Cyclopamine analogs bearing exocyclic methylenes are highly potent and acid-stable inhibitors of hedgehog signaling

Article abstract of DOI:10.3762/bjoc.9.267

The chemical synthesis and biological evaluation of new cyclopamine analogs bearing exocyclic methylenes in different positions is described. Bis-exo-cyclopamine 6 was identified as a potent inhibitor of the Gli1-dependent luciferase expression in Shh-LIGHTII cells. An extension of this study to F-ring-modified structures shows the necessity of a rigidly positioned nitrogen atom for bioactivity as well as the presence of the C21 methyl group for acid stability and bioactivity.

Full text of DOI:10.3762/bjoc.9.267

Cyclopamine analogs bearing exocyclic methylenes
are highly potent and acid-stable inhibitors of
hedgehog signaling
Johann Moschner1, Anna Chentsova1, Nicole Eilert1, Irene Rovardi1,
Philipp Heretsch*2 and Athanassios Giannis*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 2328–2335.
1Universität Leipzig, Institut für Organische Chemie, Johannisallee 29,
D-04103 Leipzig, Germany and 2Rice University, Department of
Chemistry, BioScience Research Collaborative, 6500 Main St.,
Houston, Texas 77030, USA
doi:10.3762/bjoc.9.267
Received: 16 August 2013
Accepted: 15 October 2013
Published: 31 October 2013
Email:
Philipp Heretsch* - heretsch@rice.edu; Athanassios Giannis* -
giannis@uni-leipzig.de
This article is part of the Thematic Series "Natural products in synthesis
and biosynthesis".
* Corresponding author
Guest Editor: J. S. Dickschat
Keywords:
© 2013 Moschner et al; licensee Beilstein-Institut.
License and terms: see end of document.
cyclopamine; hedgehog signaling pathway; natural products; steroidal
alkaloids; structure–activity relationship
Abstract
The chemical synthesis and biological evaluation of new cyclopamine analogs bearing exocyclic methylenes in different positions
is described. Bis-exo-cyclopamine 6 was identified as a potent inhibitor of the Gli1-dependent luciferase expression in Shh-
LIGHTII cells. An extension of this study to F-ring-modified structures shows the necessity of a rigidly positioned nitrogen atom
for bioactivity as well as the presence of the C21 methyl group for acid stability and bioactivity.
Introduction
Hedgehog signaling is involved in embryonic development and [13,14], colon [15], rhabdomyosarcoma [16], lymphoma [17-
plays an important role in the maintenance of stem cells, tissue 19], multiple myeloma [17,20], and chronic myeloic leukemia
repair and regeneration in adult organisms [1-4]. The erroneous [21-23]. More recently, other diseases like diabetes [24,25],
activation of hedgehog signaling is tightly associated with the neurodegenerative disorders [26], and trisomy [27,28], have
occurrence of basal cell carcinoma and medulloblastoma [5]. In been linked with hedgehog signaling.
addition, several other tumors are co-dependent on hedgehog
signaling, examples for this type are cancers of the skin [6,7], Cyclopamine (1, see Figure 1) was the first inhibitor of the
brain [8,9], lung [10], pancreas [11], breast [12], prostate hedgehog signaling pathway to be identified. As a highly selec-
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Beilstein J. Org. Chem. 2013, 9, 2328–2335.
tive inhibitor of the transmembrane protein Smoothened (Smo), removal of the so-obtained acetate (Et3SiH, BF3·Et2O, −78 °C
an integral component of hedgehog signaling, it presents an to −20 °C, 79%). Removal of the benzyl ether, reduction of the
attractive target for medicinal and pharmaceutical research azide moiety and concomitant alkylation was then all effected in
[29,30]. Unfortunately, its direct development into a drug is one pot by using Raney-nickel in EtOH to give analog 8, an
hampered by its low metabolic stability (decomposition at F-ring-opened exo-cyclopamine derivative in 27% overall yield
pH < 3) [31] and rather moderate potency (IC50 ~ 5 µM). from 3. Alternatively, the use of previously devised conditions
for benzyl deprotection (DDQ, DCE/pH 7 phosphate buffer,
45 °C, 78%) and then sodium borohydride-mediated reduction
of the azide (EtOH, 65 °C, 62%) cleanly furnished primary
amine 9, an exo-cyclopamine derivative with no F-ring (35%
overall yield from 3).
A second set of F-ring-modified exo-cyclopamine derivatives
was obtained starting from previously described 12-ß-triethylsi-
lyloxy compound 10 (see Scheme 2) [32]. To construct the
spirolactone 12, a three-step process consisting of (1) allylation
(allylcerium chloride, THF, 0 °C, 93%), (2) hydroboration/oxi-
Figure 1: Structures of cyclopamine and exo-cyclopamine.
dation (9-BBN, THF, 70 °C; then NaBO3, H2O, 50 °C, 91%)
and finally, (3) oxidative cyclization (BAIB, cat. TEMPO,
We previously reported the first chemical synthesis of CH2Cl2, 25 °C, 73%) was employed. Deprotection of the silyl
cyclopamine (1) starting from dehydroepiandrosterone and ether (HF, MeCN, 25 °C, 87%) and base-induced rearrange-
utilizing the C–H-functionalization logic and a biomimetic ment of the steroid skeleton via the corresponding triflate
skeleton rearrangement [32,33]. Furthermore, quantum mechan- (Tf2O, pyridine, 0 °C→50 °C, 46%) gave C-nor-D-homo-
ical calculations guided our design and synthesis of exo- derivative 13 which, in turn, was transformed into lactone azide
cyclopamine (2, see Figure 1), a ten-fold more potent and acid- 14 by using Evans’ conditions (LDA, THF, −78 °C to −30 °C,
stable analog with an exo-methylene unit at C13–C18 [34]. 10 min; then trisylazide, THF, −78 °C, 1.5 h, 43%) [35]. From
Herein, we describe a comprehensive study of cyclopamine 14 two additional exo-cyclopamine derivatives became acces-
analogs bearing exo-methylene units in different positions and sible. Partial reduction to the lactol (DIBAl-H, THF, −78 °C to
extent this rational to F-ring-modified structures.
−65 °C, 88%) and Horner–Wadsworth–Emmons reaction by
using previously reported dimethyl {3-[(4-methoxybenzyl)oxy]-
2-oxopropyl}phosphonate [32], (Ba(OH)2, THF/H2O, 80 °C,
Results and Discussion
Our synthetic approach to the analogs described here started 50%) led to 15 which, in turn, was methylenated by employing
from previously reported azide 3 which was converted in six the Peterson protocol (TMSCH2CeCl2, THF, −78 °C; then
steps [32] to the protected bis-exo-methylene compound 4. TMEDA; then HF, MeCN, 25 °C, 64%) [36]. Towards this end
Carefully chosen conditions for the deprotection of the benzyl the azide moiety was reduced and monoprotection of the
ether (DDQ, DCE/pH 7 phosphate buffer, 40 °C, 86%) and the so-obtained amine as a sulfonamide (LiAlH4, THF,
benzenesulfonylamine (sodium naphthalenide, DME, −78 °C, 0 °C→25 °C; then benzenesulfonyl chloride, Et3N, DMF, 0 °C,
79%) allowed for the isolation of bis-exo-cyclopamine 6 in 18% 69%) gave 17. Deprotetion of the 4-methoxybenzyl ether
overall yield from 3. The hydrogenation of intermediate 4 by (DDQ, DCE/pH 7 phosphate buffer, 25 °C, 59%) and cycliza-
using Wilkinson’s catalyst in benzene yielded previously tion under Mitsunobu conditions (n-Bu3P, DEAD, toluene,
described exo-cyclopamine 2 and its C25 epimer 5. Deprotec- 0 °C→25 °C, 93%) yielded piperidine 18. Deprotection of the
tion with Raney-nickel (EtOH, 78 °C) and then sodium naph- benzyl ether by using previously devised conditions (DDQ,
thalenide (DME, −78 °C, 41% over two steps) furnished 25-epi- DCE/pH 7 phosphate buffer, 44 °C, 70%) and the benzenesul-
exo-cyclopamine 5 in 3% overall yield from 3.
fonylamine (sodium naphthalenide, DME, −78 °C, 95%) gave
20-demethyl-bis-exo-cyclopamine 19 in 7% overall yield
A first set of exo-cyclopamine analogs with a modified F-ring starting from 14.
was obtained starting again from azide 3 (see Scheme 1).
Reduction of the lactone moiety to give a tetrahydrofuran was Starting from azido lactone 14 (see Scheme 2) a partial reduc-
accomplished in three steps including (1) partial reduction to tion to the lactol (DIBAl-H, THF, −78 °C to −65 °C, 88%) and
the lactol (DIBAl-H, THF, −78 °C to −60 °C, 95%), (2) acetyla- the Horner–Wadsworth–Emmons reaction by using triethyl
tion (Ac2O, pyridine, cat. DMAP, quant.) and (3) reductive phosphonoacetate (Ba(OH)2, THF/H2O, 80 °C, 55%) led to
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Beilstein J. Org. Chem. 2013, 9, 2328–2335.
Scheme 1: Synthesis of 25-epi-exo-cyclopamine 5, bis-exo-cyclopamine 6, and derivatives 8 and 9. Reaction conditions: (a) DIBAl-H, THF, −78 °C to
−65 °C, 4 h, 95%; (b) Ac2O, pyridine, DMAP (cat.), 25 °C, 20 h, quant; (c) BF3·Et2O, Et3SiH, CH2Cl2, −78 °C to −20 °C, 7 h, 79%; (d) Rh(PPh3)3Cl,
benzene, H2, 25 °C, 24 h, quant. (3:1 dr); (e) Raney-nickel (W2), EtOH, 78 °C, 5 min; (f) sodium naphthalenide, DME, −78 °C, 30 min, 41% over two
steps; (g) DDQ, DCE/phosphate buffer (pH 7), 40 °C, 95 min, 86%; (h) sodium naphthalenide, DME, −78 °C, 1 h, 79%; (i) Raney-nickel (W2), EtOH,
37%; (j) DDQ, DCE/phosphate buffer (pH 7), 45 °C, 1.5 h, 78%, (k) NaBH4, EtOH, 65 °C, 5 d, 62%.
ethyl ester 20. Reduction of the azide moiety in 20 was carried constitutive Renilla luciferase reporter [37]. The compounds
out by using Staudinger’s protocol (Ph3P, THF/H2O, 50 °C). were tested in a concentration range from 0.01 μM to 10 μM.
Immediate protection of the obtained amine (benzenesulfonyl While analogs 8, 9, and 19 showed no activity (data not shown)
chloride, Et3N, CH2Cl2, 40 °C, 97% over 2 steps) gave sulfony- in this concentration range, 5, 6, and 23 were active with
lamide 21. Reduction of the ester in 21 to the primary alcohol 25-epi-exo-cyclopamine 5 having an IC50 of 3.29 ± 0.31 μM,
(DIBAl-H, THF, −78 °C to −40 °C, 97%) and cyclization by F-nor-20,25-bis-demethyl-exo-cyclopamine 23 of 6.4 ± 0.9 μM,
employing Mitsunobu conditions (n-Bu3P, DEAD, toluene, and bis-exo-cyclopamine 6 being the most active with an IC50
0 °C→25 °C, 81%) yielded pyrrolidine 22. Previously devised of 0.20 ± 0.01 μM (see Figure 2).
conditions for the deprotection (1. DDQ, DCE/pH 7 phosphate
buffer, 40 °C; 2. sodium naphthalenide, DME, −78 °C, 46%) Given the negative test results of compounds 8 and 9 it becomes
finally afforded F-nor-20,25-bis-demethyl-exo-cyclopamine 23 evident that the F-ring is necessary for bioactivity. The piperi-
in 9% overall yield starting from 14.
dine moiety provides a rather rigidly placed nitrogen atom.
Nevertheless, a pyrrolidine as in compound 23 still provides the
Next, we examined all synthesized compounds for their ability correct orientation of the nitrogen atom. Despite compounds 8
to inhibit the Gli1-dependent luciferase expression in Shh- and 9 being inactive in the assay, both derivatives induced cyto-
LIGHTII cells, a clonal mouse fibroblast cell line which stably toxicity in the concentration range tested. Very subtle changes
incorporates a Gli-dependent firefly luciferase reporter and a of the conformation of the piperidine ring significantly change
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Beilstein J. Org. Chem. 2013, 9, 2328–2335.
Scheme 2: Synthesis of 20-demethyl-bis-exo-cyclopamine 19 and F-nor-20,25-bis-demethyl-exo-cyclopamine 23. Reaction conditions: (a) allyl-
cerium chloride, THF, 0 °C, 30 min, 93%; (b) 9-BBN, THF, 70 °C, 6 h; then NaBO3, 50 °C, 12 h, 91%; (c) BAIB, TEMPO, CH2Cl2, 25 °C, 3 h, 73%; (d)
HF 50 wt % in H2O, MeCN, 20 min, 25 °C, 87%; (e) Tf2O, pyridine, 0 °C→50 °C, 1.5 h; then additional Tf2O, 0 °C→50 °C, 2 h, 46%; (f) LDA, THF,
−78 °C to −30 °C, 10 min; then trisylazide, THF, −78 °C, 1.5 h, 43%; g) DIBAl-H, THF, −78 °C to −65 °C, 2 h, 88%; (h) dimethyl (3-((4-
methoxybenzyl)oxy)-2-oxopropyl)phosphonate, Ba(OH)2, THF/H2O, 80 °C, 13 h, 50%; (i) (trimethylsilyl)methylcerium chloride, THF, −78 °C, 30 min;
then TMEDA, −78 °C, 15 min; then HF 50 wt % in H2O, MeCN, 25 °C, 10 min, 64%; (j) LiAlH4, THF, 0 °C→25 °C, 13 h; then benzenesulfonyl chlo-
ride, Et3N, DMF, 0 °C, 25 min, 69%; (k) DDQ, CH2Cl2/phosphate buffer (pH 7), 25 °C, 2 h, 59%; (l) n-Bu3P, DEAD, toluene, 0 °C→25 °C, 12 h, 93%;
(m) DDQ, DCE/phosphate buffer (pH 7), 44 °C, 50 min, 70%; (n) sodium naphthalenide, DME, −78 °C, 30 min, 95%; (o) triethyl phosphonoacetate,
Ba(OH)2, THF, 80 °C, 12 h, 55%; (p) PPh3, THF/H2O, 50 °C, 24 h; then benzenesulfonyl chloride, Et3N, CH2Cl2, 40 °C, 5 h, 97%; (q) DIBAl-H, THF,
−78 °C to −40 °C, 3 h, 97%; (r) n-Bu3P, DEAD, toluene, 0 °C→25 °C, 24 h, 81%; (s) DDQ, DCE/phosphate buffer (pH 7), 40 °C, 30 min, 62%; (t)
sodium naphthalenide, DME, −78 °C, 40 min, 74%.
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Beilstein J. Org. Chem. 2013, 9, 2328–2335.
Figure 2: IC50 values of Shh inhibition by compounds 5, 6 and 23 in a Gli1-reporter gene assay. Data were obtained from three independent experi-
ments and represent the mean ± standard deviation.
bioactivity: While 25-epi-exo-cyclopamine 5 shows reduced compounds 19 and 23 showed decomposition. This experiment
activity in comparison to exo-cyclopamine 2, bis-exo- emphasizes the importance of the C-21 methyl group for the
cyclopamine 6 is the most active compound tested in this study. stability of exo-cyclopamine derivatives. All synthesized com-
Furthermore, the methyl group at C-20 seems to have a pounds, the number of steps required, and the respective overall
pronounced effect on the bioactivity, with 20-demethyl-bis-exo- yield starting from 3 or 14, as well as their biological activity
cyclopamine 19 being completely inactive in the tested concen- and stability under acidic conditions are summarized in Table 1.
tration range.
Conclusion
Finally, we studied the stability of all newly synthesized com- In conclusion, we succeeded in identifying the new
pounds towards acidic conditions. Therefore, they were exposed cyclopamine derivative bis-exo-cyclopamine 6 which surpasses
to a pH of approximately 1 (MeOH, 1 M HCl) for 24 h. After the biological potency of the parent compound by the 25-fold
evaporation of all volatiles, 1H NMR spectra were acquired and and is stable at pH 1. Further insights were gained into the
compared to the initially obtained spectra of the pure com- structure–activity relationship of F-ring-modified analogs of
pounds. While compounds 5, 6, 8, and 9 remained unchanged, cyclopamine and the necessity of the C-21 methyl group for
Table 1: Synthesized exo-cyclopamine derivatives, number of steps, yields, results of their biological testing, and stability towards acid.
Compound
Number of steps
from 3 or 14
Overall
yield
Potency in
Gli-assay
Stability towards
acid (pH ~1, 24 h)
9 steps from 3
3%
3.29 ± 0.31 µM
stable
5: 25-epi-exo-cyclopamine
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Table 1: Synthesized exo-cyclopamine derivatives, number of steps, yields, results of their biological testing, and stability towards acid. (continued)
8 steps from 3
18%
0.2 ± 0.01 µM
stable
6: bis-exo-cyclopamine
4 steps from 3
27%
> 10 µM
stable
8
5 steps from 3
35%
> 10 µM
stable
9
8 steps from 14
7%
> 10 µM
decomp.
19: 20-demethyl-bis-exo-cyclopamine
7 steps from 14
9%
6.40 ± 0.90 µM
decomp.
23: F-nor-20,25-bis-demethyl-exo-cyclopamine
Previously synthesized and biologically evaluated:
9 steps from 3
7%
0.5 µM [34]
stable
2: exo-cyclopamine
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Table 1: Synthesized exo-cyclopamine derivatives, number of steps, yields, results of their biological testing, and stability towards acid. (continued)
10 steps from 3
5%
5 µM [34]
decomp.
1: cyclopamine
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Supporting Information File 1
Experimental details and analytical data of all synthesized
compounds are provided.
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(http://www.beilstein-journals.org/bjoc)
The definitive version of this article is the electronic one
which can be found at:
doi:10.3762/bjoc.9.267
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