Radester, a novel inhibitor of the Hsp90 protein folding machinery

Article abstract of DOI:10.1021/ol050580a

(Chemical Equation Presented) The antitumor antibiotics radicicol and geldanamycin are potent inhibitors of the Hsp90 protein folding machinery. Radester is a hybrid composed of radicicol's resorcinol ring and geldanamycin's quinone through an isopropyl ester. Radester was prepared, and the cytotoxicity of it and the corresponding hydroquinone were determined in MCF-7 breast cancer cells to be 13.9 and 7.1 μM, respectively. Protein degradation assays were performed on Hsp90-dependent client proteins, Her-2 and Raf, to correlate Hsp90 inhibition to cytotoxicity.

Full text of DOI:10.1021/ol050580a

ORGANIC
LETTERS
2005
Vol. 7, No. 11
2157-2160
Radester, a Novel Inhibitor of the Hsp90
Protein Folding Machinery
Gang Shen and Brian S. J. Blagg*
Department of Medicinal Chemistry and The Center for Protein Structure and
Function, The UniVersity of Kansas, 1251 Wescoe Hall DriVe, Malott 4070,
Lawrence, Kansas 66045-7562
bblagg@ku.edu
Received March 16, 2005
ABSTRACT
The antitumor antibiotics radicicol and geldanamycin are potent inhibitors of the Hsp90 protein folding machinery. Radester is a hybrid
composed of radicicol’s resorcinol ring and geldanamycin’s quinone through an isopropyl ester. Radester was prepared, and the cytotoxicity
of it and the corresponding hydroquinone were determined in MCF-7 breast cancer cells to be 13.9 and 7.1
µM, respectively. Protein degradation
assays were performed on Hsp90-dependent client proteins, Her-2 and Raf, to correlate Hsp90 inhibition to cytotoxicity.
Hsp90 (90 kDa heat shock protein) is a molecular chaperone
responsible for the conformational maturation of numerous
oncogenic proteins.1 Inhibition of Hsp90 turns the protein
folding machinery into a catalyst for protein degradation.
Recent studies have demonstrated that Hsp90 multiprotein
complexes from tumor cells have higher affinity for ligands
than Hsp90 in normal cells, because malignant cells are
highly dependent upon the Hsp90 protein folding machinery
for the maturation of mutated and over expressed client
proteins that are vital to cell proliferation and growth.2 In
fact, proteins represented in all six hallmarks of cancer are
dependent on the Hsp90 maturation process. Consequently,
Hsp90 has emerged as a promising biological target for the
treatment of cancer, because inhibition of Hsp90 results in
a combinatorial blockade of multiple signaling cascades that
are essential to tumor cell survival.3
and the other in the C-terminal region.4 The energy derived
from ATP hydrolysis is used to fold the nascent polypeptide
into a biologically active protein. Disruption of Hsp90’s
ATPase activity results in the destabilization of multiprotein
complexes and subsequent degradation of the client via the
ubiquitin-proteasome pathway.5
Known inhibitors of Hsp90 manifest their activity by
binding to the N-terminal ATP binding pocket and preventing
Hsp90-catalyzed hydrolysis of ATP. Such inhibitors include
the antitumor antibiotics geldanamycin (GDA), a 17-allyl-
amino derivative of GDA (17-AAG), radicicol (RDC), and
the synthetic ATP analogue PU3 (Figure 1).6 The IC50’s as
determined in MCF-7 cells are 49 nM, 23 nM,7 and 50 µM
for GDA, RDC, and PU3, respectively.
(4) Marcu, M. G.; Chadli, A.; Bouhouche, I.; Catelli, N.; Neckers, L.
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Hsp90 is an ATP-dependent protein with two nucleotide-
binding domains, one of which is located in the N-terminus
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1280.
(3) Neckers, L.; Lee, Y.-S. Nature 2003, 425, 357.
10.1021/ol050580a CCC: $30.25
© 2005 American Chemical Society
Published on Web 05/03/2005

key hydrogen bond network with conserved water molecules
coordinated through Asp93 in the region that typically binds
the adenine ring of ATP. The carbamate on GDA provides
similar interactions with this portion of the binding pocket.
The quinone ring of GDA interacts with Asp54, Lys58,
Lys112 and other amino acids, while the epoxide on RDC
maintains only one hydrogen bond with Lys58. As a
consequence of increasing interactions with Hsp90, we
proposed that a molecule containing the quinone ring of GDA
and the resorcinol ring of RDC would provide a new scaffold
for the development of Hsp90 inhibitors. This chimera or
hybrid of radicicol and geldanamycin we named radana-
mycin ester (Radester, Figure 3).
Figure 1. Known inhibitors of Hsp90.
Although 17-AAG has entered phase I clinical trials8,9 for
the treatment of several cancers, the quinone ring is redox-
active. GDA has been shown to generate superoxide radicals
in cells, which can lead to cell death without interfering
directly with Hsp90.10 In vivo, RDC is rapidly converted to
inactive metabolites that have little or no affinity for Hsp90.11
Consequently, researchers throughout industry and academia
have pursued the development of new Hsp90 inhibitors
without these detrimental properties.6
Figure 3. Hsp90 inhibitors.
The previously solved co-crystal structures of RDC and
GDA bound to Hsp90 (Figure 2) clearly demonstrated that
Radester not only contains the quinone ring of GDA and
the resorcinol ring of RDC but also maintains the isopropyl
ester that is present on RDC and the same substituent
attached to the GDA quinone. Because this molecule can be
prepared in a minimal number of steps, we envision radester
as a starting point for the construction of Hsp90 inhibitors
with tunable properties that can be enhanced by derivatization
of either the quinone ring to produce non-redox-active
analogues or modification of the resorcinol ring to incorpo-
rate additional hydrophobic moieties that can project into
the π-rich lipophilic pocket formed by amino acids Phe138,
Trp162, and Tyr139.
The synthesis of radester began by preparation of the
resorcinolic moiety. Chlorination of methyl 2,4-dihydroxy-
benzoate (2) with sulfuryl chloride furnished 3, which was
hydrolyzed to furnish acid 4 in good yield, Scheme 1.
Figure 2. Superimposed co-crystal structures of GDA (yellow)
and RDC (magenta) with Hsp90.
Scheme 1. Synthesis of 5-Chloro-2,4-dihydroxybenzoic Acid
the resorcinol ring of RDC and the quinone of GDA bound
in opposite orientations.12 The resorcinol moiety provides a
(8) Sausville, E. A.; Tomaszewski, J. E.; Ivy, P. Curr. Cancer Drug.
Targets 2003, 3, 377.
(9) Adams, J.; Elliott, P. J. Oncogene 2000, 19, 6687. (b) Neckers, L.
Trends Mol. Med. 2002, 8, S55.
(10) Dikalov, S.; Landmesser, U.; Harrison, D. G. J. Biol. Chem. 2002,
277, 25480.
(11) Geng, X.; Yang, Z.-Q.; Danishefsky, S. J. Synlett 2004, 8, 1325.
2158
Org. Lett., Vol. 7, No. 11, 2005

In light of previous work by our laboratory that suggested
the hydroquinone was more potent than the corresponding
quinone,13 we designed a synthetic sequence that would allow
for the preparation of radester quinone and the corresponding
hydroquinone. As such, we chose to mask the hydroquinone
as the bis(methoxymethylene)ether, which could be removed
to provide the hydroquinone and upon subsequent oxidation
afford the corresponding quinone.
Scheme 3. Synthesis of Radester
The quinone precursor was prepared from 2-methoxy-1,4-
bis(methoxymethyleneoxy)-benzene14 (5, Scheme 2). Treat-
Scheme 2. Synthesis of MOM-Protected Precursor to the
Quinone
ment of this protected hydroquinone with n-butyllithium in
the presence of N,N,N′,N′-tetramethylethylenediamine af-
forded the lithium anion, which upon addition of propylene
oxide provided the secondary alcohol, 6, in good yield.
Nitration of the aromatic ring containing the acid-labile
protecting groups proved to be problematic under normal
nitration conditions. However, success was granted by
treatment of the aromatic ring with ammonium nitrate and
trifluoroacetic anhydride,15 which afforded the nitrated
product in good yield. However, under these conditions the
secondary alcohol was converted to the trifluoroacetyl ester,
which was hydrolyzed by the addition of lithium hydroxide
to furnish 7.
tion of the hydroquinone required stoichiometric palladium
on carbon and oxygen to give radester 1.17
Cell proliferation studies with 11 and 1 were performed
in the MCF-7 breast cancer cell line. As demonstrated by
Figures 4A and 4B, the IC50 for 11 and 1 was 7.1 ( 0.3 µM
and 13.9 ( 1.4 µM, respectively.
Coupling of 4 with 7 proved to be difficult, and after
considerable optimization, 8 was best provided by treatment
with dicyclohexylcarbodiimide and N,N-(dimethylamino)-
pyridine, Scheme 3. The aromatic nitro substituent was
converted to aniline 9 without reduction of the aryl-chloride
bond in the presence of platinum (IV) oxide and hydrogen.
Addition of phenolformate16 to 9 gave the N-formylated
product, 10, which mimicked the same amide functionality
found in GDA. Following the procedure of Andrus and co-
workers,14 the bis(methoxymethyleneoxy) groups were cleaved
upon exposure to in situ-derived trimethylsilyl iodide to
provide the hydroquinone precursor (11) to radester. Oxida-
Figure 4. Cytotoxicity profiles for 11 and 1 in MCF-7 breast cancer
cells (µM). The data represent the average of three experiments.
(12) Roe, S. M.; Prodromou, C.; O’Brien, R.; Ladbury, J. E.; Piper, P.
W.; Pearl, L. H. J. Med. Chem. 1999, 42, 260.
(13) Clevenger, R. C.; Blagg, B. S. J. Org. Lett. 2004, 6, 4459.
(14) Andrus, M. B.; Hicken, E. J.; Meredith, E. L.; Simmons, B. L.;
Cannon, J. F. Org. Lett. 2003, 5, 3859.
To determine whether the observed cytotoxicity was
(15) Crivello, J. V. J. Org. Chem. 1981, 46, 3056.
(16) Yale, H. L. J. Org. Chem. 1971, 36, 3238.
related to Hsp90 inhibition, 11 and 1 were incubated with
Org. Lett., Vol. 7, No. 11, 2005
2159

MCF-7 breast cancer cells for 24 h. As can be seen from
Western blot analyses of the protein lysates, both 11 and 1
resulted in the concentration-dependent degradation of Hsp90
client proteins, Her-2 and Raf (Figure 5). Hsp70 levels
and G3130, respectively (Figure 6). Together, these data
support our hypothesis that the quinone-binding region of
Hsp90 plays an important role in ligand recognition and
binding, despite its close proximity to the aqueous media
(vida infra the co-crystal structure of the Hsp90 N-terminal
domain bound to inhibitors).
Figure 6. Hsp90 inhibitors with a hydrogen bond donor in the
“quinone” binding region of the N-terminus.
These studies support our hypothesis that chimeric mol-
ecules derived from the two most potent natural product
inhibitors, RDC and GDA, represent a promising new class
of Hsp90 inhibitors. Additional structure-activity relation-
ship studies are needed to provide non-redox-active ana-
logues and chimeric molecules with greater inhibitory
activity. Such studies are currently underway and will be
reported in due course.
Figure 5. Western blot analyses of Hsp90 client protein degrada-
tion assays. Concentration of inhibitors (in µM) are denoted above
each lane.
increased in a concentration-dependent manner, which is
consistent with other Hsp90 inhibitors.6 Since actin is not
dependent upon the Hsp90 protein folding machinery, actin
levels remained unchanged.
Acknowledgment. The authors gratefully acknowledge
support of this project by the NIH COBRE RR017708, The
University of Kansas Center for Research, and The Univer-
sity of Kansas General Research Fund. G.S. is the recipient
of a Susan G. Komen Breast Cancer Foundation Dissertation
Award.
These results suggest the importance of a hydrogen bond
donor in the region that typically binds the triphosphate
moiety of Hsp90’s natural substrate, ATP. After our initial
publication disclosing radamide,13 researchers at Kosan
Biosciences18 and Novartis19 also reported molecules with
hydrogen bond donors in the same proximity, KOSN1559
Supporting Information Available: Experimental pro-
cedures and characterization for all compounds in this letter.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(17) Luly, J. R.; Rapoport, H. J. Org. Chem. 1984, 49, 1671.
(18) Patel, K.; Piagentini, M.; Rascher, A.; Tian, Z.-Q.; Buchanan, G.
O.; Regentin, R.; Hu, Z.; Hutchinson, C. R.; McDaniel, R. Chem. Biol.
2004, 11, 1625.
(19) Kreusch, A.; Han, S.; Brinker, A.; Zhou, V.; Choi, H.-S.; He, Y.;
Lesley, S. A.; Caldwell, J.; Gu, X.-J. Bioorg. Med. Chem. Lett. 2005, 15,
1475.
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