Studies directed toward the elucidation of the pharmacophore of steroid-based sonic hedgehog signaling inhibitors

Article abstract of DOI:10.1021/ol202020c

Previous work from our laboratory has established that the readily available steroid-based analog 2 of cyclopamine 1 is, like 1, a highly potent inhibitor of Hedgehog signaling. The first structure-activity relationship studies on 2, i.e., the synthesis and biological evaluation of both the C-17 epi analog 4 and the C-3 deoxy analog 11, both of which are more potent than cyclopamine 1, are described. The implications of these results for the emerging pharmacophore of these Sonic Hedgehog signaling inhibitors are discussed.

Full text of DOI:10.1021/ol202020c

ORGANIC
LETTERS
2011
Vol. 13, No. 19
5140–5143
Studies Directed toward the Elucidation of
the Pharmacophore of Steroid-Based
Sonic Hedgehog Signaling Inhibitors
ꢀ
ꢀ
Andre K. Isaacs, Chaomei Xiang, Valerie Baubet, Nadia Dahmane,* and
Jeffrey D. Winkler*
Department of Chemistry, The University of Pennsylvania, Philadelphia,
Pennsylvania 19104, United States, and The Wistar Institute, Philadelphia,
Pennsylvania 19104, United States
winkler@sas.upenn.edu; ndahmane@wistar.org
Received July 26, 2011
ABSTRACT
Previous work from our laboratory has established that the readily available steroid-based analog 2 of cyclopamine 1 is, like 1, a highly potent
inhibitor of Hedgehog signaling. The first structureꢀactivity relationship studies on 2, i.e., the synthesis and biological evaluation of both the C-17
epi analog 4 and the C-3 deoxy analog 11, both of which are more potent than cyclopamine 1, are described. The implications of these results for
the emerging pharmacophore of these Sonic Hedgehog signaling inhibitors are discussed.
The alkaloid teratogen cyclopamine 1 has emerged as an
important lead structure in the development of cancer
chemotherapeutic agents that act via inhibition of the Sonic
Hedgehog (SHH) signaling pathway,1 specifically at the level
of the transmembrane protein Smoothened (SMO).2 The
teratogenicity associated with cyclopamine has not ham-
pered interest in the development of this important SHH
signaling inhibitor.3 Cyclopamine 1 has been shown to be
effective against a number of human cancers, including basal
cell carcinomas4 and brain tumors, i.e., medulloblastomas
and gliomas.5 Activation of the SHH signaling pathway has
also been linked to melanoma6 and lung adenocarcinoma,7
as well as prostate,8 smallcelllung,9 and pancreatic cancers.10
However, the considerable cost of the natural product
which is isolated from the California corn lily, Veratrum
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Barrett, A.; Beyna, M.; Datta, M.; Datta, S.; Ruiz, A. Proc. Natl. Acad.
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M.; Briggs, K.; Parker, A.; Shimada, Y.; Eshleman, J.; Watkins, D.;
Beachy, P. Nature 2003, 425, 846–51.
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M.; Sydor, J.; Tong, J.; Palombella, V.; McGovern, K.; Adams, J.
J. Med. Chem. 2008, 51, 6646–6649.
(3) (a) Firestone, A.; Chen, J. Small Molecule Inhibitors of the
Hedgehog Pathway. Hedgehog Signaling Activation in Human Cancer
and Its Clinical Implications; Springer: New York, 2011; pp 163ꢀ168; (b)
Gould, A.; Missailidis, S. Mini-Rev. Med. Chem. 2011, 11, 200–213.
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Nature 1997, 389, 876–81.
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Eberhart, C.; Watkins, D.; Chen, J.; Taipale, J.; Olson, J.; Beachy, P.
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r
10.1021/ol202020c
Published on Web 09/09/2011
2011 American Chemical Society

californicum,11 and the metabolic instability observed in
vivo (t1/2 ca. 30 s in the presence of stomach acid)12 have
precluded its development as a clinical candidate. Instead,
the development of cyclopamine mimics has been a subject
of intense study.13 We have recently reported that the
replacement of the C-nor-D-homo ring system of 1 with
the ABCD steroidal system in 2 leads to cyclopamine
analogs with activity comparable to that of 1 in two
different cellular assays.14 We describe herein our preli-
minary results directed toward the identification of the
pharmacophore that is responsible for the potent activity
of the metabolically stable cyclopamine analog 2 and
related structures.
system, with the F-ring nitrogen atom on the R-face of the
steroid plane relative to the C-3β hydroxyl group, as
highlighted in the three-dimensional model of 1.15 In
contrast, the tetrahydrofuran ring of 3 (oxygen in red) lies
in the steroid plane and the nitrogen atom of 3 is on the β-
face of the steroid plane, as illustrated in the three-dimen-
sional model of 3.
The energy-minimized structures in Figure 1 suggest an
important role for the C-17 stereochemistry common to
both 1 and 2, which, unlike 3, share the orientation of the
C-17 oxygen substituent on the β-face of the steroid plane.
In contrast, the C-17 oxygen atom of 4, the C-17 epimer of
2, is oriented on the R-face of the steroid plane, which leads
to the orientation of the F-ring nitrogen atom of 4 on the
β-face of the steroid plane, the same orientation that is
found in tomatidine 3, a naturally occurring compound
which displays no activity as a Hedgehog signaling inhi-
bitor. To test the hypothesis that the three-point recogni-
tion of the C-3 oxygen, C-17 oxygen, and C-21 nitrogen
heteroatoms as oriented in 1 and 2 is required for recogni-
tion at SMO, we have synthesized 4, the C-17 epimer of 2,
as outlined in Scheme 1.
Scheme 1. Synthesis of C-17 Epi Analog 4
Figure 1. Structures and energy minimized models of cyclopa-
mine 1, steroid-based analog 2, tomatidine 3, and C-17-epi
analog 4.
Dehydration of 514 selectively afforded Z-alkene 6, the
stereochemistry of which was established by H NMR
spectroscopy.16 Epoxidation of 6 led to the exclusive
formation of epoxide 7, via reaction of 6 from the sterically
less hindered R-face. While reaction of 7 with hydride
donors did not lead to the desired epoxide ring opening
The difference in biological activity between cyclopamine
1 and the close structural analog tomatidine 3 (Figure 1; no
SHH inhibition with 3) has been attributed to the differ-
ence in the orientation of the nitrogen atom (blue) relative
to the steroid plane in 1 and 3. The C-nor-D-homo frame-
work of 1 can thus be viewed as a scaffold that orients the
E/F heterobicyclic moiety orthogonal to the steroidal ring
1
(15) Keeler, R. Lipids 1978, 13, 708–15.
(13) Dolgin, E. Nat. Med. 2011, 17, 523.
(14) Winkler, J.; Isaacs, A.; Holderbaum, L.; Tatard, V.; Dahmane,
N. Org. Lett. 2009, 11, 2824–7.
(16) (a) Benn, W.; Dodson, R. J. Org. Chem. 1964, 29, 1142. (b)
Mackella, F.; Slomp, G. Steroids 1968, 11, 787. (c) Tanabe, M.; Peters.,
R. J. Org. Chem. 1971, 36, 2403.
Org. Lett., Vol. 13, No. 19, 2011
5141

to give 8, the C-17 epimer of 5, we were delighted to find
that exposure of 7 to samarium diiodide afforded the
desired product 8 (65% yield), based on Kato’s work on
the deoxygenation of pyridine methanols.17 Exposure of 8
to the BuchwaldꢀHartwig cyclization conditions em-
ployed to generate 214 led, after deprotection of the silyl
ether, to the formation of 4, the C-17 epimer of 2.
To determine whether 4 inhibits SHH signaling, we first
examined the effect of 4 on the signaling pathway using
SHH-Light2 cells: this cell line is a 3T3 clone that stably
expresses a GLI-dependent reporter.18 Treatment of these
cells with recombinant SHH activates GLI-dependent fire-
fly luciferase expression, and this SHH-induced activation is
inhibited when cells are also treated with cyclopamine 1
(Figure 2).18 Treatment of SHH-Light2 cells with SHH in
combination with 4 (5 μM) led to a ca. 3-fold increase in
signaling inhibition relative to that observed with cyclopa-
mine 1. These data show that 4 is a more potent inhibitor of
SHH signaling than cyclopamine 1.
for recognition of these cyclopamine analogs at SMO,
suggesting that the C-3 oxygen functionality may not
be required for recognition at SMO.
To establish the role, if any, of the C-3 oxygen
functionality that is present in 2 and 4 on the biological
activity of these estrone-derived analogs of cyclopa-
mine 1, we have prepared the C-3 deoxy compound 11,
the synthesis of which is shown in Scheme 2. Addition
of the conjugate base of 3-bromopicoline to the known
3-deoxyestrone19 gave 10, which on reaction under
BuchwaldꢀHartwig conditions afforded 11, the C-3
deoxy analog of 2 (Figure 1).
Scheme 2. Synthesis of C-3 Deoxy Analog 11
Figure 2. Luciferase Based Assay for SHH Activity. SHH-Light2
cells were treated as described.18 GLI-binding site luciferase
activities were measured using the luciferase reporter assay
system with the luciferase kit (Promega). Treatment of SHH-
Light2 cells with recombinant SHH (600 ng, R&D) resulted in
the strong induction of reporter activity, which was largely
blocked by cotreatment either with cyclopamine 1 or with 4 at
5 μM (in DMSO), both P < 0.01 [SHH vs SHH þ 1; SHH vs
SHH þ 4].
This result contradicts our hypothesis that the three-
point recognition of the C-3 oxygen and each of the
other E and F ring heteroatom functionalities in 2 is
required for recognition at SMO, the cellular target of
cyclopamine, since structures with either orientation at
C-17, i.e., both 2 and 4, are potent inhibitors of SHH
signaling. The relative orientations of the tetrahydro-
furan oxygen and pyridine nitrogen relative to the
steroid plane do not appear to be important features
Figure 3. Luciferase Based Assay for SHH Activity. SHH-Light2
cells were treated as described.18 Treatment of SHH-Light2 cells
with recombinant SHH (600 ng, R&D) resulted in the strong
induction of reporter activity, which was blocked by cotreat-
ment either with cyclopamine 1 or with 11 at 5 μM, both P <
0.001 [SHH vs SHH þ 1; SHH vs SHH þ 11].
(17) Kato, Y.; Mase., T. Tetrahedron Lett. 1999, 40, 8823–8826.
(18) Taipale, J.; Chen, J.; Cooper, M.; Wang, B.; Mann, R.;
Milenkovic, L.; Scott, M.; Beachy, P. Nature 2000, 406, 1005–1009.
(19) Nicolaou, K. C.; Barnette, W.; Ma, P. J. Org. Chem. 1980, 45,
1463–1470.
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Org. Lett., Vol. 13, No. 19, 2011

Biological evaluation of 11 using the same luciferase
assay described for 4 (Figure 2) reveals that it is a potent
inhibitor of SHH signaling. In this assay, the C-3 deoxy
analog 11 (Figure 3) leads to a strong inhibition of SHH
signaling activity (80% inhibition at 5 μM; compared to
70% inhibition in the same assay with 2).14 We have also
demonstrated that 11 acts on the SHH pathway, as there is
a significant decrease of the expression of GLI1, a trans-
ducer and a target of this pathway, following treatment of
human medulloblastoma cells with 11 (Figure 4).
Figure 5. Analog 11 reduces DAOY medulloblastoma cell
viability. DAOY human medulloblastoma cells were treated
with carrier DMSO (Control), cyclopamine 1 (10 μM), or 11
(10 μM) for 3 days. The histogram measures cell viability
assessed by the MTT assay (absorbance at 570 nm) (asterisk
indicates p < 0.05). Similar results were obtained with
U87GBM cells (not shown).
The remarkable potency of the C-3 deoxy analog 11
suggests that the two-point interaction (of the C-3β hydroxyl
and the EF heterobicyclic ring) suggested by the three-dimen-
sional structures of cyclopamine 1 (Figure 1) is not a critical
recognition feature for biological activity, thereby pointing the
way to further simplification of the structure of the cyclopa-
mine analogs. Further studies on the synthesis and biological
evaluation of such structures are currently underway in our
laboratory, and our results will be reported in due course.
Figure 4. 11 decreases GLI1 mRNA levels in human MB DAOY
cells. DAOY cells were treated either with cyclopamine 1 or with
11 (10 μM) for 4 h, and levels of GLI1 mRNA were assessed by
quantitative RT-PCR and normalized over the expression of
GAPDH. The control level is set arbitrarily at 1 for comparison,
both P < 0.001 [control vs 1; control vs 11].
Acknowledgment. Wegratefully acknowledgethe finan-
cial support of the NIH (CA-134983) to J.D.W. and N.D.
We have also demonstrated that 11 has a potent inhibi-
tory effect on brain tumor cell growth in vitro (Figure 5),
further validating this approach to the development of
steroid-based cyclopamine mimics as brain cancer che-
motherapeutic candidates.
Supporting Information Available. Full experimental
details, characterization data, and NMR spectra of all
compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
Org. Lett., Vol. 13, No. 19, 2011
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