Synthesis of cyclopamine using a biomimetic and diastereoselective approach

Article abstract of DOI:10.1002/anie.200902520

From Homer to hedgehog: Cyclopamine, the first inhibitor of the hedgehog signaling pathway, causes cyclopia in embryos butin adults it is a potent anticancer drug. A concise biomimetic and diastereoselective synthesis of cyclopamine (2) starting from comm

Full text of DOI:10.1002/anie.200902520

Angewandte
Chemie
DOI: 10.1002/anie.200902520
Cyclopamine
Synthesis of Cyclopamine Using a Biomimetic and Diastereoselective
Approach**
Athanassios Giannis,* Philipp Heretsch, Vasiliki Sarli, and Anne Stꢀßel
Dedicated to Philip A. Beachy, Lynn F. James, and Richard F. Keeler
The hedgehog signaling (Hh) pathway is a key regulator in
embryogenesis and in the maintenance and generation of cells
in adults.[1] Since aberrant activation of the Hh pathway leads
to malignancies including basal cell carcinoma, medulloblas-
toma, rhabdomyosarcoma, and prostate, pancreatic and
breast cancers, inhibition of Hh signaling provides a route
to novel anticancer therapies.[2,3] The Veratrum alkaloid
cyclopamine was the first known inhibitor of Hh signaling;
it influences the balance between active and inactive forms of
the protein Smoothened. Though high demand exists for
cyclopamine as well as for metabolically more stable and
potent analogues, an efficient chemical synthesis of this
steroidal alkaloid is still missing.
We report herein an expeditious synthesis of cyclopamine
starting from commercially available dehydroepiandroster-
one and making use of biomimetic and diastereoselective
transformations. While one total synthesis strategy can be
envisioned based on previous publications,[4–6] this approach is
tedious, non-stereoselective, and low yielding. Our synthesis,
Scheme 1. Structure and retrosynthetic analysis of cyclopamine (1).
Bn=benzyl.
which entails a copper-mediated regioselective C–H activa-
tion/hydroxylation, cationic ring contraction/expansion, and
finally an Alder-ene reaction, enables the rapid construction
of this unusual molecule. This strategy also provides access to
various cyclopamine analogues that do not exist in nature
which might be useful in investigations of the biological
activity of this unique natural product.
Cyclopamine (1; Scheme 1) was first reported in 1957,
when sheepherders in Idaho, USA, were alarmed by the
unsettling observation of newborn lambs with a single eye
located, cyclops-like, in the middle of their foreheads.[7]
Intense investigation led to the finding that the ingestion of
the corn lily (Veratrum californicum) by pregnant sheep
caused the birth of lambs with cyclopia. The Veratrum
alkaloid cyclopamine was identified as the causative agent.
The malformation cyclopia has been known in humans
since ancient times. It has been observed in severe cases of
holoprosencephaly (HPE), a malformation of the brain in
which the prosencephalon fails to cleave, resulting in undi-
vided or incompletely divided cerebral hemispheres, telen-
cephalon and diencephalon, and olfactory and optic nerves.[8]
Associated with the brain abnormalities is a spectrum of
midline craniofacial anomalies, including ocular hypotelorism
and interorbital proboscis (ethmocephaly).[9] The etiology of
HPE is heterogeneous and can include genetic and/or
teratogenic factors.
[*] Prof. Dr. A. Giannis, Dipl.-Chem. P. Heretsch, Dipl.-Chem. A. Stꢀßel
Institut fꢁr Organische Chemie
Only recently was the cellular target of cyclopamine
elucidated. This molecule binds to the plasma membrane
protein smoothened, induces a conformational change, and
inhibits the hedgehog signaling pathway.[3]
Cyclopamine (1) displays a plethora of unusual structural
features, and therefore its synthesis poses significant chal-
lenges. Among them are the C-nor-D-homo steroid substruc-
ture and a highly substituted furan ring, which is attached
through a spiro linkage to the D ring and also fused to a
piperidine unit having a basic secondary nitrogen atom.
Additionally, cyclopamine is quite acid sensitive: at pH < 3 or
upon treatment of 1 with Lewis acids the furan ring is cleaved
and the D ring aromatizes to form the toxic veratramine.
Fakultꢂt fꢁr Chemie und Mineralogie, Universitꢂt Leipzig
Johannisallee 29, 04103 Leipzig (Germany)
Fax: (+49)341-973-6599
E-mail: giannis@uni-leipzig.de
Dr. V. Sarli
Laboratory of Organic Chemistry, Department of Chemistry,
University of Thessaloniki, 54124 Thessaloniki (Greece)
[**] We thank Dr. Lothar Hennig for recording NMR spectra and for his
help in interpreting the 2D NMR spectra. The Deutsche For-
schungsgemeinschaft (DFG) is acknowledged for partial financial
support. P.H. is a fellow of Fonds der Chemischen Industrie.
Supporting information for this article (including the syntheses and
spectroscopic data for the compounds shown) is available on the
WWW under http://dx.doi.org/10.1002/anie.200902520.
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Retrosynthetic considerations (Scheme 1) led us to define
of the 12b-hydroxy group as a triethylsily ether (!7), the
construction of the lactone was the next objective.
three objectives of paramount importance to our synthesis:
1) creation of the correct ABCD framework, 2) installation of
the furan unit (ring E), and 3) synthesis of the piperidine ring
(ring F).
The diastereoselective addition of lithium triethylsilyl
propargylate to 7 gave the tertiary alcohol, which in turn was
monodesilylated by treatment with acetic acid, reduced with
the Lindlar catalyst to yield the cis isomer exclusively, and
finally oxidatively cyclized by reaction with a catalytic
Although the total synthesis of the C-nor-D-homo steroid,
for example from the (+)-Wieland–Miescher ketone, was a
possibility we chose a semisynthetic route to this substructure
for the sake of brevity and efficiency. We presumed that the
cationic rearrangement of 12b-hydroxy steroids into their C-
nor-D-homo counterparts[10] was a much easier approach.
Since 12b-hydroxy steroids are rare and a properly function-
alized one (like 4) resembling the cyclopamine ABCD
skeleton was not available, we envisioned the hydroxylation
of commercially available steroids (e.g. dehydroepiandroster-
one 5) at position by 12 position by C–H activation. Fortu-
nately Schꢀnecker et al. recently published a method using
copper salts and molecular oxygen for this purpose.[11] We
planned that the furan with its spiro connection to the D ring
would be installed by addition of a functionalized C nucleo-
phile to the 17-keto group of 4, thereby establishing the
correct substitution at the C17 quaternary center in form of a
tertiary alcohol. Oxidative cyclization could yield a lactone
(as in 3) as the furan precursor. This functionality was thought
to be most suitable for further elaboration into the piperidine
amount
of
2,2,6,6-tetramethylpiperidinoxy
radical
(TEMPO) and stoichiometric [bis(acetoxy)iodo]benzene
(BAIB) to give the butenolide 8. Treatment of 8 with the
anion formed from trimethyl trithioorthoformate and n-
butyllithium[13] followed by careful reduction with Raney
nickel gave the properly functionalized lactone 9 as a single
diastereoisomer (54% yield over six steps).
However, lactone 9 is available in similar yield by a more
concise three-step synthesis. Treatment of the ketone with 2-
methyl-1-propenylcerium chloride,[14] hydroboration/oxida-
tion of the resulting alkenes with 9-borabicyclo[3.3.1]nonane
(9-BBN) and sodium perborate, and finally oxidative cycliza-
tion of the intermediate diols using TEMPO/BAIB gave the
lactones 9 and 11 as a mixture of epimers at C20. These in turn
were easily separable by chromatography. Thus the desired
20R lactone 9 was obtained in 42% yield in only three steps.
Furthermore the 20S-configurated lactone 11 was isolated in
30% yield. We used this much shorter sequence to access
quantities of the lactone of up to 10 g per batch.
Before the construction of the piperidine commenced, it
was time to rearrange the steroid skeleton. Cleavage of the
triethylsilylether with hydrofluoric acid yielded the free
alcohol, which was subjected to the action of trifluorometh-
anesulfonic anhydride (Tf2O) in pyridine (Scheme 3). At
elevated temperature this Wagner–Meerwein-type rearrange-
ment gave the desired C-nor-D-homo skeleton in nearly
quantitative yield as a 7:3 mixture of regioisomers 12 and 14
which were easily separable by chromatography. Though the
isomer 12 with the exocyclic double bond dominated in the
reaction mixture, we continued the next steps of the synthesis
before attempting conversion into the endocyclic isomer.
ꢀ
ꢀ
by proper C N and C C bond-forming reactions and for the
final piperidine formation (ring F) by nucleophilic substitu-
tion.
Starting from commercially available 5 protection of the
hydroxy moiety as a benzyl ether by reaction with Dudleyꢁs
pyridinium triflate[12] and formation of the 2-picolylimine at
position 7 by treatment of the ketone with 2-picolylamine
under azeotropic removal of water afforded 6 in high yield
and set the stage for the projected hydroxylation at posi-
tion 12 (Scheme 2). Using tetrakis(acetonitrilo)copper(I)
hexafluorophosphate and molecular oxygen, we were able
to introduce the 12b-hydroxy group with complete regio- and
diastereoselectivity in an encouraging yield. After protection
Scheme 2. Synthesis of 12b-hydroxy lactones 9 and 11: Reaction conditions: a) 2-benzyloxymethylpyridinium triflate, MgO, PhCF3, 858C; b) 2-
picolyl amine, pTsOH (2.5 mol%), toluene, reflux, 90% (95% b.r.s.m.) over two steps; c) [Cu(MeCN)4]PF6, acetone, then O2 (1 atm), then
NH4OH, then HOAc, MeOH, 48% (57% brsm); d) TESOTf, 2,6-lutidine, CH2Cl2, 08C, 93%; e) triethylsilylpropargyllithium, THF, ꢀ158C;
f) HOAc, THF, H2O; g) Lindlar catalyst (3 mol%), pyridine, THF; h) BAIB, TEMPO (20 mol%), CH2Cl2, 64% (77% b.r.s.m.) over four steps;
i) tris(methylthio)methyllithium, THF, ꢀ788C; j) Raney nickel (W2), THF, H2O, 69% over two steps; k) 1-methyl-2-propenylcerium chloride, THF,
08C, 95%; l) 9-BBN, THF, reflux, then NaBO3, H2O, 508C; m) BAIB, TEMPO (20 mol%), CH2Cl2, 77% over two steps, d.r. 6:4. brsm=based on
recovered starting material, OTf=triflate, pTsOH=p-toluenesulfonic acid, TES=triethylsilyl
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ketone 18 was transformed into an alkene by a two-step
Peterson olefination sequence (including reaction with (tri-
methylsily)methylcerium chloride[17] then elimination by brief
treatment with diluted hydrofluoric acid). Subsequent Stau-
dinger reduction[18] of the azide and protection of the amine
formed with benzenesulfonyl chloride furnished the sulfona-
mide 19, which in turn was treated with 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ) to give the corresponding
allyl alcohol (Scheme 4). Ring closure by a modified Mitsu-
nobu protocol[19] proceeded with high efficiency and furnished
the piperidine in a convincing yield (57% over six steps).
Scheme 3. Synthesis of azido lactoles 13, 15, and 16. Reaction
conditions: a) HF, MeCN, H2O, 85%; b) Tf2O, pyridine, 08C!508C,
94% (7:3 ratio of 12 and 14); c) LDA, THF, ꢀ788C, then trisyl azide,
ꢀ788C, then HOAc, KOAc, ꢀ788C!228C, 78% (d.r. 8:1); d) DIBAH,
THF, ꢀ788C!ꢀ658C, 82%; e) LDA, THF, ꢀ788C, then trisyl azide,
ꢀ788C, then HOAc,ꢀ788C!228C, 93% (d.r. 5:4); f) DIBAH, THF,
ꢀ788C!ꢀ658C, 95%; g) LDA, THF, ꢀ788C, then trisyl azide, ꢀ788C,
then HOAc, ꢀ788C!228C, 75% (d.r. 3:1); h) DIBAH, THF, ꢀ788C!
ꢀ658C, 90%.
Scheme 4. Synthesis of cyclopamine (1). Reaction conditions: a) 17,
Ba(OH)2, THF, H2O, then 13, 48% (59% b.r.s.m.); b) (trimethylsilyl)-
methylcerium chloride, THF, ꢀ788C, then HF, MeCN, H2O; c) PPh3,
THF, H2O, 508C, then BsCl, Et3N, CH2Cl2, 408C, 80% over two steps;
d) DDQ, pH 7.0 buffer, CH2Cl2; e) Bu3P, ADDP, toluene; f) [RhCl-
(PPh3)3], H2 (1 atm), benzene, 71% (d.r. 3:1) over three steps;
g) N-sulfinylbenzenesulfonamide, benzene, 608C, then Raney nickel
(W2), H2 (5 atm), benzene, 57%; h) Raney nickel (W2), EtOH, 788C;
i) sodium naphthalenide, DME, ꢀ788C, 79% over two steps. ADDP=
azodicarboxylic dipiperidide, Bs=benzenesulfonyl, DME=dimethoxy-
ethane, PMB=para-methoxybenzyl.
To incorporate the nitrogen in the molecule, we formed
the lactone enolate by treatment with excess lithium diiso-
propylamide (LDA) at ꢀ308C and reacted it with trisyl
azide[15] to give the azidolactones in 75% yield as 3:1 mixture
of diastereomers, which were separable by chromatography.
Since the undesired azide could be isomerized into the desired
one by treatment with 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) in CH2Cl2, no material was lost at this stage.
ꢀ
Completely regioselective hydrogenation of the C25 C27
To construct the furan motif we considered a tandem
double bond using Wilkinsonꢁs catalyst gave the protected
cyclopamine 20 with an exocyclic double bond between C13
and C18. Fortunately this isomer could be converted into
protected cyclopamine 21 by treatment with N-sulfinylbenze-
nesulfonamide[20] in an Alder-ene reaction followed by
desulfurization of the corresponding intermediate using
excess Raney nickel.[21] Under the adopted reaction condi-
Horner–Wadsworth–Emmons
reaction/intramolecular
Michael addition between b-ketophosphonate 17 (its syn-
thesis is given in the Supporting Information) and azidolactol
15 (accessible from the azidolactone by treatment with
diisobutylaluminum hydride (DIBAH) at ꢀ658C). After
extensive experimentation we realized that neither azidolac-
ꢀ
ꢀ
tol 15 (with a C12 C13 double bond) nor azidolactol 16
tions neither the C5 C6 double bond nor the benzyl ether
(directly synthesized from lactone 9 without rearrangement)
gave the desired product even at elevated temperatures and
prolonged reaction times. Much to our relief the regioisomer
protecting group was affected. This two-step preparation
proceeded in a good yield of 57%. Final removal, first of the
benzyl ether (Raney nickel in refluxing ethanol) and then of
the benzenesulfonamide using excess sodium naphthalenide
at ꢀ788C[22] gave the natural product, which was spectroscopi-
cally identical to a previously reported sample[23] in every
respect.
ꢀ
12 with a C13 C18 double bond after conversion into the
azidolactol 13 gave the desired furan 18 as a single product in
good yield under mild reaction conditions (base: barium
hydroxide, solvent system: THF/water[16]).
With the properly functionalized furan in hand, we
focussed on the construction of the piperidine ring. The 25-
We have developed a concise and efficient strategy
(20 steps, 1% overall yield) for the synthesis of cyclopamine.
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Communications
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Its features are a regio- and stereoselective hydroxylation by
ꢀ
C H activation, a biomimetic ring contraction/expansion, a
tandem Horner–Wadsworth–Emmons reaction/Michael addi-
tion, and an Alder-ene reaction. Since we also devised
methods to access several diastereoisomers and isomers
with an exocyclic double bond, we are confident that
implementation of this synthetic approach will broaden our
knowledge on cyclopamineꢁs structure–activity relationships
and will furnish analogues with fine-tuned bioactivity. Studies
and biological evaluations are currently being conducted and
will be reported in due course.
Received: May 12, 2009
Revised: June 30, 2009
Published online: July 9, 2009
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Keywords: alkaloids · antitumor agents · biomimetic synthesis ·
cyclopamine · natural products
.
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