 Hz, 2H),
6.81 (s, 1H), 6.46 (d, J = 2.2 Hz, 1H), 6.10 (d, J = 2.3 Hz, 1H), 4.31
(m, 1H), 3.96 (m, 1H), 3.79 (m, 1H), 3.08 (m, 1H), 2.62 (td,
J = 12.7, 2.9 Hz, 1H), 1.98 (s, 3H), 1.73 (m, 2H), 1.46 (m, 1H), 1.37
(m, 1H); LC–MS (ESI): m/z 483 [M+H]+; HRMS (ESI): m/z calcd for
C23H22N4O8+H+ 483.1511, found 483.1517.
5.1.27. N-Benzyl-5-[2,4-dihydroxy-6-(4-nitrophenoxy)phenyl]-
isoxazole-3-carboxamide (13)
1H NMR (400 MHz, DMSO-d6) d 10.72 (s, 1H), 10.31 (s, 1H), 9.23
(t, J = 6.2 Hz, 1H), 8.21 (d, J = 9.2 Hz, 2H), 7.11 (d, J = 9.2 Hz, 2H),
6.83 (s, 1H), 6.45 (d, J = 2.2 Hz, 1H), 6.10 (d, J = 2.2 Hz, 1H), 4.40
(d, J = 6.2 Hz, 2H); LC–MS (ESI): m/z 448 [M+H]+; HRMS (ESI): m/z
calcd for C23H17N3O7+H+ 448.1140, found 448.1145.
5.1.34. N-(1-Benzylpiperidin-4-yl)-5-[2,4-dihydroxy-6-(4-
nitrophenoxy)phenyl]-isoxazole-3-carboxamide (29)
1H NMR (400 MHz, DMSO-d6) d 10.74 (br s, 1H), 10.33 (s, 1H),
8.21 (d, J = 9.3 Hz, 2H), 7.18–7.63 (br s, 5H),7.09 (d, J = 9.3 Hz,
2H), 6.80 (s, 1H), 6.47 (d, J = 2.2 Hz, 1H), 6.10 (d, J = 2.2 Hz, 1H),
3.70 (m, 1H), 2.80 (m, 2H), 2.21 (s, 3H), 2.01 (m, 2H), 1.72 (m,
2H), 1.61 (m, 2H); LC–MS (ESI): m/z 531 [M+H]+; HRMS (ESI): m/
z calcd for C28H26N4O7+H+ 531.1875, found 531.1881.
5.1.28. N-(Cyclohexylmethyl)-5-[2,4-dihydroxy-6-(4-
nitrophenoxy)phenyl]-isoxazole-3-carboxamide (14)
1H NMR (400 MHz, DMSO-d6) d 10.71 (s, 1H), 10.30 (s, 1H), 8.60
(t, J = 6.1 Hz, 1H), 8.21 (d, J = 9.3 Hz, 2H), 7.10 (d, J = 9.3 Hz, 2H),
6.79 (s, 1H), 6.45 (d, J = 2.3 Hz, 1H), 6.10 (d, J = 2.2 Hz, 1H), 3.04
(t, J = 6.5 Hz, 2H), 1.64 (m, 5H), 1.50 (m, 1H), 1.14 (m, 4H), 0.87
(m, 1H); LC–MS (ESI): m/z 454 [M+H]+; HRMS (ESI): m/z calcd for
5.1.35. 1-[2-Hydroxy-4,6-bis(methoxymethoxy)
phenyl]ethanone (49)
To a stirred solution of dimethoxymethane (66 mL, 745 mmol)
and zinc bromide (447 mg, 1.12 mmol) in DCM (580 mL) was
added dropwise acetyl chloride (53 mL, 745 mmol) during 30 min
maintaining the temperature below 30 °C. After stirring 3 h at
room temperature, the solution was diluted with DCM (1.2 L), then
cooled at 5 °C before the portion wise addition of 1-(2,4,6-trihydr-
oxyphenyl)ethanone (48, 50.0 g, 298 mmol) followed by the drop-
wise addition of N,N-diisopropylethylamine (208 mL, 1.19 mol).
After 1 h the ice bath was removed and the temperature was al-
lowed to rise to room temperature. The resulting cloudy solution
was stirred overnight, and then was washed with NH4Cl saturated
solution, followed by washing with 10% citric acid solution. After
drying over Na2SO4, the solvent was removed to give 49 (78.0 g,
100%). 1H NMR (400 MHz, DMSO-d6) d 13.31 (s, 1H), 6.24 (d,
J = 2.3 Hz, 1H), 6.19 (d, J = 2.3 Hz, 1H), 6.19 (d, J = 2.3 Hz, 1H),
5.30 (s, 2H), 5.22 (s, 2H), 3.44 (s, 3H), 3.38 (s, 3H), 2.60 (s, 3H);
LC–MS (ESI): m/z 257 [M+H]+; HRMS (ESI): m/z calcd for
C
23H23N3O7+H+ 454.1609, found 454.1608.
5.1.29. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy) phenyl]-N-
(piperidin-4-ylmethyl)-isoxazole-3-carboxamide
trifluoroacetate (15)
Compound 15 was obtained from tert-butyl 4-{[({5-[2,4-dihy-
droxy-6-(4-nitrophenoxy)
bonyl)amino]methyl}
phenyl]-isoxazol-3-yl}car-
piperidine-1-carboxylate (prepared
according to the method described above) after treatment with
50% CF3COOH in DCM. 1H NMR (400 MHz, DMSO-d6) d 10.74 (s,
1H), 10.33 (s, 1H), 8.77 (t, J = 6.1 Hz, 1H), 8.42 (d, J = 9.6 Hz, 1H),
8.22 (d, J = 9.3 Hz, 2H), 8.07 (d, J = 9.9 Hz, 1H), 7.10 (d, J = 9.3 Hz,
2H), 6.80 (s, 1H), 6.46 (d, J = 2.3 Hz, 1H), 6.11 (d, J = 2.2 Hz, 1H),
3.24 (m, 2H), 3.13 (t, J = 6.3 Hz, 2H), 2.82 (m, 2H), 1.82 (m, 1H),
1.76 (m, 2H), 1.28 (m, 2H); LC–MS (ESI): m/z 455 [M+H]+; HRMS
(ESI): m/z calcd for C22H22N4O7+H+ 455.1562, found 455.1564.
C
12H16O6+H+ 257.1020, found 257.1019.
5.1.30. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy) phenyl]-N-[(1-
methylpiperidin-4-yl)methyl]-isoxazole-3-carboxamide (16)
1H NMR (400 MHz, DMSO-d6) d 10.75 (s, 1H), 10.34 (s, 1H), 8.77
(t, J = 6.0 Hz, 1H), 8.22 (m, 2H), 7.10 (m, 2H), 6.80 (s, 1H), 6.46 (d,
J = 2.2 Hz, 1H), 6.11 (d, J = 2.3 Hz, 1H), 3.26–3.46 (m, 2H), 3.12 (t,
J = 6.3 Hz, 2H), 2.88 (m, 2H), 2.25 (s, 3H), 1.79 (m, 2H), 1.74 (m,
1H), 1.29 (m, 2H); LC–MS (ESI): m/z 469 [M+H]+; HRMS (ESI): m/
z calcd for C23H24N4O7+H+ 469.1718, found 469.1722.
5.1.36. 1-[2,4-Bis(methoxymethoxy)-6-(4-
nitrophenoxy)phenyl]ethanone (50)
To a stirred solution of 49 (78.0 g, 298 mmol) in DMSO (500 mL)
4-nitro-1-fluorobenzene (46.3 g, 328 mmol) was added, followed
by water (40 mL) and K2CO3 (45.3 g, 328 mmol). After stirring for
15 min at room temperature, the resulting suspension was heated
for 6 h at 55 °C. After cooling, the dark solution was diluted with
EtOAc (2.0 L) and thoroughly washed with 10% citric acid solution,
then with brine and dried over Na2SO4. The solvent was removed
and the residue was purified by flash chromatography (eluent:
cyclohexane/EtOAc 3:1) to provide 50 (56.17 g, 50%). 1H NMR
(400 MHz, DMSO-d6) d 8.17–8.32 (m, 2H), 6.98–7.23 (m, 2H),
6.76 (d, J = 2.1 Hz, 1H), 6.47 (d, J = 2.1 Hz, 1H), 5.28 (s, 2H), 5.19
(s, 2H), 3.41 (s, 3H), 3.37 (s, 3H), 2.38 (s, 3H); LC–MS (ESI): m/z
378 [M+H]+; HRMS (ESI): m/z calcd for C18H19NO8+H+ 378.1184,
found 378.1187.
5.1.31. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy) phenyl]-N-(1-
methylpiperidin-4-yl)-isoxazole-3-carboxamide (18)
1H NMR (400 MHz, DMSO-d6) d 10.71 (br s, 1H), 10.31 (br s, 1H),
8.54 (d, J = 7.9 Hz, 1H), 8.22 (m, 2H), 7.10 (m, 2H), 6.80 (s, 1H), 6.46
(d, J = 2.2 Hz, 1H), 6.10 (d, J = 2.2 Hz, 1H), 3.70 (m, 1H), 2.80 (m,
2H), 2.21 (s, 3H), 2.01 (m, 2H), 1.72 (m, 2H), 1.61 (m, 2H); LC–MS
(ESI): m/z 455 [M+H]+; HRMS (ESI): m/z calcd for C22H22N4O7+H+
455.1562, found 455.1563.
5.1.32. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy) phenyl]-N-
(1,2,2,6,6-pentamethylpiperidin-4-yl)-isoxazole-3-carboxamide
(19)
5.1.37. Ethyl 5-[2,4-bis(methoxymethoxy)-6-(4-
nitrophenoxy)phenyl]-isoxazole-3-carboxylate (51)
To a stirred solution of sodium tert-butoxide (31.5 g, 328 mmol)
in THF (250 mL) at ꢀ10 °C diethyl oxalate (60.5 mL, 447 mmol) in
1H NMR (400 MHz, DMSO-d6) d 10.75 (br s, 1H), 10.33 (br s, 1H),
8.17–8.27 (m, 2H), 7.04–7.15 (m, 2H), 6.80 (s, 1H), 6.47 (d,
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.09.018

M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
13
250 mL of precooled THF was added dropwise. After 30 min a solu-
tion of 50 (56.0 g, 149 mmol) in THF (350 mL) was added dropwise.
The reaction mixture was stirred for 1 h at ꢀ10 °C and then for fur-
ther 2 h at room temperature. The solution was poured into a 10%
citric acid solution (600 mL) and thoroughly extracted with EtOAc.
After washing with brine and drying over Na2SO4, the solvent was
removed to provide a yellowish residue that was taken up in light
petrol ether to remove the excess of diethyl oxalate to afford the
quite pure ethyl (2E)-4-[2,4-bis(methoxymethoxy)-6-(4-nitro-
phenoxy)phenyl]-2-hydroxy-4-oxobut-2-enoate, which was used
directly in the next synthetic step. To a stirred solution of the crude
(71.0 g, 149 mmol) in THF (250 mL) and 95° ethanol (500 mL) so-
dium acetate (24.4 g, 298 mmol) and hydroxylamine hydrochlo-
ride (22.8 g, 328 mmol) were added at room temperature. After
2 h, 6 M HCl solution was added dropwise to reach pH 2. After stir-
ring for 24 h, saturated solution of Na2HPO4 was added to reach pH
6 and the suspension taken up in EtOAc was thoroughly washed
with brine and dried over Na2SO4. The solvent was removed and
the residue was crystallized from a mixture of MTBE (240 mL)
and hexane (180 mL), to provide 51 (35.3 g, 50%, 2 steps). 1H
NMR (400 MHz, DMSO-d6) d 8.18–8.35 (m, 2H), 7.06–7.24 (m,
2H), 6.96 (s, 1H), 6.89 (d, J = 2.2 Hz, 1H), 6.62 (d, J = 2.2 Hz, 1H),
5.33 (s, 2H), 5.26 (s, 2H), 4.34 (q, J = 7.1 Hz, 2H), 3.39 (s, 3H), 3.38
(s, 3H), 1.29 (t, J = 7.1 Hz, 3H); LC–MS (ESI): m/z 475 [M+H]+; HRMS
(ESI): m/z calcd for C22H22N2O10+H+ 475.1347, found 475.1346.
(d, J = 7.9 Hz, 1H), 8.22 (d, J = 9.3 Hz, 2H), 7.10 (d, J = 9.3 Hz, 2H),
6.80 (s, 1H), 6.45 (d, J = 2.2 Hz, 1H), 6.10 (d, J = 2.2 Hz, 1H), 3.69 (m,
1H), 2.80 (m, 2H), 2.31 (t, J = 7.0 Hz, 2H), 2.15–2.28 (m, 2H), 1.94
(td, J = 11.7, 1.8 Hz, 2H), 1.70 (m, 2H), 1.61 (m, 2H), 1.55 (qd,
J = 11.8, 3.2 Hz, 2H); LC–MS (ESI): m/z 551 [M+H]+; HRMS (ESI): m/
z calcd for C25H25F3N4O7+H+ 551.1748, found 551.1745.
5.1.41. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy) phenyl]-N-[1-
(propan-2-yl)piperidin-4-yl]-isoxazole-3-carboxamide (22)
To a stirred solution of 17 (100 mg, 0.23 mmol) and acetone
(0.033 mL, 0.46 mmol) in DMF (3 mL) was added portionwise tet-
ramethylammonium triacetoxyborohydride (121 mg, 0.46 mmol).
After stirring at room temperature overnight, the solution was ta-
ken up in EtOAc and thoroughly washed with brine. After drying
over Na2SO4, the solvent was removed and the residue was purified
by flash chromatography (gradient elution from 10% to 20% of
MeOH in DCM) to provide 22 (81 mg, 73%). 1H NMR (400 MHz,
DMSO-d6) d 10.77 (br s, 1H), 10.35 (br s, 1H), 8.63 (br s, 1H), 8.22
(m, 2H), 7.10 (m, 2H), 6.81 (s, 1H), 6.49 (d, J = 2.2 Hz, 1H), 6.11
(d, J = 2.2 Hz, 1H), 3.79 (m, 1H), 2.94 (br s, 3H), 1.56–1.87 (m,
6H), 1.04 (br s, 3H); LC–MS (ESI): m/z 483 [M+H]+; HRMS (ESI):
m/z calcd for C24H26N4O7+H+ 483.1875, found 483.1872.
The following compounds 23–28 were prepared according to
the method described above using the suitable aldehyde or ketone.
5.1.42. N-(1-Cyclohexylpiperidin-4-yl)-5-[2,4-dihydroxy-6-(4-
nitrophenoxy)phenyl]-isoxazole-3-carboxamide (23)
5.1.38. tert-Butyl 4-[({5-[2,4-bis(methoxymethoxy)-6-(4-
nitrophenoxy)phenyl]-isoxazol-3-
yl}carbonyl)amino]piperidine-1-carboxylate (52)
1H NMR (400 MHz, DMSO-d6) d 10.72 (br s, 1H), 10.30 (br s, 1H),
8.52 (br s, 1H), 8.22 (m, 2H), 7.10 (m, 2H), 6.80 (s, 1H), 6.46 (d,
J = 2.2 Hz, 1H), 6.10 (d, J = 2.2 Hz, 1H), 3.69 (br s, 1H), 2.85 (br s,
2H), 2.27 (br s, 3H), 1.73 (m, 5H), 1.57 (m, 3H), 1.20 (m, 2H),
1.07 (m, 1H); LC–MS (ESI): m/z 523 [M+H]+; HRMS (ESI): m/z calcd
for C27H30N4O7+H+ 523.2188, found 523.2190.
51 (5.0 g, 10.5 mmol) and tert-butyl 4-aminopiperidine-1-car-
boxylate (8.0 g, 40 mmol) were dissolved in DCM (30 mL). The sol-
vent was removed and the reaction mixture was stirred at 80 °C for
24 h. After cooling to room temperature, the residue was purified
by Biotage SP1 flash chromatography (hexane/EtOAc 80:20) to af-
ford 52 (6.1 g, 97%). 1H NMR (400 MHz, DMSO-d6) d 8.65 (d,
J = 8.2 Hz, 1H), 8.22 (d, J = 9.3 Hz, 2H), 7.13 (d, J = 9.3 Hz, 2H),
6.88 (d, J = 2.2 Hz, 1H), 6.86 (s, 1H), 6.62 (d, J = 2.3 Hz, 1H), 5.33
(s, 2H), 5.25 (s, 2H), 3.82–3.98 (m, 3H), 3.39 (s, 3H), 3.38 (s, 3H),
2.79 (br s, 2H), 1.71 (m, 2H), 1.39 (s, 9H), 1.41 (qd, J = 12.2,
4.4 Hz, 2H); LC–MS (ESI): m/z 629 [M+H]+; HRMS (ESI): m/z calcd
for C30H36N4O11+H+ 629.2454, found 629.2451.
5.1.43. N-(1-Cyclopentylpiperidin-4-yl)-5-[2,4-dihydroxy-6-(4-
nitrophenoxy)phenyl]-isoxazole-3-carboxamide (24)
1H NMR (400 MHz, DMSO-d6) d 10.70 (br s, 1H), 10.31 (br s, 1H),
8.53(d, J = 8.7 Hz, 1H), 8.22 (m, 2H), 7.10(m, 2H), 6.80(s, 1H), 6.46 (d,
J = 2.2 Hz, 1H), 6.10 (d, J = 2.2 Hz, 1H), 3.71 (m, 1H), 2.94 (br s, 2H),
1.97 (br s, 2H), 1.17–1.82 (m, 13H); LC–MS (ESI): m/z 509 [M+H]+;
HRMS(ESI): m/z calcdfor C26H28N4O7+H+ 509.2031, found509.2028.
5.1.39. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy) phenyl]-N-
5.1.44. N-(1-Cycloheptylpiperidin-4-yl)-5-[2,4-dihydroxy-6-(4-
nitrophenoxy)phenyl]-isoxazole-3-carboxamide (25)
1H NMR (400 MHz, DMSO-d6) d 10.73 (br s, 1H), 10.32 (br s, 1H),
8.54 (br s, 1H), 8.22 (m, 2H), 7.10 (m, 2H), 6.80 (s, 1H), 6.46 (d,
J = 2.3 Hz, 1H), 6.10 (d, J = 2.3 Hz, 1H), 3.71 (br s, 1H), 2.77 (br s,
2H), 2.21 (br s, 1H), 1.24–1.88 (m, 16H); LC–MS (ESI): m/z 537
[M+H]+; HRMS (ESI): m/z calcd for C28H32N4O7+H+ 537.2344, found
537.2347.
(piperidin-4-yl)-isoxazole-3-carboxamide hydrochloride (17)
To a solution of 52 (2.0 g, 3.2 mmol) in ethanol (8 mL) was
added 4 M HCl in dioxane (8 mL) at room temperature. The solu-
tion was set aside for 5 h then the solvent was evaporated off
and the residue taken up in diethyl ether to provide 17 (1.41 g,
93%). 1H NMR (400 MHz, DMSO-d6) d 10.77 (s, 1H), 10.35 (s, 1H),
8.82 (d, J = 7.8 Hz, 1H), 8.60 (br s, 1H), 8.34 (br s, 1H), 8.22 (m,
2H), 7.19 (m, 2H), 6.83 (s, 1H), 6.49 (d, J = 2.2 Hz, 1H), 6.11 (d,
J = 2.2 Hz, 1H), 4.05 (m, 1H), 3.25 (m, 2H), 2.98 (m, 2H), 1.91 (m,
2H), 1.72 (m, 2H); LC–MS (ESI): m/z 441 [M+H]+; HRMS (ESI): m/
z calcd for C21H20N4O7+H+ 441.1405, found 441.1401.
5.1.45. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy)phenyl]-N-(1’-
methyl-1,4’-bipiperidin-4-yl)-isoxazole-3-carboxamide (26)
1H NMR (400 MHz, DMSO-d6) d 8.45 (d, J = 8.1 Hz, 1H), 8.21 (d,
J = 9.3 Hz, 2H), 7.08 (d, J = 9.3 Hz, 2H), 6.86 (s, 1H), 6.37 (br s, 1H),
5.98 (br s, 1H), 3.66 (m, 1H), 2.82 (m, 2H), 2.76 (m, 2H), 2.12 (m,
2H), 2.11 (s, 3H), 1.80 (td, J = 11.9, 1.9 Hz, 2H), 1.59–1.73 (m, 4H),
1.50 (qd, J = 11.9, 3.9 Hz, 2H), 1.39 (qd, J = 12.1, 3.3 Hz, 2H); LC–
MS (ESI): m/z 538 [M+H]+; HRMS (ESI): m/z calcd for C27H31N5O7+-
H+ 538.2297, found 538.2295.
5.1.40. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy) phenyl]-N-[1-
(4,4,4-trifluorobutyl)piperidin-4-yl]-isoxazole-3-carboxamide
(21)
A solution of 17 (100 mg, 0.23 mmol), N,N-diisopropylethyl-
amine (0.157 mL, 0.92 mmol) and 1-bromo-4,4,4-trifluorobutane
(0.036 mL, 0.27 mmol) in DMF (3 mL) was stirred at room tempera-
ture overnight. The solution was taken up in EtOAc and thoroughly
washed with brine, then dried over Na2SO4. After removal of the sol-
vent, the residue was purified by flash chromatography (eluent:
DCM/MeOH/acetone 90:5:5) to provide 21 (56 mg, 44% yield). 1H
NMR (400 MHz, DMSO-d6) d 10.72 (br s, 1H), 10.30 (br s, 1H), 8.51
5.1.46. 5-[2,4-Dihydroxy-6-(4-nitrophenoxy) phenyl]-N-[1-(1,4-
dioxaspiro[4.5]dec-8-yl)piperidin-4-yl]-isoxazole-3-
carboxamide (27)
1H NMR (400 MHz, DMSO-d6) d 10.71 (br s, 1H), 10.30 (br s, 1H),
8.52 (br s, 1H), 8.22 (m, 2H), 7.10 (m, 2H), 6.80 (s, 1H), 6.46 (d,
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.09.018

14
M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
J = 2.2 Hz, 1H), 6.10 (d, J = 2.2 Hz, 1H), 3.83 (s, 4H), 3.67 (br s, 1H),
2.80 (br s, 2H), 2.18 (br s, 2H), 1.41–1.75(m, 11H); LC–MS (ESI): m/z
581 [M+H]+; HRMS (ESI): m/z calcd for C29H32N4O9+H+ 581.2242,
found 581.2238.
51%, 2 steps). 1H NMR (400 MHz, DMSO-d6) d 10.19 (br s, 1H),
9.80 (br s, 1H), 8.52 (d, J = 8.1 Hz, 1H), 6.90 (m, 2H), 6.77 (s, 1H),
6.74 (m, 2H), 6.14 (d, J = 2.2 Hz, 1H), 5.65 (d, J = 2.2 Hz, 1H),
3.72 (m, 1H), 2.87 (s, 6H), 2.75 (m, 2H), 2.15 (s, 3H), 1.91 (m,
2H), 1.70 (m, 2H), 1.61 (m, 2H); LC–MS (ESI): m/z 453 [M+H]+;
HRMS (ESI): m/z calcd for C24H28N4O5+H++H+ 453.2133, found
453.2131.
5.1.47. N-[1-(Cyclohexylmethyl)piperidin-4-yl]-5-[2,4-
dihydroxy-6-(4-nitrophenoxy)phenyl]-isoxazole-3-
carboxamide (28)
1H NMR (400 MHz, DMSO-d6) d 10.75 (br s, 1H), 10.34 (s, 1H),
8.22 (d, J = 9.3 Hz, 1H), 7.10 (d, J = 9.3 Hz, 2H), 6.82 (s, 1H), 6.48
(d, J = 2.1 Hz, 1H), 6.11 (d, J = 2.1 Hz, 1H), 2.71–4.24 (m, 7H),
0.73–2.06 (m, 15H); LC–MS (ESI): m/z 537 [M+H]+; HRMS (ESI):
m/z calcd for C28H32N4O7+H++H+ 537.2344, found 537.2344.
5.1.51. 5-{2,4-Dihydroxy-6-[4-(pyrrolidin-1-
yl)phenoxy]phenyl}-N-(1-methylpiperidin-4-yl)-isoxazole-3-
carboxamide (32)
To stirred solution of 54 (153 mg, 0.3 mmol), 2,5-dimethoxytet-
rahydrofuran (0.077 mL, 0.6 mmol) and 2.5 M sulfuric acid solution
(0.3 mL, 0.75 mmol) in MeOH/tetrahydrofuran (1:1, 5 mL) at 5 °C
was added portion wise sodium borohydride (46 mg, 1.2 mmol).
After stirring overnight at room temperature, the solvent was re-
moved and the residue taken up in EtOAc was washed with a sat-
urated solution of sodium hydrogen carbonate. After drying over
Na2SO4, the solvent was removed. The residue was purified by Bio-
tage SP1 flash chromatography (gradient elution from 2% to 10% of
MeOH in DCM) to afford 5-{2,4-bis(methoxymethoxy)-6-[4-(pyrr-
olidin-1-yl)phenoxy]phenyl}-N-(1-methylpiperidin-4-yl)-isoxaz-
ole-3-carboxamide (66 mg, 0.11 mmol), which was dissolved in
ethanol (0.25 mL) and treated with 4 N HCl in dioxane (0.25 mL).
The reaction mixture was stirred overnight, evaporated to small
volume and diluted with EtOAc. After washing with saturated solu-
tion of sodium hydrogen carbonate, the organic phase was dried
over Na2SO4 and the solvent removed. The residue was purified
by flash chromatography (eluent: DCM/MeOH/NH3 aq 80:20:2) to
provide 32 (47 mg, 33%, 2 steps). 1H NMR (400 MHz, DMSO-d6) d
8.51 (d, J = 8.1 Hz, 1H), 6.89 (m, 2H), 6.77 (s, 1H), 6.54 (m, 2H),
6.11 (d, J = 2.2 Hz, 1H), 5.61 (d, J = 2.2 Hz, 1H), 3.70 (m, 1H), 3.20
(m, 4H), 2.73 (m, 2H), 2.14 (s, 3H), 1.94 (m, 6H), 1.71 (m, 2H),
1.61 (m, 2H); LC–MS (ESI): m/z 479 [M+H]+; HRMS (ESI): m/z calcd
for C26H30N4O5+H+ 479.2289, found 479.2284.
5.1.48. 5-[2,4-Bis(methoxymethoxy)-6-(4-
nitrophenoxy)phenyl]-N-(1-methylpiperidin-4-yl)-isoxazole-3-
carboxamide (53)
51 (5.0 g, 10.5 mmol) and 1-methylpiperidin-4-amine (3.6 g,
31.5 mmol) were dissolved in DCM (30 mL). The solvent was re-
moved and the reaction mixture was stirred at 80 °C for 9 h. After
cooling to room temperature, the residue was purified by Biotage
SP1 flash chromatography ((gradient elution from 0% to 10% of
MeOH in DCM) to afford 53 (4.6 g, 80%). 1H NMR (400 MHz,
DMSO-d6) d 8.66 (d, J = 7.8 Hz, 1H), 8.22 (m, 2H), 7.10 (m, 2H),
6.89 (d, J = 2.2 Hz, 1H), 6.85 (s, 1H), 6.63 (d, J = 2.2 Hz, 1H), 5.33
(s, 2H), 5.25 (s, 2H), 3.77 (br s, 1H), 3.39 (s, 3H), 3.38 (s, 3H), 2.90
(br s, 2H), 2.13–2.38 (m, 7H), 1.74 (m, 2H), 1.66 (m, 2H); LC–MS
(ESI): m/z 543 [M+H]+; HRMS (ESI): m/z calcd for C26H30N4O9+H+
543.2086, found 543.2083.
5.1.49. 5-[2-(4-Aminophenoxy)-4,6-
bis(methoxymethoxy)phenyl]-N-(1-methylpiperidin-4-yl)-
isoxazole-3-carboxamide (54)
To a stirred solution of 53 (2.41 g, 4.44 mmol) in a mixture of
dioxane/water (5:1, 20 mL) were added portion wise zinc powder
(575 mg, 8.88 mmol) and ammonium chloride (3.06 g, 44.4 mmol).
After stirring at 100 °C for 3 h, the reaction mixture was cooled and
diluted with EtOAc. After washing with brine, the organic phase
was dried over Na2SO4 and the solvent removed to provide 54
(1.93 g, 85%). 1H NMR (400 MHz, DMSO-d6) d 8.59 (d, J = 7.8 Hz,
1H), 6.85 (s, 1H), 6.73 (m, 2H), 6.63 (d, J = 2.2 Hz, 1H), 6.57 (m,
2H), 6.06 (d, J = 2.2 Hz, 1H), 5.33 (s, 2H), 5.25 (s, 2H), 3.77 (br s,
1H), 3.39 (s, 3H), 3.38 (s, 3H), 2.90 (br s, 2H), 2.13–2.38 (m, 7H),
1.74 (m, 2H), 1.66 (m, 2H); LC–MS (ESI): m/z 513 [M+H]+; HRMS
(ESI): m/z calcd for C26H32N4O7+H+ 513.5654, found 513.5651.
5.1.52. 5-{2,4-Dihydroxy-6-[4-(propan-2-ylamino)
phenoxy]phenyl}-N-(1-methylpiperidin-4-yl)-isoxazole-3-
carboxamide (31)
To a stirred solution of 54 (512 mg, 1.0 mmol) and acetone
(0.147 mL, 2.0 mmol) in a mixture of DMF (5 mL) and AcOH
(0.27 mL) was added portion wise tetramethylammonium tri-
acetoxyborohydride (526 mg, 2.0 mmol) at room temperature.
After stirring for 1 h, the solution was diluted with EtOAc and
thoroughly washed with saturated solution of sodium hydrogen
carbonate. After drying over Na2SO4, the solvent was removed
to afford 5-{2,4-bis(methoxymethoxy)-6-[4-(propan-2-ylami-
no)phenoxy]phenyl}-N-(1-methylpiperidin-4-yl)-isoxazole-3-car-
boxamide, which was used directly in the next synthetic step. To
a stirred solution of the crude in ethanol (2.5 mL) was added 4 N
HCl in dioxane (2.5 mL). The reaction mixture was stirred over-
night, evaporated to small volume and diluted with EtOAc. After
washing with saturated solution of sodium hydrogen carbonate,
the organic phase was dried over Na2SO4 and the solvent re-
moved. The residue was purified by flash chromatography (elu-
ent: DCM/MeOH/NH3 aq 90:10:2) to provide 31 (303 mg, 65%,
2 steps). 1H NMR (400 MHz, DMSO-d6) d 10.15 (s, 1H), 9.78 (s,
1H), 8.56 (d, J = 8.1 Hz, 2H), 6.77 (m, 2H), 6.76 (s, 1H), 6.56 (m,
2H), 6.11 (d, J = 2.2 Hz, 1H), 5.65 (d, J = 2.2 Hz, 1H), 5.30 (d,
J = 7.9 Hz, 1H), 3.76 (m, 1H), 3.49 (m, 1H), 3.49 (m, 1H), 2.84
(m, 2H), 2.24 (br s, 3H), 1.76 (m, 2H), 1.65 (m, 2H), 1.12 (d,
J = 6.2 Hz, 6H); LC–MS (ESI): m/z 467 [M+H]+; HRMS (ESI): m/z
calcd for C25H30N4O5+H+ 467.2289, found 467.2289.
5.1.50. 5-{2-[4-(Dimethylamino)phenoxy]-4,6-
dihydroxyphenyl}-N-(1-methylpiperidin-4-yl)-isoxazole-3-
carboxamide (30)
To a stirred solution of 54 (512 mg, 1.0 mmol) and 37% HCHO
solution (1.92 mL, 24 mmol) in a mixture of DCM/DMF (2:1,
5 mL) and AcOH (0.27 mL) was added portionwise tetramethylam-
monium triacetoxyborohydride (1.42 g, 5.4 mmol) at room tem-
perature. After stirring for 1 h, the solution was diluted with
EtOAc and thoroughly washed with saturated solution of sodium
hydrogen carbonate. After drying over Na2SO4, the solvent was re-
moved
to
afford
5-{2-[4-(dimethylamino)phenoxy]-4,6-
bis(methoxymethoxy)phenyl}-N-(1-methylpiperidin-4-yl)-isoxaz-
ole-3-carboxamide, which was used directly in the next synthetic
step. To a stirred solution of the crude in ethanol (2.5 mL) was
added 4 N HCl in dioxane (2.5 mL). The reaction mixture was stir-
red overnight, evaporated to small volume and diluted with EtOAc.
After washing with saturated solution of sodium hydrogen carbon-
ate, the organic phase was dried over Na2SO4 and the solvent re-
moved. The residue was purified by flash chromatography
(eluent: DCM/MeOH/NH3 aq 90:10:2) to provide 30 (230 mg,
The following compounds 33–34 were prepared according to
the method described above using the suitable ketone.
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.09.018

M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
15
5.1.53. 5-{2-[4-(Cyclobutylamino)phenoxy]-4,6-
dihydroxyphenyl}-N-(1-methylpiperidin-4-yl)-isoxazole-3-
carboxamide (33)
5.1.57. 5-{2-[4-(Dimethylamino)phenoxy]-4,6-
dihydroxyphenyl}-N-[1-(4,4,4-trifluorobutyl)piperidin-4-yl]-
isoxazole-3-carboxamide (36)
1H NMR (400 MHz, DMSO-d6) d 10.15 (br s, 1H), 9.78 (s, 1H),
8.53 (d, J = 8.1 Hz, 1H), 6.89 (d, J = 1.5 Hz, 1H), 6.80 (m, 2H), 6.76
(s, 1H), 6.51 (d, J = 8.9 Hz, 2H), 6.11 (d, J = 2.1 Hz, 1H), 5.81 (d,
J = 6.9 Hz, 1H), 5.64 (d, J = 2.1 Hz, 1H), 3.78 (m, 1H), 3.71 (m, 1H),
2.77 (m, 1H), 2.32 (m, 2H), 2.17 (s, 3H), 1.90–2.03 (m, 2H), 1.76–
1.85 (m, 2H), 1.41–1.74 (m, 6H); LC–MS (ESI): m/z 479 [M+H]+;
A solution of 35 (102 mg, 0.2 mmol), N,N-diisopropylethyl-
amine (0.136 mL, 0.8 mmol) and 1-bromo-4,4,4-trifluorobutane
(0.049 mL, 0.36 mmol) in DMF (3 mL) was stirred at room temper-
ature overnight. The solution was taken up in EtOAc and thor-
oughly washed with brine, then dried over Na2SO4. After removal
of the solvent, the residue was purified by flash chromatography
(eluent: DCM/MeOH 90:10) to provide 36 (50 mg, 45% yield). 1H
NMR (400 MHz, DMSO-d6) d 10.19 (s, 1H), 9.80 (s, 1H), 8.53 (d,
J = 6.9 Hz, 1H), 6.90 (m, 2H), 6.77 (s, 1H), 6.74 (m, 2H), 6.14 (d,
J = 2.2 Hz, 1H), 5.65 (d, J = 2.2 Hz, 1H), 3.74 (m, 1H), 2.87 (s, 6H),
2.84 (m, 2H), 2.19–2.38 (m, 6H), 1.97 (br s, 1H), 1.53–1.80 (m,
6H); LC–MS (ESI): m/z 549 [M+H]+; HRMS (ESI): m/z calcd for C27-
H31F3N4O5+H+ 549.2320, found 549.2319.
HRMS (ESI): m/z calcd for
479.2284.
C
26H30N4O5+H+ 479.2289, found
5.1.54. 5-{2,4-Dihydroxy-6-[4-(tetrahydro-2H-pyran-4-
ylamino)phenoxy]phenyl}-N-(1-methylpiperidin-4-yl)-
isoxazole-3-carboxamide (34)
1H NMR (400 MHz, DMSO-d6) d 10.15 (s, 1H), 9.78 (s, 1H), 8.56
(d, J = 8.1 Hz, 2H), 6.77 (m, 2H), 6.76 (s, 1H), 6.56 (m, 2H), 6.11 (d,
J = 2.2 Hz, 1H), 5.65 (d, J = 2.2 Hz, 1H), 5.30 (d, J = 7.9 Hz, 1H), 3.89
(m, 2H), 3.83 (m, 2H), 3.76 (m, 1H), 3.66 (m, 1H), 2.84 (m, 2H), 1.90
(m, 2H), 1.76 (m, 2H), 1.65 (m, 2H), 1.47 (m, 2H), 1.12 (d, J = 6.2 Hz,
6H); LC–MS (ESI): m/z 509 [M+H]+; HRMS (ESI): m/z calcd for C27-
H32N4O6+H+ 509.2395, found 509.2395.
5.1.58. N-(1-Cyclohexylpiperidin-4-yl)-5-{2-[4-
(dimethylamino)phenoxy]-4,6-dihydroxyphenyl}-isoxazole-3-
carboxamide (37)
To a stirred solution of 35 (102 mg, 0.2 mmol) and cyclohexa-
none (0.041 mL, 0.4 mmol) in DMF (3 mL) was added portion wise
tetramethylammonium triacetoxyborohydride (105 mg, 0.4 mmol).
After stirring at room temperature overnight, the solution was ta-
ken up in EtOAc and thoroughly washed with brine. After drying
over Na2SO4, the solvent was removed and the residue was purified
by flash chromatography (gradient elution from 10% to 20% of
MeOH in DCM) to provide 37 (68 mg, 65%). 1H NMR (400 MHz,
DMSO-d6) d 11.98 (s, 1H), 10.22 (s, 1H), 9.82 (s, 1H), 8.77 (br s,
1H), 6.90 (m, 2H), 6.78 (s, 1H), 6.74 (m, 2H), 6.15 (d, J = 2.2 Hz,
1H), 5.65 (d, J = 2.2 Hz, 1H), 3.95 (m, 1H), 2.87 (s, 6H), 1.53–2.03
(m, 9H), 0.90–1.42 (m, 6H); LC–MS (ESI): m/z 521 [M+H]+; HRMS
(ESI): m/z calcd for C24H26N4O7+H+ 521.2759, found 521.2763.
The following compound 38 was prepared according to the
method described above using the suitable ketone.
5.1.55. tert-Butyl 4-{[(5-{2-[4-(dimethylamino) phenoxy]-4,6-
bis(methoxymethoxy)phenyl}-isoxazol-3-
yl)carbonyl]amino}piperidine-1-carboxylate (55)
To a stirred solution of 52 (3.05 g, 4.85 mmol) in a mixture of
dioxane/water (5:1, 33 mL) were added portion wise zinc powder
(1.26 g, 19.4 mmol) and ammonium chloride (3.35 g, 48.5 mmol).
After stirring at 100 °C for 3 h, the reaction mixture was cooled
and diluted with EtOAc. After washing with brine, the organic
phase was dried over Na2SO4 and the solvent removed to afford
tert-butyl
4-[({5-[2-(4-aminophenoxy)-4,6-bis(methoxyme-
thoxy)phenyl]-isoxazol-3-yl}carbonyl)amino]piperidine-1-carbox-
ylate, which was used directly in the next synthetic step. To a
stirred solution of the crude and 37% HCHO solution (9 mL,
120 mmol) in a mixture of DCM/DMF (2:1, 33 mL) and AcOH
(2.8 mL) tetramethylammonium triacetoxyborohydride (6.9 g,
26.15 mmol) was added portionwise at room temperature. After
stirring for 1 h, the solution was diluted with EtOAc and thor-
oughly washed with saturated solution of sodium hydrogen car-
bonate, and dried over Na2SO4. The solvent was removed and the
residue was purified by flash chromatography (eluent: hexane/
EtOAc 2:3) to afford 55 (2.28 g, 75%, 2 steps). 1H NMR (400 MHz,
DMSO-d6) d 8.67 (d, J = 8.1 Hz, 1H), 6.92 (m, 2H), 6.87 (s, 1H),
6.74 (m, 2H), 6.62 (d, J = 2.2 Hz, 1H), 6.06 (d, J = 2.2 Hz, 1H), 5.24
(s, 2H), 5.11 (s, 2H), 3.83–4.09 (m, 3H), 3.35 (s, 3H), 3.29 (br s,
2H), 3.33 (s, 3H), 2.88 (s, 6H), 2.81 (br s, 2H), 1.75 (m, 2H), 1.46
(m, 2H), 1.40 (s, 9H); LC–MS (ESI): m/z 627 [M+H]+; HRMS (ESI):
m/z calcd for C32H42N4O9+H+ 627.3025, found 627.3021.
5.1.59. 5-{2-[4-(Dimethylamino)phenoxy]-4,6-
dihydroxyphenyl}-N-[1-(1,4-dioxaspiro[4.5]dec-8-yl)piperidin-
4-yl]-isoxazole-3-carboxamide (38)
1H NMR (400 MHz, DMSO-d6) d 10.22 (s, 1H), 9.83 (s, 1H), 9.38
(br s, 1H), 8.88 (br s, 1H), 6.90 (m, 2H), 6.79 (s, 1H), 6.74 (m, 2H),
6.15 (d, J = 2.2 Hz, 1H), 5.65 (d, J = 2.2 Hz, 1H), 4.04 (br s, 1H),
3.87 (s, 4H), 3.06–3.50 (br s, 5H), 2.87 (s, 6H), 1.48–2.10
(m,12H); LC–MS (ESI): m/z 579 [M+H]+; HRMS (ESI): m/z calcd
for C31H38N4O7+H+ 579.2814, found 579.2813.
5.2. NMR FAXS screening method
Fluorine NMR experiments were carried out at 564 MHz, using
an Inova 600 instrument (Varian, Palo Alto, USA) equipped with a
19F-1H probe and with an autosampler. Samples for NMR screening
were prepared in 50 mM Hepes buffer, pH 7.2, containing 100 mM
5.1.56. 5-{2-[4-(Dimethylamino)phenoxy]-4,6-
KCl, 5 mM MgCl2, 10 lM EDTA and 8% D2O. A library of 300 fluo-
dihydroxyphenyl}-N-(piperidin-4-yl)-isoxazole-3-carboxamide
dihydrochloride (35)
rine-containing molecules was initially tested in mixtures (5–10
fluorine fragments in each mixture) against Hsp90 for the identifi-
cation of potential spy molecules.16 Spies are fluorine bearing
(weak) ligands, whose displacement from a macromolecular target
by a stronger ligand (screening compound) is reported by a fluo-
rine signal intensification associated to the recovered unbound
To a stirred solution of 55 (2.0 g, 3.19 mmol) in ethanol (7.9 mL)
was added 4 N HCl in dioxane (7.9 mL). The reaction mixture was
stirred overnight, the solvent removed and the residue was tritu-
rated with diethyl ether and filtered to provide 35 (1.54 g, 95%).
1H NMR (400 MHz, DMSO-d6) d 10.40 (br s, 1H), 9.99 (br s, 1H),
8.84 (d, J = 7.7 Hz, 1H), 8.74 (d, J = 13.9 Hz, 2H), 8.48 (d,
J = 10.0 Hz, 1H), 7.19 (br s, 2H), 7.01 (br s, 2H), 6.80 (s, 1H), 6.27
(br s, 1H), 5.76 (br s, 1H), 4.04 (m, 1H), 3.28 (m, 2H), 2.90–3.06
(m, 8H), 1.94 (m, 2H), 1.77 (m, 2H); LC–MS (ESI): m/z 439
[M+H]+; HRMS (ESI): m/z calcd for C23H26N4O5+H+ 439.1976, found
439.1975.
state of the spy molecule. Mixtures were tested at 50
tration using 19F one dimensional T2 filter experiments recorded in
the absence and presence of 1.5 M Hsp90. As a result, 4-(3,4-dihy-
lM concen-
l
dro-2H-1,5-benzodioxepin-7-yl)-6-(trifluoromethyl)pyrimidin-2-
amine was identified as a suitable spy molecule. In addition, a mol-
ecule that does not interact with the receptor was selected from
the library of 300 fluorine (CF and CF3) compounds and used as
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.09.018

16
M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
control molecule. Typical screening samples against Hsp90 con-
tained 0.016 mg/mL (0.6 M) Hsp90 (25,600 Da), 6 M of the
spy molecule (KD 7.5 M, value measured using a competition
binding FP assay), 6 M of control molecule, and 50 M (0.2%
DMSO-d6) of the tested fragments. FAXS experiments were per-
formed using the Carr–Purcell–Meibom–Gill (CPMG) spin-echo
scheme29 with length of 240 and 480 ms before the acquisition
period. Spectra were collected at 293 K using 128 transients. Inten-
sity reduction of the signal of the spy molecule in the presence of
the protein due to shortening of its transverse relaxation is a mar-
ker of the interactions of the molecule with the protein. Intensity
recovery of the signal in the presence of a competitive ligand that
displaced the spy molecule is an indication of specific binding.
BT474 breast cancer cells. Cellular Her2 levels were measured by
immunocytochemistry, and quantified using an ArrayScan vTi
instrument (Cellomics Thermo Scientific). IC50 values represent
the compound concentration at which cellular Her2 signal is
diminished by 50% compared with untreated controls.
l
a
l
l
l
l
5.7. High throughput solubility
Solubility at pH 7 was performed as previously described.34
5.8. Hsc70 FP displacement assay
The FP specificity assay for Hsc70-FL was set up using a com-
mercially available probe N6-(6-amino)hexyl-ATP ATTO-590-ATP
(JenaBioScience–Germany). For competition experiments, to the
mixture containing Hsc70-FL and the fluorescent probe at a final
concentrations of 600 and 20 nM, respectively, the appropriate
compound solutions in 100% DMSO (2% final concentration) were
added. The signal did not show any time dependency. Data were
collected using an excitation and emission wavelength at 590
and 630 nm, respectively, and then analyzed as above described
for Hsp90 FP displacement assay. For the Hsc70 FP assay the Z0 va-
lue was higher than 0.8 and the CV of the standard control was
5.1% (N = 2).
5.3. Crystallographic methods
Crystallization studies were performed using the N-terminal
domain of Hsp90a (aa. 9-236). Crystals of the N-terminal domain
of Hsp90 in complex with compounds 1, 3, 18 were grown from
a solution of 20–25% MPEG 2000 K, 0.2 M MgCl2, 0.1 M cacodylate
pH 6.5–7.2 at 4 °C. The protein was concentrated at 25 mg/mL and
compound was added to a nominal concentration of 2 mM. For
data collection, the crystals were transferred to drops containing
the equivalent mother liquor with 25% glycerol. Diffraction data
for compounds 1 and 3 were collected in house using a Rigaku
Micromax-007 HF X-ray generator and Mar345 Image Plate Detec-
tor (Marresearch) while data collection for compound 18 was car-
ried out at the ESRF (Grenoble, France) on beamline ID23-2. Data
were processed using the HKL package(Otwinowski, 1997).30 Mod-
el building was done using Coot31(Emsley,2004) and refinement
was done with RefMac.32(Murshudov, 1997) The coordinates have
been deposited in the Protein Data Bank with code 4bqg, 4bqj and
4b7p for 1, 3 and 18, respectively, together with structure factors.
5.9. Kinase assays
Interrogation for potential kinase inhibitory effects was per-
formed on an internally developed Kinase Selectivity Screening
(KSS) panel, designed to represent the overall diversity of the ki-
nome, as described in detail in a recent publication.35 The panel in-
cludes: ABL, ACK1, AKT1, Alk, AUR1, AUR2, BRK, CDC7, CDK2/CYCA,
CHK1, CK2alpha/beta, eEF2K, EGFR1, ERK2, FAK, FGFR1, Flt3,
GSK3beta, Haspin V473-K798, IGFR1, IKK2, IR, JAK1, JAK2, JAK3,
KIT, LCK, MELK, MET, MK2, MPS1, MST4, NEK6, NIM, P38alpha,
PAK4, PDGFRb, PDK1, PERK, PIM1, PIM2, PKAalpha, PKCbetaII,
PLK1, RET, SULU1, SYK, TRKA, TYK2, VEGFR2, VEGFR3, ZAP70.
5.4. Hsp90. FP displacement assay
The FP assay was set up using as probe a reduced FITC-Geldana-
mycin (RFG) purchased from InvivoGen (San Diego, California).
FITC-Geldanamycin was reduced as previously described17 prior
to use and then conserved in aliquots at ꢀ80°°C. In the experimen-
tal conditions used, the Z0 value was higher than 0.6 and the CV of a
standard control was of 23.9% (N = 57). For compounds Kd deter-
mination the mixtures containing Hsp90 and RFG at a final concen-
tration of 5 and 0.5 nM, respectively were incubated for 3 h, and
then compound serially diluted solutions in 100% DMSO were
5.10. In vivo pharmacokinetics
The pharmacokinetic profiles of compounds were investigated
in overnight fasted male Nu/Nu mice following a single dose given
intravenously (iv). The vehicle used was 10% Tween 80 in 5% dex-
trose solution. A total of three mice were treated. Blood samples of
each mouse were collected from the saphenous vein at predose,
0.083, 0.5, 1, 6, and 24 h postdosing. Samples were centrifuged at
10,000g for 3 min at 4 °C and the plasma was stored at ꢀ80 °C until
analysis. Samples were analyzed by LC/MS/MS technique.
added to the mixtures at a final volume of 80 lL (final DMSO con-
centration 2%). The plate was incubated for 18 h at room tempera-
ture in the dark and then the FP signal was detected. Data were
collected using a Tecan Safire2 reader, with excitation and emis-
sion wavelength at 470 and 525 nm, respectively, and then fitted
using the program Dynafit version 3.28.039.33
5.11. In vivo pharmacology
5.5. In vitro pharmacology. A2780 cells proliferation assay
Evaluation of antitumor efficacy was performed as previously
described.34
Cells were seeded into 96- or 384-wells plates at final concen-
tration ranging from 10,000 to 30,000 cells per cm2 in appropriate
medium plus 10% FCS. After 24 h cells were treated using serial
dilution of compounds in two replicates. At 72 h after the treat-
ment the amount of cells were evaluated using the Cell Titer-Glo
assay (Promega). Inhibitory activity was evaluated comparing trea-
ted versus control data using sigmoidal equation on the Assay Ex-
plorer (Symix) program.
5.12. Cell based assays
The mechanism of action of the compounds was investigated
using cell lines treated with compounds at the indicated concen-
tration or with just DMSO as a control for 24 h. Client protein deg-
radation was determined by Western blot analysis.
Immunoblotting was done according to standard procedures and
using the following antibodies: anti-Hsp70, anti-Hsp90 (Enzo Life
Sciences, Farmingdale, NY), anti-ERK, anti-p-Thr202/204 ERK,
anti-AKT, anti-pSer473 AKT. The SuperSignal chemiluminescence
kit (Pierce) was used for detection.
5.6. Her2 degradation assay
Her2 degradation cellular activity of Hsp90 inhibitors was as-
sessed by measuring the induced loss of Her2 protein levels in
Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.09.018

M. G. Brasca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
17
Hinkley, L.; Jenks, M.; Geng, L.; Lewis, M.; Otto, J.; Pronk, B.; Verleysen, K.; Hall,
S. E. J. Med. Chem. 2009, 52, 4288.
Acknowledgments
14. Caldas-Lopes, E.; Cerchietti, L.; Ahn, J. H.; Clement, C. C.; Robles, A. I.; Rodina,
A.; Moulick, K.; Taldone, T.; Gozman, A.; Guo, Y.; Wu, N.; de Stanchina, E.;
White, J.; Gross, S. S.; Ma, Y.; Varticovski, L.; Melnick, A.; Chiosis, G. Proc. Natl.
Acad. Sci. U.S.A. 2009, 106, 8368.
15. (a) Baker, M. Nat. Rev. Drug Discovery 2013, 12, 5; (b) De Kloe, G. E.; Bailey, D.;
Leurs, R.; Esch, I. J. P. Drug Discovery Today 2009, 14, 630.
We thank Mauro Paolucci for binding assays, Massimiliano Ger-
mani for PK/PD studies, the group of Assay Development and Bio-
chemical Screening for the biochemical assay on a kinase panel,
and the Experimental Therapy group for in vivo experiments.
16. (a) Dalvit, C.; Flocco, M.; Veronesi, M.; Stockman, B. J. Comb. Chem. High
Throughput Screening 2002, 5, 605; (b) Dalvit, C.; Fagerness, P. E.; Hadden, D. T.
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Supplementary data
17. Lundgren, K.; Zhang, H.; Brekken, J.; Huser, N.; Powell, R. E.; Timple, N.; Busch,
D. J.; Neely, L.; Sensintaffar, J. L.; Yang, Y. C.; McKenzie, A.; Friedman, J.;
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.09.018.
18. Wright, L.; Barril, X.; Dymock, B.; Sheridan, L.; Surgenor, A.; Beswick, M.;
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Please cite this article in press as: Brasca, M. G.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.09.018