Identification of potent water soluble purine-scaffold inhibitors of the heat shock protein 90

Article abstract of DOI:10.1021/jm0508078

Hsp90 is a chaperone protein that allows cancer cells to tolerate the many components of dysregulated pathways. Its inactivation may result in targeting multiple molecular alterations and, thus, in reverting the transformed phenotype. The PU-class, a purine-scaffold Hsp90 inhibitor series, has been reported to be potent and selective against Hsp90 both in vitro and in vivo models of cancer. Here, a series of this class was synthesized and evaluated as inhibitors of the chaperone. The structure-activity relationship and selectivity for tumor Hsp90 of compounds within the series is presented. The study identifies water soluble derivatives (> 5 mM in PBS pH 7.4) of nanomolar potency (IC₅₀ ～ 50 nM) in cellular and animal models of cancer. Binding affinities of these compounds for Hsp90 correlate well with their biological activities. When administered in vivo to mice bearing MDA-MB-468 human breast cancer xenografted tumors, these agents result in pharmacologically relevant concentrations and, accordingly, in modulation of Hsp90-client proteins in tumors.

Full text of DOI:10.1021/jm0508078

J. Med. Chem. 2006, 49, 381-390
381
Identification of Potent Water Soluble Purine-Scaffold Inhibitors of the Heat Shock Protein 90
Huazhong He,†,¶ Danuta Zatorska,†,¶ Joungnam Kim,†,¶ Julia Aguirre,† Laura Llauger,† Yuhong She,† Nian Wu,†
Robert M. Immormino,‡ Daniel T. Gewirth,‡, and Gabriela Chiosis*,†,§
Department of Medicine and Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center,
New York, New York 10021, and Department of Biochemistry, Duke UniVersity Medical Center, Durham, North Carolina 27710
ReceiVed August 13, 2005
Hsp90 is a chaperone protein that allows cancer cells to tolerate the many components of dysregulated
pathways. Its inactivation may result in targeting multiple molecular alterations and, thus, in reverting the
transformed phenotype. The PU-class, a purine-scaffold Hsp90 inhibitor series, has been reported to be
potent and selective against Hsp90 both in vitro and in vivo models of cancer. Here, a series of this class
was synthesized and evaluated as inhibitors of the chaperone. The structure-activity relationship and
selectivity for tumor Hsp90 of compounds within the series is presented. The study identifies water soluble
derivatives (>5 mM in PBS pH 7.4) of nanomolar potency (IC50 ∼ 50 nM) in cellular and animal models
of cancer. Binding affinities of these compounds for Hsp90 correlate well with their biological activities.
When administered in vivo to mice bearing MDA-MB-468 human breast cancer xenografted tumors, these
agents result in pharmacologically relevant concentrations and, accordingly, in modulation of Hsp90-client
proteins in tumors.
Introduction
A recent trend in cancer therapy has been to develop agents
that “target” a single molecular alteration. As most cancers are
a result of multiple transformation-specific regulatory alterations,
targeting one abnormality may be insufficient in reversing the
transformed phenotype. Chaperones are proteins that allow
cancer cells to tolerate the components of dysregulated pathways
that otherwise would be lethal; thus, their inactivation may result
in targeting multiple molecular alterations.1 One such chaperone
with important roles in maintaining cell-specific transformation
is the heat shock protein 90 (Hsp90).2 Its biological role is
mediated by the ability of the chaperone to interact with client
substrates. Binding and release of Hsp90 substrates is regulated
by the activity of the N-terminal ATPase domain, which binds
and hydrolyses ATP to mediate a series of association-
dissociation cycles between Hsp90 and client proteins.3 The
activity of Hsp90 is further regulated by binding of cochaperones
which promote the interconversion of the ATP- and ADP-bound
states and modulate the formation of client-specific complexes.4
Although Hsp90 is highly expressed in both normal and
transformed cells, its usefulness as a therapeutic target results
from its distinctive activation in normal and stressed environ-
ments. Recent evidence suggests that in a number of tumor cell
lines, Hsp90 may be exclusively complexed with cochaperones
in a state of high affinity for ATP/ADP or ligands of this
regulatory pocket (i.e., ATPase inhibitor drugs), whereas in
normal tissues, Hsp90 may exist primarily in a latent, uncom-
plexed, low-affinity state.5 The shift in equilibrium between the
“latent” and “activated” states may be dictated by the amount
of “stress” on the system, i.e., mutated and dysregulated proteins,
hypoxia, low nutrient concentration environment, in other words,
the amount and degree of transformation in the cells.2d These
suggest that is possible to interfere with Hsp90 function (i.e.,
Figure 1. Schematic representation of the purine-scaffold Hsp90
inhibitor class, and its representative derivatives, compounds 1, 2, and
3.
by inhibiting its ATPase activity) and disrupt only critical
chaperone functions in a transformed cell without affecting
normal cell functions. Therefore, Hsp90 ATPase inhibitor
concentrations may be identified that will affect cancer cells
without being toxic to normal cells. Moreover, the ATP-
regulatory pocket of Hsp90 is unique. While bound to Hsp90
the nucleotide adopts a bent shape found only in ATPases
belonging to the GHKL family (G ) DNA gyrase subunit B,
H ) Hsp90, K ) histidine kinases, and L ) MutL). These
enzymes share the same left-handed â-R-â fold, called the
Bergerat fold. This fold is not observed in the high-affinity
binding sites of kinases (which adopt a P-loop motif) or in other
chaperones such as Hsp70 (which adopts an actin fold).6 These
observations suggest that it is possible to discover compounds
of high selectivity for Hsp90 by identifying those that specif-
ically bind to Hsp90 via the N-terminal ATPase pocket. Making
use of this specific fold, we were able to design a class of purine-
scaffold derivatives (PU-class, Figure 1) with Hsp90 inhibitory
activities.7 The first synthesized derivative of this class, 1 (PU3,
see Figure 1), bound Hsp90 with moderate affinity and exhibited
biological activities in the 50 µM concentration range.8 Further
efforts focused at improving the potency of this agent have led
* Corresponding author. Phone: 212-639-8929. Fax: 212-794-6236.
E-mail: chiosisg@mskcc.org.
† Program in Molecular Pharmacology and Chemistry, Memorial Sloan-
Kettering Cancer Center.
‡ Department of Biochemistry, Duke University Medical Center.
Hauptman-Woodward Research Institute, Buffalo, NY 14203.
§ Department of Medicine, Memorial Sloan-Kettering Cancer Center.
¶ These authors equally contributed to the work.
10.1021/jm0508078 CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/08/2005

382 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
He et al.
Scheme 1a
derivative 7. The active species for this iodination is probably
the in situ formed iodine trifluoroacetate that can act as a very
reactive electrophile, allowing iodination at room temperature.14
The bromo derivative 8 was obtained as previously described,
by coupling 4 with 5-bromo-6-iodo-benzo[1,3]dioxole.10 These
arylsulfanyl adenines were further alkylated at the position 9-N
with the tosylate of pent-4-ynyl alcohol in the presence of Cs2-
CO3 to give derivatives 9 and 10.15 The choice of pent-4-ynyl
comes from its being previously determined to be favored by
the Hsp90 pocket.9a To increase the water solubility profile of
these compounds we probed the introduction of a chain
containing an amine functionality instead of pent-4-ynyl.
Previous attempts to introduce such chains have led to com-
pounds that retained binding to Hsp90 but were surprisingly
inactive in cellular models, likely due to reduced membrane
permeability potential.9a Preserving the favorable linearity of
the first three carbon atoms of the pent-4-ynyl chain9 and
replacing the terminal alkyne with isopropylamine, derivatives
11 and 12 were synthesized. The 3-isopropylamino-propyl chain
was introduced by alkylation of 7 with the tosylate of the Boc-
protected 3-isopropylpropanol under basic conditions (Cs2CO3),
followed by TFA induced Boc deprotection16 to yield 11.
Alternatively, the Mitsunobu conditions10,17 were utilized to react
8 with 3-bromo-1-propanol. This product was further alkylated
by reacting with excess isopropylamine18 to result in derivative
12.
a Reagents and conditions: (a) NaOt-Bu, neocuproine, CuI, DMF, 110
°C, 58%; (b) NIS, CF3COOH, acetonitrile, rt, 55%; (c) pent-4-ynyl tosylate,
Cs2CO3, DMF, 80 °C, 55-65%; (d) N-Boc-protected 3-isopropylamino-
propyl tosylate, Cs2CO3, DMF, 80 °C, 25%; (e) CF3COOH/CH2Cl2, rt, 98%;
(f) BrCH2CH2CH2OH, PPh3, DBAD, toluene/CH2Cl2, rt; (g) i-PrNH2, 1,4-
dioxane, 100 °C.
If the linker was methylene (X2 ) CH2, Figure 1), synthesis
commenced by coupling 2,4,5,6-tetraaminopyrimidine (14) with
the acid fluoride of the corresponding carboxylic acid (Scheme
2).9a The acid fluoride 13 was generated by treating 3,4-
(methylenedioxy)phenylacetic acid with cyanuric fluoride and
pyridine in CH2Cl2.9a,19 Following a quick water wash, the
resulting acid fluoride was used in the next step without further
purification. The amide 15 resulting from the pyrimidine 14-
acid fluoride 13 coupling was cyclized to compound 16 by
heating in alcoholic NaOMe.9a Transformation of the C2-amino
group to fluorine (X1 ) NH2 to F) was conducted by a modified
Schiemann diazotization-fluorodediazoniation of the amino
to the synthesis of several compounds with improved activity
in both biochemical and cellular assays.9 One such compound,
2 (PU24FCl, Figure 1), is a selective inhibitor of tumor Hsp90
and exhibits antitumor activities in both in vitro and in vivo
models of cancer.5b The biological effects of 2 are demonstrated
in the 2-6 µM concentration range. Recently we have disclosed
the synthesis of several 8-arylsulfanyl, -sulfoxyl, and -sulfonyl
adenine derivatives of the PU-class.10 The effort has identified
derivative 3 (PU-H58, Figure 1) as a specific tumor Hsp90
inhibitor exhibiting its biological effects in the high nanomolar
concentration range. Here we present the synthesis and evalu-
ation of several novel PU-class derivatives designed by the
rational assembly of favorable substituents identified from our
previous SAR efforts.
20
derivative in HF/pyridine in the presence of NaNO2 to yield
the corresponding adenine derivatives 17. We and others9 have
previously determined that fluorine in this position in general
augmented the potency of the resulting purines if the linker was
methylene (X2 ) CH2, Figure 1), likely by increasing the
hydrogen donor ability of C6 NH2. No beneficial effect was
observed by F’s presence in this position if the linker was sulfur
(X2 ) S, Figure 1).9c,10 Further selective halogenation of 17
using either of NIS, NBS, or NCS14,21 led to the corresponding
iodo, bromo, and chloro derivatives 18-20. These were
alkylated with pent-4-ynyl tosylate in the presence of Cs2CO3
to result in derivatives 21-23. For the synthesis of the water
soluble derivative 25, the iodo derivative 18 was first alkylated
with 1,3-dibromopropane in the presence of Cs2CO3. Interest-
ingly, formation of dimer was not detected in this reaction. The
resulting bromine was further alkylated in the presence of excess
isopropylamine to give 25.
Results and Discussions
Chemistry. Previous chemistry efforts have identified both
a methylene and a sulfur linker (X2 ) CH2 or S, Figure 1) as
permissive for activity.9,10 These also suggested 4,5-methyl-
enedioxy as a favorable substituent when accompanied by
bromine in C2′ on the right side aryl (see Figure 1).10 Thus,
present modifications maintain the permissive characteristics and
focus at further modifications meant to increase potency and
water solubility.
For derivatives containing a sulfur linker (X2 ) S, Figure
1), synthesis was carried out using a method previously
described by He et al and employed the copper catalyzed
coupling of 8-mercaptoadenine (4) with the aryliodide 5
(Scheme 1).11 The reaction occurred in anhydrous DMF at 110
°C under nitrogen to generate the product 6 in 58% yield.
Aryliodide 5 was generated from the commercially available
amine by its conversion via the diazonium salt to the corre-
sponding iodide,12 while 8-mercaptoadenine was obtained by
condensation of 3,4,5-triaminopyrimidine with CS2.10,13 The
8-arylsulfanyl adenine 6 was further halogenated selectively at
position 2 of the aryl moiety using NIS as a source of
electrophilic iodine and TFA as a catalyst,10,14 to result in
For derivatives bearing 3,4-methoxy substituents on the right
side aryl (31-33), synthesis was carried out in a manner similar
to that of derivatives 21-23, using as starting points both the
cyanuric fluoride 26 obtained from 3,4-dimethoxyphenylacetic
acid and the tetraaminopyrimidine 14 (Scheme 3).
Biological Testing. Compounds synthesized above were
tested in a biochemical assay and also in cellular assays that
probe for cellular fingerprints of Hsp90 inhibition.2,10 The
biochemical assay tests competitive binding of compounds to

Purine-Scaffold Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 383
Scheme 2a
a Reagents and conditions: (a) K2CO3, DMAP, DMF, 120 °C; (b) NaOMe, MeOH, i-BuOH, 105 °C; (c) HF/pyridine, NaNO2, rt, 50%; (d) NCS, DMF,
rt, 50%; (e) NBS, DMF, rt, 40%; (f) NIS, CF3COOH, DMF, rt, 20%; (g) pent-4-ynyl tosylate, Cs2CO3, DMF, 50 °C, 30-90%; (h) Cs2CO3, DMF, 50 °C,
95%; (i) i-PrNH2, rt, 90%.
Scheme 3a
degradative potential of the above-synthesized purines. Hsp90
inhibitors are also known to cause cells lacking functional Rb
to progress normally through G1 and arrest in mitosis.24 Thus,
another assay used here to test cellular Hsp90 inhibition relies
on assessing the antimitotic potential of synthesized purines.
The assay is a microtiter immunoassay and uses an antibody
against a mitotically phosphorylated form of nucleolin to detect
cells in mitosis.10,26
In addition, the cytotoxicity of these agents against SKBr3
and MDA-MB-468 breast cancer cells was determined. A
selected number of derivatives were also tested for possible
toxicity against normal lung fibroblast cells (MRC-5).
Several observations may be inferred from these tests. First,
in the 2-halo-4,5-methylenedioxy series, activity decreased in
the order I, Br, Cl (see 9 vs 10 and 21 vs 22 vs 23, Table 1 and
11 vs 12, Table 2). Second, no significant difference was
observed in activity when comparing methylene to sulfur linker
(X2 ) CH2 vs S, Figure 1) derivatives that carry identical
substituents on the right side aryl (see 9 vs 21 and 10 vs 22,
Table 1; 11 vs 25, Table 2). Third, substitution of the pent-4-
ynyl chain with 3-isopropylamino-propyl had no unfavorable
effect on the biological activity of these derivatives while
substantially increasing their water solubility (see 9 vs 11, 10
vs 12 and 21 vs 25). Fourth, it is likely that the 2-halo-4,5-
methylenedioxy series accommodates the right side aryl moiety
into the Hsp90 hydrophobic pocket differently from the 2-halo-
4,5-dimethoxy series. This is suggested by the diminished or
complete lack of biological activity observed in the 2-halo-4,5-
dimethoxy series and also by the fact that, contrary to the 2-halo-
4,5-methylenedioxy series, activity in this series decreased in
the order Cl, Br, I (see 33 vs 32 vs 31, Table 1). Such trend
was previously observed in the 2-halo-3,4,5-trimethoxy deriva-
tive series.9a Fifth, trends in binding affinity for Hsp90 in the
two cancer cell lines translated well into Hsp90-related biologi-
cal activities in those cells. To exemplify, in the breast cancer
cell line SKBr3, Hsp90 binding translated into inhibition of
growth and Her2 degradation, both biological effects occurring
with similar IC50’s. Likewise, in the breast cancer cell line
a Reagents and conditions: (a) K2CO3, DMAP, DMF, 120 °C, 90%; (b)
NaOMe, MeOH, i-BuOH, 105 °C, 85%; (c) HF/pyridine, NaNO2, rt, 50%;
(d) NIS, CF3COOH, DMF, rt, 20%; (e) NBS, DMF, rt, 54%; (f) NCS, DMF,
rt, 50%; (g) pent-4-ynyl tosylate, Cs2CO3, DMF, 50 °C, 50-90%.
Hsp90 found in cell-specific complexes and uses a fluorescence
polarization method.22,26 When cell lysates are used instead of
recombinant protein, the assay measures binding to average
Hsp90 population found in cell-specific complexes.5b
The cellular assays measure two specific biological effects
observed upon addition of known Hsp90 inhibitors to cancer
cells: (a) degradation of the tyrosine kinase Her223 and (b)
mitotic block in retinoblastoma gene product (Rb)-defective
cells.24 Overexpression of the receptor tyrosine kinase Her2 in
SKBr3 breast cancer cells leads to Akt activation which in turn
promotes cell survival. Hsp90 uniquely stabilizes Her2 via
interaction with its kinase domain, and an Hsp90 inhibitor
induces Her2 degradation by disrupting the Her2/Hsp90 as-
sociation.23 Her2 inactivation/degradation in SKBr3 is detri-
mental to the cell and leads to its death. Thus Her2 degradation
in these cells is a functional read-out of Hsp90 inhibition.
Correlation between Her2 degradation and cytotoxicity is
indicative of selective biological effect in this cell via Hsp90.
We have previously reported a fast microtiter immunoassay able
of quantifying cellular levels of Her2 following drug treat-
ments.25,26 This assay is used here to differentiate the Her2-

384 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
He et al.
Table 1. Substituent Effects on the Activity of 9-(Pent-4-ynyl)-adenine Derivatives as Inhibitors of Hsp90
SKBr3
MDA-MB-468
EC50
IC50 growth
inhibitiona
IC50 Her2
EC50
IC50 growth
inhibitiona
IC50
cpds
X1
X2
X3
X4, X5
Hsp90a
degradationa
Hsp90a
antimitotica
9
H
H
F
F
F
F
F
F
S
S
I
Br
I
Br
Cl
I
Br
Cl
-OCH2O-
-OCH2O-
-OCH2O-
-OCH2O-
-OCH2O-
OMe, OMe
OMe, OMe
OMe, OMe
10.8 ( 2
50.4 ( 4
22.3 ( 2
56.5 ( 2
77.2 ( 1
>15000
>15000
4600 ( 20
90 ( 2.0
100 ( 10
365 ( 45
90 ( 10
210 ( 10
300 ( 15
>50000
10.9 ( 1
77.1 ( 3
23.7 ( 1
30.5 ( 4
51.2 ( 5
>15000
>15000
4500 ( 70
170 ( 10
560 ( 66
150 ( 10
350 ( 30
440 ( 15
>50000
184 ( 11
530 ( 44
180 ( 15
310 ( 30
450 ( 20
>50000
>50000
>50000
10
21
22
23
31
32
33
300 ( 50
90 ( 30
CH2
CH2
CH2
CH2
CH2
CH2
215 ( 55
250 ( 30
>50000
55000 ( 2300
20600 ( 6700
>50000
30000 ( 1000
>50000
27300 ( 1200
a All values are in nanomol per liter and represent the average of n ) 3 ( standard deviation.
Table 2. Effects of a Water-Solubilizing Chain at Position 9-N on the Activity of PU-Class Derivatives as Inhibitors of Hsp90
SKBr3
MDA-MB-468
EC50
IC50 growth
inhibitiona
IC50 Her2
EC50
IC50 growth
inhibitiona
IC50
cpds
X1
X2
X3
Hsp90a
degradationa
Hsp90a
antimitotica
11
12
25
H
H
F
S
S
CH2
I
Br
I
16.1 ( 1
38.8 ( 3
50.4 ( 4
50 ( 5
142 ( 22
45 ( 6
50 ( 6
205 ( 15
80 ( 10
10.2 ( 3
55.4 ( 1
39.1 ( 1
58 ( 2.5
270 ( 20
90 ( 5.0
70 ( 5.5
375 ( 24
100 ( 20
a All values are in nanomol per liter and represent the average of n ) 3 ( standard deviation.
Table 3. Selectivity for Transformed versus Normal Cells in a Series of Purine-Scaffold Hsp90 Inhibitors
EC50
Hsp90
lunga
EC50
Hsp90
hearta
EC50
Hsp90
SKBr3a
lung/
SKBr3
heart/
SKBr3
IC50
MRC-5/
SKBr3
cpds
MRC-5a
11
12
25
2200 ( 400
5200 ( 210
2400 ( 240
6000 ( 200
13300 ( 210
6900 ( 150
16.1 ( 1
38.8 ( 3
50.4 ( 4
136
134
48
370
343
140
1000 ( 14
ND
>5000
∼20
ND
>50
a All values are in nanomol per liter and represent the average of n ) 3 ( standard deviation.
MDA-MB-468, Hsp90 binding correlated with growth inhibition
and antimitotic activity (see Tables 1 and 2).
soluble at these concentrations and required administration in
a mixture of 1:1:1 DMSO/EtOH/water.5b To date, the only other
reported Hsp90 inhibitor to be water soluble is the geldanamycin
derivative, 17DMAG.27a
The two most active derivatives, 11 and 25, were further
tested for specificity toward transformed cells (Table 3). Binding
affinities of these compounds for average population Hsp90
complexes found in normal tissues (lung and heart) and, in
addition, their cytotoxicities against MRC-5 normal lung
fibroblast cells were determined. Compounds were found to bind
Hsp90 from normal tissues with 2-log weaker affinities when
compared to that of Hsp90 from SKBr3 cells (columns 6 and
7, Table 3). This specificity translated into over 50-fold
selectivity (column 8, Table 3) in inhibiting the growth of
transformed cells compared to that of cultured normal fibroblast
cells. No significant cell death was observed in the purine-
scaffold treated MRC-5 cells even at the highest tested
concentrations.
To probe if in vivo administration of these agents resulted in
pharmacologically relevant concentrations, we used mice xe-
nografted with MDA-MB-468 human breast cancer tumors.
Previously, we have reported a tumor accumulation profile for
derivative 2.5b Administered intraperitoneally to mice bearing
MCF-7 breast cancer xenografted tumors, 2 accumulated inside
tumors to micromolar concentrations, and these levels were
sustained at 24 h postadministration. Similar tumor retention
profiles were later demonstrated for both 17AAG and 17DMAG,
suggesting this behavior to be a consequence of a higher affinity
state of Hsp90 in tumors.27 A similar tumor retention profile
was observed here with 12sat 24 h, the biologically active
concentration of 300 nM (∼IC50, see Table 2) was seen in
tumors at the administered dose of 25 mg/kg (Figure 2A). An
increase in dose to 50 mg/kg or higher, led to concentrations
of 12 in tumors above the IC90 value. At 24 h postadministration,
12 could not be detected in plasma (not shown). Compound 12
When formulated as hydrochloric or phosphoric acid salts,
derivatives 11, 12, and 25 were soluble in phosphate buffered
saline (PBS) pH 7.4 to form 10-40 mM stocks, suggesting that
their water solubility was above 5 mg/mL. This is a substantial
improvement over compounds 2 and 3 that were not water

Purine-Scaffold Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 385
Figure 2. Pharmacodynamic effects of derivative 12. The phosphate salt of 12 was administered intraperitoneally to MDA-MB-468 tumor-bearing
mice at the indicated doses. Mice (n ) 2) were sacrificed at 24 h postadministration, and tumors were harvested. Tumors were halved and processed
accordingly. Inhibitor levels were analyzed by LC-MS/MS (A) while protein levels analyzed by Western blot (B). Data are presented as (A) drug
concentration in the tumor at 24 h postadministration and (B) percent control ) protein expression in treated mice/protein expression in control
mice (administered PBS only) × 100 and plotted against administered dose of 12.
and 2 mM DTT (Fisher Biotech, Fair Lawn, NJ) were freshly added.
GM-BODIPY was synthesized as previously reported22a and was
dissolved in DMSO to form 10 µM solutions. Cell lysates were
prepared rupturing cellular membranes by freezing at -70 °C and
dissolving the cellular extract in HFB with added protease and
phosphotase inhibitors. Organs were harvested from a healthy
mouse and homogenized in HFB. Saturation curves were recorded
in which GM-BODIPY (5 nM) was treated with increasing amounts
of cellular lysates. The amount of lysate that resulted in polarization
(mP) readings corresponding to 20 nM recombinant Hsp90R was
chosen for the competition study. For the competition studies, each
96-well contained 5 nM fluorescent GM, cellular lysate (amounts
as determined above and normalized to total Hsp90 as determined
by Western blot analysis using as standard Hsp90 purified from
HeLa cells (Stressgen no. SPP-770) and tested inhibitor (initial stock
in DMSO) in a final volume of 100 µL. The plate was left on a
shaker at 4 °C for 24 h, and the FP values in mP were recorded.
EC50 values were determined as the competitor concentrations at
which 50% of the fluorescent GM was displaced.
Cell Culture. The human breast cancer cell lines SKBr3 and
MDA-MB-468 were a gift from Dr. Neal Rosen (MSKCC). Cells
were maintained in 1:1 mixture of DME/F12 supplemented with 2
mM glutamine, 50 units/mL penicillin, 50 units/mL streptomycin,
and 10% heat-inactivated fetal bovine serum (Gemini Bioproducts
no. 100-10b) and incubated at 37 °C, 5% CO2. Normal lung
fibroblast cells were obtained from the American Type Culture
Collection (Manassas, VA) maintained in minimum essential
medium (MEM) adjusted to contain 2 mM L-glutamine, 1.5 g/L
sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM
sodium pyruvate, 10% fetal bovine serum, 1% penicillin and
streptomycin and incubated at 37 °C, 5% CO2.
Growth Assays. Growth inhibition studies were performed using
the sulforhodamine B assay as previously described.28 In summary,
experimental cultures were plated at 6000, 8000, and 10 000 cells/
well for SKBr3, MDA-MB-468, and MRC-5, respectively, in
microtiter plates (Nunc no. 167008). One column of wells was left
without cells to serve as the blank control. Cells were allowed to
attach overnight. The following day, growth medium having either
drug or DMSO (or PBS for 11, 12, and 25) at twice the desired
initial concentration was added to the plate in triplicate and was
serially diluted at a 1:1 ratio in the microtiter plate. After 72 h of
growth, the cell number in treated versus control wells was
estimated after treatment with 50% trichloroacetic acid and staining
with 0.4% sulforhodamine B in 1% acetic acid. The IC50 was
calculated as the drug concentration that inhibits cell growth by
50% compared with control growth.
was further tested for its effects on pharmacodynamic markers,
such as Raf-1 kinase. Hsp90 stabilizes the kinase and maintains
it in a ready-to-be-activated conformation. Inhibition of Hsp90
leads to the disruption of the complex and further ubiquitiny-
lation and degradation of Raf-1 by the proteasome.2 Thus Raf-1
degradation in these tumors is a functional read-out of Hsp90
inhibition. We have previously shown for 2 a correlation
between compound tumor accumulation profile and its effects
on Raf-1 protein.5b In concordance, 12 induced a dose-dependent
degradation of Raf-1, with maximal effects seen at 50 mg/kg
(Figure 2B). The compound had no effect on PI3 kinase (p85
unit) expression, protein unaffected by Hsp90 inhibitors.2,8
Conclusions
In summary, we have presented the synthesis and evaluation
of several novel purine-class Hsp90 inhibitors. The study has
aided in the identification of derivatives with nanomolar
biological activities in both cellular and animal models of cancer.
It also extended our understanding into the structural elements
that allow for high affinity binding to Hsp90. In this regard, it
highlighted the importance of substituents on the right side aryl
moiety. These have a major impact not only on orienting the
moiety inside the hydrophobic pocket but also on reducing the
rotational freedom around the C-S or C-C bond and, thus, on
alleviating entropic penalties upon binding. In addition, the study
has identified position 9-N as permissive for the attachment of
functionalities that increase water solubility. SAR studies in the
series have revealed a high correlation between binding to Hsp90
and biological activities, suggesting Hsp90 inhibition as the
major mode of action of these compounds. As previously seen
with lead purine-skeleton Hsp90 inhibitors, a tumor retention
profile is also characteristic for these novel derivatives. Phar-
macologically relevant concentrations are maintained in tumors
at 24 h, leading to extended modulation of Hsp90-client
proteins in tumors. The higher affinity that these compounds
exhibit for tumor cell as compared to normal cell Hsp90 is a
likely explanation for the behavior.
Experimental Section
Hsp90 Competition Assay.22,26 Fluorescence polarization mea-
surements were performed on an Analyst AD instrument (Molecular
Devices, Sunnyvale, CA). Measurements were taken in black 96-
well microtiter plates (Corning no. 3650). The assay buffer (HFB)
contained 20 mM HEPES (K) pH 7.3, 50 mM KCl, 5 mM MgCl2,
20 mM Na2MoO4, 0.01% NP40. Before each use, 0.1 mg/mL
bovine gamma globulin (BGG) (Panvera Corporation, Madison, WI)
Her2 Assay.25,26 SKBr3 cells were plated in black, clear-bottom
microtiter plates (Corning no. 3603) at 3000 cells/well in growth
medium (100 µL) and allowed to attach for 24 h at 37 °C and 5%
CO2. Growth medium (100 µL) with drug or vehicle (DMSO) was
carefully added to the wells, and the plates were placed at 37 °C

386 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
He et al.
and 5% CO2. Following 24 h of incubation with drugs, wells were
washed with ice-cold Tris buffer saline (TBS) containing 0.1%
Tween 20 (TBST) (200 µL). A house vacuum source attached to
an eight-channel aspirator was used to remove the liquid from the
plates. Further, methanol (100 µL at -20 °C) was added to each
well, and the plate was placed at 4 °C for 10 min. Methanol was
removed by washing with TBST (2 × 200 µL). After further
incubation at room temperature for 2 h with SuperBlock (Pierce
37535) (200 µL), anti-Her-2 (c-erbB-2) antibody (Zymed Labora-
tories no. 28-0004) (100 µL, 1:200 in SuperBlock) was placed in
each well. The plate was incubated overnight at 4 °C. For control
wells, 1:200 dilution of a normal rabbit IgG (Santa Cruz no. SC-
2027) in Superblock was used. Each well was washed with TBST
(2 × 200 µL) and incubated at room temperature for 2 h with an
anti-rabbit HRP-linked antibody (Sigma, A-0545) (100 µL, 1:2000
in SuperBlock). Unreacted antibody was removed by washing with
TBST (3 × 200 µL), and the ECL Western blotting reagent
(Amersham no. RPN2106) (100 µL) was added. The plate was
immediately read in an Analyst AD plate reader (Molecular
Devices). Each well was scanned for 0.1 s. Readings from wells
containing only control IgG and the corresponding HRP-linked
secondary antibody were set as background and deducted from all
measured values. Luminescence readings resulted from drug-treated
cells versus untreated cells (vehicle treated) were quantified and
plotted against drug concentration to give the IC50 values as the
concentration of drug that caused 50% decrease in luminescence.
Antimitotic Assay.26 Black, clear-bottom microtiter 96-well
plates (Corning Costar no. 3603) were used to accommodate
experimental cultures. MDA-MB-468 cells were seeded in each
well at 8000 cells per well in growth medium (100 µL) and allowed
to attach overnight at 37 °C and 5% CO2. Growth medium (100
µL) with drug or vehicle (DMSO) was gently added to wells, and
the plates were incubated at 37 °C and 5% CO2 for 24 h. Wells
were washed with ice-cold TBST (2 × 200 µL). A house vacuum
source attached to an eight-channel aspirator was used to remove
the liquid from the 96-well plates. Ice-cold methanol (100 µL) was
added to each well, and the plate was placed at 4 °C for 5 min.
Methanol was removed by suction and plates were washed with
ice-cold TBST (2 × 200 µL). Plates were further incubated with
SuperBlock blocking buffer (Pierce no. 37535) (200 µL) for 2 h at
room temperature. The Tg-3 antibody (gift of Dr. Peter Davies,
Albert Einstein College of Medicine) diluted 1:200 in SuperBlock
was placed in each well (100 µL) except the control column that
was treated with control antibody (mouse IgM, NeoMarkers, NC-
1030-P). After 72 h, wells were washed with ice-cold TBST (2 ×
200 µL). The secondary antibody (goat antimouse IgM, Southern-
Biotech no. 1020-05) was placed in each well at 1:2000 dilution in
SuperBlock and incubated on a shaker at room temperature for 2
h. Unreacted antibody was removed by washing the plates with
ice-cold TBST (3 × 200 µL) for 5 min on a shaker. The ECL
Western Blotting detection reagents 1 and 2 in a 1:1 mix (100 µL)
were placed in each well and the plates were read immediately in
an Analyst AD plate reader (Molecular Devices). Luminescence
readings were imported into SOFTmax PRO 4.3.1. Antimitotic
activity was defined as a concentration-dependent increase in
luminescence readings in compound-treated wells as compared to
DMSO-only treated wells. Luminescence readings resulting from
drug-treated cells versus untreated cells (vehicle treated) were
quantified and plotted against drug concentration to give the IC50
values as the concentration of drug that caused a doubling in
luminescence.
conjugated corresponding secondary antibody. ECL (Amersham
Life Science, Inc.) was performed according to the manufacturer’s
instructions. Blots were visualized by autoradiography and the
protein quantified using BioRad Gel Doc 1000 software. The second
half of each tumor was processed for LC-MS/MS analysis. Tumors
were rinsed with saline isotonic solution and dried with gauze. A
selected portion of tumor was weighed and then homogenized in
mobile phase (acetonitrile (ACN)/0.1% formic acid ) 1.2/2.8, v/v).
Haloperidol was added as internal standard. Compounds were
extracted in methylene chloride. The organic layer was separated,
then speedily dried under vacuum and reconstituted in the mobile
phase. Concentrations of compound 12 were determined and
quantitated by a multiple reaction monitoring (MRM) mode (m/z
467-407.8) using a tandem high-performance liquid chromatog-
raphy-mass/mass spectrometry (HPLC-MS/MS) at the Analytical
Core Facility of Memorial Sloan-Kettering Cancer Center. The API
4000 LC-MS/MS (Applied Biosystems) was coupled with a
Shimadzu LC system and a 96-well plate autosampler. A Gemini
C18 column (5µ particle size, 50 mm × 4.6 mm i.d.) was used for
the LC separation. The analyte was eluted under an isocratic
condition for 4 min at a flow rate of 0.4 mL/min.
General Chemical Procedures. All commercial chemicals and
solvents are reagent grade and were used without further purifica-
tion. The identity and purity of each product was characterized by
MS, HPLC, TLC, and NMR. 1H NMR/13C NMR spectra were
recorded on a Bruker 400 MHz instrument. Low-resolution mass
spectra (MS) were recorded in the positive or negative ion mode
under electrospray ionization (ESI). High-performance liquid chro-
matography analyses were performed on a Waters 2996 instrument
with a photodiode array detector (read at 254 nm) and a reversed-
phase column (Higgins; HAISIL HL C18 5µm) (method a) and,
additionally, a Waters 2695 separation module with a Waters 996
photodiode array detector and a Waters micromass ZQ and a
reversed-phase column (Varian; Microsorb 100-5 C18 150 × 2)
(method b). Method a: 0.1% TFA in water/acetonitrile at the
indicated ratio. Method b: 0.05% TFA in water/0.04% TFA in
acetonitrile at the indicated gradient. Characterization data for
previously unknown compounds were determined from a single
run with isolated yields. Reactions were monitored by thin-layer
chromatography on 0.25 mm silica gel plates and visualized with
UV light. Column chromatography was performed using silica gel
(Fisher 170-400 mesh). Analytical thin-layer chromatography
(TLC) was performed on E. Merck precoated silica gel 60 F254
.
All reactions were conducted under inert atmosphere.
8-Mercaptoadenine (4). A mixture of 4,5,6-triaminopyrimidine
sulfate (5.35 g, 24 mmol), NaHCO3 (10.08 g, 120 mmol), H2O (90
mL), CS2 (14.43 mL, 20.88 g, 240 mmol), and EtOH (45 mL) was
refluxed for 3 days. After cooling, the excess CS2 was evaporated
in vacuo. Solids were filtered off, and HOAc (4 mL) was added to
the filtrate. The product as a white precipitate was collected, dried
(4.0 g, 100% yield), and further used without additional purification.
1H NMR (400 MHz, DMSO-d6) δ 13.02 (s, 1 H), 12.02 (s, 1 H),
8.05 (s, 1 H), 6.73 (bs, 2 H); MS m/z 168.0 (M - H)+.
5-Iodo-benzo[1,3]dioxole (5).29,30 Benzo[1,3]dioxol-5-ylamine
(5 g, 36.46 mmol) was dissolved in concentrated HCl (32 mL) and
ice (96 g). To it, a chilled solution of NaNO2 (5.3 g, 76.8 mmol)
in water (30 mL) was added over a 15 min period. The mixture
was stirred for an additional 10 min. Urea (1.66 g, 27.6 mmol) and
10 min later, KI (17.9 g) in water (20 mL) were further added, and
the mixture was stirred overnight. Following completion, the
reaction mixture was extracted in CH2Cl2, dried over MgSO4, and
filtered through a silica gel column using hexane as eluent to provide
Tumor Analysis. Tumors were harvested at the indicated time
and immediately flash frozen in liquid nitrogen. One-half of each
tumor was homogenized in SDS lysis buffer (50 mM Tris pH 7.4,
2% SDS). Protein concentrations were determined using the BCA
kit (Pierce) according to the manufacturer’s instructions. Protein
lysates (20-100 µg) were electrophoretically resolved on denaturing
7% SDS-PAGE, transferred to nitrocellulose membrane, and
probed with the following primary antibodies: anti-Raf-1 (Santa
Cruz) and anti-PI3K (p85) (Upstate Biotechnologies). Membranes
were then incubated with a 1:5000 dilution of a peroxidase-
1
the desired aryliodide (6.9 g, 76.7% yield). H NMR (400 MHz,
CDCl3) δ 7.11-7.08 (m, 2H), 6.56 (d, J ) 8.0 Hz, 1H), 5.91 (s,
2H).
5-Bromo-6-iodo-benzo[1,3]dioxole.30 To a solution of 5-bromo-
benzo[1,3]dioxole (10.1 g, 50 mmol) in AcOH (75 mL) was added
ICI (10.1 g, 62.5 mmol) in AcOH31 (12.5 mL). The reaction mixture
was stirred at 80 °C for 18 h and then poured into H2O and extracted
twice with CH2Cl2. The organic extracts were washed consequently
with aqueous NaHSO3, NaHCO3, and H2O, dried over MgSO4, and

Purine-Scaffold Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 387
then evaporated in vacuo. The reaction crude was purified by silica
gel chromatography (hexanes) to give the desired iodo compound
(3.8 g, 23.2% yield). H NMR (400 MHz, CDCl3) δ 7.23 (s, 1H),
7.07 (s, 1H), 5.97 (s, 2H).
(t, J ) 6.5 Hz, 2H), 2.45 (s, 3H), 1.91-1.87 (m, 2H), 1.42 (s, 9H),
1.08 (d, J ) 6.7 Hz, 6H); MS m/z 372.2 (M + H)+.
1
8-(6-Iodo-benzo[1,3]dioxol-5-ylsulfanyl)-9-(3-isopropylamino-
propyl)adeni ne (11). A solution of 7 (125 mg, 303 µmol), 3-(tert-
butoxycarbonyl-isopropyl-amino)-propyl tosylate (269 mg, 726
µmol), and Cs2CO3 (99 mg, 303 µmol) in anhydrous DMF (2 mL)
was stirred at 80 °C for 24 h. The solvent was removed under
vacuum and the crude purified by preparatory thin-layer chroma-
tography on silica gel (CHCl3/MeOH/NH4OH at 10:1:0.5) to afford
the 9-N-alkylated compound. This was placed in TFA (1 mL) at 0
°C for 1.5 h to remove the Boc protecting group and yield 11 (30
8-(Benzo[1,3]dioxol-5-ylsulfanyl)adenine (6). 8-Mercapto-
adenine (100 mg, 0.60 mmol), neocuproine hydrate (14 mg, 0.06
mmol), CuI (11 mg, 0.06 mmol), NaO-t-Bu (115 mg, 1.2 mmol),
5-iodo-benzo[1,3]dioxole (770 mg, 1.8 mmol), and DMF (4 mL)
were charged in a nitrogen box. The vessel was sealed with Teflon
tape, placed in an oil bath (110 °C), and magnetically stirred for
24 h. The solvent was removed under high vacuum and the crude
purified by column chromatography on silica gel (EtOAc/CH2Cl2/
1
mg, 19.3% yield). H NMR (400 MHz, CDCl3) δ 8.31 (s, 1H),
1
7.29 (s, 1H), 6.88 (s, 1H), 6.10 (bs, 2H), 5.96 (s, 2H), 4.29 (t, J )
7.0 Hz, 2H), 2.75-2.69 (m, 1H), 2.58 (t, J ) 6.8 Hz, 2H), 2.02-
1.95 (m, 2H), 1.03 (d, J ) 6.2 Hz, 6H); 13C NMR (100 MHz,
CDCl3) δ 154.6, 152.9, 151.6, 149.2, 148.9, 146.2, 127.9, 120.1,
119.2, 112.2, 102.2, 91.1, 48.7, 43.9, 41.7, 30.3, 22.9; MS m/z 513.2
(M + H)+. HPLC: (a) 98.9% (65% water/35% acetonitrile); (b)
95.0% (from 20% to 40% acetonitrile).
MeOH at 2:2:1) to afford 6 (99.6 mg, 58% yield). H NMR (400
MHz, DMSO-d6) δ 8.20 (s, 1H), 7.72 (bs, 2H), 7.17 (s, 1H), 7.10
(d, J ) 8.2 Hz, 1H), 7.00 (d, J ) 8.0 Hz, 1H), 6.09 (s, 2H); MS
m/z 288.1 (M + H)+.
8-(6-Iodo-benzo[1,3]dioxol-5-ylsulfanyl)adenine (7). A solution
of 6 (201 mg, 0.7 mmol), acetonitrile (8.75 mL), TFA (270 µL,
400 mg), and NIS (945 mg, 4.2 mmol) was stirred at room
temperature for 24 h. The solvent was removed under high vacuum
and the crude purified by column chromatography on silica gel
(EtOAc/CH2Cl2 at 1:1 then EtOAc/CH2Cl2/MeOH at 2:2:1) to yield
8-(6-Bromo-benzo[1,3]dioxol-5-ylsulfanyl)-9-(3-isopropylamino-
propyl)ade nine (12). A solution of 8 (513 mg, 1.4 mmol), PPh3
(808 mg, 3.08 mmol), 3-bromo-1-propanol (253 mg, 165 µL, 1.82
mmol), DBAD (1612 mg, 7 mmol) in toluene (43.8 mL), and CH2-
Cl2 (8.75, mL) was stirred at room temperature for 20 min. The
reaction mixture was loaded to a silica gel column (CHCl3 then
CHCl3/EtOAc/hexanes/i-propyl alcohol at 4:4:2:1) to provide the
9-N-alkylated compound, 8-(6-bromo-benzo[1,3]dioxol-5-ylsul-
fanyl)-9-(3-bromo-propyl)adenine) (142.6 mg, 21% yield). A solu-
tion of this product (142.6 mg, 0.29 mmol) in 1,4-dioxane (12 mL)
and i-propylamine (3 mL) was stirred at 100 °C for 2.5 h. The
solvent was removed under vacuum and the crude purified by
preparatory thin-layer chromatography on silica gel (CHCl3/MeOH/
NH4OH at 10:1:0.5 then EtOAc/CH2Cl2/MeOH at 2:2:1) to afford
12 (51 mg, 8% yield). 1H NMR (400 MHz, CDCl3) δ 8.30 (s, 1H),
7.04 (s, 1H), 6.81(s, 1H), 6.48 (bs, 2H), 5.94 (s, 2H), 4.29 (t, J )
7.0 Hz, 2H), 2.74-2.68 (m, 1H), 2.57 (t, J ) 6.8 Hz, 2H), 2.02-
1.95 (m, 2H), 1.02 (d, J ) 6.0 Hz, 6H); 13C NMR (100 MHz,
CDCl3) δ 154.8, 152.9, 151.5, 148.8, 148.0, 145.2, 123.8, 120.0,
116.7, 113.2, 112.2, 102.3, 48.6, 43.8, 41.7, 30.2, 22.8; MS m/z
465.0 (M + H)+. HPLC: (a) 99.1% (65% water/35% acetonitrile);
(b) 98.0% (from 20% to 40% acetonitrile).
8-(Benzo[1,3]dioxol-5-ylmethyl)-2-aminoadenine (16). 2,4,5,6-
Tetraaminopyrimidine sulfate (6 g, 25 mmol) was dissolved in hot
NaOH (3.6 g, 90 mmol) in water (36 mL). The solution was slowly
cooled to result in the formation of yellow crystals of free base
(14). These were collected, dried, and further used without
additional purification. To a solution of 3,4-(methylenedioxy)-
phenylacetic acid (3.0 g, 16.6 mmol) in CH2Cl2 (19 mL) under
inert atmosphere were added cyanuric fluoride (2.2 mL, 16.7 mmol)
and pyridine (1.5 mL, 18.3 mmol). The reaction mixture was stirred
for 1.5 h at room temperature and then diluted in CH2Cl2 (200 mL)
and washed once with water (5 mL). The organic layer was removed
to yield the acid fluoride 13. To this crude were added 14 (3.3 g,
23.85 mmol), K2CO3 (3.95 g, 28.62 mmol), DMAP (0.21 g, 1.75
mmol), and DMF (65 mL). The reaction was heated with stirring
at 120 °C for 2 h. Following cooling, the solids were filtered off
and washed with MeOH. Solvent was removed in vacuo to yield
the crude amide 15 (3.5 g, 69% yield). MS m/z 303.1 (M + H)+.
To this material were added isobutyl alcohol (12 mL) and a 25%
solution of NaOMe in MeOH (12 mL), and the resulting solution
was heated at 105 °C for 4.5 h. Following cooling, the pH of the
solution was adjusted to 7 by addition of concentrated HCl. The
solvent was removed under vacuum and the crude purified by silica
gel column chromatography (CHCl3/7N NH3 in MeOH at 5:1) to
yield 16 (1.4 g, 25.7%). 1H NMR (400 MHz, DMSO-d6) δ 6.85 (s,
1H), 6.83 (d, J ) 8.0 Hz, 1H), 6.72 (d, J ) 8.0 Hz, 1H), 6.59 (s,
2H), 5.95 (s, 2H), 5.59 (s, 2H), 3.89 (s, 2H); MS m/z 285.0 (M +
H)+.
1
7 (159 mg, 55% yield). H NMR (400 MHz, CDCl3/methanol-d4)
δ 8.18 (s, 1H), 7.44 (s, 1H), 7.18 (s, 1H), 6.07 (s, 2H); MS m/z
413.9 (M + H)+.
8-(6-Bromo-benzo[1,3]dioxol-5-ylsulfanyl)adenine (8). 8-Mer-
captoadenine (602 mg, 3.6 mmol), neocuproine hydrate (81 mg,
0.36 mmol), CuI (69 mg, 0.36 mmol), NaO-t-Bu (692 mg, 7.2
mmol), 5-bromo-6-iodo-benzo[1,3]dioxole (3.53 g, 10.8 mmol), and
anhydrous DMF (24 mL) were charged in a nitrogen box. The
vessel was sealed with Teflon tape, placed in an oil bath (110 °C),
and magnetically stirred for 24 h. The solvent was removed under
high vacuum and the crude purified by column chromatography
on silica gel (EtOAc/CH2Cl2/MeOH at 2:2:1) to provide the product
1
(1.29 g, 97%). H NMR (400 MHz, acetone-d6) δ 8.07 (s, 1H),
7.28 (s, 1H), 7.15 (s, 1H), 7.08 (bs, 2H), 6.13 (s, 2H); MS m/z
366.0 (M + H)+.
8-(6-Iodo-benzo[1,3]dioxol-5-ylsulfanyl)-9-(pent-4-ynyl)ade-
nine (9). A solution of 7 (40 mg, 96.8 µmol), Cs2CO3 (31.5 mg,
96.8 µmol), and pent-4-ynyl tosylate (28 mg, 114 µmol) in
anhydrous DMF (1 mL) was stirred at 80 °C for 30 min. The solvent
was removed under high vacuum and the crude purified by
preparatory thin-layer chromatography to give the desired product
(25 mg, 53.9%). 1H NMR (400 MHz, CDCl3/methanol-d4) δ 8.23
(s, 1H), 7.38 (s, 1H), 7.04 (s, 1H), 6.05 (s, 2H), 4.32 (t, J ) 7.3
Hz, 2H), 2.33-2.31 (m, 2H), 2.12-2.04 (m, 3H); 13C NMR (100
MHz, CDCl3/methanol-d4) δ 151.1, 149.6, 149.2, 147.5, 125.7,
119.4, 113.6, 102.4, 93.8, 82.1, 69.4, 42.8, 28.1, 15.8; MS m/z 480.0
(M + H)+. HPLC: (a) 98.5% (65% water/35% acetonitrile); (b)
97.7% (from 35% to 95% acetonitrile).
3-(tert-Butoxycarbonyl-isopropyl-amino)-propyl tosylate. A
solution of 3-bromo-1-propanol (5 g, 0.036 mol) in isopropylamine
(9 mL, 0.11 mol) was heated overnight at 50 °C with stirring.
Solvent was removed under vacuum to give the product, 3-iso-
propyl-amino-propanol, as a white solid. To this were added di-
tert-butyl dicarbonate (10 g, 0.05 mol) and triethylamine (11 mL,
0.08 mol), and the resulting solution was stirred at room temperature
overnight. Following solvent removal, the crude was purified by
column chromatography on silica gel (CH2Cl2, then CH2Cl2/acetone
at 3:1) to provide the 3-(tert-butoxycarbonyl-isopropyl-amino)-
1
propanol (5.8 g, 75%). H NMR (400 MHz, CDCl3) δ 3.93 (bs,
1H), 3.58 (m, 2H), 3.33 (m, 2H), 1.67 (m, 2H), 1.48 (s, 9H), 1.16
(d, J ) 6.9 Hz, 6H); MS m/z 218.1 (M + H)+. A solution of 3-(tert-
butoxycarbonyl-isopropyl-amino)-propanol (3.5 g, 0.016 mol),
p-toluenesulfonyl chloride (3.7 g, 0.019 mol), and pyridine (1.6
mL, 0.019 mol) in CH2Cl2 (50 mL) was stirred overnight at room
temperature. Following solvent removal, the product (2.3 g, 40%)
was isolated by column chromatography on silica gel (hexanes/
CH2Cl2/EtOAc at 5:4:1). 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J
) 8.2 Hz, 2H), 7.35 (d, J ) 8.2 Hz, 2H), 4.06-4.03 (m, 3H), 3.09
8-Benzo[1,3]dioxol-5-ylmethyl-2-fluoroadenine (17). To a cooled
(0 °C) solution of 16 (1.48 g, 5.2 mmol) in HF/pyridine (3.64 mL),
NaNO2 (0.47 g, 6.76 mmol) was slowly added. The reaction was

388 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
He et al.
brought to room temperature and further stirred for 1 h. Following
dilution with CH2Cl2 (38 mL), the excess HF was quenched by
stirring for an additional 1 h with CaCO3 (0.95 g) and water (5
mL). The mixture was dried in vacuo and subsequently purified
by silica gel column chromatography (CHCl3/MeOH/NH4OH at
5:1:0.5) to yield 17 (0.9 g, 60% yield). 1H NMR (400 MHz, DMSO-
d6) δ 7.59 (bs, 2H), 6.94-6.90 (m, 3H), 6.81 (d, J ) 8.0 Hz, 1H),
6.03 (s, 2H), 4.06 (s, 2H); MS m/z 288.0 (M + H)+.
2-Fluoro-8-(6-iodo-benzo[1,3]dioxol-5-ylmethyl)adenine (18).
A solution of 17 (50 mg, 0.17 mmol), NIS (94 mg, 0.4 mmol), and
TFA (20 mg, 13.4 µL, 0.17 mmol) in CH2Cl2 (200 µL) was stirred
at room temperature overnight. After solvent removal, the desired
product 18 (6 mg, 8.5%) was purified by silica gel column
chromatography (CHCl3/EtOAc at 9:1 to 4:6). 1H NMR (400 MHz,
DMSO-d6) δ 7.6 (bs, 2H), 7.38 (s, 1H), 6.95 (s, 1H), 6.03 (s, 2H),
4.12 (s, 2H); MS m/z 414.1 (M + H)+.
2-Fluoro-8-(6-bromo-benzo[1,3]dioxol-5-ylmethyl)adenine (19).
A solution of 17 (45 mg, 0.157 mmol) and NBS (56 mg, 0.314
mmol) in DMF (0.5 mL) was stirred at room temperature for 1.5
h. Following solvent removal, product (20 mg, 34.8%) was collected
through silica gel column purification (CHCl3/EtOAc at 9:1 to 4:6).
1H NMR (400 MHz, acetone-d6) δ 7.51 (bs, 2H), 7.21 (s, 1H),
6.98 (s, 1H), 6.06 (s, 2H), 4.13 (s, 2H); MS m/z 366.0 (M + H)+.
2-Fluoro-8-(6-iodo-benzo[1,3]dioxol-5-ylmethyl)-9-(3-iso-
propylamino-prop yl)adenine (25). A solution of 18 (300 mg,
0.726 mmol), Cs2CO3 (285 mg, 0.87 mmol), and 1,3-dibromopro-
pane (370 µL, 3.63 mmol) in anhydrous DMF (5 mL) was stirred
at 50 °C for 2 h. Following solvent removal, product (330 mg,
85%) was collected through silica gel column purification (CHCl3
then EtOAc/hexanes/CHCl3/i-PrOH at 4:2:4:0.4). MS m/z 534.0 (M
+ H)+. To this product, i-PrNH2 (10 mL) was added in excess,
and the resulting solution was stirred at room temperature for 1 h.
Excess amine was removed and product (230 mg, 75%) collected
through silica gel column purification (CHCl3/EtOAc/i-PrOH/NH4-
OH at 4:4:2:0.3). 1H NMR (400 MHz, CDCl3) δ 7.29 (s, 1H), 6.59
(s, 1H), 5.94 (s, 2H), 5.89 (bs, 2H), 4.25 (s, 2H), 4.11 (t, J ) 7.0
Hz, 2H), 2.73-2.60 (m, 1H), 2.55 (t, J ) 6.8 Hz, 1H), 1.93-1.86
(m, 2H), 1.03-1.02 (d, J ) 6.0 Hz, 6H); 13C NMR (100 MHz,
methanol-d4) δ 160.0, 158.4, 157.2, 152.4, 151.3, 149.4, 148.4,
133.1, 118.7, 110.6, 102.4, 88.5, 42.8, 40.1, 38.8, 27.6, 19.4; MS
m/z 513.2 (M + H)+. HPLC: (a) 98.5% (60% water/40%
acetonitrile); (b) 97.2% (from 20% to 50% acetonitrile).
2-Amino-8-(3,4-dimethoxy-benzyl)adenine. Starting from 3,4-
dimethoxyphenylacetic acid (3.0 g, 15.3 mmol) and 2,4,5,6-
tetraaminopyrimidine (4.1 g, 29 mmol) and following the procedure
for the synthesis of 16, the desired product was obtained (1.8 g,
39.2%). 1H NMR (400 MHz, DMSO-d6) δ 6.90 (s, 1H), 6.85 (d, J
) 8.0 Hz, 1H), 6.74 (d, J ) 8.0 Hz, 1H), 6.44 (bs, 2H), 5.51 (bs,
2H), 3.88 (s, 2H), 3.70 (s, 3H), 3.68 (s, 3H); MS m/z 301.2 (M +
H)+.
2-Fluoro-8-(6-chloro-benzo[1,3]dioxol-5-ylmethyl)adenine (20).
A solution of 17 (20 mg, 0.07 mmol) and NCS (35.6 mg, 0.27
mmol) in anhydrous DMF (0.4 mL) was stirred at room temperature
for 2.5 h. Following solvent removal, the product (11 mg, 48.8%)
was collected through silica gel column purification (CHCl3/EtOAc
2-Fluoro-8-(3,4-dimethoxy-benzyl)adenine (27). Starting from
2-amino-8-(3,4-dimethoxy-benzyl)adenine (0.66 g, 2.2 mmol) and
following the procedure for the synthesis of 17, the desired product
1
at 9:1 to 5:5). H NMR (400 MHz, DMSO-d6) δ 7.40 (bs, 2H),
6.97 (s, 1H), 6.89 (s, 2H), 5.97 (s, 2H), 4.04 (s, 2H); MS m/z 322.1
1
(M + H)+.
was obtained (0.34 g, 51%). H NMR (400 MHz, DMSO-d6) δ
7.61 (bs, 2H), 7.03 (s, 1H), 6.94 (d, J ) 8.3 Hz, 1H), 6.84 (d, J )
8.6 Hz, 1H), 4.07 (s, 2H), 3.80 (s, 3H), 3.77 (s, 3H); MS m/z 304.0
(M + H)+.
2-Fluoro-8-(6-iodo-benzo[1,3]dioxol-5-ylmethyl)-9-(pent-4-
ynyl)adenine (21). A solution of 18 (6 mg, 0.0145 mmol), Cs2-
CO3 (5 mg, 0.0145 mmol), and pent-4-ynyl tosylate (4.5 mg, 0.189
mmol) in anhydrous DMF (200 µL) was stirred at 60 °C for 1.5 h.
Following solvent removal, product (5.9 mg, 84.9%) was collected
through silica gel column purification (EtOAc/hexanes/CHCl3/i-
PrOH at 10:20:20:1). 1H NMR (400 MHz, CDCl3) δ 7.29 (s, 1H),
6.59 (s, 1H), 5.94 (s, 2H), 5.83 (bs, 2H), 4.26 (s, 2H), 4.11 (t, J )
7.4 Hz, 2H), 2.26-2.19 (m, 2H), 2.00 (t, J ) 2.5 Hz, 1H), 1.98-
1.94 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 150.9, 148.9, 147.8,
131.5, 118.8, 109.4, 101.9, 88.1, 82.3, 69.9, 42.3, 39.2, 28.2, 15.9;
MS m/z 480.0 (M + H)+. HPLC: (a) 95.5% (60% water/40%
acetonitrile); (b) 95.0% (from 35% to 55% acetonitrile).
2-Fluoro-8-(6-bromo-benzo[1,3]dioxol-5-ylmethyl)-9-(pent-4-
ynyl)adenine (22). A solution of 19 (20 mg, 55 µmol), Cs2CO3
(18 mg, 55 µmol), and pent-4-ynyl tosylate (17 mg, 72 µmol) in
anhydrous DMF (138 µL) was stirred at 60 °C for 2 h. Following
solvent removal, the product (13 mg, 54.7%) was collected through
silica gel column purification (EtOAc/hexanes/CHCl3/i-PrOH at 10:
20:20:1). 1H NMR (400 MHz, CDCl3) δ 7.05 (s, 1H), 6.60 (s, 1H),
6.15 (bs, 2H), 5.96 (s, 2H), 4.28 (s, 2H), 4.13 (t, J ) 7.5 Hz, 2H),
2.25-2.21 (m, 2H), 2.00 (t, J ) 2.6 Hz, 1H), 1.98-1.92 (m, 2H);
13C NMR (100 MHz, CDCl3) δ 157.6, 156.0, 152.6, 150.2, 147.5,
127.6, 116.4, 114.1, 112.5, 109.5, 101.6, 81.9, 69.5, 41.9, 33.7,
27.8, 15.5; MS m/z 432.0 (M + H)+. HPLC: (a) 99.0% (60% water/
40% acetonitrile); (b) 98.5% (from 35% to 55% acetonitrile).
2-Fluoro-8-(6-chloro-benzo[1,3]dioxol-5-ylmethyl)-9-(pent-4-
ynyl)adenine (23). A solution of 20 (11 mg, 0.034 mmol), Cs2-
CO3 (11 mg, 0.034 mmol), and pent-4-ynyl tosylate (10.5 mg, 0.044
mmol) in anhydrous DMF (85 µL) was stirred at 50 °C for 1 h.
Following solvent removal, the product (4.2 mg, 31.9%) was
collected through silica gel column purification (EtOAc/hexanes/
CHCl3/i-PrOH at 10:20:20:1). 1H NMR (400 MHz, CDCl3) δ 6.89
(s, 1H), 6.61 (s, 1H), 5.98 (bs, 2H), 5.96 (s, 2H), 4.27 (s, 2H), 4.13
(t, J ) 7.5 Hz, 2H), 2.24-2.10 (m, 2H), 2.00-1.91 (m, 3H); 13C
NMR (100 MHz, CDCl3) δ 1597, 158.0, 156.3, 150.6, 147.7, 147.2,
126.2, 125.3, 110.0, 102.0, 82.3, 69.9, 42.2, 31.4, 28.1, 15.8; MS
m/z 388.1 (M + H)+. HPLC: (a) 98.1% (65% water/35%
acetonitrile); (b) 97.0% (from 35% to 45% acetonitrile).
2-Fluoro-8-(2-iodo-4,5-dimethoxy-benzyl)adenine (28). A so-
lution of 27 (50 mg, 0.165 mmol), NIS (74 mg, 0.33 mmol), and
TFA (18.8 mg, 12.7 µL, 0.165 mmol) in acetonitrile (120 µL) was
stirred at room temperature for 24 h. Following solvent removal,
the product (12 mg, 16.9%) was collected through silica gel column
purification (CHCl3/MeOH/AcOH at 80:1:0.5 to 30:1:0.5). MS m/z
430.1 (M + H)+.
2-Fluoro-8-(2-bromo-4,5-dimethoxy-benzyl)adenine (29). A
solution of 27 (65 mg, 0.226 mmol) and NBS (80 mg, 0.45 mmol)
in DMF (0.75 mL) was stirred at room temperature for 2.5 h.
Following solvent removal, the product (8.2 mg, 53.6%) was
collected through silica gel column purification (CHCl3/MeOH/
AcOH at 80:1:0.5 to 30:1:0.5). 1H NMR (400 MHz, acetone-d6) δ
7.13 (s, 1H), 7.09 (s, 1H), 6.80 (bs, 2H), 4.26 (s, 2H), 3.84 (s, 3H),
3.78 (s, 3H); MS m/z 382.0 (M + H)+.
2-Fluoro-8-(2-chloro-4,5-dimethoxy-benzyl)adenine (30). A
solution of 27 (40 mg, 0.132 mmol) and NCS (77.8 mg, 0.58 mmol)
in anhydrous DMF (0.7 mL) was stirred at room temperature for
5.5 h. Following solvent removal, the product (22 mg, 49.4%) was
collected through silica gel column purification (CHCl3/EtOAc at
8:2 to 4:6). MS m/z 338.0 (M + H)+.
2-Fluoro-8-(2-iodo-4,5-dimethoxy-benzyl)-9-(pent-4-ynyl)-
adenine (31). A solution of 2-fluoro-8-(2-iodo-4,5-dimethoxy-
benzyl)adenine (12 mg, 0.028 mmol), Cs2CO3 (9 mg, 0.028 mmol),
and pent-4-ynyl tosylate (8.6 mg, 7 µL, 0.036 mmol) in anhydrous
DMF (80 µL) was stirred at 50 °C for 1 h. Following solvent
removal, the product (13.7 mg, 99%) was collected through silica
gel column purification (CHCl3/EtOAc/hexanes/i-PrOH at
1
20:10:20:1). H NMR (400 MHz, CDCl3) δ 7.27 (s, 1H), 6.65 (s,
1H), 5.94 (bs, 2H), 4.29 (s, 2H), 4.13 (t, J ) 7.3 Hz, 2H), 3.87 (s,
3H), 3.73 (s, 3H), 2.26-2.22 (m, 2H), 2.00 (t, J ) 2.6 Hz, 1H),
1.97-1.90 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 156.5, 153.2,
151.3, 150.0, 149.1, 130.9, 121.9, 112.6, 88.5, 82.5, 70.0, 56.4,
56.2, 42.6, 39.2, 28.4, 16.1; MS m/z 496.2 (M + H)+. HPLC: (a)
99.9% (60% water/40% acetonitrile); (b) 96.8% (from 35% to 55%
acetonitrile).

Purine-Scaffold Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 389
(8) Chiosis, G.; Timaul, M. N.; Lucas, B.; Munster, P. N.; Zheng, F. F.;
Sepp-Lorenzino, L.; Rosen, N. A small molecule designed to bind
to the adenine nucleotide pocket of Hsp90 causes Her2 degradation
and the growth arrest and differentiation of breast cancer cells. Chem.
Biol. 2001, 8, 289-299.
(9) (a) Chiosis, G.; Lucas, B.; Shtil, A.; Huezo, H.; Rosen, N. Develop-
ment of a purine-based novel class of Hsp90 binders that inhibit the
proliferation of cancer cells and induce the degradation of Her2
tyrosine kinase. Bioorg. Med. Chem. 2002, 10, 3555-3564. (b)
Chiosis, G.; Rosen, N. PCT Int. Appl. W. O. 20020236075, 2002.
(c) Kasibhatla, S. R.; Hong, K.; Zhang, L.; Biamonte, M. A.; Boehm,
M. F.; Shi, J.; Fan, J. PCT Int. Appl. W. O. 2003037860, 2003. (d)
Dymock, B.; Barril, X.; Beswick, M.; Collier, A.; Davies, N.;
Drysdale, M.; Fink, A.; Fromont, C.; Hubbard, R. E.; Massey, A.;
Surgenor, A.; Wright, L. Adenine derived inhibitors of the molecular
chaperone HSP90-SAR explained through multiple X-ray structures.
Bioorg. Med. Chem. Lett. 2004, 14, 325-328.
(10) Llauger, L.; He, H.; Kim, J.; Aguirre, J.; Rosen, N.; Peters, U.; Davies,
P.; Chiosis, G. 8-Arylsulfanyl and 8-arylsulfoxyl adenine derivatives
as inhibitors of the heat shock protein 90. J. Med. Chem. 2005, 48,
2892-2905.
(11) He, H.; Llauger, L.; Rosen, N.; Chiosis, G. General method for the
synthesis of 8-arylsulfanyl adenine derivatives. J. Org. Chem. 2004,
69, 3230-3232.
(12) Manka, J. T.; McKenzie, V. C.; Kaszynski, P. Azo group-assisted
nucleophilic aromatic substitutions in haloarene derivatives: prepara-
tion of substituted 1-iodo-2,6-bispropylthiobenzenes. J. Org. Chem.
2004, 69, 1967-1971.
(13) Naidis, F. B.; Kolesova, M. B.; Aleksander, K. L.; Smirnova, N. V.;
Pernikoza, V. G. Method of obtaining 8-bromoadenine. Izobreteniya
1995, 1, 242-243.
(14) Castanet, A.-S.; Colobert, F.; Broutin, P.-E. Mild and regioselective
iodination of electron-rich aromatics with N-iodosuccinimide and
catalytic trifluoroacetic acid. Tetrahedron Lett. 2002, 43, 5047-5048.
(15) Tipson, R. S. On esters of p-toluenesulfonic acid. J. Org. Chem. 1944,
9, 235-241.
(16) Tarbell, D. S.; Yamamoto, Y.; Pope, B. M. New method to prepare
N-t-butoxycarbonyl derivatives and the corresponding sulfur ana-
logues from di-tert-butyl dicarbonate or di-tert-butyl dithiol dicar-
bonates and amino acids. Proc. Natl. Acad. Sci. U.S.A. 1972, 69,
730-732.
2-Fluoro-8-(2-bromo-4,5-dimethoxy-benzyl)-9-(pent-4-ynyl)-
adenine (32). A solution of 8-(2-bromo-4,5-dimethoxy-benzyl)-2-
fluoroadenine (13 mg, 0.034 mmol), Cs2CO3 (11 mg, 0.034 mmol),
and pent-4-ynyl tosylate (10 mg, 9 µL, 0.044 mmol) in anhydrous
DMF (80 µL) was stirred at 60 °C for 30 min. Following solvent
removal, the product (8.2 mg, 53.6%) was collected through silica
gel column purification (CHCl3/EtOAc/hexanes/i-PrOH at
1
20:10:20:1). H NMR (400 MHz, CDCl3) δ 7.06 (s, 1H), 6.67 (s,
1H), 5.92 (bs, 2H), 4.31 (s, 2H), 4.14 (t, J ) 7.4 Hz, 2H), 3.88 (s,
3H), 3.75 (s, 3H), 2.25-2.20 (m, 2H), 1.99 (t, J ) 2.6 Hz, 1H),
1.96-1.89 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 160.0, 158.3,
156.7, 153.4, 151.3, 149.4, 127.2, 117.3, 115.9, 114.5, 113.2, 82.7,
70.2, 56.62, 56.56, 42.7, 34.3, 30.1, 28.5, 16.3; MS m/z 447.9 (M
+ H)+. HPLC: (a) 99.0% (60% water/40% acetonitrile); (b) 98.8%
(from 35% to 55% acetonitrile).
2-Fluoro-8-(2-chloro-4,5-dimethoxy-benzyl)-9-(pent-4-ynyl)-
adenine (33). A solution of 8-(2-chloro-4,5-dimethoxy-benzyl)-2-
fluoroadenine (22 mg, 0.065 mmol), Cs2CO3 (21 mg, 0.065 mmol),
and pent-4-ynyl tosylate (20 mg, 17.3 µL, 0.085 mmol) in
anhydrous DMF (170 µL) was stirred at 50 °C for 2 h. Following
solvent removal, the product (14 mg, 53.8%) was collected through
silica gel column purification (CHCl3/EtOAc/hexanes/i-PrOH at 20:
10:20:1). 1H NMR (400 MHz, CDCl3) δ 6.91 (s, 1H), 6.67 (s, 1H),
6.01 (bs, 2H), 4.31 (s, 2H), 4.14 (t, J ) 7.5 Hz, 2H), 3.87 (s, 3H),
3.75 (s, 3H), 2.24-2.20 (m, 2H), 2.01-1.99 (m, 1H), 1.97-1.88
(m, 2H); 13C NMR (100 MHz, CDCl3) δ 1596, 157.9, 156.3, 152.3,
148.9, 146.0, 124.9, 112.7, 82.3, 69.9, 56.3, 56.2, 42.2, 31.2, 28.1,
15.9; MS m/z 404.1 (M + H)+. HPLC: (a) 95.1% (65% water/
35% acetonitrile); (b) 96.7% (from 35% to 45% acetonitrile).
Acknowledgment. This work was supported by AACR-
Cancer Research and Prevention Foundation, the Susan G.
Komen Breast Cancer Foundation, Mr. William H. Goodwin
and Mrs. Alice Goodwin, and the Commonwealth Cancer
Foundation for Research and the Experimental Therapeutics
Center of Memorial Sloan-Kettering Cancer Center.
(17) (a) Mitsunobu, O. The use of diethyl azodicarboxylate and triph-
enylphosphine in synthesis and transformation of natural products.
Synthesis 1981, 1, 1-28. (b) Lucas, B.; Rosen, N.; Chiosis, G. Facile
synthesis of 9-alkyl-8-benzyl-9H-purin-6-ylamine derivatives. J.
Comb. Chem. 2001, 3, 518-520.
Supporting Information Available: Results from spectral data,
HPLC, and LC-MS analyses. This material is available free of
charge via the Internet at http://pubs.acs.org.
(18) Salvatore, R. N.; Nagle, A. S.; Jung, K. W. Cesium effect: high
chemoselectivity in direct N-alkylation of amines. J. Org. Chem.
2002, 67, 674-683.
(19) Olah, G. A.; Nojima, M.; Kerekes, I. Synthetic methods and reactions;
IV.1 Fluorination of carboxylic acids with cyanuric fluoride. Synthesis
1973, 8, 487-488.
(20) (a) Fukuhara, T.; Yoneda, N.; Suzuki, A. A facile preparation of
fluoropyridines from aminopyridines via diazotization and fluoro-
dediazoniation in HF or HF-pyridine solutions. J. Fluorine Chem.
1988, 38, 435-438. (b) Thompson, A. M.; Showalter, H. D. H.;
Denny, W. A. Synthesis of 7-substituted 3-aryl-1,6-naphthyridin-2-
amines and 7-substituted 3-aryl-1,6-naphthyridin-2(1H)-ones via
diazotization of 3-aryl-1,6-naphthyridine-2,7-diamines. J. Chem. Soc.,
Perkin Trans. 1 2000, 1843-1852.
(21) (a) Day, R. A.; Blake, J. A.; Stephens, C. E. Convenient and improved
halogenation of 3,5-diarylisoxazoles using N-halosucinimides. Syn-
thesis 2003, 10, 1586-1590. (b) Tanemura, K.; Suzuki, T.; Nishida,
Y.; Satsumababayashi, K.; Horaguchi, T. Halogenation of aromatic
compounds by N-chloro-, N-bromo-, and N-iodosuccinimide. Chem.
Lett. 2003, 32, 932-934.
(22) (a) Llauger-Bufi, L.; Felts, S. J.; Huezo, H.; Rosen, N.; Chiosis, G.
Synthesis of novel fluorescent probes for the molecular chaperone
Hsp90. Bioorg. Med. Chem. Lett. 2003, 13, 3975-3978. (b) Kim,
J.; Felts, S.; Llauger, L.; He, H.; Huezo, H.; Rosen, N.; Chiosis, G.
Development of a fluorescence polarization assay for the molecular
chaperone Hsp90. J. Biomol. Screening 2004, 9, 375-381.
(23) Mimnaugh, E. G.; Chavany, C.; Neckers, L. Polyubiquitination and
proteasomal degradation of the p185c-erbB-2 receptor protein-
tyrosine kinase induced by geldanamycin. J. Biol. Chem. 1996, 271,
22796-22801.
(24) Srethapakdi, M.; Liu, F.; Tavorath, R.; Rosen, N. Inhibition of Hsp90
function by ansamycins causes retinoblastoma gene product-depend-
ent G1 arrest. Cancer Res. 2000, 60, 3940-3946.
(25) Huezo, H.; Vilenchik, M.; Rosen, N.; Chiosis, G. Microtiter cell-
based assay for detection of agents that alter cellular levels of Her2
and EGFR. Chem. Biol. 2003, 10, 629-634.
References
(1) Mosser, D. D.; Morimoto, R. I. Molecular chaperones and the stress
of oncogenesis. Oncogene 2004, 23, 2907-2918.
(2) (a) Kamal, A.; Thao, L.; Sensintaffar, J.; Zhang, L.; Boehm, M. F.;
Fritz, L. C.; Burrows, F. J. Therapeutic and diagnostic implications
of Hsp90 activation. Trends Mol. Med. 2004, 10, 283-290. (b)
Neckers, L. Hsp90 inhibitors as novel cancer chemotherapeutic
agents. Trends Mol. Med. 2002, 8, S55-S61. (c) Workman, P.;
Maloney, A. HSP90 as a new therapeutic target for cancer therapy:
the story unfolds. Expert Opin. Biol. Ther. 2002, 2, 3-24. (d) Chiosis,
G.; Vilenchik, M.; Kim, J.; Solit, D. Hsp90: the vulnerable
chaperone. Drug DiscoVery Today 2004, 9, 881-888.
(3) (a) Panaretou, B.; Prodromou, C.; Roe, S. M.; O’Brien, R.; Ladbury,
J. E.; Piper, P. W.; Pearl, L. H. ATP binding and hydrolysis are
essential to the function of the Hsp90 molecular chaperone in vivo.
EMBO J. 1998, 17, 4829-4836. (b) Obermann, W. M.; Sondermann,
H.; Russo, A. A.; Pavletich, N. P.; Hartl, F. U. In vivo function of
Hsp90 is dependent on ATP binding and ATP hydrolysis. J. Cell
Biol. 1998, 143, 901-910.
(4) (a) Wegele, H.; Muller, L.; Buchner, J. Hsp70 and Hsp90-a relay
team for protein folding. ReV. Physiol. Biochem. Pharmacol. 2004,
151, 1-44. (b) Prodromou, C.; Pearl, L. H. Structure and functional
relationships of Hsp90. Curr. Cancer Drug Targets 2003, 5, 301-
323.
(5) (a) Kamal, A.; Thao, L.; Sensintaffar, J.; Zhang, L.; Boehm, M. F.;
Fritz, L. C.; Burrows, F. J. A high-affinity conformation of Hsp90
confers tumour selectivity on Hsp90 inhibitors. Nature 2003, 425,
407-410. (b) Vilenchik, M.; Solit, D.; Basso, A.; Huezo, H.; Lucas,
B.; He, H.; Rosen N.; Spampinato, C.; Modrich, P.; Chiosis, G.
Targeting wide-range oncogenic transformation via PU24FCl, a
specific inhibitor of tumor Hsp90. Chem. Biol. 2004, 11, 787-797.
(6) Chene, P. ATPases as drug targets: learning from their structure.
Nat. ReV. Drug DiscoVery 2002 9, 665-673.
(7) Chiosis, G.; Lucas, B.; Huezo, H.; Solit, D.; Basso, A.; Rosen, N.
Development of purine-scaffold small molecule inhibitors of Hsp90.
Curr. Cancer Drug Targets 2003, 3, 363-368.

390 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
He et al.
(26) Chiosis, G.; Rosen, N.; Huezo, H. Assays for identification of
bioactive compounds that interact with heat shock protein 90. PCT
Int. Appl. W. O. 2005012482, 2005.
allylamino-17-demethoxygeldanamycin, an inhibitor of heat shock
protein 90. J. Natl. Cancer Inst. 1999, 91, 1940-1949.
(29) Krishna Mohan, K. V. V.; Narender, N.; Kulkarni, S. J. Simple and
regioselective oxyiodination of aromatic compounds with ammonium
iodide and oxone. Tetrahedron Lett. 2004, 45, 8015-8018.
(30) Orito, K.; Hatakeyama, T.; Takeo, M.; Suginome, H. Iodination of
alkyl aryl ethers by mercury(II) oxide-iodine reagent in dichloro-
methane. Synthesis 1995, 10, 1273-1277.
(31) Hubig, S. M.; Jung, W.; Kochi, J. K. Cation radicals as intermediates
in aromatic halogenation with iodine monochloride: Solvent and salt
effects on the competition between chlorination and iodination. J.
Org. Chem. 1994, 59, 6233-6244.
(27) (a) Eiseman, J. L.; Lan, J.; Lagattuta, T. F.; Hamburger, D. R.; Joseph,
E.; Covey, J. M.; Egorin, M. J. Pharmacokinetics and pharmaco-
dynamics of 17-demethoxy 17-[[(2-dimethylamino)ethyl]amino]-
geldanamycin (17DMAG, NSC 707545) in C.B-17 SCID mice
bearing MDA-MB-231 human breast cancer xenografts. Cancer
Chemother. Pharmacol. 2005, 55, 21-32. (b) Banerji, U.; Walton,
M.; Raynaud, F.; Grimshaw, R.; Kelland, L.; Valenti, M.; Judson,
I.; Workman, P. Pharmacokinetic-pharmacodynamic relationships for
the heat shock protein 90 molecular chaperone inhibitor 17-
allylamino, 17-demethoxygeldanamycin in human ovarian cancer
xenograft models. Clin. Cancer Res. 2005, 11, 7023-7032.
(28) Kelland, L. R.; Sharp, S. Y.; Rogers, P. M.; Myers, T. G.; Workman,
P. DT-diaphorase expression and tumor cell sensitivity to 17-
JM0508078