Design, synthesis, and evaluation of small molecule Hsp90 probes

Article abstract of DOI:10.1016/j.bmc.2011.03.013

A number of compounds from different chemical classes are known to bind competitively to the ATP-pocket of Hsp90 and inhibit its chaperone function. The natural product geldanamycin was the first reported inhibitor of Hsp90 and since then synthetic inhibitors from purine, isoxazole and indazol-4-one chemical classes have been discovered and are currently or soon to be in clinical trials for the treatment of cancer. In spite of a similar binding mode to Hsp90, distinct biological profiles were demonstrated among these molecules, both in vitro and in vivo. To better understand the molecular basis for these dissimilarities, we report here the synthesis of chemical tools for three Hsp90 inhibitor classes. These agents will be useful for probing tumor-by-tumor the Hsp90 complexes isolated by specific inhibitors. Such information will lead to better understanding of tumor specific molecular markers to aid in their clinical development. It will also help to elucidate the molecular basis for the biological differences observed among Hsp90 inhibitors.

Full text of DOI:10.1016/j.bmc.2011.03.013

Bioorganic & Medicinal Chemistry 19 (2011) 2603–2614
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and evaluation of small molecule Hsp90 probes
Tony Taldone a, Danuta Zatorska a, Pallav D. Patel a, Hongliang Zong b, Anna Rodina a,
James H. Ahn a, Kamalika Moulick a, Monica L. Guzman b, Gabriela Chiosis a,
⇑
a Program in Molecular Pharmacology and Chemistry and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, United States
b Division of Hematology and Oncology, Weill Cornell Medical College, New York, NY 10065, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of compounds from different chemical classes are known to bind competitively to the
ATP-pocket of Hsp90 and inhibit its chaperone function. The natural product geldanamycin was the first
reported inhibitor of Hsp90 and since then synthetic inhibitors from purine, isoxazole and indazol-4-one
chemical classes have been discovered and are currently or soon to be in clinical trials for the treatment
of cancer. In spite of a similar binding mode to Hsp90, distinct biological profiles were demonstrated
among these molecules, both in vitro and in vivo. To better understand the molecular basis for these dis-
similarities, we report here the synthesis of chemical tools for three Hsp90 inhibitor classes. These agents
will be useful for probing tumor-by-tumor the Hsp90 complexes isolated by specific inhibitors. Such
information will lead to better understanding of tumor specific molecular markers to aid in their clinical
development. It will also help to elucidate the molecular basis for the biological differences observed
among Hsp90 inhibitors.
Received 22 September 2010
Revised 26 February 2011
Accepted 6 March 2011
Available online 12 March 2011
Keywords:
Heat shock protein 90
PU-H71
NVP-AUY922
SNX-2112
Cancer
Docking
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
differences are the kinetics of client protein modulation and
in vivo pharmacodynamic profiles which both can have a determining
Heat shock protein 90 (Hsp90) is a molecular chaperone with
key roles in folding and maintaining the conformational integrity
of its client proteins through its ATPase activity.1,2 Because many
of these proteins (i.e., Her2, Raf-1, Akt, Cdk4, Polo-1 kinase, cMet,
mutant B-Raf, mutant p53, AR, ER, Bcr–Abl, HIF-1 alpha, hTERT)
are associated with signaling pathways involved in cell regulation,
it plays an important role in maintaining the transformed pheno-
type.3 As such, Hsp90 has become one of the highly pursued
molecular targets in cancer therapy, with efforts in the develop-
ment of Hsp90-inhibitory agents in neurodegenerative diseases
and pathogenic resistance (i.e., viral, fungal, bacterial) closely fol-
lowing behind.3,4 A search on esp@cenet patent database returned
over 700 hits in September 2010, with several applications claim-
ing distinct compositions of matter that interact with Hsp90.
At least five distinct Hsp90 inhibitor chemotypes are currently
in clinical studies, with many others following in late-stage IND
evaluation or different stages of pre-clinical evaluation.4,5 While
the interest in Hsp90 inhibitors has spread fast, knowledge on
the target has however, lagged behind. It is known that while all
current Hsp90 inhibitors in advanced stage (clinical or late-stage
IND) inhibit Hsp90 by binding to its N-terminal site regulatory
pocket, evidence suggests important distinctions in the in vitro
and in vivo behavior of these agents.6–9 Among most critical
effect on the clinical efficacy and the therapeutic window of the
Hsp90-agents.6–9
In addition, with the advancement of Hsp90 inhibitors into sev-
eral cancers, and potentially other diseases, it will be important to
identify tumor- and disease-specific Hsp90 clients and implement
these in the clinic as potential molecular markers to provide proof
that the target, Hsp90, is inhibited. Identification of proper clinical
biomarkers has become of utmost importance. The cost of success-
ful anticancer drug development to the stage of approval has esca-
lated recently to more than $800 million, and having a properly
chosen pharmacodynamic disease biomarker can increase the effi-
cacy of the process. In particular, it can be a useful indicator of drug
activity and accelerate the ability to make go-no go decisions early
in the clinical process.10
Most molecules currently known to inhibit Hsp90 function
(Fig. 1) mimic or adopt scaffolds based on those of geldanamycin11
(GM, ansamycin class), PU-H7112 (purine class), NVP-AUY9227
(isoxazole class) and SNX-211213 (indazol-4-one class). Geldana-
mycin was the first identified Hsp90 inhibitor and as such has
served a central role in the study of Hsp90 biology. Originally, it
was believed to inhibit Src kinase directly, but by using geldana-
mycin covalently bound to Affi-GelÒ 10 solid support, it was later
shown to bind to Hsp90 and inhibit heterocomplex formation with
Src.11 Biotinylated geldanamycin was also synthesized.14
Here, we report on the design and synthesis of molecules based
on purine, isoxazole and indazol-4-one chemical classes attached
⇑
Corresponding author. Tel.: +1 646 888 2235; fax: +1 646 422 0416.
E-mail address: chiosisg@mskcc.org (G. Chiosis).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.013

2604
T. Taldone et al. / Bioorg. Med. Chem. 19 (2011) 2603–2614
to contain a distal amine functionality. The site of linker attach-
ment to PU-H71 was aided by the co-crystal structure of it bound
to the N-terminal domain of human Hsp90 (PDB ID: 2FWZ). This
O
O
NH2
N
MeO
I
O
N
N
a
O
N
structure shows that the purine’s N9 amine makes no direct con-
tact with the protein and is directed towards solvent (Fig. 2A).16
As well, a previous SAR indicated that this is an attractive site since
it was previously used for the introduction of water solubilizing
groups.12 Compound 5 (PU-H71-C6 linker) was designed and
docked onto the Hsp90 active site (Fig. 2A). All the interactions
of PU-H71 were preserved, and the computer model clearly
showed that the linker oriented towards the solvent exposed
region. Therefore, compound 5 was synthesized as the immediate
precursor for attachment to solid support (see Chemistry, Scheme
1). In the FP assay, 5 retained affinity for Hsp90 (IC50 = 19.8 nM
compared to 22.4 nM for PU-H71, Table 1) which then enabled
us to move forward with confidence towards the synthesis of solid
support immobilized PU-H71 probe (6) by attachment to Affi-GelÒ
10 (Scheme 1).
S
N
H
O
H3CO
OH
H3CO
NH
OCONH2
GM
PU-H71
O
H2N
O
H
N
N
OH
HO
N
N
We also designed a biotinylated derivative of PU-H71. One
advantage of the biotinylated agent over the solid supported
agents is that they can be used to probe binding directly in cells
or in vivo systems. The ligand-Hsp90 complexes can then be cap-
tured on biotin-binding avidin or streptavidin containing beads.
Typically this process reduces the unspecific binding associated
with chemical precipitation from cellular extracts. Alternatively,
for in vivo experiments, the presence of active sites (in this case
Hsp90), can be detected in specific tissues (i.e., tumor mass in
cancer) by the use of a labeled-streptavidin conjugate (i.e., FITC-
streptavidin). Biotinylated PU-H71 (7) was obtained by reaction
HN
F3C
O
OH
NVP-AUY922
Figure 1. Structures of Hsp90 inhibitors.
N
O
O
SNX-2112
to Affi-GelÒ 10 beads and on the synthesis of a biotinylated purine
compound. These are chemical tools to investigate and understand
the molecular basis for the distinct behavior of Hsp90 inhibitors.
They can be also used to better understand Hsp90 tumor biology
by examining bound client proteins and co-chaperones. Under-
standing the tumor specific clients of Hsp90 most likely to be mod-
ulated by each Hsp90 inhibitor could lead to a better choice of
pharmacodynamic markers, and thus a better clinical design. Not
lastly, understanding the molecular differences among these
Hsp90 inhibitors could result in identifying characteristics that
could lead to the design of an Hsp90 inhibitor with most favorable
clinical profile.
of
2
with biotinyl-3,6,9-trioxaundecanediamine (EZ-LinkÒ
Amine–PEO3–Biotin) (Scheme 2). Compound 7 retained affinity
for Hsp90 (IC50 = 67.1 nM) and contains an exposed biotin capable
of interacting with streptavidin for affinity purification.
From the available co-crystal structure of NVP-AUY922 with
Hsp90
a
(PDB ID: 2VCI, Fig. 2B) and co-crystal structures of related
, as well as from SAR, it was
3,4-diarylpyrazoles with Hsp90
a
evident that there was a considerable degree of tolerance for sub-
stituents at the para-position of the 4-aryl ring.7,17–19 Because the
4-aryl substituent is largely directed towards solvent and substitu-
tion at the para-position seems to have little impact on binding
affinity, we decided to attach the molecule to solid support at this
position. In order to enable attachment, the morpholine group was
changed to the 1,6-diaminohexyl group to give 10 as the immedi-
ate precursor for attachment to solid support. Docking 10 onto the
active site (Fig. 2B) shows that it maintains all of the interactions of
NVP-AUY922 and that the linker orients towards the solvent
exposed region. When 10 was tested in the binding assay it also
retained affinity (IC50 = 7.0 nM compared to 4.1 nM for NVP-
AUY922, Table 1) and was subsequently used for attachment to
solid support (see Chemistry, Scheme 3).
2. Design of Hsp90 probes and precursor evaluation
The attachment of small molecules to a solid support is a very
useful method to probe their target and the target’s interacting
partners. Indeed, as mentioned above, geldanamycin attached to
solid support enabled for the identification of Hsp90 as its target.11
Perhaps the most crucial aspects in designing such chemical
probes are determining the appropriate site for attachment of
the small molecule ligand, and designing an appropriate linker
between the molecule and the solid support. Our strategy to design
Hsp90 chemical probes entails several steps. First, in order to val-
idate the optimal linker length and its site of attachment to the
Hsp90 ligand, the linker-modified ligand was docked onto an
Although a co-crystal structure of SNX-2112 with Hsp90 is not
publicly available, that of a related tetrahydro-4H-carbazol-4-one
(27) bound to Hsp90a
(PDB ID: 3D0B, Fig. 2C) is.20 This, along with
appropriate X-ray crystal structure of Hsp90
a
. Second, the linker-
the reported SAR for 27 suggests linker attachment to the hydroxyl
of the trans-4-aminocylohexanol substituent. Direct attachment of
6-amino-caproic acid via an ester linkage was not considered
desirable because of the potential instability of such bonds in
lysate mixtures due to omnipresent esterases. Therefore, the
hydroxyl was substituted with amino to give the trans-1,4-diamin-
ocylohexane derivative 18 (Scheme 4). Such a change resulted in
nearly a 14-fold loss in potency as compared to SNX-2112
(Table 1). 6-(Boc-amino)caproic acid was attached to 18 and
following deprotection, 20 was obtained as the immediate precur-
sor for attachment to beads (see Chemistry, Scheme 4). Docking
suggested that 20 interacts similarly to 27 (Fig. 2C) and that the
linker orients towards the solvent exposed region. Compound 20
modified ligand was evaluated in a fluorescent polarization (FP)
assay that measures competitive binding to Hsp90 derived from
a cancer cell extract. This assay uses Cy3b-labeled geldanamycin
as the FP-optimized Hsp90 ligand.15 These steps are important to
ensure that the solid support immobilized molecules maintain a
strong affinity for Hsp90. Finally, the linker-modified small mole-
cule was attached to the solid support, and its interaction with
Hsp90 was validated by incubation with an Hsp90-containing cell
extract.
We chose Affi-GelÒ 10 (BioRad) for ligand attachment. These
agarose beads have an N-hydroxysuccinimide ester at the end of
a 10C spacer arm, and in consequence, each linker was designed

T. Taldone et al. / Bioorg. Med. Chem. 19 (2011) 2603–2614
2605
A
B
C
NH2
O
HN
N
H3CO
O
27
Figure 2. (A) Interactions of Hsp90
a
(PDB ID: 2FWZ) with PU-H71 (ball and stick model) and compound 5 (tube model). (B) Interactions of Hsp90
a
(PDB ID: 2VCI) with
NVP-AUY922 (ball and stick model) and compound 10 (tube model). (C) Interactions of Hsp90
a (PDB ID: 3D0B) with compound 27 (ball and stick model) and compound 20
(tube model). Hydrogen bonds are shown as dotted yellow lines and important active site amino acid residues and water molecules are represented as sticks.
NH2
N
I
NH2
N
NH2
N
I
I
N
N
O
N
N
N
N
O
S
O
N
N
a
b
S
S
O
O
N
H
O
1
NH(CH2)6NHBoc
2
Br
4
NH2
N
N
I
NH2
N
N
N
O
N
S
N
c
d
O
HN(CH2)6HN
N
S
I
NH(CH2)6NH2
O
O
5
6
Scheme 1. Synthesis of PU-H71 beads (6). Reagents and conditions: (a) Cs2CO3, 1,3-dibromopropane, DMF, rt; (b) NH2(CH2)6NHBoc (3), DMF, rt, 24 h; (c) TFA, CH2Cl2, rt; (d)
Affigel-10, DIEA, DMAP, DMF.
was determined to have good affinity for Hsp90 (IC50 = 24.7 nM
compared to 15.1 nM for SNX-2112 and 210.1 nM for 18, Table
1) and to have regained almost all of the affinity lost by 18. The
difference in activity between 18 and both 20 and SNX-2112 is
well explained by our binding model, as compounds 20 (–C@O,
Fig. 2C) and SNX-2112 (–OH, Figure not shown) form a hydrogen

2606
T. Taldone et al. / Bioorg. Med. Chem. 19 (2011) 2603–2614
Table 1
Binding affinity for Hsp90 from SKBr3 cellular extracts
at this position are disfavored. The amide bond of 20 converts the
basic amino of 18 into a non-basic amide group capable of acting as
an H-bond acceptor to Lys 58, similarly to the hydroxyl of
SNX-2112.
Compound
IC50 (nM)
GM
PU-H71
5
7
15.4
22.4
19.8
67.1
4.1
3. Chemistry
NVP-AUY922
10
SNX-2112
18
20
7.0
Synthesis of PU-H71 beads (6) is shown in Scheme 1 and
commences with the 9-alkylation of 8-arylsulfanylpurine (1)12 with
1,3-dibromopropane to afford 2 in 35% yield. The low yield obtained
in the formation of 2 can be primarily attributed to unavoidable
competing 3-alkylation. Five equivalents of 1,3-dibromopropane
were used to ensure complete reaction of 1 and to limit other
undesirable side-reactions, such as dimerization, which may also
contribute to the low yield. 2 was reacted with tert-butyl 6-amin-
ohexylcarbamate (3) to give the Boc-protected amino purine 4 in
15.1
210.1
24.7
bond with the side-chain amino of Lys 58. 18 contains a strongly
basic amino group and is incapable of forming a hydrogen bond
with Lys 58 side chain (NH2, Figure not shown). This is in good
agreement with the observation of Huang et al.13 that basic amines
NH2
N
I
N
N
O
N
S
O
NH2
N
I
N
N
O
N
a
S
HN
O
O
Br
O
H
NH
2
S
O
O
N
H
H
HN
O
PU-H71-biotin 7
Scheme 2. Synthesis of PU-H71-biotin (7). Reagents and conditions: (a) EZ-LinkÒ amine–PEO3–biotin, DMF, rt.
O
NH(CH2)6NHBoc
NH(CH2)6NH2
BnO
HO
BnO
a
b
NHEt
O
NHEt
O
NHEt
O
BnO
HO
OBn O
O
O
N
N
N
9
10
8
NH(CH2)6NH
HO
c
NHEt
O
HO
O
N
11
Scheme 3. Synthesis of NVP-AUY922 beads (11). Reagents and conditions: (a) NH2(CH2)6NHBoc (3), NaCNBH3, AcOH, MeOH, rt; (b) BCl3, CH2Cl2, rt; (c) Affigel-10, DIEA,
DMAP, DMF.

T. Taldone et al. / Bioorg. Med. Chem. 19 (2011) 2603–2614
2607
O
b
a
O
S
O
S
NH
N
NHNH2
+
O
O
O
O
13
12
14
Br
N
NC
CN
H
N
H
N
d
c
NH2
N
N
N
N
F3C
O
F3C
O
F3C
15
O
16
17
O
H2N
H2N
O
O
H
H
N
N
O
g
e
f
NHBoc
NH2
NH
N
N
N
N
F3C
F3C
O
19
18
H2N
O
H2N
O
H
N
H
N
O
O
h
NH2
NH
NH
NH
N
N
N
N
F3C
F3C
O
O
21
20
Scheme 4. Synthesis of SNX-2112 beads (21). Reagents and conditions: (a) p-toluene sulfonic acid, toluene, reflux, 1.5 h; (b) trifluoroacetic anhydride, Et3N, THF, 55 °C, 3 h,
then methanol/NaOH rt, 3 h; (c) 2-bromo-4-fluorobenzonitrile, NaH, DMF, 90 °C, 5.5 h.; (d) trans-1,4-diaminocyclohexane, NaOtBu, Pd2(dba)3, DavePhos, DME, 50 °C,
overnight; (e) DMSO, EtOH, NaOH, H2O2, rt, 3 h.; (f) 6-(Boc-amino)caproic acid, EDCI, DMAP, rt, 2 h; (g) TFA, CH2Cl2, rt; (h) Affigel-10, DIEA, DMAP, DMF.
90% yield. Deprotection with TFA followed by reaction with Affi-
GelÒ 10 resulted in 6. Biotinylated PU-H71 (7) was also synthesized
by reacting 2 with EZ-LinkÒ Amine–PEO3–Biotin (Scheme 2).
Synthesis of NVP-AUY922 beads (11) from aldehyde 87 is shown
in Scheme 3. Compound 9 was obtained from the reductive amina-
tion of 8 with 3 in 75% yield with no detectable loss of the Boc
group. In a single step, both the Boc and benzyl protecting groups
were removed with BCl3 to give isoxazole 10 in 78% yield, which
was then reacted with Affi-GelÒ 10 to give 11.
Synthesis of SNX-2112 beads (21) is shown in Scheme 4, and
while compounds 17 and 18 are referred to in the patent litera-
ture,21,22 neither is adequately characterized, nor are their synthe-
ses fully described. Therefore, we feel that it is worth describing
the synthesis in detail. Tosylhydrazone 14 was obtained in 89%
yield from the condensation of tosyl hydrazide (12) with dimedone
(13). The one-pot conversion of 14 to tetrahydroindazolone 15
occurs following base promoted cyclocondensation of the
intermediate trifluoroacyl derivative generated by treatment with
trifluroacetic anhydride in 55% yield. Compound 15 was reacted
with 2-bromo-4-fluorobenzonitrile in DMF to give 16 in 91% yield.
It is interesting to note the regioselectivity of this reaction as
arylation occurs selectively at N1. In computational studies of
indazol-4-ones similar to 15, both 1H and 2H-tautomers are known
to exist in equilibrium, however, because of its higher dipole mo-
ment the 1H tautomer is favored in polar solvents.23 The amination
of 16 with trans-1,4-diaminocyclohexane was accomplished under
Buchwald conditions24 using tris(dibenzylideneacetone)dipalla-
dium [Pd2(dba)3] and 2-dicyclohexylphosphino-20-(N,N-dimethyl-
amino)biphenyl (DavePhos) to give nitrile 17 (24%) along with
amide 18 (17%) for a combined yield of 41%. Following complete
hydrolysis of 17, 18 was coupled to 6-(Boc-amino)caproic acid
with EDCI/DMAP to give 19 in 91% yield. Following deprotection,
20 was obtained which was then reacted with Affi-GelÒ 10 to give
21.
Several methods were employed to measure the progress of the
reactions for the synthesis of the final probes. UV monitoring of the
liquid was used by measuring a decrease in kmax for each com-
pound. In general, it was observed that that there was no further
decrease in the kmax after 1.5 h, indicating completion of the reac-
tion. TLC was employed as a crude measure of the progress of the
reaction whereas LC–MS monitoring of the liquid was used to con-
firm complete reaction. While on TLC the spot would not disappear

2608
T. Taldone et al. / Bioorg. Med. Chem. 19 (2011) 2603–2614
H2N
O
H2N
O
O
CN
N
CN
N
H
N
H
N
H
N
Br
OTHP
OTHP
OH
b
c
a
N
N
N
N
N
N
F3C
F3C
F3C
F3C
O
O
O
16
25
26
SNX-2112
Scheme 5. Synthesis of SNX-2112. Reagents and conditions: (a) O-THP-trans-cylohexanolamine (24), NaOtBu, Pd2(dba)3, DavePhos, DME, 60 °C, 3.5 h; (b) DMSO, EtOH, 5 N
NaOH, H2O2, rt, 4 h; (c) PPTS, EtOH, 65 °C, 17 h.
since excess compound was used (1.2 equiv), a clear decrease in
intensity indicated progress of the reaction.
A
The synthesis and full characterization of the Hsp90 inhibitors
PU-H7112 and NVP-AUY9227 have been reported elsewhere.
SNX-2112 had previously been mentioned in the patent litera-
ture,21,22 and only recently has it been fully characterized and its
synthesis adequately described.13 At the time this research project
began specific details on its synthesis were lacking. Additionally,
we had difficulty reproducing the amination of 16 with trans-
4-aminocyclohexanol under conditions reported for similar com-
pounds [Pd(OAc)2, DPPF, NaOtBu, toluene, 120 °C, microwave]. In
our hands, only trace amounts of product were detected at best.
Changing catalyst to PdCl2, Pd(PPh3)4 or Pd2(dba)3 or solvent to
DMF or 1,2-dimethoxyethane (DME) or base to K3PO4 did not
result in any improvement. Therefore, we modified this step and
were able to couple 16 to trans-4-aminocyclohexanol tetrahydro-
pyranyl ether (24) under Buchwald conditions24 using Pd2(dba)3
and DavePhos in DME to give nitrile 25 (28%) along with amide
26 (17%) for a combined yield of 45% (Scheme 5). These were the
conditions used to couple 16 to trans-1,4-diaminocyclohexane,
and similarly some of 25 was hydrolyzed to 26 during the course
of the reaction. Because for our purpose it was unnecessary, we
did not optimize this reaction for 25. We surmised that a major
hindrance to the reaction was the low solubility of trans-4-amino-
cyclohexanol in toluene and that using the THP protected alcohol
24 at the very least increased solubility. SNX-2112 was obtained
and fully characterized (1H, 13C NMR, MS) following removal of
the THP group from 26.
Hsp90
PU-beads (µL)
B
C
Hsp90
c-Kit
IGF-IR
PU-H71
(5 µM)
-
+
-
-
-
-
Lysate (µg)
Hsp90
Bcr-Abl
Raf-1
Figure 3. (A) Hsp90 in K562 extracts (250
PU-, SNX- and NVP-beads or Control-beads (80
2-methoxyethylamine, an Hsp90-inert molecule. Proteins in pull-downs were
analyzed by Western blot. (B) In MDA-MB-468 cell extracts (300 g), PU-beads
lg) was isolated by precipitation with
l
l). Control-beads contain
l
4. Biological evaluation
isolate Hsp90 in complex with its onco-client proteins, c-Kit and IGF-IR. To evaluate
the effect of PU-H71 on the steady-state levels of Hsp90 onco-client proteins, cells
Next, we investigated whether the synthesized beads retained
interaction with tumor Hsp90. Agarose beads covalently attached
to either of PU-H71, NVP-AUY922, SNX-2112 or 2-methoxyethyl-
amine (PU-, NVP-, SNX-, control-beads, respectively), were
incubated with K562 chronic myeloid leukemia (CML) or MDA-
MB-468 breast cancer cell extracts. As seen in Figure 3A, the
Hsp90 inhibitor, but not the control-beads, efficiently isolated
Hsp90 in the cancer cell lysates. Control-beads contain an Hsp90
inactive chemical (2-methoxyethylamine) conjugated to Affi-GelÒ
10 (see Section 6) providing an experimental control for potential
unspecific binding of the solid support to proteins in cell extracts.
Further, to probe the ability of these chemical tools to isolate
genuine Hsp90 client proteins in tumor cells, we incubated
PU-H71 attached to solid support (6) with cancer cell extracts.
We were able to demonstrate dose-dependent isolation of
Hsp90/c-Kit and Hsp90/IGF-IR complexes in MDA-MB-468 cells
( Fig. 3B) and of Hsp90/Bcr-Abl and Hsp90/Raf-1 complexes in
K562 cells ( Fig. 3C). These are Hsp90-dependent onco-proteins
were treated for 24 h with PU-H71 (5
lM). (C) In K562 cell extracts, PU-beads
(40 l) isolate Hsp90 in complex with the Raf-1 and Bcr–Abl onco-proteins.
l
Lysate = endogenous protein content; PU- and Control-beads indicate proteins
isolated on the particular beads. The data are consistent with those obtained from
multiple repeat experiments (n ꢁ 2).
with important roles in driving the transformed phenotype in
triple-negative breast cancers and CML, respectively.3,25,26 In
accord with an Hsp90 mediated regulation of c-Kit and IGF-IR,
treatment of MDA-MB-468 cells with PU-H71 led to a reduction
in the steady-state levels of these proteins (Fig. 3B, compare Lysate,
ꢀ and + PU-H71). Using the PU-beads (6), we were recently able to
isolate and identify novel Hsp90 clients, such as the transcriptional
repressor BCL-6 in diffuse large B-cell lymphoma27 and JAK2 in
mutant JAK2 driven myeloproliferative disorders.28 We were also
able to identify Hsp90 onco-clients specific to triple-negative
breast cancer.9 In addition to shedding light on the mechanisms
of action of Hsp90 in these tumors, the identified proteins are

T. Taldone et al. / Bioorg. Med. Chem. 19 (2011) 2603–2614
2609
In another application of the chemical tools designed here, we
show that PU-H71-biotin (7) can also be used to specifically detect
Hsp90 when expressed on the cell surface (Fig. 4B). Hsp90, which is
mainly a cytosolic protein, has been reported in certain cases to
translocate to the cell surface. In breast cancer, for example, mem-
brane Hsp90 is involved in aiding cancer cell invasion.36 Specific
detection of the membrane Hsp90 in live cells is possible by the
use of PU-H71-biotin (7) because, while the biotin conjugated
Hsp90 inhibitor may potentially enter the cell, the streptavidin
conjugate used to detect the biotin, is cell impermeable.
IP: streptavidin beads
PU-H71-biotin
A
B
Lysate
htau
Hsp90
c1 s1 c2 s2
c1 c1 s1 c2 s2
PU-H71-biotin vs. D-biotin
Figure 4B shows that PU-H71-biotin but not D-biotin can detect
Hsp90 expression on the surface of leukemia cells.
70
60
50
40
30
20
10
0
1 µM
10 µM
5. Conclusions
In conclusion, we have prepared useful chemical tools based on
three different Hsp90 inhibitors, each of a different chemotype.
These were prepared either by attachment onto solid support, such
as PU-H71 (purine), NVP-AUY922 (isoxazole) and SNX-2112 (inda-
zol-4-one)-beads, or by biotinylation (PU-H71-biotin). The utility
of these probes was demonstrated by their ability to efficiently iso-
late Hsp90 and, in the case of PU-H71 beads (6), isolate Hsp90
onco-protein containing complexes from cancer cell extracts.
Available co-crystal structures and SAR were utilized in their
design, and docking to the appropriate X-ray crystal structure of
-10
-20
iotin
b
-
D-biotin
PU
Figure 4. (A) Hsp90-containing protein complexes from the brains of JNPL3 mice,
an Alzheimer’s disease transgenic mouse model, isolated through chemical
Hsp90a used to validate the site of attachment of the linker. These
precipitation with beads containing
construct or control streptavidin-immobilized
a
streptavidin-immobilized PU-H71-biotin
-biotin. Aberrant tau species are
D
are important chemical tools in efforts towards better understand-
ing Hsp90 biology and towards designing Hsp90 inhibitors with
most favorable clinical profile.
indicated by arrow. c1, c2 and s1, s2, cortical and subcortical brain homogenates,
respectively, extracted from 6-month-old female JNPL3 mice (Right). Western blot
analysis of brain lysate protein content (Left). (B) Cell surface Hsp90 in MV4-11
leukemia cells as detected by PU-H71-biotin. The data are consistent with those
obtained from multiple repeat experiments (n ꢁ 2).
6. Experimental
6.1. General
1H and 13C NMR spectra were recorded on a Bruker 500 MHz
instrument. Chemical shifts were reported in d values in ppm
downfield from TMS as the internal standard. 1H data were
reported as follows: chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling
constant (Hz), integration. 13C chemical shifts were reported in d
values in ppm downfield from TMS as the internal standard. Low
resolution mass spectra were obtained on a Waters Acquity Ultra
Performance LC with electrospray ionization and SQ detector.
High-performance liquid chromatography analyses were per-
formed on a Waters Autopurification system with PDA, MicroMass
ZQ and ELSD detector and a reversed phase column (Waters
important tumor specific onco-clients and will be introduced as
biomarkers in monitoring the clinical efficacy of PU-H71 and
Hsp90 inhibitors in these cancers during clinical studies.
Similar experiments were possible with PU-H71-biotin (7),
which selectively isolated Hsp90 in complex with mutant tau spe-
cies in brain homogenates obtained from Alzheimer’s disease
transgenic mice (Fig. 4A). The protein tau when of aberrant activity,
such as in Alzheimer’s disease and in a cluster of tauopathies
termed ‘frontotemporal dementia and parkinsonism linked to
chromosome 17’, is regulated by Hsp90.29–31
It is important to note that previous attempts to isolate Hsp90/
client protein complexes using a solid support immobilized GM
were of little success.32 In that case, the proteins bound to Hsp90
were washed away during the preparative steps. To prevent the
loss of Hsp90-interacting proteins, the authors had to subject the
cancer cell extracts to cross-linking with DSP, a homobifunctional
amino-reactive DTT-reversible cross-linker, suggesting that unlike
PU-H71, GM is unable to stabilize Hsp90/client protein interac-
tions. We observed a similar profile when using beads with GM
directly covalently attached to the Affi-GelÒ 10 resin (Chiosis
et al, unpublished results). Crystallographic and biochemical inves-
tigations suggest that GM preferentially interacts with Hsp90 in an
apo, open-conformation, that is, unfavorable for certain client pro-
tein binding33–35 providing a potential explanation for the limited
ability of GM-beads to capture Hsp90/client protein complexes. It
is currently unknown what Hsp90 conformations are preferred
by the other Hsp90 chemotypes, but with the NVP- and SNX-beads
also available, as reported here, similar evaluations are now possi-
ble, leading to a better understanding of the interaction of these
agents with Hsp90, and of the biological significance of these
interactions.
X-Bridge C18, 4.6 ꢂ 150 mm,
5 lm) using a gradient of (a)
H2O + 0.1% TFA and (b) CH3CN + 0.1% TFA, 5–95% b over 10 min
at 1.2 mL/min. Column chromatography was performed using
230–400 mesh silica gel (EMD). All reactions were performed un-
der argon protection. Affi-GelÒ 10 beads were purchased from
Bio-Rad (Hercules, CA). EZ-LinkÒ Amine–PEO3–Biotin was pur-
chased from Pierce (Rockford, Il). PU-H7112 and NVP-AUY9227
were synthesized according to previously published methods.
GM was purchased from Aldrich.
6.2. Synthesis
6.2.1. 9-(3-Bromopropyl)-8-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-
9H-purin-6-amine (2)
Compound 112 (0.500 g, 1.21 mmol) was dissolved in DMF
(15 mL). Cs2CO3 (0.434 g, 1.33 mmol) and 1,3-dibromopropane
(1.22 g, 0.617 mL, 6.05 mmol) were added and the mixture was
stirred at rt for 45 min. Then additional Cs2CO3 (0.079 g,
0.242 mmol) was added and the mixture was stirred for 45 min.

2610
T. Taldone et al. / Bioorg. Med. Chem. 19 (2011) 2603–2614
Solvent was removed under reduced pressure and the resulting
residue was chromatographed (CH2Cl2/MeOH/AcOH, 120:1:0.5–
80:1:0.5) to give 0.226 g (35%) of 2 as a white solid. 1H NMR
(CDCl3/MeOH-d4) d 8.24 (s, 1H), 7.38 (s, 1H), 7.03 (s, 1H), 6.05 (s,
2H), 4.37 (t, J = 7.1 Hz, 2H), 3.45 (t, J = 6.6 Hz, 2H), 2.41 (m, 2H);
MS (ESI): m/z 534.0/536.0 [M+H]+.
DMF (3 ꢂ 50 mL), Felts buffer (3 ꢂ 50 mL) and i-PrOH (3 ꢂ
50 mL). The beads 6 were stored in i-PrOH (beads/i-PrOH (1:2),
v/v) at ꢀ80 °C.
6.2.6. PU-H71-biotin (7)
Compound 2 (4.2 mg, 0.0086 mmol) and EZ-LinkÒ Amine–
PEO3–Biotin (5.4 mg, 0.0129 mmol) in DMF (0.2 mL) was stirred
at rt for 24 h. The reaction mixture was concentrated and the res-
idue chromatographed [CHCl3/MeOH–NH3 (7 N), 5:1] to give
1.1 mg (16%) of 7. 1H NMR (CDCl3) d 8.30 (s, 1H), 8.10 (s, 1H),
7.31 (s, 1H), 6.87 (s, 1H), 6.73 (br s, 1H), 6.36 (br s, 1H), 6.16 (br
s, 2H), 6.00 (s, 2H), 4.52 (m, 1H), 4.28–4.37 (m, 3H), 3.58–3.77
(m, 10H), 3.55 (m, 2H), 3.43 (m, 2H), 3.16 (m, 1H), 2.92 (m, 1H),
2.80 (m, 2H), 2.72 (m, 1H), 2.66 (m, 2H), 2.17 (t, J = 7.0 Hz, 2H),
2.04 (m, 2H), 1.35–1.80 (m, 6H); MS (ESI): m/z 872.2 [M+H]+.
6.2.2. tert-Butyl 6-aminohexylcarbamate (3)37
1,6-Diaminohexane (10 g, 0.086 mol) and Et3N (13.05 g,
18.13 mL, 0.129 mol) were suspended in CH2Cl2 (300 mL). A solu-
tion of di-tert-butyl dicarbonate (9.39 g, 0.043 mol) in CH2Cl2
(100 mL) was added dropwise over 90 min at rt and stirring contin-
ued for 18 h. The reaction mixture was added to a seperatory
funnel and washed with water (100 mL), brine (100 mL), dried over
Na2SO4 and concentrated under reduced pressure. The resulting
residue was chromatographed [CH2Cl2/MeOH–NH3 (7 N),
70:1–20:1] to give 7.1 g (76%) of 3. 1H NMR (CDCl3) d 4.50 (br s,
1H), 3.11 (br s, 2H), 2.68 (t, J = 6.6 Hz, 2H), 1.44 (s, 13H), 1.33 (s,
4H); MS (ESI): m/z 217.2 [M+H]+.
6.2.7. tert-Butyl 6-(4-(5-(2,4-bis(benzyloxy)-5-isopropylphenyl)-
3-(ethylcarbamoyl)isoxazol-4-yl)benzylamino)hexylcarbamate
(9)
AcOH (0.26 g, 0.25 mL, 4.35 mmol) was added to a mixture of 87
6.2.3. tert-Butyl 6-(3-(6-amino-8-(6-iodobenzo[d][1,3]dioxol-5-
ylthio)-9H-purin-9-yl)propylamino)hexylcarbamate (4)
(0.5 g, 0.87 mmol), 3 (0.56 g, 2.61 mmol), NaCNBH3 (0.11 g,
1.74 mmol), CH2Cl2 (21 mL) and 3 Å molecular sieves (3 g). The
reaction mixture was stirred for 1 h at rt. It was then concentrated
under reduced pressure and chromatographed [CH2Cl2/MeOH–NH3
(7 N), 100:1–60:1] to give 0.50 g (75%) of 9. 1H NMR (CDCl3) d7.19–
7.40 (m, 12H), 7.12–7.15 (m, 2H), 7.08 (s, 1H), 6.45 (s, 1H), 4.97 (s,
2H), 4.81 (s, 2H), 3.75 (s, 2H), 3.22 (m, 2H), 3.10 (m, 3H), 2.60 (t,
J = 7.1 Hz, 2H), 1.41–1.52 (m, 13H), 1.28–1.35 (m, 4H), 1.21 (t,
J = 7.2 Hz, 3H), 1.04 (d, J = 6.9 Hz, 6H); MS (ESI): m/z 775.3 [M+H]+.
Compound 2 (0.226 g, 0.423 mmol) and 3 (0.915 g, 4.23 mmol)
in DMF (7 mL) was stirred at rt for 24 h. The reaction mixture was
concentrated and the residue chromatographed [CHCl3/MeOH/
MeOH–NH3 (7 N), 100:7:3] to give 0.255 g (90%) of 4. 1H NMR
(CDCl3) d 8.32 (s, 1H), 7.31 (s, 1H), 6.89 (s, 1H), 5.99 (s, 2H), 5.55
(br s, 2H), 4.57 (br s, 1H), 4.30 (t, J = 7.0 Hz, 2H), 3.10 (m, 2H),
2.58 (t, J = 6.7 Hz, 2H), 2.52 (t, J = 7.2 Hz, 2H), 1.99 (m, 2H), 1.44
(s, 13H), 1.30 (s, 4H); 13C NMR (125 MHz, CDCl3) d 156.0, 154.7,
153.0, 151.6, 149.2, 149.0, 146.3, 127.9, 120.1, 119.2, 112.4,
102.3, 91.3, 79.0, 49.8, 46.5, 41.8, 40.5, 31.4, 29.98, 29.95, 28.4,
27.0, 26.7; HRMS (ESI) m/z [M+H]+ calcd for C26H37IN7O4S,
670.1673; found 670.1670; HPLC: tR = 7.02 min.
6.2.8. 4-(4-((6-Aminohexylamino)methyl)phenyl)-5-(2,4-dihy-
droxy-5-isopropylphenyl)-N-ethylisoxazole-3-carboxamide (10)
To a solution of 9 (0.5 g, 0.646 mmol) in CH2Cl2 (20 mL) was
added a solution of BCl3 (1.8 mL, 1.87 mmol, 1.0 M in CH2Cl2) and
this was stirred at rt for 10 h. Saturated NaHCO3 was added and
CH2Cl2 was evaporated under reduced pressure. The water was care-
fully decanted and the remaining yellow precipitate was washed a
few times with EtOAc and CH2Cl2 to give 0.248 g (78%) of 10. 1H
NMR (CDCl3/MeOH-d4) d 7.32 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.1 Hz,
2H), 6.94 (s, 1H), 6.25 (s, 1H), 3.74, (s, 2H), 3.41 (q, J = 7.3 Hz, 2H),
3.08 (m, 1H), 2.65 (t, J = 7.1 Hz, 2H), 2.60 (t, J = 7.1 Hz, 2H),
1.40–1.56 (m, 4H), 1.28–1.35 (m, 4H), 1.21 (t, J = 7.3 Hz, 3H), 1.01
(d, J = 6.9 Hz, 6H); 13C NMR (125 MHz, CDCl3/MeOH-d4) d 168.4,
161.6, 158.4, 157.6, 155.2, 139.0, 130.5, 129.5, 128.71, 128.69,
127.6, 116.0, 105.9, 103.6, 53.7, 49.2, 41.8, 35.0, 32.7, 29.8, 27.6,
27.2, 26.4, 22.8, 14.5; HRMS (ESI) m/z [M+H]+ calcd for
6.2.4. N1-(3-(6-Amino-8-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-
9H-purin-9-yl)propyl)hexane-1,6-diamine (5)
Compound 4 (0.310 g, 0.463 mmol) was dissolved in 15 mL of
CH2Cl2/TFA (4:1) and the solution was stirred at rt for 45 min.
Solvent was removed under reduced pressure and the residue
chromatographed [CH2Cl2/MeOH–NH3 (7 N), 20:1–10:1] to give
0.37 g of a white solid. This was dissolved in water (45 mL) and so-
lid Na2CO3 added until pH ꢃ12. This was extracted with CH2Cl2
(4 ꢂ 50 mL) and the combined organic layers were washed with
water (50 mL), dried over Na2SO4, filtered and concentrated under
reduced pressure to give 0.200 g (76%) of 5. 1H NMR (CDCl3) d 8.33
(s, 1H), 7.31 (s, 1H), 6.89 (s, 1H), 5.99 (s, 2H), 5.52 (br s, 2H), 4.30 (t,
J = 6.3 Hz, 2H), 2.68 (t, J = 7.0 Hz, 2H), 2.59 (t, J = 6.3 Hz, 2H), 2.53 (t,
J = 7.1 Hz, 2H), 1.99 (m, 2H), 1.44 (s, 4H), 1.28 (s, 4H); 13C NMR
(125 MHz, CDCl3/MeOH-d4) d 154.5, 152.6, 151.5, 150.0, 149.6,
147.7, 125.9, 119.7, 119.6, 113.9, 102.8, 94.2, 49.7, 46.2, 41.61,
41.59, 32.9, 29.7, 29.5, 27.3, 26.9; HRMS (ESI) m/z [M+H]+ calcd
for C21H29IN7O2S, 570.1148; found 570.1124; HPLC: tR = 5.68 min.
C28H39N4O4, 495.2971; found 495.2986; HPLC: tR = 6.57 min.
6.2.9. NVP-AUY922-Affi-Gel 10 beads (11)
Compound 10 (46.4 mg, 0.094 mmol) was dissolved in DMF
(2 mL) and added to 5 mL of Affi-Gel 10 beads (prewashed,
3 ꢂ 8 mL DMF) in a solid phase peptide synthesis vessel. 45
ll of
N,N-diisopropylethylamine and several crystals of DMAP were
added and this was shaken at rt for 2.5 h. Then 2-methoxyethyl-
6.2.5. PU-H71-Affi-Gel 10 beads (6)
amine (17.7 mg, 21 ll, 0.235 mmol) was added and shaking was
Compound 4 (0.301 g, 0.45 mmol) was dissolved in 15 mL of
CH2Cl2/TFA (4:1) and the solution was stirred at rt for 45 min. Sol-
vent was removed under reduced pressure and the residue dried
under high vacuum overnight. This was dissolved in DMF
(12 mL) and added to 25 mL of Affi-Gel 10 beads (prewashed,
continued for 30 min. Then the solvent was removed and the beads
washed for 10 min each time with CH2Cl2 (3 ꢂ 8 mL), DMF
(3 ꢂ 8 mL), Felts buffer (3 ꢂ 8 mL) and i-PrOH (3 ꢂ 8 mL). The
beads 11 were stored in i-PrOH (beads/i-PrOH, (1:2), v/v) at ꢀ80 °C.
3 ꢂ 50 mL DMF) in a solid phase peptide synthesis vessel. 225
l
l
6.2.10. N0-(3,3-Dimethyl-5-oxocyclohexylidene)-4-methylbenz-
enesulfonohydrazide (14)38
of N,N-diisopropylethylamine and several crystals of DMAP were
added and this was shaken at rt for 2.5 h. Then 2-methoxyethyl-
amine (0.085 g, 97 ll, 1.13 mmol) was added and shaking was
continued for 30 min. Then the solvent was removed and the beads
washed for 10 min each time with CH2Cl2/Et3N (9:1, 4 ꢂ 50 mL),
10.00 g (71.4 mmol) of dimedone (13), 13.8 g (74.2 mmol) of
tosyl hydrazide (12) and p-toluene sulfonic acid (0.140 g,
0.736 mmol) were suspended in toluene (600 mL) and this was
refluxed with stirring for 1.5 h. While still hot, the reaction mixture

T. Taldone et al. / Bioorg. Med. Chem. 19 (2011) 2603–2614
2611
was filtered and the solid was washed with toluene (4 ꢂ 100 mL),
ice-cold ethyl acetate (2 ꢂ 200 mL) and hexane (2 ꢂ 200 mL) and
dried to give 19.58 g (89%) of 14 as a solid. TLC (100% EtOAc)
Rf = 0.23; 1H NMR (DMSO-d6) d 9.76 (s, 1H), 8.65 (br s, 1H), 7.69
(d, J = 8.2 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 5.05 (s, 1H), 2.39 (s,
3H), 2.07 (s, 2H), 1.92 (s, 2H), 0.90 (s, 6H); MS (ESI): m/z 309.0
[M+H]+.
1.95 (d, J = 11.8 Hz, 2H), l.20–1.42 (m, 4H), 1.14 (s, 6H); MS (ESI):
m/z 464.4 [M+H]+; HPLC: tR = 7.05 min.
6.2.15. tert-Butyl 6-(trans-4-(2-carbamoyl-5-(6,6-dimethyl-4-
oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)-
phenylamino)cyclohexylamino)-6-oxohexylcarbamate (19)
To a mixture of 18 (30 mg, 0.0647 mmol) in CH2Cl2 (1 ml) was
added 6-(Boc-amino)caproic acid (29.9 mg, 0.1294 mmol), EDCI
(24.8 mg, 0.1294 mmol) and DMAP (0.8 mg, 0.00647 mmol). The
reaction mixture was stirred at rt for 2 h then concentrated under
reduced pressure and the residue purified by preparatory TLC [hex-
ane/CH2Cl2/EtOAc/MeOH–NH3 (7 N), 2:2:1:0.5] to give 40 mg
(91%) of 19. 1H NMR (CDCl3/MeOH-d4) d 7.63 (d, J = 8.4 Hz, 1H),
6.75 (d, J = 1.7 Hz, 1H), 6.61 (dd, J = 8.4, 2.0 Hz, 1H), 3.75 (m, 1H),
3.31 (m, 1H), 3.06 (t, J = 7.0 Hz, 2H), 2.88 (s, 2H), 2.50 (s, 2H),
2.15 (m, 4H), 2.03 (d, J = 11.5 Hz, 2H), 1.62 (m, 2H), l.25–1.50 (m,
17H), 1.14 (s, 6H); 13C NMR (125 MHz, CDCl3/MeOH-d4) d 191.5,
174.1, 172.3, 157.2, 151.5, 150.3, 141.5, 140.6 (q, J = 39.4 Hz),
130.8, 120.7 (q, J = 268.0 Hz), 116.2, 114.2, 109.5, 107.3, 79.5,
52.5, 50.7, 48.0, 40.4, 37.3, 36.4, 36.0, 31.6, 31.3, 29.6, 28.5, 28.3,
25.7, 25.4; HRMS (ESI) m/z [M+Na]+ calcd for C34H47F3N6O5Na,
699.3458; found 699.3472; HPLC: tR = 9.10 min.
6.2.11. 6,6-Dimethyl-3-(trifluoromethyl)-6,7-dihydro-1H-indazol-
4(5H)-one (15)
To 5.0 g (16.2 mmol) of 14 in THF (90 mL) and Et3N (30 mL) was
added trifluoroacetic anhydride (3.4 g, 2.25 mL, 16.2 mmol) in one
portion. The resulting red solution was heated at 55 °C for 3 h.
After cooling to rt, methanol (8 mL) and 1 M NaOH (8 mL) were
added and the solution was stirred for 3 h at rt. The reaction mix-
ture was diluted with 25 mL of saturated NH4Cl, poured into a
seperatory funnel and extracted with EtOAc (3 ꢂ 50 mL). The com-
bined organic layers were washed with brine (3 ꢂ 50 mL), dried
over Na2SO4 and concentrated under reduced pressure to give a
red oily residue which was chromatographed (hexane/EtOAc,
80:20–60:40) to give 2.08 g (55%) of 15 as an orange solid. TLC
(hexane/EtOAc, 6:4) Rf = 0.37; 1H NMR (CDCl3) d 2.80 (s, 2H), 2.46
(s, 2H), 1.16 (s, 6H); MS (ESI): m/z 231.0 [MꢀH]ꢀ.
6.2.16. 2-(trans-4-(6-Aminohexanamido)cyclohexylamino)-4-
(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-
1H-indazol-1-yl)benzamide (20)
6.2.12. 2-Bromo-4-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-
4,5,6,7-tetrahydro-1H-indazol-1-yl)benzonitrile (16)
To a mixture of 15 (0.100 g, 0.43 mmol) and NaH (15.5 mg,
0.65 mmol) in DMF (8 mL) was added 2-bromo-4-fluorobenzoni-
trile (86 mg, 0.43 mmol) and heated at 90 °C for 5 h. The reaction
mixture was concentrated under reduced pressure and the residue
chromatographed (hexane/EtOAc, 10:1–10:2) to give 0.162 g (91%)
of 16 as a white solid. 1H NMR (CDCl3) d 7.97 (d, J = 2.1 Hz, 1H),
7.85 (d, J = 8.4 Hz, 1H), 7.63 (dd, J = 8.4, 2.1 Hz, 1H), 2.89 (s, 2H),
2.51 (s, 2H), 1.16 (s, 6H); MS (ESI): m/z 410.0/412.0 [MꢀH]ꢀ.
Compound 19 (33 mg, 0.049 mmol) was dissolved in 1 mL of
CH2Cl2/TFA (4:1) and the solution was stirred at rt for 45 min. Sol-
vent was removed under reduced pressure and the residue purified
by preparatory TLC [CH2Cl2/MeOH–NH3 (7 N), 6:1] to give 24 mg
(86%) of 20. 1H NMR (CDCl3/MeOH-d4) d 7.69 (d, J = 8.4 Hz, 1H),
6.78 (d, J = 1.9 Hz, 1H), 6.64 (dd, J = 8.4, 1.9 Hz, 1H), 3.74 (m, 1H),
3.36 (m, 1H), 2.92 (t, J = 7.5 Hz, 2H), 2.91 (s, 2H), 2.51 (s, 2H),
2.23 (t, J = 7.3 Hz, 2H), 2.18 (d, J = 10.2 Hz, 2H), 2.00 (d, J = 9.1 Hz,
2H), 1.61–1.75 (m, 4H), l.34–1.50 (m, 6H), 1.15 (s, 6H); 13C NMR
(125 MHz, MeOH-d4) d 191.2, 173.6, 172.2, 151.8, 149.7, 141.2,
139.6 (q, J = 39.5 Hz), 130.3, 120.5 (q, J = 267.5 Hz), 115.5, 114.1,
109.0, 106.8, 51.6, 50.0, 47.8, 39.0, 36.3, 35.2, 35.1, 31.0, 30.5,
26.8, 26.7, 25.4, 24.8; HRMS (ESI) m/z [M+H]+ calcd for
6.2.13. 2-(trans-4-Aminocyclohexylamino)-4-(6,6-dimethyl-4-
oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)-
benzonitrile (17)
A mixture of 16 (0.200 g, 0.485 mmol), NaOtBu (93.3 mg,
0.9704 mmol), Pd2(dba)3 (88.8 mg, 0.097 mmol) and DavePhos
(38 mg, 0.097 mmol) in 1,2-dimethoxyethane (15 mL) was de-
gassed and flushed with argon several times. trans-1,4-Diaminocy-
clohexane (0.166 g, 1.456 mmol) was added and the flask was
again degassed and flushed with argon before heating the reaction
mixture at 50 °C overnight. The reaction mixture was concentrated
under reduced pressure and the residue purified by preparatory
TLC (CH2Cl2/MeOH–NH3 (7 N), 10:1) to give 52.5 mg (24%) of 17.
Additionally, 38.5 mg (17%) of amide 18 was isolated for a total
yield of 41%. 1H NMR (CDCl3) d 7.51 (d, J = 8.3 Hz, 1H), 6.81 (d,
J = 1.8 Hz, 1H), 6.70 (dd, J = 8.3, 1.8 Hz, 1H), 4.64 (d, J = 7.6 Hz,
1H), 3.38 (m, 1H), 2.84 (s, 2H), 2.81 (m, 1H), 2.49 (s, 2H), 2.15 (d,
J = 11.2 Hz, 2H), 1.99 (d, J = 11.0 Hz, 2H), 1.25–1.37 (m, 4H), 1.14
(s, 6H); MS (ESI): m/z 446.3 [M+H]+.
C29H40F3N6O3, 577.3114; found 577.3126; HPLC: tR = 7.23 min.
6.2.17. SNX-2112-Affi-Gel 10 beads (21)
Compound 19 (67 mg, 0.0992 mmol) was dissolved in 3.5 mL of
CH2Cl2/TFA (4:1) and the solution was stirred at rt for 20 min. Sol-
vent was removed under reduced pressure and the residue dried
under high vacuum for 2 h. This was dissolved in DMF (2 mL)
and added to 5 mL of Affi-Gel 10 beads (prewashed, 3 ꢂ 8 mL
DMF) in a solid phase peptide synthesis vessel. 45 ll of N,N-diiso-
propylethylamine and several crystals of DMAP were added and
this was shaken at rt for 2.5 h. Then 2-methoxyethylamine
(18.6 mg, 22 ll, 0.248 mmol) was added and shaking was contin-
ued for 30 min. Then the solvent was removed and the beads
washed for 10 min each time with CH2Cl2 (3 ꢂ 8 mL), DMF
(3 ꢂ 8 mL) and i-PrOH (3 ꢂ 8 mL). The beads 21 were stored in
i-PrOH (beads/i-PrOH, (1:2), v/v) at ꢀ80 °C.
6.2.14. 2-(trans-4-Aminocyclohexylamino)-4-(6,6-dimethyl-4-
oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)-
benzamide (18)
6.2.18. N-Fmoc-trans-4-aminocyclohexanol (22)39
A solution of 17 (80 mg, 0.18 mmol) in DMSO (147
ll), EtOH
To
a solution of trans-4-aminocyclohexanol hydrochloride
(590 l), 5 N NaOH (75 l) and H2O2 (88 l) was stirred at rt for
l
l
l
(2.0 g, 13.2 mmol) in dioxane/water (26:6.5 mL) was added Et3N
(1.08 g, 1.49 mL, 10.7 mmol) and this was stirred for 10 min. Then
Fmoc-OSu (3.00 g, 8.91 mmol) was added over 5 min and the
resulting suspension was stirred at rt for 2 h. The reaction mixture
was concentrated to ꢃ5 mL, then some CH2Cl2 was added. This was
filtered and the solid was washed with H2O (4 ꢂ 40 mL) then dried
to give 2.85 g (95%) of 22 as a white solid. Additional 0.100 g (3%)
3 h. The reaction mixture was concentrated under reduced pres-
sure and the residue purified by preparatory TLC [CH2Cl2/MeOH–
NH3 (7 N), 10:1] to give 64.3 mg (78%) of 18. 1H NMR (CDCl3) d
8.06 (d, J = 7.5 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 6.74 (d, J = 1.9 Hz,
1H), 6.62 (dd, J = 8.4, 2.0 Hz, 1H), 5.60 (br s, 2H), 3.29 (m, 1H),
2.85 (s, 2H), 2.77 (m, 1H), 2.49 (s, 2H), 2.13 (d, J = 11.9 Hz, 2H),

2612
T. Taldone et al. / Bioorg. Med. Chem. 19 (2011) 2603–2614
of 22 was obtained by extracting the filtrate with CH2Cl2
(2 ꢂ 100 mL), drying over Na2SO4, filtering and removing solvent
for a combined yield of 98%. TLC (hexane/EtOAc, 20:80) Rf = 0.42;
1H NMR (CDCl3) d 7.77 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 7.4 Hz, 2H),
7.40 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 4.54 (br s, 1H), 4.40
(d, J = 5.6 Hz, 2H), 4.21 (t, J = 5.6 Hz, 1H), 3.61 (m, 1H), 3.48 (m,
1H), 1.9–2.1 (m, 4H), 1.32–1.48 (m, 2H), 1.15–1.29 (m, 2H); MS
(ESI): m/z 338.0 [M+H]+.
2.0 Hz, 1H), 5.68 (br s, 2H), 4.72 (m, 1H), 3.91 (m, 1H), 3.70 (m,
1H), 3.50 (m, 1H), 3.34 (m, 1H), 2.85 (s, 2H), 2.49 (s, 2H), 2.05–
2.19 (m, 4H), 1.33–1.88 (m, 10H), 1.14 (s, 6H); MS (ESI): m/z
547.4 [MꢀH]ꢀ.
6.2.23. 4-(6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetra-
hydro-1H-indazol-1-yl)-2-(trans-4-hydroxycyclohexylamino)-
benzamide (SNX-2112)
Compound 26 (140 mg, 0.255 mmol) and pyridinium p-toluene-
sulfonate (6.4 mg, 0.0255 mmol) in EtOH (4.5 mL) was heated at
65 °C for 17 h. The reaction mixture was concentrated under re-
duced pressure and the residue purified by preparatory TLC [hex-
ane/CH2Cl2/EtOAc/MeOH–NH3 (7 N), 2:2:1:0.5] to give 101 mg
(85%) of SNX-2112. 1H NMR (CDCl3) d 8.10 (d, J = 7.4 Hz, 1H),
7.52 (d, J = 8.4 Hz, 1H), 6.75 (d, J = 1.3 Hz, 1H), 6.60 (dd, J = 8.4,
1.6 Hz, 1H), 5.97 (br s, 2H), 3.73 (m, 1H), 3.35 (m, 1H), 2.85 (s,
2H), 2.48 (s, 2H), 2.14 (d, J = 11.8 Hz, 2H), 2.04 (d, J = 11.1 Hz,
2H), 1.33–1.52 (m, 4H), 1.13 (s, 6H); 13C NMR (125 MHz, CDCl3/
6.2.19. N-Fmoc-trans-4-aminocyclohexanol tetrahydropyranyl
ether (23)
1.03 g (3.05 mmol) of 22 and 0.998 g (1.08 mL, 11.86 mmol) of
3,4-dihydro-2H-pyran (DHP) was suspended in dioxane (10 mL).
Pyridinium p-toluenesulfonate (0.153 g, 0.61 mmol) was added
and the suspension stirred at rt. After 1 h additional DHP
(1.08 mL, 11.86 mmol) and dioxane (10 mL) were added and stir-
ring continued. After 9 h additional DHP (1.08 mL, 11.86 mmol)
was added and stirring continued overnight. The resulting solution
was concentrated and the residue chromatographed (hexane/
EtOAc, 75:25–65:35) to give 1.28 g (100%) of 23 as a white solid.
TLC (hexane/EtOAc, 70:30) Rf = 0.26; 1H NMR (CDCl3) d 7.77 (d,
J = 7.5 Hz, 2H), 7.58 (d, J = 7.5 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.31
(dt, J = 7.5, 1.1 Hz, 2H), 4.70 (m, 1H), 4.56 (m, 1H), 4.40 (d,
J = 6.0 Hz, 2H), 4.21 (t, J = 6.1 Hz, 1H), 3.90 (m, 1H), 3.58 (m, 1H),
3.45–3.53 (m, 2H), 1.10–2.09 (m, 14H); MS (ESI): m/z 422.3
[M+H]+.
MeOH-d4)
d 191.0, 171.9, 151.0, 150.0, 141.3, 140.3 (q,
J = 39.6 Hz), 130.4, 120.3 (q, J = 270.2 Hz), 115.9, 113.7, 109.2,
107.1, 69.1, 52.1, 50.2, 40.1, 37.0, 35.6, 33.1, 30.2, 28.0; MS (ESI):
m/z 463.3 [MꢀH]ꢀ, 465.3 [M+H]+; HPLC: tR = 7.97 min.
6.2.24. Preparation of control-beads
DMF (8.5 mL) was added to 20 mL of Affi-Gel 10 beads (pre-
washed, 3 ꢂ 40 mL DMF) in a solid phase peptide synthesis vessel.
2-Methoxyethylamine (113 mg, 129 ll, 1.5 mmol) and several
6.2.20. trans-4-Aminocylohexanol tetrahydropyranyl ether (24)
1.28 g (3.0 mmol) of 23 was dissolved in CH2Cl2 (20 mL) and
piperidine (2 mL) was added and the solution stirred at rt for 5 h.
Solvent was removed and the residue was purified by chromatog-
raphy [CH2Cl2/MeOH–NH3 (7 N), 80:1–30:1] to give 0.574 g (96%)
of 24 as an oily residue which slowly crystallized. 1H NMR (CDCl3)
d 4.70 (m, 1H), 3.91 (m, 1H), 3.58 (m, 1H), 3.49 (m, 1H), 2.69 (m,
1H), 1.07–2.05 (m, 14H); MS (ESI): m/z 200.2 [M+H]+.
crystals of DMAP were added and this was shaken at rt for 2.5 h.
Then the solvent was removed and the beads washed for 10 min
each time with CH2Cl2 (4 ꢂ 35 mL), DMF (3 ꢂ 35 mL), Felts buffer
(2 ꢂ 35 mL) and i-PrOH (4 ꢂ 35 mL). The beads were stored in
i-PrOH (beads/i-PrOH (1:2), v/v) at ꢀ80 °C.
6.3. Competition assay
For the competition studies, fluorescence polarization (FP)
assays were performed as previously reported.15 Briefly, FP mea-
surements were performed on an Analyst GT instrument
(Molecular Devices, Sunnyvale, CA). Measurements were taken
in black 96-well microtiter plates (Corning # 3650) where both
the excitation and the emission occurred from the top of the
6.2.21. 4-(6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tet-
rahydro-1H-indazol-1-yl)-2-(trans-4-(tetrahydro-2H-pyran-2-
yloxy)cyclohexylamino)benzonitrile (25)
A
mixture of 16 (0.270 g, 0.655 mmol), NaOtBu (0.126 g,
1.31 mmol), Pd2(dba)3 (0.120 g, 0.131 mmol) and DavePhos
(0.051 g, 0.131 mmol) in 1,2-dimethoxyethane (20 mL) was de-
gassed and flushed with argon several times. Compound 24
(0.390 g, 1.97 mmol) was added and the flask was again degassed
and flushed with argon before heating the reaction mixture at
60 °C for 3.5 h. The reaction mixture was concentrated under re-
duced pressure and the residue purified by preparatory TLC [hex-
ane/CH2Cl2/EtOAc/MeOH–NH3 (7 N), 7:6:3:1.5] to give 97.9 mg
(28%) of 25. Additionally, 60.5 mg (17%) of amide 26 was isolated
for a total yield of 45%. 1H NMR (CDCl3) d 7.52 (d, J = 8.3 Hz, 1H),
6.80 (d, J = 1.7 Hz, 1H), 6.72 (dd, J = 8.3, 1.8 Hz, 1H), 4.72 (m, 1H),
4.67 (d, J = 7.6 Hz, 1H), 3.91 (m, 1H), 3.68 (m, 1H), 3.50 (m, 1H),
3.40 (m, 1H), 2.84 (s, 2H), 2.49 (s, 2H), 2.06–2.21 (m, 4H), 1.30–
1.90 (m, 10H), 1.14 (s, 6H); MS (ESI): m/z 529.4 [MꢀH]ꢀ.
wells. A stock of 10 lM GM-cy3B was prepared in DMSO and
diluted with Felts buffer (20 mM Hepes (K), pH 7.3, 50 mM KCl,
2 mM DTT, 5 mM MgCl2, 20 mM Na2MoO4, and 0.01% NP40 with
0.1 mg/mL BGG). To each 96-well were added 6 nM fluorescent
GM (GM-cy3B), 3
inhibitor (initial stock in DMSO) in a final volume of 100
l
g SKBr3 lysate (total protein), and tested
l HFB
l
buffer. Drugs were added in triplicate wells. For each assay, back-
ground wells (buffer only), tracer controls (free, fluorescent GM
only) and bound GM controls (fluorescent GM in the presence
of SKBr3 lysate) were included on each assay plate. GM was used
as positive control. The assay plate was incubated on a shaker at
4 °C for 24 h and the FP values in mP were measured. The fraction
of tracer bound to Hsp90 was correlated to the mP value and
plotted against values of competitor concentrations. The inhibitor
concentration at which 50% of bound GM was displaced was ob-
tained by fitting the data. All experimental data were analyzed
using SOFTmax Pro 4.3.1 and plotted using Prism 4.0 (Graphpad
Software Inc., San Diego, CA).
6.2.22. 4-(6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetra-
hydro-1H-indazol-1-yl)-2-(trans-4-(tetrahydro-2H-pyran-2-yl-
oxy)cyclohexylamino)benzamide (26)
A solution of 25 (120 mg, 0.2264 mmol) in DMSO (220
ll), EtOH
(885 l), 5 N NaOH (112 l) and H2O2 (132 l) was stirred at rt for
l
l
l
4 h. Then 30 mL of brine was added and this was extracted with
EtOAc (5 ꢂ 15 mL), dried over Na2SO4, filtered and concentrated
under reduced pressure. The residue was purified by preparatory
TLC [hexane/CH2Cl2/EtOAc:MeOH–NH3 (7 N), 7:6:3:1.5] to give
102 mg (82%) of 26. 1H NMR (CDCl3) d 8.13 (d, J = 7.4 Hz, 1H),
7.50 (d, J = 8.4 Hz, 1H), 6.74 (d, J = 1.9 Hz, 1H), 6.63 (dd, J = 8.4,
6.4. Chemical Precipitation, Western blotting and Flow
Cytometry
The leukemia cell lines K562 and MV4-11 and the breast cancer
cell line MDA-MB-468 were obtained from the American Type Cul-
ture Collection. Cells were cultured in RPMI (K562), in Iscove’s

T. Taldone et al. / Bioorg. Med. Chem. 19 (2011) 2603–2614
2613
modified Dulbecco’s media (MV4-11) or in DME/F12 (MDA-MB-
468) supplemented with 10% FBS, 1% -glutamine, 1% penicillin
grid box. The option to dock ligands similar in size to the workspace
ligand was selected for determining grid sizing.
L
and streptomycin, and maintained in a humidified atmosphere of
5% CO2 at 37 °C. Cells were lysed by collecting them in Felts buffer
(HEPES 20 mM, KCl 50 mM, MgCl2 5 mM, NP40 0.01%, freshly pre-
Next, the extra precision (XP) Glide docking method was used to
flexibly dock compounds PU-H71 and 5 (to 2FWZ), NVP-AUY922
and 10 (to 2VCI), and 20 and 27 (to 3D0B) into their respective bind-
ing site. Although details on the methodology used by Glide are de-
scribed elsewhere,42–44 a short description about parameters used
is provided below. The default setting of scale factor for van der
Waals radii was applied to those atoms with absolute partial
charges less than or equal to 0.15 (scale factor of 0.8) and 0.25 (scale
factor of 1.0) electrons for ligand and protein, respectively. No con-
straints were defined for the docking runs. Upon completion of each
docking calculation, at most 100 poses per ligand were allowed to
generate. The top-scored docking pose based on the Glide scoring
function45 was used for our analysis. In order to validate the XP
Glide docking procedure the crystallographic bound inhibitor
(PU-H71 or NVP-AUY922 or 27) was extracted from the binding site
and re-docked into its respective binding site. There was excellent
agreement between the localization of the inhibitor upon docking
and the crystal structure as evident from the 0.098 Å (2FWZ),
0.313 Å (2VCI) and 0.149 Å (3D0B) root mean square deviations.
Thus, the present study suggests the high docking reliability of
Glide in reproducing the experimentally observed binding mode
for Hsp90 inhibitors and the parameter set for the Glide docking
reasonably reproduces the X-ray structure.
pared Na2MoO4 20 mM, pH 7.2–7.3) with added 10 lg/lL of prote-
ase inhibitors (leupeptin and aprotinin), followed by three
successive freeze (in dry ice) and thaw steps. Total protein concen-
tration was determined using the BCA kit (Pierce) according to the
manufacturer’s instructions.
Hsp90 inhibitor beads or control-beads containing an Hsp90
inactive chemical (2-methoxyethylamine) conjugated to agarose
beads were washed three times in lysis buffer. The bead conjugates
(80
ll or as indicated) were then incubated overnight at 4 °C with
cell lysates (250
l
g), and the volume was adjusted to 200–300
ll
with lysis buffer. Following incubation, bead conjugates were
washed five times with the lysis buffer and analyzed by Western
blot, as indicated below.
For treatment with PU-H71, cells were grown to 60–70% conflu-
ence and treated with inhibitor (5 lM) for 24 h. Protein lysates
were prepared in 50 mM Tris pH 7.4, 150 mM NaCl and 1% NP-40
lysis buffer.
For Western blotting, protein lysates (10–50 lg) were electro-
phoretically resolved by SDS/PAGE, transferred to nitrocellulose
membrane and probed with a primary antibody against Hsp90
(1:2000, SMC-107A/B, StressMarq), anti-IGF-IR (1:1000, 3027, Cell
Signaling) and anti-c-Kit (1:200, 612318, BD Transduction Labora-
tories). The membranes were then incubated with a 1:3000 dilu-
Acknowledgements
tion of
a corresponding horseradish peroxidase conjugated
This work was supported in part by W.H. Goodwin and
A. Goodwin and the Commonwealth Cancer Foundation for
Research, The Experimental Therapeutics Center of Memorial
Sloan-Kettering Cancer Center (MSKCC), the Translational and
Integrative Medicine Research Fund of MSKCC, SPORE Pilot Award
and Research & Therapeutics Program in Prostate Cancer, 1R01CA
155226-01, U01AG032969-01A1, R21AG028811, 3P30CA008748-
44S2, Leukemia and Lymphoma Society LLS#6114-10, the Byrne
Fund, the Geoffrey Beene Cancer Research Center of MSKCC
(G.C.), Hirshberg Foundation for Pancreatic Cancer Research
(G.C.), grant UL1RR024996 of the Clinical and Translational Science
Center at Weill Cornell Medical College and Susan G. Komen for the
Cure (G.C. and T.T.). We also thank Dr. George Sukenick and Dr. Hui
Liu of the NMR Analytical Core Facility at MSKCC for expert mass
spectral analysis.
secondary antibody. Detection was performed using the ECL-
Enhanced Chemiluminescence Detection System (Amersham
Biosciences) according to manufacturer’s instructions.
To detect the binding of PU-H71 to cell surface Hsp90, MV4-11
cells at 500,000 cells/ml were incubated with the indicated concen-
trations of PU-H71-biotin or -biotin as control for 2 h at 37 °C fol-
D
lowed by staining of phycoerythrin (PE) conjugated streptavidin
(SA) (BD Biosciences) in FACS buffer (PBS + 0.5% FBS) at 4 °C for
30 min. Cells were then analyzed using the BD-LSRII flow cytome-
ter. Mean fluorescence intensity (MFI) was used to calculate the
binding of PU-H71-biotin to cells and values were normalized to
the MFI of untreated cells stained with SA-PE.
6.5. Docking
Molecular docking computations were carried out on a HP work-
station xw8200 with the Ubuntu 8.10 operating system using Glide
5.0.40 The coordinates for the Hsp90
a complexes with bound inhib-
References and notes
itor PU-H71 (PDB ID: 2FWZ), NVP-AUY922 (PDB ID: 2VCI) and 27
(PDB ID: 3D0B) were downloaded from the RCSB Protein Data Bank.
For docking experiments, compounds PU-H71, NVP-AUY922, 5, 10,
20 and 27 were constructed using the fragment dictionary of Mae-
stro 8.5 and geometry-optimized using the Optimized Potentials
for Liquid Simulations-All Atom (OPLS-AA) force field41 with the
steepest descent followed by truncated Newton conjugate gradient
protocol as implemented in Macromodel 9.6, and were further sub-
jected to ligand preparation using default parameters of LigPrep 2.2
utility provided by Schrödinger LLC. Each protein was optimized for
subsequent grid generation and docking using the Protein Prepara-
tion Wizard provided by Schrödinger LLC. Using this tool, hydrogen
atoms were added to the proteins, bond orders were assigned, water
molecules of crystallization not deemed to be important for ligand
binding were removed, and the entire protein was minimized. Par-
tial atomic charges for the protein were assigned according to the
OPLS-2005 force field. Next, grids were prepared using the Receptor
Grid Generation tool in Glide. With the respective bound inhibitor in
place, the centroid of the workspace ligand was chosen to define the
1. Panaretou, B.; Prodromou, C.; Roe, S. M.; O’Brien, R.; Ladbury, J. E.; Piper, P. W.;
Pearl, L. H. EMBO 1998, 17, 4829.
2. Obermann, W. M. J.; Sondermann, H.; Russo, A. A.; Pavletich, N. P.; Hartl, F. U. J.
Cell Biol. 1998, 143, 901.
3. Whitesell, L.; Lindquist, S. L. Nat. Rev. Cancer 2005, 5, 761.
4. Taldone, T.; Sun, W.; Chiosis, G. Bioorg. Med. Chem. 2009, 17, 2225.
5. Janin, Y. Drug Discovery Today 2010, 15, 342.
6. Solit, D. B.; Zheng, F. F.; Drobnjak, M.; Munster, P. N.; Higgins, B.; Verbel, D.;
Heller, G.; Tong, W.; Cardon-Cardo, C.; Agus, D. B.; Scher, H. I.; Rosen, N. Clin.
Cancer Res. 2002, 8, 986.
7. Brough, P. A.; Aherne, W.; Barril, X.; Borgognoni, J.; Boxall, K.; Cansfield, J. E.;
Cheung, K.-M. J.; Collins, I.; Davies, N. G. M.; 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. Y.; Surgenor, A.; Walmsley, D. L.; Webb, P.; Wood, M.; Workman,
P.; Wright, L. J. Med. Chem. 2008, 51, 196.
8. Chandarlapaty, S.; Sawai, A.; Ye, Q.; Scott, A.; Silinski, M.; Huang, K.; Fadden, P.;
Partdrige, J.; Hall, S.; Steed, P.; Norton, L.; Rosen, N.; Solit, D. B. Clin. Cancer Res.
2008, 14, 240.
9. 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.
USA 2009, 106, 8368.

2614
T. Taldone et al. / Bioorg. Med. Chem. 19 (2011) 2603–2614
10. Carden, C. P.; Sarker, D.; Postel-Vinay, S.; Yap, T. A.; Attard, G.; Banerji, U.;
Garrett, M. D.; Thomas, G. V.; Workman, P.; Kaye, S. B.; de Bono, J. S. Drug
Discovery Today 2010, 15, 88.
26. Law, J. H.; Habibi, G.; Hu, K.; Masoudi, H.; Wang, M. Y.; Stratford, A. L.; Park, E.;
Gee, J. M.; Finlay, P.; Jones, H. E.; Nicholson, R. I.; Carboni, J.; Gottardis, M.;
Pollak, M.; Dunn, S. E. Cancer Res. 2008, 68, 10238.
11. Whitesell, L.; Mimnaugh, E. G.; De Costa, B.; Myers, C. E.; Neckers, L. M. Proc.
Natl Acad. Sci. USA 1994, 91, 8324.
27. Cerchietti, L. C.; Lopes, E.-C.; Yang, S. N.; Hatzi, K.; Bunting, K. L.; Tsikitas, L. A.;
Mallik, A.; Robles, A. I.; Walling, J.; Varticovski, L.; Shaknovich, R.; Bhalla, K. N.;
Chiosis, G.; Melnick, A. Nat. Med. 2009, 15, 1369.
28. Marubayashi, S.; Koppikar, P.; Taldone, T.; Abdel-Wahab, O.; West, N.;
Bhagwat, N.; Lopes-Vazquez, E. C.; Ross, K. N.; Gönen, M.; Gozman, A.; Ahn,
J.; Rodina, A.; Ouerfelli, O.; Yang, G.; Hedvat, C.; Bradner, J. E.; Chiosis, G.;
Levine, R. L. J. Clin. Invest. 2010, 120, 3578.
29. Luo, W.; Dou, F.; Rodina, A.; Chip, S.; Kim, J.; Zhao, Q.; Moulick, K.; Aguirre, J.;
Wu, N.; Greengard, P.; Chiosis, G. Proc. Natl Acad. Sci. USA 2007, 104, 9511.
30. Luo, W.; Rodina, A.; Chiosis, G. BMC Neurosci. 2008, 9(Suppl 2), S7.
31. Luo, W.; Sun, W.; Taldone, T.; Rodina, A.; Chiosis, G. Mol. Neurodegener. 2010, 5,
24.
12. He, H.; Zatorska, D.; Kim, J.; Aguirre, J.; Llauger, L.; She, Y.; Wu, N.; Immormino,
R. M.; Gewirth, D. T.; Chiosis, G. J. Med. Chem. 2006, 49, 381.
13. 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. J. Med. Chem. 2009, 52, 4288.
14. Clevenger, R. C.; Raibel, J. M.; Peck, A. M.; Blagg, B. S. J. J. Org. Chem. 2004, 69,
4375.
15. Du, Y.; Moulick, K.; Rodina, A.; Aguirre, J.; Felts, S.; Dingledine, R.; Fu, H.;
Chiosis, G. J. Biomol. Screen. 2007, 12, 915.
32. Tsaytler, P. A.; Krijgsveld, J.; Goerdayal, S. S.; Rüdiger, S.; Egmond, M. R. Cell
Stress Chaperones 2009, 14, 629.
16. Immormino, R. M.; Kang, Y.; Chiosis, G.; Gewirth, D. T. J. Med. Chem. 2006, 49,
4953.
33. Roe, S. M.; Prodromou, C.; O’Brien, R.; Ladbury, J. E.; Piper, P. W.; Pearl, L. H. J.
Med. Chem. 1999, 42, 260.
17. 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. Bioorg. Med. Chem. Lett. 2005, 15, 3338.
18. 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. J. Med. Chem. 2005, 48, 4212.
19. Barril, X.; Beswick, M. C.; Collier, A.; Drysdale, M. J.; Dymock, B. W.;
Fink, A.; Grant, K.; Howes, R.; Jordan, A. M.; Massey, A.; Surgenor, A.;
Wayne, J.; Workman, P.; Wright, L. Bioorg. Med. Chem. Lett. 2006, 16,
2543.
34. Stebbins, C. E.; Russo, A. A.; Schneider, C.; Rosen, N.; Hartl, F. U.; Pavletich, N. P.
Cell 1997, 89, 239.
35. Nishiya, Y.; Shibata, K.; Saito, S.; Yano, K.; Oneyama, C.; Nakano, H.; Sharma, S.
V. Anal. Biochem. 2009, 385, 314.
36. Sidera, K.; Patsavoudi, E. Cell Cycle 2008, 7, 1564.
37. Hansen, J. B.; Nielsen, M. C.; Ehrbar, U.; Buchardt, O. Synthesis 1982, 404.
38. Hiegel, G. A.; Burk, P. J. Org. Chem. 1973, 38, 3637.
39. Crestey, F.; Ottesen, L. K.; Jaroszewski, J. W.; Franzyk, H. Tetrahedron Lett. 2008,
49, 5890.
40. Schrodinger, L. L. C., New York.
20. Barta, T. E.; Veal, J. M.; Rice, J. W.; Partridge, J. M.; Fadden, R. P.; Ma, W.; Jenks,
M.; Geng, L.; Hanson, G. J.; Huang, K. H.; Barabasz, A. F.; Foley, B. E.; Otto, J.;
Hall, S. E. Bioorg. Med. Chem. Lett. 2008, 18, 3517.
21. Serenex; Huang, K.; Ommen, A. J.; Barta, T. E.; Hughes, P. F.; Veal, J. M.; Ma, W.;
Smith, E. D.; Woodward, A. R.; McCall, W. S., WO-2008130879A2 2008.
22. Serenex; Huang, K.; Ommen, A. J.; Barta, T. E.; Hughes, P. F.; Veal, J. M.; Ma, W.;
Smith, E. D.; Woodward, A. R.; McCall, W. S., US-20080269193A1 2008.
23. Claramunt, R. M.; López, C.; Pérez-Medina, C.; Pinilla, E.; Torres, M. R.; Elguero,
J. Tetrahedron 2006, 62, 11704.
41. Jorgensen, W. L.; Maxwell, D.; Tirado-Rives, J. J. Am. Chem. Soc. 1996, 118,
11225.
42. Patel, P. D.; Patel, M. R.; Kaushik-Basu, N.; Talele, T. T. J. Chem. Inf. Model. 2008,
48, 42.
43. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.;
Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.;
Shenkin, P. S. J. Med. Chem. 2004, 47, 1739.
44. Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W.
T.; Banks, J. L. J. Med. Chem. 2004, 47, 1750.
24. Old, D. W.; Wolfe, J. P.; Buchwald, S. L. J. Am. Chem. Soc. 1998, 120, 9722.
25. Hurvitz, S. A.; Finn, R. S. Future Oncol. 2009, 5, 1015.
45. Eldridge, M. D.; Murray, C. W.; Auton, T. R.; Paolini, G. V.; Mee, R. P. J. Comput.
Aided Mol. Des. 1997, 11, 425.