Design, synthesis, and biological evaluation of conformationally constrained cis-amide HSP90 inhibitors

Article abstract of DOI:10.1021/ol900783m

Conformationally constrained cis-amide chimeric inhibitors of Hsp90 have been synthesized and evaluated for their Hsp90 inhibitory activity. These new compounds exhibited Hsp90 ATPase inhibition and induced Hsp90-dependent client protein degradation in a

Full text of DOI:10.1021/ol900783m

ORGANIC
LETTERS
2009
Vol. 11, No. 11
2353-2356
Design, Synthesis, and Biological
Evaluation of Conformationally
Constrained cis-Amide Hsp90 Inhibitors
Adam S. Duerfeldt, Gary E. L. Brandt, and Brian S. J. Blagg*
Department of Medicinal Chemistry, The UniVersity of Kansas, 1251 Wescoe Hall
DriVe, Malott 4070, Lawrence, Kansas 66045-7562
bblagg@ku.edu
Received April 10, 2009
ABSTRACT
Conformationally constrained cis-amide chimeric inhibitors of Hsp90 have been synthesized and evaluated for their Hsp90 inhibitory activity.
These new compounds exhibited Hsp90 ATPase inhibition and induced Hsp90-dependent client protein degradation in a dose-dependent
manner. Biological data reported herein suggests that amide bond isomerization of geldanamycin derivatives plays an important role in affinity
for the heteroprotein complex present in cancer cells.
The 90 kDa heat shock proteins (Hsp90) are ATP-dependent
molecular chaperones overexpressed in response to cellular
stress and necessary for the folding and rematuration of
nascent and denatured polypeptides, respectively.1-4 Two
natural products, geldanamycin5 (GDA, Figure 1) and
radicicol6 (RDC), bind the Hsp90 N-terminal binding pocket
and competitively inhibit ATP binding resulting in degrada-
tion of Hsp90-dependent client proteins via the ubiquitin-
proteasome pathway.7 Hsp90 clientele play key roles in all
six hallmarks of cancer;8 therefore, inhibition of the Hsp90
protein folding machinery results in simultaneous disruption
of all six mechanisms of oncogenesis.9,10 Consequently, not
only has Hsp90 emerged as a promising anticancer target,11
but GDA and RDC have proven to represent excellent
models for which the development of new Hsp90 inhibitors
can be pursued for drug development and mechanistic
studies.12
Although RDC and GDA bind Hsp90 with high affinity,
their modes of binding and inhibitory activities are different.
(1) Freeman, B. C.; Morimoto, R. I EMBO J. 1996, 15, 2969
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L. M. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 8324.
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(6) Schulte, T. W.; Akinaga, S.; Soga, S.; Sullivan, W.; Stensgard, B.;
Toft, D.; Neckers, L. M. Cell Stress Chaperones 1998, 3, 100.
(12) Hadden, M. K.; Lubbers, D. J.; Blagg, B. S. J. Curr. Top. Med.
Chem. 2006, 6, 1173.
10.1021/ol900783m CCC: $40.75
Published on Web 05/12/2009
 2009 American Chemical Society

Radicicol exists in the same bent conformation when bound
and unbound to Hsp90 and produces a favorable entropy of
8.3 cal/mol upon binding. Not surprisingly, the predisposition
of RDC to the bent conformation is believed to be responsible
for its similar activity in both cellular and recombinant
assays.13
ever, to the best of our knowledge, no analogues have been
synthesized that exhibit a predisposed cis-amide functionality.
Figure 1. Inhibitors of Hsp90.
Figure 2. GDA conformations in solution (left) and bound to Hsp90
(right).
In contrast to the bent, cis-amide conformation of GDA
when bound to Hsp90, both solution and crystal structures
have demonstrated that this natural product exists in an
extended, trans-amide conformation when unbound to
Hsp90 (Figure 2).14 Multiple studies have demonstrated
that prior to binding Hsp90, GDA must undergo two
conformational changes; the ansa ring must rotate over
the benzoquinone moiety and the amide bond must
isomerize from trans to cis by rotation about C1-N22 and
C20-N22 (Figure 1). The first event is reported to occur
spontaneously; however, isomerization of the amide bond
is estimated to exceed 20 kcal/mol.15 Accordingly,
isothermal titration calorimetry (ITC) experiments have
shown that GDA exhibits an entropic penalty of -6.4 cal/
mol upon binding Hsp90.16,17
Recently, chimeric inhibitors of Hsp90 were disclosed
that contained both the quinone ring from GDA and the
resorcinol moiety of RDC in an attempt to mimic the
hydrogen bonding interactions exemplified by the two
natural products when bound to the Hsp90 N-terminal
nucleotide-binding pocket. This approach has produced
novel scaffolds for Hsp90 inhibition; however, none of
the reported analogues exhibit conformational character-
istics exhibited by either natural product when bound to
Hsp90.21-23 Analysis of the seco derivative, radamide (RDA,
Figure 1), revealed the potential to introduce conformational
aspects of both natural products when bound to Hsp90.
Herein we present the first series of conformationally
constrained chimeric Hsp90 inhibitors exhibiting both a cis-
amide moiety and a bent conformation.
Retrosynthetically, we envisioned the desired analogues
could be obtained via a Horner-Wadsworth-Emmons
(HWE) olefination reaction between cyclic phosphonate
1 and homologated aldehydes, 2 and 3 (Scheme 1).
Compound 1 was proposed to result from tandem reduc-
tion/intramolecular cyclization of compound 4, which
could be obtained from commercially available 4-benzy-
loxy-3-methoxybenzaldehyde in four steps. Aldehydes 2
and 3 could be prepared directly from 521 and 6,
respectively.
As a consequence of these thermodynamic data, Santi
and co-workers hypothesized that GDA analogues con-
taining a predisposed cis-amide bond will result in ∼1000-
fold increase in Hsp90 affinity through reduction of
entropic penalties. Such postulations have inspired sub-
sequent studies aimed at determining the effect of trans/
cis isomerization of the GDA-amide moiety.18-20 How-
(13) Roe, S. M.; Prodromou, C.; O’ Brien, R.; Ladbury, J. E.; Piper,
P. W.; Pearl, L. H. J. Med. Chem. 1999, 42, 260.
(14) Thepchatri, P.; Eliseo, T.; Cicero, D. O.; Myles, D.; Snyder, J. P.
J. Am. Chem. Soc. 2007, 129, 3127.
(15) Lee, Y.; Marcu, M. G.; Neckers, L. Chem. Biol. 2004, 11, 991.
(16) Chiosis, G.; Huezo, H.; Rosen, N.; Mimnaugh, E.; Whitesell, L.;
Neckers, L. Mol. Cancer Ther. 2003, 2, 123
(17) Schulte, T. W.; Neckers, L. M. Cancer Chemother. Pharmacol.
1998, 42, 273
(18) Onuoha, S. C.; Mukund, S. R.; Coulstock, E. T.; Sengerova, B.;
Shaw, J.; McLaughlin, S. H.; Jackson, S. E. J. Mol. Biol. 2007, 372, 287
(19) Onodera, H.; Kaneko, M.; Takahashi, Y.; Uochi, Y.; Funahashi,
J.; Nakashima, T.; Soga, S.; Suzuki, M.; Ikeda, S.; Yamashita, Y.; Rahayu,
.
Commencing with commercially available 4-benzyloxy-
3-methoxybenzaldehyde (7, Scheme 2), phenol 8 was formed
Via Dakin oxidation.24 Treatment of 8 with diazophosphonate
9 resulted in the phenolic ether 10 upon a rhodium carbenoid
.
.
E. S.; Kanda, Y.; Ichimura, M. Bioorg. Med. Chem. Lett. 2008, 18, 1577
.
(21) Clevenger, R. C.; Blagg, B. S. J. Org. Lett. 2004, 6, 4459–4462
(22) Shen, G.; Blagg, B. S. J. Org. Lett. 2005, 7, 2157
(23) Shen, G.; Wang, M.; Welch, T. R.; Blagg, B. S. J. J. Org. Chem.
2006, 71, 7618
(24) Li, S.; Chen, L.; Tsai, J.; Tzeng, J.; Tsai, I.; Chi, E. Tetrahedron
Lett. 2007, 48, 2139.
.
(20) Zhang, M. Q.; Gaisser, S.; Nur-E-Alam, M.; Sheehan, L. S.;
Vousden, W. A.; Gaitatzis, N.; Peck, G.; Coates, N. J.; Moss, S. J.; Radzom,
M.; Foster, T. A.; Sheridan, R. M.; Gregory, M. A.; Roe, S. M.; Prodromou,
C.; Pearl, L.; Boyd, S. M.; Wilkinson, B.; Martin, C. J. J. Med. Chem.
.
.
2008, 51, 5494
.
2354
Org. Lett., Vol. 11, No. 11, 2009

Scheme 1
.
Retrosynthetic Analysis for the Synthesis of
cis-Amide Chimeric Analogues
Scheme 2. Synthesis of cis-Amide Cyclic Phosphonate
products 19 and 20, which were subjected to chiral HPLC
to afford the enantiopure analogues 21-24 (Scheme 4).
Scheme 3. Synthesis of Homologated Aldehydes
mediated O-H insertion.25 Regioselective nitration of 10 was
accomplished under mild conditions enlisting ammonium
nitrate and trifluoroacetic anhydride. Treatment of 4 with
tin(II) chloride under refluxing conditions resulted in not only
reduction of the nitro group to the corresponding aniline but
also cyclization to give the desired lactam, 1. The protected
resorcinol precursors were prepared by treatment of 5 with
lithium diisopropylamide at -78 °C to generate the benzylic
anion, which was quenched upon addition of dimethylfor-
mamide to afford the aldehyde product, 2, or with allyl
bromide to give 6 (Scheme 3). Oxidation of 6 with osmium
tetroxide gave the corresponding diol, which was cleaved
in situ with sodium periodate to yield homologated aldehyde
3.
With the synthons in hand, the fragments were joined Via
a Horner-Wadsworth-Emmons olefination reaction (3:1,
E/Z) and subsequent removal of the protecting groups with
tetrabutylammonium fluoride gave compounds 11-14
(Scheme 4).
Originally, we were interested in the saturated analogues,
but realizing the olefinated intermediates exhibited higher
conformational rigidity than the saturated analogues, we
attempted to selectively remove the benzyl ether in the
presence of the unsaturated amide with FeCl3, Pd(OAc)2/
Et3SiH/Et3N, and NaI/TMSCl. However, none of these
conditions afforded the desired products. Finally, conditions
were employed utilizing aluminum(III) chloride in anisole
that effectively cleaved the benzyl ether without alteration
of the R,ꢀ-unsaturated amide to afford compounds 15-18
(Scheme 4).
After completing the synthesis of 10 analogues, biological
investigation was undertaken. Antiproliferation studies with
compounds 15-24 were conducted with MCF7 and SKBr3
breast cancer cell lines. As shown in Table 1, the E-olefin is
more active than the Z-olefin for both linker lengths and the
(+)-enantiomer is more active than the (-)-enantiomer.
To evaluate this series for inhibition of ATPase activity,26
recombinant yeast Hsp90 was overexpressed and purified.27
The purified protein was incubated with ATP in the presence
of 15, 17, 21, and 23. As shown in Table 1, the IC50 values
were similar to those found in the antiproliferation studies,
suggesting equipotency of these analogues for the hetero-
protein complex present in cancer cells versus the purified
homodimer representative of normal cells.
Standard hydrogenation conditions with palladium on
carbon under a hydrogen atmosphere yielded racemic
(26) Avila, C.; Kornilayev, B. A.; Blagg, B. S. J. Bioorg. Med. Chem.
2006, 14, 1134.
(27) Richeter, K.; Muschler, P.; Hainzl, O.; Buchner, J. J. Biol. Chem.
2001, 276, 33689.
(25) Haigh, D. Tetrahedron 1994, 50, 3177.
Org. Lett., Vol. 11, No. 11, 2009
2355

heteroprotein complex present in cancer cells versus purified
recombinant Hsp90, which is reminiscent of the form found
in normal cells.15,28,29 In addition, the data suggest the
heteroprotein complex is more sensitive to amide isomer-
ization and conformational predisposition. Further SAR
studies and cocrystallization experiments are ongoing in an
effort to delineate the interactions of this class of compounds
with Hsp90 and the results from such studies will be reported
in due course.
Scheme 4. Fragment Coupling and Deprotection
Table 1. Anti-Proliferation and ATPase Activity of cis-Amide
Analogues
a
IC50 (µM)
compd
15
16
17
MCF7
SKBr3
ATPase
3.2 ( 0.1
78.9 ( 11.3
1.5 ( 0.1
1.8 ( 0.1
57.1 ( 8.4
1.2 ( 0.1
2.4 ( 0.1
1.6 ( 0.0
18
19
9.8 ( 1.9
4.8 ( 1.1
15.9 ( 4.1
7.3 ( 0.2
20
6.3 ( 0.9
7.8 ( 0.2
21
22
23
3.6 ( 0.1
14.9 ( 0.2
2.7 ( 0.5
2.1 ( 0.2
15.4 ( 0.8
2.1 ( 0.7
1.5 ( 0.1
1.2 ( 0.0
24
78.0 ( 4.4
18.6 ( 0.9b
9.8 ( 0.1 nM
47.7 ( 2.6 nM
15.4 ( 0.8
23.7 ( 1.7b
8.5 ( 1.1 nM
37.5 ( 4.0 nM
To confirm the cytotoxic nature of these compounds
resulted from Hsp90 inhibition, compounds 17 and 23 were
incubated with MCF7 cells for 24 h, and Western blot
analyses were performed with the cell lysates (see Supporting
Information). As expected, the immunoblots confirmed
concentration-dependent degradation of Hsp90-dependent
client proteins, Her-2 and Raf.
radamide
GDA
RDC
5.9c
2.5 ( 0.4
0.4 ( 0.0
a IC50 ) concentration needed to produce 50% inhibition. b Hadden,
M. K.; Blagg, B. S. J. J. Org. Chem. 2009, in press. c Obtained from ref
21.
In conclusion, we have designed, synthesized, and pro-
vided biological data for a series of analogues that not only
contain hydrogen-bonding moieties exhibited by GDA and
RDC, but also exhibit the conformational biases adopted by
the natural products when bound to Hsp90 Via inclusion of
a bent conformation and a cis-amide. In accord with Santi
and co-workers, introduction of a cis-amide moiety increased
Hsp90 inhibitory activity (∼10-fold). As indicated by the
biological results, introduction of both the cis-amide and the
bent conformation to the chimeric inhibitors does not
influence activity against purified recombinant Hsp90; how-
ever, the activity against cancer cells increased ∼10-fold with
respect to the seco derivative, radamide.
Acknowledgment. We gratefully acknowledge the Pris-
inzano laboratory for instrumentation use. We thank the
Madison and Lila Self Graduate Fellowship and the Ameri-
can Foundation of Pharmaceutical Education (A.F.P.E.) for
financial support. This project was funded by NIH/NCI
CA109265.
Supporting Information Available: Experimental pro-
cedures and characterization for all compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL900783M
(28) Chiosis, G.; Neckers, L. ACS Chem. Biol. 2006, 1, 279
(29) Kamal, A.; Thao, L.; Sensintaffar, J.; Zhang, L.; Boehm, M. F.;
Fritz, L. C.; Burrows, F. J. Nature 2003, 425, 407
.
These results coincide with the ideas posed by multiple
groups, whom suggest a mechanistic difference between the
.
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Org. Lett., Vol. 11, No. 11, 2009