Evaluation of 8-arylsulfanyl, 8-arylsulfoxyl, and 8-arylsulfonyl adenine derivatives as inhibitors of the heat shock protein 90

Article abstract of DOI:10.1021/jm049012b

Hsp90 is a chaperone protein with important roles in maintaining transformation and in elevating the survival and growth potential of cancer cells. Currently there is an increasing interest in developing inhibitors of this protein as anticancer therapeutics. One of such inhibitors, the purine-scaffold class, has been reported to be potent and selective against Hsp90 both in vitro and in vivo models of cancer. Here, a series of 8-arylsulfanyl, -sulfoxyl, and -sulfonyl adenine members of the purine 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. Our results suggest that 8-arylsulfanyl adenine derivatives are good inhibitors of chaperone activity, whereas oxidation of the sulfides to sulfoxides or sulfones leads to compounds of decreased activity. The study identifies derivative 11v as the most potent Hsp90 inhibitor of the purine-scaffold series published to date (EC₅₀ = 30 nM), and also as the compound of this class with highest selectivity for tumor vs normal cell Hsp90 (700 to 3000-fold). Most rewardingly, this work has allowed for the identification of Hsp90 inhibitors with selective affinities for Hsp90-client protein complexes, derivatives that may represent useful pharmacological tools in dissecting Hsp90-regulated processes.

Full text of DOI:10.1021/jm049012b

2892
J. Med. Chem. 2005, 48, 2892-2905
Evaluation of 8-Arylsulfanyl, 8-Arylsulfoxyl, and 8-Arylsulfonyl Adenine
Derivatives as Inhibitors of the Heat Shock Protein 90
Laura Llauger,†,| Huazhong He,†,| Joungnam Kim,† Julia Aguirre,† Neal Rosen,†,§ Ulf Peters,† Peter Davies,‡ 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 Pathology, Albert Einstein College of Medicine, Bronx, New York 10461
Received December 3, 2004
Hsp90 is a chaperone protein with important roles in maintaining transformation and in
elevating the survival and growth potential of cancer cells. Currently there is an increasing
interest in developing inhibitors of this protein as anticancer therapeutics. One of such
inhibitors, the purine-scaffold class, has been reported to be potent and selective against Hsp90
both in vitro and in vivo models of cancer. Here, a series of 8-arylsulfanyl, -sulfoxyl, and -sulfonyl
adenine members of the purine 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. Our results suggest that 8-arylsulfanyl adenine derivatives are
good inhibitors of chaperone activity, whereas oxidation of the sulfides to sulfoxides or sulfones
leads to compounds of decreased activity. The study identifies derivative 11v as the most potent
Hsp90 inhibitor of the purine-scaffold series published to date (EC50 ) 30 nM), and also as the
compound of this class with highest selectivity for tumor vs normal cell Hsp90 (700 to 3000-
fold). Most rewardingly, this work has allowed for the identification of Hsp90 inhibitors with
selective affinities for Hsp90-client protein complexes, derivatives that may represent useful
pharmacological tools in dissecting Hsp90-regulated processes.
Introduction
The chaperone heat shock protein 90 (Hsp90) is an
emerging target in cancer treatment due to its impor-
tant roles in maintaining transformation and in in-
creasing the survival and growth potential of cancer
cells.1 Hsp90 function is regulated by a pocket in the
N-terminal region of the protein that binds and hydro-
lyzes ATP.1a Occupancy of this pocket by high affinity
ligands prevents the dissociation of Hsp90 client pro-
Figure 1. Natural product Hsp90 inhibitors.
teins from the chaperone complex, and as a conse-
quence, the trapped proteins do not achieve their
mature functional conformation and are degraded by the
exhibits dose and schedule dependent liver toxicity
limited solubility and cumbersome formulation. It also
proteasome. Protein clients of Hsp90 are mostly kinases,
steroid receptors, and transcriptional factors involved
in driving multistep malignancy and, in addition, mu-
tated oncogenic proteins required for the transformed
phenotype. Examples include Her2, Raf-1, Akt, Cdk4,
cMet, mutant p53, ER, AR, mutant BRaf, Bcr-Abl, Flt-
3, Polo-1 kinase, HIF-1 alpha, and hTERT.1c-e Degrada-
tion of these proteins by Hsp90 inhibitors leads to cell-
specific growth arrest and apoptosis in cancer cells in
culture and to tumor growth inhibition or regression in
animal models. One such inhibitor, 1 (17-allyl-amino-
desmethoxy-geldanamycin; 17AAG, see Figure 1) has
entered clinical trials in cancer patients in the US and
UK and has shown early evidence of therapeutic activity
when administered alone or in combination with doc-
etaxel.2 Despite these early promising results, 1 has
several potential limitations. Most prominent are its
believed to be caused by the benzoquinone functionality.2a
A structurally unrelated natural product, 2 (radicicol;
RD, see Figure 1) has biological activity similar to that
of 1 but is not hepatotoxic,3 yet no derivative of this class
has made it into clinic. Making use of the peculiar bent
shape of Hsp90 inhibitors and of existent Hsp90 crystal
data, we were able to design a class of purine-scaffold
derivatives with Hsp90 inhibitory activities.4 The first
synthesized derivative of this class, 3 (PU3, see Figure
2), bound Hsp90 with moderate affinity and elicited
cellular effects that mimic 1 addition.5 Preliminary
efforts focused at improving the potency of this agent
have mostly focused on modifying the left side of the
scaffold (see Figure 2) and have led to the synthesis of
several compounds with improved activity in both
biochemical and cellular assays.6 One such compound,
4 (PU24FCl, Figure 2), is a potent and selective inhibitor
of tumor Hsp90 and exhibits antitumor activities in both
in vitro and in vivo models of cancer.7 Other researchers
have adopted our purine-scaffold and furthered struc-
ture-activity relationship (SAR) studies, their efforts
resulting in compounds with higher potency over 4 in
in vitro models of cancer.8,9 Although a significant
* Corresponding author. Tel: 212-639-8929, Fax: 212-717-3267,
e-mail: chiosisg@mskcc.org.
† Program in Molecular Pharmacology and Chemistry, Memorial
Sloan-Kettering Cancer Center.
§ Department of Medicine, Memorial Sloan-Kettering Cancer Center.
‡ Department of Pathology, Albert Einstein College of Medicine.
| These authors equally contributed to the work.
10.1021/jm049012b CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/25/2005

Purine-Scaffold Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2893
phenyl moiety. Here we present the synthesis of such
8-arylsulfanyl, -sulfoxyl, and -sulfonyl adenine deriva-
tives, respectively, and their structure-activity rela-
tionship as inhibitors of Hsp90.
Results and Discussions
Chemistry. Formation of a sulfur link between
adenine and the phenyl ring (X3 ) S, Figure 2) may be
obtained either by nucleophilic attack of the arylthiolate
anion on 8-bromoadenine (Step a, Scheme 1)9,10 or by
the copper-catalyzed coupling of aryliodides with mer-
captoadenine (Step b, Scheme 1).11 Our method for the
formation of 8-arylsulfanyl adenine derivatives (10)
from 8-mercaptoadenine (7) and aryl iodides (8) used
CuI/neocuproine as catalyst and NaOt-Bu/DMF as the
base/solvent combination.11c The reaction occurred in
anhydrous DMF at 110 °C under nitrogen to generate
the products in good yields. If bromine or chlorine were
present on the aryl moiety, the coupling required milder
basic condition and utilized Na3PO4 or K2CO3 instead
of NaOt-Bu. Although less attractive due to thiophenols’
limited commercial availability, stench, and tendency
to quickly oxidize, coupling of 8-bromoadenine (5) with
thiophenols 6 in the presence of a base has also been
used. When not commercially available, thiophenols
could be generated by a modified Leuckart thiophenol
reaction12 starting from the corresponding arylamine.
In a first step, the arylamines were converted via the
aryl diazonium salt to aryl xanthates which afforded
thiophenols on reduction with LiAlH4 or on warming
with a base solution. The 8-arylsulfanyl adenines 10
obtained in these coupling reactions were further alky-
lated at the position 9-N with butyl, pent-4-ynyl, or
2-isopropoxy-ethyl chains. The presence of these chains
at the 9-N position was previously determined to be
favored by the Hsp90 pocket.6 Their introduction was
carried out using a Mitsunobu type reaction13b between
the alcohol and the respective 8-arylsulfanyl adenines
10 in toluene/CH2Cl2 to result in the corresponding 9-N-
alkyl-8-arylsulfanyl adenines 11. Alternatively, deriva-
tives 10 were alkylated using tosylates of the corre-
sponding alcohols14 in the presence of Cs2CO3 or K2CO3
in DMF. Formation of the 3-N (5-30%) and 7-N (1-
10%) isomers was observed by these two procedures;
however, these byproducts could be removed by column
chromatography.6,13b Treatment of 10 with base and an
alkylating agent tended to result in higher percentage
of 3- and 7-N isomers compared to alcohol treatment
under the Mitsunobu conditions. If further introduction
Figure 2. Schematic representation of the purine-scaffold
Hsp90 inhibitor class, and its representative derivatives,
compounds 3 and 4.
number of derivatives has been created by these com-
bined efforts, the nature and position of substituents
on the right side aryl moiety (X1 and X2, Figure 2) has
not been sufficiently investigated. A crystal structure
of the purine derivative 3 in complex with Hsp90R8
proposes that its right side aromatic moiety is involved
in π-stacking interactions with a phenylalanine residue.
It also suggests that the ligand establishes close hydro-
phobic interactions with the pocket in this region, and
thus subtle modifications on the aryl moiety may lead
to a dramatic SAR. A more detailed analysis of favorable
substituents in this area is thus warranted. Identifica-
tion of such substitutions is unfortunately not straight-
forward using the existent chemical methodology when
X3 (see Figure 2) is methylene. The use of phenyl acetic
acid derivatives6 in the formation of the purine scaffold
limits the number of alternatives as few such reagents
are commercially available. Our efforts4,6 paralleled by
work Kasibhatla et al.9 have focused on changing the
nature of the linker between the purine and aryl
moieties (X3, Figure 2), searching for compounds with
retained activity while chemically more flexible. At-
tempts to change the nature of the linker from carbon
to oxygen or nitrogen have led to compounds with
diminished biological activity.6,9 Interestingly, introduc-
tion of sulfur in this position resulted in compounds with
retained activity against Hsp90.9 As a higher diversity
of reagents is available for building the S-linker (X3 )
S) derivatives, this observation prompts for a detailed
exploration of favorable X1 and X2 substitutions on the
Scheme 1a
a Reagents and conditions: (a) K2CO3, DMF, 90-120 °C, 29-85%; (b) NaOt-Bu, K2CO3 or Na3PO4, neocuproine, CuI, DMF, 110 °C,
40-80%; (c) ROH, PPh3, DBAD, toluene:CH2Cl2, rt, 40-70%; (d) ROTs, Cs2CO3 or K2CO3, DMF, 80 °C, 10-80%; (e) HCl, t-BuOOH,
MeOH, reflux, 31-66%; (f) NIS, CF3COOH, acetonitrile, rt, 69%. Method A: a, c; Method B: a, c, e; method C: b, d; Method D: a, f, c.

2894 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Llauger et al.
Scheme 2a
a Reagents and conditions: (a) CS2, NaHCO3, H2O/EtOH, reflux, 97%; (b) ArI, NaOt-Bu, CuI, ethylene glycol, 130 °C, 60%; (c) HF-
pyridine, NaNO2, -40 °C to room temperature, 15-20%; (d) ROH, PPh3, DBAD, toluene:CH2Cl2, rt, 50%; (e) ROTs, Cs2CO3, DMF, 80 °C,
50%; (f) HCl, t-BuOOH, 1,4-dioxane or i-PrOH, 80 °C, 20-70%; (g) NBS, DMF, rt, 80%; (h) ArSH, K2CO3, DMF, 130 °C, 85%. Method E:
a, b, c, d; Method F: a, b, c, d, f; Method G: a, b, e, f, c; Method H: e, g, h.
of chlorine on the aryl moiety was desired (X2 ) Cl,
Figure 2), the 9-N-alkyl-8-arylsulfanyl adenines 11 were
subjected to positive chlorine species generated in situ
from HCl/t-BuOOH to result in derivatives 12.15 It is
believed that chlorination in this reagent conditions
proceeds via the formation of tert-butylhypochlorite. The
reaction resulted preferentially in ortho-chlorinated
products. If iodine was further added to the aryl moiety
(X2 ) I, Figure 2), derivatives 10 were reacted with
N-iodosuccinimide in trifluoroacetic acid followed by 9-N
alkylation under the Mitsunobu conditions to generate
derivatives 13.16 The active species for this iodination
is probably the in situ formed iodine trifluoroacetate
that can act as a very reactive electrophile, allowing
iodination in 1 h at room temperature.
This reaction gave significantly higher yields over the
previously published method9 using HBF4/iso-amyl
nitrite, likely to be due at least in part to the more
anhydrous nature of this solvent system which reduces
the proportion of hydrolysis. Synthesis of derivatives
containing both an unsaturated chain at 9-N (i.e. pent-
4-ynyl) and fluorine at the C2 position of the adenine
moiety (X4 ) F) required a different strategy to avoid
fluorine addition to the triple bond. Compound 16 was
first subjected to introduction of fluorine at C2 to result
in the corresponding 2-fluoro-8-arylsulfanyl adenine 19.
Surprisingly, conducting the reaction at room-temper-
ature resulted in addition of fluorine at both C2 and
C6 of the purine moiety. This problem was averted by
lowering the reaction temperature to -40 °C. Addition
of fluorine at position C2 significantly increased the
solubility of these purines in organic solvents. Thus,
further alkylation could be easily conducted using the
Mitsunobu reaction in toluene:CH2Cl2 to result in
derivatives 20. Introduction of chlorine (X2 ) Cl), again
with HCl/t-BuOOH, led to formation of 21. Alterna-
tively, synthesis of 2-fluoro-9-N-alkyl-8-arylsulfanyl ad-
enines 20 was started with the commercially available
2-fluoroadenine (22) (Scheme 2). This reagent was first
9-N alkylated by Cs2CO3/tosylate treatment followed by
8-bromination with N-bromosuccinimide (NBS)19 to
result in 2-fluoro-9-N-alkyl-8-bromo adenine 23. Its
coupling with thiophenols generated the 2-fluoro-9-N-
alkyl-8-arylsulfanyl adenines 20.
Syntheses of 8-arylsulfoxyl adenine derivatives 27
and 8-arylsulfonyl adenine derivatives 28 (Scheme 3)
were previously reported by Llauger et al.20 Briefly,
synthesis started with the preparation of C6-NH2 triph-
enylphosphine protected21 9-N-alkyl-8-arylsulfanyl ad-
enine derivatives 24 in a two-step (alkylation-protec-
tion) one-pot reaction from the corresponding 8-arylsul-
fanyl adenines 10 using the Mitsunobu conditions13b
followed by addition of an excess of PPh3 and di-tert-
butyl azodicarboxylate (DBAD). Oxidation of 24 with
OXONE in the presence of alumina22 allowed for
monitoring the reaction to either sulfoxide (25) or
sulfone (26). Deprotection of the C6-NH2 triphenylphos-
phine group was conveniently conducted in refluxing
AcOH/EtOH21 to result in good yields in the correspond-
Several derivatives were prepared by the introduction
of fluorine at position C2 of the adenine moiety (Scheme
2). We and others6,8,9 have previously determined that
fluorine in this position in general increased the solubil-
ity and/or potency of the resulting purines. For the
preparation of such derivatives, synthesis commenced
with the condensation of the commercially available
2,4,5,6-tetraaminopyrimidine sulfate (14) with carbon
disulfide in a refluxing solution of NaHCO3 in aqueous
EtOH.17 The resulting 2-amino-8-mercaptoadenine (15)
was further coupled with aryl iodides in the presence
of NaOt-Bu and CuI in ethylene glycol11b to give
2-amino-8-arylsulfanyl adenines 16. The reaction did
not proceed using the method published by He et al.11c
as 15 did not dissolve in DMF or most organic solvents
for that matter. Unsaturated chains at position 9-N
were introduced at this stage to result in the 2-amino-
9-N-alkyl-8-arylsulfanyl adenines 17. The reaction re-
quired the use of K2CO3 or Cs2CO3 and an alkylating
agent in DMF due to the poor solubility of 16 in the
Mitsunobu reaction solvent. If further, introduction of
chlorine at X2 on the aryl moiety was desired, com-
pounds 17 were subjected to HCl/t-BuOOH to result
preferentially in ortho-chlorinated compounds (18).
Transformation of the C2-amino group to fluorine (X4
) NH2 to F) was conducted by diazotization-fluorod-
ediazoniation of the amino derivative in HF/pyridine in
6,18
the presence of NaNO2
to yield the corresponding
2-fluoro-9-N-alkyl-8-arylsulfanyl adenine derivatives 21.

Purine-Scaffold Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2895
Scheme 3a
a Reagents and conditions: (a) OXONE, alumina, CH2Cl2, rt, 35-70%; (b) AcOH, EtOH, reflux, 60-70%; (c) HCl, t-BuOOH, MeOH, 80
°C, 43%.
ing 8-arylsulfoxyl adenine derivatives 27 and 8-aryl-
ortho- and para-positions (for methoxy substituent, 11e1
vs 11a vs 11f). Effects were substituent-dependent -
methoxy (11a) and hydroxymethyl (11c) were not al-
lowed in the ortho-position while trifluoromethoxy (11b)
retained activity. Chlorine (11g), methoxy (11f), and
methylcarbonyl (11h) were permitted in the para-
position; however, a larger and more rigid substituent
such as pyrrolyl (11i) abolished activity. For a two-
substituent analysis, derivatives with combinations 2,4;
2,5; and 3,5 were synthesized. A preference for 2,5-
substituents was evident, with the 2-iodo-5-methoxy
combination (11q) most favored as previously reported
by Kasibhatla et al.9 Changing methoxy into trifluoro-
methyl at position 5 decreased activity by 10-fold (11o
vs 11r). In an iso-subtituent series of 2,5-derivatives,
methoxy (11o) was favored over chlorine (11n), which
was better than methyl (11m). In a series in which
position 5 was kept constant as methoxy and position 2
was varied, activity changed in the order: methoxy )
chlorine (11o and 12a) < bromine (11p) < iodine (11q).
These compounds exhibited similar cellular potencies
in the 1 to 4 µM range and were the only ones of the
2,5-series to block MDA-MB-468 cells in mitosis (Table
1, column 7). For a three-substituent analysis, deriva-
tives with combinations 2,4,5 and 3,4,5 were synthe-
sized. Not surprisingly, as observed in the methylene
linker series,6 3,4,5-trimethoxy (11w1) was a favorable
combination of whose activity was increased by addition
of chlorine in ortho-position (12b) and almost abolished
by larger moieties such as iodine (13). Building onto the
2,5-position led to 11v, a nanomolar potency compound
in both biochemical and cellular assays (Table 1;
sulfonyl adenine derivatives 28. The latter could be
chlorinated at C′2 (X2 ) Cl) by HCl/t-BuOOH treatment
to result in derivatives 29.
Biological Testing. Compounds synthesized above
were tested in a biochemical assay and also in cellular
assays that probe for cellular fingerprints of Hsp90
inhibition.1 The biochemical assay tests competitive
binding of compounds to recombinant Hsp90R protein
and also Hsp90 found in cell specific complexes and uses
a fluorescence polarization method.7,23 When using cell
lysates instead of recombinant protein, the assay mea-
sures binding to average Hsp90 population found in cell
specific complexes.7
The cellular assays measure two specific biological
effects observed upon addition of known Hsp90 inhibi-
tors to cancer cells: (a) degradation of the tyrosine
kinase Her224 and (b) mitotic block in RB-defective
cells.25 Overexpression of the receptor tyrosine kinase
Her2 in SKBr3 breast cancer cells leads to Akt activa-
tion 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 association.24 We have pre-
viously reported a fast microtiter immunoassay able of
quantifying cellular levels of Her2 following drug treat-
ments.26 This assay is used here to differentiate the
Her2-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.25 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.27 This antibody (Tg-3), origi-
nally described as a marker of Alzheimer’s disease, is
highly specific for mitotic cells, Tg-3 immunofluores-
cence being >50-fold more intense in mitotic cells than
in interphase cells.28
In addition, the cytotoxicity of these agents against
SKBr3 breast cancer cells was determined. A selected
number of most active purines was also tested for
possible toxicity against a normal cell line, renal proxi-
mal tubular epithelial cells (RPTEC).7
Structure-Activity Relationships. A comprehen-
sive analysis of substituents on the right side aryl
moiety (X1 and X2) was conducted to determine favor-
able combinations (Table 1). In a first permutation, the
effect of a single substituent on activity was studied.
As previously reported for derivatives containing a
methylene linker6,8,9 (X3 ) CH2, Figure 2), monosubsti-
tution in the meta-position was also favored here over
Hsp90R
Her2
EC50
IC50
) 30 ( 0.5 nM, IC50
) 300 ( 5 nM;
SKBr3
) 200 ( 10 nM). This compound is the most
potent purine-scaffold derivative published to date.
Derivative 11q previously reported to degrade Her2 in
the breast cancer cell line MCF7 (a cell line with
medium expression of Her2) with an IC50 of 300 nM,9
was less active in the Her2 overexpressing cell line
SKBr3, an IC50 of 810 ( 30 nM being determined in
our hands (Table 1).
Interestingly, for many derivatives presented in Table
1, moderate Hsp90R binding affinity translated into
Her2 degradation potential and inhibition of growth in
SKBr3 cells but did not result in antimitotic activity
(11b, 11e1, 11g, 11k, 11n, 11r, 11u, and 11w1). In
three separate cases (11j, 11m, and 13), no cellular ac-
tivity was observed despite moderate binding, possibly
due to poorer permeability profiles of these compounds
into cells.
We tested further if previous observations made in
the methylene linker series translated to the sulfur link-
er series (X3 ) CH2 vs S, Figure 2). For this purpose, iso-

2896 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Llauger et al.
Table 1. Effect of 8-Aryl Substituents on the Activity of 9-(Pent-4-ynyl)-8-arylsulfanyl- Adenines as Inhibitors of Hsp90
compd
method
Ar
EC50 Hsp90Ra
IC50 Her2a
IC50 SKBr3a
antimitotic
11a
11b
11c
11d
11e1
11f
11g
11h
11i
C
C
C
C
A
C
C
C
C
C
C
C
C
C
A
B
C
A
C
C
C
C
C
A
B
D
2-OMe-Ph
34.9 ( 4.4
5.6 ( 0.6
>400
108 ( 10
86.0 ( 4.2
>500
122 ( 20
36.2 ( 1.6
41.4 ( 4.8
30.2 ( 3.7
>150
121 ( 20
>150
31.8 ( 3.4
>500
467 ( 50
44.1 ( 3.9
1.9 ( 0.1
3.2 ( 0.8
1.1 ( 0.01
0.57 ( 0.12
21.5 ( 2.1
144 ( 31
34.4 ( 4.5
48.4 ( 2.5
0.2 ( 0.01
27.2 ( 2.3
9.4 ( 0.9
>75
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
+
+
-
-
-
-
+
-
+
-
2-OCF3-Ph
97.0 ( 6.3
>400
2-CH2OH-Ph
2-Cl-Ph
85.1 ( 24.2
42.2 ( 21.0
0.97 ( 0.01
19.1 ( 5.4
3.4 ( 1.1
195 ( 13
28.3 ( 6.3
75.0 ( 8.8
44.7 ( 8.3
>150
311 ( 21
>150
39.2 ( 2.9
355 ( 15
147 ( 21
26.5 ( 2.1
3.2 ( 0.3
3.5 ( 0.7
1.45 ( 0.15
0.81 ( 0.03
35.9 ( 2.7
60.5 ( 12.7
>100
58.0 ( 1.4
0.3 ( 0.05
22.3 ( 0.8
9.1 ( 2.6
100.0 ( 10
3-OMe-Ph
4-OMe-Ph
4-Cl-Ph
4-COMe-Ph
4-Pyrrolyl-Ph
2,4-OMe2-Ph
2,4-Me2-Ph
2.7 ( 1.0
>100
11j
11k
11l
2.0 ( 0.2
8.6 ( 1.2
2,4-Cl2-Ph
37.5 ( 2.3
13.4 ( 7.9
2.1 ( 0.7
11m
11n
11o
12a
11p
11q
11r
11s
11t
11u
11v
11w1
12b
13
2,5-Me2-Ph
2,5-Cl2-Ph
2,5-OMe2-Ph
2-Cl-5-OMe-Ph
2-Br-5-OMe-Ph
2-I-5-OMe-Ph
2-Cl-5-CF3-Ph
3,5-Cl2-Ph
0.19 ( 0.02
0.20 ( 0.06
0.07 ( 0.004
0.05 ( 0.005
3.7 ( 0.1
30.4 ( 4.4
10.6 ( 0.1
8.5 ( 0.1
0.03 ( 0.005
1.5 ( 0.3
0.18 ( 0.05
3.1 ( 0.4
3,5-Me2-Ph
2,4,5-Cl3-Ph
2-Br-4,5-(OCH2O)-Ph
3,4,5-OMe3-Ph
2-Cl-3,4,5-OMe3-Ph
2-I-3,4,5-OMe3-Ph
a All values are in µM and represent the average of n ) 3 ( standard deviation.
Table 2. Structure-Activity Relationship Evaluation in a Series of 2-X4-9-N-Alkyl-8-Arylsulfanyl Adenines
a
compd method
X4
R
Ar
EC50 Hsp90Ra
IC50 Her2
IC50 SKBr3a antimitotic
11e1
11e2
11e3
11w1
11w2
11w3
20a
A
A
A
A
A
A
E
H
B
H
pent-4-ynyl
butyl
3-OMe-Ph
3-OMe-Ph
0.97 ( 0.1
4.6 ( 0.6
2.8 ( 0.5
1.5 ( 0.3
3.1 ( 0.7
3.4 ( 1.5
1.5 ( 0.6
2.5 ( 0.2
0.85 ( 0.6
>100
0.12 ( 0.03
0.16 ( 0.05
2.4 ( 0.1
5.0 ( 1.1
2.1 ( 0.3
90.7 ( 10.7
15.4 ( 2.2
85.1 ( 8.8
28.3 ( 6.3
53.7 ( 1.5
47.6 ( 4.8
22.3 ( 0.8
88.5 ( 17.6
65.3 ( 5.7
20.9 ( 2.5
23.5 ( 1.7
22.8 ( 1.2
>150
1.3 ( 0.4
2.2 ( 0.6
26.3 ( 2.1
58.3 ( 2.2
13.8 ( 3.3
314 ( 15
47.8 ( 3.2
122 ( 8
36.2 ( 1.6
37.7 ( 2.1
42.3 ( 2.7
27.2 ( 2.3
65.4 ( 3.8
66.7 ( 5.7
18.2 ( 2.8
41.2 ( 9.3
14.7 ( 3.6
>150
-
-
-
-
-
-
-
-
-
-
+
+
-
-
-
+
+
+
H
H
H
H
H
F
2-isopropoxy-ethyl 3-OMe-Ph
pent-4-ynyl
butyl
3,4,5-OMe3-Ph
3,4,5-OMe3-Ph
2-isopropoxy-ethyl 3,4,5-OMe3-Ph
pent-4-ynyl 3,4,5-OMe3-Ph
2-isopropoxy-ethyl 3,4,5-OMe3-Ph
20b
F
H
12d
17a
butyl
2-Cl-3,4,5-OMe3-Ph
b
-
NH2 pent-4-ynyl
3,4,5-OMe3-Ph
21a
F
G
G
B
B
F
pent-4-ynyl
2-Cl-3,4,5-OMe3-Ph
1.8 ( 0.2
3.3 ( 1.5
20.7 ( 1.3
16.1 ( 0.7
10.1 ( 0.6
130 ( 5
21b
F
2-isopropoxy-ethyl 2-Cl-3,4,5-OMe3-Ph
21c
F
butyl
2-Cl-3,4,5-OMe3-Ph
12c
H
H
H
H
H
2-isopropoxy-ethyl 2,4-Cl2-5-OMe-Ph
12e
2-isopropoxy-ethyl 2,6-Cl2-3,4,5-OMe3-Ph
c
10r
-
H
H
H
2-Cl-5-CF3-Ph
2,4,5-Cl3-Ph
2,5-Cl2-Ph
c
10u
10n
-
36.0 ( 0.2
190 ( 15
c
-
a All values are in µM and represent the average of n ) 3 ( standard deviation. b Obtained by a, b, d; Scheme 2. c Obtained by b;
Scheme 1.
series with the 9-N alkyl chain as sole variable were
synthesized (Table 2). Three well-studied chains,6,8,9
butyl, pent-4-ynyl, and 2-isopropoxy-ethyl were chosen
for the case study. In the methylene linker series, butyl
was found to be least favored while appending the pent-
4-ynyl resulted in highest potency. Activity in the sulfur
linker series declined in a similar fashion, irrespective
of substituents on the aryl moiety (11e2 < 11e3 < 11e1
and 11w2 < 11w3 < 11w1). Addition of fluorine at
position C2 of the adenine moiety (X4 ) F, Figure 2)
was also probed. Previous observations made in the
methylene linker series6 pointed to an increase in
solubility and potency by exchanging the hydrogen at
this position with fluorine and complete abolishment of
activity when the position is occupied by an amino
group. In the sulfur series case, however, addition of

Purine-Scaffold Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2897
Table 3. Effect of Sulfur’s Oxidation State on the Activity of 9-Alkyl-8-Arylsulfanyl Adenines as Inhibitors of Hsp90
compd
X3
R
Ar
EC50 Hsp90Ra
IC50 Her2a
IC50 SKBr3a
antimitotic
12d
29
S
butyl
butyl
2-Cl-3,4,5-OMe3-Ph
2-Cl-3,4,5-OMe3-Ph
3-OMe-Ph
0.85 ( 0.6
4.8 (d 1.1
1.5 ( 0.7
3.5 ( 0.3
4.7 ( 0.9
76.5 ( 1.9
2.3 ( 0.3
>50
22.8 ( 1.2
12.4 ( 2.8
31.8 ( 0.6
70.3 ( 0.6
57.2 ( 4.4
110.3 ( 15.7
43.9 ( 4.5
157 ( 20
170 ( 12
125 ( 11
315 ( 28
105 ( 15
>500
14.7 ( 3.6
36.7 ( 3.7
25.3 ( 2.6
63.4 ( 2.3
53.0 ( 3.6
43.9 ( 3.7
66.2 ( 2.8
41.2 ( 1.9
57.6 ( 5.1
57.2 ( 4.9
85.8 ( 6.1
68.4 ( 5.7
68.8 ( 2.9
-
-
-
-
-
-
-
-
-
-
-
-
-
SO2
SO
27a
27b
27c
27d
27e
28a
28b
28c
28d
28e
28f
pent-4-ynyl
2-isopropoxy-ethyl
butyl
SO
3-OMe-Ph
SO
3-OMe-Ph
SO
butyl
3,4,5-OMe3-Ph
3,4,5-OMe3-Ph
3-OMe-Ph
SO
2-isopropoxy-ethyl
pent-4-ynyl
2-isopropoxy-ethyl
butyl
SO2
SO2
SO2
SO2
SO2
SO2
3-OMe-Ph
3-OMe-Ph
3,4,5-OMe3-Ph
3,4,5-OMe3-Ph
3,4,5-OMe3-Ph
>50
>50
butyl
pent-4-ynyl
2-isopropoxy-ethyl
>50
>50
>50
a All values are in µM and represent the average of n ) 3 ( standard deviation.
Table 4. Parallel Analysis of Linker’s Effect on Activity (X3 ) CH2 vs S)
EC50 Hsp90
SKBr3a
EC50 Hsp90
MDA-MB-468a IC50 Her2a IC50 SKBr3a antimitotic
compd
X3
X4
R
Ar
11w1
30
11w3
31
20a
32
21a
4
S
CH2
S
CH2
S
CH2
S
CH2
H
H
H
H
F
pent-4-ynyl
pent-4-ynyl
2-isopropoxy-ethyl 3,4,5-OMe3-Ph
2-isopropoxy-ethyl 3,4,5-OMe3-Ph
pent-4-ynyl
pent-4-ynyl
pent-4-ynyl
pent-4-ynyl
3,4,5-OMe3-Ph
3,4,5-OMe3-Ph
4.2 ( 0.2
3.6 ( 0.3
5.3 ( 1.7
5.1 ( 0.9
2.0 ( 0.7
1.6 ( 0.5
2.8 ( 0.7
3.5 ( 0.4
4.3 ( 0.6
5.8 ( 0.5
2.2 ( 0.3
1.4 ( 0.3
0.29 ( 0.03
0.23 ( 0.02
22.3 ( 0.8
14.7 ( 2.7
65.3 ( 5.7
28.7 ( 6.2
20.9 ( 2.5
10.5 ( 1.5
1.3 ( 0.4
27.2 ( 2.3
40.5 ( 4.8
66.7 ( 5.7
71.1 ( 0.7
18.2 ( 2.8
16.8 ( 2.4
1.8 ( 0.2
-
+
-
+
-
+
+
+
3,4,5-OMe3-Ph
3,4,5-OMe3-Ph
2-Cl-3,4,5-OMe3-Ph 0.09 ( 0.03
2-Cl-3,4,5-OMe3-Ph 0.17 ( 0.09
F
F
F
1.5 ( 0.5
1.8 ( 0.4
a All values are in µM and represent the average of n ) 3 ( standard deviation.
fluorine did not seem to affect binding to Hsp90R nor
cellular activity in the Her2 degradation and growth
inhibition assays (20a vs 11w1, and 20b vs 11w3). As
anticipated, biological activity was abolished by the
introduction of NH2 in this position (X4 ) NH2) (17a).6
Introduction of chlorine in the ortho-position of the aryl
ring (X2 ) Cl) increased potency (3-fold in 21c vs 11w2),
as seen in the methylene series.6 Adding both fluorine
at position C2 of adenine (X4 ) F) and chlorine in the
ortho-position of the aryl moiety (X2 ) Cl) increased
activity by over 10-fold (21a vs 11w1, and 21b vs 11w3).
Antimitotic activity was observed in these series only
for the high potency compounds 21a and 21b (Table 2,
column 9). Derivatives 12c, 12e, and 21c although
exhibited good Her2 degradation and growth inhibition
potentials, had no antimitotic activity.
The nonalkylated series (compounds 10, Scheme 1)
showed no significant potency in the binding assay or
the cellular assays probing for Her2 degradation and
growth inhibition (not shown). Paradoxically, 10n, 10r,
and 10u were active in the Tg-3 assay at concentrations
that did not induce other Hsp90-related events (see
Table 2). These compounds have a common substituent
pattern on the aryl moiety, with chlorine in position 2
(X2 ) Cl) while position 5 is occupied by a variable
substituent (X1 ) variable in C′5). As previously ob-
served in the methylene class (X3 ) CH2), the 3- and
7-N isomers obtained as byproducts from alkylation of
10 did not exhibit any notable binding to Hsp90.6
The oxidation state of sulfur in the linker was also
modified to probe its effect on activity (Table 3). Several
sulfoxides and sulfones were synthesized for this pur-
pose. Sulfones (28a-f) were in general inactive in the
binding assay and the Her2 degradation assay but
retained modest cytotoxicity, likely via a different
mechanism. Sulfoxides of the 3-methoxyphenyl series
(27a-c) were of comparable activity to their corre-
sponding sulfides (27a vs 11e1, 27b vs 11e3, 27c vs
11e2). No correlation was found in the 3,4,5-methox-
yphenyl series (27d vs 11w2, and 27e vs 11w3).
Addition of chlorine to position C2′ (X2 ) Cl) of the 3,4,5-
methoxyphenyl series resulted in sulfones of retained
activity (29 vs 12d).
To gain a better understanding on the effect of sub-
stituting the methylene linker for sulfur (X3 ) CH2 vs
S), we subjected several of these derivatives to a parallel
analysis. In general, no significant differences in activity
between the S and CH2 series were observed (Table 4).
The most potent of the analyzed pairs (21a vs 4)
behaved practically identical in the Hsp90-related as-
Hsp90 MDA-MB-468
says (EC50Hsp90 SKBr3 ) 90 ( 30 nM, EC50
Her2
SKBr3
) 290 ( 30 nM, IC50
) 1300 ( 40 nM; IC50
)
Hsp90 SKBr3
1800 ( 200 nM vs EC50
) 170 ( 90 nM,
EC Hsp90 MDA-MB-468 ) 230 ( 20 nM, IC50Her2 ) 1500 (
50

2898 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Llauger et al.
Table 5. Selectivity for Transformed vs Normal Cells in a Series of Purine-Scaffold Hsp90 Inhibitors
EC50 Hsp90 EC50 Hsp90 EC50 Hsp90 EC50 Hsp90
compd
braina
lunga
hearta
ND
ND
SKBr3a
brain/SKBr3 lung/SKBr3 heart/SKBr3 IC50 RPTECa RPTEC/SKBr3
11w1
31
ND
ND
4.2 ( 0.2
3.6 ( 0.3
ND
ND
ND
ND
>500
212
ND
ND
414
226
890
3265
61
120 ( 15
>200
4.4
ND
ND
>100
11o
11p
11q
11v
12a
12b
21a
21b
30.5 ( 2.7
14.4 ( 3.3
30.5 ( 5.1
>75
66.3 ( 24.4 0.16 ( 0.04
190
110
1010
2000
144
128
298
163
31.4 ( 5.2
8.1 ( 1.6
3.3 ( 0.2
4.1 ( 0.9
28.7 ( 5.3
54.5 ( 4.2
28.1 ( 2.2
47.2 ( 1.9
16.5
7.4
27.6 ( 1.5 29.5 ( 1.7
29.7 ( 3.3 26.7 ( 3.1
0.13 ( 0.05
0.03 ( 0.01
0.02 ( 0.06
0.31 ( 0.06
0.33 ( 0.05
990
4.1
20.5
9
5.8
15.6
14.3
40.2 ( 25.3 14.7 ( 1.7 65.3 ( 9.9
735
44.9 ( 14.1
42.4 ( 4.9
26.9 ( 7.7
76.7 ( 30.3
9.6 ( 3.4 18.9 ( 0.2
31
>75
18.2 ( 2.1
>200
2400
160
55
590
57
217 ( 50
>75
53.1 ( 20.5 0.09 ( 0.03
26.7 ( 8.1 0.47 ( 0.02
a All values are in µM and represent the average of n ) 3 ( standard deviation.
SKBr3
50 nM; IC50
) 1800 ( 400 nM). One paradoxical
ents in positions 2,4,5 as most favored in regard to
binding into the Hsp90 ATP-pocket and warrants
further explorations in these series. It also discovers
derivative 11v as the most potent Hsp90 inhibitor of
the purine-scaffold series published to date, and also
the compound of these series with highest selectivity
for tumor vs normal cell Hsp90. Most rewardingly, this
work has allowed the identification of Hsp90 inhibitors
that may have selective affinities for certain Hsp90-
client protein complexes. Derivatives with high Hsp90
binding affinity such as 11o, 11p, 11q, 11v, 12a, 12b,
21a, and 21b (EC50s from 30 to 300 nM) induce Her2
degradation and inhibition of growth in SKBr3 cells and
also exhibit antimitotic activity in MDA-MB-468 cells,
these events occurring with similar potencies. However,
among the moderate affinity binders, derivatives were
identified that degrade Her2 with corresponding poten-
cies but do not affect at similar concentrations the cell
cycle distribution in RB-defective cells. The Hsp90 client
protein of whose inactivation by Hsp90 inhibitors is
responsible for the block of these cells in mitosis is
currently unknown.25 It is conceivable that selective
disruption of complexes between Hsp90 and this protein
may be the basis for the differential activity shown by
purine-scaffold compounds in RB-positive and RB-
defective cells. Due to the selectivity profile, these
derivatives may be useful pharmacological tools in
dissecting Hsp90-regulated processes and in identifying
the mitotic checkpoint protein regulated by Hsp90.
Thus, a parallel analysis of cells treated with 20a and
32 might be useful in elucidating these phenomena.
observation was however made in series with lower
binding affinity for Hsp90. While the methylene bridge
compounds (X3 ) CH2) exhibited antimitotic activity (30,
31, and 33), their corresponding sulfur bridge deriva-
tives (11w1, 11w3, and 20a) (X3 ) S), although similar
in Hsp90R binding affinity and Her2 degradation po-
tential, were inactive in the Tg-3 assay. Differential cell
uptake or possible compound metabolism (i.e. S-linker
oxidation) is not the base for this discrepancy; both the
S- and CH2-linker compounds inhibited the growth of
SKBr3 and MDA-MB-468 cells with similar potencies
(not shown). In addition, these compounds were not
differentiated by binding affinities toward average
population Hsp90 complexes present in SKBr3 and
MDA-MB-468 cells (Table 4, columns 6 and 7). These
compounds may have selective affinities for Hsp90-
client specific complexes that regulate selective path-
ways in the two cancer cell lines. Since our binding
assays are not as sophisticated as to detect client-speci-
fic binding in cellular mixtures, the mechanism causing
such differences in behavior remains to be elucidated.
Several active derivatives were tested for specificity
toward transformed cells (Table 5). Binding affinities
of selected compounds for average population Hsp90
complexes found in normal tissues (brain, lung, and
heart) and in addition, their cytotoxicities against RPTEC
normal cells were determined. Compounds were found
to bind Hsp90 from normal tissues with 1- to 3-log weak-
er affinities when compared to Hsp90 from SKBr3 cells
(30-140-fold for the least selective compound 12a to
730-3200-fold for derivative 11v). This specificity trans-
lated into 5 to 100-fold selectivity (column 10, Table 5)
in inhibiting the growth of transformed cells compared
to cultured normal epithelial cells (RPTEC tested). No
significant cell death was observed in the purine-scaffold
treated RPTEC cells even at the highest tested concen-
trations.
Experimental Section
Hsp90 Competition Assay. Fluorescence polarization
measurements were performed on an Analyst AD instrument
(Molecular Devices, Sunnyvale, CA). Measurements were
taken in black 96-well microtiter plates (Corning # 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) and 2 mM DTT (Fisher Biotech,
Fair Lawn, NJ) were freshly added. GM-BODIPY was syn-
thesized as previously reported23a and was dissolved in DMSO
to form 10 µM solutions. Recombinant Hsp90R was purchased
from Stressgen Bioreagents (cat. No. SPP-776), (Victoria,
Canada). Cell lysates were prepared rupturing cellular mem-
branes by freezing at - 70 °C and dissolving the cellular
extract in HFB with added protease and phosphotase inhibi-
tors. Organs were harvested from a healthy mouse and
homogenized in HFB. To verify that murine Hsp90 in trans-
formed cells has similar affinity to human transformed cell
Hsp90, we tested binding of these agents to Hsp90 from Baf3
cells, a murine hematopoietic cell line, and verified that indeed
the two species Hsp90s have similar affinities for ATP-pocket
Conclusions
Synthesis of several 8-arylsulfanyl, -sulfoxyl, and
-sulfonyl adenine derivatives has allowed for detailed
analyses of permissible modifications of the right-side
aryl moiety and for the examination of structure-
activity relationships of these derivatives as inhibitors
of Hsp90. Our results suggest that 8-arylsulfanyl ad-
enine derivatives retain the activity of the methylene
linker compounds (X3 ) CH2) and are chemically flexible
allowing for extensive SAR studies. Oxidation of the
sulfur linker to sulfoxides or sulfones, however, leads
to a decrease in activity. The study identifies substitu-

Purine-Scaffold Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2899
ligands. Thus, we conclude that the use in binding experiments
of murine normal organs instead of normal human epithelial
cells is acceptable. It is also representative of a closer-to-reality
system as the mouse organs are truly normal while the
cultured normal epithelial cells are engineered to grow in
tissue culture settings. 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 recom-
binant 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 nor-
malized to total Hsp90 as determined by Western blot analysis
using as standard Hsp90 purified from HeLa cells (Stressgen#
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 7
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.
background and deducted from all measured values. Lumi-
nescence readings resulted from drug-treated cells versus
untreated cells (vehicle treated) were quantified and plotted
against drug concentration to give the EC50 values as the
concentration of drug that caused 50% decrease in luminescence.
Antimitotic Assay. Black, clear-bottom microtiter 96-well
plates (Corning Costar #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 the 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 #37535) (200 µL) for 2 h
at RT. The Tg-3 antibody (gift of Dr. 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 Anti-Mouse IgM,
SouthernBiotech #1020-05) was placed in each well at 1:2000
dilution in SuperBlock and incubated on a shaker at RT 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 1:1 mix
(100 µL) was placed in each well, and the plates were read
immediately in an Analyst AD plate reader (Molecular De-
vices). 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.
General Chemical Procedures. All commercial chemicals
and solvents are reagent grade and were used without further
purification. The identity and purity of each product was
characterized by MS, HPLC, TLC, IR, 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 ion mode under electron-spray ionization (ESI).
High performance liquid chromatography analyses were per-
formed on a Waters 2996 instrument with a photodiode array
detector (read at 265 nm) and a reverse-phase column (Hig-
gins; 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 reverse-
phase column (Varian; Microsorb 100-5 C18 150 × 2) (meth-
ods (b) and (c)). Method (a): 0.1% TFA in water-acetonitrile
in the indicated ratio; method (b): 0.05% TFA in water-0.04%
TFA in acetonitrile; method (c): 0.05% TFA in water-0.04%
TFA in acetonitrile gradient (35% acetonitrile over 18 min,
35-95% acetonitrile over 6 min, 95% acetonitrile over 9 min).
Infrared spectra (IR) were obtained on a Perkin-Elmer FT-IR
model 1600 spectrometer. Characterization data for previously
unknown compounds were determined from a single run with
isolated yields. Reactions were monitored by thin-layer chro-
matography on 0.25-mm silica gel plates and visualized with
UV light. Column chromatography was performed using silica
gel (Fisher 170-400 mesh) or alumina (Fisher 60-325 mesh).
Oxidation reactions with OXONE were carried out in the
presence of the Fisher alumina (A540; 80-200 mesh). Analyti-
cal thin-layer chromatography (TLC) was performed on E.
Merck precoated silica gel 60 F254. Waters Sep-Pak Vac 6 cm3
(500 mg) C18 cartridges were used for the purification of
compounds 16. All reactions were conducted under inert
atmosphere except of those in aqueous media.
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 supplement-
ed with 2 mM glutamine, 50 units/mL penicillin, 50 units/mL
streptomycin, and 10% heat inactivated fetal bovine serum
(Gemini Bioproducts #100-10b) and incubated at 37 °C,
5% CO2.
Growth Assays. Growth inhibition studies were performed
using the sulforhodamine B assay as previously described.29
In summary, experimental cultures were plated in microtiter
plates (Nunc #167008). One column of wells was left without
cells to serve as the blank control. Cells were allowed to attach
overnight. On the following day, growth medium having either
drug or DMSO 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. Normal human renal proximal
tubular epithelial (RPTEC) cells were purchased pre-seeded
in 96-well plates (Clonetics, CC-3190). Upon receipt, cells were
placed in a humidified incubator at 37 °C, 5% CO2, and allowed
to equilibrate for 3 h. Media was removed by suction and
replaced with fresh media provided by the manufacturer. Cells
were then treated with either drugs or DMSO for 72 h, and
the IC50 values were determined as described above.
Her2 Assay. SKBr3 cells were plated in black, clear-bottom
microtiter plates (Corning #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 (DM-
SO) was carefully added to the wells, and the plates were
placed at 37 °C and 5% CO2. Following 24 h 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 re-
move 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 RT for 2 h
with SuperBlock (Pierce 37535) (200 µL), anti-Her-2 (c-erbB-
2) antibody (Zymed Laboratories #28-004) (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 #SC-2027) in Superblock was used.
Each well was washed with TBST (2 × 200 µL) and incubated
at RT 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#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
3,4,5-Trimethoxy-benzenethiol (6).30 To 3,4,5-trimethoxy-
aniline (2 g, 10.9 mmol) at 0 °C was added a concentrated
solution of HCl (3 mL, 0.27 mL/mmol) and H2O (7.7 mL)
followed by NaNO2 (932 mg, 13.1 mmol). The resulting solution

2900 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Llauger et al.
1
was poured over potassium ethyl xanthogenate (5.35 g, 32.7
mmol) in H2O (6.2 mL) and stirred at 50 °C for 40 min. The
reaction mixture was brought to room temperature, diluted
with EtOAc (80 mL), and washed with 10% NaOH, followed
by H2O until the pH reached 7. The organic fraction was dried
over Na2SO4 and the solvent evaporated under high vacuum.
The residue was purified by column chromatography on silica
gel (CH2Cl2) to furnish the xanthogenate intermediate (1.82
g, 58% yield). This was taken up in anhydrous THF (30 mL).
To the resulting solution was slowly added LiAlH4 (1 g, 25
mmol), and the mixture was stirred for 1 h at reflux temper-
ature. Following cooling to room temperature, the reaction was
quenched with ice cold water (50 mL) and 10% H2SO4 (5 mL)
and extracted with CHCl3. The organic phase was dried over
Na2SO4 and evaporated to give the desired thiophenol (1.18
g, 93% yield). 1H NMR (CDCl3) δ 6.53 (s, 2H), 3.84 (s, 6H,
OCH3), 3.82 (s, 3H, OCH3), 3.46 (s, 1H, SH).
Procedures for the Formation of 8-Arylsulfanylad-
enine Derivatives 10: Scheme 1, Synthetic Step (b).
8-Mercaptoadenine (7) (50.2 mg, 0.30 mmol), neocuproine
hydrate (6.8 mg, 0.03 mmol), CuI (5.7 mg, 0.03 mmol), NaO-
t-Bu (57.6 mg, 0.6 mmol), the corresponding aryl iodide (0.90
mmol), and anhydrous DMF (2 mL) were charged in a nitrogen
box. The reaction vessels were sealed with Teflon tape, placed
in an oil bath (110 °C) and magnetically stirred for 24 h. The
reaction mixture was then cooled to room temperature and
DMF was removed in vacuo. The crude material was purified
by silica gel flash chromatography eluting with a gradient of
CHCl3:NH4OH at 10:0.5 to CHCl3:MeOH:NH4OH at 10:1:0.5
to afford the desired product.
8-(2-Trifluoromethoxy-phenylsulfanyl)adenine (10b).
Yield, 45%. 1H NMR (400 MHz, DMSO-d6) δ 8.11 (s, 1H), 7.46
(s, 2H), 7.39-7.31 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ
152.4, 145.8, 131.7, 129.5, 128.5, 121.5, 121.3, 118.8; MS m/z
327.9 (M + H)+.
8-(4-Acetyl-phenylsulfanyl)adenine (10h). Yield, 75%.
1H NMR (400 MHz, DMSO-d6) δ 8.13, 7.91-7.89 (d, 2H), 7.43-
7.41 (d, 2H), 7.36 (s, 2H), 2.53 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 197.0, 152.7, 139.6, 135.3, 129.2, 128.3, 26.7; MS
m/z 285.07 (M-H)-.
8-(4-Pyrrol-1-yl-phenylsulfanyl)adenine (10i). Yield, 78%.
1H NMR (400 MHz, DMSO-d6) δ 8.1 (s, 1H), 7.63-7.61 (d, 2H),
7.53 (s, 2H), 7.37 (s, 2H), 7.22 (s, 2H), 6.26 (s, 2H), 5.74 (s,
2H); 13C NMR (100 MHz, DMSO-d6) δ 152.4, 132.5, 131.5,
120.1, 118.9, 110.9, 40.1, 39.9, 39.7, 39.5, 39.3, 39.1, 38.8; MS
m/z 308.9 (M + H)+.
K2CO3 as base. Yield, 56%. H NMR (400 MHz, DMSO-d6) δ
8.12 (s, 1H), 8.00 (s, 1H), 7.60 (s, 1H), 7.37 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) δ 132.5, 132.3, 132.2, 131.4, 131.2, 130.8;
MS m/z 345.9 (M + H)+. HPLC: (a) 99.9% (65% water - 35%
acetonitrile); (b) 99.4%.
8-(7-Bromo-2,3-dihydro-benzo[1,4]dioxin-6-ylsulfanyl)-
1
adenine (10v). Yield, 66%. H NMR (400 MHz, DMSO-d6) δ
8.08 (s, 1H), 7.35 (s, 1H), 7.19 (s, 2H), 7.04 (s, 1H), 6.10 (s,
2H); 13C NMR (100 MHz, DMSO-d6) δ 152.0, 149.0, 147.8,
113.2, 112.9, 102.6; MS m/z 366.0 (M + H)+.
Formation of 10a, 10c, 10d, 10f, 10g, 10j, 10k, 10r, and
10t has followed a similar protocol. Synthesis and character-
ization of these derivatives has been described elsewhere.11c
Scheme 1, Synthetic Step (a): 8-(3-Methoxy-phenyl-
sulfanyl)adenine (10e). Adenine (1.1 g, 8.1 mmol) was added
to a solution of bromine (1.7 mL, 33.1 mmol) in water (244
mL), and the resulting mixture was stirred overnight at room
temperature. The solvent was evaporated to dryness, and the
brominated product 5 was used further without additional
purification. Separately, K2CO3 (2.25 g, 16.3 mmol) was added
to a solution of 3-methoxy-benzenethiol (1.5 mL, 11.8 mmol)
in DMF (30 mL), and the reaction mixture was stirred for 1 h
at room temperature. To it, a solution of 5 in DMF (81 mL)
was dropwise added. The temperature was raised to 120 °C
and stirring continued for an additional 14 h. Following cooling
and solvent evaporation under high vacuum, the crude mate-
rial was purified by column chromatography on silica gel
(CHCl3:MeOH 97:3) to give 10e (1.9 g, 85% yield). 1H NMR
(400 MHz, DMSO-d6) δ 13.05 (bs, 1H, NH), 8.16 (s, 1H, H-2),
7.34 (m, 3H), 7.05-6.94 (m, 3H, Ar and NH2), 3.79 (s, 3H,
OCH3); 13C NMR (100 MHz, DMSO-d6) δ 163.0, 154.8, 152.6
(C-2), 133.2, 130.5, 122.3, 115.7, 113.6, 55.3 (OCH3); MS (EIS)
m/z 273.9 (M + 1)+.
8-(2,5-Dimethoxy-phenylsulfanyl)adenine (10o). Follow
the procedure for 10e. Yield, 29%. 1H NMR (400 MHz, DMSO-
d6) δ 13.28 (bs, 1H, NH), 8.08 (s, 1H, H-2), 7.24 (bs, 2H, NH2),
7.01 (m, 1H), 6.88 (m, 1H), 6.61 (bs, 1H), 3.74 (s, 3H, OCH3),
3.63 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 162.9,
159.1, 155.0, 153.5, 153.1, 152.6 (C-2), 150.6, 142.1, 116.0,
113.1, 112.7, 56.5 and 55.5 (OCH3); MS (EIS) m/z 304.1 (M +
1)+.
8-(3,4,5-Trimethoxy-phenylsulfanyl)adenine (10w). Fol-
low the procedure for 10e except the reaction temperature is
90 °C instead of 120 °C. Yield, 65%. 1H NMR (400 MHz,
DMSO-d6) δ 13 (bs, 1H, NH), 8.09 (s, 1H, H-2), 7.26 (bs, 2H,
NH2), 6.87 (s, 2H), 3.75 (s, 6H, OCH3), 3.66 (s, 3H, OCH3); 13
C
8-(2,4-Dichloro-phenylsulfanyl)adenine (10l). Yield, 44%.
1H NMR (400 MHz, DMSO-d6) δ 8.12 (s, 1H), 7.74-7.73 (d,
1H), 7.41-7.38 (q, 1H), 7.33 (s, 2H), 7.24-7.22 (d, 1H); 13C
NMR (100 MHz, DMSO-d6) δ 154.7, 152.3, 141.5, 134.7, 133.4,
132.9, 132.3, 131.1, 129.4, 128.4, 127.7, 127.2; MS m/z 311.9
(M + H)+.
8-(2,5-Dimethyl-phenylsulfanyl)adenine (10m). Yield,
76%. 1H NMR (400 MHz, DMSO-d6) δ 8.07 (s, 1H), 7.18-7.09
(m, 5H), 2.27 (s, 3H), 2.21 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 152.3, 136.2, 130.7, 129.5, 20.4, 19.8; MS m/z 271.9 (M +
H)+.
8-(2,5-Dichloro-phenylsulfanyl)adenine (10n). Yield,
55%. 1H NMR (400 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.59-7.57
(d, 1H), 7.40-7.38 (m, 3H), 7.24 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) δ 154.8, 152.2, 141.3, 134.3, 132.5, 131.4, 130.9,
129.6, 128.9; MS m/z 311.9 (M + H)+. HPLC: (a) 98.5% (65%
water - 35% acetonitrile); (b) 99.0%.
NMR (100 MHz, DMSO-d6) δ 153.9, 152.0 (C-2), 138.1, 124.6,
109.9, 60.1 and 56.1 (OCH3); MS (EIS) m/z 334.0 (M + 1)+.
Scheme 1, Synthetic Step (c): Alkylation via the Mitsun-
obu reaction was conducted as previously described.13b Briefly,
to a solution of the sulfide 10 (1 equiv) in toluene:CH2Cl2 (6
mL/mmol) (5:1) were added 1.3 equiv of the corresponding
alcohol, 2.2 equiv of PPh3, and 5 equiv of di-tert-butyl azodi-
carboxylate (DBAD). The reaction mixture was stirred at room
temperature for 30 min, concentrated in vacuo, and taken up
in CH2Cl2. The organic layer was washed with brine, dried
over anhydrous Na2SO4, and evaporated under high vacuum.
The crude material was purified by column chromatography
on silica gel eluting with CHCl3:Hexanes:EtOAc:i-PrOH (2:2:
1:0.1) to give the 9-N-alkylated products 11 in 40-70% yields.
9-(Pent-4-ynyl)-8-(3-methoxy-phenylsulfanyl)adenine
(11e1). Yield, 70%. IR (film) νmax 3300-2835, 1641, 1590, 1573,
1479, 1320 (S); 1H NMR (400 MHz, CDCl3) δ 8.33 (s, 1H, H-2),
7.24 (t, J ) 8.3 Hz, 1H), 6.96 (dd, J ) 2 and 4 Hz, 1H), 6.83
(m, 1H), 6.11 (bs, 2H, NH2), 4.30 (t, J ) 7.3 Hz, 2H, NCH2),
3.76 (s, 3H, OCH3), 2.24 (dt, J ) 2.5 and 6.9 Hz, 2H), 1.98 (m,
3H, CH and CH2); 13C NMR (100 MHz, CDCl3) δ 160.2, 154.7,
153.2 (C-2), 151.5, 145.3, 132.3, 130.4, 122.7, 120.0, 116.2,
113.8, 82.4 (C quaternary), 69.4 (CH), 55.4 (OCH3), 42.9
(NCH2), 28.2, 16.0; MS (EIS) m/z 340.1 (M + 1)+. HPLC: (a)
99.0% (70% water - 30% acetonitrile); (c) 99.0%.
8-(2-Bromo-5-methoxy-phenylsulfanyl)adenine (10p).
Yield, 66%. 1H NMR (400 MHz, Acetone-d6/ DMSO-d6) δ 8.12
(s, 1H), 7.58-7.56 (d, J ) 8.79 Hz, 1H), 7.26 (s, 2H), 6.86-
6.83 (dd, J ) 8.69, J ) 2.42, 1H), 6.72 (s, 1H), 3.66 (s, 3H); 13
C
NMR (100 MHz, Acetone-d6/ DMSO-d6) δ 159.2, 152.7, 133.8,
114.4, 55.4; MS m/z 351.9 (M + H)+.
8-(3,5-Dichloro-phenylsulfanyl)adenine (10s). Yield, 72%.
1H NMR (400 MHz, DMSO-d6) δ 8.12 (s, 1H), 7.55 (s, 1H), 7.45
(s, 2H), 7.37 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 154.8,
152.2, 136.5, 134.7, 127.7, 127.3; MS m/z 311.9 (M + H)+.
8-(2,4,5-Trichloro-phenylsulfanyl)adenine (10u). Use
9-(Pent-4-ynyl)-8-(2,5-dimethoxy-phenylsulfanyl)ade-
nine (11o).9 Yield, 45%. HPLC: (a) 95.0% (70% water - 30%
acetonitrile); (c) 97.3%.

Purine-Scaffold Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2901
9-(Pent-4-ynyl)-8-(3,4,5-trimethoxy-phenylsulfanyl)ad-
enine (11w1).9 Yield, 33%. HPLC: (a) 98.4% (70% water -
30% acetonitrile); (c) 99.0%.
1H), 7.32-7.28 (m, 1H), 7.22-7.20 (m, 1H), 6.93-6.91 (m, 2H),
5.73 (s, 2H), 4.35-4.31 (t, 2H), 3.84 (s, 3H), 2.27-2.22 (dt, 2H),
2.07-2.00 (m, 2H), 1.96-1.95 (t, 1H); 13C NMR (100 MHz,
DMSO-d6) δ 157.4, 154.3, 152.9, 151.5, 145.9, 132.0, 129.7,
121.5, 120.1, 119.5, 111.4, 82.5, 69.2, 56.0, 42.9, 28.3, 16.0; MS
m/z 340.1 (M + H)+. HPLC: (a) 98.2% (75% water - 25%
acetonitrile); (b) 97.7%.
9-(Pent-4-ynyl)-8-(2-trifluoromethoxy-phenylsulfanyl)-
adenine (11b). Yield, 40%. 1H NMR (400 MHz, CDCl3) δ 8.35
(s, 1H), 7.35-7.33 (m, 2H), 7.26-7.22 (m, 2H), 5.77 (s, 2H),
4.35-4.31 (t, 2H), 2.26-2.22 (m, 2H), 2.05-1.98 (m, 2H), 1.96-
1.95 (t, 1H); 13C NMR (100 MHz, CDCl3) δ 154.6, 153.3, 151.6,
147.1, 143.7, 131.8, 129.4, 127.7, 125.6, 121.7, 121.3, 120.3,
82.2, 69.4, 42.9, 28.3, 15.9; MS m/z 393.9 (M + H)+. HPLC:
(a) 99.5% (75% water - 25% acetonitrile); (b) 99.1%.
9-(Pent-4-ynyl)-8-(2-hydroxymethyl-phenylsulfanyl)-
adenine (11c). Yield, 56%. 1H NMR (400 MHz, CDCl3/MeOD-
d4) δ 8.19 (s, 1H), 7.68 (d, 1H), 7.53-7.50 (t, 2H), 7.36-7.32
(t, 1H), 4.85 (s, 2H), 4.37-4.33 (t, 2H), 2.35-2.31 (m, 2H),
2.11-2.04 (m, 3H); 13C NMR (100 MHz, CDCl3/MeOD-d4) δ
151.8, 150.8, 147.8, 144.1, 134.6, 130.1, 128.7, 128.4, 126.1,
81.9, 69.3, 62.3, 42.6, 27.8, 15.5; MS m/z 340.0 (M + H)+.
HPLC: (a) 99.3% (75% water - 25% acetonitrile); (b) 99.7%.
9-(Pent-4-ynyl)-8-(2-chloro-phenylsulfanyl)adenine
(11d). Yield, 22%.1H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H),
7.45-7.43 (d, 1H), 7.24-7.13 (m, 3H), 5.68 (s, 2H), 4.35-4.31
(m, 2H), 2.27-2.23 (m, 2H), 2.05-1.98 (m, 2H), 1.97-1.96 (t,
1H); 13C NMR (100 MHz, CDCl3) δ 154.6, 153.4, 151.7, 144.2,
133.9, 131.4, 131.0, 130.2, 123.0, 127.7, 82.3, 69.5, 43.1, 28.4,
16.1; MS m/z 344.0 (M + H)+. HPLC: (a) 98.5% (75% water -
25% acetonitrile); (b) 99.0%.
9-Butyl-8-(3-methoxy-phenylsulfanyl)adenine (11e2).9
Yield, 50%. HPLC: (a) 99.5% (70% water - 30% acetonitrile);
(c) 99.0%.
9-Butyl-8-(3,4,5-trimethoxy-phenylsulfanyl)adenine
(11w2). Yield, 50%. IR (film) νmax 3313-2932, 1649, 1582,
1498, 1406 (S); 1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H, H-2),
6.71 (s, 2H), 5.65 (s, 2H, NH2), 4.20 (t, J ) 7.6 Hz, 2H, NCH2),
3.83 (s, 3H, OCH3), 3.80 (s, 6H, CH3), 1.70 (m, 2H), 1.32 (six,
J ) 7.4 Hz, 2H), 0.91 (t, J ) 7.4 Hz, 3H, CH3); 13C NMR (100
MHz, CDCl3) δ 155.0, 154.1, 153.3 (C-2), 151.9, 146.5, 139.0,
125.4, 120.3, 109.4, 61.3 and 56.7 (OCH3), 44.0 (NCH2), 32.1,
20.4, 14.0 (CH3); MS (EIS) m/z 390.1 (M + 1)+. HPLC: (a)
99.2% (70% water - 30% acetonitrile); (c) 99.0%.
9-(2-Isopropoxy-ethyl)-8-(3-methoxy-phenylsulfanyl)-
adenine (11e3). Yield, 35%. IR (film) νmax 3315-2869, 1642,
1591, 1573, 1478, 1320 (S); 1H NMR (400 MHz, CDCl3) δ 8.33
(s, 1H, H-2), 7.22 (t, J ) 8.3 Hz, 1H), 6.95 (m, 2H), 6.81 (dd, J
) 2 and 8.3 Hz, 1H), 5.98 (s, 2H, NH2), 4.42 (t, J ) 6 Hz, 2H,
NCH2), 3.76 (s, 3H, OCH3), 3.69 (t, J ) 6 Hz, 2H), 3.46 (six, J
) 6.1 Hz, 1H, CH), 1.03 (d, J ) 6.1 Hz, 6H, CH3); 13C NMR
(100 MHz, CDCl3) δ 160.1, 154.7, 153.1 (C-2), 151.5, 146.3,
133.0, 130.3, 122.6, 120.1, 115.9, 113.7, 72.2 (CH), 65.4, 55.3
(OCH3), 43.9 (NCH2), 21.8 (CH3); MS (EIS) m/z 360.1 (M +
1)+. HPLC: (a) 99.4% (70% water - 30% acetonitrile); (c)
99.0%.
9-(2-Isopropoxy-ethyl)-8-(3,4,5-trimethoxy-phenylsul-
fanyl)adenine (11w3). Yield, 49%. IR (film) νmax 3312-2934,
1651, 1582, 1498, 1309 (S); 1H NMR (400 MHz, CDCl3) δ 8.25
(s, 1H, H-2), 6.66 (s, 2H), 6.04 (s, 2H, NH2), 4.36 (t, J ) 6 Hz,
2H, NCH2), 3.74 (s, 3H, OCH3), 3.73 (s, 6H, CH3), 3.61 (t, J )
6 Hz, 2H), 3.38 (m, 1H, CH), 0.81 (d, J ) 5.8 Hz, 6H, CH3);
13C NMR (100 MHz, CDCl3) δ 154.6, 153.6, 152.9 (C-2), 151.4,
147.0, 138.4, 125.5, 120.0, 108.8, 72.2 (CH), 65.4, 60.8 and 56.2
(OCH3), 43.8 (NCH2), 21.8 (CH3); MS (EIS) m/z 420.1 (M +
1)+. HPLC: (a) 98.3% (70% water - 30% acetonitrile); (c) 99.0%.
9-(Pent-4-ynyl)-8-(2-iodo-3,4,5-trimethoxy-phenylsulfa-
nyl)adenine (13). A mixture of 10w (135 mg, 0.4 mmol), N-
iodosuccinimide (192 mg, 0.8 mmol), and trifluoroacetic acid
(31 µL, 0.4 mmol) in acetonitrile (1.6 mL) was stirred for 1 h
at room temperature. Following solvent removal, the reaction
crude material was washed with an aqueous solution of sodium
bisulfite, extracted in CH2Cl2, and dried on anhydrous sodium
sulfate. The crude solid was added to a solution of PPh3 (160
mg, 0.6 mmol), DBAD (325 mg, 1.4 mmol), and 4-pentyn-1-ol
(35 µL, 0.4 mmol) in toluene:CH2Cl2 (8.3:1.7 mL), and the re-
action mixture was stirred at room temperature for 30 min.
Following solvent removal, the solid mass was taken up in
CH2Cl2 washed with brine and dried over anhydrous Na2SO4.
The crude material was purified by column chromatography
on silica gel eluting with CHCl3:Hexanes:EtOAc:i-PrOH (2:2:
1:0.1) to give 13 (43 mg, 20% yield). IR (film) νmax 3299-2936,
1644, 1595, 1471, 1370 (S); 1H NMR (400 MHz, CDCl3) δ 8.34
(s, 1H, H-2), 6.73 (s, 1H), 6.11 (bs, 2H, NH2), 4.33 (t, J ) 7.2
Hz, 2H, NCH2), 3.89 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.68
(s, 3H, OCH3), 2.26 (dt, J ) 2.4 and 7 Hz, 2H), 2.02 (m, 2H),
1.98 (t, J ) 2.4 Hz, 1H, CH); 13C NMR (100 MHz, CDCl3) δ
154.7, 154.3, 154.1, 153.2 (C-2), 151.5, 145.9, 142.1, 131.0,
120.2, 111.0, 89.4, 82.3 (C quaternary), 69.5 (CH), 61.0-60.9
and 56.2 (OCH3), 43.1 (NCH2), 28.4, 16.1; MS (EIS) m/z 526.0
(M + 1)+. HPLC: (a) 98.7% (70% water - 30% acetonitrile);
(c) 95.0%.
9-(Pent-4-ynyl)-8-(4-methoxy-phenylsulfanyl)ade-
1
nine (11f). Yield, 77%. H NMR (400 MHz, CDCl3) δ 8.29 (s,
1H), 7.48-7.46 (d, 2H), 6.91-6.89 (d, 2H), 5.75 (s, 2H), 4.32-
4.28 (2H), 3.80 (s, 3H), 2.28-2.25 (m, 2H), 2.02-2.05 (m, 3H);
13C NMR (100 MHz, CDCl3) δ 152.7, 134.4, 115.2, 82.5, 69.4,
55.4, 42.7, 28.1, 16.1; MS m/z 340.1 (M + H)+. HPLC: (a) 98.3%
(75% water - 25% acetonitrile); (b) 98.8%.
9-(Pent-4-ynyl)-8-(4-chloro-phenylsulfanyl)adenine (11
g). Yield, 21%. 1H NMR (400 MHz, CDCl3) δ 8.33 (s, 1H), 7.41-
7.39 (d, 2H), 7.34-7.31 (d, 2H), 5.68 (s, 2H), 4.33-4.29 (t, 2H),
2.28-2.24 (m, 2H), 2.05-1.97 (m, 3H); 13C NMR (100 MHz,
CDCl3) δ 154.3, 152.9, 151.6, 145.7, 134.8, 132.6, 129.8, 129.1,
120.1, 82.4, 69.5, 42.9, 28.2, 16.0; MS m/z 344.1 (M + H)+.
HPLC: (a) 98.0% (75% water - 25% acetonitrile); (b) 97.1%.
9-(Pent-4-ynyl)-8-(4-acetyl-phenylsulfanyl)adenine
1
(11h). Yield, 48%. H NMR (400 MHz, CDCl3) δ 8.37 (s, 1H),
7.91-7.89 (d, 2H), 7.42-7.39 (d, 2H), 5.70 (s, 2H), 4.34-4.30
(t, 2H), 2.58 (s, 3H), 2.26-2.22 (m, 2H), 2.04-1.96 (m, 3H);
13C NMR (100 MHz, CDCl3) δ 154.7, 153.6, 151.6, 143.7, 138.3,
136.1, 129.3, 129.0, 82.3, 69.5, 43.0, 28.3, 26.6, 16.0; MS m/z
352.0 (M + H)+. HPLC: (a) 99.6% (75% water - 25%
acetonitrile); (b) 99.8%.
9-(Pent-4-ynyl)-8-(4-pyrrol-1-yl-phenylsulfanyl)ade-
1
nine (11i). Yield, 50%. H NMR (400 MHz, CDCl3) δ 8.32 (s,
1H), 7.55-7.53 (d, 2H), 7.39-7.37 (d, 2H), 7.07-7.06 (d, 2H),
6.36-6.35 (d, 2H), 5.76 (s, 2H), 4.35-4.31 (t, 2H), 2.29-2.25
(m, 2H), 2.05-1.98 (m, 3H); 13C NMR (100 MHz, CDCl3) δ
154.3, 152.8, 151.6, 146.3, 140.8, 133.1, 126.8, 121.1, 119.0,
111.1, 82.4, 69.5, 42.8, 28.2, 16.0; MS m/z 375.0 (M + H)+.
HPLC: (a) 99.0% (75% water - 25% acetonitrile); (b) 99.6%.
9-(Pent-4-ynyl)-8-(2,4-dimethoxy-phenylsulfanyl)ade-
1
nine (11j). Yield, 10%. H NMR (400 MHz, CDCl3) δ 8.29 (s,
1H), 7.42-7.40 (d, 1H), 6.52-6.49 (m, 2H), 5.54 (s, 2H), 4.35-
4.32 (t, 2H), 3.82 (s, 3H), 3.79 (s, 3H), 2.29-2.25 (m, 2H), 2.09-
2.02 (m, 2H), 1.99-1.98 (t, 1H); 13C NMR (100 MHz, CDCl3) δ
162.3, 153.9, 152.4, 151.6, 135.8, 105.6, 99.5, 82.7, 69.2, 56.0,
55.5, 42.7, 28.3, 16.1; MS m/z 370.0 (M + H)+. HPLC: (a) 99.2%
(75% water - 25% acetonitrile); (b) 98.3%.
Scheme 1, Synthetic Step (d): A mixture of 8-arylsulfanyl
adenine 10 (100 µmol), Cs2CO3 (100 µmol), and pent-4-ynyl
4-methylbenzenesulfonate (120 µmol) in DMF (1.3 mL) under
nitrogen protection was heated at 80 °C for 30 min. Following
solvent removal, the crude material was purified by prepara-
tory TLC with CHCl3:MeOH:NH4OH at 10:1:0.5 or CHCl3:
MeOH:AcOH at 10:1:0.5 to provide the corresponding 9-alkyl-
8-arylsulfanyladenine derivatives 11.
9-(Pent-4-ynyl)-8-(2,4-dimethyl-phenylsulfanyl)ade-
nine (11k). Yield, 54%. 1H NMR (400 MHz, CDCl3) δ 8.28 (s,
1H), 7.25-7.23 (d, 1H), 7.08 (s, 1H), 6.98-6.96 (d, 1H), 5.96
(s, 2H), 4.28-4.25 (t, 2H), 2.40 (s, 3H), 2.30 (s, 3H), 2.28-2.24
(m, 2H), 2.05-1.98 (m, 3H); 13C NMR (100 MHz, CDCl3) δ
9-(Pent-4-ynyl)-8-(2-methoxy-phenylsulfanyl)adenine
(11a). Yield, 50%. 1H NMR (400 MHz, DMSO-d6) δ 8.32 (s,

2902 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Llauger et al.
154.2, 152.5, 151.6, 146.7, 140.0, 139.3, 133.0, 131.8, 127.8,
125.6, 119.9, 82.5, 69.3, 42.7, 28.1, 21.0, 20.6, 16.0; MS m/z
338.0 (M + H)+. HPLC: (a) 97.7% (75% water - 25%
acetonitrile); (b) 97.1%.
9-(Pent-4-ynyl)-8-(2,4-dichloro-phenylsulfanyl)-
adenine (11l). Yield, 44%. 1H NMR (400 MHz, CDCl3) δ 8.34
(s, 1H), 7.48-7.47 (d,1H), 7.19-7.18 (m, 2H), 5.69 (s, 2H),
4.35-4.32 (t, 2H), 2.29-2.25 (m, 2H), 2.07-2.00 (m, 2H), 1.98-
1.97 (t, 1H); 13C NMR (100 MHz, CDCl3) δ 152.2, 150.6, 144.7,
136.2, 135.4, 133.8, 129.9, 127.8, 127.6, 81.6, 69.1, 42.6, 27.8,
15.3; MS m/z 377.9 (M + H)+. HPLC: (a) 98.7% (75% water -
25% acetonitrile); (b) 99.1%.
9-(Pent-4-ynyl)-8-(2,5-dimethyl-phenylsulfanyl)ade-
nine (11m). Yield, 47%. 1H NMR (400 MHz, CDCl3) δ 8.29 (s,
1H), 7.16-7.14 (d, 1H), 7.11 (s, 1H), 7.06-7.04 (d, 1H), 5.98
(s, 2H), 4.29-4.26 (t, 2H), 2.39 (s, 3H), 2.27-2.24 (m, 5H),
2.04-1.97 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 154.3, 152.6,
151.6, 146.3, 136.8, 136.6, 132.8, 130.8, 129.8, 129.1, 120.0,
82.4, 69.3, 42.7, 28.1, 20.8, 20.2, 16.0; MS m/z 338.0 (M + H)+.
HPLC: (a) 99.8% (75% water - 25% acetonitrile); (b) 99.9%.
9-(Pent-4-ynyl)-8-(2,5-dichloro-phenylsulfanyl)-
adenine (11n). Yield, 48%. 1H NMR (400 MHz, CDCl3) δ 8.37
(s, 1H), 7.36-7.34 (d, 1H), 7.19-7.17 (q, 1H), 7.14-7.13 (d,
1H), 6.19 (s, 2H), 4.37-4.34 (t, 2H), 2.29-2.25 (m, 2H), 2.08-
2.01 (m, 2H), 1.98-1.97 (t, 1H); 13C NMR (100 MHz, CDCl3) δ
154.9, 153.5, 151.5, 142.8, 133.5, 133.2, 131.9, 131.0, 130.1,
128.9, 82.1, 69.5, 43.1, 28.4, 16.0; MS m/z 377.8 (M + H)+.
HPLC: (a) 99.7% (75% water - 25% acetonitrile); (b) 99.8%.
9-(Pent-4-ynyl)-8-(2-bromo-5-methoxy-phenylsulfany-
l)adenine (11p). Yield, 60%.1H NMR (400 MHz, CDCl3/
MeOD-d4) δ 8.24 (s, 1H), 7.58-7.56 (d, J ) 8.79 Hz, 1H), 6.89
(d, J ) 2.89 Hz, 1H), 6.85-6.82 (dd, J ) 8.80 Hz, J ) 2.87 Hz,
1H), 4.37-4.33 (t, J ) 7.40 Hz, 2H), 3.76 (s, 3H), 2.31-2.27
(dd, J ) 6.99 Hz, J ) 2.55 Hz, 2H), 2.09-2.02 (m, 3H); 13C
NMR (100 MHz, CDCl3/MeOD-d4) δ 159.1, 152.3, 150.7, 145.2,
133.9, 132.0, 118.3, 115.5, 115.4, 81.7, 69.2, 55.1, 42.7, 27.9,
15.5; MS m/z 418.1 (M + H)+. HPLC: (a) 99.5% (75% water -
25% acetonitrile); (b) 99.8%.
d4) δ 150.9, 149.7, 148.0, 146.9, 121.3, 119.1, 113.8, 113.4,
102.4, 81.9, 69.3, 42.7, 28.0, 15.6; MS m/z 432.0 (M + H)+.
HPLC: (a) 98.7% (75% water - 25% acetonitrile); (b) 98.9%.
2,6-Diamino-8-mercaptoadenine (15). To a solution of
2,4,5,6-tetraaminopyrimidine sulfate (14) (1 g, 4.1 mmol) in
EtOH (7.6 mL) and H2O (15.3 mL) were added CS2 (2.5 mL,
41.6 mmol) and NaHCO3 (1.7 g, 20.2 mmol). The resulted
mixture was refluxed for 24 h. Following cooling to room
temperature, the solution was acidified with AcOH to pH 4-5.
The formed yellow precipitate was filtered, washed three times
with H2O, and dried on high vacuum. The product was
obtained as a pale-yellow powder (720 mg, 97% yield). 1H NMR
(400 MHz, DMSO-d6) δ 12.37 (bs, 1H, NH), 11.55 (bs, 1H, SH),
6.28 (bs, 2H, NH2), 5.84 (bs, 2H, NH2); 13C NMR (100 MHz,
DMSO-d6) δ 163.9 (C-4), 160.3 (C-2), 151.9 (C-8), 148.1 (C-6),
101.4 (C-5); MS (EIS) m/z 182.9 (M + 1)+.
2-Amino-8-(3,4,5-trimethoxy-phenylsulfanyl)adenine
(16). A solution of 15 (500 mg, 2.74 mmol), CuI (53.3 mg, 0.27
mmol), NaOt-Bu (544 mg, 5.49 mmol), and 2-iodo-3,4,5-
trimethoxybenzene (1.72 g, 5.73 mmol) in ethylene glycol (8.2
mL) was heated to 130 °C and stirred under inert atmosphere
for 15 h. Following cooling to room temperature, the crude
material was purified by column chromatography on silica gel
(CHCl3:MeOH at 97:3). Excess ethylene glycol was removed
by passing the mixture through a short Sep-Pak C18 cartridge
and eluting with H2O:MeOH at 4:1 to afford 16 (580 mg, 60%
1
yield). H NMR (400 MHz, DMSO-d6) δ 12.49 (bs, 1H, NH),
6.80 (bs, 1H, H-2), 6.72 (s, 2H), 5.80 (bs, 2H, NH2), 3.72 (s,
6H, OCH3), 3.63 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-
d6) δ 160.2, 155.4, 153.9, 153.2, 137.2, 127.8, 114.8, 107.7, 60.0
and 56.0 (OCH3); MS (EIS) m/z 348.8 (M + 1)+.
2-Amino-9-butyl-8-(3,4,5-trimethoxy-phenylsulfanyl)-
adenine (17c). To a solution of 16 (101 mg, 0.28 mmol) in
DMF (0.7 mL) were added Cs2CO3 (94 mg, 0.28 mmol) and
butyl 4-methylbenzenesulfonate (86 mg, 0.38 mmol). The
reaction mixture was heated to 80 °C and stirred for 5 h.
Following solvent removal, the residue was purified by column
chromatography on silica gel with CHCl3:Hexanes:EtOAc:
MeOH at 2:2:1:0.1 to afford 17c (46 mg, 42% yield). MS (EIS)
m/z 405.2 (M + 1)+.
2-Amino-9-(2-isopropoxy-ethyl)-8-(3,4,5-trimethoxy-
phenylsulfanyl)adenine (17b). Following the general method
for the preparation of 17c, a mixture of Cs2CO3 (280 mg, 0.86
mmol), 2-isopropoxy-ethyl 4-methylbenzenesulfonate (289 mg,
1.2 mmol), and 16 (300 mg, 0.86 mmol) in DMF (2.1 mL)
afforded 17b (182 mg, 49% yield). 1H NMR (400 MHz, CDCl3)
δ 6.62 (s, 2H), 5.73 (bs, 2H, NH2), 4.93 (bs, 2H, NH2), 4.26 (t,
J ) 6.4 Hz, 2H, NCH2), 3.80 (s, 3H, OCH3), 3.78 (s, 6H, CH3),
3.58 (t, J ) 6.4 Hz, 2H), 3.45 (quin, J ) 6.1 Hz, 1H, CH), 1.04
(d, J ) 6.1 Hz, 6H, CH3); 13C NMR (100 MHz, CDCl3) δ 162.5,
160.0, 158.8, 155.4, 153.6, 141.8, 128.6, 107.3, 72.1 (CH), 65.4,
60.8 and 56.2 (OCH3), 43.3 (NCH2), 21.9 (CH3); MS (EIS) m/z
435.1 (M + 1)+.
9-(Pent-4-ynyl)-8-(2-chloro-5-trifluoromethyl-phenyl-
sulfanyl)adenine (11r). Yield, 40%. 1H NMR (400 MHz,
CDCl3) δ 8.36 (s, 1H), 7.59-7.57 (m, 2H), 7.51-7.48 (d, 1H),
5.86 (s, 2H), 4.39-4.36 (t, 2H), 2.29-2.23 (m, 2H), 2.07-2.04
(m, 2H), 1.97-1.96 (m, 1H); 13C NMR (100 MHz, CDCl3) δ
154.6, 153.5, 151.6, 143.1, 132.7, 130.7, 128.2, 125.8, 120.5,
82.1, 69.5, 43.1, 28.4, 16.0; MS m/z 412.0 (M + H)+. HPLC:
(a) 99.0% (75% water - 25% acetonitrile); (b) 98.7%.
9-(Pent-4-ynyl)-8-(3,5-dichloro-phenylsulfanyl)-
adenine (11s). Yield, 30%. 1H NMR (400 MHz, CDCl3) δ 8.36
(s, 1H), 7.29-7.28 (m, 3H), 5.96 (s, 2H), 4.35-4.31 (t, 2H),
2.29-2.25 (m, 2H), 2.06-1.98 (m, 3H); 13C NMR (100 MHz,
CDCl3) δ 154.8, 153.5, 151.6, 143.4, 135.8, 134.6, 128.4, 128.1,
82.3, 69.6, 42.9, 28.2, 15.9; MS m/z 377.8 (M + H)+. HPLC:
(a) 98.4% (75% water - 25% acetonitrile); (b) 97.9%.
9-(Pent-4-ynyl)-8-(3,5-dimethyl-phenylsulfanyl)ade-
2-Amino-9-(pent-4-ynyl)-8-(3,4,5-trimethoxy-phenylsul-
fanyl)adenine (17a). Following the general method for the
preparation of 17c, a mixture of Cs2CO3 (72 mg, 0.22 mmol),
pent-4-ynyl 4-methylbenzenesulfonate (70 mg, 0.29 mmol), and
16 (50 mg, 0.14 mmol) in DMF (0.35 mL) afforded 17a (54
mg, 90% yield). IR (film) νmax 3304-2937, 1632, 1585, 1497,
1
nine (11t). Yield, 36%. H NMR (400 MHz, CDCl3) δ 8.33 (s,
1H), 7.03 (s, 2H), 6.92 (s, 1H), 5.99 (s, 2H), 4.31-4.28 (t, 2H),
2.27 (s, 6H), 2.25-2.21 (m, 2H), 2.00-1.93 (m, 3H); 13C NMR
(100 MHz, CDCl3) δ 154.6, 153.0, 151.6, 145.9, 139.4, 130.3,
130.2, 128.6, 120.0, 82.5, 69.3, 42.9, 28.1, 21.2, 16.0; MS m/z
338.1 (M + H)+. HPLC: (a) 99.8% (75% water - 25%
acetonitrile); (b) 99.6%.
9-(Pent-4-ynyl)-8-(2,4,5-trichloro-phenylsulfanyl)ade-
nine (11u). Yield, 46%. 1H NMR (400 MHz, CDCl3/MeOD-d4)
δ 8.26 (s, 1H), 7.63 (s, 1H), 7.50 (s, 1H), 4.38-4.35 (t, J ) 7.3
Hz, 2H), 2.32-2.28 (m, 2H), 2.09-2.02 (m, 3H); 13C NMR (100
MHz, CDCl3/MeOD-d4) δ 154.4, 152.7, 150.9, 143.8, 134.1,
133.7, 133.4, 131.9, 131.3, 129.4, 81.8, 69.4, 42.9, 28.1, 15.7;
MS m/z 411.9 (M + H)+. HPLC: (a) 98.5% (75% water - 25%
acetonitrile); (b) 97.1%.
1
1408, 1232, 1126; H NMR (400 MHz, CDCl3) δ 6.62 (s, 2H),
5.44 (bs, 2H, NH2), 4.74 (bs, 2H, NH2), 4.15 (t, J ) 7.4 Hz, 2H,
NCH2), 3.82 (s, 3H, OCH3), 3.79 (s, 6H, CH3), 2.21 (dt, J ) 2.5
and 7.1 Hz, 2H), 1.97 (t, J ) 2.5 Hz, 1H, CH), 1.93 (quin, J )
7.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 160.1, 155.4, 153.7,
153.2, 141.1, 138.0, 127.0, 115.1, 107.4, 82.7 (C quaternary),
60.8 and 56.2 (OCH3), 60.2 (CH), 42.4 (NCH2), 28.0, 16.0; MS
(EIS) m/z 415.1 (M + 1)+. HPLC: (a) 99.0% (70% water - 30%
acetonitrile); (c) 98.5%.
9-Butyl-8-(2-chloro-3,4,5-trimethoxy-benzenesulfanyl)-
adenine (12d). To a cooled mixture (0 °C) of 11w2 (14 mg,
36 µmol) and concentrated HCl (18 µL, 0.5 mL/mmol) in MeOH
(1 mL) was added a 70% aqueous solution of tert-butyl hydro-
peroxide (t-BuOOH) (30 µL, 0.2 mmol). The mixture was first
stirred at 0 °C for 5 min and then at 80 °C overnight. Following
9-(Pent-4-ynyl)-8-(6-bromo-benzo[1,3]dioxol-5-ylsulfa-
nyl)adenine (11v). Yield, 48%.1H NMR (400 MHz, CDCl3/
MeOD-d4) δ 8.22 (s, 1H), 7.17 (s, 1H), 7.00 (s, 1H), 6.06 (s,
2H), 4.35-4.31 (t, J ) 7.26 Hz, 2H), 4.12 (s, 2H), 2.33-2.30
(m, 2H), 2.08-2.05 (m, 3H). 13C NMR (100 MHz, CDCl3/MeOD-

Purine-Scaffold Hsp90 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2903
solvent evaporation, the product was purified by column
chromatography on silica gel (CHCl3:Hexanes:EtOAc:i-PrOH
2:2:1:0.5) to afford compound 12d (10 mg, 66% yield). 1H NMR
(400 MHz, CDCl3) δ 8.32 (s, 1H, H-2), 7.72 (s, 1H), 5.90 (bs,
2H, NH2), 4.22 (t, J ) 7.5 Hz, 2H, NCH2), 3.90 (s, 3H, OCH3),
3.87 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 1.72 (m, 2H), 1.33 (m,
2H), 0.91 (t, J ) 7.3 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3)
δ 154.7, 153.0 (C-2), 152.9, 151.9, 151.2, 145.8, 144.2, 125.1,
122.0, 123.0, 111.6, 61.6 and 56.7 (OCH3), 44.2 (NCH2), 32.2,
20.3, 14.0 (CH2); MS (EIS) m/z 424.0 (M + 1)+. HPLC: (a)
98.5% (70% water - 30% acetonitrile); (c) 97.2%.
9-(Pent-4-ynyl)-8-(2-chloro-3,4,5-trimethoxy-benzene-
sulfanyl)adenine (12b).9 Following the general method for
the preparation of 12d, 11w1 afforded 12b. Yield, 21%.
HPLC: (a) 95.1% (70% water - 30% acetonitrile); (c) 95.0%.
9-(2-Isopropoxy-ethyl)-8-(2,4-dichloro-5-methoxy-ben-
zenesulfanyl)adenine (12c). Following the general method
for the preparation of 12d, 11e3 afforded 12c. Yield, 53%. 1H
NMR (400 MHz, CDCl3) δ 8.36 (s, 1H, H-2), 7.44 (s, 1H), 7.09
(s, 1H), 5.55 (bs, 2H, NH2), 4.48 (t, J ) 5.6 Hz, 2H, NCH2),
3.81 (s, 3H, OCH3), 3.74 (t, J ) 5.6 Hz, 2H), 3.49 (d, J ) 6.1
Hz, 1H, CH), 1.04 (d, J ) 6.1 Hz, 6H, CH3); 13C NMR (100
MHz, CDCl3) δ 154.1 (C-2), 153, 147, 131.0, 130.2, 126.9, 123.6,
116.0, 72.3 (CH), 65.6, 56.5 (OCH3), 44.1 (NCH2), 21.8 (CH2);
MS (EIS) m/z 428.0 (M + 1)+. HPLC: (a) 90.2% (70% water -
30% acetonitrile); (c) 91.0%.
9-(2-Isopropoxy-ethyl)-8-(2,6-dichloro-3,4,5-trimethoxy-
benzenesulfanyl)adenine (12e). Following the general
method for the preparation of 12d, 11w3 afforded 12e. Yield,
44%. 1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H, H-2), 5.60 (bs,
2H, NH2), 4.49 (t, J ) 5.5 Hz, 2H, NCH2), 3.99 (s, 3H, OCH3),
3.90 (s, 6H, OCH3), 3.76 (t, J ) 5.5 Hz, 2H), 3.52 (m, J ) 6.1
Hz, 1H, CH), 1.11 (d, J ) 6.1 Hz, 6H, CH3); 13C NMR (100
MHz, CDCl3) δ 153.7, 151.9, 149.7, 130.0, 124.1, 72.5 (CH),
65.8, 61.4 and 61.2 (OCH3), 44.0 (NCH2), 21.9 (CH2); MS (EIS)
m/z 487.9 (M + 1)+. HPLC: (a) 98.0% (60% water - 40%
acetonitrile); (c) 98.0%.
2-Amino-9-butyl-8-(2-chloro-3,4,5-trimethoxy-phenyl-
sulfanyl)adenine (18c). Following the general method for the
preparation of 12d, 17c afforded 18c. Yield, 38%. 1H NMR (400
MHz, CHCl3) δ 6.45 (s, 1H), 5.61 (bs, 2H, NH2), 4.82 (bs, 2H,
NH2), 4.06 (t, J ) 7.5 Hz, 2H, NCH2), 3.91 (s, 3H, OCH3), 3.85
(s, 3H, OCH3), 3.64 (s, 3H, OCH3), 1.65 (m, 2H), 1.30 (m, 2H),
0.88 (t, J ) 7.3 Hz, 3H, CH3); 13C NMR (100 MHz, CHCl3) δ
160.0, 155.3, 153.5, 152.6, 150.5, 142.8, 140.1, 127.3, 119.8,
115.5, 108.8, 61.2 and 56.2 (OCH3), 43.3 (NCH2), 31.7, 19.8,
13.6 (CH2); MS (EIS) m/z 439.2 (M + 1)+.
2873, 1648, 1594, 1573, 1479, 1312, 1149, 1112 (SO), 1013
1
(SO); H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H, H-2), 7.71 (s,
1H), 5.89 (bs, 2H, NH2), 4.57 (t, J ) 7.8 Hz, 2H, NCH2), 4.00-
387 (3s, 9H, OCH3), 1.85 (m, 2H), 1.40 (m, J ) 7.5 Hz, 2H),
0.95 (t, J ) 7.3 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ
156.5, 155.2 (C-2), 151.9, 151.0, 150.8, 148.5, 145.8, 131.1,
121.0, 119.0, 109.5, 61.4-61.3 and 56.6 (OCH3), 44.9 (NCH2),
32.4, 20.0, 13.6; MS (EIS) m/z 456.1 (M + 1). HPLC: (a) 99.7%
(70% water - 30% acetonitrile); (c) 99.0%.
Method for the Fluorination of the Adenine Moiety
at C2: 2-Fluoro-9-butyl-8-(2-chloro-3,4,5-trimethoxy-phe-
nylsulfanyl)adenine (21c). To a cooled solution (0 °C) of 18c
(11.3 mg, 0.02 mmol) in HF/pyridine (18 µL, 0.7 mL/mmol)
was slowly added NaNO2 (2.2 mg, 0.03 mmol). The resulted
mixture was stirred at room temperature for 1 h and then
quenched by stirring for 1 h with 14 mg of CaCO3 in CH2Cl2
(75 µL). The crude material was taken up in CH2Cl2, washed
with water, and dried over anhydrous Na2SO4. Following
solvent removal, the residue was purified on a preparative
silica gel plate (CHCl3:Hexanes:EtOAc:i-PrOH at 2:2:1:0.1) to
afford 21c (1.9 mg, 17% yield). IR (film) νmax 3318-2953, 1657,
1604, 1583, 1479, 1385, 1111, 1015; 1H NMR (400 MHz, CDCl3)
δ 6.72 (s, 1H), 5.83 (bs, 2H, NH2), 4.18 (t, J ) 7.5 Hz, 2H,
NCH2), 3.92 (s, 3H, OCH3), 3.89 (s, 3H, CH3), 3.74 (s, 3H, CH3),
1.72 (m, 2H), 1.32 (m, 2H), 0.92 (t, J ) 7.4 Hz, 3H, CH3); 13C
NMR (100 MHz, CDCl3) δ 160.1, 158.0, 156.1, 152.5, 150.8,
143.9, 124.6, 111.2, 61.2 and 56.3 (OCH3), 43.9 (NCH2), 31.7,
29.7, 19.7 (CH3); MS (EIS) m/z 442.2 (M + 1)+. HPLC: (a)
95.9% (60% water - 40% acetonitrile); (c) 98.0%.
2-Fluoro-9-(2-isopropoxy-ethyl)-8-(2-chloro-3,4,5-tri-
methoxy-phenylsulfanyl)adenine (21b). Following the
method for the preparation of 21c, 18b afforded 21b. Yield,
11%. IR (film) νmax 3322-2870, 1648, 1603, 1584, 1478, 1384,
1110, 1013; 1H NMR (400 MHz, CDCl3) δ 6.80 (s, 2H), 6.14
(bs, 2H, NH2), 4.42 (t, J ) 5.5 Hz, 2H, NCH2), 3.88-3.89 and
3.76 (3s, 9H, OCH3), 3.71 (t, J ) 5.5 Hz, 2H), 3.50 (quin, J )
6 Hz, 1H, CH), 1.04 (d, J ) 6 Hz, 6H, CH3). 13C NMR (100
MHz, CDCl3) δ 158.0, 156.1 (JC-F ) 20 Hz), 152.4, 150.7, 143.7,
128.6, 125.4, 122.0, 111.3, 72.2 (CH), 65.3, 61.2 and 56.3
(OCH3), 44.1 (NCH2), 21.8 (CH3); MS (EIS) m/z 471.9 (M +
1)+. HPLC: (a) 98.6% (60% water - 40% acetonitrile); (c)
95.0%.
2-Fluoro-8-(3,4,5-trimethoxy-phenylsulfanyl)adenine
(19). To a cooled solution (-40 °C) of 16 (303 mg, 0.87 mmol)
in HF/pyridine (0.4 mL, 0.5 mL/mmol) was slowly added
NaNO2 (68 mg, 0.95 mmol), and the mixture was stirred for
15 min at -30 °C. The reaction was quenched by stirring for
1 h at room temperature with CaCO3 (380 mg) in CH2Cl2 (2
mL). The slurry was taken up in CH2Cl2, washed with water,
and dried over anhydrous Na2SO4. Following solvent removal,
the product was further used without additional purification
2-Amino-9-(2-isopropoxy-ethyl)-8-(2-chloro-3,4,5-tri-
methoxy-phenylsulfanyl)adenine (18b). Following the gen-
eral method for the preparation of 12d, 17b afforded 18b.
Yield, 71%. 1H NMR (400 MHz, DMSO-d6) δ 9.00 (bs, 2H,
NH2), 7.61 (bs, 2H, NH2), 6.81 (s, 1H, H-2), 4.21 (t, J ) 5.2
Hz, 2H, NCH2), 3.80 and 3.76 (2s, 9H, OCH3), 3.57 (t, J ) 5.2
Hz, 2H), 3.43 (m, J ) 6 Hz, 1H, CH), 0.94 (d, J ) 6 Hz, 6H,
CH3); 13C NMR (400 MHz, DMSO-d6) δ 150.3, 150.0, 149.6,
142.8, 140.9, 125.8, 119.5, 116.9, 110.8, 108.0, 71.2 (CH), 54.6,
61.0-60.8 and 56.3 (OCH3), 43.7 (NCH2), 21.7 (CH2); MS (EIS)
m/z 469.1 (M + 1)+.
1
(120 mg, 39% yield). H NMR (400 MHz, DMSO-d6) δ 13.34
(bs, 1H, NH), 7.77 (bs, 2H, NH2), 6.83 (s, 2H), 3.76 (s, 6H,
OCH3), 3.67 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6) δ
159.6, 157.6, 153.4, 109.2, 60.0 and 56.1 (OCH3); MS (EIS) m/z
352.0 (M + 1)+.
2-Fluoro-9-(pent-4-ynyl)-8-(3,4,5-trimethoxy-phenyl-
sulfanyl)adenine (20a). Following the general method for
the preparation of 11e1, a mixture of DBAD (365 mg, 1.55
mmol), PPh3 (181 mg, 0.68 mmol), 4-pentnyn-1-ol (39 µL, 0.41
mmol), and 19a (120 mg, 0.34 mmol) afforded 20a (68.5 mg,
48% yield). IR (film) νmax 3303-2941, 1664, 1582, 1499, 1359
(S), 1128, 1002; 1H NMR (400 MHz, CDCl3) δ 6.65 (s, 2H), 6.03
(bs, 2H, NH2), 4.20 (t, J ) 7.5 Hz, 2H, NCH2), 3.80 (s, 3H,
OCH3), 3.77 (s, 6H, OCH3), 2.18 (dt, J ) 2.6 and 7 Hz, 2H),
1.92 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 158.0, 156.1 (JC-F
) 20 Hz), 153.8, 153.1, 146.3, 138.8, 124.7, 118.1, 109.1, 82.3
(C quaternary), 69.5 (CH), 60.9 and 56.3 (OMe3), 43.0 (NCH2),
28.0, 16.0; MS (EIS) m/z 418.1 (M + 1)+. HPLC: (a) 97.2%
(70% water - 30% acetonitrile); (c) 98.0%.
9-(Pent-4-ynyl)-8-(2-chloro-5-methoxy-phenylsulfany-
l)adenine (12a). Following the general method for the
1
preparation of 12d, 11e1 afforded 12a. Yield, 31%. H NMR
(400 MHz, CDCl3) δ 8.33 (s, 1H, H-2), 7.24 (t, J ) 8.2 Hz, 1H),
6.95 (d, J ) 2 Hz, 1H), 6.83 (dd, J ) 2 and 8.2 Hz, 1H), 6.11
(bs, 2H, NH2), 4.30 (t, J ) 7.3 Hz, 2H, NCH2), 3.76 (s, 3H,
OCH3), 2.24 (dt, J ) 2.5 and 7 Hz, 2H), 1.99 (m, 3H); 13C NMR
(100 MHz, CDCl3) δ 158.7, 155.5, 154.5, 153.2 (C-2), 132.0,
130.7, 125.4, 116.8, 114.6, 82.3, 69.5 (CH), 55.3 (OCH3), 43.1
(NCH2), 28.4, 16.0; MS (EIS) m/z 374.0 (M + 1)+. HPLC: (a)
96.0% (70% water - 30% acetonitrile); (c) 95.5%.
9-Butyl-8-(2-chloro-3,4,5-trimethoxy-benzenesulfonyl)-
adenine (29). Following the general method for the prepara-
tion of 12d, a mixture of t-BuOOH (30 µL, 0.22 mmol), HCl
(18 µL, 0.5 mL/mmol) and 28d (15 mg, 0.11 mmol) in MeOH
(1 mL) afforded 29 (7 mg, 43% yield). IR (film) νmax 3317-
2-Fluoro-9-(pent-4-ynyl)-8-(2-chloro-3,4,5-trimethoxy-
phenylsulfanyl)adenine (21a). Following the general method
for the preparation of 12d but using 1,4-dioxane as solvent,
20a afforded 21a. Yield, 19%. IR (film) νmax 3307-2850, 1649,

2904 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Llauger et al.
1605, 1584, 1478, 1385, 1110, 1013; 1H NMR (400 MHz, CDCl3)
δ 6.81 (s, 1H), 6.16 (bs, 2H, NH2), 4.33 (t, J ) 7.4 Hz, 2H,
NCH2), 3.91 and 3.90 (2s, 6H, OCH3), 3.77 (s, 3H, CH3), 2.27
(dt, J ) 2.6 and 7 Hz, 2H), 2.03 (m, J ) 7 Hz, 2H), 1.98 (t, J
) 2.6 Hz, 1H, CH); 13C NMR (100 MHz, CDCl3) δ 158.1, 155.8
(JC-F ) 20 Hz), 152.6, 150.9, 146.0, 144.3, 123.4, 122.6, 117.3,
112.0, 82.0 (C quaternary), 69.7 (CH), 61.2 and 56.3 (OCH3),
43.3 (NCH2), 28.1, 16.0; MS (EIS) m/z 452.1 (M + 1)+. HPLC:
(a) 96.2% (60% water - 40% acetonitrile); (c) 95.0%.
cancer chemotherapeutic agents. Trends Mol. Med. 2002, 8,
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wide-range oncogenic transformation via PU24FCl, a specific
inhibitor of tumor Hsp90. Chem. Biol. 2004, 11, 787-797.
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molecular chaperone HSP90-SAR explained through multiple
X-ray structures. Bioorg. Med. Chem. Lett. 2004, 14, 325-328.
(b) Wright, L.; Barril, X.; Dymock, B.; Sheridan, L.; Surgenor,
A.; Beswick, M.; Drysdale, M.; Collier, A.; Massey, A.; Davies,
N.; Fink, A.; Fromont, C.; Aherne, W.; Boxall, K.; Sharp, S.;
Workman, P.; Hubbard, R. E. Structure-activity relationships
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Izobreteniya 1995, 1, 242-243. (b) Laxer, A.; Major, D. T.;
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General Method for the Synthesis of 8-Arylsulfanyl Adenine
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2-Fluoro-8-bromo-9-(2-isopropoxy-ethyl)adenine (23).
To a solution of 2-fluoroadenine (22) (20 mg, 0.13 mmol) in
DMF (0.33 mL) were added at room-temperature Cs2CO3 (43
mg, 0.13 mmol) and 2-isopropoxy-ethyl 4-methylbenzenesul-
fonate (44 mg, 0.17 mmol). The reaction mixture was heated
to 80 °C and stirred for 5 h. The solvent was evaporated and
the residue purified by column chromatography on silica gel
(CHCl3:MeOH at 98:2) to afford 2-fluoro-9-(2-isopropoxy-ethyl)-
1
adenine (19 mg, 61% yield). H NMR (400 MHz, DMSO-d6) δ
8.03 (s, 1H), 7.72 (bs, 2H, NH2), 4.17 (t, J ) 5.4 Hz, 2H, NCH2),
3.67 (t, J ) 5.4 Hz, 2H), 3.49 (m, J ) 6.0 Hz, 1H, CH), 0.98 (d,
J ) 6.0 Hz, 6H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 159.6,
157.5, 156.1 (JC-F ) 21 Hz), 150.8 (JC-F ) 20 Hz), 141.6, 116.9,
70.9 (CH), 65.1, 43.4 (NCH2), 21.8 (CH3); MS (EIS) m/z 438.0
(M - 1)-. N-bromosuccinimide (382 mg, 2.1 mmol) was added
to a solution of 2-fluoro-9-(2-isopropoxy-ethyl)adenine (257 mg,
1.1 mmol) in DMF (6 mL), and the resulting mixture was
stirred for 11 h at room temperature. Following solvent re-
moval under high vacuum, the crude material was purified
by column chromatography on silica gel (EtOAc:MeOH:NH4-
OH at 18:1:0.2) to furnish 23 (84 mg, 25% yield). 1H NMR (400
MHz, CDCl3) δ 8.34 (s, 1H), 5.59 (bs, 2H, NH2), 4.41 (t, J )
5.9 Hz, 2H, NCH2), 3.81 (t, J ) 5.8 Hz, 2H), 3.55 (m, J ) 6.1
Hz, 1H, CH), 1.08 (d, J ) 6.1 Hz, 6H, CH3); MS (EIS) m/z 299.9
(M + 2)+.
2-Fluoro-9-(2-isopropoxy-ethyl)-8-(3,4,5-trimethoxy-
phenylsulfanyl)adenine (20b). To 3,4,5-trimethoxythiophe-
nol (6) (35 mg, 0.17 mmol) in DMF (1 mL) was added K2CO3
(44 mg, 0.31 mmol), and the resulting solution was stirred for
1 h at room temperature. Following the addition of 23 (50 mg,
0.16 mmol), the reaction mixture was heated to 130 °C and
stirred for 4 h. The solvent was removed under high vacuum
and the crude material purified by column chromatography
on silica gel (Hexanes:CH2Cl2:EtOAc at 2:1:2) to afford 20b
(27 mg, 40% yield). IR (film) νmax 3322-2868, 1649, 1582, 1498,
1
1367, 1127, 1004; H NMR (400 MHz, CDCl3) δ 6.64 (s, 2H),
6.25 (bs, 2H, NH2), 4.30 (t, J ) 5.9 Hz, 2H, NCH2), 3.75 (s,
3H, OCH3), 3.74 (s, 6H, OCH3), 3.59 (t, J ) 5.9 Hz, 2H), 3.41
(quin, J ) 6 Hz, 1H, CH), 0.97 (d, J ) 6 Hz, 3H, CH3); 13C
NMR (100 MHz, CDCl3) δ 160.1, 156.2 (JC-F ) 20.5 Hz), 153.7,
147.2, 138.5, 125.4, 118.0, 108.9, 72.7 (CH), 65.2, 60.8 and 56.3
(OCH3), 44.0 (NCH2), 21.8; MS (EIS) m/z 438.1 (M + 1)+.
HPLC: (a) 98.1% (60% water - 40% acetonitrile); (c) 96.0%.
Acknowledgment. This work was supported by
AACR-Cancer Research and Prevention Foundation,
NIH/NCI, Mr. William H. Goodwin and Mrs. Alice
Goodwin the Commonwealth Cancer Foundation for
Research, The Experimental Therapeutics Center of
Memorial Sloan-Kettering Cancer Center, and a gener-
ous donation by the Taub Foundation.
(15) Barhate, N. B.; Gajare, A. S.; Wakharkar, R. D.; Ashutosh, V.
B. Simple and practical halogenation of arenes, alkenes and
alkynes with hydrohalic acid/H2O2 (or TBHP). Tetrahedron,
1999, 55, 11127-11142.
(16) Castanet, A.-S.; Colobert, F.; Broutin, P.-E. Mild and regiose-
lective iodination of electron-rich aromatics with N-iodosuccin-
imide and catalytic trifluoroacetic acid. Tetrahedron Lett. 2002,
43, 5047-5048.
(17) VanAllan, J. A.; Deacon, B. D. 2-Mercaptobenzimidazole. Org.
Synth. 1945, 4, 569.
Supporting Information Available: Spectral data, HPLC,
and LC-MS analyses are available free of charge via the
Internet at http://pubs.acs.org.
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