Fluorine- and rhenium-containing geldanamycin derivatives as leads for the development of molecular probes for imaging Hsp90

Article abstract of DOI:10.1039/c2ob25744k

Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone responsible for protein quality control in cells. Hsp90 has been shown to be overexpressed in many human cancers. This has prompted extensive research on Hsp90 inhibitors as novel antic

Full text of DOI:10.1039/c2ob25744k

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PAPER
Fluorine- and rhenium-containing geldanamycin derivatives as leads for the
development of molecular probes for imaging Hsp90†
Frank Wuest,*a Vincent Bouvet,a BaoChan Maib and Paul LaPointeb
Received 18th April 2012, Accepted 6th July 2012
DOI: 10.1039/c2ob25744k
Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone responsible for protein quality
control in cells. Hsp90 has been shown to be overexpressed in many human cancers. This has prompted
extensive research on Hsp90 inhibitors as novel anticancer agents and, more recently, the development of
molecular probes for imaging Hsp90 expression in vivo. This work describes the development of various
fluorine-containing and rhenium-containing geldanamycin derivatives as leads for the development of
corresponding 18F-labeled and 99mTc-labeled PET and SPECT probes for molecular imaging of Hsp90
expression. All compounds were evaluated in an in vitro ATPase activity assay using Hsp90 isoform
Hsp82p. Fluorobenzoylated geldanamycin derivative 5 displayed comparable inhibitory potency like
parent compound geldanamycin.
proliferation, survival and immortalization, invasion and meta-
Introduction
stasis, and angiogenesis.1d,e,2a,3 Therefore, combinatorial inhi-
Heat shock protein 90 (Hsp90) is an ATP-dependent molecular
chaperone which belongs to the ATPase/kinase protein superfam-
ily. Hsp90 contains a Bergerat ATP-binding fold responsible for
the regulation of signalling protein function and protein quality
control, including protein folding and protein degradation.1 Over
the last 15 years, Hsp90 has gained considerable attention as a
highly promising anticancer drug target. Hsp90 is largely in a
latent state in normal cells but becomes overexpressed in cancer
cells.2 Hsp90 plays a central role in the conformational matu-
ration, the controlling of function, trafficking and turnover of
many oncogenic proteins also referred to as client proteins,
which are involved in the genesis and progression of cancer.
Hsp90 influences the activity and stability of oncogenic client
proteins that function as key regulators in cellular growth, differ-
entiation, and apoptotic pathways. Such important oncogenic
client proteins include p53, Raf-1, ErbB2, Akt, Bcr-Abl,
MEK-1, HIF-1α and cdk4. More than 100 of the known Hsp90
client proteins regulate multiple signal transduction pathways
that are aberrantly activated in many human cancers and
connected to the hallmarks of cancer like cell signalling,
bition of several oncogenic pathways in cancer cells should give
a powerful anticancer effect and limit the development of drug
resistance. The potential value of Hsp90 as an anticancer target
is further enhanced by the fundamental structural difference of
the Hsp90 protein complex in cancer cells compared to normal
cells. In cancer cells, Hsp90 is present in an activated multi-
chaperone complex which binds significantly more tightly to
Hsp90 inhibitors than Hsp90 in normal cells.4 Moreover, Hsp90
is constitutively expressed at 2- to 10-fold higher levels in
cancer cells compared to normal cells.2 Another factor in the
cancer selectivity of Hsp90 inhibitors is the stress
response. Malignant cells are likely to become dependent on
Hsp90 due to the stressful conditions present in tumors like
deregulated oncogenic signaling, hypoxia, acidosis, and nutrient
deprivation.4b
Considerable progress has been made in understanding the
three-dimensional molecular structure of Hsp90, which led to
insights of the mechanism of Hsp90 action provided by X-ray
crystallography and electron microscopy.5
The recognition of Hsp90 as an important drug target for the
development of anticancer drugs has led to the design, synthesis
and evaluation of numerous Hsp90 inhibitors. Several very good
review articles summarize the structural diversity of Hsp90
inhibitors.6
aDepartment of Oncology, Faculty of Medicine and Dentistry, University
of Alberta, 11560 University Ave, Edmonton AB T6G 1Z2, Canada.
E-mail: wuest@ualberta.ca; Fax: +1 780 432 8483;
Tel: +1 780 989 8150
Inhibitors of the ATP-pocket of Hsp90 can be classified into
natural product-based compounds like geldanamycin (GA),
17-AAG, 17-DMAG and radicicol, and synthetic drugs based on
purines (PUH71), pyrazoles (VER-49009), and isoxazoles
(NVP-AUY922). The structures of prominent Hsp90 inhibitors
are given in Fig. 1.
bDepartment of Cell Biology, Faculty of Medicine and Dentistry,
University of Alberta, Edmonton AB T6G 2H7, Canada.
E-mail: pglapoin@ualberta.ca; Fax: +1 780 492 0450;
Tel: +1 780 492 1804
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob25744k
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diagnosis, monitoring of disease progression, and evaluation of
therapeutic interventions. Only a few reports have been pub-
lished in the literature dealing with the synthesis and evaluation
of radiolabeled Hsp90 inhibitors. First reports describe labeling
of GA and GA-derivatives with radioiodine isotopes I-131 and
I-125.12 Another series of reports deals with the radiolabeling
and radiopharmacological evaluation of radioiodinated purine
derivatives [124I]PUH71, [124I]PUDZ13, and [124I]PUDZ13.13
The short-lived positron emitter fluorine-18 (18F, t1/2
=
109.8 min) and the gamma emitter technetium-99m (99mTc, t1/2
= 6.0 h) are among the most widely used radioisotopes for the
preparation of molecular probes for PET-imaging (18F) and
SPECT-imaging (99mTc). Fluorine-18 chemistry is mostly based
on organic chemistry based on no-carrier-added (n.c.a.) [18F]-
fluoride,14 whereas radiochemistry with radiometal technetium-
99m exploits various coordination chemistry tools for the for-
mation of 99mTc-based radiotracers.15 This work describes the
design, synthesis and evaluation of a series of fluorine- and
rhenium-containing geldanamycin derivatives as leads for the
development of 18F-labeled and 99mTc-labeled geldanamycin
derivatives as PET and SPECT probes, respectively, for molecu-
lar imaging of Hsp90 expression.
Fig. 1 Structures of prominent Hsp90 inhibitors.
About 10 Hsp90 inhibitors, both natural product-based and
synthetic drugs, have reached advanced clinical trials.7
Geldanamycin (GA), a naturally occurring benzoquinone
ansamycin, was among the first Hsp90 inhibitors to study Hsp90
function. GA was originally isolated from the broth of Strepto-
myces hygroscopicus in the 1970s and characterized as an anti-
biotic. The antitumor activity of GA was reported in the early
1990s. GA binds to the N-terminal ATP-binding site of Hsp90
locking the protein into the “intermediate complex” which
results in the ubiquitin-mediated proteolytic degradation of client
proteins.3,8 However, early clinical studies revealed some un-
desirable properties of GA as an anti-cancer agent such as hepato-
toxicity, cellular instability, and poor solubility.
Results and discussion
Synthesis of fluorine- and rhenium-containing GA derivatives
The design of fluorine- and rhenium-containing GA derivatives
was based on three different synthesis routes taking into account
the special radiochemistry requirements involving n.c.a. [18F]
fluoride and 99mTc, and organic chemistry of benzoquinone GA.
GA is a member of the ansamycin natural product family which
displays a methoxy quinone moiety and the ansa ring as key
structural motifs. Chemistry of GA is especially controlled by
the methoxy quinone motif. The methoxy quinone moiety in GA
undergoes rapid Michael addition with primary amines followed
by an elimination reaction to furnish the corresponding vinylo-
gous amine products. Thus, the principal design and synthesis of
fluorine- and rhenium-containing GA derivatives as target com-
pounds was based on the treatment of GA with various primary
amines to give (1) respective fluorine-containing GA derivatives
directly as the first synthesis route or a primary amine group-con-
taining GA intermediate, which was further converted into the
final (2) fluorine- and (3) rhenium-containing compounds using
bioconjugation chemistry with prosthetic groups or coordination
chemistry as the second and third synthesis route, respectively.
The first set of compounds was prepared through reaction of
fluorine-containing primary amines with GA to afford com-
pounds 1, 2 and 3. Fluorine-containing amines like 2-fluoro-
ethylamine, 4-fluorobenzylamine and 2,2,2,-trifluoroethylamine
represent suitable building blocks for the radiosynthesis of
corresponding compounds labeled with the short-lived positron
emitter 18F. Radiolabeled 2-[18F]fluoroethylamine16 and 4-[18F]-
fluorobenzylamine17 as well as N-dibenzyl protected 2,2,2,-tri-
[18F]fluoroethylamine18 have been used for the radiosynthesis of
various 18F-labeled compounds. The synthesis of fluorine-
containing GA derivatives 1, 2 and 3 is given in Scheme 1.
Treatment of GA with an excess of the respective primary
amine in DMSO afforded corresponding vinylogous amine
This led to the development of a number of GA derivatives
with improved toxicity and solubility profile, better bioavailabil-
ity and increased metabolic stability. Among those GA deriva-
tives, 17-allylamino-17-demethoxy-geldanamycin (17-AAG)
and
17-(2-dimethylaminoethyl)-17-demethoxygeldanamycin
(17-DMAG) are currently in different stages of various clinical
trials for cancer treatment. The chemical structures of GA and
GA derivatives 17-AAG and 17-DMAG are displayed in Fig. 1.
Structure–activity analyses of modified GA derivatives demon-
strated that position 17 on the quinone moiety of GA is particu-
larly tolerant to modifications without substantial loss of
biological activity. The tolerance of the 17 position for diverse
substitution patterns is consistent with observed structures of
GA–Hsp90 complexes showing the 17 position of GA exposed
to the less sterically demanding outside. Consequently, numerous
reports in the literature have described the synthesis and evalu-
ation of GA derivatives modified in position 17 of the
molecule.9
Current medicinal chemistry is increasingly implementing
molecular imaging techniques for the characterization and evalu-
ation of novel molecular targets, and the assessment of the
pharmacologic profile of novel drugs.10 Among the plethora of
molecular imaging techniques, especially radionuclide-based
imaging methodologies positron emission tomography (PET)
and single photon emission computed tomography (SPECT)
allow the exact elucidation of biological and physiological pro-
cesses at the cellular and molecular level in vivo.11 The develop-
ment of molecular probes for PET and SPECT targeting Hsp90
in vivo would allow assessment of functional HSP90 expression
in cancer. Molecular probes for non-invasive imaging of Hsp90
expression would have significant impact on early tumor
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Scheme 1 Reagents and conditions for compounds 1, 2 and 3: (a)
DMSO, R-NH2, r.t., 2 h.
products 1 and 2 in high chemical yields of 89%, whereas com-
pound 3 could be isolated in only 38% yield. The obtained
lower yield of compound 3 can be attributed to the lower nucleo-
philicity of the primary amine group in 2,2,2,-trifluoroethyl-
amine. Formation of compounds 1, 2 and 3 could easily be
monitored through the colour change of the reaction mixture
from yellow to purple after the addition of the primary amines to
GA in DMSO. The purple colour is indicative of the vinylogous
amine motif in compounds 1, 2 and 3.
Scheme 2 Reagents and conditions for compounds 5 and 7: (a)
CH2Cl2, NH2–(CH2)5–NH2, r.t., 2 h; (b) SFB; (c) (Boc-aminooxy)acetic
acid, EDCl; (d) HCl; (e) FBA.
The synthesis of fluorobenzoylated GA derivative 5 and
fluorobenzylidene-oxime 7 according to the prosthetic group
approach is depicted in Scheme 2.
A second set of reactions was based on the attachment of
various fluorine-containing prosthetic groups to GA. The use of
18F-labeled prosthetic groups is a frequently employed approach
in 18F chemistry for the labelling of peptides and protein with
18F under mild conditions. The used prosthetic groups differ in
the complexity of their radiosynthesis, labeling yield, and
efficiency and chemoselectivity of conjugation to biomacromole-
cules. Among the arsenal of available 18F-labeled prosthetic
groups, acylation agent succinimidyl-4-[18F]fluorobenzoate
([18F]SFB) and oxime-forming agent 4-[18F]fluorobenzaldehyde
([18F]FBA) are widely and frequently used in 18F chemistry for
bioconjugation to peptides and proteins.19 SFB can be attached
to primary amine groups via an acylation reaction with the active
ester moiety, whereas FBA undergoes “click chemistry”-like
reactions with amino-oxy compounds to form the corresponding
oximes.
The application of prosthetic groups SFB and FBA for bio-
conjugation reactions to GA requires the presence of a primary
amine group in the case of SFB, and an amino-oxy group in the
case of FBA. For this purpose, GA was converted into 5-amino-
pentyl group-containing GA derivative 4, which was used as a
starting material for the synthesis of fluoro-benzoylated GA com-
pound 5 and fluorobenzylidene-oxime 7.
Treatment of GA with a 10-fold molar excess of 1,5-diamino-
pentane in CH2Cl2 afforded 5-aminopentyl-substituted GA
derivative 4 in almost quantitative yield after purification using
column chromatography. Amine 4 was treated with a 1.4 molar
excess of SFB to provide fluorobenzoylated compound 5 in 59%
yield after purification. In a second acylation reaction, amine 4
was treated with (Boc-aminooxy)acetic acid in the presence of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCl) as a
carboxyl group activating agent in CH2Cl2 to form the corres-
ponding amide 6 in very good chemical yields of 89%.
Removal of the Boc-protecting group in amide 6 with HCl
and subsequent reaction of the free amino-oxy intermediate
with FBA according to a click chemistry reaction gave fluoro-
benzylidene-oxime 7 in a high yield of 83% for both steps.
Fluorobenzylidene-oxime 7 was formed in the E-configuration
exclusively.
The third synthesis route was directed to the preparation
of a rhenium-tricarbonyl complex with GA. Rhenium is used
as a surrogate for radioactive technetium. The coordination
chemistry of rhenium and technetium is similar, and radio-
pharmacological profiles of 99mTc- and radio-rhenium complexes
are comparable in vivo, except for some differences in lipophili-
city. Therefore, we set up the synthesis of a GA derivative
containing a rhenium-complex moiety as a surrogate for a
99mTc-labeled compound.
As a member of the group 7 of the Periodic Table, technetium
and rhenium are typical transition metal elements with rich and
diverse coordination chemistry. The chemistry of technetium and
rhenium is well explored, and numerous excellent books, mono-
graphs and reviews have been published to address aspects of
technetium and rhenium coordination chemistry extensively.20
Recently, organometallic Tc-tricarbonyl complexes have
brought new interest in the design of novel 99mTc radiopharma-
ceuticals. The facile synthesis route to the {99mTc(CO)3}+ core,
its water solubility, and the robust chemical character and kinetic
inertness of the {99mTc(CO)3}+ core offer important advantages
for the preparation of radiopharmaceuticals. Various bifunctional
chelators for the {99mTc(CO)3}+ core have been developed.
Single amino acid chelates (SAAC) based on pyridyl, imidazole,
and/or carboxylate derivatized amino acids were shown to be
particularly useful for tripodal chelation of the {99mTc(CO)3}+
core.21
We performed the synthesis of SAAC 12 containing a pyridine
nitrogen/amine nitrogen/carboxylate set of donor groups for
efficient tripodal chelation of a {Re(CO)3}+ core as a surrogate
for radioactive {99mTc(CO)3}+. SAAC 13 contains an additional
primary amine group required for the Michael reaction with the
methoxy quinone motif in GA, and an ethylene glycol chain to
lower the lipophilicity of the quite lipophilic {Re(CO)3}+ core.
The complete multi-step synthesis route for the preparation of
SAAC 13 and its attachment to GA to afford compound 14 is
displayed in Scheme 3.
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Scheme 4 Reagents and conditions for compound 15: (a)
[NEt4]2[ReBr3(CO)3], CH2Cl2, 1.5 h.
Scheme 3 Reagents and conditions for compound 12: (a) glycine-tert-
butyl ester, toluene, reflux, 2 h; (b) NaBH4, MeOH, r.t., 1 h; (c) 2-(2-
(2-iodoethoxy)ethoxy)ethanol, NaCO3, acetone–water, reflux, 5 h; (d)
MsCl, TEA, THF, 0 °C to r.t., overnight; (e) NaN3, CH3CN, reflux, 4 h;
(f) TFA, CH2Cl2, r.t., overnight; (g) 10% Pd/C, 1.8 bar H2, MeOH, r.t.,
2 h; (h) GA, compound 13, DMSO, r.t., 4 h.
The multi-step reaction sequence commenced with a reductive
amination reaction using commercially available pyridine-2-car-
baldehyde and glycine-tert-butyl ester, which afforded amine 9
in 56% yield. Secondary amine 9 was alkylated with 2-(2-(2-
iodoethoxy)-ethoxy)ethanol in 25% isolated yield to afford com-
pound 10. 2-(2-(2-Iodoethoxy)ethoxy)ethanol was prepared
according to the literature procedure reported by Ishow et al.22
Compound 10 was converted into azide 11 through treatment
with MsCl followed by the reaction with an excess of sodium
azide. Azide 11 was prepared in 80% yield for both steps.
Removal of the tert-butyl group in compound 11 with TFA
afforded compound 12 in 82% yield. Final reduction of the azide
group in compound 12 using Pd/C and H2 gave tripodal chelator
13 in 76% yield after purification with column chromatography.
Chelator 13 was further attached to GA according to an estab-
lished Michael reaction protocol to afford compound 14 in 88%
isolated yield.
Tricarbonylrhenium moieties are readily available as stable
[NEt4]2[ReBr3(CO)3] complexes, which can be prepared in
multigram quantities starting from commercially available
NH4[ReO4].23 Formation of tricarbonylrhenium(I) complex 15
through reaction of equimolar amounts of [NEt4]2[ReBr3(CO)3]
with tripodal ligand 14 is displayed in Scheme 4. The two nitro-
gen donor groups and the carboxylate group in compound 14 are
capable of replacing all three bromine atoms in complex
[NEt4]2[ReBr3(CO)3] to afford neutral tricarbonylrhenium(I)
complex 15 in 87% yield.
Fig. 2 ATPase activity assay using yeast Hsp90 isoform Hsp82p.
ATPase rates are expressed as a percentage of the unstimulated rate of
Hsp82p.
shown to be specific for Hsp90. The ATP-binding site in Hsp90
as demonstrated by various structural and biochemical studies is
also the binding site for GA. GA binds to the ATP-binding site
of yeast Hsp90 with a KD of 0.5 μM, making GA a potent inhibi-
tor of ATP binding in Hsp90 protein.
In the absence of client proteins, Hsp90 maintains inherent
ATPase activity which is significantly reduced by Hsp90 inhibi-
tors. Inhibition of inherent ATPase activity of Hsp90 was used to
evaluate fluorine-containing compounds 1, 2, 3, 5, 7 and
rhenium-tricarbonyl-containing compound 15 in an in vitro
ATPase activity assay. Hsp90-specific inhibitor GAwas used as a
reference in the assay. Co-chaperone Aha1 was used as an activa-
tor of ATPase activity. Aha1 is known to stimulate ATPase
activity upon binding to Hsp90.25 All compounds were used in
an inhibitor concentration of 1.0 μM. The results of the ATPase
assay using Hsp90 isoform Hsp82p are displayed in Fig. 2.
The activating effect of co-chaperone Aha1 towards ATPase
activity of Hsp90 is clearly demonstrated. ATPase activity of
Hsp90 was increased 3.5-fold upon activation with co-chaperone
Aha1 when compared with unstimulated Hsp90. No effect upon
reduction of ATPase activity was observed in the control exper-
iment with DMSO, whereas treatment of Aha1 activated Hsp90
with 1.0 μM of inhibitor GA led to a significant 80% reduction
of ATPase activity. This is in agreement with the known high
Biological evaluation of novel GA derivatives using an in vitro
ATPase assay
The Saccharomyces cerevisiae Hsp90 isoform Hsp82 was used
to assay APTase activity.24 The observed ATPase activity was
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inhibitory potency of GA towards the ATP-binding site of
Hsp90.
and converted to the TMS scale. Mass spectra were recorded
using a Micromass ZabSpec Hybrid Sector-TOF by positive
mode electrospray ionization. Flash chromatography was con-
ducted using MERCK silica gel (mesh size 230–400 ASTM).
Thin-layer chromatography (TLC) was performed on Merck
silica gel F-254 aluminum plates, with visualization under UV
light (254 nm). HPLC analyses were performed with a Beckman
HPLC system on a semi-preparative Luna C18 column (100 Å,
10 μm, 250 × 10 mm) using gradient elution at a flow rate of
3 mL min−1 and UV detection at 254 nm. The elution started
with an acetonitrile–water gradient from 15/85 to 50/50 (v/v) for
8 min, followed by an 8 min gradient elution from 50/50 to
70/30 (v/v) and a final 14 min elution at 70/30 (v/v).
Within the series of fluorine-containing GA derivatives, only
fluorobenzoylated compound 5 displayed comparable ATPase
inhibitory potency as found for parent compound GA. Com-
pound 5 led to a 75% reduction of ATPase activity at 1 μM con-
centration of the inhibitor. Tricarbonyl-rhenium complex 15 and
fluorobenzyl-containing GA derivative 2 were the least active
compounds in the ATPase assay at this inhibitor concentration.
Treatment of Aha1 activated Hsp90 with both compounds at a
concentration of 1.0 μM resulted in a 25–35% reduction of
ATPase activity. Fluorine-containing compounds 1, 3, and 7
reduced ATPase activity by 60%, 65%, and 50%, respectively.
These findings are in agreement with various structure–
activity relationship studies of 17-aminogeldanamycin deriva-
tives.9 The ATP-binding site of Hsp90 accommodates small lipo-
philic substituents in position 17 of the GA scaffold as
demonstrated for fluoroethyl and trifluoroethyl group-containing
compounds 1 and 3. Larger groups directly attached to the GA
scaffold like a fluorobenzyl group in compound 2 significantly
reduced inhibitory potency compared to parent compound GA.
Low inhibitory potencies were also found for fluorobenzylidene-
oxime 7 and, more profound, for tricarbonyl-rhenium complex
15. Both compounds contain a linker to separate the fluorine-
and tricarbonyl-rhenium-containing motifs from the GA scaf-
fold. An adverse effect was observed for fluorobenzoylated com-
pound 5, which exhibited comparable Hsp90 ATPase inhibitory
potency as GA. This clearly favors compound 5 as a lead for the
development of an 18F-labeled molecular probe.
All chemicals were obtained from commercial suppliers
(reagent grade) and used without further purification. Geldana-
mycin was purchased in multi-gram quantities from LC
Laboratories.
2-(2-(2-Iodoethoxy)ethoxy)ethanol,22 succinimidyl-4-fluoro-
benzoate (SFB),26 and [NEt4]2[ReBr3(CO)3]24 were prepared
according to literature procedures.
Chemical syntheses
17-(2-Fluoroethylamino)-17-demethoxy-geldanamycin 1. GA
(50 mg, 89.18 μmol), 2-fluoroethylamine hydrochloride
(88.6 mg, 0.89 mmol) and triethylamine (124 μL, 0.89 mmol)
were stirred in DMSO (1 mL) for 2 h at room temperature. The
reaction mixture was poured into 1 N HCl and extracted with
CHCl3. After drying with Na2SO4 and evaporation of the
solvent under reduced pressure the residue was purified by flash
chromatography (10% MeOH–CHCl3) to give 47 mg (89%) of
the desired compound. HPLC analysis: tR = 17.25 min.
1H-NMR (400 MHz, CDCl3): δ 0.99 (m, 6H, 2× CH3), 1.80 (s,
3H, CH3), 2.02 (s, 3H, CH3), 2.31 (m, 1H), 2.72 (m, 2H), 3.27
(s, 3H, OCH3), 3.36 (s, 3H, OCH3), 3.46 (m, 1H), 3.57 (d, J =
8.6 Hz, 1H), 3.87 (m, 2H), 4.31 (d, J = 10.1 Hz, 1H), 4.65 (dt, J
= 46.9 Hz, J = 4.7 Hz, 2H), 5.19 (s, 1H), 5.85 (d, J = 10.9 Hz,
1H), 5.87 (dd, J = 9.4 Hz, J = 3.1 Hz, 1H), 6.39 (t, J = 5.5 Hz,
1H), 6.58 (t, J = 11.7 Hz, 1H), 6.94 (d, J = 11.7 Hz, 1H), 7.30
(s, 1H), 9.11 (s, 1H); 19F-NMR (376 MHz, CDCl3): δ −225.9
(m). LR-MS m/z (ESI): 614.3 [M + Na]+. HR-MS m/z (ESI)
C30H42FN3O8Na ([M + Na+]) calcd 614.2848, found 614.2838.
Summary and conclusions
The synthesis of a series of fluorine- and rhenium-containing
GA derivatives as leads for the development of respective 18F-
and 99mTc-labeled molecular probes for Hsp90 imaging has been
accomplished. All compounds were prepared based on the treat-
ment of GA with primary amines to furnish corresponding viny-
logous amine products. Design and synthesis of fluorine- and
rhenium-containing GA derivatives as described in this work
was directed to the special requirements encountered for the
preparation of respective molecular probes labeled with 18F and
99mTc. All described synthesis routes are easily applicable to
radiosyntheses with short-lived positron-emitter 18F and gamma-
emitter 99mTc. This especially counts for fluorobenzoylated com-
pound 5, which displayed the highest Hsp90 ATPase inhibitory
potency comparable to that of GA. This makes compound 5 a
promising lead for the development of an 18F-labeled molecular
probe for Hsp90 imaging in vivo.
17-(4-Fluorobenzylamino)-17-demethoxy-geldanamycin
2.
GA (54 mg, 96.3 μmol) was dissolved in DMSO (1.0 mL).
4-Fluorobenzylamine (55 μL, 482 μmol) was added and the
mixture was stirred at room temperature for 30 min. The reaction
mixture was diluted with 1 N HCl and extracted with CHCl3.
After drying with Na2SO4 and evaporation of the solvent under
reduced pressure the residue was purified by flash chromato-
graphy (10% MeOH–CHCl3) to give 56 mg (89%) of the
Radiosynthesis and radiopharmacological evaluation of
18F-labeled compound 5 employing prosthetic group [18F]SFB
as a radiolabel is currently in progress.
1
desired compound. HPLC analysis: tR = 22.92 min. H-NMR
(400 MHz, CDCl3): δ 1.01 (m, 6H, 2× CH3), 1.80 (s, 3H, CH3),
2.03 (s, 3H, CH3), 2.43 (m, 1H), 2.68 (d, J = 13.3 Hz, 1H), 2.75
(m, 1H), 3.28 (s, 3H, OCH3), 3.37 (s, 3H, OCH3), 3.45 (m, 1H),
3.58 (m, 1H), 4.15 (bs, 1H), 4.32 (d, J = 9.4 Hz, 1H), 4.66 (ddd,
J = 57.0 Hz, J = 14.1 Hz, J = 5.5 Hz, 2H), 5.19 (s, 1H), 5.86 (d,
J = 10.2 Hz, 1H), 5.87 (dd, J = 9.4 Hz, J = 3.1 Hz, 1H), 6.38 (t,
J = 5.5 Hz, 1H), 6.59 (t, J = 11.7 Hz, 1H), 6.96 (d, J = 11.7 Hz,
Experimental
General methods
1H-NMR and 13C-NMR and 19F-spectra were recorded on a
Varian Inova-400 at 400 MHz, 100 MHz and 376 MHz, respect-
ively. Chemical shifts (δ) were determined relative to the solvent
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1H), 7.09 (t, J = 8.6 Hz, 2H), 7.24–7.28 (m, 2H), 7.31 (s, 1H),
9.15 (s, 1H); 19F-NMR (376 MHz, CDCl3): δ −113.2 (m).
LR-MS m/z (ESI): 676.3 [M + Na]+. HR-MS m/z (ESI):
C35H44FN3O8Na ([M + Na]+) calcd 676.3005, found 676.2991.
LR-MS m/z (ESI): 775.4 [M + Na]+. HR-MS m/z (ESI)
C40H53N4O9Na ([M + Na]+) calcd 775.3689, found 775.3678.
17-(tert-Butyl
2-(((5-aminopentyl)-2-oxoethoxycarbamate))-
6. 17-(5-Amino-pentyl-
amino)-17-demethoxy-geldanamycin
amino)-17-demethoxy-geldanamycin 4 (60 mg, 95 μmol), (Boc-
aminooxy)acetic acid (18.2 mg, 95 μmol), and EDCl (22.8 mg,
119 μmol) were stirred in 1.0 mL of dry CH2Cl2 at room temp-
erature for 1 h. The reaction mixture was diluted with CH2Cl2
(25 mL) and extracted with 0.1 N HCl. The organic layer was
dried and the solvent was evaporated. The residue was purified
with flash chromatography (10% MeOH–CHCl3) to afford
17-(2,2,2-Trifluoroethylamino)-17-demethoxy-geldanamycin
3. GA (100 mg, 178 μmol) was dissolved in DMSO (1.0 mL).
2,2,2-Trifluoroethylamine (72 μL, 901 μmol) was added and the
mixture was stirred at room temperature for 30 min. The reaction
mixture was diluted with 1 N HCl and extracted with CHCl3.
After drying with Na2SO4 and evaporation of the solvent under
reduced pressure the residue was purified by flash chromato-
graphy (10% MeOH–CHCl3) to give 42 mg (38%) of the
1
68 mg (89%) of compound 6. H-NMR (400 MHz, CDCl3): δ
1
0.96 (d, J = 6.6 Hz, 3H), 1.00 (d, J = 7.0 Hz, 3H), 1.23 (t, J =
7.0 Hz, 2H), 1.47 (m, 9H), 1.61–1.78 (m, 8H), 1.80 (m, 4H),
2.12 (s, 3H), 2.40 (m, 1H), 2.66 (d, J = 13.4 Hz, 1H), 2.72 (m,
1H), 3.26 (s, 3H, OCH3), 3.36 (s, 3H, OCH3), 3.41–3.49 (m,
4H), 3.52–3.58 (m, 2H), 4.32 (m, 1H), 5.17 (s, 1H), 5.83 (d, J =
10.5 Hz, 1H), 5.87 (d, J = 12.0 Hz, 1H), 6.27 (m, 1H), 6.58 (t,
J = 11.5 Hz, 1H), 6.94 (d, J = 11.4 Hz, 1H), 7.26 (s, 1H), 7.70
(m, 1H), 8.22 (bs, 1H), 9.18 (s, 1H). m/z (ESI) C40H61N5O12Na
([M + Na]+) calcd 826.4214, found 826.4208.
desired compound. HPLC analysis: tR = 18.68 min. H-NMR
(400 MHz, CDCl3): δ 0.99 (m, 6H, 2× CH3), 1.79 (s, 3H, CH3),
2.02 (s, 3H, CH3), 2.19 (m, 1H), 2.74 (m, 1H), 3.27 (s, 3H,
OCH3), 3.36 (s, 3H, OCH3), 3.43 (m, 1H), 3.57 (m, 1H), 4.13
(m, 1H), 4.30 (m, 2H), 5.19 (s, 1H), 5.83 (d, J = 10.6 Hz, 1H),
5.87 (d, J = 10.6 Hz, 1H), 5.97 (t, J = 7.0 Hz, 1H), 6.57 (t, J =
11.7 Hz, 1H), 6.94 (d, J = 11.1 Hz, 1H), 7.26 (s, 1H), 7.31 (s,
1H), 9.15 (s, 1H); 19F-NMR (376 MHz, CDCl3): δ −72.0 (t, J =
8.5 Hz). LR-MS m/z (ESI): 650.3 [M + Na]+. HR-MS m/z (ESI)
C30H40F3N3O8Na ([M + Na]+) calcd 650.2660, found 650.2647.
17-(2-(((4-Fluorobenzylidene)amino)oxy)acetamido)pentyl-
amino-17-demethoxy-geldanamycin 7. Compound 6 (60 mg,
74.7 μmol) was dissolved in 4.0 N HCl in dioxane. The reaction
mixture was stirred at room temperature for 1 h. After removal of
the solvent under reduced pressure, the residue was re-dissolved
in DMF. 4-Fluorobenzaldehyde was added, and the reaction
mixture was stirred for 2 h at room temperature. The reaction
mixture was diluted with 1.0 N HCl, and the mixture was
extracted with CH2Cl2. The solvent was evaporated and the
residue purified with flash chromatography (10% MeOH–
CHCl3) to afford 48 mg (80%) of compound 7. HPLC analysis:
tR = 18.25 min. 1H-NMR (400 MHz, CDCl3): δ 0.96 (d, J = 6.6 Hz,
3H), 1.00 (d, J = 7.0 Hz, 3H), 1.24 (t, J = 7.0 Hz, 2H),
1.41–1.74 (m, 8H), 1.79 (m, 4H), 2.02 (s, 3H), 2.17 (m, 1H),
2.42 (m, 1H), 2.66 (d, J = 13.4 Hz, 1H), 2.72 (m, 1H), 3.26 (s,
3H, OCH3), 3.36 (s, 3H, OCH3), 3.42–3.49 (m, 4H), 3.52–3.58
(m, 2H), 4.30 (d, J = 9.4 Hz, 1H), 5.18 (s, 1H), 5.29 (s, 1H),
5.83 (d, J = 10.5 Hz, 1H), 5.87 (d, J = 12.0 Hz, 1H), 6.29 (m,
1H), 6.58 (t, J = 11.5 Hz, 1H), 6.94 (d, J = 11.4 Hz, 1H), 7.26
(m, 1H), 9.18 (s, 1H); 19F-NMR (376 MHz, CDCl3): δ −110.9
(m). LR-MS m/z (ESI): 882.4 [M + Na]+. HR-MS m/z (ESI)
C42H56FN5O10Na ([M + Na]+) calcd 832.3909, found 832.3905.
17-(5-Aminopentylamino)-17-demethoxy-geldanamycin
4.
GA (100 mg, 178 μmol) was dissolved in CH2Cl2 (10.0 mL).
1,5-Diaminopentane (210 μL, 1.78 mmol) was added and the
mixture was stirred at room temperature for 1 h. The purple reac-
tion mixture was washed thoroughly several times with saturated
CaCl2 solution, followed by water and brine. The organic layer
was dried, and the solvent was evaporated to give 106 mg (95%)
1
of compound 4 as a purple solid. H-NMR (400 MHz, CDCl3):
δ 1.01 (m, 6H, 2× CH3), 1.48 (m), 1.57 (m), 1.70(m), 1.79 (s,
3H, CH3), 2.02 (s, 3H, CH3), 2.68 (d, J = 13.3 Hz, 1H), 2.73 (t,
J = 6.5 Hz, 2H), 3.27 (s, 3H, OCH3), 3.36 (s, 3H, OCH3), 3.46
(m, 1H), 3.56 (m, 1H), 4.31 (d, J = 9.4 Hz, 1H), 5.18 (s, 1H),
5.29 (s, 1H), 5.84 (d, J = 10.2 Hz, 1H), 5.87 (d, J = 9.4 Hz, 1H),
6.29 (t, J = 5.7 Hz, 1H), 6.58 (t, J = 11.7 Hz, 1H), 6.95 (d, J =
11.7 Hz, 1H), 7.27 (t, J = 8.6 Hz, 2H), 9.18 (s, 1H). m/z (ESI)
C33H50N4O8Na ([M + Na]+) calcd 653.3526, found 653.3521.
17-(4-Fluorobenzamido)pentyl)amino)-17-demethoxy-gelda-
namycin
5. N-Succinimidyl-4-fluorobenzoate (6.25 mg,
26.0 μmol) dissolved in 500 μL of dry CH2Cl2 was added to a
solution of compound 4 (12.5 mg, 18.9 μmol) in 500 μL of dry
CH2Cl2. The reaction mixture was stirred for 2 h at 25 °C. The
mixture was then evaporated to dryness and purified by column
chromatography (10% MeOH–CHCl3) to afford 8.5 mg of com-
[(Pyridin-2-ylmethyl)-amino]-acetic acid tert-butyl ester
9. Pyridine-2-carbaldehyde 8 (0.84 mL, 8.83 mmol) and
glycine-tert-butyl ester (1.0 g, 8.83 mmol) were refluxed in
toluene (10 mL) for 2 h. The solvent was evaporated and the
residue was dissolved in MeOH (10 mL). NaBH4 (333 mg,
8.8 mmol) was added and the mixture was stirred at room temp-
erature for 1 h. After the addition of acetone (2 mL) and stirring
for an additional 30 min, the solvent was evaporated and the
residue was purified by column chromatography (10% MeOH–
CHCl3) to give 1.1 g (56%) of the desired product 9 as an oil.
1H-NMR (CDCl3, 400 MHz): δ 1.46 (s, 9H, CH3), 3.34 (s, 2H,
CH2), 3.93 (s, 2H, CH2), 7.15 (ddd, J = 7.7 Hz, J = 4.8 Hz, J =
0.7 Hz, 1H, Ar-H), 7.33 (d, J = 7.7 Hz, 1H, Ar-H), 7.63 (td, J =
7.7 Hz, J = 1.8 Hz, 1H, Ar-H), 8.55 (m, 1H, Ar-H); 13C-NMR
1
pound 5 (59% yield). HPLC analysis: tR = 18.33 min. H-NMR
(400 MHz, CDCl3): δ 0.96 (d, J = 6.6 Hz, 3H), 1.00 (d, J = 7.0
Hz, 3H), 1.48–1.53 (m, 3H), 1.65–1.80 (m, 12H), 2.12 (s, 3H),
2.39 (dd, J = 10.7 Hz, J = 13.8 Hz, 1H), 2.67 (d, J = 13.4 Hz,
1H), 2.39 (dd, J = 10.7 Hz, J = 13.8 Hz, 1H), 2.73 (M, 1H),
3.27 (s, 3H, OCH3), 3.36 (s, 3H, OCH3), 3.45–3.50 (m, 4H),
3.56–3.58 (m, 2H), 4.30 (d, J = 5.0 Hz, 1H), 5.19 (s, 1H), 5.85
(d, J = 10.5 Hz, 1H), 5.89 (d, J = 12.0 Hz, 1H), 6.09 (t, J = 3
Hz, 1H), 6.25 (s, 1H), 6.58 (t, J = 11.5 Hz, 1H), 6.95 (d, J =
11.4 Hz, 1H), 7.09–7.13 (m, 2H), 7.29 (s, 1H), 7.75–7.79 (m,
2H), 9.17 (s, 1H); 19F-NMR (376 MHz, CDCl3): δ −108.2 (m).
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6724–6731 | 6729

View Article Online
(CDCl3, 100 MHz): δ 28.10, 51.26, 54.63, 81.15, 121.95,
122.07, 136.44, 149.33, 159.34, 171.45; LR-MS (ESI) 223
[M − H]+.
2H, CH2), 7.28 (m, 1H, Ar-H), 7.30 (s, 1H, Ar-H), 7.73 (td, J =
7.7 Hz, J = 1.8 Hz, 1H, Ar-H), 8.58 (m, 1H, Ar-H); 13C-NMR
(CDCl3, 100 MHz): δ 20.84, 50.62, 54.53, 58.30, 60.34, 68.89,
70.02, 70.41, 70.50, 123.01, 137.66, 148.51, 157.19, 176.08;
LR-MS (ESI) 324 [M − H]+.
({2-[2-(2-Hydroxy-ethoxy)-ethoxy]-ethyl}-pyridin-2-yl-methyl-
amino)-acetic acid tert-butyl ester 10. A mixture of [(pyridin-2-
ylmethyl)-amino]-acetic acid tert-butyl ester
9
(150 mg,
({2-[2-(2-Amino-ethoxy)-ethoxy]-ethyl}-pyridin-2-ylmethyl-
amino)-acetic acid 13. ({2-[2-(2-Azido-ethoxy)-ethoxy]-ethyl}-
pyridin-2-ylmethyl-amino)-acetic acid 12 (600 mg, 2.05 mmol)
and 10% Pd/C (60 mg) were stirred in MeOH (150 mL) over H2
(1.8 bar) for 2 h. The reaction mixture was filtered through celite
to remove Pd/C. The residue was evaporated under reduced
pressure to give amine 13 (450 mg, 76%) as a yellow-brown oil.
1H-NMR (CDCl3, 400 MHz): δ 2.66 (t, J = 4.7 Hz, 2H, CH2),
3.17 (t, J = 4.7 Hz, 2H, CH2), 3.19 (s, 2H, CH2), 3.47–3.57 (m,
6H), 3.63–3.67 (m, 2H), 3.79 (s, 2H, CH2), 3.81 (m, 2H, CH2),
7.19–7.25 (m, 2H, Ar-H), 7.66 (td, J = 7.8 Hz, J = 1.8 Hz, 1H,
Ar-H), 8.56 (m, 1H, Ar-H). LR-MS (ESI) 298.4 [M + H]+.
0.67 mmol), 2-[2-(2-iodo-ethoxy)-ethoxy]-ethanol (348 mg,
1.35 mmol) and Na2CO3 (300 mg) was refluxed in acetone
(3 mL) and water (1 mL) for 5 h. The solvent was reduced and
the residue was purified by column chromatography (10%
MeOH–CHCl3) to give 60 mg (25%) of compound WUE16 as a
pale yellow oil. 1H-NMR (CDCl3, 400 MHz): δ 1.45 (s, 9H,
CH3), 2.93 (t, J = 5.7 Hz, 2H, CH2), 3.38 (s, 2H, CH2),
3.55–3.63 (m, 8H), 3.71 (t, J = 4.5 Hz, 2H, CH2), 4.00 (s, CH2),
7.13 (ddd, J = 7.7 Hz, J = 4.8 Hz, J = 1.0 Hz, 1H, Ar-H), 7.52
(d, J = 7.7 Hz, 1H, Ar-H), 7.64 (td, J = 7.7 Hz, J = 1.8 Hz, 1H,
Ar-H), 8.50 (m, 1H, Ar-H); 13C-NMR (CDCl3, 100 MHz): δ
28.16, 53.28, 56.28, 60.43, 61.64, 69.70, 70.23, 70.30, 72.63,
80.97, 121.95, 122.96, 136.58, 148.95, 159.62, 170.88; LR-MS
(ESI) 355 [M − H]+.
17-({2-[2-(2-Amino-ethoxy)-ethoxy]-ethyl}-pyridin-2-yl-methyl-
amino)-acetic
acid-17-demethoxy-geldanamycin
14. GA
(123 mg, 220 μmol) and {2-[2-(2-amino-ethoxy)-ethoxy]-
ethyl}-pyridin-2-ylmethyl-amino)-acetic acid 13 (330 mg,
1.1 mmol) were stirred in DMSO (4 mL) for 4 h at room temp-
erature. The reaction mixture was diluted with saturated CaCl2-
solution and extracted with CHCl3. The organic layer was
washed with water, dried (Na2SO4) and evaporated under
reduced pressure. The residue was purified by flash chromato-
graphy (20% MeOH–CHCl3 containing 0.5% TFA) to give
160 mg (85%) of the desired compound as a purple foam.
1H-NMR (400 MHz, CD3OD): δ 0.96 (d, J = 3.9 Hz, 3H), 0.97
(d, J = 3.9 Hz, 3H), 1.72 (s, 3H, CH3), 2.00 (s, 3H, CH3), 2.30
(m, 1H), 2.73 (m, 1H), 3.29 (s, 3H, OCH3), 3.33 (s, 3H, OCH3),
3.42 (t, J = 4.7 Hz, 2H), 3.65 (m, 4H), 3.72 (m, 2H), 3.82 (t, J =
4.7 Hz, 2H), (m, 2H), 4.09 (s, 2H), 4.54 (d, J = 7.8 Hz, 1H),
4.61 (s, 2H), 5.23 (s, 1H), 5.55 (d, J = 9.4 Hz, 1H), 5.87 (m,
1H), 6.61 (t, J = 10.9 Hz, 1H), 6.99 (s, 1H), 7.13 (d, J =
10.9 Hz, 1H), 7.60 (dd, J = 7.0 Hz, J = 5.5 Hz, 1H), 7.66 (d, J =
7.8 Hz, 1H), 8.10 (td, J = 7.8 Hz, J = 1.6 Hz, 1H), 8.68 (d, J =
4.7 Hz, 1H). LR-MS (ESI positive): m/z (%) = 848.61 (100)
(M + Na).
({2-[2-(2-Azido-ethoxy)-ethoxy]-ethyl}-pyridin-2-ylmethyl-
amino)-acetic acid tert-butyl ester 11. A mixture of ({2-[2-(2-
hydroxy-ethoxy)-ethoxy]-ethyl}-pyridin-2-ylmethyl-amino)-acetic
acid tert-butyl ester 10 (1.56 g, 4.4 mmol) and triethylamine
(1.23 mL, 8.8 mmol) in THF (50 mL) was cooled to 0 °C. MsCl
(681 μL, 8.8 mmol) was added dropwise while stirring. The
mixture was slowly warmed up to room temperature and stirred
overnight. After the addition of water (100 mL) the mixture was
extracted with EtOAc. The organic layer was flushed through a
short silica-gel plug. After evaporation of the solvent the mesy-
late was obtained as a brown-yellow oil (1.68 g), which was
used without further purification in the next step. The mesylate
was dissolved in acetonitrile (50 mL). After the addition of
sodium azide (1.43 g, 22 mmol) the mixture was refluxed for
4 h. Water (100 mL) was added and the mixture was extracted
with EtOAc. The organic layer was flushed through a short
silica-gel plug. After evaporation of the solvent the desired azide
11 was obtained as a brown-yellow oil, which was sufficiently
pure. Yield: 1.34 g (80%) for two steps. 1H-NMR (CDCl3,
400 MHz): δ 1.46 (s, 9H, CH3), 2.93 (t, J = 5.7 Hz, 2H, CH2),
3.36 (t, J = 5.5 Hz, 2H, CH2), 3.41 (s, 2H, CH2), 3.56–3.68 (m,
8H), 3.99 (s, CH2), 7.14 (ddd, J = 7.7 Hz, J = 4.8 Hz, J = 1.0 Hz,
1H, Ar-H), 7.54 (d, J = 7.7 Hz, 1H, Ar-H), 7.64 (td, J = 7.7 Hz,
J = 1.8 Hz, 1H, Ar-H), 8.52 (m, 1H, Ar-H); 13C-NMR (CDCl3,
100 MHz): δ 28.16, 50.60, 53.33, 56.56, 60.62, 69.95, 70.02,
70.29, 70.58, 80.83, 121.87, 122.90, 136.39, 148.97, 159.78,
170.81; LR-MS (ESI) 380 [M − H]+.
Tricarbonyl-{17-({2-[2-(2-amino-ethoxy)-ethoxy]-ethyl}-
pyridin-2-yl-methyl-amino)-acetic acid-17-demethoxy-geldana-
mycin}rhenium(I)
15. 17-({2-[2-(2-Amino-ethoxy)-ethoxy]-
ethyl}-pyridin-2-ylmethyl-amino)-acetic acid-17-demethoxy-gel-
danamycin 14 (20 mg, 24.2 μmol) and [Et4N][Re(CO)3Br3]
(16.3 mg, 24.2 mmol) were stirred in CH2Cl2–MeOH (1 : 1)
(4 mL) for 2 h at room temperature. The solvent was removed
under reduced pressure and the residue was purified by flash
chromatography (10% MeOH–CHCl3) to give 23 mg (87%) of
({2-[2-(2-Azido-ethoxy)-ethoxy]-ethyl}-pyridin-2-ylmethyl-
amino)-acetic acid 12. ({2-[2-(2-Azido-ethoxy)-ethoxy]-ethyl}-
pyridin-2-ylmethyl-amino)-acetic acid tert-butyl ester (1.3 g,
3.5 mmol) was stirred overnight in methylene chloride (10 mL)
and FTA (1 mL) at room temperature. The solvent was reduced
and the residue was purified by column chromatography (15%
MeOH–CHCl3 containing 1% HOAc) to give 930 mg (82%) of
compound 12 as a yellow-brown oil. 1H-NMR (CDCl3,
400 MHz): δ 3.00 (t, J = 5.3 Hz, 2H, CH2), 3.39 (t, J = 5.0 Hz,
2H, CH2), 3.57 (s, 2H, 2H, CH2), 3.58–3.67 (m, 8H), 4.18 (s,
the rhenium complex as a purple glass. HPLC analysis: tR
=
1
16.26 min. H-NMR (400 MHz, CDCl3): δ 0.96 (d, J = 6.5 Hz,
3H), 0.99 (d, J = 6.5 Hz, 3H), 1.79 (s, 3H, CH3), 2.02 (s, 3H,
CH3), 3.27 (s, 3H, OCH3), 3.35 (s, 3H, OCH3), 3.43 (m, 1H),
3.48–3.57 (m, 2H), 3.70 (m, 6H), 3.83 (m, 2H), 3.89–4.00 (m,
2H), 4.31 (dd, J = 10.1 Hz, J = 3.1 Hz, 1H), 4.56–4.68 (m, 2H),
5.18 (d, J = 6.2 Hz, 1H), 5.87 (t, J = 10.9 Hz, 1H), 6.58 (t, J =
10.9 Hz, 1H), 6.75 (dt, J = 17.9 Hz, J = 5.5 Hz, 1H), 6.95 (bd,
J = 10.9 Hz, 1H), 7.19 (d, J = 2.3 Hz, 1H), 7.41 (dt, J = 7.8 Hz,
6730 | Org. Biomol. Chem., 2012, 10, 6724–6731
This journal is © The Royal Society of Chemistry 2012

View Article Online
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J = 4.7 Hz, 1H), 7.54 (dd, J = 10.1 Hz, J = 7.8 Hz, 1H), 7.93
(m, 1H), 8.80 (bt, J = 4.7 Hz, 1H), 9.16 (d, J = 2.3 Hz, 1H).
LR-MS (ESI positive): m/z (%) = 1116.21 (60), 1118.20 (100)
(M + Na). HR-MS m/z (ESI) C45H58N5O15Na[187Re] ([M +
Na]+) calcd 1118.3379, found 1118.3368.
Biological evaluation
ATPase activity assays were carried out using an enzyme-
coupled system as previously described.24 All reactions were
carried out in 100 μL volumes in a 96-well plate and in triplicate.
Average values of those triplicates are shown with error
expressed as standard error of the mean. All reactions contained
0.5 μM Hsp82p, 1 μM Aha1p, 31.4 mM Hepes pH 7.2, 1 mM
sodium chloride, 19.3 mM potassium acetate, 5 mM magnesium
chloride, 1 mM dithiothreitol, 0.3 mM NADH, 2 mM ATP,
1 mM phosphoenol pyruvate, and 2.5 μL of pyruvate kinase–
lactate dehydrogenase (Sigma) in 100 μL. Drugs were used at
10 μM final concentrations where indicated.
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14 M. C. Lasne, C. Perrio, J. Rouden, L. Barré, D. Roeda, F. Dollé and
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