Development of a purine-scaffold novel class of hsp90 binders that inhibit the proliferation of cancer cells and induce the degradation of Her2 tyrosine kinase

Article abstract of DOI:10.1016/S0968-0896(02)00253-5

The first published synthesis and characterization of a purine-scaffold library of hsp90 inhibitors is presented. The purine-scaffold represents a platform for the creation of easily synthesizable and derivatizable soluble molecules that are amenable for oral administration. The most active compound of the series (71) exhibits binding to hsp90 comparable to the natural product derivative 17AAG that is now in Phase I clinical trial as a cancer therapeutic. 71 Induces the degradation of Her2 tyrosine kinase and arrests the MCF-7 breast cancer cell line at low micromolar concentrations (IC₅₀=2 μM).

Full text of DOI:10.1016/S0968-0896(02)00253-5

Bioorganic & Medicinal Chemistry10 (2002) 3555–3564
Development of a Purine-Scaffold Novel Class of Hsp90 Binders
that Inhibit the Proliferation of Cancer Cells and Induce the
Degradation of Her2Tyrosine Kinase
Gabriela Chiosis,a,b,* Brian Lucas,a,y Alexander Shtil,a,{ Henri Huezoa and Neal Rosena,b
aProgram in Cell Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021, USA
bDepartment of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021, USA
Received 28 February2002; accepted 23 May2002
Abstract—The first published synthesis and characterization of a purine-scaffold library of hsp90 inhibitors is presented. The
purine-scaffold represents a platform for the creation of easilysynthesizable and derivatizable soluble molecules that are amenable
for oral administration. The most active compound of the series (71) exhibits binding to hsp90 comparable to the natural product
derivative 17AAG that is now in Phase I clinical trial as a cancer therapeutic. 71 Induces the degradation of Her2 tyrosine kinase
and arrests the MCF-7 breast cancer cell line at low micromolar concentrations (IC50=2 mM).
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
degradation of certain proteins that undergo mutation
in cancer, such as v-src, bcr-abl and p53, but have little
or no effect on their normal counterparts.15ꢀ17 Most
cancer cells treated with ansamycins or RD block in G1,
phenomenon that is followed bymorphological and
functional differentiation.18,19
The hsp90 molecular chaperones playa keyrole in reg-
ulating the physiology of cells exposed to environmental
stress and in maintaining the malignant phenotype in
tumor cells.1ꢀ4 These proteins possess an ATP/ADP
binding pocket at their N-terminal that is well-con-
served among the four familymembers: Hsp90 a and
Hsp90 b (cytoplasm), Grp94 (endoplasmic reticulum)
and Trap-1 (mitochondria).5ꢀ7 Geldanamycin and her-
bimycin (GM, HA) (Fig. 1) and the unrelated com-
pound radicicol (RD) were found to bind to this
N-terminal pocket of Hsp90.8ꢀ10 Addition of these
compounds to cells induces the proteasomal degrada-
tion of a small subset of proteins involved in signal
transduction (i.e., steroid receptors, Raf kinase, Akt,
certain transmembrane tyrosine kinases). The met and
Her2 tyrosine kinase were shown to be the most sensi-
tive targets described.11ꢀ13 Her2 has oncogenic proper-
ties and is overexpressed in manyhuman tumors,
including approximately30% of human breast can-
cers.14 In addition, these molecules cause the selective
The ansamycin derivative 17AAG (Fig. 1) is currently in
earlyclinical trial in cancer patients. 17AAG is a potent
anticancer drug, but it is poorlysoluble and hard to
formulate, and it was shown to induce hepatotoxicity
related to the presence of the benzoquinone moietyin its
structure.20 There is no synthetic route available for the
construction of this molecule and the drug is obtainable
onlybyfermentation. Radicicol has no activityin ani-
mals because of its instability. Oxime-derivatives of RD,
like 6-O-[2-(2-pyrrolidonyl)-ethyl] radicicol oxime, were
found to have potent activityin vitro and in vivo and
were extensivelystudied in Japan. 17,21ꢀ23 Unfortunately,
the compounds induced severe cataracts in animals,
again for reasons unrelated to hsp90 binding.
We have used the structural requirements for Hsp90 a
binding and designed a small molecule PU3 (Fig. 1) that
exhibited qualitative effects on cellular protein expres-
sion, proliferation, and differentiation verysimilar to
those induced byGM and RD. PU3 displayed a relative
binding affinityfor hsp90 of 15–20 mM. Addition of
PU3 to cells caused the degradation of Her2 kinase,
*Corresponding author. Tel.: +1-212-639-8929; fax: +1-212-717-
3627; e-mail: chiosisg@mskmail.mskcc.org
{Present address: Laboratoryof Tumor Cell Genetics, N. Blokhin
Cancer Center, Moscow 115478, Russia.
{Present address: Department of Chemistry, University of Wisconsin-
Madison, 1101 UniversityAvenue, Madison, WI 53706, USA.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00253-5

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G. Chiosis et al. / Bioorg. Med. Chem. 10 (2002) 3555–3564
2,4,5,6-tetraaminopyrimidine sulfate to give the corre-
sponding 5-N-acylated aminopyrimidines. These were
cyclized in NaOMe/MeOH to construct the purine
building blocks 2 and 3, respectively(Scheme 1). Several
9-alkyl-8-benzyl-9H-purin-6-ylamines (4–46) created by
9-N-alkylation of 2 are depicted in Fig. 2.25 The
2-amino group of 3 was initiallyconverted to fluorine
via a diazotization reaction in non-aqueous media using
tert-butyl nitrite (TBN) in HF/pyridine26 and subse-
quently alkylated to give the 2-fluoro-9-alkyl-8-
(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamines 47–59
(Scheme 2). The alkylation was performed using the
Mitsunobu reaction, methodologythat can accom-
modate a large arrayof primaryand secondaryalco-
hols. Contraryto alkylation of 2 where the 3-N product
was observed as an undesired product, alkylation of the
fluoro derivative gave cleanlythe 9-N alkyl purine. The
2-fluoro derivatives were further converted to the
2-alkoxypurines 60 and 61 byrefluxing in MeOH or
EtOH and NaOMe, respectively.27 Iodine was introduced
in that position using isoamyl nitrite in diiodomethane
generating 62.28,29 The 2-iodo derivative was transformed
to the cyano-derivative 63 with tri-n-butylcyanostannane
and tetrakis(triphenylphosphine)palladium(0) in DMF
and to the vinyl-derivative 64 with vinyltributyltin and
(MeCN)2PdCl2.30
Figure 1. Schematic representation of the known hsp90 binders.
estrogen receptor and Raf kinase. The proteins affected
byPU3 were identical to those affected byansamycins
and RD. Exposure of breast cancer cells to PU3 resulted
in growth inhibition and profound morphologic chan-
ges. Cessation of proliferation was accompanied by
induction of a more mature, epithelial morphology
consistent with breast differentiation.24 Although the
activityof this molecule is lower compared to the nat-
ural products, the structural skeleton of PU3 allows for
extensive chemical modifications in the pursuit of com-
pounds with better activity, solubility and therapeutic
index. Here we describe the first published synthesis and
characterization of a small libraryof derivatives of PU3
that resulted in a compound with 30-times better cel-
lular effects than our lead, PU3, and which displays a
relative binding affinityfor hsp90 of 0.55 mM, a value
similar to 17AAG.
The benzyl moiety was enriched in electron-donating
groups in order to increase its interaction with the
pocket lysine (Scheme 3). Chlorine and bromine
were added via a radicalic reaction using t-butyl
hydroperoxide and the corresponding HX.31 In the
case of chlorine onlymonosubstitution was observed
(65), while bromine gave the monosubstituted (66)
and a small amount of di-bromosubstituted product
(67).
The nature and length of the bridge between the purine
and the phenyl ring were additionally modified
(Scheme 4). As starting material we utilized 8-bromo-
adenine. This was 9-N alkylated and consequently,
reacted at high temperature with the aniline-, phenol-
or benzyl-derivative to give the corresponding products
68–70.
Results and Discussion
Chemistry
The assemblyof the fullysubstituted derivatives 71 and
72 commenced with 3 (Scheme 5). Chlorine was added
initiallyvia a radicalic reaction to give 73. Subsequently,
the 2-amino group was transformed to fluorine and the
resulting purine (74) was alkylated via the Mitsunobu
reaction.
The
commerciallyavailable
3,4,5-trimeth-oxy
phenylacetic acid (1, Scheme 1) was used as the starting
point for building a PU3 class library. In general, the
carboxylic acid was converted to the acid fluoride and
then coupled with 4,5,6-triaminopyrimidine sulfate or
SAR and Biological Results
All compounds were tested for their abilityto compete
with geldanamycin for hsp90 binding. To assess their
cellular effect we tested the efficacyof the compounds to
arrest the growth of the MCF-7 breast cancer cell line
and to induce the degradation of the oncogenic Her2
tyrosine kinase. The M logP value was calculated for all
compounds and the ‘rule of 5’ was kept as a guideline in
the choice for derivatization.32ꢀ34
Scheme 1. Reagents: (a) cyanuric fluoride, pyridine, DCM; (b) 4,5,6-
triaminopyrimidine sulfate, DIEA, DMF for 2 and 2,4,5,6-tetra-
aminopyrimidine sulfate, aqueous NaOH for 3; (c) NaOMe, MeOH.

G. Chiosis et al. / Bioorg. Med. Chem. 10 (2002) 3555–3564
3557
Figure 2. Representative purines obtained by modifying the nature and length of the 9-N butyl chain of PU3.
Scheme 2. Reagents: (a) HF, pyridine, t-butyl nitrite; (b) R1OH, PPh3, diethyl azodicarboxylate or di-tert-butyl azodicarboxylate, toluene/DCM;
(c) R2OH, NaOMe; (d) CH2I2, isoamyl nitrite; (e) Bu3SnCN, Pd[PPh3]4; (f) (MeCN)2PdCl2, Bu3(vinyl)Sn.
Scheme 3. Reagents: (a) HX, t-butylhydroperoxide.
Effect of the 9-alkyl chain on activity
observation resulted from testing these derivatives is
The varietyof unbranched and branched, linear and
cyclic, saturated and unsaturated primary alcohols and
several secondaryalcohols available for the 9- N-alkyl-
ation reaction allowed for the introduction of a high
degree of diversityat that position. A first important
that no compound that has a substituent on C1, such as
29, 30, 31, 33, and 34 (i.e., resulted byalkylation with
secondaryalcohols) is active. No binding of these deri-
vatives to hsp90 or anycellular effect is observed at
concentrations as high as 250 mM. Secondly, none of the

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G. Chiosis et al. / Bioorg. Med. Chem. 10 (2002) 3555–3564
Table 1.
EC50 hsp90
IC50 MCF-7
IC50 Her2
M logP
4
5
6
7
8
9
NA
4.5
10.8
13
11.8
20.4
114
52.1
15.7
16.2
47.8
32
70
80
47
50
62
98
69
160
62
75
111
46
40
47
64
41
51
24
72
65
39
92
NA
70
50
55
50
100
50
>100
70
70
120
60
50
50
70
30
70
20
100
85
1.34
1.59
1.83
2.06
2.28
2.06
2.51
2.51
2.29
2.06
2.29
2.29
2.73
2.21
2.21
1.98
1.98
2.21
0.99
1.09
1.55
1.55
10
11
13
15
16
17
18
22
23
24
25
26
36
37
38
41
>100
9.5
7.5
10.6
NA
1.5
NA
5.5
1.7
3.4
Scheme 4. Reagents: (a) 3,4,5-trimethoxyaniline; (b) 3,4,5-trimethoxy-
phenol; (c) 3,4,5-trimethoxybenzyl alcohol.
45
70
All values are in mM. See Experimental for assayprotocols. Standard
deviations for binding assays were ꢁ35% of the mean or less, while
for growth arrest and Her2 protein degradation of ꢁ20%. The values
are the mean of two experiments.
NA, not available.
moietyleads to decreased or completelyabolished
activity. However, fluorine in general increases the
potency and water solubility of the 9-alkyl-8-benzyl-
9H-purin-6-ylamine derivatives (Table 2). Fluorine is a
small electron-withdrawing atom and its addition to the
purine moietyis verylikelyto improve the H-donor
Scheme 5. Reagents: (a) HCl, t-butylhydroperoxide; (b) HF, pyridine,
t-butyl nitrite; (c) R3OH, PPh3, di-tert-butyl azodicarboxylate, tolu-
ene/DCM.
abilityof C6 NH .
2
Effect of the addition of halogens to the
trimethoxyphenyl moiety
aromatic moietycontaining chains such as in derivatives
27 and 28 allowed for activity. Compounds 43, 44, and
45 containing the piperidinyl-, morpholinyl- and pyrro-
lidinyl-acetamides, respectively, and 39 and 40 contain-
ing the oxetane ring-derivatives, have no activityin the
binding and cellular assays. Somewhat surprising was
the cellular inefficacyof compound 21 that contains a
tertiaryamine in the 9-N chain. Although it bound
hsp90 with a potencycomparable to PU3, it shows no
effect when added to cancer cells. It is possible that the
presence of the tertiaryamine limits the cellular uptake
of 21. Compounds 12, 14, 19, 31, and 46 are insoluble in
the assaymedia and their further characterization was
stopped.
Additional electron-donating functionalities on the tri-
methoxyphenyl ring should theoretically improve the
efficacyof binding to hsp90 byincreasing the interaction
of this moietywith the Lys residue found at the top of
the ADP-binding pocket. We found that both bromine
and chlorine improve potency(Table 3). Although bro-
mine is expected to have a greater effect, it is possible
that due to its large size it is more difficult to accom-
modate inside the pocket. If two bromines are added to
the molecule, binding to hsp90 in abolished, again
probablydue to steric effects ( 67). Addition of one Cl to
the trimethoxyphenyl moiety improves activity over
PU3 bya factor of 2 ( 65).
Several chains are favorable for activity(Table 1). The
greatest gain in activityis obtained bythe replacement
of the butyl chain of PU3 (7) with either a pent-4-ynyl
(26) or a 2-isopropoxy-ethyl (38) chain. These modifi-
cations improve binding to hsp90 byalmost an order of
magnitude.
Effect of the nature and length of the bridge between the
purine and trimethoxyphenyl moieties on activity
None of the effected modifications at this position ren-
ders active compounds. This observation points to the
importance of the angle between the purine and tri-
methoxyphenyl moieties. It is possible that the replace-
ment of the bridge C with O or N changes the tilt of the
trimethoxyphenyl ring and therefore makes the inter-
action of this moietywith the top Lys of the pocket
unfavorable.
Effect of the substitution at position 2of the purine
moiety on activity
Addition of cyano, vinyl, iodo, methoxy, ethoxy or
amino-functionalityat the C2 position of the purine

G. Chiosis et al. / Bioorg. Med. Chem. 10 (2002) 3555–3564
3559
Table 2.
Table 3.
EC50 hsp90 IC50 MCF-7 IC50 Her2 M logP
EC50 hsp90
IC50 MCF-7
IC50 Her2
M logP
47
48
49
50
51
52
53
54
55
56
57
58
59
5.3
3.5
8.9
13.8
15
25
24
36
64
44
16
33
45
25
37
11
41
23
30
25
35
65
40
15
30
50
20
45
5
1.94
2.17
2.40
1.20
2.40
1.66
2.09
2.32
2.09
2.32
2.32
2.40
1.66
65
66
67
4.6
11.7
>100
19
25
30
25
50
>100
2.28
2.40
2.73
All values are in mM. See Experimental for assayprotocols. Standard
deviations for binding assays were ꢁ35% of the mean or less, while
for growth arrest and Her2 protein degradation of ꢁ20%. The values
are the mean of two experiments.
Table 4.
EC50 hsp90
IC50 MCF-7
IC50 Her2
M logP
71
72
0.45
0.52
2
5.4
2
3
2.54
1.88
All values are in mM. See Experimental for assayprotocols. Standard
deviations for binding assays were ꢁ35% of the mean or less, while
for growth arrest and Her2 protein degradation of ꢁ20%. The values
are the mean of two experiments.
1.3
9.8
18
Conclusions
A libraryof approximately70 derivatives based on our
designed compound PU3 and tailored to meet the ‘rule
of 5’ requirements was synthesized. This is the first
biased libraryof small molecule inhibitors of hsp90
described in literature. Our most potent compound to
date, 71, is 30-fold more active than PU3 and binds
hsp90 with affinities comparable to the natural product
17AAG. Addition of 71 to the breast cancer cell line
MCF-7 induces growth arrest and leads to the degra-
dation of the oncogenic Her2 tyrosine kinase at low
micromolar (IC50=2 mM) concentrations. The skeleton
is easilyamenable for derivatization and one would pre-
dict that sensitive further modifications would bring
activityin the nanomolar range. Additionally, the mol-
ecules are water soluble at the tested concentrations and
are amenable for oral administration. Such molecules
could have considerable advantages as drugs including
the fact that oral availabilitywould eliminate the incon-
veniences of prolonged intravenous administration.
3.5
10.4
0.7
9.2
6.8
40
20
All values are in mM. See experimental section for assayprotocols.
Standard deviations for binding assays were ꢁ35% of the mean or
less, while for growth arrest and Her2 protein degradation of ꢁ20%.
The values are the mean of two experiments.
Experimental
General
Effect of the sum of favorable substitutions on activity
All commercial chemicals and solvents are reagent
grade and were used without further purification unless
otherwise specified. All reactions except those in aqu-
eous media were carried out with the use of standard
techniques for the exclusion of moisture. The Argonaut
Quest 210 synthesizer was used for reactions performed
in parallel. Reactions were monitored bythin-laeyr
chromatographyon 0.25-mm silica gel plates and
visualized with UV light or cerium ammonium molyb-
date in aqueous H2SO4.
The favorable substitutions made on the 9-alkyl-8-ben-
zyl-9H-purin-6-ylamines are additive. Introduction of
the best alkyl chains at the position 9-N of purine, of
fluorine at position C2 of purine and of chlorine at
position 2 of the trimethoxyphenyl ring improves activ-
ityover PU3 bya factor of 30 (Table 4). These com-
pounds exhibit a potencyof binding to hsp90 equal to
the natural product derivative 17AAG35ꢀ38 that is now
in Phase I clinical trial. 71 has a molecular weight of
434, the sum of Ns and Os is eight, has one hydrogen
bond donor (NH2) and a calculated M logP32,33 value of
2.54. Based on these characteristics, it is likelythat 71
could have favorable pharmacokinetics as predicted by
Lipinski’s ‘rule of 5’.34
Final compounds were purified on an ISCO Combi-
Flash system and separation of the components was
monitored byUV at 254 nm. The identityand purityof
each product was characterized byMS, HPLC, TLC

3560
G. Chiosis et al. / Bioorg. Med. Chem. 10 (2002) 3555–3564
1
1
and H NMR. H NMR spectra were recorded on a
Bruker 400 MHz instrument. Splitting patterns are
designed as s, singlet; d, doublet; t, triplet; m, multiplet.
Low-resolution mass spectra (MS) were recorded in the
positive ion mode under electron-sprayionization (ESI).
2-Fluoro-9-propyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-
1
6-ylamine (47). Yield, 66%. Rf 0.38. H NMR (CDCl3,
400 MHz) d 6.41 (s, 2H), 6.01 (b s, 2H), 4.15 (s, 2H),
3.93 (t, J=7.6 Hz, 2H), 3.80 (s, 3H), 3.79 (s, 6H), 1.65–
1.60 (m, 2H), 0.85 (t, J=7.4 Hz, 3H); 13C NMR
(CDCl3, 100 MHz) 159.6, 157.7, 156.3, 156.1, 153.6,
152.9, 152.8, 151.0, 137.2, 131.0, 116.7, 105.4, 60.9, 56.2,
44.7, 34.9, 22.8, 11.1. MS (ESI) m/z 376.0 (M+1).
8-(3,4,5-Trimethoxy-benzyl)-9H-purine-2,6-diamine (3).
To trimethoxyphenyl acetic acid (1.0 g, 4.4 mmol) in
CH2Cl2 (20 mL) (under inert atmosphere) was added
cyanuric fluoride (371 mL, 4.4 mmol) and pyridine (356
mL, 4.4 mmol). The mixture was stirred for 1 h at room
temperature, after which, an additional 30 mL of
CH2Cl2 was added. The resulting solution was washed
once with water (5 mL), and the acid fluoride resulting
from the removal of the solvent was taken up in DMF
(10 mL) and used in the next step. Separately, 2,4,5,6-
tetraaminopyrimidine sulfate (0.9 g, 3.8 mmol) was dis-
solved in water (40 mL) that contained NaOH (456 mg,
11.4 mmol). The resulting solution was heated to 70 ꢂC
and the acid fluoride was added dropwise over a 20 min
period. The reaction mixture was stirred for 1.5 h at
70 ꢂC and then concentrated to dryness. To the crude
material was added MeOH (16 mL) and a 25% solution
of NaOMe in MeOH (16 mL) and the resulting solution
was heated at 90 ꢂC for 18 h. Following cooling, the pH
of the solution was adjusted to 7 byaddition of con-
centrated HCl. The aqueous solution was removed and
the crude taken up in DCM (100 mL) and MeOH (50
mL). The undissolved solids were filtered off and the
product (470 mg, 37%) was purified on a silica gel col-
umn with EtOAc/CH2Cl2/MeOH at 4:4:2. 1H NMR
(DMSO-d6, 400 MHz) 11.90 (b s, 1H), 6.62 (s, 2H), 6.49
(b s, 2H), 5.55 (b s, 2H), 3.91 (s, 2H), 3.73 (s, 6H), 3.55
(s, 3H). MS (ESI) m/z 330.9 (M+1). Rf (hexane/EtOAc/
CH2Cl2/MeOH at 10:5:5:2) 0.12.
2-Fluoro-9-butyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-
1
ylamine (48). Yield, 60%. Rf 0.38. H NMR (CDCl3,
400 MHz) d 6.41 (s, 2H), 5.96 (b s, 2H), 4.15 (s, 2H),
3.95 (t, J=7.7 Hz, 2H), 3.81 (s, 3H), 3.79 (s, 6H), 1.56–
1.52 (m, 2H), 1.29–1.23 (m, 2H), 0.85 (t, J=7.3 Hz, 3H);
13C NMR (CDCl3, 100 MHz) 159.8, 157.7, 156.3, 156.1,
153.6, 152.9, 152.8, 151.0, 137.3, 131.0, 116.7, 105.4,
60.9, 56.2, 43.1, 34.9, 31.5, 19.9, 13.6. MS (ESI) m/z
389.9 (M+1).
2-Fluoro-9-pentyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-
1
6-ylamine (49). Yield, 65% Rf 0.39. H NMR (CDCl3,
400 MHz) d 6.40 (s, 2H), 6.15 (b s, 2H), 4.15 (s, 2H),
3.95 (t, J=7.9 Hz, 2H), 3.80 (s, 3H), 3.79 (s, 6H), 1.59–
1.51 (m, 2H), 1.26–1.18 (m, 4H), 0.81 (t, J=7.1 Hz, 3H);
13C NMR (CDCl3, 100 MHz) 159.9, 157.7, 156.3, 156.1,
153.6, 152.9, 152.8, 150.9, 137.2, 131.0, 116.7, 105.4,
60.9, 56.1, 43.3, 34.9, 29.2, 28.8, 22.2, 15.3, 13.8. MS
(ESI) m/z 404.0 (M+1).
2-Fluoro-9-(2-methoxy-ethyl)-8-(3,4,5-trimethoxy-ben-
1
zyl)-9H-purin-6-ylamine (50). Yield, 28%. Rf 0.37. H
NMR (CDCl3, 400 MHz) d6.43 (s, 2H), 5.79 (b s, 2H),
4.25 (s, 2H), 4.16 (t, J=5.1 Hz, 2H), 3.81 (s, 3H), 3.80
(s, 6H), 3.58 (t, J=5.2 Hz, 2H), 3.26 (s, 3H). MS (ESI)
m/z 391.9 (M+1).
2-Fluoro-8-(3,4,5-trimethoxy-benzyl)-9H-purin-6-ylamine
(FAC). To 3 (500 mg, 1.5 mmol) was slowlyadded a
70% solution of HF in pyridine (2 mL), pyridine (8
mL), followed by t-butyl nitrite (200 mL, 2.0 mmol). The
mixture was stirred for 2 h and then quenched overnight
with 8 g CaCO3 in water (15 mL) and MeOH (10 mL).
The solution was concentrated to dryness and the
resulting crude was taken up in MeOH (30 mL) and
CH2Cl2 (10 mL). The insoluble solids were filtered off
and the solvent was removed to give the crude product.
This was purified on a silica gel column eluting with hex-
ane/DCM/EtOAc/MeOH at 10:5:5:0.75 (240 mg, 50%).
1H NMR (DMSO-d6, 400MHz) d 7.41 (b s, 2H), 6.46 (s,
2H), 3.86 (s, 2H), 3.57 (s, 6H), 3.23 (s, 3H). MS (ESI) m/ z
333.9 (M+1). Rf (hexane/EtOAc/CH2Cl2/MeOH at
10:5:5:2) 0.40.
2-Fluoro-9-[(S)-2-methyl-butyl]-8-(3,4,5-trimethoxy-ben-
1
zyl)-9H-purin-6-ylamine (51). Yield, 68%. Rf 0.41. H
NMR (CDCl3, 400 MHz) d 6.38 (s, 2H), 6.23 (b s, 2H),
4.15 (s, 2H), 3.87–3.73 (m, 2H), 3.81 (s, 3H), 3.79 (s,
6H), 1.95–1.85 (m, 1H), 1.29–1.13 (m, 2H), 0.85 (t,
J=7.3 Hz, 3H), 0.78 (d, J=6.7 Hz, 3H); 13C NMR
(CDCl3, 100 MHz) 159.9, 157.7, 156.4, 156.2, 153.7,
153.5, 153.4, 151.3, 137.2, 131.0, 116.7, 105.4, 60.9,
56.1, 48.9, 35.0, 26.8, 16.6, 11.2. MS (ESI) m/z 404.0
(M+1).
2-Fluoro-9-(2-isopropoxy-ethyl)-8-(3,4,5-trimethoxy-ben-
1
zyl)-9H-purin-6-ylamine (52). Yield, 86%. Rf 0.42. H
NMR (CDCl3, 400 MHz) cdd 6.43 (s, 2H), 5.91 (b s,
2H), 4.29 (s, 2H), 4.14 (t, J=5.2 Hz, 2H), 3.81 (s, 3H),
3.80 (s, 6H), 3.61 (t, J=5.2 Hz, 2H), 3.48–3.42 (m, 1H),
1.06 (d, J=6.1 Hz, 6H). MS (ESI) m/z 420.0 (M+1).
General method for the 9-N-alkylation of purines
2-Fluoro-9-but-3-enyl-8-(3,4,5-trimethoxy-benzyl)-9H-
purin-6-ylamine (53). Yield, 42%. Rf 0.40. 1H NMR
(CDCl3, 400 MHz) d 6.41 (s, 2H), 5.86 (b s, 2H), 5.69–
5.62 (m, 1H), 5.00 (d, J=9.6 Hz, 1H), 4.91 (dd, J=9.6,
1.6 Hz, 1H), 4.16 (s, 2H), 4.04 (t, J=7.4 Hz, 2H), 3.81
(s, 3H), 3.79 (s, 6H), 2.33–2.27 (m, 2H); 13C NMR
(CDCl3, 100 MHz) d 159.6, 157.5, 156.3, 156.1, 153.9,
152.9, 152.8, 151.0, 137.3, 133.7, 131.3, 118.5, 105.8,
61.2, 56.5, 35.4, 33.8. MS (ESI) m/z 388.1 (M+1).
The alkylation was performed via a Mitsunobu reaction
as described before.25 Essentially, to FAC in toluene/
CH2Cl2 at 5:1 was added 1.3 equiv of the corresponding
alcohol, 2 equiv PPh3, 3 equiv di-tert-butyl azodi-
carboxylate and the resulting solution was stirred at
room temperature for 10 min to 1 h (conversion was
monitored byTLC with hexane/EtOAc/CH Cl2/MeOH
2
at 10:5:5:1) to give:

G. Chiosis et al. / Bioorg. Med. Chem. 10 (2002) 3555–3564
3561
2-Fluoro-9-pent-4-enyl-8-(3,4,5-trimethoxy-benzyl)-9H-
purin-6-ylamine (54). Yield, 86%. Rf 0.41. 1H NMR
(CDCl3, 400 MHz) d 6.41 (s, 2H), 5.85 (b s, 2H), 5.73–
5.63 (m, 1H), 5.00 (d, J=3.1 Hz, 1H), 4.96 (s, 1H), 4.29
(s, 2H), 3.96 (t, J=7.7 Hz, 2H), 3.81 (s, 3H), 3.80 (s,
6H), 2.04–1.99 (m, 2H), 1.67 (m, 2H). MS (ESI) m/z
402.1 (M+1).
2-Ethoxy-9-butyl-8-(3,4,5-trimethoxy-benzyl)-9H-purin-
6-ylamine (61). To a solution of 48 (20 mg, 0.051
mmol) in EtOH (2 mL) was added NaOMe (15 mg, 0.28
mmol) and the mixture was refluxed for 2 h. After
cooling and neutralization with HCl, the solution was
concentrated to dryness. The product (12 mg, 56%) was
1
purified as described above. Rf 0.26. H NMR (CDCl3,
400 MHz) d 6.41 (s, 2H), 6.15 (b s, 2H), 4.40–4.35 (m,
2H), 4.15 (s, 2H), 3.94 (t, J=7.5 Hz, 2H), 3.80 (s, 3H),
3.79 (s, 6H), 1.56–1.52 (m, 2H), 1.39 (t, J=7.1 Hz, 3H),
1.27–1.22 (m, 2H), 0.85 (t, J=7.3 Hz, 3H). MS (ESI)
m/z 415.8 (M+1).
2-Fluoro-9-but-3-ynyl-8-(3,4,5-trimethoxy-benzyl)-9H-
purin-6-ylamine (55). Yield, 36%. Rf 0.39. 1H NMR
(CDCl3, 400 MHz) d 6.43 (s, 2H), 6.02 (b s, 2H), 4.25 (s,
2H), 4.15 (t, J=7.0 Hz, 2H), 3.81 (s, 3H), 3.80 (s, 6H),
2.53 (dd, J=6.9, 2.6 Hz, 2H), 2.00 (t, J=2.6 Hz, 1H);
13C NMR (CDCl3, 100 MHz) d 159.9, 157.7, 156.3,
156.1, 153.7, 152.9, 152.8, 151.3, 137.3, 130.8, 116.7,
105.5, 79.8, 71.2, 60.9, 56.2, 41.6, 34.9, 19.3. MS (ESI)
m/z 386.1 (M+1).
2-Iodo-9-pent-4-ynyl-8-(3,4,5-trimethoxy-benzyl)-9H-
purin-6-ylamine (62). To 3 (200 mg, 0.61 mmol) in tolu-
ene (20 mL) and CH2Cl2 (4 mL) was added PPh3 (330
mg, 1.3 mmol), 4-pentyne-1-ol (75 mL, 0.8 mmol) and
di-t-butyl azodicarboxylate (600 mg, 2.5 mmol). The
mixture was stirred at room temperature for 2 h. The
product was isolated bycolumn chromatographyon a
silica gel column eluting with hexane/CHCl3/EtOAc/
EtOH at 10:8:4:4 (120 mg solid, 49%). 1H NMR
(CDCl3, 400 MHz) d 6.43 (s, 2H), 5.41 (b s, 2H), 4.69
(b s, 2H), 4.15 (s, 2H), 4.11 (t, J=7.2 Hz, 2H), 3.81 (s,
3H), 3.80 (s, 6H), 2.18–2.14 (m, 2H), 2.00 (t, J=2.5
Hz, 1H), 1.88–1.81 (m, 2H). MS (ESI) m/z 396.6
(M+1). To this (50 mg, 0.13 mmol) was added CH2I2
(2.5 mL) and isoamyl nitrite (100 mL, 0.78 mmol) and
the resulting solution was heated at 80 ꢂC for 1 h. After
cooling, the solution was concentrated and then added
to a silica gel column. The product was isolated (40
mg, 61%) using eluent hexane:CHCl3:EtOAc:EtOH at
2-Fluoro-9-pent-3-ynyl-8-(3,4,5-trimethoxy-benzyl)-9H-
purin-6-ylamine (56). Yield, 25%. Rf 0.41. 1H NMR
(CDCl3, 400 MHz) d 6.44 (s, 2H), 5.79 (b s, 2H), 4.25 (s,
2H), 4.10 (t, J=7.0 Hz, 2H), 3.81 (s, 9H), 2.49–2.45 (m,
2H), 1.71 (s, 3H). MS (ESI) m/z 400.0 (M+1).
2-Fluoro-9-pent-4-ynyl-8-(3,4,5-trimethoxy-benzyl)-9H-
purin-6-ylamine (57). Yield, 76%. Rf 0.41. 1H NMR
(CDCl3, 400 MHz) d 6.45 (s, 2H), 6.00 (b s, 2H), 4.20 (s,
2H), 4.11 (t, J=7.2 Hz, 2H), 3.81 (s, 3H), 3.80 (s, 6H),
2.21–2.17 (m, 2H), 2.01 (t, J=2.5 Hz, 1H), 1.92–1.85
(m, 2H); 13C NMR (CDCl3, 100 MHz) d 159.9, 157.7,
156.2, 156.0, 153.8, 152.9, 152.8, 151.3, 137.2, 130.7,
116.4, 105.2, 82.4, 69.4, 60.6, 55.9, 41.7, 34.4, 27.5, 15.4.
MS (ESI) m/z 400.1 (M+1).
1
10:4:4:0.75. H NMR (CDCl3, 400 MHz) 6.42 (s, 2H),
6.07 (b s, 2H), 4.21 (s, 2H), 4.11 (t, J=7.3 Hz, 2H),
3.80 (s, 3H), 3.79 (s, 6H), 2.21–2.16 (m, 2H), 2.01 (t,
J=2.5 Hz, 1H), 1.92–1.85 (m, 2H). MS (ESI) m/z 508.0
(M+1). Rf (hexane/EtOAc/CH2Cl2/MeOH at 10:5:5:1)
0.42.
2-Fluoro-9-cyclobutylmethyl-8-(3,4,5-trimethoxy-benzyl)-
1
9H-purin-6-ylamine (58). Yield, 33%. Rf 0.42. H NMR
(CDCl3, 400 MHz) d 6.40 (s, 2H), 5.82 (b s, 2H), 4.15 (s,
2H), 3.99 (d, J=4.4 Hz, 2H), 3.81 (s, 3H), 3.79 (s, 6H),
2.71–2.67 (m, 1H), 1.94–1.76 (s, 6H). MS (ESI) m/z
402.1 (M+1).
2-Cyano-9-pent-4-ynyl-8-(3,4,5-trimethoxy-benzyl)-9H-
purin-6-ylamine (63). A solution of 62 (20 mg, 0.04
mmol), Pd(PPh3)4 (7 mg, 6.3ꢃ10ꢀ3 mmol) and tribu-
tyltin cyanide (14 mg, 0.043 mmol) in dry DMF (2.5
mL) was heated at 180 ꢂC for 6 h. Following cooling
and removal of the solvent, the product (13 mg, 81%)
was isolated bycolumn chromatography(hexane/
2-Fluoro-9-(tetrahydrofuran-2-ylmethyl)-8-(3,4,5-trime-
thoxy-benzyl)-9H-purin-6-ylamine (59). Yield, 53%. Rf
0.36. 1H NMR (CDCl3, 400 MHz) d 6.42 (s, 2H), 6.02 (b s,
2H), 4.34–4.23 (m, 2H), 4.19–4.13 (m, 2H), 3.99–3.95 (m,
1H), 3.86–3.79 (m, 10H), 3.74–3.70 (m, 1H), 2.04–2.00 (m,
1H), 1.86–1.76 (m, 2H), 1.61–1.58 (m, 1H); 13C NMR
(CDCl3, 100 MHz) d 159.8, 157.7, 156.3, 156.1, 153.5,
152.9, 152.8, 151.0, 137.3, 131.4, 116.7, 105.7, 68.3, 60.8,
56.2, 46.7, 34.6, 28.9, 25.7. MS (ESI) m/z 417.9 (M+1).
1
CHCl3/EtOAc/EtOH at 10:8:4:0.75). H NMR (CDCl3,
400 MHz) 6.46 (s, 2H), 5.90 (b s, 2H), 4.25 (s, 2H), 4.20
(t, J=7.4 Hz, 2H), 3.81 (s, 3H), 3.80 (s, 6H), 2.24–2.20
(m, 2H), 2.01 (t, J=2.5 Hz, 1H), 1.92–1.85 (m, 2H). MS
(ESI) m/z 407.2 (M+1). Rf (hexane/EtOAc/CH2Cl2/
MeOH at 10:5:5:1) 0.39.
2-Methoxy-9-butyl-8-(3,4,5-trimethoxy-benzyl)-9H-
purin-6-ylamine (60). To a solution of 48 (20 mg, 0.051
mmol) in MeOH (1 mL) was added a 25% solution of
NaOMe in MeOH (1.5 mL). The resulting mixture was
heated at 85ꢂC for 2 h. Subsequent to cooling, the mixture
was neutralized with 4 N HCl and then concentrated to
dryness. The crude was purified on a silica gel column with
hexane/EtOAc/DCM/MeOH at 10:5:5:1 to give a solid (11
mg, 51%). Rf 0.25. 1H NMR (CDCl3, 400MHz) d 6.40 (s,
2H), 5.55 (b s, 2H), 4.11 (s, 2H), 3.92–3.90 (m, 5H), 3.80 (s,
3H), 3.78 (s, 6H), 1.58–1.51 (m, 2H), 1.27–1.19 (m, 2H),
0.85 (t, J=7.2 Hz, 3H). MS (ESI) m/z 402.0 (M+1).
9-Pent-4-ynyl-8-(3,4,5-trimethyl-benzyl)-2-vinyl-9H-
purin-6-ylamine (64). A solution of 62 (20 mg, 0.04
mmol), (MeCN)2PdCl2 (0.5 mg, 2ꢃ10ꢀ3 mmol) and
tributylvinyltin (12.5 mL, 0.041 mmol) in dryDMF (3
mL) was heated at 100 ꢂC for 1 h. Following cooling
and removal of the solvent, the product (15 mg, 92%)
was isolated bycolumn chromatography(hexane/
CHCl3/EtOAc/EtOH at 10:8:4:1). 1H NMR (CDCl3,
400 MHz) d 6.73 (dd, J=17.2, 10.5 Hz, 1H), 6.47 (dd,
J=17.2, 1.9 Hz, 1H), 5.56 (dd, J=10.5, 1.9 Hz, 1H),

3562
G. Chiosis et al. / Bioorg. Med. Chem. 10 (2002) 3555–3564
5.42 (b s, 2H), 4.23 (s, 2H), 4.16 (t, J=7.2 Hz, 2H), 3.81
(s, 3H), 3.79 (s, 6H), 2.21–2.17 (m, 2H), 2.02 (t, J=2.5
Hz, 1H), 1.96–1.89 (m, 2H). MS (ESI) m/z 401.8
(M+1). Rf (hexane/EtOAc/CH2Cl2/MeOH at 10:5:5:1)
=0.39.
9-Butyl-8-(3,4,5-trimethoxy-phenoxy)-9H-purin-6-yla-
mine (69). A mixture of trimethoxyphenol (75 mg, 0.4
mmol), t-BuOK (34 mg, 0.3 mmol), Cu powder (10 mg,
0.64 mmol) and 8-bromo-9-N-butyladenine (26 mg, 0.1
mmol) was heated for 1 h at 140 ꢂC. The product (13 mg
solid, 35%) was isolated bycolumn chromatography
1
9-Butyl-8-(2-chloro-3,4,5-trimethoxy-benzyl)-9H-purin-6-
ylamine (65). To 7 (37 mg, 0.1 mmol) in MeOH (3 mL)
was added concentrated HCl (33 mL, 0.4 mmol). The
solution was cooled to 0 ꢂC and a 90% aqueous solution
of t-butyl hydroperoxide (44 mL, 0.4 mmol) was added.
The resulting solution was refluxed overnight. Follow-
ing cooling and removal of the solvent, the product (31
mg solid, 72%) was isolated bycolumn chromato-
graphy(elute with hexane/EtOAc/CH 2Cl2/MeOH at
10:5:5:1.5). 1H NMR (CDCl3, 400 MHz) d 8.32 (s,
1H), 6.50 (s, 1H), 5.99 (b s, 2H), 4.31 (s, 2H), 4.03
(t, J=7.5 Hz, 2H), 3.90 (s, 3H), 3.84 (s, 3H), 3.67 (s,
3H), 1.62–1.54 (m, 2H), 1.33–1.23 (m, 2H), 0.85 (t,
J=7.3 Hz, 3H); 13C NMR (CDCl3, 100 MHz) d
152.3, 150.1, 142.5, 128.9, 119.5, 108.4, 61.1, 56.1,
42.9, 31.8, 31.4, 19.9, 13.6. MS (ESI) m/z 406.0
(M+1). Rf (hexane/EtOAc/CH2Cl2/MeOH at 10:5:5:1)
0.41.
(hexane/CH2Cl2/EtOAc/MeOH at 10:5:5:1). H NMR
(CDCl3, 400 MHz) d 8.29 (s, 1H), 6.58 (s, 2H), 5.25 (b s,
2H), 4.16 (t, J=7.3 Hz, 2H), 3.85 (s, 6H), 3.84 (s, 3H),
1.90–1.82 (m, 2H), 1.46–1.37 (m, 2H), 0.95 (t, J=7.4
Hz, 3H). MS (ESI) m/z 374.1 (M+1).
9-Butyl-8-(3,4,5-trimethoxy-benzyloxy)-9H-purin-6-yla-
mine (70). A solution of benzyl alcohol (250 mL, 2.5
mmol) in 25% NaOMe in MeOH (50 mL) was stirred
for 5 min. Following the removal of methanol,
8-bromo-9-N-butyladenine (26 mg, 0.096 mmol) and Cu
powder (10 mg, 0.16 mmol) was added, and the mixture
was heated for 2 min at 180 ꢂC. The product was puri-
fied on silica gel column with hexane/CH2Cl2/EtOAc/
MeOH at 10:5:5:1 (5.5 mg,14%). 1H NMR (CDCl3,
400 MHz) d 8.25 (s, 1H), 6.69 (s, 2H), 5.45 (s, 2H), 5.36
(b s, 2H), 4.03 (t, J=7.3 Hz, 2H), 3.86 (s, 6H), 3.85 (s,
3H), 1.79–1.72 (m, 2H), 1.41–1.23 (m, 2H), 0.98 (t,
J=7.4 Hz, 3H). MS (ESI) m/z 388.3 (M+1).
9-Butyl-8-(2-bromo-3,4,5-trimethoxy-benzyl)-9H-purin-
6-ylamine (66) and 9-butyl-8-(2,6-dibromo-3,4,5-trime-
thoxy-benzyl)-9H-purin-6-ylamine (67). To 7 (37 mg, 0.1
mmol) in MeOH (3 mL) was added a 48% aqueous
solution of HBr (45 mL, 0.4 mmol). The mixture was
cooled to 0 ꢂC, and t-butyl hydroperoxide (44 mL, 0.4
mmol) was added dropwise. The resulting solution was
stirred for 30 min at 0 ꢂC and then refluxed for an
additional h. Following cooling and removal of the sol-
vent, the mixture was separated bycolumn chromato-
graphy(elute with hexane/EtOAC/CH 2Cl2/MeOH at
10:5:5:1.5) to give predominantlymonobrominated
product (31 mg, 69%) and a trace of dibrominated
product (2.3 mg, 4.4%). 66: 1H NMR (CDCl3,
400 MHz) d 8.31 (s, 1H), 6.49 (s, 1H), 5.87 (b s, 2H),
4.35 (s, 2H), 4.03 (t, J=7.3 Hz, 2H), 3.89 (s, 3H), 3.84
(s, 3H), 3.67 (s, 3H), 1.62–1.54 (m, 2H), 1.34–1.26 (m,
2H), 0.85 (t, J=7.4 Hz, 3H). MS (ESI) m/z 452.0
(M+1). Rf (hexane/EtOAc/CH2Cl2/MeOH at 10:5:5:1)
8-(2-Chloro-3,4,5-trimethoxy-benzyl)-9H-purin-2,6-dia-
mine (73). To a slurryof 3 (1 g, 3.0 mmol) in MeOH
(50 mL) was added concentrated HCl (1.5 mL, 18
mmol). The resulting solution was cooled to 0 ꢂC and a
70% aqueous solution of t-butyl hydroperoxide (2. 5
mL, 8 mmol) was slowlyadded. The mixture was stirred
for 30 min at 0 ꢂC and then refluxed for 20 h. The sol-
vent was removed under high vacuum to give clean
1
product (1.09 g, 98%). H NMR (DMSO-d6, 400 MHz)
d 8.62 (b s, 2H), 8.36 (b s, 2H), 4.14 (s, 2H), 3.73 (s,
3H), 3.71 (s, 3H), 3.68 (s, 3H). MS (ESI) m/z 364.8
(M+1).
8-(2-Chloro-3,4,5-trimethoxy-benzyl)-2-fluoro-9H-purin-
6-ylamine (74). To a 70% solution of HF in pyridine
(6.5 mL) was added (under inert atmosphere) pyridine
(13.5 mL), followed by 73 (1 g, 2.7 mmol). The mixture
was stirred for 5 min and consequently, t-butyl nitrite
(450 mL, 3.5 mmol) was slowlyadded. Stirring con-
tinued for another 30 min. The reaction was quenched
byaddition of CaCO 3 (16.5 g) in water (10 mL): MeOH
(10 mL) and stirring for 2 h. The solution was con-
centrated and the resulting slurrywas taken up in
CH2Cl2 (50 mL)/MeOH (50 mL). The insoluble solids
were filtered off and washed with CH2Cl2/MeOH at 1:1
(2ꢃ25 mL). Following solvent removal, the product was
purified on a silica gel column eluting with hex-
ane:EtOAc/CH2Cl2/MeOH at 10:5:5:1 (370 mg solid,
1
0.42. 67: H NMR (CDCl3, 400 MHz) d 8.28 (s, 1H),
4.61 (s, 2H), 4.35 (t, J=7.4 Hz, 2H), 3.99 (s, 3H), 3.96
(s, 6H), 1.96–1.84 (m, 2H), 1.53–1.46 (m, 2H), 1.05 (t,
J=7.4 Hz, 3H). MS (ESI) m/z 529.9 (M+1). Rf (hex-
ane/EtOAc/CH2Cl2/MeOH at 10:5:5:1) 0.44.
9-Butyl-N*8*-(3,4,5-trimethoxy-phenyl)-9H-purine-6,8-
diamine (68). A mixture of 8-bromo-9-N-butyladenine
(50 mg, 0.186 mmol) and trimethoxyaniline (120 mg,
0.65 mmol) was heated at 160 ꢂC for 30 min. Following
cooling, the solid was taken up in CH2Cl2 (20 mL) and
MeOH (5 mL) and anyinsoluble solids were filtered off.
The product (57 mg solid, 83%) was purified on a silica
gel column eluting with CH2Cl2/EtOAc/MeOH at 7:4:1.
1H NMR (CDCl3, 400 MHz) d 8.20 (s, 1H), 6.82 (s, 2H),
6.48 (b s, 1H), 5.42 (b s, 2H), 4.06 (t, J=7.3 Hz, 2H),
3.83 (s, 6H), 3.80 (s, 3H), 1.80–1.73 (m, 2H), 1.40–1.30
(m, 2H), 0.92 (t, J=7.4 Hz, 3H). MS (ESI) m/z 373.3
(M+1).
1
37%). H NMR (DMSO-d6, 400 MHz) d 12.9 (s, 1H),
7.61 (b s, 2H), 7.03 (s, 1H), 4.23 (s, 2H), 3.85 (s, 6H),
3.82 (s, 3H). MS (ESI) m/z 367.7 (M+1).
2-Fluoro-9-pent-4-ynyl-8-(2-chloro-3,4,5-trimethoxy-ben-
1
zyl)-9H-purin-6-ylamine (71). (308 mg, 76%). H NMR
(CDCl3, 400 MHz) d 6.46 (s, 1H), 6.17 (b s, 2H), 4.31 (s,
2H), 4.12 (t, J=7.3 Hz, 2H), 3.91 (s, 3H), 3.86 (s, 3H),
3.72 (s, 3H), 2.19 (dd, J=6.65, 2.5 Hz, 2H), 1.97 (t,

G. Chiosis et al. / Bioorg. Med. Chem. 10 (2002) 3555–3564
3563
J=2.5 Hz, 1H), 1.94–1.87 (m, 2H); 13C NMR (CDCl3,
100 MHz) d 159.8, 157.7, 156.2, 152.8, 152.4, 150.5,
150.2, 142.6, 128.7, 119.7, 116.8, 108.5, 82.2, 69.8, 61.1,
56.2, 42.1, 31.5, 28.1, 15.7. MS (ESI) m/z 434.1 (M+1).
Rf (hexane/EtOAc/CH2Cl2/MeOH at 10:5:5:1) 0.42.
staining with 0.4% sulforhodamine B in 1% acetic acid.
The IC50 is calculated as the drug concentration that
inhibits cell growth by50% compared with control
growth.
Binding studies: solid-phase competition assays. GM was
immobilized on Affigel 10 resin (BioRad) as descri-
bed.15,31 The GM-beads were washed with TEN buffer
(50 mM Tris–HCl pH 7.4, 1 mM EDTA, 1% NP-40)
containing protease inhibitors and then blocked for 45
min at 4 ꢂC with 0.5% BSA in TEN buffer. Hsp90 pro-
tein from Stressgen (SPP-770) was incubated with or
without drugs for 17 min on ice. To each sample were
added 20 mL GM-beads and the mixtures were rotated
at 4 ꢂC for 1 h followed bythree washes with 500 mL ice
cold TEN each. The GM-beads bound protein was
eluted from the solid phase byheating in 65 mL 1ꢃSDS.
A 40 mL aliquot was applied to the SDS/PAGE gel and
visualized byimmunoblotting with anti-hsp90 (Neo-
Markers#RB-118) and the corresponding HRP-linked
secondaryantibod.y Blots were visualized byauto-
radiographyand the protein quantified using BioRad
Gel Doc 1000 software. The EC50 was calculated as the
drug concentration needed to compete with the immo-
bilized GM for 50% of the hsp90 protein.
2-Fluoro-9-(2-isopropoxy-ethyl)-8-(2-chloro-3,4,5-trime-
thoxy-benzyl)-9H-purin-6-ylamine (72). 1H NMR
(CDCl3, 400 MHz) d 6.49 (s, 2H), 6.02 (b s, 2H), 4.40 (s,
2H), 4.22 (t, J=5.2 Hz, 2H), 3.88 (s, 3H), 3.85 (s, 6H),
3.67 (t, J=5.2 Hz, 2H), 3.53–3.45 (m, 1H), 1.06 (d,
J=6.1 Hz, 6H); 13C NMR (100 MHz, CDCl3) d 159.7,
157.7, 156.1, 152.7, 152.4, 150.5, 150.2, 142.6, 128.5,
119.7, 108.5, 82.2, 72.5, 66.6, 61.1, 56.4, 43.6, 34.9, 34.3,
22.2. MS (ESI) m/z 454.2 (M+1). Rf (hexane/EtOAc/
CH2Cl2/MeOH at 10:5:5:1) 0.43.
Cell culture. The human cancer cell line MCF-7 was
obtained from the American Type Culture Collection
(Manassas, VA, USA) and maintained in 1:1 mixture of
DME:F12 supplemented with 2 mM glutamine, 50 u/
mL penicillin, 50 u/mL streptomycin and 5% heat
inactivated fetal bovine serum (Gemini Bioproducts)
and incubated at 37 ꢂC in 5% CO2.
Protein assays. Cells were grown to 60–70% confluence
and exposed to drugs or DMSO vehicle for the indi-
cated time periods. Lysates were prepared using 50 mM
Tris pH 7.4, 2% SDS and 10% glycerol lysis buffer.
Protein concentration was determined using the BCA
kit (Pierce Chemical Co.), according to the manu-
facturers instructions. Clarified protein lysates (20–50
mg) were electrophoreticallyresolved on denaturing
SDS-PAGE, transferred to nitrocellulose and probed
with the anti-Her2 (C-18) primaryantibody(Santa Cruz
Biotechnology) and the corresponding HRP-linked sec-
ondaryantibod.y Blots were visualized byauto-
radiographyand the protein quantified using BioRad
Gel Doc 1000 software. The IC50 was calculated as the
drug concentration needed to degrade 50% of the total
Her2 protein.
M logP values were calculated using the algorithm
implemented in Sybyl 6.7.
Acknowledgements
This work was supported bythe Susan G. Komen
Foundation (G.C.), NIH/NCI (1U01 CA91178-01)
(G.C. and N.R.) and a generous donation bythe Taub
Foundation.
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