Novel 3,4-isoxazolediamides as potent inhibitors of chaperone heat shock protein 90

Article abstract of DOI:10.1021/jm201155e

A structural investigation on the isoxazole scaffold led to the discovery of 3,4-isoxazolediamide compounds endowed with potent Hsp90 inhibitory properties. We have found that compounds possessing a nitrogen atom directly attached to the C-4 heterocycle ring possess in vitro Hsp90 inhibitory properties at least comparable to those of the structurally related 4,5-diarylisoxazole derivatives. A group of compounds from this series of diamides combine potent binding affinity and cell growth inhibitory activity in both series of alkyl- and aryl- or heteroarylamides, with IC₅₀ in the low nanomolar range. The 3,4-isoxazolediamides were also very effective in causing dramatic depletion of the examined client proteins and, as expected for the Hsp90 inhibitors, always induced a very strong increase in the expression levels of the chaperone Hsp70. In vivo studies against human epidermoid carcinoma A431 showed an antitumor effect of morpholine derivative 73 comparable to that induced by the reference compound 10. (Figure presented)

Full text of DOI:10.1021/jm201155e

Article
pubs.acs.org/jmc
Novel 3,4-Isoxazolediamides as Potent Inhibitors of Chaperone Heat
Shock Protein 90†
Riccardo Baruchello,‡ Daniele Simoni,*,‡ Giuseppina Grisolia,‡ Giuseppina Barbato,‡ Paolo Marchetti,‡
Riccardo Rondanin,‡ Stefania Mangiola,‡ Giuseppe Giannini,*,§ Tiziana Brunetti,§ Domenico Alloatti,§
Grazia Gallo,§ Andrea Ciacci,§ Loredana Vesci,§ Massimo Castorina,§ Ferdinando M. Milazzo,§
Maria L. Cervoni,§ Mario B. Guglielmi,§ Marcella Barbarino,§ Rosanna Fodera,§ Claudio Pisano,§
̀
and Walter Cabri§
‡Dipartimento di Scienze Farmaceutiche, Universita
̀
degli Studi di Ferrara, I-44121 Ferrara, Italy
§R&D Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., I-00040 Pomezia, Roma, Italy
S
* Supporting Information
ABSTRACT: A structural investigation on the isoxazole scaffold led to the discovery of 3,4-
isoxazolediamide compounds endowed with potent Hsp90 inhibitory properties. We have found that
compounds possessing a nitrogen atom directly attached to the C-4 heterocycle ring possess in vitro
Hsp90 inhibitory properties at least comparable to those of the structurally related 4,5-diarylisoxazole
derivatives. A group of compounds from this series of diamides combine potent binding affinity and cell
growth inhibitory activity in both series of alkyl- and aryl- or heteroarylamides, with IC50 in the low
nanomolar range. The 3,4-isoxazolediamides were also very effective in causing dramatic depletion of
the examined client proteins and, as expected for the Hsp90 inhibitors, always induced a very strong
increase in the expression levels of the chaperone Hsp70. In vivo studies against human epidermoid
carcinoma A431 showed an antitumor effect of morpholine derivative 73 comparable to that induced by
the reference compound 10.
Hsp90 derived from tumor cells has particularly high ATPase
INTRODUCTION
■
activity with higher binding affinity to Hsp90 inhibitors than
the latent form in normal cells, allowing selective targeting of
Hsp90 inhibitors to tumor cells with little inhibition of Hsp90
function in normal cells.9 In addition, Hsp90 has also been
recently identified as an important extracellular mediator for
tumor invasion.20−22 Thus, Hsp90 is considered a major thera-
peutic target for anticancer drug development because inhi-
bition of a single target represents attack on all of the hallmark
traits of cancer. Since the discovery that two natural com-
pounds, geldanamycin (1a) and radicicol (2) (Figure 1), were
able to inhibit Hsp90 function through binding to the ATP
binding pocket in its N-terminal domain, the interest for Hsp90
inhibitors has grown. The natural antibiotic geldanamycin was
shown to exhibit potent antitumor activity against human
cancer cells,23 but significant toxicities prevented its clinical
development.24
The first-in-class Hsp90 inhibitor to enter clinical trials was
the geldanamycin analogue 17-allylaminogeldanamycin (17-
AAG, 1b, Figure 1). Even though high in vitro activity char-
acterizes this geldanamycin derivative, its interest is shadowed
by poor solubility coupled to hepatotoxicity properties. Some
of these problems have been partially solved by the discovery
of 17-dimethylaminoethylamino-17-demethoxygeldanamycin
(17-DMAG, 1c, Figure 1). Infinity is developing two drug
Heat shock proteins (Hsp’s) play a key role in cell protection
against various cell stress factors (e.g., toxic xenobiotic,
chemotherapy, radiation), acting as a protective factor against
the misfolding of essential proteins involved in maintaining
cell functionality. Hsp90 proteins, members of these molecular
chaperones, are proteins that play a key role in the conforma-
tional maturation, stability, and function of so-called “client”
proteins.1−3 The inhibition of Hsp90 triggers the disruption
of the Hsp90−client protein complex, and subsequently its
proteasome-mediated degradation causes loss of function and
inhibition of cell growth. Many Hsp90 client proteins are
overexpressed in cancer, often in mutated forms, and are
responsible for unrestricted cancer cell proliferation and survival.
Interestingly, heat shock protein 90 has emerged as an important
target in several diseases.4−9 In particular, the role played by
Hsp90 in regulating and maintaining the transformed phenotype
in cancers and neurodegenerative diseases has been recently
identified, as well as its roles in fungal and viral infections.10−12
Inhibition of the ATPase activity of Hsp90 disrupts an
ongoing “folding” cycle, involving multiple cochaperone
proteins, and in turn leads to destabilization, ubiquitination,
and ultimately proteasomal degradation of client proteins.13−15
Hsp90 is normally expressed in normal cells, representing 1−2%
of the total intracellular protein, from which about 3% is found
in the nucleus with effects on the regulation of several nuclear
events.16 Under stress conditions, such as in tumors, its levels
increase to 4−6% of the whole proteomic load of the cell.17−19
Received: August 30, 2011
Published: November 9, 2011
© 2011 American Chemical Society
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Figure 1. Hsp90 inhibitors: natural compounds and some drugs currently in clinical trials.
candidates in its Hsp90 chaperone inhibitor program:
retaspimycin hydrochloride (IPI-504, 1d, Figure 1), an intrave-
nously administered small molecule, and IPI-493 (1e, Figure 1),
which is administered orally.25 Radicicol (2, Figure 1), a natural
macrocyclic antifungal antibiotic, was found to inhibit Hsp90
protein by interacting at the same site of action of
geldanamycin.26 However, because of its intrinsic chemical
instability, it is deprived of in vivo activity. After pioneering
studies with natural products geldanamycin and radicicol, many
selective Hsp90 inhibitors from various institutions have been
disclosed and entered into clinical trials.27,28 The purine scaffold
is another important class of Hsp90 inhibitors.29,30 This class of
derivatives was devised by structural homology with ATP.
Among the many inhibitors developed within this family,
BIIB021 (3), investigated by Chiosis and co-workers,30 and
CUDC-305 (4), developed by Curis Inc. (Figure 1), have
entered into clinical trials.31 The o-aminobenzoic acid derivative
SNX-2112 (5a) and its prodrug SNX-5422 (PF-04929113)
(5b) (Figure 1), proposed by Serenex Inc., are also under
clinical investigation.32 High-throughput screening campaigns
permitted the discovery of benzoisoxazole derivative 6, having a
resorcinol moiety in position 3, that has also been disclosed as a
potent Hsp90 inhibitor.33 Other resorcinols, AT13387 (7) by
Astex and ganetespib by Synta Pharmaceuticals Corp. (chemical
structure not yet disclosed), entered recently into clinical
trials.34−36
Myrexis has reported MPC-3100 (8) (Figure 1), an orally
bioavailable small-molecule Hsp90 inhibitor, as well as its more
water-soluble prodrug MPC-0767.37,38 Among the different
classes of Hsp90 inhibitors, researchers from Vernalis Ltd. and
Institute of Cancer Research (U.K.) have disclosed the 4,5-
diarylpyrazoles,39 3-aryl-4-carboxamide pyrazoles,40 4,5-diary-
lisoxazoles,41 3,4-diarylpyrazole derivatives,42,43 and thieno[2,3-
d]pyrimidines.39,42,44,45 Investigations on 4,5-diarylisoxazoles 9a
(VER-50589, Figure 2) generated Hsp90 inhibitors that proved
Figure 2. Hsp90 inhibitors with a 4,5-diarylisoxazole/pyrazole scaffold.
to be from 5 to 50 times more active at inhibiting cancer cell
proliferation compared to the previously reported 4,5-diaryl-
pyrazoles 9b (VER-49009, Figure 2). Thus, the 4,5-diarylisoxazole
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scaffold bearing a resorcinol at position 5 was considered a
prerequisite for the activity on Hsp90, and compound 10 (VER-
52296/NVP-AUY922, Figure 2), currently in phase II clinical
trials, belongs to this class of compounds. However, to date,
no Hsp90 inhibitors in clinical trials fully satisfy the requisites
of safety and stability. Some of the drugs under clinical investi-
gation have showed toxicity toward liver, eyes, stomach−intestine,
and heart.35,46 Therefore, the need for potent and selective Hsp90
inhibitors still remains an interesting and promising goal.
Table 1. Data for Isoxazole-3,4-diamides: Binding on Hsp90
by a Fluorescence Polarization Assay (FP Assay) and
Cytotoxicity on NCI-H460 Non-Small-Cell Lung Carcinoma
Cells
In continuation of our antitumor drug-discovery project47 we
have recently undertaken a detailed structural investigation on a
new class of 3,4-isoxazolediamides 11 (Figure 3). We mainly
Figure 3. General structure of new Hsp90 inhibitors used in these
study.
focused our attention on the C-4 position of the isoxazole
scaffold where we found that compounds possessing a nitrogen
atom directly attached to the heterocycle ring possess in vitro
Hsp90 inhibitory properties comparable to those of the
structurally related 4,5-diarylisoxazoles. Herein, we will give
full details on the synthesis and pharmacological character-
ization of a new class 3,4-isoxazolediamides as potent Hsp90
inhibitors. A group of compounds that combine notable
binding properties and potent cell growth inhibitory activity
will be described. Additionally, the in vivo activity of the 3,4-
isoxazolediamides 49 and 73 against human epidermoid
carcinoma A431 will be also reported.
CHEMISTRY
■
The synthetic route used for the preparation of amides 15−
73 (see Tables 1, 3, and 4) is shown in Scheme 1. The 4-
nitroisoxazoles 13a−d were synthesized by chemoselective
nitration of the isoxazoles 12a−d41 using concentrated nitric
acid and acetic anhydride. Compounds 12b,c were prepared
starting from the appropriate ester derivative with the following
sequence of reactions (as in Supporting Information): (a)
hydrolysis of ester moiety, (b) activation of resulting carboxylic
acid, and (c) subsequent reaction with the proper commercially
amines. Reduction of the nitro compounds 13a−d to give 14a−
d in good yields was done using zinc powder and ammonium
chloride in a mixture of water and tetrahydrofuran as solvents.
Reaction of the amines 14a−d with the proper commercially
available acyl chloride gave the corresponding dibenzylated
amides that after deprotection of phenolic groups with boron
trichloride furnished the desired amides 15−73.
The morpholine derivatives 77a−e and 79a,b and the
piperazine derivative 79c were synthesized as reported in
Scheme 2. The alcohols 74a−e were simply prepared starting
from the amine 14d by reaction with the adequate acyl
chloride. The alcohols 74a−e were then activated as the
corresponding mesylate derivatives by reaction with mesyl
chloride in methylene chloride prior to being reacted with
morpholine to give 76a−e. Removal of benzylic protecting
groups using boron trichloride furnished the final 77a−e
in good yields. Similarly, removal of benzylic groups from
74a−e gave the deprotected derivatives 75a−e. The derivatives
78a−c were obtained by reaction of 14a,d with acryloyl
chloride, and the resulting acryloyl amides were then reacted
through a Michael addition reaction with morpholine or
1-methylpiperazine. Removal of the benzylic protecting groups
by means of boron trichloride yielded the desired compounds
79a−c.
The synthesis of amines 81a−d is described in Scheme 3.
The amine 14a was first debenzylated using boron trichloride.
The reductive amination reaction of the resulting resorcinol
derivative 80 by means of sodium cyanoborohydride and a
proper aldehyde or ketone furnished the desired amines 81a−d
in moderate to good yields.
RESULTS AND DISCUSSION
In our project, we systematically investigated possible
modifications on the isoxazole scaffold, focusing mainly our
■
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a
Scheme 1
a
Reagents and conditions: (a) conc HNO3/Ac2O; (b) Zn, H2O/THF, NH4Cl; (c) (i) R2COCl, TEA, DCM; (ii) BCl3, DCM, 0 °C.
a
Scheme 2
a
Reagents and conditions: (a) (i) acyl chloride, TEA, DCM; (ii) BF3·Et2O, DCM, 0 °C; (b) (i) mesyl chloride, DIPEA, DCM; (ii) morpholine,
70 °C; (c) BCl3, DCM, 0 °C; (d) (i) acryloyl chloride, TEA, DCM; (ii) morpholine or 1-methylpiperazine, reflux.
attention on the C-4 position. As reported above, among the
structural classes of compounds that have provided interesting
results as potential Hsp90 inhibitors, the 4,5-diarylisoxazoles
are of remarkable importance having recently led to 10 (VER-
52296/NVP-AUY922, Figure 2) currently in phase II clinical
trials.27 Crystal structures of Hsp90 N-terminal domain in
complex with several inhibitors have been well described in
literature: in Figure 4A the active site of Hsp90 cocrystallized
with 10 (PDB code 2VCI) is reported.41 A network of
hydrogen-bonding interactions involving the resorcinol moiety
and the heterocyclic ring with Asp93, Thr184, and a cluster of
structurally conserved and highly ordered water molecules
characterize the key interactions of the compound with protein.
A bulky hydrophobic group interacts with the lipophilic pocket
created by Phe138, Met98, Val150, Leu103, Leu107, Trp162,
and Tyr139; additional hydrogen bonding interactions involve
the 3-amide group with Gly97 and Lys58. We hypothesized
that the introduction of a second amide group at C-4, as shown
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a
Scheme 3
a
Reagents and conditions: (a) BCl3, DCM, 0 °C; (b) (i) appropriate aldehyde or ketone, MeOH, AcOH 0,1%; (ii) NaCNBH3.
aromatic portion. The key interactions of the OH-resorcinol
groups and the C-3 amide still remain identical. Thus, the idea
of an additional H-bond donor interaction in the active site,
mediated by an amide moiety at position 4 of the isoxazole ring,
was considered as the basic structural motif for the construction
of a series of 3,4-isoxazolediamides48 as those reported in
Tables 1, 3, and 4. The resorcinol portion and the C-3 amide
moiety were maintained unaltered considering the privileged
role offered by these fragments in the interaction with the
Hsp90 protein in both radicicol and 4,5-diarylisoxazole series of
compounds.41
The feasibility of the project was first investigated by
preparing a small explorative series of compounds bearing a 5-
chlororesorcinol residue at the C-5 position. Meanwhile the
amides at C-3 and C-4 of the isoxazole ring were varied as
shown in Table 1. The 5-chloro substitution was at the
beginning chosen for ease of synthesis; moreover, 5-
unsubstituted resorcinols are known to be less active with
respect to the substituted ones.42
All the synthesized compounds were tested for their potential
Hsp90 inhibitory properties. Measurements of binding were by
a fluorescence polarization (FP) assay, and inhibition of cell
growth proliferation of the NCI-H460 tumor cell line was
determined.
It was apparent from the results shown in Table 1 that the
strategy of the C-4 amide moiety could lead to compounds
useful as Hsp90 inhibitors. All the synthesized compounds,
apart from the fluorinated derivative 25, showed binding
affinity in the range of 37−250 nM. Compounds 19, 21, 23,
and 79a also showed cytotoxic activity in the submicromolar
range (130−750 nM). The fluorinated compound 24,
synthesized to further reinforce the H-bond with Lys58,
contrary to our expectation, showed equivalent binding and
cellular activity, thus demonstrating no advantage compared
with the related ethylamide residue 16. The other fluorinated
derivative 25, where the hydrogen on the C-3 amide was
missing, was inactive, reinforcing the importance of the
presence of a hydrogen in this position for its H-bond
capabilities and minimal steric hindrance. The C-4-aminoalkyl
derivatives 81a−d (Table 2) were synthesized and screened to
better substantiate our structure−activity investigation. Amine
compounds 81a,b (Table 2) showed a drop in binding affin-
ity when compared with the parent amides 19 and 16.
Furthermore, the observed drop with regard to affinity was
corroborated by the absence of cytotoxicity. A loss of a putative
interaction of C-4 amide carbonyl with Lys58 as shown in the
Figure S1 of compound 81b could explain in some way the
Figure 4. (A) VER-52296/NVP-AUY922 (compound 10) cocrystal-
lized in Hsp90 active site (PDB code 2VCI). Color scheme is by atom
type: gray, VER-52296/NVP-AUY922; cyan, protein residues involved
in nonpolar interactions; green, protein residues involved in polar
interactions. H-Bonds are shown as dotted lines. (B) Docking pose of
compound 49 in Hsp90 active site. Color scheme is by atom type:
gray, 49; cyan, protein residues involved in nonpolar interactions;
green, protein residues involved in polar interactions. H-Bonds are
shown as dotted lines.
for compound 49 (Figure 4B), could play an additional role in
the interaction with the protein, producing an extra interaction
with Lys58, as well as a concomitant reorientation of the
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Interestingly, the morpholine water solubilizing group in
compounds 49, 73, 77a,d,e and 79b was well tolerated as
demonstrated by the binding and cell growth inhibiting
properties of such compounds. Unfortunately, a group of
compounds (i.e., 32−42, 44, 45, 48, 50−54, 60−63, 65−67,
69, 70, 73, 75a−e, and 77b,c) despite possessing an excellent
binding profile showed only poor cell proliferation inhibitory
properties. Such behavior can be accountable to permeability
issues across cell membranes. Indeed, some compounds
presented a large polar surface (i.e., 35−39, 44, 54, 60−62,
and 73; Table S1) or positively charged appendages at pH 7.4
(i.e., 33, 34, 40, and 41) as demonstrated by the LogD7.4
parameter disclosed in Table S1. Instead, a lipophilicity index of
3.2 ± 0.7 (i.e., log P parameter in Table S1), calculated using
ACD/Labs software, does correlate to a high cytotoxicity
profile (i.e., 27, 28, 31, 43, 46, 47, 55, 57, and 58).
Table 2. Data for the 4-Aminoisoxazoles: Binding on Hsp90
(FP Assay) and Cytotoxicity on NCI-H460 Non-Small-Cell
Lung Carcinoma Cells
To validate that the antiproliferative activity of these
compounds was indeed related to the inhibition of Hsp90, a
few selected compounds (27, 28, 31, 49, 55, and 57) were
further tested for their effects on the expression of some typical
Hsp90 client proteins in the A431 (human epidermoid
carcinoma) cell line. The examined client proteins (Akt,
Cdk4, and EGFR) and Hsp70 were evaluated by means of
the classical Western blotting analysis on total protein extracts
of A431 cells, following a 24 h exposure to various concen-
trations of the test compounds. All tested compounds were very
effective, causing dramatic depletion of the examined client
proteins and, as expected for the Hsp90 inhibitors, always
inducing a very strong increase in the expression levels of the
chaperone Hsp70 (Figure 5 and Figures S2−S4). The depletion
was always dose dependent and was achieved with potency
similar to that of the reference compound (17-DMAG). The
only partial exception was represented by compound 55, which
caused a marked depletion of EGFR and CDK4 client proteins
but, at the same time, showed only a marginal effect on the
expression of Hsp70. These results therefore support the prem-
ise that the effects on cell growth of the 3,4-isoxazolediamides
were a consequence of their Hsp90 chaperone inhibitory
properties.
Finally, 12 compounds (see Tables 3 and 4) were selected
for in vivo study against human epidermoid carcinoma A431
tumor xenografts (Table S3 show the results of cytotoxicity
assay of the compounds tested in vivo on A431 cell lines). All
compounds were administered intraperitoneally once daily at
their maximum tolerated doses (i.e, doses that produced a body
weight loss of 7−8%) without any sign of toxicity. Compound
73 showed an antitumor effect (tumor volume inhibition
of 48%) comparable to that of the reference compound 10
(Table 5 and Figure 6).49 In contrast, compound 49, struc-
turally related to the reference compound 10 (NVP-AUY922),
showed a reduced in vivo activity. The ability of 73 to affect in
vivo the expression of typical Hsp90 client proteins was
assessed in A431 tumor xenografts 2 h after the last treatment.
As shown in Figure 7, 73 induced a strong degradation of three
typical “client” proteins (EGFR, Akt, and CDK-4) and signif-
icantly increased the expression levels of Hsp70, with a potency
comparable to that of the reference inhibitor 10 (NVP-
AUY922). Thus, although in vitro activity of 73 is somehow
moderate, the compound still shows a very good in vivo activity
profile among those tested.
decrease of binding affinity. Anyway, the loss of cytotoxic
activity shown by all the C-4 amines 81a−d allowed us to view
this type of substitution with little interest.
The interesting activity shown by some chlororesorcinol
diamides gave confirmation of the idea that manipulation
around the C-4 region of the isoxazole scaffold may
significantly alter the binding of the compounds, providing an
interesting opportunity for the construction of novel series of
Hsp90 inhibitors. We further explored the potential of the new
scaffold, investigating a series of additional substitutions at the
C-4 amide portion. Given that substitution of the chlorine atom
on the resorcinol moiety with an isopropyl group had proved to
improve cytotoxicity,41 isopropyl analogues have been synthe-
sized. We carried out virtual docking experiments, using the
Hsp90−10 X-ray complex structure and a virtual library of 3,4-
isoxazolediamides bearing diverse C-4 substituents. Such a
library was prepared by “reacting” the debenzylated inter-
mediate 14d with 350 commercially available acyl chlorides or
carboxylic acids having molecular weights between 100 and
350. The resulting 3,4-isoxazolediamides were filtered accord-
ing to the rule of 5 < 2 (except 35−37 with two violations),
subjected to a conformational analysis, and then docked in the
active site of the enzyme. Compounds with scoring value higher
than that of the simplest analogue (acetamide derivative 26)
were selected for synthesis. Scoring values for synthesized
compounds are reported in Table S1. Biological evaluation of a
library of 60 isopropyl derivatives (Tables 3 and 4) showed
binding affinity in the range of 1−260 nM. Several compounds
combined notable binding properties with potent cell growth
inhibitory activity, i.e., IC50 values in the low nanomolar range:
26, 27, 31, 43 (Table 3) in the class of alkyl and cycloalkyl
derivatives; 46, 47, 49 in the class of phenyl derivatives; 55−58,
68, 71, and 72 in the class of heterocyclic derivatives (Table 4).
The antiproliferative effects of compounds 28 and 30 were also
noteworthy (16 and 42 nM, respectively) notwithstanding a
decrease of their binding activity.
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Table 3. Data for Isoxazole-4-alkyl- and Cycloalkylamides: Binding on Hsp90 (FP Assay) and Cytotoxicity on NCI-H460
b
Non-Small-Cell Lung Carcinoma Cells
a
b
For the detection limits of the assay, see Chiosis et al.50 The asterisk (∗) indicates that the compound was submitted to in vivo studies.
endowed with potent Hsp90 inhibitory activity. The C-4 amide
appendage could be either an alkyl or aryl moiety. A group of
amides showed affinity for the target comparable to that shown
by 4,5-diaryl products 9a,b and 10. Compounds of interest that
combine potent binding and cell growth activity were obtained
in a series of alkyl- and cycloalkyldiamides (26, 27, 31, and 43),
as well as in a series of the aryl and heteroaryl derivatives
CONCLUSION
■
We postulated that the introduction of a second amide group at
the C-4 position of the 4,5-diarylisoxazoles scaffold could
represent an interesting opportunity for the construction of a
novel class of Hsp90 inhibitors. We have performed a detailed
investigation and elaboration of the isoxazole scaffold, leading
to the discovery of a novel class of 3,4-isoxazolediamides
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Table 4. Data for Isoxazole-4-aryl- and Heteroarylamides: Binding on Hsp90 (FP Assay) and Cytotoxicity on NCI-H460
b
Non-Small-Cell Lung Carcinoma Cells
a
b
For the detection limits of the assay, see Chiosis et al.50 The asterisk (∗) indicates that the compound was submitted to in vivo studies.
(46, 47, 49, 55−58, 68, 71, and 72). The antiproliferative
effects of compounds 28 and 30 (16 and 42 nM, respectively)
were notable notwithstanding the compounds having rather
moderate binding affinities.
Analysis of the degradation of the client proteins in A431
cells proved that the antiproliferative effects of the diamides
were indeed related to the inhibition of Hsp90. As expected for
Hsp90 inhibitors, the tested diamides induced a very strong
increase in the expression levels of the chaperone Hsp70.
Twelve compounds with a different range of in vitro profile
were selected to be studied in vivo against human epidermoid
carcinoma A431. Compound 73 revealed an antitumor effect
(tumor volume inhibition of 48%) comparable to that of the
reference compound 10.
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Figure 5. Analysis of Hsp90 client protein levels (EGFR, Akt, CDK-4) and Hsp70 in A431 tumor cells treated with compound 49. Total cellular
extracts were obtained 24 h after treatment. Actin is shown as a control for protein loading. Results of densitometry analysis were reported as
normalized (to β-actin) ratios.
Table 5. Antitumor Activity of Compounds 49 and 73 in
Comparison with 10 (NVP-AUY922) against A431
Epidermoid Carcinoma
drug treatment
dose/route (mg/kg)
TVI ± SE (%)
BWL (%)
10
49
73
60/ip
60/ip
60/ip
48 ± 5
33 ± 4
48 ± 4
8
2
7
Figure 6. Antitumor activity of the compounds 49 and 73 in
comparison with that of compound 10 (NVP-AUY922) against A431
epidermoid carcinoma xenografted in CD1 nude mice.
Figure 7. Analysis of Hsp90 client protein levels (EGFR, Akt, CDK-4,
and Hsp70) in A431 tumor xenografts, following treatment with 73
and 10 (NVP-AUY922). Total proteins were purified 2 h after the last
treatment. Actin is shown as a control for protein loading. A repre-
sentative blot is shown.
EXPERIMENTAL SECTION
■
Tumor Xenograft Model. Experiments were carried out at
8−11 weeks old (Harlan, Italy). Mice were maintained in laminar flow
rooms with constant temperature and humidity. Experimental
Sigma-Tau (Rome, Italy) using female athymic nude CD-1 mice,
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protocols were approved by the Ethic Committee for Animal
Experimentation.
determination of the protein concentration by Bradford protein
assay (Thermo Scientific, Rockford, IL, U.S.), equal amounts of
cellular extracts were separated by SDS gel electrophoresis (SDS−
PAGE), transferred onto nitrocellulose membranes, and probed with
the following primary antibodies: anti-EGFR (Upstate Biotechnology,
Millipore Corporate, Billerica, MA, U.S.), anti-Cdk4 (Santa Cruz
Biotechnology Inc., CA, U.S.), anti-Akt (Cell Signaling Technology,
Inc., MA, U.S.), anti-Hsp70 (BRM-22), and anti-actin (Sigma
Chemical Co., St. Louis, MO, U.S.).
Immunoreactive bands were revealed by horseradish peroxidase
conjugated secondary antibodies, using an enhanced chemilumines-
cence detection reagent (ECL Plus, GE Healthcare Bio-Sciences,
Uppsala, Sweden) and a specific image system (STORM 860;
Molecular Dynamics, Sunnyvale, CA, U.S.). Protein loading
equivalence was corrected in relation to the expression of β-actin.
For quantification of signals, blots were subjected to densitometry
analysis.
A431 epidermoid carcinoma cells (3 × 106/mouse) were inoculated
subcutaneously (sc) in the right flank of CD1 nude mice. Drug
treatments started 3 days after tumor injection according to the
schedule qd×5/w×2w. To evaluate the antitumor activity, tumor
diameters were measured biweekly with a Vernier caliper. The formula
TV (mm3) = [length (mm) × width (mm)2]/2 was used, where the
width and the length are the shortest and the longest diameters of each
tumor, respectively. When tumors reached a volume of 1 cm3, mice
were sacrificed by cervical dislocation. Body weight was recorded every
day throughout the study. Toxicity of the molecules was determined as
follows: body weight loss percent (% BWLmax) = 100 − [(mean BWx/
mean BW1) × 100], where BWx is the mean BW at the day of maximal
loss during the treatment and BW1 is the mean BW on the first day of
treatment. Lethal toxicity was also evaluated. Doubling time (DT) of
control tumors was also calculated.
In order to assess the effect in vivo of 73, compared to the reference
compound 10 (NVP-AUY922), on the expression of typical Hsp90
client proteins, A431 tumor xenografts (3 samples/group) were
excised 2 h after the last treatment and then total proteins were
extracted through the homogenization of tumor samples in T-PER
tissue protein extraction reagent (Pierce, Rockland, IL, U.S.),
supplemented with 10 μg/mL protease inhibitor cocktail (Sigma
Chemical Co., St. Louis, MO, U.S.). Determination of the protein
concentration and Western blotting analysis were finally performed as
described in the section Client Protein Degradation Assay.
Chemistry. 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
spectrometer, and peak positions are given in parts per million
downfield 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 Finnigan
MAT 95 instrument with BE geometry.
Thin layer chromatography (TLC) was carried out using Merck
precoated silica gel F-254 plates. Flash chromatography was done
using Merck silica gel 60 (0.063−0.200 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. All the final products
undergoing biological testing were analyzed in a Jasco HPLC system
consisting of two PU-2080 pumps and an MD-2010 detector. A purity
threshold of 98% was set up. Microanalysis of all new synthesized
compounds indicated values were within ±0.4% of the calculated
value.
General Procedure for the Synthesis of Nitroisoxazoles
13a−d. A suspension of adequate isoxazole derivative 12a−d (2.2
mmol) in Ac2O (20 mL) was cooled to 0 °C, and concentrated HNO3
(0.26 mL, 4.3 mmol) was added dropwise under stirring, the
temperature being maintained between 0 and 5 °C. After the addition
was complete, the mixture was stirred for 70 h at 5−10 °C and then
poured into ice and extracted with DCM (3 × 40 mL). The extract
was dried and concentrated under vacuo. The yellow solid obtained
was triturated with Et2O and filtered to give the desired nitro
compound 13a−d.
5-[2,4-Bis(benzyloxy)-5-chlorophenyl]-4-nitroisoxazole-3-
carboxylic Acid Ethylamide (13a). Yield 73%. Yellow solid. 1H
NMR (200 MHz CDCl3) δ: 1.26 (t, J = 7.4 Hz, 3H), 3.46−3.55 (m,
2H), 5.0 (s, 2H), 5.10 (s, 2H), 6.57 (m, 2H), 7.23−7.29 (m, 2H),
7.32−7.37 (m, 8H), 7.66 (s, 1H). m/z 508.5/510.4 [M + H]+.
5-[2,4-Bis(benzyloxy)-5-chlorophenyl]-N-(2,2,2-trifluoroeth-
yl)-4-nitroisoxazole-3-carboxamide (13b). Yellow solid. 1H
NMR (400 MHz CDCl3) δ: 4.07−4.15 (m, 2H), 5.01 (s, 2H), 5.10
(s, 2H), 6.59, (s, 1H), 6.93, (br, 1H), 7.22−7.27 (m, 2H), 7.32−7.40
(m, 8H), 7.68 (s, 1H). m/z 562.5/564.4 [M + H]+.
Cellular Sensitivity to Drugs. In non-small-cell lung carcinoma
cells (NCI-H460) and in epidermoid carcinoma cells (A431), 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 untreated control cells.
Binding on Hsp90 by a Fluorescence Polarization Assay.50
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 −20 °C until use. Hsp90, purchased from Stressgen
(catalog no. SPP-776, Victoria, BC, Canada), was previously dissolved
in assay buffer (HFB) to form 2.2 μM stock solutions and kept at
−80 °C until use.
The compounds were previously dissolved in DMSO to obtain
stock solutions and kept at −20 °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. Before each
use, 0.1 mg/mL bovine γ globulin and 2 mM DTT were freshly added.
Fluorescence polarization (FP) was performed in Opti-Plate-96F well
plates (Perkin-Elmer, Zaventem, Belgium) using a plate reader (Wallac
Envision 2101 multilabel reader, Perkin-Elmer, Zaventem, Belgium).
To evaluate the binding affinity of the molecules, an amount of 50 μL
of the GM-FTC solution (5 nM) was added to 30 nM Hsp90 in the
presence of 5 μL of the test compounds 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 concentration 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 performed using Prism GraphPad software
program (GraphPad Software, Inc., San Diego, CA, U.S.)
Client Protein Degradation Assay. Client protein degradation
was determined by Western blotting. Twenty-four hours after seeding
on Petri dishes, A431 (human epidermoid carcinoma) cells were
treated, for 24 h in complete medium, with various concentrations of
test compounds depending on their relative potency. 17-DMAG (at
0.2 μM) was used as internal reference inhibitor. Following treatments,
cells were rinsed twice with ice-cold PBS and lysed in RIPA buffer
supplemented with protease and phosphatase inhibitors. After
5-[2,4-Bis(benzyloxy)-5-chlorophenyl]-4-nitroisoxazol-3-yl)-
(3,3-difluoroazetidin-1-yl)methanone (13c). Yellow solid. 1H
NMR (400 MHz CDCl3) δ: 4.52−4.58 (m, 4H), 4.99 (s, 2H), 5.14
(s, 2H), 6.62, (s, 1H), 7.25−7.27, (m, 2H), 7.35−7.41 (m, 8H), 7.66
(s, 1H). m/z 556.5/558.4 [M + H]+.
5-[2,4-Bis(benzyloxy)-5-isopropylphenyl]-4-nitroisoxazole-
1
3-carboxylic Acid Ethylamide (13d). Yellow solid. H NMR (200
MHz CDCl3) δ: 1.22−1.26 (m, 9H), 3.24−3.38 (m, 1H), 3.43−3.57
(m, 2H), 5.02 (s, 4H), 6.54 (s, 1H), 6.59 (br, 1H), 7.30−7.39 (m,
10H), 7.46 (s, 1H). m/z 516.5 [M + H]+.
8601
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General Procedure To Obtain the 4-Aminoisoxazoles 14a−
d. A solution of the appropriate nitro derivative 13a−d (1.97 mmol)
in THF (7 mL) was added to a solution of NH4Cl (2.7 g, 50 mmol) in
water (15 mL). Zinc dust (4 g, 61 mmol) was then added portionwise
over 15 min with stirring at 0 °C. After 30 min at 0 °C, the mixture
was filtered and the resulting cake was rinsed with MeOH. The
combined filtrate was evaporated in vacuo to give the desired amino
compounds 14a−d.
4-Amino-5-[2,4-bis(benzyloxy)-5-chlorophenyl]isoxazole-3-
carboxylic Acid Ethylamide (14a). Yield 82%. Yellow solid. 1H
NMR (200 MHz CDCl3) δ: 1.24 (t, J = 7.2 Hz, 3H), 3.38−3.53 (m,
2H), 5.02 (s, 2H), 5.15 (s, 2H), 6.64 (s, 1H), 6.79 (br, 1H), 7.35−7.42
(m, 10H), 7.64 (s, 1H). m/z 478.3/480.4 [M + H]+.
(br, 1H), 9.81 (s, 1H), 9.90 (br, 1H). m/z 348.5 [M + H]+. Anal.
(C17H21N3O5) C, H, N.
5 - ( 2 , 4 - D i h y d r o x y - 5 - i s o p r o p y l p h e n y l ) - 4 - ( 2 , 2 -
dimethylpropionylamino)isoxazole-3-carboxylic Acid Ethyl-
1
amide (27). White solid. H NMR (400 MHz CD3OD) δ: 1.18−
1.24 (m, 18H), 3.17−3.24 (m, 1H), 3.48 (q, J = 6.8 Hz, 2H), 6.47 (s,
1H), 7.27 (s, 1H). m/z 390.5 [M + H]+. Anal. (C20H27N3O5) C, H, N.
4-(Cyclohexanecarbonylamino)-5-(2,4-dihydroxy-5-
isopropylphenyl)isoxazole-3-carboxylic Acid Ethylamide
(28). 1H NMR (300 MHz) δ: 1.06 (t, J = 5.1 Hz, 3H), 1.08 (d, J =
7.2 Hz, 6H), 1.10−1.36 (m, 5H), 1.57−1.77 (m, 5H), 2.23 (m, 1H),
3.06 (m, 1H), 3.19 (m, 2H), 6.47 (s, 1H), 7.09 (s, 1H), 8.46 (t, J = 6.3
Hz, NH), 8.97 (s, OH), 9.77 (s, NH), 9.89 (s, OH). m/z 438.0 [M +
Na]+. Anal. (C22H29N3O5) C, H, N.
5-[2,4-Bis(benzyloxy)-5-chlorophenyl)-4-amino-N-(2,2,2-
trifluoroethyl)isoxazole-3-carboxamide (14b). Yield 77%. Yel-
5-(2,4-Dihydroxy-5-isopropylphenyl)-4-[(cis-4-
methoxycyclohexanecarbonyl)amino]isoxazole-3-carboxylic
Acid Ethylamide (31). 1H NMR (300 MHz) δ: 1.07 (t, J = 5.2 Hz,
3H), 1.09 (d, J = 7.3 Hz, 6H), 1.37 (m, 2H), 1.49 (m, 2H), 1.61 (m,
2H), 1.80 (m, 2H), 2.26 (m, 1H), 3.06 (m, 1H), 3.14 (s, 3H), 3.16 (m,
2H), 3.29 (bs, 1H), 6.47 (s, 1H), 7.09 (s, 1H), 8.47 (t, J = 6.2 Hz, 1H),
8.9 (s, 1H), 9.76 (s, 1H), 9.85 (s, 1H). m/z 468.3 [M + Na]+. Anal.
(C23H31N3O6) C, H, N.
(1S,3R)-3-[5-(2,4-Dihydroxy-5-isopropylphenyl)-3-ethylcar-
bamoylisoxazol-4-ylcarbamoyl]cyclopentanecarboxylic Acid
Methyl Ester (43). 1H NMR (300 MHz) δ: 1.06 (t, J = 7.0 Hz,
3H), 1.08 (d, J = 6.7 Hz, 6H), 1.76−1.90 (m, 4H), 2.03−2.12 (m,
2H), 2.75−2.79 (m, 2H), 3.06 (hept, J = 7.0 Hz, 1H), 3.19 (q, J = 7.0
Hz, 2H), 3.56 (s, 3H), 6.48 (s, 1H), 7.08 (s, 1H), 8.49 (t, J = 5.5 Hz,
1H), 9.12 (bs, 1H), 9.78 (bs, 2H). m/z 459.9 [M + H]+. Anal.
(C23H29N3O7) C, H, N.
1
low solid. H NMR (200 MHz CDCl3) δ: 3.97−4.14 (m, 2H), 4.35
(br, 2H), 5.04 (s, 2H), 5.16 (s, 2H), 6.65, (s, 1H), 7.08, (br, 1H),
7.31−7.44 (m, 10H), 7.65 (s, 1H). m/z 532.3/534.3 [M + H]+.
5-[2,4-Bis(benzyloxy)-5-chlorophenyl]-4-aminoisoxazol-3-
yl)-(3,3-difluoroazetidin-1-yl)methanone (14c). Yield 65%. Yel-
1
low solid. H NMR (200 MHz CDCl3) δ: 4.43−4.55 (m, 4H), 4.83−
4.95 (m, 2H), 5.03 (s, 2H), 5.15 (s, 2H), 6.64, (s, 1H), 7.29−7.43 (m,
10H), 7.64 (s, 1H). m/z 526.4/528.5 [M + H]+.
4-Amino-5-[2,4-bis(benzyloxy)-5-isopropylphenyl]-
isoxazole-3-carboxylic Acid Ethylamide (14d). Yield 72%.
1
Yellow solid. H NMR (200 MHz CDCl3), δ: 1.21−1.28 (m, 9H),
3.28−3.38 (m, 1H), 3.39−3.53 (m, 2H), 4.38 (br, 2H), 5.05 (s, 2H),
5.08 (s, 2H), 6.61 (s, 1H), 6.83 (br, 1H), 7.33−7.42 (m, 10H), 7.45 (s,
1H). m/z 486.6 [M + H]+.
General Procedure for Preparation of 4-Isoxazole Amides
15−73. A solution of the appropriate amine 14a−d (1.45 mmol) and
TEA (1.74 mmol, 0.24 mL) in DCM was added dropwise to a solution
of the corresponding acyl chloride (1.45 mmol). The mixture was
stirred for 5 h, diluted with DCM, and washed with 1 N HCl. The
organic extract was dried and filtered. Solvents were removed in vacuo
to give the crude residue that was used without further purification.
The amide thus obtained (0.35 mmol) was dissolved in DCM
(10 mL) and was cooled under an inert atmosphere at 0 °C, and BCl3
in 1 M DCM (1.05 mmol, 1.05 mL) was added dropwise. The mixture
was stirred at 0 °C for 20 min. The cooling bath was then removed
and the mixture left for a further 50 min at room temperature. The
mixture was cooled again and then quenched by cautious addition of
saturated aqueous NaHCO3 solution (20 mL). The DCM was
removed in vacuo, and water (20 mL) was added. The mixture was
then extracted with EtOAc (200 mL). The organic layers were washed
with water (2 × 30 mL), saturated aqueous NaCl solution (50 mL)
and then dried over Na2SO4. Crude products were purified by flash
chromatography on silica gel (yield, 35−60%).
5 - ( 2 , 4 - D i h y d r o x y - 5 - i s o p r o p y l p h e n y l ) - 4 - ( 4 -
methoxybenzoylamino)isoxazole-3-carboxylic Acid Ethyl-
amide (46). White solid. 1H NMR (400 MHz) δ: 1.04−1.28
(m, 9H), 3.03−3.08 (m, 1H), 3.19−3.23 (m, 2H), 3.82 (s, 3H), 6.47
(s, 1H), 7.02 (d, J = 8.8 Hz, 2H), 7.23 (s, 1H), 7.87 (d, J = 8.8 Hz,
2H), 8.61 (t, J = 5.6 Hz, 1H), 9.56 (br, 1H), 9.82 (s, 1H), 10.08 (br,
1H). m/z 440.4 [M + H]+. Anal. (C23H25N3O6) C, H, N.
5-(2,4-Dihydroxy-5-isopropylphenyl)-4-(4-morpholin-4-
ylmethylbenzoylamino)isoxazole-3-carboxylic Acid Ethyl-
amide (49). 1H NMR (300 MHz) δ: 1.04 (d, J = 7.2 Hz, 6H),
1.07 (t, J = 7.0 Hz, 3H), 3.18−3.22 (m, 4H), 3.02−3.09 (m, 3H), 3.78
(m, 2H), 3.89 (m, 2H), 4.38 (bs, 2H), 6.52 (s, 1H), 7.21 (s, 1H), 7.73
(d, J = 7.3 Hz, 2H), 7.96 (d, J = 7.3 Hz, 2H), 8.64 (t, J = 5.8 Hz, 1H),
9.76 (s, 1H), 9.86 (s, 1H), 10.13 (s, 1H), 11.2 (s, 1H). m/z 530.8 [M +
Na]+. Anal. (C27H32N4O6 HCl) C, H, N.
5-(2,4-Dihydroxy-5-isopropylphenyl)-4-(5-ethylisoxazole-
3-carbonyl)aminoisoxazole-3-carboxylic Acid Ethylamide
1
(55). White solid. H NMR (400 MHz CD3OD) δ: 1.18−1.31 (m,
12H), 2.80−2.86 (m, 2H), 3.16−3.22 (m, 1H), 3.39 (q, J = 7.2 Hz,
2H), 6.47 (s, 1H), 6.48 (s, 1H), 7.33 (s, 1H). m/z 429.0 [M + H]+.
Anal. (C21H24N4O6) C, H, N.
5-(5-Chloro-2,4-dihydroxyphenyl)-4-[(3-methylthiophene-2-
carbonyl)amino]isoxazole-3-carboxylic Acid Ethylamide
1
(19). Light-yellow solid. H NMR (400 MHz) δ: 1.09 (t, J = 6.8
5-(2,4-Dihydroxy-5-isopropylphenyl)-4-[(3-methylthio-
Hz, 3H), 2.43 (s, 3H), 3.20−3.25 (m, 2H), 6.65 (s, 1H), 7.01 (d, J =
5.2 Hz, 1H), 7.44 (s, 1H), 7.66 (d, J = 5.2 Hz, 1H), 8.58 (t, J = 5.6 Hz,
1H), 9.29 (br, 1H), 10.70 (s, 2H). m/z 422.1/424.1 [M + H]+. Anal.
(C18H16ClN3O5S) C, H, Cl, N, S.
phene-2-carbonyl)amino]isoxazole-3-carboxylic Acid Ethyl-
1
amide (57). White solid. H NMR (400 MHz) δ: 1.07−1.11 (m,
9H), 2.44 (s, 3H), 3.05−3.11 (m, 1H), 3.19−3.25 (m, 2H), 6.52 (s,
1H), 7.01 (d, J = 5.2 Hz, 1H), 7.22 (s, 1H), 7.66 (d, J = 5.2 Hz, 1H),
8.66 (t, J = 5.6 Hz, 1H), 9.12 (s, 1H), 9.89 (s, 1H), 10.22 (br, 1H). m/
z 430.6 [M + H]+. Anal. (C21H23N3O5S) C, H, N, S.
5 - ( 5 - C h l o r o - 2 , 4 - d i h y d r o x y p h e n y l ) - 4 - ( 2 , 2 -
dimethylpropionylamino)isoxazole-3-carboxylic Acid Ethyla-
1
mide (21). White solid. H NMR (400 MHz) δ: 1.06 (t, J = 7.2 Hz,
4-[(5-Bromofuran-2-carbonyl)amino]-5-(2,4-dihydroxy-5-
isopropylphenyl)isoxazole-3-carboxylic Acid Ethylamide
(58). 1H NMR (300 MHz) δ: 1.05 (d, J = 7.0 Hz, 6H), 1.07 (t, J =
7.6 Hz, 3H), 3.05 (hept, J = 6.7 Hz, 1H), 3.21 (quint, J = 7.6 Hz, 2H),
6.47 (s, 1H), 6.78 (d, J = 3.7 Hz, 1H), 7.17 (s, 1H), 7.24 (d, J = 3.7
Hz, 1H), 8.62 (t, J = 5.7 Hz, 1H), 9.75 (bs, 1H), 9.81 (s, 1H), 10.08
(bs, 1H). m/z 477.3/479.3 [M − H]−. Anal. (C20H20BrN3O6) C,
H, N.
5-(2,4-Dihydroxy-5-isopropylphenyl)-4-[(furan-2-carbonyl)-
amino]isoxazole-3-carboxylic Acid Ethylamide (71). 1H NMR
(300 MHz) δ: 1.06 (d, J = 7.0 Hz, 6H), 1.08 (t, J = 7.5 Hz, 3H), 3.06
(m, 1H), 3.21 (m, 2H), 6.49 (s, H), 6.65 (dd, J = 1.6 Hz, J = 3.5 Hz,
1H), 7.19 (s, 1H), 7.2 (d, J = 3.5 Hz, 1H), 7.88 (d, J = 1.6 Hz, 1H),
3H), 1.14 (s, 9H), 3.19−3.24 (m, 2H), 6.66 (s, 1H), 7.34 (s, 1H), 8.58
(t, J = 5.6 Hz, 1H), 8.83 (s, 1H), 10.50 (br, 1H), 10.70 (s, 1H). m/z
382.2/384.2 [M + H]+. Anal. (C17H20ClN3O5) C, H, Cl, N.
4-[(Adamantane-1-carbonyl)amino]-5-(5-chloro-2,4-
dihydroxyphenyl)isoxazole-3-carboxylic Acid Ethylamide
(23). White solid. 1H NMR (400 MHz) δ: 1.09 (t, J = 7.2 Hz,
3H), 1.65−1.69 (m, 6H), 1.81−1.82 (m, 6H), 1.99 (s, 3H), 3.18−3.24
(m, 2H), 6.67 (s, 1H), 7.34 (s, 1H), 8.74 (t, J = 5.6 Hz, 1H), 10.55 (br,
2H). m/z 460.2/462.3 [M + H]+. Anal. (C23H26ClN3O5) C, H, Cl, N.
4-Acetylamino-5-(2,4-dihydroxy-5-isopropylphenyl)-
isoxazole-3-carboxylic Acid Ethylamide (26). White solid. 1H
NMR (400 MHz) δ: 1.07−1.12 (m, 9H), 3.06−3.13 (m, 1H), 3.21−
3.26 (m, 2H), 6.49 (s, 1H), 7.11 (s, 1H), 8.55 (t, J = 6.0 Hz, 1H), 9.15
8602
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8.62 (t, J = 5.4 Hz, 1H), 9.5 (s, 1H), 9.83 (s, 1H), 10.2 (s, 1H). m/z
400.3 [M + H]+. Anal. (C20H21N3O6) C, H, N.
(8) Solit, D. B.; Chiosis, G. Development and application of Hsp90
inhibitors. Drug Discovery Today 2008, 13c, 38−43.
5 - ( 2 , 4 - D i h y d r o x y - 5 - i s o p r o p y l p h e n y l ) - 4 - ( 5 -
(morpholinomethyl)isoxazole-3-carbonylamino)isoxazole-3-
carboxylic Acid Ethylamide (73). 1H NMR (400 MHz CD3OD),
δ: 1.18−1.26 (m, 9H), 2.52−2.54 (m, 4H), 3.17−3.25 (m, 1H), 3.39
(q, J = 7.2 Hz, 2H), 3.68−3.70 (m, 4H), 3.78 (s, 1H), 6.47 (s, 1H),
6.72 (s, 1H), 7.34 (s, 1H). m/z 499.9 [M + H]+. Anal. (C24H29N5O7)
C, H, N.
(9) Prodromou, C.; Peral, L. H. Structure and functional relationship
of the Hsp90. Curr. Cancer Drug Targets 2003, 3, 301−323.
(10) Cowena, L. E.; Singha, S. D.; Kohler, J. R.; Collins, C.; Zaas, A.
̈
K.; Schell, W. A.; Aziz, H.; Mylonakis, E.; Perfect, J. R.; Whitesellf, L.;
Lindquistf, S. Harnessing Hsp90 function as a powerful, broadly
effective therapeutic strategy for fungal infectious disease. Proc. Natl.
Acad. Sci.U.S.A. 2009, 106, 2818−2823.
(11) Connor, J. H.; McKenzie, M. O.; Parks, G. D.; Lyles, D. S.
Antiviral activity and RNA polymerase degradation following Hsp90
inhibition in a range of negative strand viruses. Virology 2007, 362,
109−119.
(12) Luo, W.; Dou, F.; Rodina, A.; Chip, S.; Kim, J.; Zhao, Q.;
Moulick, K.; Aguirre, J.; Wu, N.; Greengard, P.; Chiosis, G. Roles of
heat-shock protein 90 in maintaining and facilitating the
neurodegenerative phenotype in tauopathies. Proc. Natl. Acad. Sci.
U.S.A. 2007, 104, 9511−9516.
(13) Zhang, H.; Burrows, F. Targeting multiple signal transduction
pathways through inhibition of Hsp90. J. Mol. Med. 2004, 82, 488−
499.
(14) Workman, P. Combinatorial attack on multistep oncogenesis by
inhibiting the Hsp90 molecular chaperone. Cancer Lett. 2004, 206,
149−157.
(15) Vilenchik, M.; Solit, D.; Basso, A.; Huezo, H.; Lucas, B.; He, H.;
Rosen, N.; Spampinato, C.; Modrich, P.; Chiosis, G. Targeting wide-
range oncogenic transformation via PU24FCl, a specific inhibitor of
tumor Hsp90. Chem. Biol. 2004, 11, 787−797.
(16) Csermely, P.; Schnaider, T.; Soti, C.; Prohaszka., Z.; Nardai, G.
The 90-kDa molecular chaperone family: structure, function, and
clinical applications. A comprehensive review. Pharmacol. Ther. 1998,
79, 129−168.
(17) Stravopodis, D. J.; Margaritis, L. H.; Voutsinas, G. E. Drug-
mediated targeted disruption of multiple protein activities through
functional inhibition of the Hsp90 chaperone complex. Curr. Med.
Chem. 2007, 14, 3122−3138.
(18) Whitesell, L.; Lindquist, S. L. HSP90 and the chaperoning of
cancer. Nat. Rev. Cancer 2005, 5, 761−772.
(19) Picard, D. Heat-shock protein 90, a chaperone for folding and
regulation. Cell. Mol. Life Sci. 2002, 59, 1640−1648.
(20) Eustace, B. K.; Sakurai, T. A.; Stewart, J. K.; Yimlamai, D.;
Unger, C.; Zehetmeier, C.; Lain, B.; Torella, C.; Henning, S. W.; Beste,
G.; Scroggins, B. T.; Neckers, L.; Ilag, L. L.; Jay, D. G. Functional
proteomic screens reveal an essential extracellular role for hsp90 alpha
in cancer cell invasiveness. Nat. Cell Biol. 2004, 6, 507−514.
(21) Koga, F.; Tsutsumi, S.; Neckers, L. M. Low dose geldanamycin
inhibits hepatocyte growth factor and hypoxia-stimulated invasion of
cancer cells. Cell Cycle 2007, 6, 1393−1402.
ASSOCIATED CONTENT
* Supporting Information
Additional experimental procedures and characterization data,
docking studies, analysis of the degradation of Hsp90 client
proteins, physicochemical parameters of 60 compounds, and
metabolism data. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*For D.S.: phone, +39-0532-455923; fax, +39-0532-455953;
e-mail, smd@unife.it. For G.G.: phone, +39-0691393640; fax,
+39-0691393638; e-mail, giuseppe.giannini@sigma-tau.it.
■
ACKNOWLEDGMENTS
■
The authors thank Isabella Lustrati, Silvio Zavatto, M. Cati,
Massimo De Santis, Federico Serafini, and Virgilio Achermann
for excellent technical assistance. This work was supported by
grants from Sigma-Tau Research Switzerland S.A. (P.O. Box
1823, Via Motta 2a-CH-6850 Mendrisio, Switzerland; phone,
+41-091640.4050).
DEDICATION
†Dedicated to the memory of Dr. Paolo Carminati.
■
ABBREVIATIONS USED
■
Hsp90, heat shock protein 90; 17-AAG, 17-allylaminogeldana-
mycin; 17-DMAG, 17-dimethylaminoethylamino-17-demethoxy-
geldanamycin; TVI, tumor volume inhibition; BWL, body weight
loss
REFERENCES
■
(1) Maloney, A.; Workman, P. Hsp90 as a new therapeutic target for
cancer therapy: the story unfolds. Expert Opin. Biol. Ther. 2002, 2,
3−24.
(22) Trepel, J.; Mollapour, M.; Giaccone, G.; Neckers, L. Targeting
the dynamic HSP90 complex in cancer. Nat. Rev. Cancer 2010, 10,
537−549.
(2) Whitesell, L.; Lindquist, S. L. Hsp90 and the chaperoning of
cancer. Nat. Rev. Cancer 2005, 5, 761−772.
(23) Whitesell, L.; Shifrin, S. D.; Schwab, G.; Neckers, L. M.
Benzoquinonoid ansamycins possess selective tumoricidal activity
unrelated to src kinase inhibition. Cancer Res. 1992, 52, 1721−1728.
(24) Supko, J. G.; Hickman, R. L.; Grever, M. R.; Malspeis, L.
Preclinical pharmacologic evaluation of geldanamycin as an antitumor
agent. Cancer Chemother. Pharmacol. 1995, 36, 305−315.
(25) Infinity Pharmaceuticals, Inc. Product Candidates. http://www.
infi.com/product-candidates-pipeline.asp.
(3) Kamal, A.; Boehm, M. F.; Burrows, F. J. Therapeutic and
diagnostic implications of Hsp90 activation. Trends Mol. Med. 2004,
10, 283−290.
(4) Pratt, W. B.; Morishima, Y.; Peng, H.-M.; Osawa, Y. Proposal for
a role of the Hsp90/Hsp70-based chaperone machinery in making
triage decisions when proteins undergo oxidative and toxic damage.
Exp. Biol. Med. 2010, 235, 278−289.
(5) Vega, V. L.; De Maio, A. Geldanamycin treatment ameliorates the
response to LPS in murine macrophages by decreasing CD14 surface
expression. Mol. Biol. Cell 2003, 14, 764−773.
(6) Poulaki, V.; Iliaki, E.; Mitsiades, N.; Mitsiades, C. S.; Paulus, Y.
N.; Bula, D. V.; Gragoudas, E. S.; Miller, J. W. Inhibition of Hsp90
attenuates inflammation in endotoxin-induced uveitis. FASEB J. 2007,
21, 2113−2123.
(7) Kumar, R.; Pavithra, S. R.; Tatu, U. Three-dimensional structure
of heat shock protein 90 from Plasmodium falciparum: molecular
modelling approach to rational drug design against malaria. J. Biosci.
2007, 32, 531−536.
(26) Sharma, S. V.; Agatsuma, T.; Nakano, H. Targeting of the
protein chaperone, HSP90, by the transformation suppressing agent,
radicicol. Oncogene 1998, 16, 2639−2645.
(27) Biamonte, M. A.; Van de Water, R.; Arndt, J. W.; Scannevin, R.
H.; Perret, D.; Lee, W.-C. Heat shock protein 90: inhibitors in clinical
trials. J. Med. Chem. 2010, 53, 3−17.
(28) Holzbeierlein, J. M.; Windsperger, A.; Vielhauer, G. Hsp90: a
drug target? Curr. Oncol. Rep. 2010, 12, 95−101.
(29) Taldone, T.; Chiosis, G. Purine-scaffold Hsp90 inhibitors. Curr.
Top. Med. Chem. 2009, 9, 1436−1446.
8603
dx.doi.org/10.1021/jm201155e | J. Med. Chem. 2011, 54, 8592−8604

Journal of Medicinal Chemistry
Article
(30) He, H.; Zatorska, D.; Kim, J.; Aguirre, J.; Llauger, L.; She, Y.;
Wu, N.; Immormino, R. M.; Gewirth, D. T.; Chiosis, G. Identification
of potent water soluble purine-scaffold inhibitors of the heat shock
protein 90. J. Med. Chem. 2006, 49, 381−390.
Y.; Surgenor, A.; Walmsley, D. L.; Webb, P.; Wood, M.; Workman, P.;
Wright, L. 4,5-Diarylisoxazole Hsp90 chaperone inhibitors: potential
therapeutic agents for the treatment of cancer. J. Med. Chem. 2008, 51,
196−218.
(42) Dymock, B. W.; Barril, X.; Brough, P. A.; Cansfield, J. E.;
Massey, A.; McDonald, E.; Hubbard, R. E.; Surgenor, A.; Roughley, S.
D.; Webb, P.; Workman, P.; Wright, L.; Drysdale, M. J. Novel potent
small-molecule inhibitors of the molecular chaperone Hsp90
discovered through structure-based design. J. Med. Chem. 2005, 48,
4212−4215.
(43) Smith, N. F.; Hayes, A.; James, K.; Nutley, B. P.; McDonald, E.;
Henley, A.; Dymock, B.; Drysdale, M. J.; Raynaud, F. I.; Workman, P.
Preclinical pharmacokinetics and metabolism of a novel diaryl pyrazole
resorcinol series of heat shock protein 90 inhibitors. Mol. Cancer Ther.
2006, 5, 1628−1638.
(31) Debiopharm Group. Pipeline. www.debiopharm.com/our-
business/pipeline.html.
(32) Huang, K. H.; Veal, J. M.; Fadden, R. P.; Rice, J. W.; Eaves, J.;
Strachan, J.-P.; Barabasz, A. F.; Foley, B. E.; Barta, T. E.; Ma, W.;
Silinski, M. A.; Hu, M.; Partridge, J. M.; Scott, A.; DuBois, L. G.;
Freed, T.; Steed, P. M.; Ommen, A. J.; Smith, E. D.; Hughes, P. F.;
Woodward, A. R.; Hanson, G. J.; McCall, W. S.; Markworth, C. J.;
Hinkley, L.; Jenks, M.; Geng, L.; Lewis, M.; Otto, J.; Pronk, B.;
Verleysen, K.; Hall, S. E. Discovery of novel 2-aminobenzamide
inhibitors of heat shock protein 90 as potent, selective and orally active
antitumor agents. J. Med. Chem. 2009, 52, 4288−4305.
(44) Brough, P. A.; Barril, X.; Borgognoni, J.; Chene, P.; Davies, N.
G.; Davis, B.; Drysdale, M. J.; Dymock, B.; Eccles, S. A.; Garcia-
Echeverria, C.; Fromont, C.; Hayes, A.; Hubbard, R. E.; Jordan, A. M.;
Jensen, M. R.; Massey, A.; Merrett, A.; Padfield, A.; Parsons, R.;
Radimerski, T.; Raynaud, F. I.; Robertson, A.; Roughley, S. D.;
Schoepfer, J.; Simmonite, H.; Sharp, S. Y.; Surgenor, A.; Valenti, M.;
Walls, S.; Webb, P.; Wood, M.; Workman, P.; Wright, L. Combining
hit identification strategies: fragment-based and in silico approaches to
orally active 2-aminothieno[2,3-d]pyrimidine inhibitors of the Hsp90
molecular chaperone. J. Med. Chem. 2009, 52, 4794−4809.
(45) Sharp, S. Y.; Boxall, K.; Rowlands, M.; Prodromou, C.; Roe, S.
M.; Maloney, A.; Powers, M.; Clarke, P. A.; Box, G.; Sanderson, S.;
Patterson, L.; Matthews, T. P.; Cheung, K. M.; Ball, K.; Hayes, A.;
Raynaud, F.; Marais, R.; Pearl, L.; Eccles, S.; Aherne, W.; McDonald,
E.; Workman, P. In vitro biological characterization of a novel,
synthetic diaryl pyrazole resorcinol class of heat shock protein 90
inhibitors. Cancer Res. 2007, 67, 2206−2216.
(46) Ohba, S.; Hirose, Y.; Yoshida, K.; Yazaki, T.; Kawase, T.
Inhibition of 90-kD heat shock protein potentiates the cytotoxicity of
chemotherapeutic agents in human glioma cells. J. Neurosurg. 2010,
112, 33−42.
(47) Simoni, D.; Romagnoli, R.; Baruchello, R.; Rondanin, R.; Rizzi,
M.; Pavani, M. G.; Alloatti, D.; Giannini, G.; Marcellini, M.; Riccioni,
T.; Castorina, M.; Guglielmi, M. B.; Bucci, F.; Carminati, P.; Pisano, C.
Novel combretastatin analogues endowed with antitumor activity. J.
Med. Chem. 2006, 49, 3143−3152.
(33) Gopalsamy, A.; Shi, M.; Golas, J.; Vogan, E.; Jacob, J.; Johnson,
M.; Lee, F.; Nilakantan, R.; Petersen, R.; Svenson, K.; Chopra, R.;
Tam, M. S.; Wen, Y.; Ellingboe, J.; Arndt, K.; Boschelli, F. Discovery of
benzisoxazoles as potent inhibitors of chaperone heat shock protein
90. J. Med. Chem. 2008, 51, 373−375.
(34) Woodhead, A. J.; Angove, H.; Carr, M. G.; Chessari, G.;
Congreve, M.; Coyle, J. E.; Cosme, J.; Graham, B.; Day, P. J.;
Downham, R.; Fazal, L.; Feltell, R.; Figueroa, E.; Frederickson, M.;
Lewis, J.; McMenamin, R.; Murray, C. W.; O’Brien, M. A.; Parra, L.;
Patel, S.; Phillips, T.; Rees, D. C.; Rich, S.; Smith, D.-M.;
Gary Trewartha, G.; Vinkovic, M.; Williams, B.; Woolford, A. J.-A.
Discovery of (2,4-dihydroxy-5-isopropylphenyl)-[5-(4-methylpiperazin-
1-ylmethyl)-1,3-dihydroisoindol-2-yl]methanone (AT13387), a novel
inhibitor of the molecular chaperone Hsp90 by fragment based drug
design. J. Med. Chem. 2010, 53, 5956−5969.
(35) Wang, Y.; Trepel, J. B.; Neckers, L. M.; Giaccone, G. STA-9090,
a small-molecule Hsp90 inhibitor for the potential treatment of cancer.
Curr. Opin. Invest. Drugs 2010, 11, 1466−1476.
(36) London, C. A.; Bear, M. D.; McCleese, J.; Foley, K. P.;
Paalangara, R.; Inoue, T.; Ying, W.; Barsoum, J. Phase I Evaluation of
STA-1474, a Pro-Drug of the Novel HSP90 Inhibitor Ganetespib, in
Dogs with Spontaneous Cancer. Presented at the 102nd Annual
Meeting of the AACR, Orlando, FL, Apr 2−6, 2011; Abstract 1282.
(37) Papac, D. I.; Patton, J. S.; Reeves, L.; Bulka, K.; DeMie, L.;
Roman, O.; Bradford, C.; Kim, S.-H.; Tangallapally, R.; Trovato, R.;
Markovitz, B.; Bajji, A.; Wettstein, D.; Baichwal, V.; Mather, G.
Comparative in Vitro and in Vivo Metabolism of MPC-3100, an Oral
Hsp90 Inhibitor, in Rat, Dog, Monkey and Human. Presented at the
102nd Annual Meeting of the AACR, Orlando, FL, Apr 2−6, 2011;
Abstract 3233.
(48) Giannini, G.; Cabri, W.; Simoni, D.; Baruchello, R.; Carminati,
P.; Pisano, C. PCT Int. Appl. WO2010000748A1, 2010. Sigma-Tau
Research Switzerland S.A. retains the property rights of all the
compounds mentioned in the paper.
(49) A preliminary metabolism study on human cryopreserved
hepatocytes, performed on four representative compounds (26, 46, 57,
73), revealed that this class of inhibitors possesses metabolic stability
higher than the reference compound 10 (Table S2).
(38) Papac, D.; Patton, J. S.; Reeves, L.; DeMie, L.; Bradford, C.;
Hachey, B.; Clemetson, B.; Christensen, C.; Kim, S.-H.; Tangallapally,
R.; Parker, D.; Trovato, R.; Kim, I. C.; Wettstein, D.; Baichwal, V. R.;
Bajji, A. Evaluation of the Pharmacokinetics and Efficacy of a Novel
Pro-Drug of the Hsp90 Inhibitor, MPC-3100, Designed with
Improved Solubility. Presented at the 102nd Annual Meeting of the
AACR, Orlando, FL, Apr 2−6, 2011; Abstract 3237.
(50) Kim, J.; Felts, S.; Llauger, L.; He, H.; Huezo, H.; Rosen, N.;
Chiosis, G. Development of a fluorescence polarization assay for the
molecular chaperone Hsp90. J. Biomol. Screening 2004, 9, 375−381.
(39) Cheung, K. M.; Matthews, T. P.; James, K.; Rowlands, M. G.;
Boxall, K. J.; Sharp, S. Y.; Maloney, A.; Roe, S. M.; Prodromou, C.;
Pearl, L. H.; Aherne, G. W.; McDonald, E.; Workman, P. The
identification, synthesis, protein crystal structure and in vitro
biochemical evaluation of a new 3,4-diarylpyrazole class of Hsp90
inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 3338−3343.
(40) Brough, P. A.; Barril, X.; Beswick, M.; Dymock, B. W.; Drysdale,
M. J.; Wright, L.; Grant, K.; Massey, A.; Surgenor, A.; Workman, P. 3-
(5-Chloro-2,4-dihydroxyphenyl)-pyrazole-4-carboxamides as inhibitors
of the Hsp90 molecular chaperone. Bioorg. Med. Chem. Lett. 2005, 15,
5197−5201.
(41) Brough, P. A.; Aherne, W.; Barril, X.; Borgognoni, J.; Boxall, K.;
Cansfield, J. E.; Cheung, K. M.; Collins, I.; Davies, N. G.; Drysdale, M.
J.; Dymock, B.; Eccles, S. A.; Finch, H.; Fink, A.; Hayes, A.; Howes, R.;
Hubbard, R. E.; James, K.; Jordan, A. M.; Lockie, A.; Martins, V.;
Massey, A.; Matthews, T. P.; McDonald, E.; Northfield, C. J.; Pearl, L.
H.; Prodromou, C.; Ray, S.; Raynaud, F. I.; Roughley, S. D.; Sharp, S.
8604
dx.doi.org/10.1021/jm201155e | J. Med. Chem. 2011, 54, 8592−8604