Divergent synthesis of a pochonin library targeting HSP90 and in vivo efficacy of an identified inhibitor

Article abstract of DOI:10.1002/anie.200800233

(Chemical Equation Presented) Make a clean breast of it: The generation of a library of pochonin Dderivatives by a solid-phase approach has led to the discovery of pochoxime (see scheme), a potent inhibitor of the heat-shock protein 90, with over 100-fold improved incellular activity. Pochoxime was found to be effective in breast tumor xenografts, leading to a reduction in the tumor size.

Full text of DOI:10.1002/anie.200800233

Communications
DOI: 10.1002/anie.200800233
Antitumor Agents
Divergent Synthesis of a Pochonin Library Targeting HSP90 and
In Vivo Efficacy of an Identified Inhibitor**
Sofia Barluenga, Cuihua Wang, Jean-Gonzague Fontaine, Kaïss Aouadi, Kristin Beebe,
Shinji Tsutsumi, Len Neckers, and Nicolas Winssinger*
The heat-shock protein 90 (HSP90) has emerged as one of the
most exciting therapeutic targets in recent years.[1,2] Despite
the seemingly ubiquitous function of this constitutively
expressed chaperone,its role in stabilizing conformationally
labile proteins has implications in pathologies ranging from
oncology to neurodegenerative diseases. Most endogenous
clients[3] of HSP90s are key regulators of cell signaling which
are destabilized and degraded in the absence of the chaper-
oning activity of HSP90. The dependence of transformed cells
on HSP90 is further heightened by the fact that many
oncogenic mutations,while increasing the activity of pro-
growth signaling pathways,are less stable than their wild-type
counterparts and have an increased dependence on the
chaperoning activity of HSP90.[4] A clinically relevant exam-
ple is the heightened dependence of drug-resistant Bcr-Abl
mutants on the activity of HSP90,and the fact that HSP90
inhibitors in combination with Abl inhibitors remain effective
against such mutants.[5,6] Accordingly,HSP90 inhibition
provides a broad and effective target for the treatment of
cancer. Furthermore,HSP90 inhibitors can act synergistically
with a cytotoxic agent.[7] HSP90 is also implicated in regulat-
Scheme 1. Structure of radicicol, geldanamycin, 17AAG, pochonin D,
and general structure of the pochonin library (5).
[*] Dr. S. Barluenga, Dr. C. Wang, J.-G. Fontaine, Dr. K. Aouadi,
Prof. N. Winssinger
Institut de Science et d’IngØnierie Supramoleculaires
UniversitØ Louis Pasteur
8 allØe Gaspard Monge, 67000 Strasbourg (France)
Fax. (+33)3-9024-5112
E-mail: winssinger@isis.u-strasbg.fr
Homepage: www-isis.u-strasbg.fr/winssinger/
Scheme 2. Reagents and conditions: a) LDA (2.0 equiv), THF, À788C,
5 min; 7 (1.0 equiv), 10 min; PS-COOH (5.0 equiv), À78 to 238C,
20 min, ca. 50%; b) NH2OCH2CHO2H (5.0 equiv), Py/AcOH 5:1, 408C,
24 h, 45–95%; c) PS-ClTr-Cl (3.0 equiv), DIPEA (6.0 equiv), CH2Cl2,
238C, 24 h; then AcOH (20 equiv), 238C, 24 h; d) TBAF (4.0 equiv),
238C, 4 h; e) 10 (5.0 equiv), Ph3P (2.0 equiv), DEAD (2.0 equiv),
toluene, 238C, 12 h, f) Grubbs II cat. (0.06 equiv), CH2Cl2, 1208C,
microwaves, 3”45 min; g) HFIP/CH2Cl2 (1:4), 238C, 3 h, 20–30% over
5 steps; h) PS-DCC (3.0 equiv), DMAP (cat.), R3-H (2.0 equiv), 238C,
72 h, ca. 75%; i) PS-SO3H (10 equiv), MeOH, 238C, 4 h, ca. 85%.
DCC=N,N’-dicyclohexylcarbodiimide, DEAD=diethylazodicarboxylate,
DIPEA=N,N-diisopropylethylamine, DMAP=4-dimethylaminopyridine,
EOM=ethoxymethyl, HFIP=hexafluoroisopropanol, LDA=lithium di-
isopropylamide, PS=polystyrene, Py=pyridine, TBAF=tetrabutylam-
monium fluoride, Tr=trityl.
K. Beebe, Dr. S. Tsutsumi, Dr. L. Neckers
National Cancer Institute, Urologic Oncology Branch
9000 Rockville Pike, Bethesda, MD 20892 (USA)
[**] This work was funded in part by a grant from the Agence National de
la Recherche (ANR) and Conectus. A BDI fellowship(J.-G.F.) is also
gratefully acknowledged. We thank Emilie Moulin for preliminary
work on this project.
Supporting information for this article (including physical charac-
terization of compounds 13 and 14) is available on the WWW under
http://www.angewandte.org or from the author.
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ing the fate of a number of conformationally unstable proteins
which are associated with the development of neurodegener-
ative diseases.[8] It has been shown that HSP90 inhibitors can
reduce protein aggregates in cellular and animal models of
Huntington disease,[9] spinal and bulbar muscular atrophy,[10]
Parkinson disease,[11] and other Tau protein related neuro-
degenerative diseases.[12]
down-regulation of the client proteins of HSP90 revealed
important structure–activity relationships and pointed to the
ketone moiety as the most favorable position for improving
the activity. Herein,we report the structure–activity relation-
ship of a focused library of this important pharmacophore
which has led to the identification of an analogue of
pochonin D that has a 100-fold improvement in cellular
activity and we report its efficacy in a breast tumor xenograft
(BT-474).
While some of the simpler resorcylides such as pocho-
nin D had good affinity for HSP90,their cellular activity was
disappointing in comparison to that of 17AAG. The ambig-
uous correlation between the affinity of 17AAG for HSP90
and its cellular activity remains a subject of intense inves-
tigation,[29–31] but can be rationalized by the kinetics of
binding.[31] Similar discrepancies between HSP90 affinity
measured by a fluorescence polarization assay and ATPase
inhibition have been noted for inhibitors based on the
resorcylides motif.[26]
Two natural products,radicicol and geldanamycin ( 1 and
2,Scheme 1),were instrumental in understanding the role of
HSP90 in oncogenic processes as well as its therapeutic
potential.[13–15] However,neither natural product has accept-
able pharmacological properties for clinical application.
Structure-based design and high-throughput screening have
led to the discovery of novel scaffolds such as purines[16,17] and
pyrazoles;[18] however,improving the pharmacological prop-
erties and potency of the natural pharmacophores remains
important. Indeed,the most advanced clinical candidate is
17AAG (3,Scheme 1),the semisynthetic derivative of
geldanamycin,which is currently in multiple phase II stud-
ies.[19] Another semisynthetic derivative with a dimethoxyhy-
droquinone functionality has recently been reported to have
better pharmacological properties than 17AAG while acting
as a prodrug.[20] Radicicol,although having a higher affinity
than geldanamycin for HSP90,suffers from two limitating
features: a strained and highly sensitive epoxide and a
conjugate diene which functions as a
Based on the observation that oxime substitutions in the
pochonin series did not affect the HSP90 activity,and inspired
by previous success with radicicol,[21–23] we developed a
divergent synthesis that provided rapid access to this class
of compounds. Readily available intermediate 6 was depro-
tonated with LDA (Scheme 2) and treated with Weinreb
Michael acceptor. Indeed,the inactiv-
ity of radicicol in animal models has
been attributed to a conjugate addi-
tion of thiol nucleophiles at the C13-
position.[21] Akinaga and co-workers
overcame this limitation by convert-
ing radicicol into an oxime,which
showed significant antitumor activity
(reduction in tumor growth) in animal
models.[21–23] Mindful of the labile
epoxide,Danishefsky and co-workers
reported a cyclopropyl analogue of
radicicol which was nearly as effective
in cellular assays; however,its efficacy
in animals has not been reported.[24,25]
More recently,Moody and co-work-
ers reported the synthesis of radicicol-
related resorcylides,and explored the
importance of the size of the macro-
cyle.[26]
We previously suggested that the
epoxide moiety of radicicol is impor-
tant as a conformational bias which
favors the bioactive conformation of
the macrocycle,and have shown that
another natural product,pochonin D
(4,Scheme 1),was also a good ligand
for HSP90.[27] Furthermore,we have
reported the use of polymer-sup-
ported reagents to synthesize a library
that extends the diversity of the
Figure 1. Biological activity (mm) of pochonin-oxime derivatives: HSP90a affinity, client depletion
(Her-2 from SKBr3 cell line), and cytotoxicity (SKBr3 and HCC1954 respectively). * denotes an
pochonins (5,Scheme 1). [28] Screening
this library for HSP90 affinity and approximate 1:1 mixture of E/Z oximes.
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amide 7. Quenching the reaction with benzoic acid resin
vehicle) or drug for 28 days following the q2d schedule
revealed a dramatic loss of cellularity in tumors obtained
from drug-treated animals. The nuclei of remaining cells were
uniformly condensed,thus suggesting the occurrence of
massive apoptosis (Figure 3,top panels). This finding was
confirmed by the high degree of nuclear TUNEL staining
seen in tumors excised from drug-treated animals (Figure 3,
bottom panels). These data suggest that tumor regression in
animals treated for 28 days with the q2d schedule may be
more dramatic than estimated from measurement of the
tumor volume,as depicted in Figure 3,since few to no viable
cells could be identified at the end of the treatment period.
In conclusion,pochoximes 13a and 13b have a higher
affinity for HSP90 and are more active in reducing the client
proteins of HSP90 than is radicicol. This is the first report of
an HSP90 inhibitor based on the resorcylic macrolide scaffold
to show a regression in tumor size,and their effectiveness at
doses below the maximum tolerated dose suggest a mean-
ingful therapeutic window. The use of polymer-bound
reagents[36] and solid-phase chemistry has facilitated the
sequestered the amine by-products and afforded 8 with
sufficient purity to be engaged directly in the formation of
an oxime on reaction with aminooxyacetic acid. After
evaporation of the solvent,the crude mixture was treated
with an acidic resin,which removed the excess hydroxylamine
and some by-products stemming from conjugate addition,to
afford 9. Intermediate 9 was then loaded onto 2-chlorotrityl
resin and the carboxylate group was deprotected with TBAF
to reveal the acid,which was subsequently engaged in a
Mitsunobu esterification with homoallylic alcohols 10 to
obtain polymer-bound intermediates 11. It is important to
note that this reaction sequence is not possible in the absence
of the oxime functionality as it leads to the formation of
coumarin.[32] The resins were then treated with the second-
generation Grubbs catalyst under microwave irradiation to
obtain the macrocycles 12 in excellent yield and purity after
cleavage from the resin with hexafluoroisopropanol (HFIP).
In contrast to TFA,these mild cleavage conditions were
found to leave the EOM groups intact,thus enabling a
subsequent selective esterification or amidation. For this
purpose,we used an immobilized carbodiimide reagent
followed by treatment with a sulfonic acid resin to obtain a
library of pochonin oximes 13.
The library was then screened for affinity to HSP90a,[33]
Her-2 (Hsp90 client) degradation,[34] and cytotoxicity against
SKBr3 and HCC1954,two breast cancer cell lines which
overexpress Her-2 (Figure 1). The most potent inhibitors
were compounds 13a and 13b,which contain the piperidine
amide moiety. It is interesting to note that the simplified
analogue lacking the chiral methyl group is as active as the
parent compound 13c,and that while the chlorine atom is
important for the activity of both radicicol and pochonin D,it
is not important for the activity of 13b. The structure–activity
data suggest that the piperidine amide has a relatively good fit
in a lipophilic pocket,as the morpholino analogue ( 13d),
piperazine analogue (13e),and simple methylamide ( 13i)
have significantly lower activity. The cyclohexylamide (13k)
or benzylamide (13j) analogues,on the other hand,were also
good ligands. Consistent with the previous radicicol oxime,[22]
it is interesting to note that there is a significant difference in
activity between the E and the Z isomers,with the E isomer
having higher activity (13a versus 13 f and 13b versus 13g).
Compound 13a was further evaluated in vivo because of its
potent activity.
Treatment of CB17/SCID mice with 13a at 100 mgkgÀ1
for five consecutive days was well tolerated,with minimal
weight loss observed. To investigate the in vivo efficacy of
13a,a xenograft bearing BT-474 (breast-tumor cell line) was
used,as this tumorgenic cell line has been shown to respond
to HSP90 inhibitors[35] in an animal model. Based on the
cellular potency of 13a,two schedules of 100 mg every other
day (q2d) or every four days (q4d) over 28 days were
investigated. Gratifyingly,treatment with 13a resulted in a
dose-dependent inhibition of the tumor growth,with an 18%
regression in the tumor volume using the q2d schedule (p =
0.0002,Figure 2a). In neither schedule was a significant
weight loss observed (Figure 2b). Histologic examination of
tumors removed from animals receiving either DMSO (as
Figure 2. a) Tumor volume (BT474) and b) animal weight following
treatment with 13a or the control vehicle (DMSO). Each point
represents the mean of measurements from five (for the vehicle) or six
(for 13a) animals. See the Supporting Information for errors and
statistical analysis.
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Received: January 16,2008
Revised: February 28,2008
Published online: April 25,2008
Keywords: antitumor agents · bioorganic chemistry ·
.
natural products · solid-phase synthesis · synthesis design
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