4,5-Diarylisoxazole Hsp90 chaperone inhibitors: Potential therapeutic agents for the treatment of cancer

Article abstract of DOI:10.1021/jm701018h

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential chemotherapeutic agents for cancer. Here, we describe the structure-based design, synthesis, structure - activity relationships and pharmacokinetics of potent small-molecule inhibitors of Hsp90 based on the 4,5-diarylisoxazole scaffold. Analogues from this series have high affinity for Hsp90, as measured in a fluorescence polarization (FP) competitive binding assay, and are active in cancer cell lines where they inhibit proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Compound 40f (VER-52296/NVP-AUY922) is potent in the Hsp90 FP binding assay (IC₅₀ = 21 nM) and inhibits proliferation of various human cancer cell lines in vitro, with GI₅₀ averaging 9 nM. Compound 40f is retained in tumors in vivo when administered i.p., as evaluated by cassette dosing in tumor-bearing mice. In a human colon cancer xenograft model, 40f inhibits tumor growth by ～50%.

Full text of DOI:10.1021/jm701018h

196
J. Med. Chem. 2008, 51, 196–218
4,5-Diarylisoxazole Hsp90 Chaperone Inhibitors: Potential Therapeutic Agents for the
Treatment of Cancer
Paul A. Brough,*,† Wynne Aherne,‡ Xavier Barril,† Jenifer Borgognoni,† Kathy Boxall,‡ Julie E. Cansfield,†
Kwai-Ming J. Cheung,‡ Ian Collins,‡ Nicholas G. M. Davies,† Martin J. Drysdale,† Brian Dymock,† Suzanne A. Eccles,‡
Harry Finch,† Alexandra Fink,† Angela Hayes,‡ Robert Howes,† Roderick E. Hubbard,† Karen James,‡ Allan M. Jordan,†
Andrea Lockie,† Vanessa Martins,‡ Andrew Massey,† Thomas P. Matthews,‡ Edward McDonald,‡ Christopher J. Northfield,†
Laurence H. Pearl,‡ Chrisostomos Prodromou,‡ Stuart Ray,† Florence I. Raynaud,‡ Stephen D. Roughley,† Swee Y. Sharp,‡
Allan Surgenor,† D. Lee Walmsley,† Paul Webb,† Mike Wood,† Paul Workman,‡ and Lisa Wright†
Vernalis Ltd., Granta Park, Great Abington, Cambridge CB21 6GB, U.K., and Cancer Research U.K. Centre for Cancer Therapeutics,
The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, U.K.
ReceiVed August 15, 2007
Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential chemotherapeutic
agents for cancer. Here, we describe the structure-based design, synthesis, structure-activity relationships
and pharmacokinetics of potent small-molecule inhibitors of Hsp90 based on the 4,5-diarylisoxazole scaffold.
Analogues from this series have high affinity for Hsp90, as measured in a fluorescence polarization (FP)
competitive binding assay, and are active in cancer cell lines where they inhibit proliferation and exhibit a
characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Compound
40f (VER-52296/NVP-AUY922) is potent in the Hsp90 FP binding assay (IC50 ) 21 nM) and inhibits
proliferation of various human cancer cell lines in vitro, with GI50 averaging 9 nM. Compound 40f is retained
in tumors in vivo when administered i.p., as evaluated by cassette dosing in tumor-bearing mice. In a human
colon cancer xenograft model, 40f inhibits tumor growth by ∼50%.
Introduction
cancer versus normal cells.17 Further validation of Hsp90 as a
protein target for drug discovery comes from pharmacological
and clinical studies with the GM analogue 17-allylaminogeldan-
amycin (17-AAG, Figure 1, 1b).18 This first-in-class Hsp90
inhibitor to enter clinical trials has generated encouraging results
in Phase-I trials.19–21 Further clinical trials have been initiated
with 17-AAG used as a single agent and in drug combinations
in various cancer types. Hsp90 inhibitors are now the focus of
significant research and development efforts,22–25 and their
ability to interfere with multiple oncogenic pathways has raised
hopes that they will show broad antitumor activity and be less
susceptible to acquired drug resistance. However, despite the
high cellular activity and clinical progression of 17-AAG, this
compound has several potential limitations, including poor
solubility, (thereby demanding special formulation), limited
bioavailability, hepatotoxicity and extensive metabolism by
polymorphic enzymes.26–28 Some of these issues have been
addressed with the development of the water-soluble ansamycin
17-dimethylaminoethylgeldanamycin (17-DMAG Figure 1, 1c)29
and the prodrug of 17-AAG, IPI-50430,31 (Figure 1), which are
reported to be in Phase-I/Ib and Phase-I/II clinical trials,
respectively. However, these issues and the inherent chemical
complexity of these ansamycin inhibitors have led to significant
efforts to identify novel small-molecule inhibitors of Hsp90.22,32–37
Research from several groups has focused on the purine
scaffold and has generated potent inhibitors such as 3 and 4
(Figure 2), which are active in cellular and animal models of
cancer.38–40 We recently disclosed the identification of 4,5-
diarylpyrazoles such as 5 (Figure 3) as a new class of Hsp90
inhibitors,41,42 and the structure-based elaboration of this hit
resulted in the amide-substituted pyrazole 6.43 Compound 6
binds to the ATP binding site of Hsp90 with an IC50 of 25 nM
in a FP assay and has antiproliferative activity against HCT116
colon cancer cells, with a GI50 of 260 nM. This activity
compares favorably with the GI50 value for 17-AAG of 160
Molecular chaperones are proteins which play a key role in
the conformational maturation, stability and function of other
“client”protein substrates within the cell.1,2 Many of the client
proteins of heat shock protein 90 (Hsp90)3–6 are involved in
signal transduction, cell cycle regulation and apoptosis, including
kinases, transcription factors and hormone receptors. The
dysregulation of pathways involving these proteins are com-
monly associated with cancer pathology,7 and Hsp90 thus plays
a key role in assisting survival, proliferation, invasion, metastasis
and angiogenesis, which represent the hallmark traits of
malignancy. However, Hsp90 is ubiquitously expressed in
normal cells, comprising 1–2% of the total cellular protein, and
has important functions in normal cell homeostasis. Conse-
quently, Hsp90 was not pursued as a drug target until the
discovery and characterization of natural product inhibitors of
Hsp90 function such as geldanamycin8–10 (GM) and radicicol11,12
(RD) (Figure 1, 1a and 2). In the cell, Hsp90 function is
complex, involving homodimerization and binding to client
proteins, co-chaperones and other accessory proteins operating
in a chaperone cycle.13 The chaperone cycle is driven by
hydrolysis of ATP to ADP and by ATP/ADP exchange,14,15
and it was shown that both GM and RD are able to inhibit Hsp90
function by binding to an ATP binding pocket in the N-terminal
domain of Hsp90.16 The subsequent loss of chaperone function
causes client proteins to be degraded by the ubiquitin-proteasome
pathway. The prevalence of a high-affinity form of Hsp90 in
tumor cells, the “addiction”of cancer cells to oncogenic client
proteins and their greater dependency on Hsp90 have been
proposed as rationales for selectivity of Hsp90 inhibitors for
* To whom correspondence should be addressed. E-mail: p.brough@
vernalis.com. Phone: 44 (0)1223 895372. Fax: 44 (0)1223 895556.
†
Vernalis Ltd.
The Institute of Cancer Research.
‡
10.1021/jm701018h CCC: $40.75
2008 American Chemical Society
Published on Web 11/20/2007

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 197
Figure 1. Natural product derived Hsp90 inhibitors.
and assessing the ability to inhibit tumor growth in the HCT116
human colon carcinoma murine xenograft model.
Chemistry
Our strategy to optimize the in vivo activity in our series was
focused on the structure-activity relationships (SAR) in three
distinct areas of the molecule and was guided by structural
information of ligands (such as 6) bound to the ATP binding site
of Hsp90R.46 The binding mode anchor of this family of com-
pounds is the resorcinol moiety, also present in radicicol.16 The
resorcinol binds deep into the pocket, where the 4′-hydroxyl
displaces one of the four water molecules that mediate substrate
binding in the adenine binding region. Both phenolic hydroxyl
groups and the N atoms of the pyrazole ring form a very tight
network of hydrogen bonds with the carboxylate of the key residue
Asp93 and surrounding polar groups or water molecules. The high
complementarities and tight packing between Hsp90 and the
inhibitor in this area leave room only for small modifications of
Figure 2. Purine class Hsp90 inhibitors.
Figure 3. Pyrazole class Hsp90 inhibitors.
nM in the same tumor cell line. Further medicinal chemistry
efforts in this area have identified 4-substituted pyrazole amides
such as 744 and piperazines 8.45 These are not as active in the
FP assay as compound 6 and, consequently, exhibit reduced
activity in tumor cell lines. Here, we report on further medicinal
chemistry studies which identified diarylisoxazoles with a greater
than 20-fold increase in their ability to inhibit cancer cell
proliferation compared with that of previously reported dia-
rylpyrazole compounds. Pharmacokinetic analyses performed
by cassette dosing in tumor-bearing animals were used to select
compounds showing the highest ratio between tumor levels and
cellular GI50. The drug discovery potential of lead compounds
was further evaluated by monitoring target modulation in vitro
Figure 4. X-ray structure of 6 bound to the ATP binding site of human
Hsp90R. Red spheres represent structurally conserved water molecules.

198 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Brough et al.
Scheme 1. Synthesis of Pyrazole Hsp90 Inhibitors 19e and 20a-i a
a Reagents and conditions: (a) AcOH, BF3 ·OEt2, 58%; (b) BnBr, MeCN, K2CO3, 90%; (c) (CO2Et)2, NaOEt, EtOH, 94%; (d) H2NNH2, AcOH, 87%, or
H2NNH2 ·HCl, EtOH, ∆, 81%; (e) NIS, CAN (1 mol %), MeCN, ∆, 98%; (f) 2 M NaOH, dioxane; 92%; (g) (COCl)2, DMF, DCM; (h) EtNH2 ·HCl, NEt3,
99%; (i) 4-formylphenylboronic acid, Pd(dppf)Cl2, K2CO3, THF, H2O, microwave, 68%; (j) 3-formylphenylboronic acid, Pd(dppf)Cl2, K2CO3, THF, H2O,
microwave, 71%; (k) R1R2NH, NaCNBH3, AcOH, MeOH; (l) H2, Pd/C, AcOH.
the inhibitor’s core, but a wide range of substituents can be placed
off of the pyrazole ring and the 5′ position of the resorcinol.
Scheme 2. Synthesis of Pyrazole Hsp90 Inhibitors 19a-d and
19f-g a
Accordingly, and bearing in mind the experience gained in our
previous work,43–45,47 the areas that we focused on were (i)
incorporation of a solubilizing group on the 4-arylpyrazole sub-
stituents, (ii) modification of the central pyrazole heterocycle, and
(iii) the optimization of the 5′ substituent on the resorcinol ring.
Figure 4 shows compound 6 bound to the Hsp90 ATP binding
site and illustrates the importance of the key interaction of the
resorcinol with Asp93 and the hydrogen bond from the 5-amide
substituent to Gly97.43,48 Further inspection of this structure, and
others of the same structural class, indicated that the meta and para
positions of the aryl group appended to the 4 position of the
pyrazole were pointing toward solvent and should therefore be
suitable sites for the incorporation of a solubilizing group. Thus,
we initiated the synthesis of pyrazole Hsp90 inhibitors (19 and
20), incorporating CH2-amine moieties as the solubilizing func-
tionality. Several routes to these targets were utilized (Schemes 1
and 2). Key intermediates were the p- and m-formylphenyl
compounds (17 and 18), serving as precursors to reductive
amination reactions (Scheme 1), or the p-aminomethyl derivative
22, as a versatile reagent for reductive amination, alkylation,
conjugate addition and amide-forming reactions. The route used
for the synthesis of 19e and 20a-i is shown in Scheme 1.
Acetophenone 10 was made by regioselective Friedel-Crafts
acylation of commercially available 4-chlororesorcinol 9. Both
phenolic hydroxyls were protected with benzyl groups, generating
11 prior to the subsequent reaction of an intermediate sodium
a Reagents and conditions: (a) 4-aminomethylphenyl boronic acid, H2O,
THF, Pd(dppf)Cl2, K2CO3, Boc2O, Et3N, DMF, ∆, 26%; (b) (i) TFA; (ii)
NaHCO3, 95%; (c) (i) vinylmethylsulfone or divinylsulfone; (ii) BCl3, DCM;
(d) (i) acetaldehyde, NaBH(OAc)3, DCE then NaBH4, EtOH; (ii) cyclo-
hexadiene, Pd/C, MeOH, 16%; (e) (i) HATU, DMF, DIPEA, 1-methyl-
piperidine-3-carboxylic acid or 2-carboxymethylpyrrolidine-1-carboxylic
acid, rt; (ii) BCl3, DCM; (f) (i) 2-bromoethanol, EtOH, DIPEA; (ii) BCl3,
DCM, 13%.

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 199
Scheme 3. Synthesis of Isoxazole Hsp90 Inhibitors a
a Reagents and conditions: (a) H2NOH·HCl, EtOH, 86%; (b) EtNH2, MeOH, ∆, 89%; (c) NBS, CAN, MeCN, ∆, 4.5 h, 74%; (d) 4-formylphenylboronic
acid, Pd(PPh3)2Cl2, NaHCO3, DMF, H2O, 73%; (e) 3-formylphenylboronic acid, Pd(PPh3)2Cl2, NaHCO3, DMF, H2O; (f) R1R2NH, NaCNBH3, AcOH,
MeOH; (g) boronate ester, DME, NaHCO3, Pd(PPh3)4; (h) BCl3, DCM, 0 °C.
Compounds 19a-d and 19f,g were made using alternative
Scheme 4. Preparation of 33 (the Isoxazole Analogue of
Pyrazole 6) a
procedures (Scheme 2) from the key intermediate aminomethyl
derivative 22. Compound 16 was cross-coupled with 4-ami-
nomethylboronic acid and protected in situ as the t-butyloxy-
carbamate derivative to aid in product isolation. Trifluoroacetic-
acid-mediated deprotection afforded the amine 22, which was
functionalized via four distinct synthetic methodologies. Di-
ethylamine 19a was prepared by reductive amination with
acetaldehyde and subsequent debenzylation by transfer hydro-
genolysis with cyclohexadiene and palladium on carbon. Sul-
fones 19c and 19d were prepared by vinylogous attack of the
amine on methylvinylsulfone and divinylsulfone, respectively.
Amides 19b and 19f were formed by a HATU49-mediated
coupling procedure, and 19g utilized bis nucleophilic displace-
ment on 2-bromoethanol. With the exception of 19a, final
products were obtained by boron trichloride-mediated depro-
tection of the dibenzyl resorcinol.
a Reagents and conditions: (a) 4-methoxyphenylboronic acid, NaHCO3,
H2O, DMF, Pd(PPh3)2Cl2, 63%; (b) BCl3, DCM, 0 °C, 65%.
enolate with diethyl oxalate, which afforded keto-enol ester 12.
Construction of the pyrazole heterocycle 13 was achieved by
reaction of 12 with hydrazine hydrate in acetic acid or hydrazine
hydrochloride in ethanol. Iodination at the 4-position of the pyrazole
was achieved with N-iodosuccinimide, and it was found that the
reaction yield was significantly improved with the addition of a
catalytic amount of ceric ammonium nitrate. Conversion of the
ethyl ester moiety of 14 to the ethylamide of 16 was achieved by
ester saponification, activation of resulting acid 15, and subsequent
reaction with ethylamine. Compound 16 underwent a palladium-
catalyzed Suzuki cross-coupling reaction with p- or m-formyl-
phenylboronic acid to generate 17 and 18, respectively. Reductive
amination with various amines, followed by hydrogenation to
remove the benzyl groups, furnished the desired amines 19e and
20 a-i.
The synthesis of the isoxazoles is analogous to that used for
the pyrazole series (Scheme 3). Keto-enol ester 12 was reacted
with hydroxylamine hydrochloride to give the isoxazole 23, and
the subsequent conversion to final compounds utilized methods
similar to those used for the corresponding pyrazoles, as outlined
in Scheme 1. The amide functionality however was introduced
directly from the ester prior to halogenation. N-bromosuccin-
imide (NBS)/ceric ammonium nitrate (CAN) proved superior
for bromination compared to an earlier method which used
bromine in acetic acid. However, compared to pyrazole bro-
mination, a relative increase in the amount of CAN was

200 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Brough et al.
Scheme 5. Conversion of Key Intermediate 34 to Isoxazole Hsp90 Inhibitors a
a Reagents and conditions: (a) NBS or NIS, CAN, MeCN, ∆; (b) 4-formylphenylboronic acid, NaHCO3, H2O, DMF, Pd(PPh3)2Cl2; (c) R1R2NH, NaCNBH3,
AcOH, MeOH; (d) BCl3, DCM, 0 °C.
Scheme 6. Synthesis of Key Isoxazole Intermediates 34a-c and 34 h-j a
a Reagents and conditions: (a) BnBr, K2CO3, MeCN, 97%; (b) NBS, DMF, rt, 97%; (c) (CO2Et)2, NaOEt, EtOH, 96%; (d) hydroxylamine hydrochloride,
EtOH, ∆, 92%; (e) EtNH2, MeOH, ∆, 80%; (f) trans-2-phenylvinylboronic acid, DMF, H2O, Pd(PPh3)2Cl2, NaHCO3, ∆, 71%; (g) PhB(OH)2, K2CO3, H2O,
THF, Pd(dppf)Cl2, ∆, 68%; (h) trans-2-(4-fluorophenyl)vinylboronic acid, DMF, H2O, Pd(PPh3)2Cl2, NaHCO3, 69%; (i) 1,4-dioxane, Pd(C), H2; (j) (i)
3-fluorostyrene, Pd(o-Tol3P)2Cl2, n-BuOH; i-Pr2NH, (ii) TsNH2NH2, NaOAc, 1,2-DME, ∆, 92%.
coupled with boronate esters having the amino functionality
Scheme 7. Synthesis of Isoxazole 45 a
already present. A modification of this route was also used for
making the isoxazole analogue (33) of VER-49009 (6) using
the intermediate 32 (Scheme 4).
The final area of interest for SAR studies described here was
the 5′ position on the resorcinol ring. Substituents at C-5′ have
the potential to induce a conformational change in the protein
by forming a helix between residues 104 and 111.50 This
structural change creates a lipophilic pocket in the ATP binding
site of the Hsp90 protein, and we were keen to explore the
a Reagents and conditions: (a) 4-fluorophenylboronic acid, NaHCO3,
H2O, DMF, Pd(dppf)Cl2, 76%; (b) BCl3, DCM, 0 °C, 65%.
influence of substituents at this position on the potency and
physicochemical properties of the Hsp90 ligands. All of the 5′
analogues were made from key intermediates 34 by the method
outlined in Scheme 5. Differing synthetic methodologies were
utilized for the synthesis of variably substituted 5′-resorcinol
derivatives of 34. For 5′-aryl and 5′-phenethyl substituents, the
5′-bromo intermediate 51 (Scheme 6) was used as a substrate
for Suzuki or Heck Pd-catalyzed coupling reactions. Styrene
necessary to effect good yields in this case. Debenzylation of
28 and 29 was accomplished with boron trichloride in preference
to hydrogenation. This change was due to concerns that
hydrogenation may cleave the isoxazole ring and the observation
of partial (ca. 5%) 5′-chlorine reduction in several reactions
using catalytic hydrogenation. Some compounds of this class
were made by a modified sequence, in which 25 was cross-

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 201
Scheme 8. Synthesis of Isoxazole Intermediate 34e a
a Reagents and conditions: (a) isobutyric acid, BF3 ·OEt2 quant; (b) (i) ethylchloroformate, NEt3, THF; (ii) NaBH4, THF/H2O; (iii) 10% aq NaOH, 98%;
(c) (i) AcOH, BF3 ·OEt2; (ii) BnBr, K2CO3, MeCN, 25%; (d) (CO2Et)2, NaOEt, EtOH, ∆, 58%; (e) hydroxylamine hydrochloride, EtOH, ∆, 74%; (f) EtNH2,
MeOH, ∆, quant.
Scheme 9. Synthesis of Isoxazole Intermediate 34f a
a Reagents and conditions: (a) Ph3PMeBr, n-BuLi, THF, 80%; (b) H2, Pd/C, EtOH, 70%; (c) AcOH, BF3 ·OEt2, 88%; (d) BnBr, K2CO3, DMF, 88%; (e)
(CO2Et)2, NaOEt, NaH, toluene, ∆, 86%; (f) hydroxylamine hydrochloride, EtOH, ∆, 91%; (g) EtNH2, MeOH, ∆, 85%.
derivatives such as 34a from Heck or Suzuki reactions of
styrenes or vinylogous boronic acids were reduced prior to
further manipulation. Compound 35c (Scheme 6) was subse-
quently used to make the 5′-phenyl derivative 45 (Scheme 7),
which was used for compound profiling comparisons. For the
isobutyl-substituted intermediate 34e, the route outlined in
Scheme 8 was used. Starting from resorcinol, a Friedel-Crafts
acylation afforded acetophenone derivative 53, which was
reduced51 to the sec-butyl compound 54 prior to a second
Friedel-Crafts acylation and benzyl protection. Construction
of the key isoxazole 34e proceeded as described above. An
acetophenone derivative was also used in the route to the
isopropyl-substituted intermediate 34f (Scheme 9). In this case
however, a Wittig reaction was followed by hydrogenation to
generate the 5′-isopropyl substituent (47–59, Scheme 9). The
reduction of the styrene functionality in 58 caused concomitant
debenzylation. However, the resorcinol 59 acylated in better
yield than the corresponding benzyl-protected substrate; there-
fore, reprotection was delayed until the subsequent step. The
5′-ethyl-substituted compounds (such as 34d) were made from
4-ethylresorcinol by the same route as that used for the
5-chlororesorcinol outlined in Scheme 3 (see also Scheme 10).
For the introduction of a 5′-tert-butyl group, Friedel-Crafts
alkylation methodology was applied (Scheme 10), utilizing the
tert-butyl cation generated from tert-butanol to afford acetophen-
one 65g. Conversion to 34g was accomplished in a similar
fashion as that described previously for compound 24 (Scheme
3).
Results and Discussion
Using the routes outlined in Schemes 1 and 2, we synthesized
a number of pyrazoles incorporating solubilizing groups on the
4-aryl group (Table 2). For substituents in the para position of
the 4-aryl group, in some cases, both enzyme potency and
cellular GI50 values (19e and 19f) were similar to those of VER-
49009 (6). However, in most instances, these diverse analogues
were less potent than 6. For compounds with aminomethyl
groups at the meta position (20a-i), none of the examples
matched the binding affinity of compound 6 in the FP assay or
in the cell growth inhibition assay with HCT116 cells. Thus,
although the crystal structure of 6 suggested that there was space
to introduce solubilizing groups off of the 4-aryl substituent, it
appeared that the regiochemistry and nature of the solubilizing

202 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Brough et al.
Scheme 10. Synthesis of Key Isoxazole Intermediates 34d and 34g a
a Reagents and conditions: (a) H2SO4, 2-methylpropan-2-ol, TFA, 75 °C, 92%; (b) AcOH, BF3 ·OEt2, 90 °C; (c) BnBr, K2CO3; (d) (CO2Et)2; NaOEt,
EtOH; (e) H2NOH·HCl, EtOH, ∆; (f) EtNH2, MeOH, ∆.
However, the advantage of the isoxazole over the pyrazole
scaffold became apparent when we looked at the activity of 6
versus 33 in cell growth inhibition assays across a range of
Table 1. Binding (FP Assay) and Cell Growth Inhibition (SRB Assay)
Data for Previously Reported Hsp90 Inhibitorsa
a
a
FP IC50
GI50
human cancer cell lines (Table 3). These data demonstrated a
trend for higher potency (with respect to cell growth inhibition)
for the isoxazole 33. The interactions of pyrazole 6 and isoxazole
33 with Hsp90 were studied previously by isothermal calori-
metry,47 which showed that the isoxazole-Hsp90 complex is
marginally more stable than the corresponding pyrazole complex
and that the difference is predominantly due to the enthalpy
difference. Kinetic analysis by surface plasmon resonance (SPR)
has shown that, although these compounds have similar Kon
values (6.4 × 105 and 8.2 × 105 M-1 s-1) for 33 and 6,
respectively, the Koff for isoxazole 33 of 4.05 × 10-4 s-1 is
10-fold slower than that of 5.64 × 10-3 s-1 for pyrazole 6.
These combined data can help account for the increased cellular
potency of the isoxazole and also help rationalize the measured
four-fold higher concentrations of 33 compared to 6 recently
shown in HCT116 cells. This was also sufficient for the
isoxazole 33 to demonstrate the first signs of in vivo efficacy
in a xenograft tumor model, as reported previously.47
compound
(µM)
HCT116 (µM)
17-AAG (1b)
1.27
0.28
0.025
0.258
0.600
0.16
5.8
0.26
11.6
6.5
5
6
7
8
a Values are reported as the mean of at least two independent
determinations.
group had an effect on how well this substitution was tolerated
in these pyrazole analogues.
Examination of the binding for the pyrazole ring in ligands
such as 6 demonstrated that the polar interactions deep inside
of the binding site are very well matched between the protein
and ligand. With this in mind, it was clear to us that very little
steric change would be tolerated in this region. The nitrogen
adjacent to the resorcinol (N1) is involved in hydrogen bonding
with the key Asp93/water network at the base of the pocket as
an H-bond acceptor. We felt that an alternative hydrogen-bond
acceptor such as oxygen might be advantageous. Pyrazoles can
exist in tautomeric forms, and when N1 is protonated, this
represents a form which is unlikely to bind well to Hsp90.
Further, the interaction between N2 and the amide backbone
carbonyl of Gly97 is not in the ideal geometry. Therefore, fixing
the position designated N1 as a 100% H-bond acceptor was
considered to be a preferred feature, with the N2 position being
less critical. We desired to achieve such a subtle change while
keeping the other carbon positions already substituted with key
functionality in a defined orientation for binding. Isoxazoles and
pyrazoles differ in that the former does not bear hydrogen on a
heteroatom and can therefore not be substituted. As a conse-
quence, the isoxazole is also smaller in size and should still be
accommodated in the binding site. In addition, there is no
possibility of the existence of isomeric forms that can exist with
the pyrazole. Evaluation of the binding of the isoxazole analogue
33 (Scheme 4) of the pyrazole 6 to Hsp90 protein gave an IC50
value of 28 nM, similar to that of compound 6 (25 nM). This
shows that the change is tolerated, with the matched pyrazole/
isoxazole 6/33 exhibiting equipotent binding in the FP assay.
The pyrazole to isoxazole scaffold change was next combined
with solubilizing group incorporation on the 4-aryl ring. Table
4. shows data for a series of diarylisoxazoles, substituted with
a range of amine-bearing substituents predominantly on the para
position of the 4-aryl ring. It was clear that the pyrazole to
isoxazole switch was very well tolerated in this series of
compounds and that the preference for substitution at the para
position of the 4-aryl ring compared to the meta position seen
with analogous pyrazole series was maintained. All of the para-
substituted examples had sub-70 nM IC50 values in the binding
assay and were at least equipotent with analogous pyrazoles
(30a vs 19d, 30b vs 19c, and 30f vs 19a). The meta-substituted
compounds 31a, 31b, and 31c were less potent, as was also
observed in the pyrazole series, but when comparing analogous
pyrazole analogues (31a vs 20a and 31c vs 20f), the isoxazoles
were more potent. Most importantly, the significant improved
inhibition of cell proliferation seen with isoxazole 33 compared
to that of pyrazole 6 was retained in this subseries (Table 4).
Thus 30d, 30f, 30g, and 30k have sub-100 nM GI50 values
against HCT116 cells.

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 203
Table 2. Binding (FP Assay) and Cell Growth Inhibition (SRB Assay) Data for Aminomethyl-Functionalized Diarylpyrazolesa
a Values are reported as the mean of at least two independent determinations.
We next turned our attention to the 5′ position of the
resorcinol. We had previously shown43 that the removal of ethyl
or chloro from this position resulted in a 20-fold decrease in
binding affinity for ligands closely related to 6. This loss in
potency is rationalized by structural information showing that
the ethyl and chloro groups are interacting at the edge of a

204 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Brough et al.
Table 3. Binding (FP Assay) and Cell Growth Inhibition (SRB Assay) Data in Various Human Cancer Cell Lines for Compounds 6 and 33a
human
Hsp90ꢀ FP
IC50 (µM)
HCT116
(colon)
GI50 (µM)
DU145
(prostate)
GI50 (µM)
PC3M
(prostate)
GI50 (µM)
SKMel28
(melanoma)
GI50 (µM)
SF268
(glioma)
GI50 (µM)
U87MG
(glioma)
GI50 (µM)
compound
number
6
33
0.025
0.028
0.36
0.12
1.2
0.23
2.2
0.22
0.48
0.045
0.32
0.034
1.2
0.028
a Values are reported as the mean of at least two independent determinations.
Table 4. Binding (FP Assay) and Cell Growth Inhibition (SRB Assay) Data for Aminomethyl-Functionalized Diarylisoxazolesa
a Values are reported as the mean of at least two independent determinations.
lipophilic pocket created by Phe138, Met98, Val150, Leu103,
Leu107, Trp162, and Tyr139. This region of the protein is also
flexible and able to accommodate larger groups by a confor-
mational movement of the loop defined by Ile104 to Ala111.50
The data presented in Table 5. clearly demonstrate that a large
steric variance is tolerated at the 5′ position. Groups such as
phenethyl (40i, 40j, and 41i) and phenyl (40c, 41c, and 41m)
retain binding affinity similar to those of the chloro analogues,
such as 30d, or small alkyl-substituted analogues, such as 42d
and 42f. Additionally, these analogues demonstrated excellent

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 205
Table 5. Effects of Modifications to the 5′ Position of the Resorcinol on Binding (FP Assay) and Cell Growth Inhibition (SRB Assay)a
a
FP
GI50
compound
number
IC50a
(HCT116)
X
amine moiety
(µM)
(µM)
40b
40d
42d
40i
-CH2CH2Ph
Et
Et
-CH2CH2(4-FPh)
-CH2CH2(4-FPh)
-CH2CH2(3-FPh)
Ph
Ph
2-FPh
2-MePh
4-FPh
isobutyl
tert-butyl
tert-butyl
isopropyl
isopropyl
isopropyl
Ph
morpholino
morpholino
NEt2
0.032
0.029
0.013
0.020
0.026
0.026
0.022
0.014
0.039
0.014
0.036
0.035
0.008
0.011
0.021
0.006
0.006
0.074
0.029
0.025
0.041
0.039
0.029
0.058
0.152
0.086
0.087
0.114
0.035
0.133
0.070
0.065
0.016
0.006
0.040
0.091
morpholino
piperidinyl
morpholino
morpholino
piperidinyl
morpholino
piperidinyl
piperidinyl
morpholino
morpholino
piperidinyl
morpholino
NEt2
41i
40j
40c
41c
40l
41m
41k
40e
40g
41g
40f
42f
41f
43c
piperidinyl
ethyl
a Values are reported as the mean of at least two independent determinations.
equivalent binding mode as we previously reported41,43,47 for
5, 6, and 33, with the same 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. The isoxazole 3-amide
group in compound 40f makes the same hydrogen-bond
interactions with the protein backbone via Gly97 as that seen
with 6 and 33, as well as a further interaction with Lys58, but
as previously reported, the latter reaction does not significantly
contribute to the binding free energy.43,48 Replacement of the
chlorine of the resorcinol ring in 6 or 30d53 by an isopropyl
group in 40f results in an additional hydrophobic interaction
with Leu107 in the flexible lipophilic pocket, and additional
hydrophobic interactions are also seen with Thr109 and Gly135
from the morpholine moiety present in 40f.
Table 6. Effect of Oxygen-methylation on Binding (FP Assay) and Cell
Growth Inhibition (SRB Assay)a
compound
number
FP
IC50 (µM)
GI50
(HCT116) (µM)
R1
R2
40d
70
71
H
Me
H
H
H
Me
Me
0.029
0.015
0.540
1.56
0.025
0.080
2.15
72
Me
32.0
It was important to establish that the new ligands retained
the well-established molecular signature of known Hsp90
inhibitors with respect to the effect of the ligands on cellular
markers. Figure 6shows that the pyrazoles 6 and 19e and the
isoxazoles 33, 30d, and 40f gave the expected depletion of the
client proteins Raf-1 and Her2 and induction of Hsp72 in
HCT116 cells in vitro, as determined by western blots at 1 and
2 × GI50 concentrations. These results support the premise that
the effects on cell growth were a consequence of Hsp90
chaperone inhibition.
a Values are reported as the mean of at least two independent
determinations.
potency in cell growth inhibition assays, as demonstrated by a
number of the compounds in Table 5, in particular, the
5′-isopropyl analogues 40f and 42f.
To evaluate the importance of the resorcinol OH moieties in
the binding of this class of inhibitor, several O-methylated
analogues of the 5′-ethyl compound 40d were synthesized. As
demonstrated in Table 6, O-methylation at C-2′ caused a large
drop in binding affinity. O-Methylation at C-4′ was tolerated,
but compound 70 had reduced potency and cellular activity and
thus demonstrated no advantage over the free resorcinol.
At all stages in the optimization process, X-ray crystal
structures were obtained with ligands bound to the N-terminal
domain of Hsp90R to confirm that the changes made in the
elaboration of 6 had not altered the binding mode of the ligands.
Figure 5shows an overlay of the X-ray structures of 6 and the
isoxazole 40f.52 The structural data for 40f demonstrate an
We had previously shown54 that the resorcinol moiety was
glucuronidated but that in vitro microsomal metabolism rates
were not predictive of the plasma clearance of this chemical
series. Preferred compounds from the foregoing research were
therefore further characterized in vivo using cassette dosing to
mice bearing subcutaneous HCT116 human colon cancer
xenografts, followed by quantitative LC/MS/MS analysis in
various tissues.54,55 The method was first validated by comparing
results for a set of five compounds dosed singly and in mixture

206 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Brough et al.
Figure 5. Overlay of X-ray structure of 6 and 40f bound to the ATP binding site of human Hsp90R. Red spheres represent structurally conserved
water molecules.
Figure 6. Effects of 17-AAG, pyrazoles 6 and 19e, and isoxazoles
30d, 33, and 40f on expression of Raf-1, Hsp72, and Her2 in HCT116
cells as determined by western blot. Compounds were dosed for 24 h
at concentrations of 1 and 2 times the GI50 value. GAPDH was used
as a loading control.
Figure 7. Cassette dosing method validation, showing the compound
tumor concentration 6 h post administration in mice bearing HCT116
by measuring tumor concentrations 6 h post administration.
subcutaneous human colon tumor xenografts dosed at 4 mg per kg i.p.
Concentrations in tumors were similar in both experiments
Compounds were dosed as single agents or were cassette dosed with
(Figure 7), with the same rank order, suggesting that compound-
compound interactions were not significant. In subsequent
studies, selected compounds were administered i.p. in cassettes
of five compounds to tumor-bearing mice at a dose of 4 mg/kg
each. Table 7. shows a summary of three cassettes dosed with
some of the most potent diarylisoxazole amides. Table 8 shows
some additional pharmacodynamic (PK) parameters for cassette
dosing experiment 3. The pharmacokinetic standard used, 33,
gave consistent results for tumor levels but exhibited variable
plasma concentrations (up to 60% between cassettes 1 and 3).
Overall, tumor-to-plasma ratios were found to vary from 0.3 to
20. Compounds with aliphatic substituents such as ethyl (40d)
and isopropyl (40f) in the 5′ position of the resorcinol had higher
tumor exposure than compounds with aromatic substituents
(such as 40b, 41i, and 41m). The 5′-phenyl derivative 43c had
mixtures of five compounds.
a comparatively high tumor concentration but still higher plasma
levels. The 5′-chloro compounds also achieved high tumor
concentrations (30f, 30g and 30c). The isopropyl analogue 40f
clearly showed the highest ratio of tumor concentration to
cellular GI50 (up to 35-fold), with a half-life of 9.5 h in the
tumor, and this compound was therefore taken forward for
further study.
Isoxazole 40f showed potent antiproliferative effects in a
range of human cancer cell lines (Table 9), and the expected
depletion of client proteins and induction of Hsp72 were
observed in all of the cell lines examined (data not shown).
Compound 40f was administered at 50 mg/kg daily i.p. to

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 207
Table 7. Pharmacokinetic Data Obtained by Cassette Dosing with Isoxazole Compoundsa
AUClast tumor
(hr*nmol/L)
AUClast plasma
(hr*nmol/L)
tumor/plasma
ratio
max fold
above GI50
cassette
compound
33
30c
45
1332.8
2522.9
597.5
2930.4
2403.5
1437.6
655.9
1750.9
668.2
685.5
1222.8
1542.3
1191.8
925.8
989.0
814.9
2547.2
618.8
1327.4
1630.4
659.5
3708.1
754.2
792.2
1.1
1.6
0.5
3.2
2.4
1.8
0.3
2.8
0.5
0.4
2.6
0.3
2.3
0.9
1.0
6.5
4.7
1.5
12.0
20.0
6.0
4.2
24.0
5.6
1.9
7.0
cassette 1
30f
30g
33
40b
40d
41i
41m
33
43c
40f
40g
41c
cassette 2
cassette 3
1733.0
1248.0
1720.0
751.1
2.8
35.0
2.9
994.8
966.7
2.8
a A graphical representation of cassette dosing PK results for cassette 3 is provided in the Supporting Information section.
Table 8. Details of Pharmacokinetic Parameters for Cassette Dosing Experiment 3
Tmax
(hr)
Cmax
(nmol/L)
AUClast
(hr*nmol/L)
HL_Lambda_z
(hr)
Vz_F_obs
(L)
CL_F_obs
(L/hr)
compound
33
1
4
1
1
1
482.1
252.5
385.4
202.9
237.4
1733.0
1248.0
1720.0
751.1
3.9
14.6
9.5
5.0
7.3
0.432
0.736
0.478
0.809
0.652
0.076
0.035
0.035
0.111
0.062
43c
40f
40g
41c
994.8
Table 9. Binding (FP Assay) and Cell Growth Inhibition (SRB Assay) in Various Human Cancer Cell Lines for Compound 40fa
human
Hsp90ꢀ
FP IC50 (µM)
HCT116
(colon
GI50 (µM)
DU145
(prostate)
GI50 (µM)
PC3M
(prostate)
GI50 (µM)
SKMel28
(melanoma)
GI50 (µM)
SF268
(glioma)
GI50 (µM)
U87MG
(glioma)
GI50 (µM)
compound
number
40f
0.021
0.016
0.005
0.006
0.005
0.006
0.008
a Values are reported as the mean of at least two independent determinations.
Figure 8. Response of HCT116 human colon carcinoma xenografts to 40f therapy (50 mg/kg i.p. q.d). (A) Mean tumor volumes (therapy vs vehicle
controls). (B) Mean weights of excised tumors after 12 days of therapy. Therapy/control ratio (T/C) ) 49.9. (C) Mouse body weights during therapy. (D)
Changes in PD markers as measured at 6, 16, and 24 h after the final dose. GAPDH was used as a loading control for the western blots.

208 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Brough et al.
mm mean diameter for therapy and 8–9 mm for cassette dosing
pharmacokinetic studies.
athymic mice bearing HCT116 human colon carcinoma xe-
nografts. The rate of tumor growth was significantly inhibited
by 40f administration (Figure 8A), and treated tumor weights
measured at day 12 were 49.8% of the control values (Figure
8B). This dose schedule was well-tolerated, with no body weight
losses (Figure 8C), and the pharmacodynamic (PD) marker
changes at the end of the study were consistent with Hsp90
inhibition (Figure 8D).
Compounds were dissolved in DMSO and diluted in sterile
saline/Tween 20 such that animals received the dose required in
0.1 mL of final solution per 10 g of body weight. Final concentra-
tions were 10% DMSO, 5% Tween 20, and 85% saline. The
compound was stable in this vehicle and, for repeat dosing in the
therapy study, was stored for 3–4 days at 4 °C between doses.
Controls received an equal volume of vehicle. For cassette dosing,
animals received 4 mg/kg of each compound. The approach used
was consistent with the guidelines for cassette dosing that we have
published previously55 and our prior experience with early
pyrazole-resorcinol Hsp90 inhibitors.54
Conclusion
A significant reduction of tumor growth in HCT116 colon
cancer xenografts was observed with the potent Hsp90 inhibitor
VER-52296/NVP-AUY92259 (40f). This compound has excel-
lent potency against Hsp90 in a FP binding assay (IC50 ) 21
nM) and inhibits proliferation in a wide range of human cancer
cell lines with an average GI50 of 9 nM. Treated cells show the
characteristic molecular signature of Hsp90 inhibition, namely,
elevation of Hsp72 and degradation of oncogenic client proteins.
The compound is retained in HCT116 xenograft tumors at
concentrations well above the GI50, with a half-life of 9.5 h
following i.p. administration at 50 mg/kg qd. The identification
of 40f as a potential therapeutic agent for treatment of cancer
was strongly influenced by X-ray structures of protein-inhibitor
complexes and was the culmination of studies that optimized
the physical and biological properties of lead compounds,
including, in particular, tumor drug levels. In addition, 40f
showed excellent efficacy in a range of subcutaneous and
orthotopic human tumor xenograft models covering major cancer
types and diverse oncogenic profiles, and these data will be
published elsewhere.56 The overall properties warrant further
studies of this compound, and an i.v. formulation of NVP-
AUY922 (40f) is currently in a Phase-I study in patients with
a range of cancer indications.
The plasma and tumors were taken as previously described54 at
0, 0.25, 0.5, 1, 2, 4, and 6 h post administration (N ) 3 animals
per time point) and snap frozen. Quantitative analysis of the plasma
and tumors was performed by LC/MS/MS. The LC system
comprised a Waters Alliance 2795 separations module (Hertford,
U.K.). Chromatography was achieved on a Synergi Polar-RP 4µ
50 × 2.1 mm column (Phenomenex, Macclesfield, U.K.) with an
A100-X precolumn filter (Anachem, Luton, Bedfordshire, UK). The
mobile phase, initially containing 0.1% formic acid and methanol,
80/20 v/v, was increased linearly to 90% methanol from 0 to 6
min, kept isocratic for 5 min, and then washed with 100% methanol
for 1 min before returning to the initial conditions. The flow rate
was 0.2 mL/min. Detection was achieved on a Micromass Quattro
Ultima triple-quadrupole mass spectrometer (Manchester, U.K.).
The electrospray interface was used in negative-ionization mode
(ES-) with a capillary voltage of 4 kV and a source temperature of
200 °C. Detection was accomplished by multiple reaction monitor-
ing of the optimized transitions and collision energy for each of
the six compounds (five compounds and an analytical internal
standard). The program WinNonlin was used to evaluate the half
life of test compounds.
Therapeutic Activity in Human Tumor Xenografts. The
compound 40f was prepared as described below. Therapy was
initiated when tumors were established with a mean diameter of
4–5 mm and continued for 12 days. The dose selected (50 mg/kg)
had been shown in pilot studies to be well-tolerated and to give
plasma levels within the therapeutic range. Tumors were measured
three times weekly and weighed when excised at the end of the
experiment. Animals were weighed daily as an indication of general
health. Tumor samples were also collected for analysis of phar-
macodynamic biomarkers, as described earlier.
Experimental Section
Fluorescence Polarization Assay. The ability of compounds to
compete with a fluorescently labeled probe for binding to full-length
human Hsp90ꢀ was determined, as described previously,57 with a
modified probe (VER-00051001). This probe binds with greater
affinity to Hsp90 than that described previously, allowing the
accurate determination of IC50 for higher-affinity compounds.
Synthetic procedures for VER-00051001 and details relating to the
FP assay can be found in the Supporting Information section.
Cell Growth and Client Protein Degradation. Growth inhibi-
tion assays using the SRB method were undertaken as previously
described.27 Client protein degradation was determined by western
blotting. In brief, HCT116 cells were exposed to compounds 6,
19e, 30d, 33, 40f, or 17-AAG at either 1× or 2× their cellular
GI50 for 24 h. Cell lysates were prepared in 50 mM tris-HCl and
2% SDS (pH 6.8), and protein levels were determined by the BCA
method (Pierce, Rockford, IL). Equal amounts of lysate were
separated by SDS-PAGE and proteins transferred to PVDF
membrane. Her2 (ab8054, Abcam), Raf-1 (sc-133, Santa Cruz
Biotechnology), and Hsp72 (SPA-810, StressGen) were determined
using ECL (GE Bioscience). GAPDH (MAB374, Chemicon
International) levels were determined to show equal protein loading.
Pharmacokinetics and Metabolism. Cassette Dosing in
Tumor-Bearing Mice. All work was performed in accordance with
the Home Office regulations under the Animals (Scientific Proce-
dures) Act 1986 and according to the UKCCCR guidelines for
animal experimentation.58 HCT116 human colon cancer cells were
cultured in DMEM plus 5% fetal calf serum. When ∼80%
confluent, they were released from plastic flasks by a short exposure
to trypsin, washed, and re-suspended in PBS. Two million cells
were injected s.c. bilaterally in the flanks of female NCr athymic
mice. Dosing commenced when tumors were well established, 4–5
Chemistry. 1-(5-Chloro-2,4-dihydroxyphenyl)ethanone (10).
Acetic acid (17.5 mL) was added dropwise to a suspension of
4-chlorobenzene-1,3-diol (42.5 g, 0.294 mol) in BF3 ·OEt2 (200 mL,
1.62 mol) under a nitrogen atmosphere. The reaction mixture was
heated at 90 °C for 3.5 h and then allowed to cool to room
temperature, causing a solid to precipitate. The mixture was poured
into a 10% w/v aqueous sodium acetate solution (700 mL). This
mixture was then stirred vigorously for 2.5 h to afford a light-
brown solid, which was filtered, washed with water, and air-dried
overnight to afford the title compound 10 (31.6 g, 58%): TLC Rf
) 0.40 (1:3 EtOAc/hexane); LCMS tR ) 1.75 min; m/z ) 185[M
1
- H]-; H NMR (400 MHz, DMSO-d6) δ 2.70 (s, 3H), 6.62 (s,
1H), 8.03 (s, 1H), 11.45 (s, 1H), 12.35 (s, 1H); 13C ΝΜR (100.6
MHz, DMSO-d6) δ 27.0 (CH3), 103.5 (CH), 111.4 (C), 113.6 (C),
132.6 (CH), 159.9 (CH), 162.1 (C), 202.1 (C); ν ) 3257, 1608
cm-1
.
1-(2,4-Bis(benzyloxy)-5-chlorophenyl)ethanone (11). Benzyl
bromide (30 mL, 0.252 mol) was added to a mixture of 1-(5-chloro-
2,4-dihydroxyphenyl)ethanone (10) (20 g, 0.107 mol) and potassium
carbonate (37 g, 0.27 mol) in acetonitrile (350 mL). The mixture
was heated at reflux for 6 h and then allowed to cool to ambient
temperature and stirred overnight. The mixture was filtered, and
the solids were washed with dichloromethane (3 × 100 mL). The
combined organic filtrates were evaporated in vacuo to leave a pale-
yellow solid, which was triturated with a mixture of hexane (350
mL)/EtOAc (15 mL) and filtered to give the title compound 11
(35.4 g, 90% yield) as an off-white solid: TLC Rf ) 0.33 (1:4

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 209
EtOAc/hexane); LCMS tR ) 2.66 min; m/z ) 389[M + Na]+,
5-(2,4-Bis(benzyloxy)-5-chlorophenyl)-4-iodo-2H-pyrazole-
3-carboxylic Acid (15). Compound 14 (37.5 g, 63.8 mmol) was
dissolved in a mixture of 1,4-dioxane (300 mL) and aqueous sodium
hydroxide solution (2.0 M, 50 mL, 100 mmol), and the mixture
was heated to reflux for 5 h. The reaction mixture was cooled to
ambient temperature and then acidified with aqueous HCl (2.0 M).
The majority of the 1,4-dioxane was removed in vacuo and the
residual mixture diluted with more water. The resulting precipitate
was filtered and dried in vacuo, and the solid was stirred with EtOH,
filtered, and then stirred with diethyl ether and filtered again. Drying
in vacuo afforded the title compound 15 (32.7 g, 92%) as a colorless
solid: LCMS tR ) 2.60 min; m/z ) 561[M + H]+; 1H NMR (400
MHz, DMSO-d6) δ 5.17 (s, 2H), 5.30 (s, 2H), 7.16 (brs, 1H),
7.29–7.40 (m, 7H), 7.41–7.47 (m, 2H), 7.48–7.51 (m, 2H), 13.8
(brs, 1H).
1
367[M + H]+; H NMR (400 MHz, DMSO-d6) δ 2.55 (s, 3H),
5.06 (s, 2H), 5.13 (s, 2H), 6.55 (s, 1H), 7.3–7.4 (m, 10H), 7.90 (s,
1H); ν ) 3035, 1660 cm-1
.
4-(2,4-Bis(benzyloxy)-5-chlorophenyl)-2-hydroxy-4-oxobut-
2-enoic Acid Ethyl Ester (12). Sodium metal (1.35 g, 58 mmol)
was cut into small pieces, washed with hexane to remove mineral
oil, and added to anhydrous EtOH (35 mL) under a nitrogen
atmosphere over a period of 20 min. The reaction mixture was
stirred for a further 10 min until all sodium had reacted. Compound
11 (10 g, 27.26 mmol) was added in portions over 5 min, and the
resulting suspension was then stirred for a further 5 min. Diethyl
oxalate (6 mL, 43 mmol) was added, resulting in a thicker yellow-
colored precipitate. The reaction mixture was heated to reflux for
4 h, affording a dark-colored homogeneous solution, which, upon
cooling, produced a solid mass to which acetic acid (6 mL) was
added. The mixture was triturated to afford a yellow solid, which
was filtered, washed sequentially with water, EtOH, and diethyl
ether, and then dried in vacuo to afford the title compound 12 (12
g, 94%) as a yellow solid. LCMS tR ) 2.93 min; m/z ) 489[M +
Na]+, 467[M + H]+; 1H NMR (400 MHz, CDCl3) δ, 1.29 (t, 3H,
J ) 7.0 Hz), 4.28 (q, 2H, J ) 7.0 Hz), 5.11 (s, 2H), 5.17 (s, 2H),
6.58 (s, 1H), 7.33–7.42 (m, 10H), 8.01 (s, 1H), 15.34 (brs, 1H);
13C ΝΜR (100.6 MHz, CDCl3) δ 14.0 (CH3), 62.2 (CH2), 99.5
(CH), 103.0 (CH), 116.1 (C), 118.4 (C), 127.0 (CH), 127.4 (CH),
128.4 (CH), 128.5 (CH), 128.8 (CH), 128.8 (CH), 132.1 (CH), 135.2
(CH), 135.4 (CH), 158.6 (C), 158.7 (C), 162.4 (C), 168.6 (C).
5-(2,4-(Bis)benzyloxy-5-chlorophenyl)-2H-pyrazole-3-carbox-
ylic Acid Ethyl Ester (13). Method a: Compound 12 (20 g, 42.8
mmol) was added portionwise to cooled glacial acetic acid (100
mL) with mechanical stirring under nitrogen, with the temperature
maintained at ∼14 °C with an ice bath. The suspension was treated
dropwise with hydrazine hydrate (2.4 mL, 47.2 mmol). The cooling
was removed at the initiation of freezing in order to maintain
stirring, and the reaction was allowed to warm to room temperature
over 3 h during continued stirring. The resultant suspension was
poured into ice/water (500 mL), and the pale-yellow precipitate
was filtered and washed with water. The solid was recrystallized
from hot EtOH to furnish the title compound 13 (17 g, 86%).
Method b: Compound 12 (18.34 g, 39.3 mmol) was added to EtOH
(350 mL) and mechanically stirred. Hydrazine hydrochloride (3.41
g, 49.1 mmol) was added, and the mixture was then heated at reflux
for 3.75 h and then allowed to cool. The resulting solid was filtered,
and the filtered solids were washed with EtOH and then diethyl
ether and dried in vacuo to afford the title compound 13 (14.78 g,
87% yield) as a yellow solid: LCMS tR ) 2.82 min; m/z ) 463[M
5-(2,4-Bis(benzyloxy)-5-chlorophenyl)-4-iodo-2H-pyrazole-
3-carboxylic Acid Ethylamide (16). Oxalyl chloride (42 µL, 0.50
mmol) was added to a suspension of compound 13 (242 mg, 0.43
mmol) in dichloromethane (10 mL). One drop of DMF was added,
and the reaction mixture was stirred at ambient temperature for
1 h; then, the solvents were removed in vacuo to afford a yellow
residue, which was suspended in dichloromethane (5 mL) and added
dropwise to a stirred solution of ethylamine hydrochloride (350
mg, 4.3 mmol) and triethylamine (600 µL, 4.3 mmol) in dichlo-
romethane (15 mL). After 1 h, the solvents were evaporated in
vacuo and the residue dissolved in EtOAc and washed sequentially
with aqueous HCl (2N), a 5% w/v Na2CO3 solution, and a saturated
aqueous NaCl solution. The organic phase was dried over Na2SO4
and filtered; the filtrate solvents were removed in vacuo to afford
title compound 16 (250 mg, 99% yield) as an off-white solid: TLC
Rf ) 0.35 (1:1 EtOAc/hexane); LCMS tR ) 2.80 min; m/z ) 588[M
1
+ H]+; H NMR (400 MHz, CDCl3) δ 1.23 (t, 3H, J ) 7.4 Hz),
3.47 (dt, 2H, J ) 7.4, 5.5 Hz), 5.01 (s, 2H), 5.17 (s, 2H), 6.66 (s,
1H), 6.91 (t, 1H, J ) 5.5 Hz), 7.25 (m, 2H), 7.31–7.45 (m, 8H),
7.88 (s, 1H), NH not observed.
5-(2,4-Bis(benzyloxy)-5-chlorophenyl)-4-(4-formylphenyl)-2H-pyrazole-
3-carboxylic Acid Ethylamide (17). Compound 16 (235 mg, 0.4
mmol) was dissolved in THF (4 mL) and water (0.4 mL).
4-Formylphenylboronic acid (120 mg, 0.8 mmol) was added,
followed by potassium carbonate (110 mg, 0.8 mmol). The mixture
was
degassed,[1,1′-bis(diphenylphosphino)ferrocene-
]dichloropalladium(II) (5 mol%) was added, and the reaction
mixture was heated in a microwave synthesizer at 160 °C for 10
min. The solvents were removed in vacuo to afford a solid residue,
which was adsorbed onto silica gel and then purified by flash
chromatography on silica gel, eluting with a gradient of 1:2 to 1:1
EtOAc/hexane. This afforded the title compound 17 (153 mg, 68%)
as a colorless solid: LCMS tR ) 2.76 min; m/z ) 566[M + H]+;
1H NMR (400 MHz, CDCl3) δ 1.06 (t, 3H, J ) 7.0 Hz), 3.20 (dt,
2H, J ) 7.0, 6.0 Hz), 4.92 (s, 2H), 5.23 (s, 2H), 7.02 (s, 1H),
7.04–7.08 (m, 2H), 7.25 (s, 1H), 7.26–7.46 (m, 11H), 7.70 (d, 2H,
J ) 8 Hz), 8.24 (brt, 1H, J ) 6 Hz), 9.95 (s, 1H), 13.45 (s, 1H).
5-(2,4-Bis(benzyloxy)-5-chlorophenyl)-4-(3-formylphenyl)-
2H-pyrazole-3-carboxylic Acid Ethylamide (18). The title com-
pound was made by same synthetic method as that used for
compound 17. Thus, 16 was reacted with 3-formylphenylboronic
acid to afford the title compound as a colorless solid: LCMS tR )
2.78 min; m/z ) 566[M + H]+.
General Procedure A: Preparation of Amines 19e and
20a-i from 17 and 18 by Reductive Amination. The appropriate
amine (0.265 mmol, 2 equiv) was added to a solution of 17 or 18
(75 mg, 0.133 mmol, 1 equiv) in MeOH (5 mL). Molecular sieves
(Aldrich, 3 Å) were added, followed by acetic acid (12 µL, 0.2
mmol) and sodium cyanoborohydride (13 mg, 0.2 mmol, 1.5 equiv).
The reaction mixture was stirred for 2–4 h (analyzed by LCMS),
and when judged complete, the solvents were removed in vacuo,
and the residue was purified by flash chromatography on silica gel
(eluting with a gradient of dichloromethane to DMAW 120).
Alternatively, the reactions underwent an aqueous workup. The
reaction mixture was quenched with MeOH or saturated NaHCO3
solution, solvents were removed in vacuo, and the residue was
1
+ H]+; H NMR (400 MHz, DMSO-d6) δ 1.27 (t, 3H, J ) 7.1
Hz), 4.24 (q, 2H, J ) 7.1 Hz), 5.29 (s, 2H), 5.30 (s, 2H), 7.10 (s,
1H), 7.16 (s, 1H), 7.35–7.44 (m, 6H), 7.45–7.49 (m, 4H), 7.96 (s,
1H); 13C ΝΜR (100.6 MHz, DMSO-d6) δ 14.3 (CH3), 61.0 (CH2),
71.2 (CH2), 71.6 (CH2), 100.1 (C), 104.9 (CH), 111.1 (C), 116.2
(C), 127.0 (CH), 128.0 (CH), 128.3 (CH), 128.8 (CH), 128.9 (CH),
129.1 (CH), 129.2 (CH), 134.9 (C), 135.8 (C), 141.4 (C), 143.3
(C), 154.5 (C), 154.8 (C), 162.2 (C).
5-(2,4-Bis(benzyloxy)-5-chlorophenyl)-4-iodo-2H-pyrazole-
3-carboxylic Acid Ethyl Ester (14). Compound 13 (30 g, 64.8
mmol) was suspended in acetonitrile (1000 mL) and treated with
N-iodosuccinimide (16 g, 71 mmol) followed by ceric ammonium
nitrate (350 mg, 0.64 mmol), and the mixture was heated to reflux
overnight. The reaction mixture was cooled to ambient temperature,
and solvents were removed in vacuo to leave a foam-like solid.
The residual solid was triturated with water and then filtered and
washed with 1% sodium thiosulfate solution and then further water.
The solid product was dried, then stirred in EtOH, and filtered,
and the filtered solids were stirred with diethyl ether and filtered.
The resultant solid was dried in vacuo to afford title the compound
14 (37.5 g, 98%) as a colorless solid: TLC Rf ) 0.56 (1:1 EtOAc/
1
hexane); LCMS tR ) 2.66 min; m/z ) 589[M + H]+; H NMR
(400 MHz, CDCl3) δ 1.42 (t, 3H, J ) 7.1 Hz), 4.43 (q, 2H, J )
7.1 Hz), 4.99 (s, 2H), 5.16 (s, 2H), 6.66 (s, 1H), 7.22–7.26 (m,
2H), 7.30–7.45 (m, 8H), 7.84 (s, 1H), NH not observed.

210 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Brough et al.
partitioned between water and EtOAc. The organic phase was
washed with saturated aqueous NaCl solution, dried with sodium
sulfate, and evaporated in vacuo. The resulting amine products were
purified as above or used directly in the debenzylation step. The
amine product of reductive amination (0.122 mmol) was dissolved
in acetic acid (5 mL), and the solution was degassed by
evacuation-nitrogen purge (two cycles). Palladium on carbon
catalyst (10%) was added, and the reaction mixture was hydroge-
nated with shaking for 16 h. The reaction mixture was filtered
through a pad of Celite 521 and the filtrate concentrated in vacuo.
Final product purification was by preparative HPLC unless other-
wise stated.
The mixture was filtered, and filtrate solvents were removed in
vacuo to give a white solid. The solid was suspended in MeOH
(10 mL), and cyclohexadiene (1 mL) was added. The mixture was
then heated to reflux for 1 h in the presence of a catalytic amount
of palladium on carbon. The reaction mixture was filtered, and the
filtrate was concentrated to give a brown solid, which was purified
by preparative HPLC to give the title compound 19a (4 mg, 16%)
as a colorless solid: LCMS tR ) 1.63 min; m/z ) 443[M + H]+;
purity > 95%;1H NMR (400 MHz, CD3OD) δ 1.16 (t, 6H, J ) 7.0
Hz), 1.18 (t, 3H, J ) 7.0 Hz), 2.73 (q, 4H, J ) 7.0 Hz), 3.34 (m,
2 H), 3.87 (s, 2 H), 6.51 (s, 1 H), 6.80 (s, 1 H), 7.16 (d, 2H, J )
7.8 Hz), 7.3 (d, 2H, J ) 7.1 Hz).
5-(5-Chloro-2,4-dihydroxyphenyl)-4-(4-morpholin-4-ylmeth-
ylphenyl)-2H-pyrazole-3-carboxylic Acid Ethylamide (19e). By
general procedure A, morpholine was reacted with 17, and benzyl
groups were removed by catalytic hydrogenation to afford the title
compound 19e (11%) as a colorless solid: LCMS tR ) 1.69 min;
5-(2,4-Bis(benzyloxy)-5-chlorophenyl)isoxazole-3-
carboxylic Acid Ethyl Ester (23). 4-(2,4-Bis(benzyloxy)-5-chlo-
rophenyl)-2-hydroxy-4-oxobut-2-enoic acid ethyl ester (12) (12.0
g, 25.7 mmol) was suspended in EtOH (230 mL), and hydroxy-
lamine hydrochloride (2.23 g, 1.25 equiv) was added. The reaction
mixture was heated to reflux for 3.5 h and then allowed to cool to
ambient temperature. The resulting suspension was filtered, washed
sequentially with EtOH (2 × 20 mL), water (2 × 20 mL), and
EtOH (2 × 20 mL), and dried in vacuo to afford the title compound
23 (10.23 g, 86%) as a flocculent light-yellow solid: TLC Rf )
0.41 (20:80 EtOAc/hexane); LCMS tR ) 2.94 min; m/z ) 466,
1
m/z ) 457[M + H]+; H NMR (400 MHz, DMSO-d6) δ 1.05 (t,
3H, J ) 7.4 Hz), 2.34 (brm, 4H), 3.18 (m, 2H), 3.41 (brs, 2H),
3.56 (brm, 4H), 6.56 (s, 1H), 6.71 (s, 1H), 7.13 (d, 2H, J ) 8.4
Hz), 7.16 (d, 2H, J ) 8.4 Hz), 8.00 (brs, 1H); 13C NMR (100.6
MHz, DMSO-d6) δ 14.9 (CH3), 33.2 (CH2), 53.2 (CH2), 62.3 (CH2),
66.2 (CH2), 103.7 (CH), 108.7 (C), 109.5 (C), 119.2 (C), 128.2
(CH), 129.8 (CH), 131.2 (CH), 131.8 (C), 135.4 (C), 137.5 (C),
143.4 (C), 153.9 (C), 155.3 (C), 162.4 (C); HRMS calcd for
C23H25ClN4O4[M + H]+ 457.1642, found 457.1626; HPLC 98.7%
(tR ) 2.87 min).
1
464[M + H]+, chlorine isotope pattern observed; H NMR (400
MHz, CDCl3) δ 1.40 (s, 3H, J ) 7.1 Hz), 4.42 (q, 2H, J ) 7.1
Hz), 5.13 (s, 2H), 5.15 (s, 2H), 6.62 (s, 1H), 7.01 (s, 1H), 7.30–7.43
(m, 10H), 8.80 (s, 1H); 13C ΝΜR (100.6 MHz, CDCl3) δ 14.1,
62.0, 71.0, 71.1, 99.8, 103.0, 109.8, 115.7, 127.0, 127.3, 128.3,
128.6, 128.6, 128.8, 128.9, 135.5, 135.7, 155.0, 156.3, 157.0, 160.2,
166.6.
5-(2,4-Bis(benzyloxy)-5-chlorophenyl)isoxazole-3-carboxylic
Acid Ethylamide (24). Compound 23 (10.0 g, 21.5 mmol) was
suspended in EtOH (120 mL), and ethylamine in MeOH solution
(2.0 M, 70 mL, 140 mmol) was added. The reaction mixture was
heated to 80 °C with stirring for 18 h, affording a yellow
homogeneous solution, which was allowed to cool to ambient
temperature. A flocculent colorless solid formed upon cooling, and
the reaction mixture was cooled further to 4 °C and then filtered.
The filtered product was washed with cold EtOH and dried in vacuo
to afford the title compound 24 (8.86 g, 89%) as a colorless solid:
TLC Rf ) 0.22 (20:80 EtOAc/hexane); LCMS tR ) 2.86 min; m/z
) 465, 463[M + H]+; 1H NMR (400 MHz, CDCl3) δ 1.25 (s, 3H,
J ) 7.4 Hz), 3.47 (dq, 2H, J ) 7.3, 5.2 Hz), 5.10 (s, 2H), 5.16 (s,
2H), 6.59 (s, 1H), 6.82 (brt, 1H, J ) 5.2 Hz) 7.08 (s, 1H), 7.30–7.43
(m, 10H), 7.96 (s, 1H).
{4-[3-(2,4-Bis(benzyloxy)-5-chlorophenyl)-5-ethylcarbamoyl-
1H-pyrazol-4-yl]benzyl}carbamic Acid tert-Butyl Ester (21).
4-Aminomethylphenylboronic acid hydrochloride (80 mg, 0.43
mmol) was added to 5-(2,4-bis(benzyloxy)-5-chlorophenyl)-4-iodo-
2H-pyrazole-3-carboxylic acid ethylamide (16) (200 mg, 0.34
mmol) and potassium carbonate (140 mg, 1.01 mmol) in a
microwave vial. THF (3 mL) and water (0.3 mL) were added, and
the mixture was degassed by bubbling nitrogen through the reaction
mixture. 1,1-Bis(diphenylphosphino)ferrocenedichloropalladium was
added (10 mol%), and the reaction mixture was heated in a
microwave synthesizer at 150 °C for 10 min. The reaction mixture
after cooling was filtered through a pad of Celite 521, and the
solvents were removed in vacuo. The residue was dissolved in THF
(5 mL), and triethylamine (142 µL) was added followed by di-
tert-butyldicarbonate (222 mg, 1.02 mmol). The reaction mixture
was heated for 10 min at 100 °C and then allowed to cool and was
partitioned between EtOAc and water. The organic phase was dried
over Na2SO4, evaporated in vacuo, and purified by flash chroma-
tography on silica gel, eluting 15–75% gradient of EtOAc in hexane,
to afford title compound 21 (60 mg, 26%) as a glass-like solid:
5-(2,4-Bis(benzyloxy)-5-chlorophenyl)-4-bromoisoxazole-3-
carboxylic Acid Ethylamide (25). N-bromosuccinimide (1.90 g,
10.7 mmol) was added to a suspension of 24 (4.50 g, 9.73 mmol)
in acetonitrile (120 mL). Ceric ammonium nitrate (2.66 g, 4.86
mmol) was added, and the resulting orange suspension was heated
to reflux for 4.5 h. The mixture was allowed to cool to room
temperature, and the acetonitrile was removed in vacuo. The residue
was partitioned between EtOAc (120 mL) and water (100 mL),
the phases were then separated, and the organic phase was washed
with water (50 mL) and saturated aqueous NaCl solution (100 mL)
and dried over MgSO4. The mixture was filtered, and the filtrate
solvents were removed in vacuo to afford an orange solid, which
was triturated with diethyl ether, filtered, and dried in vacuo to
give the title compound 25 (3.90 g, 74%) as a cream-colored solid:
Rf ) 0.22 (30:70 EtOAc/hexane); LCMS tR ) 2.93 min; m/z )
1
LCMS tR ) 2.8 min; m/z ) 667[M + H]+; H NMR (400 MHz,
CDCl3) δ 1.23 (t, 3H, J ) 7.2 Hz), 1.47 (s, 9H), 3.36 (m, 2H),
4.34 (brd, 2H, J ) 5.8 Hz), 4.85 (brs, 1H), 4.94 (s, 2H), 5.07 (s,
2H), 6.56 (s, 1H), 6.6 (brs, 1H), 7.06 (s, 1H), 7.15–7.30 (m, 8H),
7.31–7.43 (m, 6H), pyrazole NH not observed.
4-(4-Aminomethylphenyl)-5-(2,4-bis-benzyloxy-5-chloro-
phenyl)-2H-pyrazole-3-carboxylic Acid Ethylamide (22). Com-
pound 21 (0.191 g, 0.0286 mmol) was dissolved in dichoromethane
(5 mL), trifluoroacetic acid (5 mL) was added, and the mixture
was stirred at room temperature for 1 h. The mixture was then
concentrated to give a yellow oil, which was partitioned between
EtOAc and saturated aqueous NaHCO3 solution. The organic phase
was dried over Na2SO4 and concentrated to give a yellow oil, which
was triturated with hexane/EtOAc mixture to afford the title
compound 22 (0.154 g, 95%) as a cream-colored powder: LCMS
tR ) 2.11 min; m/z ) 568[M + H]+.
5-(5-Chloro-2,4-dihydroxyphenyl)-4-(4-diethylaminomethyl-
phenyl)-2H-pyrazole-3-carboxylic Acid Ethylamide (19a). Com-
pound 22 (0.03 g, 0.056mMol) was dissolved in dichloroethane
(10 mL). To this mixture were added sodium triacetoxyborohydride
(0.034 mg, 0.16 mmol) and acetaldehyde (0.07 mL, 0.16 mmol).
The mixture was stirred at room temperature for 1 h, and then,
sodium borohydride (0.01 g, 0.26 mmol) in EtOH (1 mL) was
added, and the mixture was stirred at room temperature for 1 h.
1
543, 541[M + H]+, chlorine isotope pattern observed; H NMR
(400 MHz, CDCl3) δ 1.26 (t, 3H); 3.50 (m, 2H); 5.07 (s, 2H); 5.12
(s, 2H); 6.62 (s, 1H); 6.74 (t, 1H), 7.28–7.43 (m, 10H); 7.52 (s,
1H).
5-(2,4-Bis(benzyloxy)-5-chlorophenyl)-4-(4-formylphenyl)-
isoxazole-3-carboxylic Acid Ethylamide (26). 4-Formylphenyl-
boronic acid (1.52 g, 10.2 mmol) was added to a mixture of 25
(4.60 g, 8.5 mmol) and NaHCO3 (2.14 g, 25.5 mmol). DMF (105
mL) was added followed by water (21 mL), and the resulting
suspension was degassed by evacuation-nitrogen purge (three
cycles) and then bubbling nitrogen gas through the reaction mix

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 211
for 10 min. Dichlorobis(triphenylphosphine)palladium(II) (5 mol%)
was added, and the reaction mixture was heated under nitrogen
atmosphere at 80 °C for 3 h. The mixture was allowed to cool, and
the majority of the solvents was removed in vacuo. The residual
mixture was partitioned between EtOAc (200 mL) and water (200
mL), and this mixture was then filtered through a pad of Celite
521. The filtrate phases were separated, and the organic phase was
washed with water (2 × 200 mL) and saturated aqueous sodium
chloride solution (250 mL) and then dried over anhydrous Na2SO4
and filtered. The filtrate solvents were evaporated in vacuo, and
the crude was product purified by trituration with diethyl ether to
afford the title compound 26 (3.51 g, 73%) as a yellow solid: LCMS
tR ) 2.91 min; m/z ) 567[M + H]+, chlorine isotope pattern
observed; 1H NMR (400 MHz, CDCl3) δ 1.23 (t, 3H, J ) 7.3 Hz),
3.44 (m, 2H), 4.59 (s, 2H), 5.05 (s, 2H), 6.44 (s, 1H), 6.81 (brt,
1H, J ) 5.3 Hz), 6.88 (m, 2H), 7.21–7.30 (m, 3H), 7.31–7.41 (7H),
7.48 (s, 1H), 7.69 (d, 2H, J ) 8.4 Hz), 9.96 (s, 1H).
5-(5-Chloro-2,4-dihydroxyphenyl)-4-[4-(1,1-dioxo-1-thiomor-
pholin-4-ylmethyl)phenyl]isoxazole-3-carboxylic Acid Ethyl-
amide (30a). Triethylamine (800 µL, 76 mmol) was added to a
solution of 4-aminomethyl phenylboronic acid (710 mg, 3.8 mmol)
in EtOH (3 mL). Divinylsulfone (380 µL, 3.8 mmol) was added,
and the mixture was then heated at 100 °C for 3 h. The EtOH was
removed in vacuo and the residue triturated with a small quantity
of water, filtered, and dried in vacuo to afford the crude boronic
acid as a solid. To the boronic acid (60 mg, 0.19 mmol) was added
25 (110 mg, 0.19 mmol), followed by DME (3 mL) and saturated
NaHCO3(aq) solution (1 mL). The mixture was degassed and
tetrakis(triphenylphosphine)palladium(0) (5 mol%) was added, and
the mixture was heated in a microwave synthesizer at 130 °C for
10 min. The reaction mixture was partitioned between dichlo-
romethane and water, and the phases were separated. The organic
phase was dried over sodium sulfate and filtered, and the filtrate
solvents were removed in vacuo to afford the crude dibenzylated
product, which was then dissolved in dichloromethane (5 mL), and
boron trichloride in dichloromethane solution (2.0 M, 5 mL, 10
mmol) was added and the mixture stirred at room temperature for
10 min. After this time, water (10 mL) was added, the mixture
was concentrated, and the residue was purified by preparative HPLC
to afford the title compound 30a (0.016 g, 17%) as a colorless solid:
LCMS tR ) 2.09 min; m/z ) 528[M + Na]+, 506[M + H]+,
chlorine isotope pattern observed; 1H NMR (400 MHz, CD3OD) δ
1.23 (t, 3H, J ) 7.3 Hz), 3.01 (m, 4H), 3.13 (m, 4H), 3.39 (q, 2H,
J ) 7.3 Hz), 3.71 (s, 2H), 6.51 (s, 1H), 7.07 (s, 1H), 7.30 (d, 2H,
J ) 8.6 Hz), 7.35 (d, 2H, J ) 8.6 Hz); 13C ΝΜR (100.6 MHz,
DMSO-d6) δ 14.3 (CH3), 33.7 (CH2), 50.2 (CH2), 50.2 (CH2), 59.1
(CH2), 103.8 (CH), 106.2 (C), 110.2 (C), 115.8 (C), 128.1 (C),
128.4 (CH), 128.6 (CH), 130.8 (CH), 137.1 (C), 155.8 (C), 155.8
(C), 157.7 (C), 159.5 (C), 164.2 (C); HRMS calcd for
C23H24ClN3O6S[M + H]+ 506.1152, found 506.1162; HPLC 97.3%
(tR ) 3.04 min).
DME (3 mL) and saturated NaHCO3(aq) solution (1 mL). The
mixture was degassed, tetrakis(triphenylphosphine)palladium(0) (5
mol%) was added, and the mixture was heated in a microwave
synthesizer at 130 °C for 10 min. The reaction mixture was
partitioned between DCM and water, and the phases were separated.
The organic phase was dried over Na2SO4 and filtered, and the
filtrate solvents were removed in vacuo to afford the crude
dibenzylated product, which was then dissolved in DCM (5 mL);
boron trichloride in DCM solution (1.0 M, 2 mL) was added, and
the mixture was stirred at room temperature for 10 min. Water (10
mL) was added, the mixture was concentrated, and the residue was
purified by preparative HPLC to give the title compound 30c (0.5
formate salt) as a colorless solid: LCMS tR ) 1.75 min; m/z )
1
473, 471[M + H]+, chlorine isotope pattern observed; H NMR
(400 MHz, CD3OD) δ 1.23 (t, 3H, J ) 7.4 Hz), 2.67 (s, 3H), 2.69
(brs, 4H), 3.00 (brs, 4H), 3.40 (q, 2H, J ) 7.4 Hz), 6.51 (s, 1H),
7.06 (s, 1H), 7.30 (d, 2H, J ) 8.6 Hz), 7.34 (d, 2H, J ) 8.6 Hz),
8.54 (s, 0.5H, formate); HRMS calcd for C24H27ClN4O4[M + H]+
471.1799, found 471.1792; HPLC 98.3% (tR ) 1.46 min).
5-(5-Chloro-2,4-dihydroxyphenyl)-4-(4-diethylaminomethyl-
phenyl)isoxazole-3-carboxylic Acid Ethylamide (30f). AcOH
(0.51 mL, 8.85 mmol) was added dropwise to a mixture of 26 (1.0
g, 1.77 mmol), diethylamine (0.55 mL, 5.3 mmol), NaCNBH3 (222
mg, 3.5 mmol), CH2Cl2 (30 mL), and 3 Å molecular sieves (1 g).
The reaction mixture was left to stir at RT under a nitrogen
atmosphere for 16 h. The mixture was then filtered through a
sintered funnel, and the filtrate was washed with saturated NaHCO3
solution (40 mL). The aqueous phase was extracted further with
CH2Cl2 (2 × 40 mL). The combined organics were dried over
Na2SO4 and evaporated in vacuo to give a crude product, which
was purified by flash chromatography on silica gel, eluting with a
gradient of 50% EtOAc/hexane - 100% EtOAc, to afford 5-(2,4-
bis(benzyloxy)-5-chlorophenyl)-4-(4-diethylaminomethylphenyl-
)isoxazole-3-carboxylic acid ethylamide (0.65 g, 59%) as a colorless
oil: LCMS tR ) 2.43 min; m/z ) 624, 626. To this product (55
mg, 0.09 mmol) in CH2Cl2 (1 mL) cooled to 0 °C under N2 was
added BCl3 (1.0 M in CH2Cl2, 0.27 mL, 0.26 mmol). The reaction
was allowed to warm to RT and was stirred for 1.5 h. Saturated
NaHCO3 solution (5 mL) was added and the CH2Cl2 removed in
vacuo. The aqueous residue was extracted with EtOAc (4 × 10
mL), passed through a hydrophobic frit, and evaporated in vacuo.
Flash chromatography on silica gel, eluting with 10% MeOH/
CH2Cl2 then 20% MeOH/CH2Cl2, afforded the title compound 30f
(0.018 g, 45%) as a yellow solid: LCMS tR ) 1.78 min; m/z )
444, 446[M + H]+; 1H NMR (400 MHz, DMSO-d6) δ 0.97 (t,
6H, J ) 7.0 Hz), 1.07 (t, 3H, J ) 7.0 Hz), 2.41–2.54 (m, 4H),
3.19–3.28 (m, 2H), 3.52 (brs, 2H), 6.61 (s, 1H), 7.07 (s, 1H), 7.18
(d, 2H, J ) 8.0 Hz), 7.25 (d, 2H, J ) 8.0 Hz), 8.89 (t, 1H, J ) 8.0
Hz), 10.15 (s, 1H), 10.67 (brs, 1H); 13C ΝΜR (100.6 MHz, DMSO-
d6) δ 11.4 (CH3), 14.3 (CH3), 33.8 (CH2), 46.12 (CH2), 56.4, 104.0
(CH), 106.2 (C), 110.2 (C), 115.8 (C), 127.7 (C), 128.4 (CH), 128.5
(CH), 130.8 (CH), 138.6 (C), 155.9 (C), 156.0 (C), 157.8 (C), 159.6
(C), 164.3 (C); HRMS calcd for C23H26ClN3O4[M + H]+ 444.1690,
found 444.1693; HPLC 96.4% (tR ) 2.31 min).
1-(2,4-Bis(benzyloxyphenyl)ethanone (47). Potassium carbonate
(90.80 g, 0.657 mol) was added to a solution of 2,4-dihydroxy-
acetophenone 46 (40.00 g, 0.263 mol) in acetonitrile (400 mL),
and the suspension was stirred at room temperature. Benzyl bromide
(78 mL, 112.00 g, 0.657 mol) was added dropwise over 10 min
and the mixture heated at reflux for 18 h. The mixture was cooled,
the solvents were evaporated in vacuo to afford a slurry, which
was partitioned between water (1 L) and EtOAc (1 L), and the
phases were separated. The aqueous layer was further extracted
with dichloromethane (250 mL), and the organic extracts were
combined, dried over MgSO4, and evaporated in vacuo. The product
was triturated with hexane, filtered, washed with cold hexane, and
dried in vacuo at 45 °C to give the title compound 47 (84.69 g,
97%) as a white powder: LCMS tR ) 2.87 min; m/z ) 333[M +
H]+; 1H NMR (400 MHz, CDCl3) δ 2.55 (s 3H), 5.08 (s, 2H),
5.11 (s, 2H), 6.63–6.60 (m, 2H), 7.44–7.35 (m, 10H), 7.85 (d, 1H,
J ) 9.3 Hz).
5-(5-Chloro-2,4-dihydroxyphenyl)-4-[4-(4-methylpiperazin-
1-ylmethyl)phenyl]isoxazole-3-carboxylic Acid Ethylamide (30c).
Methyl iodide (250 µL, 4.6 mmol) was added to a mixture of 1-(4-
bromobenzyl)piperazine (1.0 g, 4.15 mmol) and potassium carbon-
ate (1.8 g, 12.45 mmol) in DMF (15 mL) and stirred overnight.
The reaction mixture was partitioned between EtOAc and water,
the phases were separated, the organic phase was dried over MgSO4,
and the filtrate solvents were evaporated in vacuo. The resulting
product (750 mg, 6.5 mmol) was dissolved in DMSO (15 mL),
and potassium carbonate (900 mg, 3.0 equiv) was added followed
by bis(pinnacolato)diboron (1.1 g, 4.5 mmol). The mixture was
degassed, 1,1′-bis(diphenylphosphinoferrocene)dichloropalladium(II)
(5 mol%) was added, and the reaction mixture was heated at 85
°C for 3 h under a nitrogen atmosphere; another 1.0 equiv of
bis(pinnacolato)diboron was added, and heating continued for 3 h.
The reaction mixture was allowed to cool and was worked up as
above. The crude product was purified by flash chromatography
on silica gel (eluting with 0–8% gradient of MeOH in DCM) to
afford the product as a colorless solid. To the boronate ester (82.5
mg (0.26 mmol) were added 27 (106 mg, 0.18 mmol) followed by

212 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Brough et al.
1-(2,4-Bis(benzyloxy)-5-bromophenyl)ethanone (48). 1-(2,4-
Bis(benzyloxyphenyl)ethanone (47) (84.60 g, 0.255 mol) was
dissolved in DMF (250 mL) and stirred at room temperature.
N-Bromosuccinimide (45.30 g, 0.255 mol) was added portionwise
over 10 min and stirred for 3 h, over which time a white precipitate
formed. The suspension was poured into water (1 L), and the
product was removed by filtration and dried in vacuo at 45 °C to
give the title compound 48 (101.37 g, 97%) as a white powder:
LCMS tR ) 2.97 min; m/z ) 411.2, 413.2[M + H]+, bromine
partitioned between dichloromethane (200 mL) and water (200 mL),
and this mixture was then filtered through a pad of Celite 521. The
filtrate phases were separated, and the organic phase was washed
with water (2 × 50 mL) and saturated aqueous NaCl solution (100
mL) and then dried over anhydrous Na2SO4 and filtered. The filtrate
solvents were evaporated in vacuo, and the crude product was
purified by trituration with a hexane/EtOAc mixture (9:1) to afford
the title compound 34a (808 mg, 71%) as a light-brown solid: TLC
Rf ) 0.40 (30:70 EtOAc/hexane); LCMS tR ) 3.13 min; m/z )
531[M + H]+; 1H NMR (400 MHz, CDCl3) δ 1.27 (t, 3H, J ) 7.3
Hz), 3.50 (dq, 2H, J ) 7.3, 5.4 Hz), 5.11 (s, 2H), 5.19 (s, 2H),
6.57 (s, 1H), 6.83 (brt, 1H, J ) 5.5 Hz), 7.10 (s, 1H), 7.17 (d, 1H,
J ) 16.5 Hz), 7.23 (m, 1H), 7.31–7.45 (m, 13H), 7.48–7.52 (m,
2H), 8.21 (s, 1H).
1
isotope pattern observed; H NMR (400 MHz, CDCl3) δ 2.52 (s,
3H), 5.07 (s, 2H), 5.14 (s, 2H), 6.52 (s, 1H), 7.42–7.33 (m, 10H),
8.06 (s, 1H).
4-(5-Bromo-2,4-diphenoxyphenyl)-2-hydroxy-4-oxobut-2-
enoic Acid Ethyl Ester (49). Sodium (8.38 g, 364.71 mmol) was
added portionwise to EtOH (350 mL) over 1 h with mechanical
stirring under nitrogen, and the resulting solution was stirred at
room temperature for 18 h. 1-(2,4-Bis(benzyloxy)-5-bromophen-
yl)ethanone (48) (49.31 g, 119.9 mmol) was added over 5 min
followed by diethyl oxalate (24.77 mL, 26.65 g, 182.4 mmol). The
suspension became pale-yellow in color. The mixture was warmed
to reflux, whereupon the solids dissolved and a dark orange solution
formed. The mixture was heated under reflux for 2 h and then
cooled to 65 °C. Glacial acetic acid (30 mL) was added, and the
hot solution was poured into 800 mL of cold water. The suspension
was stirred while cooling, and the cooled suspension was filtered,
washed with water (500 mL), and dried in vacuo at 45 °C to give
the title compound 49 (59.59 g, 96%) as a golden-yellow powder:
LCMS tR ) 3.12 min; m/z ) 511.2, 513.2[M + H]+, bromine
5-(2,4-Bis(benzyloxy)-5-phenethylphenyl)isoxazole-3-carbox-
ylic Acid Ethylamide (34b). Compound 34a (690 mg, 1.30 mmol)
was dissolved in 1,4-dioxane, and 10% palladium on carbon catalyst
(50 mg) was added. The reaction mixture was hydrogenated at
atmospheric pressure for a total of 4 h and 45 min, with further
catalyst (50 mg) added at 1.5 and 4.0 h. The reaction mixture was
filtered through a pad of Celite 521, and the filter cake was washed
with 1,4 dioxane and dichloromethane. The combined filtrate
solvents were removed in vacuo to leave a cream-colored solid,
which was adsorbed onto silica gel and purified by flash chroma-
tography on silica gel (20 g), eluting with a gradient of 10–50%
EtOAc in hexane. This afforded the title compound 34a (609 mg,
88%) as a pale-yellow solid: TLC Rf ) 0.43 (30:70 EtOAc/hexane);
LCMS tR ) 3.12 min; m/z ) 533[M + H]+; 1H NMR (400 MHz,
DMSO-d6) δ 1.09 (t, 3H, J ) 7.1 Hz), 2.79–2.89 (m, 4H), 3.25
(dq, 2H, J ) 7.1, 5.8 Hz), 5.23 (s, 2H), 5.35 (s, 2H), 6.85 (s, 1H),
7.08 (s, 1H), 7.13–7.19 (m, 2H), 7.21–7.27 (m, 2H), 7.34–7.46 (m,
6H), 7.47–7.53 (m, 4H), 7.66 (s, 1H), 8.75 (brt, 1H, J ) 5.8 Hz).
5-(2,4-Bis(benzyloxy)-5-phenethylphenyl)-4-bromoisoxazole-
3-carboxylic Acid Ethylamide (35b). N-bromosuccinimide (207
mg, 1.16 mmol) was added to a stirred suspension of 34b (564
mg, 1.06 mmol) in acetonitrile (230 mL). Ceric ammonium nitrate
(290 mg, 0.53 mmol) was added, and the mixture was heated to
reflux for 30 min. Acetonitrile was removed in vacuo, and the
residue was partitioned between EtOAc (50 mL) and water (50
mL). The organic phase was washed with saturated aqueous NaCl
solution and dried over Na2SO4 and filtered. The filtrate solvents
were removed in vacuo to afford a yellow oil, which was purified
by flash chromatography on silica gel (20 g), eluting with a gradient
of 10–30% EtOAc in hexane. This afforded the title compound
35b (326 mg, 53%) as a colorless solid: LCMS tR ) 3.09 min; m/z
1
isotope pattern observed; H NMR (400 MHz, CDCl3) δ 1.27 (t,
3H, J ) 7.1 Hz), 4.27 (q, 2H, J ) 7.1 Hz), 5.11 (s, 2H), 5.16 (s,
2H), 6.54 (s, 1H), 7.42–7.34 (m, 11H), 8.17 (s, 1H), 13.5–13.0
(brs, 1H).
5-(2,4-Bis(benzyloxy)-5-bromophenyl)isoxazole-3-carboxylic
Acid Ethyl Ester (50). Hydroxylamine hydrochloride (1.41 g, 20.4
mmol) was added to a solution of 49 (8.69 g, 17 mmol) in EtOH
(170 mL), and the mixture was heated at reflux for 1 h and 10
min. The reaction mixture was allowed to cool to room temperature
and was then filtered; the filtered solids were washed with water
(2 × 30 mL) and EtOH (2 × 15 mL) and dried in vacuo to afford
the title compound 50 (7.98 g, 92%) as a flocculent yellow solid:
TLC Rf ) 0.63 (30:70 EtOAc/hexane); LCMS tR ) 3.11 min; m/z
) 508, 510[M + H]+, bromine isotope pattern observed; 1H NMR
(400 MHz, CDCl3) δ 1.40 (t, 3H, J ) 7.2 Hz), 4.42 (q, 2H, J )
7.2 Hz), 5.13 (s, 2H), 5.15 (s, 2H), 6.58 (s, 1H), 7.00 (s, 1H),
7.32–7.44 (m, 10H), 8.15 (s, 1H); 13C ΝΜR (100.62 MHz, CDCl3)
δ 14.1, 62.0, 71.0, 71.0, 99.5, 102.9, 103.8, 110.4, 126.9, 127.3,
128.3, 128.6, 128.7, 128.9, 131.5, 135.4, 135.6, 155.7, 156.9, 157.3,
160.2.
1
) 613, 611[M + H]+; H NMR (400 MHz, DMSO-d6) δ 1.11 (t,
3H, J ) 7.3 Hz), 2.82 (m, 4H), 3.27 (q, 2H, J ) 7.3 Hz), 5.24 (s,
2H), 5.25 (s, 1H), 7.09 (s, 1H), 7.12–7.10 (m, 3H), 7.22–7.27 (m,
2H), 7.29 (s, 1H), 7.30–7.50 (m, 10H), 8.93 (t, 1H, J ) 5.8 Hz);
13C ΝΜR (100.62 MHz, CDCl3) δ 14.4, 31.2, 33.7, 35.5, 69.8,
70.1, 90.9, 99.0, 99.9, 106.0, 122.3, 127.9, 128.1, 128.3, 128.3,
128.5, 128.6, 131.3, 136.5, 136.7, 141.6, 156.1, 158.0, 157.6, 159.6,
166.8.
5-(2,4-Bis(benzyloxy)-5-bromophenyl)isoxazole-3-carboxylic
Acid Ethylamide (51). Ethylamine in MeOH solution (2.0M, 65
mL, 130 mmol) was added to a suspension of 50 (7.89 g, 15.5
mmol) in EtOH (80 mL), and the mixture was heated to reflux for
18 h, affording a homogeneous solution which was allowed to cool
to ambient temperature. A colorless precipitate formed, which was
filtered, washed with EtOH, and dried in vacuo to afford the title
compound 51 (6.31 g, 80%) as a colorless solid: TLC Rf ) 0.39
(30:70 EtOAc/hexane); LCMS tR ) 2.98 min; m/z ) 509, 507[M
5-(2,4-Dihydroxy-5-phenethylphenyl)-4-(4-morpholin-4-yl-
methylphenyl)isoxazole-3-carboxylic Acid Ethylamide (40b).
This compound was made using the procedure described for 30c,
utilizing 5-(2,4-bis(benzyloxy)-5-phenethylphenyl)-4-bromoisox-
azole-3-carboxylic acid ethylamide (35b) and 4-[4-(4,4,5,5-tetram-
ethyl-[1,3,2]dioxaborolan-2-yl)benzyl]morpholine. Thus, 35b (374
mg, 0.576 mmol) was reacted with boronate ester (215 mg, 0.72
mmol), and the resulting crude product after workup was purified
by flash chromatography on silica gel, eluting with a gradient of
50–100% EtOAc in hexane. Deprotection with BCl3, as described
previously, gave a crude product, which was purified by flash
chromatography on silica gel, eluting with 10% MeOH in EtOAc,
to afford title compound 40b as a light-brown oil. The HCl salt
was made by trituration with 1.0 M HCl in ether solution, affording
the title compound 40b·HCl (19 mg, 6%) as a light-brown solid:
1
+ H]+, bromine isotope pattern observed; H NMR (400 MHz,
CDCl3) δ 1.25 (t, 3H, J ) 7.3 Hz), 3.48 (dt, 2H, J ) 7.3, 5.3 Hz),
5.10 (s, 2H), 5.16 (s, 2H), 6.56 (s, 1H), 6.82 (brt, 1H, J ) 5.4 Hz),
7.08 (s, 1H) 7.30–7.42 (m, 10H), 8.12 (s, 1H).
5-(2,4-Bis(benzyloxy)-5-styrylphenyl)isoxazole-3-carboxylic
Acid Ethylamide (34a). trans-2-Phenylvinylboronic acid (472 mg,
3.2 mmol) was added to 51 (1.079 g, 2.13 mmol) and NaHCO3
(536 mg, 6.39 mmol). Water (5 mL) and DMF (25 mL) were added,
and the mixture was degassed by evacuation-nitrogen purge
followed by bubbling nitrogen gas through the reaction mixture
for 5 min. Dichlorobis(triphenylphosphine)palladium(II) chloride
(149 mg, 5 mol%) was added, and the reaction mixture was heated
at 80 °C for 7 h. The mixture was allowed to cool, and the majority
of solvents was removed in vacuo. The residual mixture was
1
LCMS tR ) 2.02 min; m/z ) 528[M + H]+; purity 90–95%; H
NMR (400 MHz, DMSO-d6) δ 1.11 (t, 3H, J ) 6.9 Hz), 2.60–2.70
(m, 4H), 3.00–3.30 (m, 4H), 3.69 (m, 2H), 3.87 (m, 2H), 4.3 (m,

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 213
2H), 6.45 (s, 1H), 6.84 (s, 1H), 7.11–7.32 (m, 6H), 7.46–7.65 (m,
3H), 8.93 (brt, 1H), 9.67 (s, 1H), 9.90 (s, 1H), 10.73 (brs, 1H);
HRMS calcd for C31H33N3O5[M + H]+ 528.2498, found 528.2486.
1-(2,4-Dihydroxyphenyl)-2-methylpropane-1-one (53). Resor-
cinol (2.42 g, 22 mmol) was taken up in BF3 ·OEt2 (15 mL), and
isobutyric acid (2.04 mL, 22 mmol) was added. The solution was
heated for 1.5 h at 90 °C and then allowed to cool to room
temperature. The solution was added dropwise to 10% NaOAc(aq)
(100 mL) and allowed to stand for 4 h before being extracted in
EtOAc. The organic phases were combined and washed with
saturated NaHCO3(aq), dried over anhydrous MgSO4, filtered, and
concentrated in vacuo to give the title compound 53 (3.96 g, 100%)
as a red oil, which was used without additional purification: LCMS
tR ) 2.28 min; m/z ) 181[M + H]+; 1H NMR (400 MHz, CDCl3)
δ 1.18 (d, 6H, J ) 6.8 Hz), 3.49 (m, 1H), 6.38 (m, 1H), 7.24 (s,
1H), 7.64 (d, 1H, J ) 9.6 Hz), 13.06 (s, 1H).
(d, 6H, J ) 6.6 Hz), 1.38 (t, 3H, J ) 7.1 Hz), 1.91 (m, 1H), 2.51
(d, 2H, J ) 7.1 Hz), 4.40 (q, 2H, J ) 7.1 Hz), 5.03 (s, 2H), 5.13
(s, 2H), 6.54 (s, 1H), 6.97 (s, 1H), 7.37 (m, 10 H), 7.71 (s, 1H).
5-(2,4-Bis(benzyloxy)-5-isobutylphenyl)isoxazole-3-carboxyl-
ic Acid Ethyl Amide (34e). Compound 57 (0.73 g, 1.52 mmol)
was dissolved in ethylamine in MeOH solution (2.0 M, 7.6 mL,
15.2 mmol) and heated in a Smith microwave synthesizer at 120
°C for 10 min. The solution was concentrated in vacuo to give the
title compound 34e (0.73 g, 100%) as a colorless solid, which was
used without additional purification: LCMS tR ) 3.11 min; m/z )
485.3[M + H]+; 1H NMR (400 MHz, CDCl3) δ 0.89 (d, 6H, J )
6.6 Hz), 1.23 (t, 3H, J ) 7.1 Hz), 1.92 (m, 1H), 2.49 (d, 2H, J )
7.3 Hz), 3.46 (dt, 2H, J ) 5.7 Hz, 7.1 Hz), 5.00 (s, 2H), 5.14 (s,
2H), 6.52 (s, 1H), 6.79 (1H, bt, J ) 5.7 Hz), 7.04 (s, 1H), 7.35 (m,
10 H), 7.67 (s, 1H).
5-(2,4-Bis(benzyloxy)-5-isobutylphenyl)-4-iodoisoxazole-3-
carboxylic Acid Ethyl Amide (35e). Compound 34e (0.63 g, 1.30
mmol) and N-iodosuccinimide (0.58 g, 2.6 mmol) were dissolved
in acetonitrile (50 mL), ceric ammonium nitrate (0.07 g, 0.13 mmol)
was added, and the solution was stirred at room temperature
overnight. The reaction mixture was concentrated in vacuo, and
the resulting gum was partitioned between EtOAc and saturated
aqueous NaCl solution. The organic phase was dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel, eluting with 4:1 hexane/
EtOAc, to give the title compound 35e (0.43 g, 54%) as an oil:
LCMS tR ) 3.09 min; m/z ) 611.2[M + H]+.
5-(2,4-Bis(benzyloxy-5-isobutylphenyl)-4-(4-formylphenyl)-
isoxazole-3-carboxylic Acid Ethyl Amide (36e). This compound
was made using the procedure described for compound 32. Thus,
compound 35e (0.42 g, 0.69 mmol) was reacted with 4-formylphen-
ylboronic acid (0.21 g, 1.38 mmol) to afford a crude product after
workup, which was purified by flash chromatography on silica gel,
eluting with 10% EtOAc in hexane, to afford the title compound
36e (4.85 g, 58%) as a yellow solid: LCMS tR ) 5.57 min (7.5
min run time); m/z ) 589.1[M + H]+; 1H NMR (400 MHz, CDCl3)
δ 0.79 (d, 6H, J ) 6.8 Hz), 1.21 (t, 3H, J ) 7.2 Hz), 1.81 (m, 1H),
2.39 (d, 2H, J ) 7.0 Hz), 3.43 (dt, 2H, J ) 3.7 Hz, 7.2 Hz), 4.67
(s, 2H), 4.96 (s, 2H), 6.41 (s, 1H), 6.87 (brt, 1H, J ) 3.7 Hz), 6.98
(m, 2H), 7.06 (s, 1H), 7.25 (m, 2 H), 7.33 (m, 6H), 7.38 (d, 2H, J
) 8.2 Hz), 7.67 (d, 2H, J ) 8.2 Hz), 9.92 (s, 1H).
5-(2,4-Dihydroxy-5-isobutylphenyl)-4-(4-morpholin-4-ylmeth-
ylphenyl)isoxazole-3-carboxylic Acid Ethylamide (40e). This
compound was made using the procedure described for compound
30f, using morpholine in reaction with compound 36e (0.13 g, 0.22
mmol). The crude product after debenzylation was purified by
preparative HPLC to afford the title compound 40e as a colorless
solid: LCMS tR ) 2.17 min; m/z ) 480[M + H]+; 1H NMR (400
MHz, CDCl3) δ 0.72 (d, 6H, J ) 6.6 Hz), 1.17 (t, 3H, J ) 7.3
Hz), 1.68 (m, 1H), 2.20 (d, 2H, J ) 7.1 Hz), 2.60 (m, 4H), 3.38
(m, 2H), 3.57 (s, 2H), 3.68 (m, 4H), 6.11 (s, 1H), 6.81 (s, 1H),
6.85 (t, 1H, J ) 5.8 Hz), 7.20 (d, 2H, J ) 8.1 Hz), 7.25 (d, 2H, J
) 8.1 Hz); 13C ΝΜR (100.6 MHz, CDCl3) 14.5 (CH3), 22.4 (CH3),
28.4 (CH), 34.5 (CH2), 38.4 (CH2), 52.9 (CH2), 62.6 (CH2), 65.7
(CH2), 104.0 (CH), 105.6 (C), 115.8 (C), 120.7 (C), 129.7 (C),
129.9 (CH), 130.2 (CH), 132.1 (CH), 133.5 (C), 153.8 (C), 156.1
(C), 157.8 (C), 159.7 (C), 167.6 (C); HRMS calcd for
C27H33N3O5[M + H]+ 480.2498, found 480.2485; HPLC 93% (tR
) 4.61 min).
4-Isobutylbenzene-1,3-diol (54). Ethyl chloroformate (6.3 mL,
66 mmol) was added slowly to a cooled (0 °C) solution of
compound 53 (3.96 g, 22 mmol) and triethylamine (9.2 mL, 66
mmol) in THF (50 mL). The mixture was warmed to ambient
temperature and stirred for 3 h before being filtered, and the solids
were washed with cold THF (10 mL). The combined filtrates were
cooled to 0 °C, and sodium borohydride (3.35 g, 88 mmol) in water
(60 mL) was added slowly. The mixture was warmed to ambient
temperature, stirred for 3 h, and diluted with water. The mixture
was twice extracted with diethyl ether, and the combined extracts
were concentrated to dryness and resuspended in 10% aqueous
sodium hydroxide solution (40 mL). After refluxing for 90 min,
the mixture was cooled, acidified with 5.0 M aqueous HCl, and
twice extracted with diethyl ether. The organic extracts were dried
over anhydrous MgSO4 and filtered, and the solvents were removed
in vacuo to give 4-isobutylbenzene-1,3-diol 54 (3.61 g, 98%) as a
1
cloudy oil, which was used without further purification: H NMR
(400 MHz, CDCl3) δ 0.89 (d, 6H, J ) 6.8 Hz), 1.85 (m, 1H), 2.37
(d, 2H, J ) 7.2 Hz), 6.31 (m, 2H), 7.24 (s, 1H), 6.87 (d, 1H, J )
8.1 Hz).
1-(2,4-Bis(benzyloxy)-5-isobutylphenyl)ethanone (55). 4-Isobu-
tylbenzene-1,3-diol (54) (3.61 g, 21.7 mmol) was dissolved in
BF3 · OEt2 (16 mL), and acetic acid (2.48 mL, 43.4 mmol) was
added. The solution was heated for 16 h at 90 °C and then allowed
to cool to room temperature. The solution was added dropwise to
10% NaOAc(aq) and allowed to stand for 4 h before being extracted
twice with diethyl ether. The organic phases were combined and
washed with saturated NaHCO3(aq), dried over anhydrous MgSO4,
filtered, and concentrated in vacuo to give 6-acetyl-4-isobutylre-
sorcinol, which was dissolved in DMF (50 mL). Potassium
carbonate (12.7 g, 92 mmol) and then benzyl bromide (11 mL, 92
mmol) were added. The suspension was heated, with stirring to
150 °C, under nitrogen for 16 h. The solution was cooled to room
temperature, filtered, and concentrated in vacuo. The resulting solid
was purified by flash column chromatography on silica gel, eluting
with hexane/EtOAc (4:1), and then recrystallized from the EtOAc/
hexane mixture to give the title compound 55 (2.14 g, 25%) as
colorless crystals: LCMS tR ) 3.03 min; m/z ) 389.3[M + H]+;
1H NMR (400 MHz, CDCl3) δ 0.91 (d, 6H, J ) 8.8 Hz), 1.85 (m,
1H), 2.41 (d, 2H, J ) 6.8 Hz), 2.54 (s, 3H), 6.36 (s, 1H), 7.39 (s,
1H).
4-(2,4-Bis(benzyloxy)-5-isobutylphenyl)-2-hydroxy-4-oxobut-
2-enoic Acid Ethyl Ester (56). This compound was made using
the procedure described for compound 12. Thus, 55 (2.14 g, 5.51
mmol) was reacted with sodium ethoxide and diethyl oxalate to
afford title compound 56 (1.55 g, 58%) as a yellow solid, used
without further purification: LCMS tR ) 3.25 min; m/z ) 489.3[M
+ H]+.
2,4-Bis(benzyloxy)-1-isopropenylbenzene (58). Methyltri-
phenylphosphonium bromide (64.99 g, 0.182 mol) was suspended
in anhydrous THF (300 mL), and the mixture was cooled to 0 °C
under a nitrogen atmosphere. n-Butyllithium (1.6 M solution in
hexanes, 114 mL, 0.182 mol) was added dropwise, and the resulting
mixture was stirred at 0 °C for 30 min. A solution of 1-(2,4-
bis(benzyloxy)phenyl)ethanone (47) (46.78 g, 0.14 mol) in anhy-
drous THF (150 mL) was then added dropwise to the reaction
mixture. When the addition was complete, the ice bath was
removed, and the reaction mixture was stirred at room temperature
under nitrogen for 18 h. MeOH (100 mL) was added to the reaction
mixture, and the resulting solution was evaporated in vacuo. Hexane
5-(2,4-Bis(benzyloxy)-5-isobutylphenyl)isoxazole-3-carboxyl-
ic Acid Ethyl Ester (57). This compound was made using the
procedure described for 23. Thus, compound 56 (0.73 g, 1.52 mmol)
was reacted with hydroxylamine hydrochloride to afford title
compound 57 (0.73 g, 74%) as a colorless solid: LCMS tR ) 3.26
1
min; m/z ) 486.3[M + H]+; H NMR (400 MHz, CDCl3) δ 0.90

214 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Brough et al.
was added to the resulting oil, and the mixture was heated to reflux
for 30 min and then filtered through a pad of Celite 521. The liquors
were combined and were evaporated in vacuo to give an oil, which
was purified by flash column chromatography on silica gel, eluting
with 5% EtOAc in hexane, to give the title compound 58 (37.15 g,
80%) as a colorless oil: TLC Rf ) 0.73 (25:75 EtOAc/hexane); 1H
NMR (400 MHz, CDCl3) δ 2.12 (s, 3H), 5.03 (s, 2H), 5.05 (s,
2H), 5.07 (brs, 2H), 6.54 (dd, 1H, J ) 8.3 Hz, 2.5 Hz), 6.59 (d,
1H, J ) 2.5 Hz), 7.14 (d, 1H, J ) 8.3 Hz). 7.29–7.44 (m, 10H).
4-Isopropylbenzene-1,3-diol (59). Compound 58 (37.15 g, 0.113
mol) was dissolved in EtOH (300 mL) and carefully added to 10%
palladium on carbon, which had been prewetted with water under
a nitrogen atmosphere. Hydrogen was introduced to the flask, and
the mixture was allowed to shake under a positive hydrogen
atmosphere for 16 h. The catalyst was filtered from the reaction
mixture, and the filtrate solvents were removed in vacuo to give
the title compound 59 (12.05 g, 70%) as a white crystalline solid:
LCMS tR ) 2.08 min; m/z ) 153.1[M + H]+; 1H NMR (400 MHz,
CDCl3) δ 1.22 (d, 6H, J ) 7.1 Hz), 3.09 (sept, 1H, J ) 7.1 Hz),
4.63 (brs, 1H), 4.74 (brs, 1H), 6.29 (d, 1H, J ) 2.5 Hz), 6.38 (dd,
1H, J ) 8.3, 2.5 Hz), 7.03 (d, 1H, J ) 8.3 Hz).
1-(2,4-Dihydroxy-5-isopropylphenyl)ethanone (60). 4-Iso-
propylbenzene-1,3-diol (59) (12.05 g, 0.079 mol) was dissolved in
BF3 ·OEt2 (60 mL, 0.486 mol) and acetic acid (9 mL, 0.157 mol)
was added. The solution was heated for 16 h at 90 °C, allowed to
cool to room temperature, added dropwise to 10% NaOAc(aq)
solution (1000 mL), and allowed to stand for 4 h before being
extracted with EtOAc (2 × 200 mL). The organic phases were
combined and washed with saturated NaHCO3(aq), dried over
MgSO4 and filtered, and the filtrate solvents were removed in vacuo.
The residual oil was triturated with cold hexane to afford the title
J ) 7.1 Hz), 3.29 (sept, 1H, J ) 6.8 Hz), 4.28 (q, 2H, J ) 7.1 Hz),
5.11 (s, 2H), 5.14 (s, 2H), 6.53 (s, 1H), 7.32–7.46 (m, 11H), 7.87
(s, 1H).
5-(2,4-Bis(benzyloxy)-5-isopropylphenyl)isoxazole-3-carbox-
ylic Acid Ethyl Ester (63). Compound 62 (14.94 g, 31.5 mmol)
was dissolved in EtOH (150 mL), hydroxylamine hydrochloride
(2.63 g, 37.8 mmol) was added, and the solution was heated to
reflux for 4 h under a nitrogen atmosphere. The reaction mixture
was cooled to room temperature and concentrated in vacuo. The
residue was partitioned between dichloromethane and saturated
NaCl(aq) solution. The organic phase was dried over MgSO4,
filtered, and concentrated in vacuo to give a yellow solid, which
was triturated with an EtOH/water mix, filtered, and dried to afford
the title compound 62 (13.5 g, 91%) as a yellow solid: LCMS tR
) 3.20 min; m/z ) 472[M + H]+; 1H NMR (400 MHz, CDCl3) δ
1.26 (d, 6H, J ) 6.8 Hz), 1.41 (t, 3H, J ) 7.1 Hz), 3.34 (sept, 1H,
J ) 6.8 Hz), 4.42 (q, 2H, J ) 7.1 Hz), 5.07 (s, 2H), 5.16 (s, 2H),
6.57 (s, 1H), 7.00 (s, 1H), 7.32–7.43 (m, 10H), 7.83 (s, 1H); 13C
NMR (100.6 MHz, DMSO-d6) δ 13.9 (CH3), 22.4 (CH3), 26.2 (CH),
61.7 (CH2), 69.8 (CH2), 70.4 (CH2), 98.8 (CH), 101.5 (CH), 107.2
(C), 124.0 (CH), 127.5 (CH), 128.0 (CH), 128.0 (CH), 128.2 (CH),
128.6 (CH), 128.6 (CH), 129.3 (C), 136.3 (C), 136.7 (C), 154.9
(C), 156.5 (C), 158.6 (C), 159.4 (C), 167.8 (C).
5-(2,4-Bis(benzyloxy)-5-isopropylphenyl)isoxazole-3-carbox-
ylic Acid Ethylamide (34f). Compound 63 (13.57 g, 28.8 mmol)
was suspended in ethylamine in MeOH solution (2.0M, 140 mL,
280 mmol) and heated to 90 °C for 1 h to afford a homogeneous
solution. The solution was allowed to cool to room temperature,
and a colorless solid precipitated. The solid was collected by
filtration, washed with cold MeOH, and dried in vacuo to afford
title compound 63 (11.5 g, 85%) as a colorless solid: LCMS tR )
3.06 min; m/z ) 471[M + H]+; 1H NMR (400 MHz, CDCl3) δ.1.25
(d, 6H, J ) 6.8 Hz), 1.26 (t, 3H, J ) 7.0 Hz), 3.33 (sept, 1H, J )
6.9 Hz), 3.49 (m, 2H), 5.03 (s, 2H), 5.17 (s, 2H), 6.55 (s, 1H),
6.81 (brt, 1H, J ) 5.7 Hz), 7.07 (s, 1H), 7.30–7.42 (m, 10H), 7.78
(s, 1H).
compound 60 (13.57 g, 88%) as an off-white solid: LCMS tR
)
1
2.63 min; m/z ) 195.1[M + H]+; H NMR (400 MHz, CDCl3) δ
1.25 (d, 6H, J ) 6.8 Hz), 2.57 (s, 3H), 3.13 (sept, 1H, J ) 6.8
Hz), 5.64 (s, 1H), 6.30 (s, 1H), 7.50 (s, 1H), 12.56 (s, 1H).
1-(2,4-Bis(benzyloxy)-5-isopropylphenyl)ethanone (61). 1-(2,4-
Dihydroxy-5-isopropylphenyl)ethanone (60) (13.57 g, 0.070 mol)
was dissolved in DMF (120 mL), and potassium carbonate (21.27
g, 0.154 mol) and benzyl bromide (18.3 mL, 0.154 mol) were added.
The resulting suspension was heated, with stirring, to 150 °C under
a nitrogen atmosphere for 16 h. The solution was cooled to room
temperature, and the mixture was poured into 1.0 M HCl(aq) (1000
mL) and then extracted with EtOAc (2 × 300 mL). The organic
phases were combined and washed with 1.0 M HCl(aq) and
saturated NaCl(aq) solution (3 × 300 mL). The organic phase was
dried over MgSO4 and filtered, and the filtrate solvents were
removed in vacuo to give a solid, which was purified by trituration
with diethyl ether/hexane (1:1) to give the title compound 61 (23.10
g, 88%) as a colorless solid: LCMS tR ) 3.03 min; m/z ) 375.2[M
5-(2,4-Bis(benzyloxy)-5-isopropylphenyl)-4-iodoisoxazole-3-
carboxylic Acid Ethylamide (35f). Compound 34f (11.94 g, 25.4
mmol) was dissolved in a mixture of anhydrous acetonitrile (150
mL) and dichloromethane (60 mL), N-iodosuccinimide (11.44 g,
50.8 mmol) was added followed by ceric ammonium nitrate (0.697
g, 1.27 mmol), and the solution was stirred at room temperature
overnight. The reaction mixture was concentrated in vacuo, and
the resulting gum was partitioned between EtOAc and saturated
NaCl(aq) solution. The organic phase was dried over MgSO4 and
filtered, and the filtrate solvents were removed in vacuo. The residue
was purified by column chromatography, eluting with 9:1 hexane/
EtOAc, to give the title compound 35f (9.1 g, 60%) as a pale-
1
yellow solid: LCMS tR ) 2.97 min; m/z ) 597.2[M + H]+; H
NMR (400 MHz, CDCl3) δ.1.23 (d, 6H, J ) 6.8 Hz), 1.26 (t, 3H,
J ) 7.3 Hz), 3.35 (sept, 1H, J ) 6.8 Hz), 3.50 (m, 2H), 5.05 (s,
2H), 5.09 (s, 2H), 6.58 (s, 1H), 6.80 (brt, 1H, J ) 5.7 Hz), (s, 1H),
7.28–7.41 (m, 11H).
1
+ H]+; H NMR (400 MHz, CDCl3) δ 1.22 (d, 6H, J ) 6.9 Hz),
2.56 (s, 3H), 3.28 (sept, 1H, J ) 6.9 Hz), 5.08 (s, 2H), 5.10 (s,
2H), 6.51 (s, 1H), 7.31–7.44 (m, 10H), 7.76 (s, 1H).
4-(2,4-Bis(benzyloxy)-5-isopropylphenyl)-2-hydroxy-4-oxo-
but-2-enoic Acid Ethyl Ester (62). Sodium hydride (60% in
mineral oil) (2.19 g, 54.9 mmol) was added to an ice-cold
homogeneous solution of 1-(2,4-bis(benzyloxy)-5-isopropylphen-
yl)ethanone (61) (13.65 g, 36.5 mmol) in toluene (200 mL). Diethyl
oxalate (5.0 mL, 36.8 mmol) was added, and the reaction mixture
was heated to reflux for 4 h. The mixture was allowed to cool to
room temperature, and 1.0 M HCl(aq) (600 mL) was added to
acidify the reaction mixture. Solvents were removed in vacuo, and
the resulting gum was partitioned between dichloromethane and
saturated NaCl(aq) solution. The phases were separated, and the
organic phase was dried over MgSO4 and filtered, and the filtrate
solvents were removed in vacuo to afford an orange gum, which
slowly crystallized. Trituration with hexane and filtration afforded
the title compound 62 (14.94 g, 86%) as a yellow-orange
5-(2,4-Bis(benzyloxy)-5-isopropylphenyl)-4-(4-formylphenyl)-
isoxazole-3-carboxylic Acid Ethylamide (36f). This compound
was made using the procedure described for compound 32. Thus,
compound 35f (9.0 g, 15.1 mmol) was reacted with 4-formylphenyl
boronic acid (4.53 g, 30.2 mmol) to afford a crude product after
workup, which was purified by flash chromatography on silica gel,
eluting with dichloromethane, to afford the title compound 36f (6.0
g, 69% yield) as a yellow solid: LCMS tR ) 2.98 min; m/z )
575.3[M + H]+; 1H NMR (400 MHz, CDCl3) δ 1.04 (d, 6H, J )
6.8 Hz), 1.08 (t, 3H, J ) 7.1 Hz), 3.16 (sept, 1H, J ) 6.8 Hz),
3.23 (m, 2H), 4.95 (s, 2H), 5.18 (s, 2H), 6.93 (s, 1H), 7.05 (m,
2H), 7.12 (s, 1H), 7.25–7.29 (m, 3H), 7.32–7.37 (m, 3H), 7.39–7.47
(m, 4H), 7.78 (d, 2H, J ) 8.3 Hz), 8.95 (brt, 1H, J ) 5.6 Hz), 9.98
(s, 1H); 13C NMR (100.6 MHz, DMSO-d6) δ 14.3 (CH3), 22.3
(CH3), 25.8 (CH), 33.8 (CH2), 69.7 (CH2), 69.9 (CH2), 98.9 (CH),
107.2 (C), 115.0 (C), 127.3 (CH), 127.5 (CH), 127.8 (CH), 127.9
(CH), 127.9 (CH), 128.2 (CH), 128.5 (CH), 128.9 (C), 129.2 (CH),
1
crystalline solid: LCMS tR ) 3.06 min; m/z ) 475[M + H]+; H
NMR (400 MHz, CDCl3) δ 1.23 (d, 6H, J ) 6.8 Hz), 1.28 (t, 3H,

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 215
129.3 (CH), 135.0 (C), 135.8 (C), 136.3 (C), 136.7 (C), 155.2 (C),
157.6 (C), 158.5 (C), 159.2 (C), 165.7 (C), 192.6 (CH).
into water (100 mL); the resulting off-white precipitate was filtered
and recrystallized from EtOH (100 mL) to yield the title compound
68d (3.51 g, 88%) as a white solid: LCMS tR ) 3.16 min; m/z )
5-(2,4-Dihydroxy-5-isopropylphenyl)-4-(4-morpholin-4-yl-
methylphenyl)isoxazole-3-carboxylic Acid Ethylamide (40f). This
compound was made using the procedure described for compound
30f, using morpholine in reaction with compound 36f. The crude
product after debenzylation was purified by flash chromatography
(eluting with a gradient of 2–4% MeOH in dichloromethane) to
afford the title compound as a colorless solid: LCMS tR ) 1.99
1
458.3[M + H]+; H NMR (400 MHz, CDCl3) δ 1.39 (t, 3H, J )
7.5), 1.56 (t, 3H, J ) 7.1 Hz), 2.84 (q, 2H, J ) 7.5 Hz), 4.58 (q,
2H, J ) 7.1 Hz), 5.22 (s, 2H), 5.32 (s, 2H), 6.73 (s, 1H), 7.16 (s,
1H), 7.48–7.58 (m, 10H), 7.95 (s, 1H); 13C NMR (100.6 MHz,
CDCl3) δ 13.9 (CH3), 14.3 (CH3), 22.3 (CH2), 61.7 (CH2), 69.7
(CH2), 70.5 (CH2), 98.6 (CH), 101.5 (CH), 107.1 (C), 125.0 (C),
126.8 (CH), 127.5 (CH), 128.0 (CH), 128.0 (CH), 128.2 (CH), 128.6
(CH), 128.7 (CH), 136.3 (C), 136.7 (C), 155.1 (C), 156.5 (C), 159.1
(C), 159.5 (C), 167.8 (C).
5-(2,4-Bis(benzyloxy)-5-ethylphenyl)isoxazole-3-carboxylic
Acid Ethylamide (34d). Compound 68d (1.8 g, 3.93mmol) was
suspended in EtOH (23 mL), ethylamine solution (2.0 M in MeOH,
15.7 mL, 31.4 mmol) was added, and the reaction mixture was
heated to reflux for 3 h. The reaction was allowed to cool to room
temperature and poured into water (50 mL), and the resulting white
precipitate was filtered, washed with water, and recrystallized from
EtOH (37 mL) to yield the title compound 34d (1.53 g, 85%) as a
colorless flaky solid: LCMS tR ) 3.01 min; m/z ) 457.3[M + H]+;
1H NMR (400 MHz, DMSO-d6) δ 1.10 (t, 3H, J ) 7.2 Hz), 1.14
(t, 3H, J ) 7.5 Hz), 2.60 (q, 2H, J ) 7.5 Hz), 3.26 (m, 2H), 5.23
(s, 2H), 5.34 (s, 2H), 6.87 (s, 1H), 7.04 (s, 1H), 7.35–7.50 (m,
10H), 7.66 (s, 1H), 8.75 (t, 1H, J ) 5.8 Hz); 13C NMR (100.6
MHz, DMSO-d6) δ 14.3 (CH3), 14.6 (CH3), 22.3 (CH2), 33.7 (CH2),
69.6 (CH2), 70.2 (CH2), 98.7 (CH), 100.7 (CH), 107.5 (C), 124.9
(C), 127.0 (CH), 127.5 (CH), 127.9 (CH), 128.0 (CH), 128.2 (CH),
128.6 (CH), 128.7 (CH), 136.4 (C), 136.7 (C), 154.9 (C), 158.4
(C), 158.95 (C), 159.4 (C), 167.0 (C).
1
min; m/z ) 466.3[M + H]+; H NMR (400 MHz, DMSO-d6) δ
0.90 (d, 6H, J ) 7.1 Hz), 1.07 (t, 3H, J ) 7.4 Hz), 2.33 (brm, 4H),
2.97 (sept, 1H, J ) 7.1 Hz), 3.22 (m, 2H), 3.42 (s, 2H), 3.55 (brm,
4H), 6.43 (s, 1H), 6.72 (s,1H), 7.18 (d, 2H, J ) 8.4 Hz), 7.23 (d,
2H, J ) 8.4 Hz), 8.83 (brt, 1H, J ) 5.9 Hz), 9.65 (s, 1H), 9.76 (s,
1H); 13C NMR (100.6 MHz, DMSO-d6) δ 14.4 (CH3), 22.4 (CH3),
25.4 (CH), 33.7 (CH2), 53.1 (CH2), 62.1 (CH2), 66.2 (CH2), 102.7
(CH), 104.5 (C), 114.7 (C), 125.6 (C), 127.7 (CH), 128.5 (C), 128.7
(CH), 128.8 (CH), 136.9 (C), 154.7 (C), 157.4 (C), 157.8 (C), 159.8
(C), 166.3 (C); HRMS calcd for C26H31N3O5[M + Na]+ 488.2161,
found 488.2162; HPLC 96% (tR ) 4.90 min).
1-(5-Ethyl-2,4-dihydroxyphenyl)ethanone (65d). 4-Ethylresor-
cinol (64) (6.23 g, 45.1 mmol) was dissolved in BF3 ·OEt2 (50 mL)
and acetic acid (5.42 g, 90.3 mmol) and stirred at 90 °C for 12 h.
The reaction was cooled to room temperature and quenched with
water (200 mL), and the reaction mixture pH was adjusted to pH
) 3 with Na2CO3. The red precipitate was filtered and recrystallized
from toluene (53 mL) to yield the title compound 65d (5.63 g,
69%) as a light-pink crystalline solid: LCMS tR ) 2.26 min; m/z
) 181.2[M + H]+; 1H NMR (400 MHz, DMSO-d6) δ 1.12 (t, 3H,
J ) 7.5 Hz), 2.48 (q, 2H, J ) 7.5 Hz), 2.52 (s, 3H), 6.29 (s, 1H),
7.58 (s, 1H), 10.61 (s, 1H), 12.49 (s, 1H).
1-(2,4-Bis(benzyloxy)-5-ethylphenyl)ethanone (66d). Com-
pound 65d (5.63 g, 31.24 mmol) was dissolved in DMF (100 mL).
K2CO3 (9.50 g, 68.7 mmol) and benzyl bromide (11.76 g, 68.7
mmol) were added, and the reaction mixture was stirred at room
temperature for 3 h. The mixture was poured into water (300 mL),
and the off-white precipitate was filtered off and recrystallized from
acetonitrile (50 mL) to yield the title compound 66d (7.85 g, 70%)
as a colorless solid: LCMS tR ) 2.98 min; m/z ) 361.2[M + H]+;
1H NMR (400 MHz, CDCl3) δ 1.12 (t, 3H, J ) 7.5 Hz), 2.49 (s,
3H), 2.56 (q, 2H, J ) 7.5 Hz), 5.01 (s, 2H), 5.02 (s, 2H), 6.43 (s,
1H), 7.26–7.35 (m, 10H), 7.63 (s, 1H); 13C NMR (100.6 MHz,
CDCl3) δ 14.2 (CH3), 22.1 (CH2), 32.0 (CH3), 69.7 (CH2), 70.5
(CH2), 98.3 (CH), 119.7 (C), 124.22 (C), 127.5 (CH), 128.0 (CH),
128.0 (CH), 128.1 (CH), 128.6 (CH), 128.6 (CH), 130.2 (CH), 136.5
(C), 136.7 (C), 158.4 (C), 160.6 (C), 196.2 (C).
5-(2,4-Bis(benzyloxy)-5-ethylphenyl)-4-iodoisoxazole-3-car-
boxylic Acid Ethylamide (35d). Compound 34d (33.96 g, 74.38
mmol) was dissolved in acetonitrile (400 mL) and dichloromethane
(250 mL). N-Iodosuccinimide (33.47 g, 148.77 mmol) and ceric
ammonium nitrate (20.39 g, 37.19 mmol) were added, and the
reaction was stirred at room temperature for 6 h. The solvent was
removed under reduced pressure, and the residue was partitioned
between dichloromethane and aqueous Na2S2O3 solution. The
organic phase was washed with water and saturated NaCl(aq) and
dried over Na2SO4. The solvents were removed in vacuo, and the
residue was triturated with EtOH to afford title compound 35d
(16.43 g, 38%) as a yellow solid: LCMS tR ) 2.96 min; m/z )
1
583.2[M + H]+; H NMR (400 MHz, CDCl3) δ 1.20 (t, 3H, J )
7.5 Hz), 1.25 (t, 3H, J ) 7.3 Hz), 2.65 (q, 2H, J ) 7.5 Hz),
3.46–3.53 (m, 2H), 5.01 (s, 2H), 5.04 (s, 2H), 6.58 (s, 1H), 6.80 (t,
1H, J ) 5.2 Hz), 7.27 (s, 1H), 7.28–7.39 (m, 10H); 13C NMR (100.6
MHz, CDCl3) δ 14.00 (CH3), 14.6 (CH3), 22.5 (CH2), 34.4 (CH2),
56.87 (C), 70.0 (CH2), 71.0 (CH2), 98.5 (CH), 107.8 (C), 125.6
(C), 127.0 (CH), 127.1 (CH), 127.9 (CH), 128.0 (CH), 128.6 (CH),
128.7 (CH), 131.3 (CH), 136.5 (C), 136.5 (C), 156.0 (C), 156.4
(C), 158.4 (C), 159.5 (C), 172.0 (C).
4-(2,4-Bis(benzyloxy)-5-ethylphenyl)-2-hydroxy-4-oxobut-2-
enoic Acid Ethyl Ester (67d). Sodium (1.5 g, 65.2 mmol) was
added in small pieces to absolute EtOH (200 mL) under nitrogen
over a period of 20–30 min. When all of the sodium had reacted,
compound 66d (7.85 g, 21.8 mmol) and diethyl oxalate (4.77 g,
32.67 mmol) were added, and the mixture was heated at reflex
for 3 h. The reaction was allowed to cool to room temperature
and quenched with 2 N aqueous hydrochloric acid (33 mL). The
yellow precipitate was filtered, washed with water, and dried in
vacuo to afford the title compound 67d (9.41 g, 94%) as a yellow
5-(2,4-Bis(benzyloxy)-5-ethylphenyl)-4-(4-formylphenyl)isox-
azole-3-carboxylic Acid Ethylamide (36d). Compound 35d (1.41
g, 2.42 mmol) was dissolved in THF (20 mL). 4-Formylphenylboronic
acid (544 mg, 3.63 mmol),[1,1′-bis(diphenylphospino)ferrocene-
]dichloropalladium(II) (0.090 g, 0.12 mmol) and NaHCO3 (1.0 M in
water, 4.84 mL, 4.84 mmol) were added, and the mixture was degassed
with nitrogen. The reaction was stirred at 80 °C for 8 h and then cooled
to room temperature. Water and EtOAc were added, the phases were
separated, and the organic phase was washed with water (2 ×) and
then saturated NaCl(aq) solution. The organic layer was dried over
Na2SO4 and filtered through a silica plug. The solvent was removed
in vacuo, and the residual solid was triturated with diethyl ether, filtered,
and dried to afford the title compound 36d (0.99 g, 74%) as a pink
solid: LCMS tR ) 2.90 min; m/z ) 561.2[M + H]+; 1H NMR (CDCl3)
δ 1.04 (t, 3H, J ) 7.5 Hz), 1.11 (t, 3H, J ) 7.0 Hz), 2.50 (q, 2H, J )
7.5 Hz), 3.32–3.41 (m, 2H), 4.55 (s, 2H), 4.89 (s, 2H), 6.31 (s, 1H),
6.72 (s, 1H), 6.85–6.87 (m, 2H), 7.12 (s, 1H), 7.15–7.17 (m, 2H),
7.23–7.32 (m, 8H), 7.59 (d, 2H, J ) 8.4 Hz), 9.86 (s, 1H); 13C NMR
(100.6 MHz, CDCl3) δ 13.9 (CH3), 14.3 (CH3), 21.9 (CH2), 33.8 (CH2),
69.5 (CH2), 69.8 (CH2), 98.7 (CH), 107.1 (C), 115.0 (C), 124.5 (C),
1
solid: LCMS tR ) 3.14 min; m/z ) 461.3[M + H]+; H NMR
(400 MHz, CDCl3) δ 1.46 (t, 3H, J ) 7.5 Hz), 1.53 (t, 3H, J )
7.1 Hz), 2.90 (q, 2H, J ) 7.5 Hz), 4.53 (q, 2H, J ) 7.1 Hz),
5.36 (s, 2H), 5.38 (s, 2H), 6.77 (s, 1H), 7.59–7.70 (m, 11H),
8.06 (s, 1H), 15.62 (s, 1H); 13C NMR (100.6 MHz, CDCl3) δ
13.8 (CH3), 14.0 (CH3), 22.1 (CH2), 61.8 (CH2), 69.9 (CH2),
70.8 (CH2), 98.5 (CH), 102.6 (CH), 115.7 (C), 125.1 (C), 127.6
(CH), 127.9 (CH), 128.1 (CH), 128.1 (CH), 128.6 (CH), 128.6
(CH), 130.2 (CH), 136.0 (C), 136.5 (C), 159.1 (C), 161.9 (C),
162.0 (C), 167.7 (C), 188.8 (C).
5-(2,4-Bis(benzyloxy)-5-ethylphenyl)isoxazole-3-carboxylic
Acid Ethyl Ester (68d). Compound 67d (4.0 g, 8.69 mmol) was
suspended in EtOH (90 mL), and hydroxylamine hydrochloride
(0.905 g, 13.0 mmol) was added. The reaction mixture was heated
to reflux for 1.5 h, allowed to cool to room temperature, and poured

216 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Brough et al.
127.2 (CH), 127.5 (CH), 127.7 (CH), 127.9 (CH), 128.2 (CH), 128.5
(CH), 129.1 (CH), 129.1 (CH), 130.3 (CH), 134.9 (C), 135.7 (C), 136.1
(C), 136.6 (C), 155.2 (C), 157.6 (C), 159.0 (C), 159.1 (C), 165.4 (C),
192.5 (CH).
saturated aqueous NaCl solution (100 mL). The organic solution
was dried over anhydrous Na2SO4 and filtered, and the filtrate
solvents were removed in vacuo to afford a yellow gum. Trituration
with hexane gave a yellow solid, which was filtered, washed with
hexane, and then dried in vacuo (at 40 °C) to afford the title
5-(2,4-Dihydroxy-5-ethylphenyl)-4-(4-morpholin-4-ylmethyl-
phenyl)isoxazole-3-carboxylic Acid Ethylamide (40d). This
compound was made using the procedure described for compound
30f, using morpholine in reaction with compound 36d. The crude
product after debenzylation was purified by preparative HPLC to
afford the title compound 40d (15.3 mg, 69%) as an off-white solid:
LCMS tR ) 1.78 min; m/z ) 452.2[M + H]+; 1H NMR (400 MHz,
DMSO-d6) δ 0.93 (t, 3H, J ) 7.5 Hz), 1.07 (t, 3H, J ) 7.2 Hz),
2.30–2.35 (m, 6H), 3.23 (m, 2H), 3.42 (s, 2H), 3.55 (m, 4H), 6.42
(d, 1H, J ) 2.4 Hz), 6.77 (s, 1H), 7.17–7.23 (m, 4H), 8.86 (t, 1H,
J ) 5.7 Hz), 9.61 (bs, 1H), 9.74 (bs, 1H); 13C NMR (100.6 MHz,
DMSO-d6) δ 14.0 (CH3), 14.4 (CH3), 21.6 (CH2), 33.8 (CH2), 53.2
(CH2), 62.1 (CH2), 66.2 (CH2), 102.6 (CH), 104.6 (C), 114.9 (C),
121.2 (C), 128.4 (C), 128.6 (2 × CH), 130.3 (CH), 136.8 (C), 154.9
(C), 157.8 (C), 158.0 (C), 159.8 (C), 166.0 (C); HRMS calcd for
C25H29N3O5[M + H]+ 452.2185, found 452.2182; HPLC 97% (tR
) 3.97 min).
1-(5-tert-Butyl-2,4-dihydroxyphenyl)ethanone (65g). Sulfuric
acid (4 mL, 75 mmol) was added to a suspension of 2,4-
dihydroxyacetophenone (46) (22.8 g, 150 mmol) in a mixture of
2-methyl-2-propanol (35 g, 470 mmol) and trifluoroacetic acid (80
mL) under a nitrogen atmosphere. The resulting suspension was
heated at 75 °C (oil bath temperature) for 3 h, generating a pale-
red solution, which was allowed to cool to ambient temperature
and then poured into an ice/water mixture (350 mL) to give a pale-
pink precipitate. The solids were removed by filtration, washed with
water (600 mL) and hexane (200 mL), and then dried in vacuo (at
40 °C) to give the title compound 65g (28.8 g, 92%) as a pale-
orange powder: LCMS tR ) 2.74 min; m/z ) 209.1[M + H]+; 1H
NMR (400 MHz, CDCl3) δ 1.39 (s, 9H), 2.58 (s, 3H), 6.27 (s,
1H), 7.59 (s, 1H).
1
compound 67g (9.1 g, 93%) as a yellow powder: H NMR (400
MHz, CDCl3) δ 1.26 (t, 3H, J ) 7.1 Hz), 1.39 (s, 9H), 4.28 (q,
2H, J ) 7.1 Hz), 5.11 (s, 2H), 5.13 (s, 2H), 6.54 (s, 1H), 7.34–7.45
(m, 11H), 7.96 (s, 1H).
5-(2,4-Bis(benzyloxy)-5-tert-butylphenyl)isoxazole-3-carbox-
ylic Acid Ethyl Ester (68g). Hydroxylamine hydrochloride (3.6
g, 52 mmol) was added to a solution of 67g (9.0 g, 18.5 mmol) in
EtOH (75 mL), and the suspension was heated at reflux for 4 h.
The resulting solution was allowed to cool and poured into water
(200 mL) to give an off-white precipitate. The solids were removed
by filtration and then dissolved in dichloromethane (150 mL). The
solution was washed with water (150 mL) and then saturated
aqueous NaCl solution (50 mL). The organic phase was dried over
anhydrous Na2SO4 and filtered, and the filtrate solvents were
removed in vacuo to afford an off-white solid, which was washed
with hexane and dried in vacuo (at 40 °C) to give the title compound
68g (8.0 g, 89%) as a pale-brown powder: LCMS tR ) 3.13 min;
1
m/z ) 486.5[M + H]+; H NMR (400 MHz, CDCl3) δ 1.41 (t,
3H, J ) 7.1 Hz), 1.41 (s, 9H), 4.43 (q, 2H, J ) 7.1 Hz), 5.09 (s,
2H), 5.15 (s, 2H), 6.59 (s, 1H), 7.00 (s, 1H), 7.33–7.43 (m, 10H),
7.93 (s, 1H).
5-(2,4-Bis(benzyloxy)-5-tert-butylphenyl)isoxazole-3-carbox-
ylic Acid Ethylamide (34g). This compound was made using the
procedure described for compound 34d. Thus, 68g (10.0 g, 20.6
mmol) was reacted with ethylamine in MeOH solution (2.0 M) to
afford the title compound as a brown solid (9.9 g, 100%): LCMS
tR ) 3.02 min; m/z ) 485.3[M + H]+; 1H NMR (400 MHz, CDCl3)
δ 1.26 (t, 3H, J ) 7.1 Hz), 1.41 (s, 9H), 3.49 (dq, 2H, J ) 7.1, 5.4
Hz), 5.06 (s, 2H), 5.15 (s, 2H), 6.57 (s, 1H), 6.85 (brt, 1H, J ) 5.4
Hz), 7.08 (s, 1H), 7.3–7.4 (m, 10H), 7.88 (s, 1H).
1-(2,4-Bis(benzyloxy)-5-tert-butylphenyl)ethanone (66g). Ben-
zyl bromide (10 mL, 84 mmol) was added to a solution of 65g
(13.5 g, 65 mmol) in DMF (50 mL). Potassium carbonate (20 g,
145 mmol) was added, and the suspension was stirred at room
temperature for 4 h. The resulting suspension was poured into water
(200 mL) to give a pale-orange precipitate. The solids were removed
by filtration, washed with water, and then dissolved in dichlo-
romethane (150 mL), and the solution was washed with water (2
× 100 mL) and saturated NaCl(aq) solution (100 mL). The solution
was dried over anhydrous, and the solvents were removed in vacuo
to afford a pale-red oil. Benzylation was not complete at this stage,
the crude product was dissolved in 2-methyl-2-propanol (100 mL),
and potassium tert-butoxide (7.5 g, 67 mmol) was added to give a
pale-yellow precipitate. Benzyl bromide (8 mL, 67 mmol) was
added, and the mixture was heated at reflux for 1 h. The resulting
suspension was allowed to cool and was poured into water (250
mL) to give a pale-orange precipitate. The solids were removed
by filtration, washed with water, dissolved in EtOAc (150 mL),
and washed sequentially with water (2 × 200 mL) and saturated
NaCl(aq) solution (100 mL). The solution was dried over anhydrous
Na2SO4 and filtered, and the filtrate solvents were removed in vacuo.
Trituration with EtOH afforded a pale-pink solid, which was
collected by filtration and dried in vacuo (at 40 °C) to give the
title compound 66g (9.1 g, 36%) as a pale-pink powder: LCMS tR
) 3.03 min; m/z ) 389.3[M + H]+; 1H NMR (400 MHz, CDCl3)
δ 1.38 (s, 9H), 2.57 (s, 3H), 5.08 (s, 2H), 5.11 (s, 2H), 6.53 (s,
1H), 7.33–7.42 (m, 10 H), 7.85 (s, 1H).
5-(2,4-Bis(benzyloxy)-5-tert-butylphenyl)-4-iodoisoxazole-3-
carboxylic Acid Ethylamide (35g). This compound was made
using the procedure described for compound 35d. Thus, 34g (9.9
g, 20.4 mmol) was reacted with NIS and CAN to afford the title
compound 35g (7.75, 62%) as an off-white powder: LCMS tR
)
1
3.07 min; m/z ) 610[M + H]+; H NMR (400 MHz, CDCl3) δ
1.26 (t, 3H, J ) 7.1 Hz), 1.41 (s, 9H), 3.50 (dq, 2H, J ) 7.1, 5.4
Hz), 5.07 (s, 4H), 6.60 (s, 1H), 6.80 (brt, 1H, J ) 5.4 Hz), 7.28–7.44
(m, 11H).
5-(2,4-Bis(benzyloxy)-5-tert-butylyphenyl)-4-(4-formylphen-
yl)isoxazole-3-carboxylic Acid Ethylamide (36g). Aqueous potas-
sium phosphate solution (25 mL, 1.2 M) was added to a solution
of 35g (6.1 g, 10 mmol) and 4-formylphenyl boronic acid (2.35 g,
15.7 mmol) in 1,4-dioxane (75 mL) under a nitrogen atmosphere.
Dichlorobis(tri-O-tolylphosphine)palladium(II) (cat.) was added, and
the mixture was heated at 100 °C (oil bath temperature) for 1 h.
The mixture was allowed to cool, and the aqueous layer was
separated and extracted with EtOAc (100 mL). The combined
organic phases were concentrated in vacuo to give a pale-brown
gum, which was purified by flash column chromatography on silica
gel, eluting with EtOAc/hexane (1:3), to give the title compound
36g (5.18 g, 88%) as a pale-yellow foam: LCMS tR ) 3.01 min;
1
m/z ) 589.4[M + H]+; H NMR (400 MHz, CDCl3) δ 1.26 (t,
3H, J ) 7.1 Hz), 1.27 (s, 9H), 3.44 (dq, 2H, J ) 7.1, 5.4 Hz), 4.67
(s, 2H), 5.00 (s, 2H), 6.44 (s, 1H), 6.82 (brt, 1H, J ) 5.4 Hz), 7.00
(m, 2H), 7.25–7.27 (m, 4H), 7.33–7.39 (m, 5H), 7.43 (d, 2H, J )
6.9 Hz), 7.72 (d, 2H, J ) 6.9 Hz), 9.96 (s, 1H).
4-(2,4-Bis(benzyloxy)-5-tert-butylphenyl)-2-hydroxy-4-oxo-
but-2-enoic Acid Ethyl Ester (67g). Sodium ethoxide (2.8 g, 41
mmol) was added to a suspension of 66g (7.8 g, 20 mmol) in EtOH
(40 mL). Diethyl oxalate (4 mL, 29.5 mmol) was added, and the
resulting suspension was heated at reflux for 2 h to give a pale-red
solution, which was allowed to cool and then poured into water
(200 mL). The mixture was acidified with hydrochloric acid (50
mL, 1.0 M) and extracted with dichloromethane (150 mL). The
organic extracts were washed with water (2 × 200 mL) and then
5-(2,4-Dihydroxy-5-ethylphenyl)-4-(4-morpholin-4-ylmethyl-
phenyl)isoxazole-3-carboxylic Acid Ethylamide (40g). This com-
pound was made using the procedure described for compound 30f,
using morpholine in reaction with compound 36g. The crude
product after debenzylation was purified by preparative HPLC to
afford the title compound 40g (21 mg, 75%) as an off-white solid
1
(formate salt): LCMS tR ) 1.97 min; m/z ) 480.5[M + H]+; H
NMR (400 MHz, DMSO-d6) δ 1.05 (t, 3H, J ) 7.2 Hz), 1.1 (s,
9H), 2.3 (brs, 4H), 3.2 (dq, 2H, J ) 7.2 Hz, 5.6 Hz), 3.45 (brs,

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 217
(5) Kamal, A.; Boehm, M. F.; Burrows, F. J. Therapeutic and diagnostic
implications of Hsp90 activation. Trends Mol. Med. 2004, 10, 283–
290.
4H), 6.45 (s, 1H), 6.7 (s, 1H), 7.15 (d, 2H, J ) 7.2 Hz), 7.25 (d,
2H, J ) 7.2 Hz), 8.8 (t, 1H, J ) 5.6 Hz); HRMS calcd for
C27H33N3O5[M + H]+ 480.2483, found 480.2500; HPLC 91% (tR
) 4.65 min).
(6) Chiosis, G.; Vilenchik, M.; Kim, J.; Solit, D. Hsp90: the vulnerable
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100, 57–70.
5-(5-Ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-morpholin-4-
ylmethylphenyl)isoxazole-3-carboxylic Acid Ethylamide (70);
5-(5-Ethyl-4-hydroxy-2-methoxyphenyl)-4-(4-morpholin-4-ylm-
ethylphenyl)isoxazole-3-carboxylic Acid Ethylamide (71); 5-(5-
Ethyl-2,4-dimethoxyphenyl)-4-(4-morpholin-4-ylmethylphenyl-
)isoxazole-3-carboxylic Acid Ethylamide (72). To an argon-
charged flask containing 5-(5-ethyl-2,4-dihydroxyphenyl)-4-(4-
morpholin-4-ylmethylphenyl)isoxazole-3-carboxylic acid ethylamide
(42d) (25 mg, 0.055 mmol) and N,N-(diisopropyl)aminometh-
ylpolystyrene[PS-DIEA] (35 mg, 3.83 mmol/g, 2.4 equiv) were
added anhydrous DCM (2.3 mL) and anhydrous MeOH (0.25 mL).
During gentle stirring, 2 M (trimethylsilyl)diazomethane in hexanes
(28 µL, 1.0 equiv) was added, and the solution was stirred overnight
at room temperature. Argon was bubbled through the solution for
10 min, the resin was filtered off, and the volatiles were removed
in vacuo. The crude residue was purified by semipreparative HPLC
to isolate the three methylated products. 70 (1.14 mg, 4%): LCMS
(method B) tR ) 5.13 min; m/z ) 466.3[M + H]+; 1H NMR (250
MHz, acetone-d6) δ 1.10 (t, 3H, J ) 7.5 Hz), 1.25 (t, 3H, J ) 7.5
Hz), 2.42 (m, 4H), 2.50 (q, 2H, J ) 7.5 Hz), 3.10–3.40 (m, 4H),
3.45 (q, 2H, J ) 7.5 Hz), 3.82 (s, 2H), 3.95 (s, 2H), 4.10–4.40 (m,
4H), 6.65 (s, 1H), 7.05 (s, 1H), 7.40 (d, 2H, J ) 8.2 Hz), 7.70 (d,
2H, J ) 8.2 Hz), 7.90 (brs, 1H), 8.75 (s, 1H); HRMS calcd for
C26H32N3O5[M + H]+, 466.2342, found 466.2342. 71 (5.5 mg,
21%): LCMS (method B) tR ) 4.88 min; m/z ) 466.3[M + H]+;
1H NMR (250 MHz, acetone-d6) δ 0.95 (t, 3H, J ) 8.4 Hz), 1.06
(t, 3H, J ) 7.1 Hz), 2.25–2.29 (m, 4H), 2.40 (q, 2H J ) 7.6 Hz),
3.26 (q, 2H, J ) 7.2 Hz) 3.27 (s, 3H), 3.35 (s, 2H), 3.46–3.3.49
(m, 4H), 6.42 (s, 1H), 6.95 (s, 1H), 7.12 (s, 4H), 8.00 (brs, 1H).
72: (1.46 mg, 5%): LCMS (method B) tR ) 5.44 min; m/z ) 480.3;
1H NMR (500 MHz, CD3OD) δ 1.08 (t, 3H, J ) 7.4 Hz), 1.19 (t,
3H, J ) 8.1 Hz), 2.48 (brs, 4H), 2.53 (q, 2H, J ) 8.1 Hz), 3.37 (q,
2H, J ) 7.4 Hz), 3.48 (s, 3H), 3.54 (s, 2H), 3.69 (t, 4H, J ) 5.4
Hz), 3.89 (s, 3H), 6.58 (s, 1H), 7.09 (s, 1H), 7.21(d, 2H, J ) 8.1
Hz), 7.29 (d, 2H, J ) 8.1 Hz); HRMS calcd for C27H34N3O5
480.2498[M + H]+, found 480.2492.
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Acknowledgment. The authors are grateful to Heather
Simmonite, Tim Haymes, and Amin Mirza for analytical
support, to Adam Hold for compound purification, and to Kate
Grant for assay determinations. This work was supported by
Cancer Research UK[CUK] program, Grant Numbers CA309/
A2187 and C309/A8274. We are also grateful to our colleagues
for valuable discussions. Paul Workman is a Cancer UK Life
Fellow.
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molecular chaperone: attacking the master regulator in cancer. Curr.
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geldanamycin (NSC 330507) by murine and human hepatic prepara-
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Supporting Information Available: Experimental procedures
for the FP assay and synthesis of the FP assay probe. Methods and
parameters relating to X-ray crystallography and protein production
are also provided as are graphical representations of PK cassette
dosing results for cassette 3. Full chemistry experimental procedures
and characterization data are presented in this section. This
information is available free of charge via the Internet at http://
pubs.acs.org.
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