Rationally designed high-affinity 2-amino-6-halopurine heat shock protein 90 inhibitors that exhibit potent antitumor activity

Article abstract of DOI:10.1021/jm050752+

Heat shock protein 90 (Hsp90) is a molecular chaperone protein implicated in stabilizing the conformation and maintaining the function of many cell-signaling proteins. Many oncogenic proteins are more dependent on Hsp90 in maintaining their conformation, stability, and maturation than their normal counterparts. Furthermore, recent data show that Hsp90 exists in an activated form in malignant cells but in a latent inactive form in normal tissues, suggesting that inhibitors selective for the activated form could provide a high therapeutic index. Hence, Hsp90 is emerging as an exciting new target for the treatment of cancer. We now report on a novel series of 2-amino-6-halopurine Hsp90 inhibitors exemplified by 2-amino-6-chloro-9-(4-iodo-3,5-dimethylpyridin- 2-ylmethyl)purine (30). These highly potent inhibitors (IC₅₀ of 30 = 0.009 μM in a HER-2 degradation assay) also display excellent antiproliferative activity against various tumor cell lines (IC₅₀ of 30 = 0.03 μM in MCF7 cells). Moreover, this class of inhibitors shows higher affinity for the activated form of Hsp90 compared to our earlier 8-sulfanylpurine Hsp90 inhibitor series. When administered orally to mice, these compounds exhibited potent tumor growth inhibition (>80%) in an N87 xenograft model, similar to that observed with 17-allylamino-17- desmethoxygeldanamycin (17-AAG), which is a compound currently in phase I/II clinical trials.

Full text of DOI:10.1021/jm050752+

J. Med. Chem. 2007, 50, 2767-2778
2767
Rationally Designed High-Affinity 2-Amino-6-halopurine Heat Shock Protein 90 Inhibitors That
Exhibit Potent Antitumor Activity
Srinivas R. Kasibhatla,*,† Kevin Hong,† Marco A. Biamonte,† David J. Busch,‡ Patricia L. Karjian,‡ John L. Sensintaffar,‡
Adeela Kamal,‡ Rachel E. Lough,§ John Brekken,§ Karen Lundgren,§ Roy Grecko,| Gregg A. Timony,§ Yingqing Ran,#
Robert Mansfield,# Lawrence C. Fritz, Edgar Ulm,∧ Francis J. Burrows,‡ and Marcus F. Boehm†
Departments of Chemistry, Molecular DiscoVery, Pharmacology, Toxicology, Pharmaceutical DeVelopment and Oncology R&D,
Biogen Idec, Inc., 5200 Research Place, San Diego, CA 92122
ReceiVed August 1, 2005
Heat shock protein 90 (Hsp90) is a molecular chaperone protein implicated in stabilizing the conformation
and maintaining the function of many cell-signaling proteins. Many oncogenic proteins are more dependent
on Hsp90 in maintaining their conformation, stability, and maturation than their normal counterparts.
Furthermore, recent data show that Hsp90 exists in an activated form in malignant cells but in a latent
inactive form in normal tissues, suggesting that inhibitors selective for the activated form could provide a
high therapeutic index. Hence, Hsp90 is emerging as an exciting new target for the treatment of cancer. We
now report on a novel series of 2-amino-6-halopurine Hsp90 inhibitors exemplified by 2-amino-6-chloro-
9-(4-iodo-3,5-dimethylpyridin-2-ylmethyl)purine (30). These highly potent inhibitors (IC50 of 30 ) 0.009
µM in a HER-2 degradation assay) also display excellent antiproliferative activity against various tumor
cell lines (IC50 of 30 ) 0.03 µM in MCF7 cells). Moreover, this class of inhibitors shows higher affinity for
the activated form of Hsp90 compared to our earlier 8-sulfanylpurine Hsp90 inhibitor series. When
administered orally to mice, these compounds exhibited potent tumor growth inhibition (>80%) in an N87
xenograft model, similar to that observed with 17-allylamino-17-desmethoxygeldanamycin (17-AAG), which
is a compound currently in phase I/II clinical trials.
Chart 1
Introduction
As evidence accumulates that many mutated or overexpressed
signaling proteins in malignant cells are dependent on the
molecular chaperone protein Hsp90 for their stabilization and
function, this chaperone has rapidly become the focus of drug
discovery research efforts.1 Indeed, Hsp90 has emerged as an
important cancer target because of its key participation in regu-
lating clinically validated oncogenic proteins such as HER-2,
ER, AR, and Bcr-Abl, as well as other signaling proteins that
play critical roles in malignancy, such as Raf-1, p-Akt, Cdk-4,
mutant p53, and Flt-3.2 These Hsp90 “client” proteins regulate
many cellular processes important to the survival of cancer cells,
including growth, cell cycle progression, and apoptosis. Another
important rationale for targeting Hsp90 is to achieve simulta-
neous destruction of several signaling proteins in tumor cells
so as to overcome the inevitable drug resistance associated with
the activation of redundant signaling pathways.3 Finally, our
recent discovery4 of the preponderance of a high affinity form
of Hsp90 in tumor cells as compared to normal cells makes the
Hsp90 pathway an even more attractive target for cancer therapy
because of the resulting increase in therapeutic index that might
be obtained from tumor cell selectivity. The Hsp90 cellular
protein family consists of four members, Hsp90R, Hsp90â,
Grp94, and Trap1. Structural studies reveal that Hsp90 is com-
posed of three highly conserved domains:5 (1) The N-terminal
lobe that contains an adenine nucleotide binding pocket which
is responsible for the ATPase activity of Hsp90,6 (2) the highly
charged “middle region” that plays an important role in
modulating the ATPase activity,7 (3) the C-terminal lobe that
contains an additional ATP binding pocket identified by its
ability to bind the antibiotic novobiocin.8 Activation of Hsp90
results in the formation of complexes with a series of cochap-
erones including Aha1, p23, Hop, Cdc37/p50, Hsp70, and Hsp40
to form a superchaperone complex that interacts with the client
proteins.4,9 Inhibition of the ATPase activity with high affinity
natural product inhibitors results in proteasome-mediated deg-
radation of the client proteins and subsequent interruption of
their signaling function.3 Several of these natural product
inhibitors of Hsp90 ATPase activity have been reported,
including the ansamycin antibiotic geldanamycin, geldanamycin
analogues, and radicicol.10 A semisynthetic product of geldana-
mycin, 17-allylamino-17-desmethoxygeldanamycin (17-AAG),
* Corresponding author. Tel: 858-401-8302. Fax: 858-795-9650.
E-mail: srinivasrao.kasibhatla@biogenidec.com.
† Department of Chemistry.
‡ Department of Molecular Discovery.
§ Department of Pharmacology.
# Department of Pharmaceutical Development.
| Department of Toxicology.
∧ Oncology R&D.
10.1021/jm050752+ CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/08/2007

2768 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Kasibhatla et al.
Scheme 1a
a Reagents and conditions: (a) K2CO3, DMF, NaI, R-L (L is a leaving
group).
phosphate-binding region, it actually projects toward a pocket
that is not occupied by any of the natural products ATP,
geldanamycin,14b or radicicol.14c In this paper, we will refer to
this pocket as the “aryl-binding pocket”. Structure-activity
relationship (SAR) studies revealed that the N(1) and 6-NH2
groups are essential for activity, since they form two strong
hydrogen bonds with the ‘hinge-region’ of Hsp90. Addition of
a 2-fluoro or 2-chloro substituent on the adenine ring improves
the potency by about 2-fold.13m The aryl group tolerates various
substitution patterns, but a 2-halo-5-methoxy13p or 2-halo-3,4,5-
trimethoxy13m was preferred. Furthermore, the aryl group must
be connected to the purine ring by a linker of exactly one atom
(CH2 or S). This places the aryl group six bonds away from the
NH2 group, and we surmised that this distance is an essential
element of the pharmacophore. Finally, the N(9) side chain
tolerates a wide range of modifications. In our design of new
Hsp90 inhibitors, we sought to capture the critical interactions
of 8-benzylpurines by rearranging the substituents around the
purine ring, as indicated in Figure 1. The first modification
entailed shifting the aryl binding moiety from the C(8) position
to the N(9) position. Taken alone, this transformation resulted
in molecules that did not inhibit Hsp90. However, when the
shift of the aryl binding moiety to the N(9) position was
combined with a commensurate shift of the NH2 group from
the 6- to the 2-position so as to re-established the overall six-
bond distance between the 6-NH2 group and the aryl group,
the newly formed 2-amino-6-halo-9-benzylpurines exhibited
outstanding Hsp90 inhibitory activity, resulting in a marked
increase in in vitro and in vivo potency.
Figure 1. Rational design of 2-amino-9-benzylpurines from 6-amino-
8-benzylpurines.
is currently in phase I/II clinical trials for cancer.11 Although
this compound represents a major advancement in Hsp90-
directed therapy, natural product derivatives based on geldana-
mycin engender a number of difficulties.12 In particular,
geldanamycin derivatives present significant formulation and
delivery challenges associated with their relative insolubility
in aqueous media. Moreover, the dose-limiting toxicities appear
to be hepatotoxicity due to the presence of the reactive quinone
moiety. These reasons have encouraged researchers to develop
second-generation small molecule inhibitors that are structurally
unrelated to the first-generation natural products. We and others
have recently identified a number of synthetic analogues of
Hsp90 inhibitors.13 The first synthetic purine-based inhibitor
analogues, designed by Chiosis et al.13q,m and represented by 1
(PU24FCl), showed low micromolar in vitro potency but still
exhibited some in vivo efficacy.13n Drysdale et al.13h described
pyrazole-based inhibitors such as compound 2, which displayed
excellent binding potency and cell-based activity.13o However,
no in vivo efficacy data on these compounds was reported.
Finally, Biamonte et al.13p and He et al.13r recently disclosed
the 8-(phenylsulfanyl)purine series, exemplified by compound
3, with ionizable groups appended at the end of the N(9)
substituents, which showed improved oral bioavailability and
measurable antitumor activity. Since compound 3 showed only
moderate efficacy in human xenograft models, we continued
with our efforts to develop inhibitors with improved in vivo
potency and selectivity toward the activated form of Hsp90.
Herein, we discuss these efforts and disclose a new series of
exceptionally potent and orally bioavailable Hsp90 inhibitors.
We also wanted to examine a broad range of 9-aryl groups
including heteroaryls in order to introduce diverse pharmaceuti-
cal properties that could yield compounds with drug-like
properties. For example, our recent experiences with 8-benzyl-
adenines showed that they lacked significant solubility in
aqueous media, which led to poor oral absorption.13p We
reasoned that 9-benzyladenines may also be insufficiently sol-
uble and further refined the design of this new series of inhibitors
by replacing the substituted phenyl ring (e.g. 2-halo-3,4,5-
phenyl) with a substituted pyridine ring.
Chemistry
We prepared 2-amino-6-halo-9-substituted purine Hsp90
inhibitor analogues simply by alkylating the 2-amino-6-halo-
purine 4 with an appropriate electrophile in the presence of
K2CO3 and NaI in warm DMF, which gave the desired N(9)
regioisomer 5 as the predominant product (Scheme 1).
The electrophiles, 2,5-dimethoxybenzyl chloride, 3,4,5-tri-
methoxybenzyl chloride, and 2-chloromethyl-4-methoxy-3,5-
dimethylpyridine 6 hydrochloride are commercially available.
Compounds 7-9 were prepared from 2-chloromethyl-4-meth-
oxy-3,5-dimethylpyridine 6 following reported procedures.15
Demethylation of the hydrochloride salt of 6 in refluxing toluene
gave 2-chloromethyl-4-hydroxy-3,5-dimethylpyridine 7, which
was deoxyhalogenated with POCl3 or POBr3 to provide the
Design Strategy
Our goal was to design a more potent series of Hsp90
inhibitors based on the reported structural information14 and on
the information gained by several laboratories13 from working
with 8-benzyl13a,g and 8-sulfanylpurines13a (Figure 1). The X-ray
structure of 1 cocrystallized with Hsp9014a indicates that it binds
to the N-terminal ATP-binding site. As expected, the adenine
ring of 1 binds in the same manner as the adenine ring of ATP.
The 9-pentynyl group emulates the ribose group and forms a
hydrophobic interaction with the side chain of Leu107. Although
the 8-benzyl group of 1 was initially designed to occupy the

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12 2769
Scheme 2a
17 with NCS or NBS or NIS gave the corresponding 2-halo-
3,4,5-trimethoxybenzyl derivatives 18a-c. Nucleophilic dis-
placement of C(6) chlorine atom in compounds 18a and 18b
provided compounds 19a-d. Methylation of 18a with AlMe3
in the presence of Pd(0) gave 6-methyl derivative 19f.17
Alkylation of 2-aminopurine with 3,4,5-trimethoxybenzyl chlo-
ride followed by bromination gave 19e. Demethylation of 20
with BBr3 provided the 4-hydroxy analogue 21, which was
alkylated with MeI or EtI in the presence of K2CO3 to give the
alkylated compounds 22 and 23. Oxidation of 20 gave the
N-oxide 24, which was treated with SOCl2 to give the
chlorinated compound 25.19
a Reagents and conditions: (a) toluene, 120 °C; (b) POCl3 or POBr3,
110 °C, 1.5 h.
Scheme 3a
Results and Discussion
The primary screening assay for structure-activity optimiza-
tion studies measured HER-2 degradation in MCF7 cells. HER-2
was chosen because it is a well-established oncology target20
that is exquisitely dependent on Hsp90. The activity of each
compound was measured by its ability to induce degradation
of HER-2 using a flow cytometric assay.4,10a The IC50 values
reported are the average of two or three experiments and are
within 30% agreement. Additional in vitro assays used to
evaluate the Hsp90 inhibitors are a cell lysate binding assay to
determine the binding affinity to the activated form of Hsp90
and a MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay4,10a to deter-
mine their cytostatic/cytotoxic activity to cancer cells.
To evaluate our design concept, we selected 2-amino-6-
chloropurine as our first adenine-binding moiety. The selection
was based on an earlier finding13a,m that the halogen at the
2-position of the purine ring is important for increased potency.
Our initial selections of N(9) substituents, which are believed
to occupy the ATP-ribose binding pocket, were 2,5-dimethoxy-
benzyl and 3,4,5-trimethoxybenzyl moieties, the same moieties
that were optimized in the 6-amino-8-phenylsulfenylpurine
series.13a,m We were gratified to see that the initial compounds
26 and 17 showed HER-2 degradation activity of 3.0 and 2.0
µM, respectively.
a Reagents and conditions: (a) mCPBA, CH2Cl2, 0 °C; (b) Br2, K2CO3,
CCl4, rt; (c) HNO3, H2SO4, 0-100 °C; (d) 10% Pd/C, EtOH, H2, 60 psi;
(e) HBF4, NaNO2, KI; (f) MeSNa, THF, 70 °C; (g) Pd[P(Ph3)] , AlMe3,
4
THF, 70 °C; (h) HBF4, NaNO2, CuCN; (i) mCPBA, CH2Cl2 (11a f 11i),
rt; (j) Ac2O, 90 °C; (k) K2CO3, MeOH, 50 °C; PPh3, CBr4, CH2Cl2, rt.
corresponding 4-halo compounds chloromethyl-4-chloro-3,5-
dimethylpyridine 8 and chloromethyl-4-bromo-3,5-dimethyl-
pyridine 9 (Scheme 2). 2-Bromomethylpyridines were prepared
from 2,3,5-trimethylpyridine-N-oxide 11b, in three to five steps
using the sequence depicted in Scheme 3. The key step involved
rearrangement of the 2-methylpyridine N-oxide derivative to
the 2-acetoxymethylpyridine derivative using acetic anhydride.16
Subsequent deacetylation and bromination with PPh3/CBr4
provided the 4-bromomethylpyridines electrophiles 13a-i. The
intermediate 2,3,5-trimethylpyridine N-oxide 11b could be
functionalized in a variety of ways. Bromination provided the
intermediate 2,3,5-trimethyl-4-bromopyridine N-oxide 11c. Treat-
ment of the 4-bromopyridine 11c with AlMe3 in the presence
of Pd(0) followed by three-step sequence depicted in Scheme
3, gave the 3,4,5-trimethylpyridine derivative 13d.17 Similarly,
reaction of 11c with MeSNa provided the 4-thiomethylpyridine
11a. Nitration of 11b gave the 4-nitropyridine 11e, which was
reduced to the 4-aminopyridine 11f and subjected to a Sand-
meyer reaction to yield the 4-iodopyridine 11g. The three-step
sequence of rearrangement, deacetylation, and deoxybromination
was applied to 11a-g to provide the bromomethylpyridines
13a-g.
The 5-chloromethylpyridine 15, a regioisomer of 6 in which
the CH and N atom are formally permuted, was prepared in
three steps starting from pyridoxine hydrochloride (Scheme 4).
The intermediate chloromethylpyridine 14 was readily prepared
by the reduction of pyridoxine hydrochloride with hydrazine
followed by chlorination with thionyl chloride using reported
procedures.18 Methylation of the 3-hydroxypyridine 14 under
Mitsunobu conditions gave the 3-methoxy-5-chloromethylpy-
ridine 15. Alkylation of 2-amino-6-chloropurine with electro-
phile 15 using the standard conditions gave compound 16.
Modifications at the C(6)-position and substitutions at N(9)
of the purine ring were made at a later stage of the synthesis
(Schemes 5 and 6). Aromatic ring halogenation of the compound
These compounds exhibited approximately 2-3-fold en-
hanced potency as compared to similar compounds prepared in
our 6-amino-8-phenylsulfanylpurine series.13p With this initial
success, we then optimized the series on the basis of HER-2
degradation activity.
We first examined the effect of halogenation on the benzyl
ring of compound 17 to determine if changes in activity were
similar to those observed in the 6-amino-8-phenylsulfenylpurine
series.13m As expected, introduction of a halogen at the 2-posi-
tion of the benzyl ring increased the potency by 5-40-fold
(compounds 18a-c, Table 1). Optimization of over 30 benzyl
derivatives indicated that the 2-halo-3,4,5-trimethoxybenzyl is
the optimal benzyl derivative at the N(9) position. To understand
the binding role of the chlorine atom at the C(6) position of the
purine ring, we next investigated the size and electronics
requirements of moieties at the C(6) position (Table 2).
Replacement of chlorine with bromine (compound 27) gave a
slight improvement in the potency. However, replacing the
chlorine with other substituents such as NH2 (19a), OCH3 (19b),
OH (19c), SH (19d), H (19e), or CH3 (19f) resulted in decreased
potency. Interestingly, the loss of potency observed with
hydrogen (19e) and methyl (19f) moieties at the C(6) position
suggested that halogen not only contributes to the hydrophobic
interactions but also provides electron-withdrawing character-
istics that effect the electrostatic interaction of the 2-NH2 group
with the adenine binding site. As these benzyl analogues (18a

2770 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Kasibhatla et al.
Scheme 4a
a Reagents and conditions: (a) 95% NH2NH2, reflux; (b) SOCl2, DMF, 80 °C; (c) PPh3, DIAD, MeOH, rt; (d) K2CO3, DMF, NaI, 2-amino-6-chloropurine,
40 °C.
Scheme 5a
a Reagents and conditions: (a) NBS or NCS or NIS, CH2Cl2, rt; (b) NH3 in MeOH, sealed-tube (19a); NaOMe, MeOH, 70 °C (19b); 1 N HCl, H2O, 100
°C (19c); thiourea, EtOH, 80 °C (19d); Pd[P(Ph)3]4, Me3Al, THF, 70 °C (19f).
Scheme 6a
Table 1. Optimization of the N(9) Benzyl Substituents
HER-2
degradation
compd
R
IC50 (µM)a
26
2,5-dimethoxy
3.0
17
3,4,5-trimethoxy
2.0
18a
18b
18c
2-chloro-3,4,5-trimethoxy
2-bromo-3,4,5-trimethoxy
2-iodo-3,4,5-trimethoxy
0.05
0.07
0.34
a The IC50 values are the average of two or three replications and are
within 30% agreement.
a Reagents and conditions: (a) BBr3, CH2Cl2, 0 °C to rt. (b) when R )
H, CH3I, or C2H5I, K2CO3, DMF, 40 °C; (c) when R ) CH3, mCPBA,
CH2Cl2, 0 °C; (d) SOCl2, 100 °C.
of aromaticity and lipophilicity while at the same time introduc-
ing aqueous solubility to the molecule. Replacement of the
benzyl group of compound 18a with a (3,5-dimethyl-4-meth-
oxypyridine)methyl group gave compound 20, which retained
the potency (HER-2 IC50 of 0.03 µM). Having identified an
active 9-heteroaryl moiety, we now focused our SAR studies
on optimizing the potency by varying substituents on the
heteroaryl ring. Starting with the 4′-position, demethylation of
the 4′-OMe of compound 20 to 4′-OH compound 21 led to a
substantial drop in potency (>3000-fold). Similarly, replacement
of the methoxy group with an ethoxy (22) or isopropyloxy group
resulted in a 50-fold potency loss (Table 3). In contrast,
introduction of a bromo (29) or iodo (30) substituent showed a
2-3-fold improvement in potency. A chlorine atom had no
effect on the potency. Although this hydrophobic binding re-
gion tolerated bulkier groups such as bromine and iodine, the
50-fold loss in potency observed with alkyl substituents such
as ethyl (22) and isopropyl (23) indicated a linear space
limitation for this domain. Further support for the presence of
a hydrophobic region was indicated by the significant loss of
and 18b) fulfilled our requirement of low nanomolar cellular
activity, we turned our attention to optimizing the pharmaco-
kinetic parameters of these analogues.
One important goal of this project was to develop an orally
administrable compound. Accordingly, compounds were initially
screened for solubility in serum and simulated gastric and
intestinal fluids. Subsequently, the pharmacokinetic properties
of the compounds were evaluated in mice at a dose of 100
mg/kg as an oral suspension formulated in PWD.21 Serum
samples were obtained during the first 2 h and concentrations
were measured by HPLC fitted with a UV detector and
quantified. Under these conditions, compound 18a was observed
only in very small amounts (<1000 ng/mL at 100 mg/kg dose)
in the plasma. We attributed the poor oral absorption of 18a to
its low aqueous solubility (<1 µg/mL). Similar results were also
observed with other benzyl derivatives, 18b and 18c. Having
had limited success with N(9) benzyl substituted analogues, we
then replaced the N(9)-benzyl group with pyridylmethyl, hoping
that the pyridine ring would maintain the proper combination

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12 2771
Table 4. In Vitro Binding Affinity
Table 2. Optimization of the C(6) Substituents
IC50 (µM)a
HER-2
HER-2
HER-2
degradation
rHsp90
binding
lysate
binding
degradation
degradation
compd
R
compd
R
X
IC50 (µM)a compd
R
X
IC50 (µM)a
17-AAG
PU24FCl
3
16
20
24
25
28
29
30
34
35
36
-
-
-
-
0.007
1.7
0.09
0.02
0.03
0.7
6.0
0.9
0.9
0.9
0.8
0.02
2.5
18b
27
19a
19b
Br Cl
Br Br
Br NH2
Br OMe
0.07
0.055
17.0
45.0
19f
19c
19d
19e
Cl Me
Cl OH
Br SH
1.8
0.7
100.0
50.0
0.15
0.018
0.02
0.03
0.025
0.01
0.002
0.003
0.035
0.04
10.0
4.0
Br
H
4-OMe
1-oxa-4-OMe
4-OMe-6-Cl
4-Cl
a The IC50 values are the average of two or three replications and are
within 30% agreement.
0.08
0.09
NAb
NA
NA
NA
NA
NA
NA
NA
0.035
0.015
0.009
0.042
0.027
>10.0
7.5
4-Br
4-I
4-Me
Table 3. Optimization of the Pyridine Ring Substituents
4-SMe
4-S(O)Me
4-H
37
a The IC50 values are the average of two or three replications and are
within 30% agreement. b NA: data not available.
Table 5. Cell Viability Data
HER-2
HER-2
degradation
degradation
compd
R
IC50 (µM)a compd
R
IC50 (µM)a
17-AAG
PU24FCl
3
16
20
21
22
23
24
25
-
-
-
-
0.007
1.7
0.09
0.02
0.03
100.0
1.5
1.5
0.08
0.09
28
29
30
31
32
33
34
35
36
37
4-Cl
4-Br
4-I
4-NO2
4-NH2
4-CN
4-Me
4-SMe
0.038
0.015
0.009
1.0
5.80
0.99
4-OMe
4-OH
4-OEt
4-O-iPr
1-Oxa-4-OMe
4-OMe-6-Cl
MTS IC50 (µM)a
0.042
0.027
compd
R
MCF7
BT474
4-S(O)Me >10.0
4-H 7.5
17-AAG
PU24FCl
3
-
-
-
-
0.01
1.2
0.5
0.15
0.1
0.1
0.5
0.1
0.03
0.03
0.3
0.01
NAb
0.1
0.05
0.1
0.15
0.5
0.3
0.05
0.03
0.08
0.09
a The IC50 values are the average of two or three replications and are
within 30% agreement.
16
20
24
25
28
29
30
34
35
4-OMe
1-oxa-4-OMe
4-OMe-6-Cl
4-Cl
4-Br
4-I
activity observed for the 4′-unsubstituted analogue 37. Strong
electron-withdrawing or -donating 4′-substituents such as CN
(33), S(O)CH3 (36), NO2 (31), or NH2 (32) also resulted in sig-
nificant loss in potency. As expected, small hydrophobic groups
such as CH3 (34) and SCH3 (35) maintained the potency. To
further probe the available space around the pyridyl ring, we
prepared 6-chloro (25) and 1-oxide (24) analogues of compound
20. The slight drop in the potency as compared to compound
20 indicates that the space around the pyridyl ring is limited.
To confirm that the degradation of HER-2 in cells occurs
via Hsp90 inhibition, we analyzed the binding interaction of
these analogues with Hsp90. The competitive binding assay
using recombinant Hsp90 and a biotinylated geldanamycin probe
showed that these compounds bound to Hsp90 within a narrow
IC50 range from 0.7 to 0.9 µM. The insensitive nature of this
assaysdue to the absence of the full complement of Hsp90
cochaperones4smade it unsuitable to generate meaningful
structure-activity relationships. As a result we switched to a
cell lysate binding assay,4 which comprised the full complement
of cochaperones complexed with Hsp90 and provided high
affinity binding activity. In this assay, our compounds showed
excellent correlation between the cell lysate binding activity and
the HER-2 degradation activity in cells (Table 4).
4-Me
4-SMe
0.5
a The IC50 values are the average of two or three replications and are
within 30% agreement. b NA: data not available.
cancer cell lines, MCF7 and BT474 (Table 5), using the MTS
assay. The IC50s ranged from 0.03 to 0.5 µM, and the results
also correlated well with those obtained from the HER-2
degradation assay as well as with the Hsp90 cell lysate
competitive binding assay.
A measure of in vitro therapeutic index for this class of
compounds was determined by comparing their activity in
normal and cancer cells using lysate binding10a and cell
proliferation/cytotoxicity assays. Relative binding affinities for
a subset of compounds were compared in cell lysates extracted
from MCF7 breast cancer cells to cell lysates extracted from
normal human dermal fibroblasts (NDF). The compounds
exhibited 7-400-fold greater affinity for the Hsp90 complex
in the cancer cell line over the normal cells (Table 6). It was
gratifying to note that the higher affinity of binding to the
activated Hsp90 complex in cancer cells translated to selective
Selected compounds were also evaluated for their ability to
inhibit the proliferation of two HER-2 expressing human breast

2772 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Kasibhatla et al.
Table 6. Cell Lysate Binding Selectivity in Normal Cells vs Tumor
Table 9. In Vivo Antitumor Activities of Selected Compounds in N87
Cells
Xenografts
IC50 (µM)a
dose
(mg/kg)
t-test
(p value)
fold
selectivity
compd
schedule
%TGIa
compd
NDF
MCF7
17-AAGb
16c
90
60
125
ip qdx5
po qdx5
po qdx5
70
83
87
0.02
0.001
0.0001
17-AAG
PU24FCl
20
29
30
1.0
18.0
0.4
0.8
1.0
0.02
2.5
0.020
0.002
0.003
50
7
20
400
333
20c
a TGI, tumor growth inhibition. b 17-AAG was given intraperitoneally
in PMSE. c Compound administered as a 0.1 N aqueous HCl solution.
a The IC50 values are the average of two or three replications and are
within 30% agreement.
These compounds were also screened for metabolic stability
in human liver microsomes (HLM). Upon incubation with
HLM, low intrinsic metabolism was observed with both
compounds, which suggests potentially high oral bioavailability
in humans.
Table 7. Selective Antiproliferative Activity in Tumor Cells vs Normal
Cells
IC50 (µM)a
selectivity
compd
RPTEC
HT29
MCF7
HT29
MCF7
In Vivo Antitumor Activity. On the basis of their encourag-
ing pharmaceutics profiles, compounds 16 and 20 were selected
for further evaluation in a mouse xenograft cancer model. The
human stomach carcinoma N87 was selected for the evaluation
due to its relatively high expression of HER-2, which is a
sensitive Hsp90 client protein. Tumor fragments were implanted
subcutaneously and treatment started once the tumor size
reached 80-100 mm3. Following once daily oral dosing of
compound 16 at 60 mg/kg and compound 20 as a mesylate salt
at 125 mg/kg on a weekly 5-day-on and 2-day-off schedule for
5 weeks, both compounds showed statistically significant tumor
growth inhibition (Table 9). Both compounds were well-
tolerated, exhibiting no treatment-related toxicity at these doses
other than minimal weight loss (3-5%) over the course of the
study.
17-AAG
0.1
0.6
5.0
1.3
0.15
2.3
0.002
0.13
0.5
0.13
0.023
0.2
0.01
0.1
0.1
0.03
0.03
0.5
50
5
10
10
7
10
6
50
43
5
20
28
29
30
35
12
5
a The IC50 values are the average of two or three replications and are
within 30% agreement.
Table 8. Mouse Oral Pharmacokinetic Properties and Stability in HLM
HLMb
% parent
remaining
Cmax
(µg/mL)
Tmax
(h)
AUC
(µg h/mL)
T1/2
(h)
compda
16
20
28.2
10.0
0.1
0.1
9.0
4.8
0.9
0.8
83
86
a Dose of 100 mg/kg as a 0.1 N aqueous HCl solution. b Drug incubated
for 1 h in HLM (human liver microsomes) at 37 °C.
Conclusion
In summary, we rationally designed and identified a novel
2-amino-6-halo-9-substituted purine scaffold that exhibits potent
Hsp90 inhibitory activity. Compounds of this structural series
also significantly bias the binding selectivity toward the activated
form of the Hsp90 complex, which is reflected in increased
tumor cell retention and, subsequently, more effective tumor
cell killing. Among these highly selective inhibitors, compounds
16 and 20 displayed a superior overall profile as measured by
in vitro potency, pharmaceutical properties, and in vivo oral
efficacy. This new series presented an additional advantage in
that it significantly simplified the synthetic chemistry by
reducing the number of synthetic steps and avoided the diffi-
cult coupling reaction between 8-bromoadenine and the thiophe-
nols.22 More detailed biochemical, biological, pharmacokinetics,
and pharmacodynamic characterizations of these compounds,
as well as additional data from a range xenograft models will
be published separately.23,24
antiproliferative activity in human breast (MCF7) and colon
(HT29) carcinoma cells over normal human renal proximal
epithelial cells (RPTEC) (Table 7). In cell growth assays, the
compounds were 5-50-fold selective in inhibiting cancer cell
growth over that of even highly proliferative untransformed
cells, suggesting that a reasonable therapeutic index could be
expected in vivo.
As these compounds bound to the N-terminal ATP binding
site of Hsp90, we were interested in evaluating the specificity
for Hsp90 over other ATP binding proteins. The selectivity
profile of compound 20 was tested at a single concentration of
10 µM in a small panel of kinases: aurora-A, CHK2, IKKR,
MAPK1, MAPK2, MEK1, PDK1, Plk3, PI3k, c-Raf, and cSrc.
No significant inhibition was observed on any of the targets
after 1 h of incubation with 10 µM ATP concentration (data
not shown).
Pharmacokinetic Studies. The compounds in Tables 3-5
that met our potency and binding criteria (<100 nM in
both) were screened for their solubility in simulated gastric
(pH 2.0) and intestinal (pH 6.5) fluids and in pooled normal
mouse serum (pH 7.4). Compounds 16 and 20 showed good
overall solubility (gastric, 451 and 240 µg/mL; intestinal, 234
and 87 µg/mL; and serum, 186 and 176 µg/mL, respectively)
and hence were selected for the mouse in vivo pharmacoki-
netic evaluation. In the pharmacokinetic study Balb/C mice were
administered a single 100 mg/kg oral dose of each compound
as a 0.1 N aqueous HCl solution (pH 1.2). Blood samples were
drawn at 5, 15, 30, 60, and 120 min, and total parent drug
concentration was determined in serum by HPLC-UV. Ter-
minal half-life and AUC values (Table 8) were estimated using
a noncompartmental method (WinNonlin, Version 4.1). Both
compounds 16 and 20 showed good oral exposure in mice.
Experimental Section
Chemistry. General Methods. All reactions were carried out
with continuous stirring under an atmosphere of nitrogen. Com-
mercial reagents and solvents were used as received without further
purification or drying. 1H and 13C NMR spectra were obtained using
a Bruker-400 spectrometer. Chemical shifts are reported in parts
per million relative to tetramethylsilane as internal standard. Thin-
layer chromatography (TLC) was performed on 250 µM silica gel
plates (Whatman 4861-820). Flash chromatography was run using
EM Science silica gel (230-400 mesh). Compound purity was
determined by HPLC analysis using a C18 reverse phase column
(Agilent Zorbax 300SB-C18; 5 µm; 4.6 mm × 150 mm) with an
Agilent 1100 series system attached to a Hewlett-Packard chro-
matograph manager. A gradient was applied between solvent A
(0.1% TFA in H2O) and solvent B (0.5% TFA in CH3CN)
increasing the proportion of A linearly from 5% (t ) 0) to 100%

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12 2773
(t ) 7.00 min), with a constant flow rate of 1 mL/min. The samples
were diluted to 0.1-1 mg/mL in MeOH or CH3CN and the injection
volumes were typically 10 µL. Elemental analyses were obtained
from Robertson Microlit Laboratories (Madison, NJ) and were
within 0.4% of theoretical values.
2-Chloromethyl-4-chloro-3,5-dimethylpyridine (8). A suspen-
sion of 715 (10 g, 58.3 mmol) in POCl3 (30 mL, 551 mmol) was
stirred at 110 °C for 1.5 h. The resulting mixture was cooled to
room temperature and poured onto ice water and the pH was slowly
adjusted to 10 with KOH. The compound was extracted into
chloroform and dried over MgSO4. The solvent was evaporated
under reduced pressure and the crude product 8 (8.1 g, 74%
recovered yield) was used in the next step without purification:
HPLC tR 5.54 min; 1H NMR (CDCl3) δ 8.24 (s, 1H), 4.71 (s, 2H),
2.48 (s, 3H), 2.36 (s, 3H).
then cooled to room temperature, poured onto ice, and extracted
with chloroform. The organic layer was washed with water followed
by saturated NaHCO3, dried over MgSO4, filtered, and concentrated
to give 11e (4.89 g, 97%): Rf (MeOH/EtOAc 1:9) 0.7; HPLC tR
1
4.75 min; H NMR (CDCl3) δ 8.08 (s, 1H), 2.50 (s, 3H), 2.27 (s,
3H), 2.23 (s, 3H); MS m/z 183.1 (M + H)+.
2,3,5-Trimethylpyridin-4-ylamine N-Oxide Hydrochloride
(11f). A suspension of 11e (4.2 g, 23 mmol) in 60 mL of conc.
HCl/EtOH (1:11) was hydrogenated over 10% Pd/C (0.42 g) at a
60 psi H2 pressure for 3 h. The reaction mixture was filtered through
Celite and evaporated to give 11f as a light yellow solid (4.30 g,
1
quantitative yield): HPLC tR 4.75 min; H NMR (DMSO-d6) δ
8.28 (s, 1H), 7.24 (s, 2H), 2.50 (s, 3H), 2.12 (s, 3H), 2.11 (s, 3H);
MS m/z 153.2 (M + H)+.
4-Iodo-2,3,5-trimethylpyridine N-Oxide (11g). To a cold (5
°C) solution of 11f (1.9 g, 10 mmol) and HBF4 (3.6 mL, 20 mmol)
in 50 mL of water was added a solution of NaNO2 (1.04 g, 20
mmol) in 5 mL of water dropwise to give a dark yellow solution.
Potassium iodide (2.3 g, 13.9 mmol) was slowly added in several
portions to the suspension and the mixture turned dark brown. The
reaction mixture was stirred at room temperature for an additional
5 min and then heated to 60 °C for 10 min. The mixture was cooled
to room temperature and the pH was adjusted to 10 with KOH.
The organics were extracted with chloroform, washed with saturated
NaHCO3 and then water, dried over MgSO4, and concentrated to
4-Bromo-2-chloromethyl-3,5-dimethylpyridine (9). A neat
mixture of 715 (8.2 g, 47.8 mmol) and POBr3 (60 g, 209 mmol)
was stirred at 130 °C for 3 h. The resulting mixture was cooled to
room temperature and poured onto ice water and the pH was slowly
adjusted to 10 with KOH. The compound was extracted into
chloroform and dried over MgSO4. The solvent was evaporated
under reduced pressure and the crude product was purified by flash
1
chromatography to give 9 (8.7 g, 78%): HPLC tR 6.03 min; H
NMR (CDCl3) δ 8.20 (s, 1H), 4.62 (s, 2H), 2.50 (s, 3H), 2.38 (s,
3H).
1
give 11g (0.37 g, 14%): HPLC tR 5.57 min; H NMR (CDCl3) δ
2,3,5-Trimethyl-4-methylsulfanylpyridine N-Oxide (11a). A
solution of 11c (1.00 g, 4.63 mmol) in THF (25 mL) was treated
in a pressure vessel with MeSNa (0.57 g, 8.13 mmol) at 110 °C
for 16 h. The vessel was cooled to room temperature and the
reaction mixture was transferred to a round-bottomed flask. The
solvent was evaporated under reduced pressure and the crude
product was purified by flash chromatography to give 11a (0.74 g,
8.07 (s, 1H), 2.62 (s, 3H), 2.56 (s, 3H), 2.38 (s, 3H); MS m/z 264.1
(M + H)+.
4-Cyano-2,3,5-trimethylpyridine N-Oxide (11h). Compound
11h was prepared in 17% yield from 11f and CuCN in HBF4
following the procedure described for 11g but replacing KI with
CuCN: HPLC tR 4.41 min; 1H NMR (CDCl3) δ 8.10 (s, 1H), 2.55
(s, 3H), 2.49 (s, 3H), 2.45 (s, 3H); MS m/z 162.2 (M + H)+.
4-Methanesulfinyl-2,3,5-trimethylpyridine N-Oxide (11i). To
solution of 2,3,5-trimethyl-4-methylsulfanylpyridine N-oxide 11a
(0.26 g, 1.4 mmol) in 10 mL of CH2Cl2 was added mCPBA (1.0 g,
4.1 mmol). After 10 min at room temperature the mixture was
extracted with dichloromethane, washed with NaHCO3 and water,
and dried over MgSO4. The solvent was removed and the residue
was purified by flash chromatography (10% MeOH/EtOAc) to give
1
87%): HPLC tR 5.30 min; H NMR (CDCl3) δ 8.07 (s, 1H), 2.57
(s, 3H), 2.52 (s, 3H), 2.42 (s, 3H), 2.23 (s, 3H).
2,3,5-Collidine N-Oxide (11b). A solution of 10 (6.10 g, 50
mmol) in dichloromethane (50 mL) was cooled in an ice bath,
treated with mCPBA (19 g, 55 mmol) in three portions, and allowed
to warm to room temperature. The mixture was extracted with
dichloromethane, washed with saturated NaHCO3 and water, and
dried over MgSO4 to give 11b (4.8 g, 70%): HPLC tR 3.96 min;
1H NMR (CDCl3) δ 8.03 (s, 1H), 6.90 (s, 1H), 2.47 (s, 3H), 2.31
(s, 3H), 2.24 (s, 3H); MS m/z 138.2 (M + H)+; Rf (20% MeOH/
EtOAc) 0.35.
4-Bromo-2,3,5-collidine N-Oxide (11c). To a suspension of 11b
(1.3 g, 10 mmol) and K2CO3 (2.9 g, 20 mmol) in 10 mL of CCl4,
was added dropwise 20 mL (20 mmol) of a 1 M bromine solution
in CCl4. The reaction mixture was heated at reflux for 2 h, cooled
to room temperature, extracted with ethyl acetate, washed with
saturated NaHCO3 (10 mL) and then water, and dried over MgSO4.
The solvent was evaporated under reduced pressure and the crude
product was purified by flash chromatography (10% MeOH/EtOAc)
to afford 11c (1.05 g, 51%): HPLC tR 5.23 min; 1H NMR (CDCl3)
δ 8.06 (s, 1H), 2.56 (s, 3H), 2.43 (s, 3H), 2.31 (s, 3H); Rf (20%
MeOH/EtOAc) 0.45.
2,3,4,5-Tetramethylpyridine N-Oxide (11d). To a mixture of
11c (2 g, 9.2 mmol) and tetrakis(triphenylphosphino)palladium (80
mg, 4 mol %) in 20 mL of dry THF was added a toluene solution
of trimethylaluminum (2 M, 7.6 mL, 15.2 mmol) under nitrogen.
The solution was heated at reflux for 4 h, diluted with toluene (20
mL), and quenched with 4 mL of methanol followed by 6 mL of
aqueous ammonium chloride (24 mmol). The resulting mixture was
further heated to reflux for 2 h, cooled, and filtered through Celite.
The filtrate was concentrated, and the residue was taken into
dichloromethane, washed with water, dried over MgSO4, filtered,
and concentrated to afford 11d (0.8 g, 57%): HPLC tR 4.18 min;
1H NMR (CDCl3) δ 8.05 (s, 1H), 2.55 (s, 3H), 2.27 (s, 3H), 2.22
(s, 3H), 2.20 (s, 3H).
1
11i (0.24 g, 86%): HPLC tR 3.07 min; H NMR (CDCl3) δ 8.07
(s, 1H), 2.93 (s, 3H), 2.58 (s, 3H), 2.52 (s, 6H).
Acetic Acid 3,5-Dimethyl-4-methylsulfanylpyridin-2-ylmethyl
Ester (12a). A solution of 11a (240 mg, 1.3 mmol) in acetic
anhydride (3 mL) was heated at reflux for 0.5 h. Excess acetic
anhydride was evaporated under reduced pressure and the residue
was purified by flash chromatography to afford 12a (292 mg,
1
quantitative yield): HPLC tR 4.34 min; H NMR (CDCl3) δ 8.27
(s, 1H), 5.20 (s, 2H), 2.57 (s, 3H), 2.46 (s, 3H), 2.25 (s, 3H), 2.10
(s, 3H).
Acetic Acid 3,5-Dimethylpyridin-2-ylmethyl Ester (12b). The
title compound 12b was prepared in quantitative yield from
compound 11b (150 mg, 1.1 mmol) using the method described
1
for the synthesis of 12a: HPLC tR 2.92 min; H NMR (CDCl3) δ
8.29 (d, J ) 1.2 Hz, 1H), 7.33 (d, J ) 1.2 Hz, 1H), 5.21 (s, 2H),
2.34 (s, 3H), 2.32 (s, 3H), 2.13 (s, 3H).
Acetic Acid 3,4,5-Trimethylpyridin-2-ylmethyl Ester (12d).
The title compound 12d was prepared in 46% yield from compound
11d using the method described for the synthesis of 12a: HPLC
1
tR 3.84 min; H NMR (CDCl3) δ 8.20 (s, 1H), 5.22 (s, 2H), 2.26
(s, 3H), 2.25 (s, 3H), 2.21 (s, 3H), 2.11 (s, 3H).
Acetic Acid 4-Iodo-3,5-dimethylpyridin-2-ylmethyl Ester
(12g). The title compound 12g was prepared in 50% yield from
11g using the method described for the synthesis of 12a: HPLC tR
1
2.91 min; H NMR (CDCl3) δ 8.26 (s, 1H), 5.32 (s, 2H), 2.47 (s,
3H), 2.41 (s, 3H), 2.24 (s, 3H); MS m/z 306.0 (M + H)+.
Acetic Acid 4-Cyano-3,5-dimethylpyridin-2-ylmethyl Ester
(12h). The title compound 12h was prepared in 63% yield from
11h using the method described for the synthesis of 12a: HPLC
2,3,5-Trimethyl-4-nitropyridine N-Oxide (11e). To a suspen-
sion of 11a (3.77 g, 28 mmol) in concentrated H2SO4 (8 mL) cooled
to 0 °C was slowly added dropwise fuming HNO3 (5 mL, 100
mmol). The resulting solution was heated at 100 °C for 24 h and
1
tR 5.37 min; H NMR (CDCl3) δ 8.47 (s, 1H), 5.25 (s, 2H), 2.57
(s, 3H), 2.53 (s, 3H), 2.14 (s, 3H); MS m/z 205.0 (M + H)+.

2774 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Kasibhatla et al.
Acetic Acid 4-Methanesulfinyl-3,5-dimethylpyridin-2-yl-
methyl Ester (12i). The title compound 12i was prepared in 76%
yield from 11i using the method described for the synthesis of
12a: HPLC tR 3.74 min; 1H NMR (CDCl3) δ 8.30 (s, 1H), 5.24 (s,
2H), 2.90 (s, 3H), 2.57 (s, 3H), 2.56 (s, 3H), 2.13 (s, 3H).
2-Bromomethyl-3,5-dimethyl-4-methylsulfanylpyridine (13a).
A mixture of 12a (0.5 g, 2.2 mmol) and K2CO3 (0.57 g, 4.4 mmol)
in methanol (5 mL) was stirred at 50 °C for 0.5 h. The solvent was
evaporated and the residue was taken into chloroform and washed
with water to give (3,5-dimethyl-4-methylsulfanylpyridin-2-yl)-
methanol (0.4 g, quantitative yield): HPLC tR 3.92 min. The crude
mixture was used without any further purification.
reduced pressure and the crude product was purified by flash
chromatography to give 16 (6.2 g, 82%): mp 210-215 °C; HPLC
1
tR 3.74 min; H NMR (CDCl3) δ 8.25 (s, 1H), 7.63 (s, 1H), 5.39
(s, 2H), 5.11 (s, 2H), 3.73 (s, 3H), 2.55 (s, 3H), 2.22 (s, 3H). Anal.
(C14H15ClN6O) C, H, N.
6-Chloro-9-(3,4,5-trimethoxybenzyl)-9H-purin-2-ylamine (17).
A suspension of 2-amino-6-chloropurine (4.8 g, 28.4 mmol), 3,4,5-
trimethoxybenzyl chloride (6.2 g, 28.7 mmol), and K2CO3 (1.0 g,
72.0 mmol) in DMF (30 mL) was heated at 70 °C for 4 h under N2
atmosphere. The solvent was removed under reduced pressure and
the residue was extracted with EtOAc, washed with water, and dried
over MgSO4. Evaporation and flash chromatography gave 17 (6.94
1
A solution of (3,5-dimethyl-4-methylsulfanylpyridin-2-yl)metha-
nol (0.4 g, 2.2 mmol) and triphenylphosphine (1.0 g, 4.0 mmol) in
dichloromethane (6 mL) was cooled to 0 °C. A solution of CBr4
(1.0 g, 3.0 mmol) in dichloromethane (2 mL) was added dropwise
and the mixture was stirred for 1 h at room temperature. The solvent
was evaporated under reduced pressure and the crude product was
purified by flash chromatography to give 13a (0.48 g, 90%): HPLC
g, 70%): HPLC tR 4.91 min; H NMR (CDCl3) δ 7.76 (s, 1H),
6.51 (s, 2H), 5.18 (s, 2H), 5.12 (s, 2H), 3.85 (s, 3H), 3.84 (s, 6H);
HRMS calcd for C15H16ClN5O3 348.0869 (M - H)-, found
348.0867.
6-Chloro-9-(2-chloro-3,4,5-trimethoxybenzyl)-9H-purin-2-
ylamine (18a). A solution of 17 (2.47 g, 7.0 mmol) and N-
chlorosuccinimide (1.0 g, 7.5 mmol) in acetic acid (40 mL) was
stirred at 90 °C for 0.5 h. The acetic acid was removed under
reduced pressure and the residue was extracted with EtOAc and
washed with water. Flash chromatography (3:1 EtOAc/hexanes)
gave the title compound 18a as a white solid (2.09 g, 77%); mp
154-155 °C; HPLC tR 5.63 min; 1H NMR (CDCl3) δ 7.83 (s, 1H),
6.68 (s, 1H), 5.51 (s, 2H), 5.23 (s, 2H), 3.94 (s, 3H), 3.90 (s, 3H),
3.80 (s, 3H). Anal. (C15H15Cl2N5O3) C, H, N.
1
tR 4.90 min; H NMR (CDCl3) δ 8.26 (s, 1H), 4.59 (s, 2H), 2.62
(s, 3H), 2.47 (s, 3H), 2.27 (s, 3H); MS m/z 246.13 (M + H)+.
2-Bromomethyl-3,5-dimethylpyridine (13b). The title com-
pound 13b was prepared in quantitative yield from 12b using the
method described for the synthesis of 13a: HPLC tR 3.90 min; 1H
NMR (CDCl3) δ 8.24 (d, J ) 1.2 Hz, 1H), 7.30 (d, J ) 1.2 Hz,
1H), 4.60 (s, 2H), 2.41 (s, 3H), 2.32 (s, 3H).
2-Bromomethyl-3,4,5-trimethylpyridine (13d). The title com-
pound 13d was prepared in 57% yield from 12d using the method
described for the synthesis of 13a: HPLC tR 3.97 min; H NMR
(CDCl3) δ 8.18 (s, 1H), 4.63 (s, 2H), 2.35 (s, 3H), 2.48 (s, 3H),
2.24 (s, 3H).
6-Chloro-9-(2-bromo-3,4,5-trimethoxybenzyl)-9H-purin-2-
ylamine (18b). To a solution of 17 (2.3 g, 6.6 mmol) in 1 M acetate
buffer (30 mL), THF (60 mL), and MeOH (60 mL) was added
bromine (1.4 g, 8.7 mmol) dropwise over a period of 5 min. The
volatile solvents were removed under reduced pressure and the
aqueous residue was extracted with EtOAc. Flash chromatography
(3:1 EtOAc/hexanes) gave the title compound 18b (2.08 g, 72%)
1
2-Bromomethyl-3,5-dimethyl-4-nitropyridine (13e). The title
compound 13e was prepared in 53% yield from 12e using the
method described for the synthesis of 13a: HPLC tR 6.21 min; 1H
NMR (CDCl3) δ 8.46 (s, 1H), 4.64 (s, 2H), 2.38 (s, 3H), 2.33 (s,
3H).
2-Bromomethyl-4-iodo-3,5-dimethylpyridine (13g). The title
compound 13g was prepared in 60% yield from 12g using the
method described for the synthesis of 13a: HPLC tR 5.95 min; 1H
NMR (CDCl3) δ 8.14 (s, 1H), 4.67 (s, 2H), 2.59 (s, 3H), 2.45 (s,
3H); MS m/z 326.07 (M + H)+.
2-Bromomethyl-4-cyano-3,5-dimethylpyridine (13h). The title
compound 13h was prepared in 30% yield from 12h using the
method described for the synthesis of 13a: HPLC tR 6.12 min; 1H
NMR (CDCl3) δ 8.45 (s, 1H), 4.61 (s, 2H), 2.64(s, 3H), 2.55 (s,
3H).
2-Bromomethyl-4-methanesulfinyl-3,5-dimethylpyridine 1-Ox-
ide (13i). The title compound 13i was prepared in 46% yield (over
two steps) from 12i using the method described for the synthesis
of 13a: HPLC tR 4.33 min. 1H NMR (CDCl3) δ 8.27 (s, 1H), 4.60
(s, 2H), 2.90 (s, 3H), 2.62 (s, 3H), 2.57 (s, 3H).
5-Chloromethyl-3-methoxy-2,4-dimethylpyridine (15). To a
cold suspension of 1418 (11.6 g, 77.9 mmol), triphenylphosphine
(26.8 g, 102 mmol), and MeOH (3.3 g, 100 mmol) in dry THF
(150 mL) was slowly added a solution of diisopropyl azodicar-
boxylate (20.2 g, 100 mmol) in 80 mL of THF over a period of
0.5 h. After 2 h at room temperature, the solvent was removed
under reduced pressure and ether (300 mL) was added to the residue
to precipitate the triphenylphosphine oxide. The triphenylphosphine
oxide was removed by filtration and the ether filtrate was subjected
to an acid-base extraction to give 6.2 g of a crude brown solid.
Filtration through a small plug of silica gel gave 15 (4.85 g, 45%):
HPLC tR 2.87; 1H NMR (CDCl3) δ 8.18 (s, 1H), 4.58 (s, 2H), 3.74
(s, 3H), 2.51 (s, 3H), 2.36 (s, 3H).
6-Chloro-9-(5-methoxy-4,6-dimethylpyridin-3-ylmethyl)-9H-
purin-2-ylamine (16). A suspension of 2-amino-6-chloropurine (4.0
g, 236.0 mmol), compound 15 (4.65 g, 250.0 mmol), and K2CO3
(1.0 g, 72.0 mmol) in DMF (30 mL) was heated at 70 °C for 4 h
under N2 atmosphere. The solvent was evaporated under reduced
pressure and the residue was extracted with EtOAc, washed with
water, and dried over MgSO4. The solvent was evaporated under
1
as a white solid: mp 184-186 °C; HPLC tR 5.74 min; H NMR
(CDCl3) δ 7.85 (s, 1H), 6.66 (s, 1H), 5.32 (s, 2H), 5.11 (s, 2H),
3.90 (s, 3H), 3.87 (s, 3H), 3.76 (s, 3H). Anal. (C15H15BrClN5O3)
C, H, N.
6-Chloro-9-(2-iodo-3,4,5-trimethoxybenzyl)-9H-purin-2-
ylamine (18c). The title compound 18c was prepared in 22% yield
by halogenation of 17 with N-iodosuccinimide using the same
procedure described for 18a: mp 187-190 °C; HPLC tR 5.89 min;
1H NMR (CDCl3) δ 7.87 (s, 1H), 6.67 (s, 1H), 5.33 (s, 2H), 5.22
(s, 2H), 3.91 (s, 3H), 3.88 (s, 3H), 3.77 (s, 3H); HRMS calcd for
C15H15ClIN5O3 475.9981 (M + H)+, found 475.9960.
9-(2-Bromo-3,4,5-trimethoxybenzyl)-9H-purine-2,6-diamine
(19a). A solution of 18b (35 mg, 0.08 mmol) in MeOH was treated
with 3 mL of 7 N NH3 in MeOH in a pressure vessel at 90 °C for
16 h. The vessel was cooled to room temperature and the reac-
tion mixture was transferred to a round-bottom flask. The sol-
vent was evaporated under reduced pressure and the crude product
was purified by preparative TLC (EtOAc:hexane 3:1) to give
1
19a (16 mg, 48%): mp >250 °C; HPLC tR 4.88 min; H NMR
(DMSO-d6) δ 7.67 (s, 1H), 6.71 (s, 2H), 6.60 (s, 1H), 5.84 (s, 2H),
5.17 (s, 2H), 3.80 (s, 3H), 3.76 (s, 3H), 3.66 (s, 3H); HRMS calcd
for C15H17BrN6O3 409.0618 (M + H)+, found 409.0621.
6-Methoxy-9-(2-bromo-3,4,5-trimethoxybenzyl)-9H-purin-2-
ylamine (19b). A solution of 18b (7.0 mg, 0.02 mmol) and
MeO-Na+ (10.0 mg, 0.18 mmol) in MeOH (1 mL) was heated to
reflux for 1 h. The mixture was quenched with 1 N NH4Cl and the
solvent was removed. The residue was taken into dichloromethane
and washed with water, dried over MgSO4, and concentrated. The
crude product was purified by preparative TLC to give 19b (3
1
mg): HPLC tR 5.23 min; H NMR (CDCl3) δ 7.69 (s, 1H), 6.58
(s, 1H), 5.33 (s, 2H), 4.88 (s, 2H), 4.11 (s, 3H), 3.93 (s, 3H), 3.89
(s, 3H), 3.74 (s, 3H); HRMS calcd for C16H18BrN5O4 424.0615
(M + H)+, found 424.0600.
2-Amino-9-(2-chloro-3,4,5-trimethoxybenzyl)-9H-purin-6-ol
(19c). A suspension of 18a (37 mg, 0.1 mmol) in 1 N HCl (2 mL)
was heated at reflux for 2 h, at which point the mixture became
homogeneous. The solvent was evaporated under reduced pressure
and the solid was washed with EtOAc to give 19c (30 mg, 85%):

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12 2775
mp 215-230 °C; HPLC tR 4.60 min; 1H NMR (DMSO-d6) δ 10.61
(s, 1H), 7.82 (s, 1H), 6.65 (s, 1H), 6.60 (s, 2H), 5.15 (s, 2H), 3.81
(s, 3H), 3.77 (s, 3H), 3.69 (s, 3H); HRMS calcd for C15H16ClN5O4
366.0964 (M + H)+, found 366.0955.
iodide (31 mg, 0.2 mmol) dropwise. After stirring at room
temperature for 0.5 h, the mixture was diluted with ethyl acetate,
washed with water, and dried over MgSO4. The solvent was
evaporated under reduced pressure and the crude product was
purified by flash chromatography to give 22 (26 mg, 50%): mp
200-210 °C; HPLC tR 4.32 min; 1H NMR (CDCl3) δ 8.21 (s, 1H),
7.90 (s, 1H), 5.34 (s, 2H), 5.12 (s, 2H), 3.90 (q, 2H), 2.31 (s, 3H)
2.26 (s, 3H), 1.44 (t, 3H). Anal. (C15H17ClN6O‚0.1 H2O) C, H, N.
6-Chloro-9-(4-isopropoxy-3,5-dimethylpyridin-2-ylmethyl)-
9H-purin-2-ylamine (23). The title compound 23 was obtained
by the reaction of 21 (50 mg, 0.16 mmol), K2CO3 (67 mg, 0.5
mmol), and isopropyl iodide (34 mg, 0.2 mmol) in DMF (3 mL)
followed by purification by flash chromatography in the similar
manner described for 22 (22 mg, 40%): mp 177-178 °C; HPLC
6-Mercapto-9-(2-bromo-3,4,5-trimethoxybenzyl)-9H-purin-2-
ylamine (19d). A suspension of 27 (0.3 g, 0.6 mmol) and thiourea
(0.2 g, 2.6 mmol) in EtOH (5 mL) was heated at reflux for 1 h.
The reaction mixture was cooled to room temperature and the
solvent was removed under reduced pressure and the solid residue
was washed with water and EtOAc to give 19d (0.17 g, 63%): mp
200-215 °C; HPLC tR 4.93 min; 1H NMR (DMSO-d6) δ 11.95 (s,
1H), 7.82 (s, 1H), 6.85 (s, 2H), 6.60 (s, 1H), 5.15 (s, 2H), 3.80 (s,
3H), 3.78 (s, 3H), 3.71 (s, 3H). Anal. (C15H16BrN5O3S) C, H, N.
9-(2-Bromo-3,4,5-trimethoxybenzyl)-9H-purin-2-ylamine (19e).
A suspension of 2-aminopurine (100 mg, 1.1 mmol), 3,4,5-
trimethoxybenzyl chloride (200 mg, 1.1 mmol), and K2CO3 (100
mg, 7.2 mmol) in DMF (3 mL) was heated at 70 °C for 4 h under
a nitrogen atmosphere. The solvent was evaporated under reduced
pressure and the residue was extracted with EtOAc, washed with
water, and dried over MgSO4. Evaporation and flash chromatog-
raphy gave 9-(3,4,5-trimethoxybenzyl)-9H-purin-2-ylamine (120
1
tR 4.57 min; H NMR (CDCl3) δ 8.17 (s, 1H), 7.89 (s, 1H), 5.32
(s, 2H), 5.06 (s, 2H), 4.20 (sept., J ) 6.2 Hz, 1H), 2.26 (s, 3H)
2.22 (s, 3H), 1.28-1.30 (d, J ) 6.2 Hz, 6H). Anal. (C16H19ClN6O)
C, H, N.
6-Chloro-9-(4-methoxy-3,5-dimethyl-N-oxypyridin-2-ylmethyl)-
9H-purin-2-ylamine (24). A solution of 20 (4.2 g, 13.0 mmol) in
dichloromethane (100 mL) was cooled with an ice-bath, treated
with mCPBA (4.2 g, 18.3 mmol) in three portions, and allowed
to warm to room temperature. The mixture was extracted
with dichloromethane, washed with saturated NaHCO3 (10 mL),
and dried over MgSO4. The solvent was evaporated under re-
duced pressure and the crude product was purified by flash
chromatography to give 24 (3.1 g, 69%): mp 190-200 °C; HPLC
1
mg, 36%): HPLC tR 5.06 min; H NMR (CDCl3) δ 8.74 (s, 1H),
7.74 (s, 1H), 5.52 (s, 2H), 5.20 (s, 2H), 5.04 (s, 2H), 3.85 (s, 3H),
3.83 (s, 6H).
The title compound 19e (17 mg, 27%) was prepared by
halogenation of 9-(3,4,5-trimethoxybenzyl)-9H-purin-2-ylamine (50
mg, 0.2 mmol) with bromine (75 mg, 0.5 mmol) using the procedure
described for 18b: mp 204-205 °C; HPLC tR 6.69 min; 1H NMR
(CDCl3) δ 8.70 (s, 1H), 7.85 (s, 1H), 6.66 (s, 1H), 5.34 (s, 2H),
5.11 (s, 2H), 3.91 (s, 3H), 3.88 (s, 3H), 3.75 (s, 3H). Anal.
(C15H17N5O3) C, H, N.
6-Methyl-9-(2-chloro-3,4,5-trimethoxybenzyl)-9H-purin-2-
ylamine (19f). To a mixture of 18a (72 mg, 0.2 mmol) and tetrakis-
(triphenylphosphino)palladium (22 mg, 0.02 mmol) in 20 mL of
dry THF was added 0.22 mL (0.44 mmol) of a 2 M solution of
trimethylaluminum in toluene under nitrogen. The solution was
heated at reflux for 4 h, diluted with toluene (5 mL), and quenched
with 0.5 mL of methanol followed by ammonium chloride (1 mmol
of 1.0 N solution). The resultant mixture was heated at reflux for
2 h and the hot mixture was filtered through Celite. The filtrate
was concentrated, and the residue was taken into dichloromethane,
washed with water, dried over MgSO4, filtered, and concentrated
to afford 19f (34 mg, 50%): mp 163-164 °C; HPLC tR 4.80 min;
1H NMR (CDCl3) 7.82 (s, 1H), 6.65 (s, 1H), 5.35 (s, 2H), 5.10 (br
s, 2H), 3.95 (s, 3H), 3.90 (s, 3H), 3.75 (s, 3H), 2.65 (s, 3H). Anal.
(C16H18ClN5O3‚0.3HCl) C, H, N.
1
tR 4.43 min; H NMR (CDCl3) δ 8.55 (s, 1H), 8.06 (s, 1H), 5.52
(s, 2H), 5.07 (s, 2H), 3.76 (s, 3H), 2.61 (s, 3H), 2.25 (s, 3H). Anal.
(C14H17ClN6O2‚0.1H2O) C, H, N.
6-Chloro-9-(6-chloro-4-methoxy-3,5-dimethylpyridin-2-yl-
methyl)-9H-purin-2-ylamine (25). A suspension of 24 (0.5 g, 1.5
mmol) in POCl3 (5 mL) was heated at 90 °C for 2 h. The resulting
mixture was cooled to room temperature and poured onto ice water
and the pH was slowly adjusted to 10 with 1 N KOH. The
compound was extracted into chloroform and dried over MgSO4.
The solvent was evaporated under reduced pressure to give 25 (90
mg, 24%): mp 212-213 °C; HPLC tR 5.74 min; 1H NMR (CDCl3)
δ 7.92 (s, 1H), 5.28 (s, 2H), 5.06 (s, 2H), 3.73 (s, 3H), 2.29 (s,
3H), 2.28 (s, 3H). Anal. (C14H14Cl2N6O) C, H, N.
6-Chloro-9-(2,5-dimethoxybenzyl)-9H-purin-2-ylamine (26).
The title compound 26 (0.7 g, 70%) was prepared by alkylation
of 2-amino-6-chloropurine (0.5 g, 3.0 mmol) with chloromethyl-
2,5-dimethoxyphenyl (0.6 g, 3.2 mmol) as the alkylating agent
using the procedure described for 17: HPLC tR 5.29 min; 1H
NMR (CDCl3) δ 7.82 (s, 1H), 6.85-6.84 (d, 1H), 6.82-6.82 (d,
2H), 5.18 (s, 2H), 5.16 (s, 2H), 3.80 (s, 3H), 3.75 (s, 3H). Anal.
(C14H14ClN5O2) C, H, N.
6-Bromo-9-(2-bromo-3,4,5-trimethoxybenzyl)-9H-purin-2-
ylamine (27). The title compound 27 (0.62 g, 73%) was pre-
pared by halogenation of 6-bromo-9-(3,4,5-trimethoxybenzyl)-9H-
purin-2-ylamine (0.7 g, 1.8 mmol) with bromine (0.6 g, 3.9
mmol) using the procedure described for 18b: HPLC tR 5.79 min;
1H NMR (CDCl3) δ 7.88 (s, 1H), 6.68 (s, 1H), 5.33 (s, 2H), 5.19
(s, 2H), 3.93 (s, 3H), 3.90 (s, 3H), 3.79 (s, 3H); HRMS calcd for
C15H15Br2N5O3 471.9614 (M + H)+, found 471.9619.
6-Chloro-9-(4-methoxy-3,5-dimethylpyridin-2-ylmethyl)-9H-
purin-2-ylamine (20). A suspension of 2-amino-6-chloropurine
(21.77 g, 128 mmol), K2CO3 (53.5 g, 387 mmol), NaI (1.92 g,
12.8 mmol), and compound 6 (29.9 g, 128 mmol) in DMF (650
mL) was heated at 40 °C with stirring under nitrogen. After 6 h,
the reaction mixture was cooled and the inorganic solids were
filtered and washed with DMF. Dilution with 700 mL of water
induced the crystallization of the desired isomer 20 (21.8 g, 63%):
1
Rf 0.20 (EtOAc); mp 192-193 °C; H NMR (DMSO-d6) 8.08 (s,
1H), 8.02 (s, 1H), 6.84 (s, 2H), 5.36 (s, 2H), 3.74 (s, 3H), 2.30 (s,
3H), 2.16 (s, 3H); 13C NMR (DMSO-d6) 163.81, 160.23, 155.01,
153.26, 149.65, 149.19, 144.65, 125.61, 123.71, 123.64, 60.30,
45.69, 13.29, and 10.65. Anal. (C14H15ClN6O) C, H, N.
6-Chloro-9-(4-chloro-3,5-dimethylpyridin-2-yl methyl)-9H-
purin-2-ylamine (28). The title compound 28 (11.7 g, 88%) was
prepared by alkylation of 2-amino-6-chloropurine (7 g, 41 mmol)
with 8 (8.2 g, 43 mmol) as the alkylating agent using the procedure
2-(2-Amino-6-chloropurin-9-ylmethyl)-3,5-dimethylpyridin-
4-ol (21). The title compound 21 (0.14 g, 47%) was prepared by
alkylation of 2-amino-6-chloropurine (0.17 g, 1.0 mmol) with
2-chloromethyl-4-hydroxy-3,5-dimethylpyridine 715 (0.2 g, 1.2
mmol) as the alkylating agent using the procedure described for
the synthesis of 17: mp 255-270 °C; HPLC tR 3.64 min; 1H NMR
(DMSO-d6) δ 10.95 (s, 1H), 8.09 (s, 1H), 7.51 (s, 1H), 6.93 (s,
2H), 5.25 (s, 2H), 2.03 (s, 3H), 1.89 (s, 3H). Anal. (C13H13ClN6O‚
0.3H2O) C, H, N.
1
described for 17: HPLC tR 5.17 min; mp 229-235 °C; H NMR
(CDCl3) δ 8.24 (s, 1H), 7.90 (s, 1H), 5.40 (s, 2H), 5.07 (s, 2H),
2.49 (s, 3H), 2.37 (s, 3H); HRMS calcd for C13H12Cl2N6 321.0428
(M - H)-, found 321.0420.
6-Chloro-9-(4-bromo-3,5-dimethylpyridin-2-ylmethyl)-9H-pu-
rin-2-ylamine (29). The title compound 29 (0.6 g, 76%) was
prepared by alkylation of 2-amino-6-chloropurine (0.36 g, 2.1
mmol) with 9 (0.55 g, 2.4 mmol) as the alkylating agent using the
procedure described for 17: mp 215-225 °C; HPLC tR 5.30 min;
1H NMR (CDCl3) δ 8.19 (s, 1H), 7.88 (s, 1H), 5.41 (s, 2H), 5.06
(s, 2H), 2.53 (s, 3H), 2.39 (s, 3H). Anal. (C13H12BrClN6) C, H, N.
6-Chloro-9-(4-ethoxy-3,5-dimethylpyridin-2-ylmethyl)-9H-pu-
rin-2-ylamine (22). To a suspension of 21 (50 mg, 0.16 mmol)
and K2CO3 (67 mg, 0.5 mmol) in DMF (5 mL) was added ethyl

2776 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Kasibhatla et al.
6-Chloro-9-(4-iodo-3,5-dimethylpyridin-2-ylmethyl)-9H-
purin-2-ylamine (30). The title compound 30 (320 mg, 70%) was
prepared by alkylation of 2-amino-6-chloropurine (190 mg, 1.1
mmol) with 13g (600 mg, 1.6 mmol) as the alkylating agent using
the same procedure described for 14: mp 195-220 °C; HPLC tR
compounds were then added at 100, 30, 10, 3, 1, or 0.3 µM dilutions
(in PBS), and the plates were mixed for 30 s on the plate shaker
and then incubated for 1 h at 37 °C. The wells were washed twice
with 200 µL of PBS, biotinylated-geldanamycin (biotin-GM) was
added, and the plates were incubated for 1 h at 37 °C. The wells
were washed again twice with 200 µL of PBS before the addition
of 20 µg/mL streptavidin-phycoerythrin (PE) (Molecular Probes,
Eugene, OR) and incubation for 1 h at 37 °C. The wells were
washed again twice with 200 µL of PBS. The relative fluorescence
units (RFU) were measured using a SpectraMax Gemini XS
spectrofluorometer (Molecular Devices, Sunnyvale, CA) with an
excitation of 485 nm and emission of 580 nm; data were acquired
using SOFTmaxPRO software (Molecular Devices Corp., Sunny-
vale, CA). The background was defined as the RFU generated from
wells that were not coated with Hsp90 but were treated with the
biotin-GM and streptavidin-PE. Percent inhibition of binding for
each sample was calculated from the background subtracted values
as follows: % binding inhibition ) [untreated RFU - sample
RFU]/untreated RFU] × 100. IC50 was defined as the concentration
of the compound at which there was 50% inhibition of the biotin-
GM binding to rHsp90.
Her-2 Degradation Assay. MCF7 breast carcinoma cells
(ATCC) were grown in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum (FBS) and 10 mM
HEPES and plated in 24-well plates (50% confluent). Twenty-four
hours later (cells are 65-70% confluent), test compounds were
added at 1000 nM and six half-log dilutions down to 3 nM and
incubated overnight for 16 h. The wells were washed with 1 mL
of PBS, and 200 µL trypsin was added to each well. After
trypsinization was complete, 50 µL of FBS was added to each well.
Then 200 µL of cells was transferred to 96-well plates The cells
were pipetted up and down to obtain a single cell suspension. The
plates were centrifuged at 2500 rpm for 1 min using a Sorvall
Legend RT tabletop centrifuge (Kendro Laboratory Products,
Asheville, NC). The cells were then washed once in PBS containing
0.2% BSA and 0.2% sodium azide (BA buffer). PE conjugated anti-
Her-2/neu antibody (Becton Dickinson, #340552) or PE conjugated
anti-keyhole limpet hemacyanin (KLH) (Becton Dickinson, #340761)
control antibody was added at a dilution of 1:20 and 1:40,
respectively (final concentration was 1 µg/mL), and the cells were
pipeted up and down to form a single cell suspension and incubated
for 15 min. The cells were washed twice with 200 µL of BA buffer,
resuspended in 200 µL of BA buffer, and transferred to FACSCAN
tubes with an additional 250 µL of BA buffer. Samples were
analyzed using a FACSCalibur flow cytometer (Becton Dickinson,
San Jose, CA) equipped with argon ion laser that emits 15 mW of
488-nm light for excitation of the phycoerythrin fluorochrome;
10 000 events were collected per sample. A fluorescence histogram
was generated and the mean fluorescence intensity (MFI) of each
sample was determined using Cellquest software. The background
was defined as the MFI generated from cells incubated with con-
trol IgG-PE and was subtracted from each sample stained with the
Her-2/neu antibody. Cells were always incubated with DMSO as
untreated controls, since the compounds were resuspended in
DMSO. Percent degradation of Her-2 was calculated as follows:
% Her-2 degradation ) (MFl Her-2 untreated cells - MFI Her-2
sample)/(MFI Her-2 untreated cells) × 100. EC50 was defined as
the concentration of the compound at which there was 50%
degradation of the Her-2/neu protein.
1
5.36 min; H NMR (CDCl3) δ 8.08 (s, 1H), 7.86 (s, 1H), 5.41 (s,
2H), 5.04 (s, 2H), 2.57 (s, 3H), 2.41 (s, 3H). Anal. (C13H12ClIN6)
C, H, N.
6-Chloro-9-(3,5-dimethyl-4-nitropyridin-2-ylmethyl)-9H-
purin-2-ylamine (31). The title compound 31 (460 mg, 69%) was
prepared by alkylation of 2-amino-6-chloropurine (350 mg, 2.0
mmol) with 13e (570 mg, 2.3 mmol) as the alkylating agent using
the procedure described for 17: mp 240-250 °C; HPLC tR 6.21
1
min; H NMR (CDCl3) δ 8.40 (s, 1H), 7.94 (s, 1H), 5.40 (s, 2H),
5.05 (s, 2H), 2.40 (s, 3H), 2.27 (s, 3H). Anal. (C13H12ClN7O2) C,
H, N.
6-Chloro-9-(3,5-dimethyl-4-aminopyridin-2-ylmethyl)-9H-
purin-2-ylamine (32). A suspension of 31 (200 mg, 0.6 mmol)
and an excess of sodium hydrosulfite (310 mg, 3.0 mmol) in
methanol was stirred for 2 days at room temperature. The inorganic
salts were filtered through Celite and concentrated, and the residue
was purified by preparative TLC (100% EtOAc) to give 32 (73
1
mg, 40%): HPLC tR 3.54 min; H NMR (CDCl3) δ 8.05 (s, 1H),
7.83 (s, 1H), 5.31 (s, 2H), 5.05 (s, 2H), 4.08 (s, 2H), 2.12 (s, 6H);
HRMS calcd for C13H14ClN7 304.1072 (M + H)+, found 304.1071.
6-Chloro-9-(3,5-dimethyl-4-cyanopyridin-2-ylmethyl)-9H-
purin-2-ylamine (33). The title compound 33 (16 mg, 57%) was
prepared by alkylation of 2-amino-6-chloropurine (16 mg, 0.1
mmol) with 13h (20 mg, 0.09 mmol) as the alkylating agent
using the procedure described for 17: mp 200-220 °C; HPLC tR
1
5.19 min; H NMR (CDCl3) δ 8.41 (s, 1H), 7.94 (s, 1H), 5.40 (s,
2H), 5.05 (s, 2H), 2.70 (s, 3H), 2.53 (s, 3H); HRMS calcd for
C14H12ClN7 312.0076 (M - H)-, found 312.0766.
6-Chloro-9-(3,4,5-trimethylpyridin-2-ylmethyl)-9H-purin-2-
ylamine (34). The title compound 34 (43 mg, 21%) was pre-
pared by alkylation of 2-amino-6-chloropurine (110 mg, 0.65 mmol)
with 13d (150 mg, 0.7 mmol) as the alkylating agent using the
same procedure described for 17: mp 200-220 °C; HPLC tR
1
3.90 min; H NMR (CDCl3) δ 8.18 (s, 1H), 7.84 (s, 1H), 5.38 (s,
2H), 5.08 (s, 2H), 2.29 (s, 3H), 2.27 (s, 3H), 2.22 (s, 3H). Anal.
(C14H15ClN6) C, H, N.
6-Chloro-9-(3,5-dimethyl-4-methylsulfanylpyridin-2-ylmethyl)-
9H-purin-2-ylamine (35). The title compound 35 (36 mg, 67%)
was prepared by alkylation of 2-amino-6-chloropurine (30 mg,
0.18 mmol) with 13a (40 mg, 0.16 mmol) as the alkylating agent
using the procedure described for 17: mp 170-180 °C; HPLC tR
1
4.61 min; H NMR (CDCl3) δ 8.24 (s, 1H), 7.87 (s, 1H), 5.36 (s,
2H), 5.00 (s, 2H), 2.61 (s, 3H), 2.47 (s, 3H), 2.26 (s, 3H). Anal.
(C14H15ClN6S) C, H, N.
6-Chloro-9-(4-methanesulfinyl-3,5-dimethylpyridin-2-ylmethyl)-
9H-purin-2-ylamine (36). The title compound 36 (12 mg, 38%)
was prepared by alkylation of 2-amino-6-chloropurine (16 mg, 0.1
mmol) with 13i (21 mg, 0.09 mmol) as the alkylating agent using
the procedure described for 17: mp 190-200 °C; HPLC tR 4.02
1
min; H NMR (CDCl3) δ 8.23 (s, 1H), 7.91 (s, 1H), 5.39 (s, 2H),
5.09 (s, 2H), 2.90 (s, 3H), 2.68 (s, 3H), 2.54 (s, 3H); HRMS calcd
for C14H15ClN6OS 349.0644 (M - H)-, found 349.0643.
6-Chloro-9-(3,5-dimethylpyridin-2-ylmethyl)-9H-purin-2-
ylamine (37). The title compound 37 (42 mg, 69%) was prepared
by alkylation of 2-amino-6-chloropurine (30 mg, 0.18 mmol) with
13b (40 mg, 0.16 mmol) as the alkylating agent using the procedure
Cell Lysate Binding Assay. MCF7 cell lysates were prepared
by douncing in lysis buffer (20 mM Hepes, pH 7.3, 1 mM EDTA,
5 mM MgCl2, 100 mM KCl) and then incubating with or without
test compound for 0.5 h at 4 °C, followed by incubation with
biotin-GM linked to BioMag streptavidin magnetic beads (Qiagen)
for 1 h at 4 °C. Tubes were placed on a magnetic rack, and the
unbound supernatant was removed. The magnetic beads were
washed three times in lysis buffer and boiled for 5 min at 95° C in
SDS-PAGE sample buffer. Samples were analyzed on SDS protein
gels, and Western blots were done for rHsp90. Bands in the Western
blots were quantitated using a Bio-Rad Fluor-S MultiImager, and
the percent inhibition of binding of rHsp90 to the biotin-GM was
1
described for 17; mp 180-190 °C; HPLC tR 3.76 min; H NMR
(CDCl3) δ 8.21 (d, J ) 1.4 Hz, 1H), 7.89 (s, 1H), 7.30 ( d, J ) 1.4
Hz, 1H), 5.32 (s, 2H), 5.05 (s, 2H), 2.36 (s, 3H), 2.29 (s, 3H).
Anal. (C13H13ClN6) C, H, N.
Biochemical Assays. rHsp90 Competitive Binding Assay. Five
micrograms of purified rHsp90 protein (Stressgen, BC, Canada,
#SPP-770) in phosphate-buffered saline (PBS) was coated on 96-
well plates by incubating overnight at 4 °C. Unbound protein was
removed, and the coated wells were washed twice with 200 µL of
PBS. DMSO controls (considered as untreated samples) or test

Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12 2777
(2) (a) Xu, W.; Mimnaugh, E.; Rosser, M. F.; Nicchitta, C.; Marcu, M.;
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calculated. The IC50 reported is the concentration of test compound
needed to cause half-maximal inhibition of binding.
MTS Assay. Measurement of Cytotoxicity of HSP90 Inhibi-
tors. Cells were seeded in 96-well plates at 2000 cells/well and
allowed to adhere overnight in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal bovine serum. The final
culture volume was 100 µL. Viable cell number was determined
by using the Celltiter 96 AQueous Non-radioactive Cell Proliferation
Assay (Promega, Madison WI). The MTS/PMS (phenazine metho-
sulfate) solution was mixed at a ratio of 20:1, and 20 µL was added
per well to 100 µL of culture medium. MTS is a tetrazolium dye
that is converted to a formazan product by dehydrogenase enzymes
of metabolically active cells, which is measured at 490 nm
absorbance after 2-4 h using a multiwell plate spectrophotometer.
Background was determined by measuring the 490 nm absorbance
of cell culture medium and MTS-PMS in the absence of cells.
The background value was subtracted from all values. Percent viable
cells was calculated as follows: % viable cells ) (A490 sample/A490
untreated) × 100. IC50 was defined as the concentration that gave rise
to 50% viable cell number.
In Vivo Studies. Mouse PK Study. Six to eight week old Balb/C
and nu/nu athymic female mice were obtained from Harlan Sprague
Dawley (Indianapolis, IN). The mice were maintained in sterilized
filter-topped cages or ventilated caging in a room with a 12 h light/
dark cycle. Irradiated pelleted food (Harlan Teklad #7912) and
autoclaved deionized water were provided ad libitum. Animals were
identified by the use of individually numbered ear tags. Experiments
were carried out under institutional guidelines for the proper and
human use of animals in research established by the Institute for
Laboratory Animal Research (ILAR). The test compounds were
formulated (10 mg/mL) and a single dose was administered orally
(po) at a dose volume of 10 mL/kg. Blood was collected from the
retroorbital sinus in a 75 mm capillary tube. The blood was
transferred to 1.5 mL conical tube and allowed to clot. Serum was
stored at -20 °C until analysis.
Mouse Tumor Study. Tumor fragments (approximately 2 mm3)
or 5 × 106 tumor cells were inoculated subcutaneously in the right
or left flank of the animal. Mice with established tumors (50-200
mm3) were selected for study (n ) 7-10/treatment group). Tumor
dimensions were measured using calipers, and tumor volumes were
calculated using the equation for an ellipsoid sphere (l × w2)/2 )
mm3, where l and w refer to the larger and smaller dimensions
collected at each measurement. The test compounds were formu-
lated and administered orally (po) at a dose volume of 10 mL/kg.
The vehicle alone was administered to control groups. Animals were
dosed 5 days per week (Monday through Friday) for 4-6
consecutive weeks. Animals were weighed and the tumors were
measured twice per week. Mice were followed until tumor volumes
in the control group reached approximately 1000 mm3 and were
sacrificed by CO2 euthanasia. The mean tumor volumes of each
group were calculated. The change in mean treated tumor volume
was divided by the change in mean control tumor volume,
multiplied by 100 and subtracted from 100% to give the tumor
growth inhibition for each group. Statistical analysis was performed
using the standard t-test and using GraphPad Prism Software.
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