Stereoselective synthesis of F-ring saturated estrone-derived inhibitors of hedgehog signaling based on cyclopamine

Article abstract of DOI:10.1021/ol2017966

Previous work in this laboratory established that the readily available F-ring aromatic analog of cyclopamine is a highly potent inhibitor of Hedgehog signaling. The synthesis and biological evaluation of two F-ring saturated analogs that are more potent

Full text of DOI:10.1021/ol2017966

ORGANIC
LETTERS
2011
Vol. 13, No. 18
4786–4789
Stereoselective Synthesis of F-Ring
Saturated Estrone-Derived Inhibitors
of Hedgehog Signaling Based on
Cyclopamine
†
‡
†
‡
ꢀ
Zhihui Zhang, Valerie Baubet, Christian Ventocilla, Chaomei Xiang,
Nadia Dahmane,*,‡ and Jeffrey D. Winkler*,†
Department of Chemistry, The University of Pennsylvania, Philadelphia,
Pennsylvania 19104, United States, and The Wistar Institute, Molecular and Cellular
Oncogenesis Program, Philadelphia, Pennsylvania 19104, United States
winkler@sas.upenn.edu; ndahmane@wistar.org
Received July 5, 2011
ABSTRACT
Previous work in this laboratory established that the readily available F-ring aromatic analog of cyclopamine is a highly potent inhibitor of
Hedgehog signaling. The synthesis and biological evaluation of two F-ring saturated analogs that are more potent than the F-ring aromatic
structure are reported.
Some time ago it was found that ingestion of the
California corn lily, veratrum californicum, by pregnant
sheep could induce cyclopia and other profound develop-
mental defects in the offspring. Keeler and co-workers
established that the active constituent in the plant is the
alkaloid cyclopamine 1 (Figure 1),1 and it was later found
that cyclopamine mediates this effect by interfering with
Sonic Hedgehog (SHH) signaling,2 specifically via the
inhibition of the transmembrane protein Smoothened
(SMO).3 SHH signaling is active in the majority of spora-
dic basal cell carcinomas4 and also in brain tumors,
including medulloblastomas and gliomas.5 This signaling
pathway has also been linked to melanoma,6 lung
adenocarcinoma,7 as well as prostate,8 small cell lung,9
and pancreatic cancer.10 As a result, the development of
inhibitors of the SHH cellular signaling pathway has
emerged as an important goal in medicinal chemistry.
The most common strategy in the pharmaceutical industry
† The University of Pennsylvania.
(6) Stecca, B.; Mas., C.; Clement, V.; Zbinden, M.; Correa, R.;
Piguet, V.; Beermann, F.; Ruiz, A. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 5895–5900.
‡ The Wistar Institute.
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N.; Radovanovic, I.; Ruiz i Altaba, A. Curr. Biol. 2007, 17, 165–172.
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M.; Weiner, H.; Ruiz i Altaba, A. Development 2001, 128, 5201–5212.
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Memoli, V.; Dmitrovsky, E.; Robbins, D. Oncogene 2007, 26, 1046–1055.
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D.; Maitra, A.; Isaacs, J.; Berman, D.; Beachy, P. Nature 2004, 431, 707–
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A.; Barrett, A.; Beyna, M.; Datta, M.; Datta, S.; Ruiz, A. Proc. Natl.
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Baylin, S. Nature 2003, 422, 313–317.
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r
10.1021/ol2017966
Published on Web 08/15/2011
2011 American Chemical Society

Figure 1. Structures of cyclopamine 1, GDC-0449 2, estrone
analog 3, and saturated analog 4.
Figure 2. Complementary approaches to the synthesis of the
F-ring saturated analog 4.
We envisioned that the EF heterocyclic moiety of the
target structure 4 could be prepared via nitrenium ion
mediated bicyclization of 5 (Figure 2). Elegant studies by
Wardrop13 have suggested that such an approach should
befeasible. Wehave initiated a moreincremental approach
to the synthesis of the EF heterobicyclic ring system, via
iodoetherification of 7 to give 6, which on treatment with
ammonia would generate the requisite EF heterobicyclic
ring system. The synthesis and reaction of the key iodo-
etherification substrate 10 is outlined in Scheme 1.
Reaction of the known epoxide 814 (Scheme 1) with the
anion formed from the t-butyldiphenylsilyl ether of 4-pen-
tynol and removal of the silyl protecting group with TBAF
provided alkynol 9. Reduction of 9 with LiAlH4 stereo-
selectively provided the (E)-alkenol that was reacted with
TsCl to generate 10. In analogy to the work of Knight15
and Lipshutz16 on the iodocyclization of homoallylic
alcohols, exposure of 10 to iodine and sodium bicarbonate
efficiently led to the anticipated trans-β-iodotetrahydro-
furan 11 as a single diastereomer.17
The stereochemical outcome of the reaction is consistent
with the model proposed by Knight, in which conforma-
tion A, which leads to the formation of the observed
product 11, is favored over conformation B, which would
lead to the formation of the diastereomeric trans-disubstituted
tetrahydrofuran 13 (only the hydrindane moiety of the
estrone framework is shown; Figure 3).
Reaction of 11 with liquid ammonia afforded a piper-
idine product via displacement of both the iodide and
tosylate moieties present in 11, followed by reductive
has involved the screening of libraries of diverse chemical
structures in the hope of discovering drug-like structures
that will interfere with SHH signaling. The most noteworthy
success to date using this approach is GDC-0449 2 (Figure 1),
a compound that is currently in phase II clinical trials.11
Using a conceptually different approach, we have re-
cently shown that designed, structurally simplified, cyclo-
pamine-like structures such as the estrone-derived analog 3
(Figure 1) are potent inhibitors of Hedgehog signaling.12
These analogs offer two important advantages over cyclo-
pamine 1: (1) they do not contain the allylic ether present in
1 that confers metabolic, that is, acid, instability and (2)
they are easily prepared (four chemical steps) from com-
mercially available steroids, that is, estrone.
There are several important structural differences be-
tween the estrone-derived lead structure 3 and cyclopa-
mine 1, notably the presence of the aromatic A and F rings
in 3 that are not present in 1 (Figure 1). We report herein
the design and synthesis of F-ring saturated analogs of 3
that are related to 4, which contains the same relative
stereochemistry at C-22 and C-23 as cyclopamine 1, and
that the SHH signaling inhibitory activity of these new
analogs is greater than that of cyclopamine 1 in medullo-
blastoma cell viability assays.
While the direct reduction of the pyridine ring in3 would
appear to be the most direct approach to the synthesis of
F-ring saturated analogs, that is, 4, we have found the
pyridine ring in 3 resistant to direct reduction without
extensive decomposition.
Instead, we have developed an alternative approach to
the synthesis of F-ring saturated analogs, that leads, inter
alia, to the synthesis of novel structural analogs lacking the
E-ring tetrahydrofuran present in both 1 and in 3. These
new compounds are highly potent inhibitors of SHH
signaling, suggesting that the tetrahydrofuran E ring of
cyclopamine is not required for biological function.
(13) Wardrop, D.; Bowen, E.; Forslund, R.;Sussman, A.; Weerasekera,
S. J. Am. Chem. Soc. 2010, 132, 1188–1189.
(14) Cook, C.; Corley, R.; Wall, M. J. Org. Chem. 1968, 33, 2789–
2793.
(15) Bedford, S.; Bell, K.; Bennett, F.; Hayes, C.; Knight, D.; Shaw,
D. J. Chem. Soc., Perkin Trans. 1 1999, 15, 2143–2153.
(16) Lipshutz, B.; Barton, J. J. Am. Chem. Soc. 1992, 114, 1084–1086.
(17) All compounds were characterized by full spectroscopic (NMR,
IR, high resolution MS) data. Yields refer to spectroscopically and
chromatographically homogeneous (>95%) materials.
(18) Taipale, J.;Chen, J.;Cooper, M.;Wang, B.;Mann, R.;Milenkovic,
L.; Scott, M.; Beachy, P. Nature 2000, 406, 1005–1009.
(11) Sheridan, C. Nat. Biotechnol. 2009, 27, 968–969.
(12) Winkler, J.; Isaacs, A.; Holderbaum, L.; Tatard, V.; Dahmane,
N. Org. Lett. 2009, 11, 2824–2827.
Org. Lett., Vol. 13, No. 18, 2011
4787

Scheme 1. Synthesis and Cyclization of E-Alkene Substrate 10
to Give 12
Scheme 2. Synthesis and Cyclization of Z-Alkene Substrate 14
to Give Pyrrolidino-Oxetane 16
bis-electrophile 15 with ammonia afforded the oxetane-
pyrrolidine 16 in 55% overall yield after O-debenzyla-
tion. The stereochemistry of 16 was unequivocally
established by X-ray crystallographic analysis of the
derived 3,5-dinitrobenzamide, confirming that the
cyclization occurred with the analogous orientation
of the Z-alkene (A0 in Scheme 2) as shown in A for
the E-alkene cyclization substrate 10 (Scheme 1). How-
ever, instead of the anticipated tetrahydrofuran pro-
duct (the result of 5-endo cyclization), based on the
work of Knight, we observed the exclusive formation of
the oxetane product 15, the result of 4-exo cyclization
of 14. The basis for this surprising regiochemical out-
come is currently under study in our laboratory.
Biological evaluation of 12 and 16 reveals that both
are highly potent inhibitors of Hedgehog signaling,
comparable to cyclopamine 1 in potency.18 We first
tested 12 and 16 for their activity on the SHH signaling
pathway by using the SHH-Light2 cells: this cell line
is a 3T3 clone that stably expresses a GLI-dependent
reporter.19 Treatment of these cells with recombinant
SHH activates GLI-dependent firefly luciferase expres-
sion and this SHH-induced activation is inhibited when
cells are also treated with cyclopamine 1.18 In this assay,
SHH-light2 cells were treated with SHH (600 ng/mL,
R&D) alone or in combination with cyclopamine 1, 12 or
16 (all at 5 μM). As illustrated in Figure 4, 12 and 16 both
lead to a strong inhibition of SHH signaling activity
comparable to that of cyclopamine.
Figure 3. Rationale for the observed formation of 11 and not 13
in the cyclization of E-alkene 10.
debenzylation to generate 12 (in nine chemical steps and
23% overall yield from estrone), the structure of which was
confirmed by X-ray crystallographic analysis of the de-
rived para-bromobenzamide. We note that 12 is epimeric
with the target structure 4 (Figure 1) at C-22, that is, cis-
instead of trans-EF ring fusion.
Although the work of Knight had demonstrated that
the iodoetherification of Z-homoallylic alcohols was
slower than that of the E-alkene substrates, we envi-
sioned that the trans EF ring fusion stereochemistry
found in cyclopamine and in 4 could result from ana-
logous reaction of 14, the Z-alkene corresponding to 10, as
outlined in Scheme 2.
Granule neuron precursors (GNPs) represent a physio-
logical system for the testing of SHH inhibition, as SHH is
a potent mitogen for postnatal mouse cerebellar GNPs,19
and cyclopamine 1 is known to block their proliferation.20
As illustrated in Figure 5, GNP proliferation in response to
SHH signaling activation is blocked with 1 as well as with
compounds 12 and 16.
In the event, Lindlar reduction of 9 (Scheme 1)
afforded 14, which on exposure to iodine-mediated
cyclization conditions afforded not the anticipated
tetrahydrofuran product analogous to 11 (Scheme 1)
but instead the oxetane 15 in 64% yield. Reaction of
(19) Dahmane, N.; Ruiz i Altaba, A. Development 1999, 26, 3089–
3100.
4788
Org. Lett., Vol. 13, No. 18, 2011

with either cyclopamine 1, 12, or 16, and that both12 and 16
are more active in reducing DAOY cell viability than 1.
Figure 4. 12 and 16 are potent inhibitors of the SHH-induced
GLI-luciferase activity in SHH-Light2 cells. The asterisk indi-
cates p-value p < 0.001.
Figure 6. Effect of 12 and 16 on cell survival of human medullo-
blastoma DAOY cells. The asterisk indicates p-value p < 0.05.
The significance of the structures of 12 and 16 for
observed potency in these diverse biological systems is
underscored by the recent disclosure of the synthesis and
biological activity of desmethylveramiline 17, which repre-
sents a des-E analog of cyclopamine 1, by Mann and
co-workers (Figure 7).22 They report that 17 is ca. 50%
less potent than 1 in the same SHH-light2 assay described
in Figure 4, in which 16 is equipotent with 1, suggesting
that the rigidity imparted by the oxetane ring is important
to the observed potency of 16.
Figure 5. 12 and 16 block SHH-induced granule neuron precursor
(GNP) proliferation. In this event, GNPs were purified from
postnatal day 5 (P5) mouse cerebella and treated with either
SHH alone or in combination with 1, 12 or 16 (all at 5 μM). BrdU
was added to the media 6 h before fixation. Percentage of brdU
positive cells were determined by counting with a fluorescence
microscope using a 20ꢀ objective (Axioskop, Zeiss). At least five
independent fields for each culture condition were counted. The
analysis was performed on three independent experiments. Statis-
tical analysis was performed with the Student t test. The asterisk
indicates p-value *p < 0.05, **p < 0.01.
Figure 7. Desmethylveramiline.
Abnormal activation of the SHH signaling pathway has
been linked to various cancers including medulloblastoma,
a malignancy of the cerebellum.21 A subset of these tumors
is thought to arise from deregulation of granule neuron
precursor (GNP) development in the cerebellum and
require SHH signaling for their growth. We thus tested
whether compounds 12 and 16 were also efficient in
reducing the viability of medulloblastoma cells using hu-
man medulloblastoma DAOY cells. DAOY cells were
treated for 72 h with either cyclopamine 1, 12 or 16 before
cell survival was assayed with a colorimetric MTT assay, in
which absorbance at 570 nm is directly proportional to the
number of living cells. As illustrated in Figure 6, there is a
significant decrease in cell survival when the cells are treated
We have developed structurally novel analogs 12 and 16
of cyclopamine 1 that exhibit enhanced potency relative to
1 in both GNP proliferation and DAOY medulloblastoma
cellular assays. Further studies on stereoisomeric analogs of 12
and 16 and related structures are currently underway in our
laboratory, and our results will be reported in due course.
Acknowledgment. We gratefully acknowledge the NIH
(CA-134983) for their generous support of this work and
the NSF for an X-ray diffractometer (CHE-0840438).
Supporting Information Available. Full experimental
details, characterization data and NMR spectra of all
compounds, and crystallographic data. This material is
available free of charge via the Internet at http://pubs.acs.org.
(20) Fernandez, C.; Tatard, V.; Bertrand, N.; Dahmane, N. Dev.
Neurosci. 2010, 32, 59–70.
(21) Ellison, D. Neuropathology 2010, 120, 305–316.
(22) Guerlet, G.; Spangenberg, T.; Mann, A.; Faure, H.; Ruat, M.
Bioorg. Med. Chem. Lett. 2011, 21, 2608–2612.
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