Concise modular asymmetric synthesis of deguelin, tephrosin and investigation into their mode of action

Article abstract of DOI:10.1002/chem.201001080

(Figure Presented) The concise nature and modularity of the synthesis described for deguelin and tephrosin (retrosynthetic analysis depicted) should facilitate access to labeled analogues to dissect the mechanism of action of this important pharmacophore.

Full text of DOI:10.1002/chem.201001080

COMMUNICATION
DOI: 10.1002/chem.201001080
Concise Modular Asymmetric Synthesis of Deguelin, Tephrosin and
Investigation into Their Mode of Action
Josꢀ Garcia,[a] Sofia Barluenga,[a] Kristin Beebe,[b] Len Neckers,[b] and
Nicolas Winssinger*[a]
Dedicated to Professor Josꢀ Barluenga on the occasion of his 70th birthday
Cubꢀ resin has been known since the 1850s[1] to contain
active ingredients useful as pesticides and insecticides.[2]
Fractionation of the active ingredients has led to the isola-
tion of biosynthetically related flavonoids and rotenoids
which have been reisolated from various botanical sources.[3]
The most prominent and active ingredients are rotenone,
deguelin and tephrosin (Figure 1). The toxic properties of
rotenone were originally ascribed to its inhibition of the
NADH/ubiquinone oxidoreductase machinery. Since the
mid 90s, several lines of evidence emerged regarding the an-
ticancer and chemopreventive properties of these com-
pounds.[4,5] It was indeed shown by Pezzuto and co-workers
that deguelin most potently inhibited the induction of orni-
thine decarboxylase[6] which is associated with tumor pro-
gression.[7,8] It was then shown that the reduction of orni-
thine decarboxylase activity is coupled to the NADH/ubiq-
uinone inhibition[9] and that rotenone binds to the PSST
subunit of this large multi-protein complex.[10] More recent-
ly, Lee and co-workers demonstrated that deguelin disrupt-
ed the interaction between Hsp90 and HIF1a and suggested
that deguelin binds to the nucleotide binding pocket of
Hsp90[11] as radicicol and geldanamycin derivatives.[12] Most
recently, deguelin and related rotenoids were identified in a
screening for modulators of oncogene-regulated hemato-
poietic differentiation using a zebrafish model supporting
the Hsp90 inhibition mechanism of deguelin.[13] Hsp90 has
emerged as an important therapeutic target.[14–16] Despite
the seemingly ubiquitous functions of this highly expressed
chaperone, its role in stabilizing conformationally labile pro-
teins has implications in diverse pathologies ranging from
cancer indications,[17,18] neurodegenerative diseases,[19–23] in-
fectious diseases,[24] and inflammation-related disorders.[25]
Our interest in Hsp90 inhibition[26,27] instigated the develop-
ment of synthetic technologies which could be used to
broaden the diversity of the rotenoids.
Figure 1. Structure of rotenone, deguelin and tephrosin.
While the first synthesis of rotenone dates back to the
1960s,[28,29] the ready availability of the natural product itself
has not stimulated significant synthetic efforts towards the
rotenoids and there is no asymmetric synthesis of deguelin
(only three total synthesis of racemic deguelin and one
formal synthesis).[30–33] Even more surprising considering its
potent biological activity, there is no reported synthesis of
tephrosin. Structure–activity studies regarding the rotenoids
has been largely confined to related natural products ex-
tracted from different sources and manipulation by semisyn-
thesis.[3] A recent study comparing the activity of deguelin,
tephrosin, rotenone and related natural products showed
tephrosin to be the most potent inhibitor of HT1080 prolif-
eration amongst the family of rotenoids and related fla-
vins.[34]
[a] Dr. J. Garcia, Dr. S. Barluenga, Prof. N. Winssinger
Institut de Science et Ingꢀnierie Supramolꢀculaires (ISIS–UMR 7006)
Universitꢀ de Strasbourg–CNRS, 8 allꢀe Gaspard Monge
67000 Strasbourg (France)
Fax : (+33)368855112
E-mail: winssinger@unistra.fr
[b] K. Beebe, Dr. L. Neckers
National Cancer Institute, Urologic Oncology Branch
9000 Rockville Pike, Bethesda, MD 20892 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201001080.
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9767

We reasoned that deguelin and tephrosin could arise syn-
thetically from a common intermediate 1 via different forms
of oxidation (dihydroxylation or epoxidation) of the conju-
gated alkene 1 which would in turn provide efficient discon-
nections of the fused ring system (Scheme 1). Further dis-
connections through a combination of a metathesis and
Heck cyclization leads to two readily available intermediates
2 and 3 which could be coupled via Mitsunobu reaction.
Indeed, some precedents for the dihydroxylation and the
Heck reaction already existed, however, the question re-
mained whether this dihydroxylation could be performed in
the presence of the other pyran unsaturation.[35]
The synthesis commenced with the derivatization of 2,4-
dihydroxybenzaldehyde (4) with 3-chloro-3-methylbutyne
(Scheme 2). A very clean conversion to the desired para-al-
kylated product was obtained due to its greater acidity using
potassium carbonate as a base. It turned out to be crucial to
include catalytic amounts of copper[36] in the reaction to ac-
centuate the reactivity of this otherwise sterically encum-
bered electrophile. The alkyne was cyclized under thermal
conditions (1658C) to obtain 5.[37] While this reaction can be
done at lower temperature in the presence of platinum di-
chloride catalyst, the thermal reaction proceeded in higher
yield and could be achieved quantitatively in only 30 min
under microwave irradiation. The aldehyde was then olefi-
nated with an excess of methylenetriphenylphosphorane to
afford intermediate 2 in 87% overall yield (three steps).
Fragment 9 was obtained in three steps from commercially
available 3,4-dimethoxyphenol (6) by nucleophilic epoxide
opening of (2R)-vinyloxirane followed by iodination (NIS,
TFA)[38] and Heck cyclization in 34% overall yield. The io-
dination proved to be the most problematic step. While the
crude NMR of the reaction showed a quantitative transfor-
mation, the product was colored and a quick filtration
through a pad of silica always led to loss of material. Other
iodination methods and alternative work-up procedures did
not improve the outcome of the reaction. The key inter-
mediates 2 and 9 were coupled under Mitsunobu conditions.
The cyclization using Grubbsꢂ second-generation cata-
lyst[39–41] afforded a quantitative transformation based on
NMR and LC/MS analysis, however, purification by silica
gel column led to loss of material. Sharpless asymmetric di-
hydroxylation[42] using the ADmix-a afforded the desired
product 10 in excellent yield as a 5:1 diastereomeric ratio
(separable by silica gel) while the ADmix-b afforded the op-
Scheme 2. Synthesis of tephrosin and deguelin. a) 3-Chloro-3-methylbut-
1-yne (1.1 equiv), K2CO3 (1.1 equiv), KI (0.10 equiv), CuI (0.05 equiv),
CH3CN, 238C, 12 h, 90%; b) m-xylene, mw, 1808C, 30 min, quant; c)
PPh3MeBr (3.3 equiv), nBuLi (3.3 equiv), THF, 08C, 1 h, 97 %; d) 7
(6.0 equiv), NaH (1.5 equiv), DMF, mw, 80 8C, 20 min, 70%; e) NIS
(1.1 equiv), TFA (0.30 equiv), CH3CN, 238C, 15 min, 60%; f) KOAc
(5.0 equiv), PdACHTNUTRGNEUNG(OAc)2 (0.1 equiv), dppe (0.20 equiv), DMF, 30 min,
1008C, 81%; g) PS-DEAD (2.0 equiv), PPh3 (2.0 equiv), NEt3.(2.0 equiv),
CH2Cl2, 238C, 24 h, 63%; h) Grubbs II (0.10 equiv), CH2Cl2, 238C, 4 h,
78%; i) ADmix-a (3.0 equiv), MeSO2NH2 (3.0 equiv), tBuOH/H2O 1:1,
238C, 12 h, 97% (5:1 d.r.); j) PS-IBX (10 equiv), CH2Cl2, 238C, 14 h,
33%; k) Zn, AcOH, mw, 88 8C, 10 min, 50%. PS-DEAD = ethoxycarbo-
nylazocarboxymethyl polystyrene; dppe
=
1,2-bis(diphenylphosphino)-
2-iodoxybenzoic acid;
ethane; NIS N-iodosuccinimide; IBX
=
=
ADmix-a = 1.6mm (DHQ)2PHAL, 500mm potassium carbonate, 500mm
potassium ferricyanide, and 0.7mm potassium osmate dihydrate.
posite diastereoisomer exclu-
sively; this suggests that the ex-
isting stereochemical informa-
tion is mismatched relatively to
the asymmetric induction of the
ADmix-a. It is noteworthy that
no product arising from dihy-
droxylation of the pyran system
was detected. Treatment of the
Scheme 1. Retrosynthetic disconnections of deguelin and tephrosin.
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Concise Modular Asymmetric Synthesis
COMMUNICATION
product 10 with IBX resin[43] afforded tephrosin along with a
byproduct originating from the cleavage of the diol in a
ratio of 8:1 (measured by LC-MS). While these products
were not separable by silica gel, tephrosin could be isolated
by HPLC albeit with a loss of material affording a 33% iso-
lated yield. It is noteworthy that treatment of 10 with Dess–
Martin reagent led exclusively to the cleavage of the diol.
Treatment of 10 with acids such as sulfonic acid resin yield-
ed a pinacol rearrangement to give the furan 11 related to
glyceollidin (Scheme 3). No trace of proton transfer which
cation. Thus, the crude Mitsunobu product was subjected to
a metathesis followed by dihydroxylation and IBX oxida-
tion. To our satisfaction, tephrosine and its diastereoisomers
emanating from the S enantiomer of 9 were the only signifi-
cant products observed by LC/MS (Figure 2) and could be
isolated in 23% overall yield.
With these products in hand we assayed their affinity for
Hsp90’s nucleotide-binding pocket using
a competition
assay[44] with fluorescently labeled 17-AAG known to target
the N-terminal nucleotide binding pocket. Contrarily to the
previous speculations based on
docking studies,[11] deguelin,
tephrosin and rotenone showed
no notable affinity in this assay
(>50 mm). To further investi-
gate potential pharmacological
similarities between deguelin
and geldanamycin derivative
17-AAG, the three rotenoids
(deguelin, tephrosin and rote-
Scheme 3. Pinacol rearrangement of intermediate 10. a) PS-SO3H (20 equiv), CH2Cl2, 238C, 2 h, quant; b)
H2NOR·HCl (10 equiv), EtOH, 408C, 4 h, (12, 90%; 13, 92%).
none) were assayed for their
ability to promote client deple-
tion in SkBr3, a breast cancer
cell line known to be responsive
would have led to deguelin was detected under a variety
conditions. Attempts to achieve this transformation via the
epoxide were unsuccessful. Nevertheless, product 11 pro-
vides an interesting diverging point into a different ring ge-
ometry and could be readily elaborated into diverse oximes
such as 12 and 13. Tephrosin could be cleanly reduced to de-
guelin under the action of zinc with acetic acid albeit with-
out reaching a complete conversion. Attempts to push the
reaction beyond 50% conversion led to the emergence of
side products. Synthetic tephrosin and deguelin had identical
spectral and chromatographic properties to the natural sam-
ples. Noting that several isolated yields were low despite ex-
cellent conversion, the reaction sequence starting from inter-
mediates 2 and racemic 9 was carried out without any purifi-
to Hsp90 inhibitors such as 17-AAG.[45] The level of ErbB2,
a client known to be sensitive to pharmacological inhibition
of Hsp90 with 17-AAG, and phosphoAkt (pAkt), a client
previously reported to be sensitive to deguelin treatment[11]
were analyzed. As shown in Figure 3, 17-AAG caused the
expected depletion of both ErbB2 and pAkt. None of the
rotenoids caused significant depletion of ErbB2 while all ro-
tenoids caused moderate depletion of pAkt with rotenone
being the most effective compound (Figure 3). While degue-
lin does not bind the N-terminal nucleotide pocket of
Hsp90, these results do not exclude that deguelin and relat-
ed rotenoids modulate the function of Hsp90 by binding to
the C-terminal domain in analogy to novobiocin.[46–48]
Indeed, it has been shown that C-terminal domain binders
Figure 2. LC-MS chromatogram of the crude product obtained from the reaction sequence starting with 2 and racemic 9 without any silica purification.
Chem. Eur. J. 2010, 16, 9767 – 9771
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N. Winssinger et al.
Figure 3. Depletion of ErbB2 and pAkt cells that were treated with the inhibitor for 18 h.
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three rotenoids (deguelin, tephrosin and rotenone) on the
Hsp90-IP6K2 association was evaluated as it is known to be
sensitive to C-terminal binders. To this end, 293T cells co-
transfected with Flag-Hsp90 and myc-IP6K2 were incubated
with 10 mm of each compound for 18 h and, upon lysis of the
cell, the integrity of the Hsp90-IP6K2 association was as-
sessed by immunoprecipitation of Hsp90 (anti-Flag anti-
body) and blotted with anti-myc antibody. None of the rote-
noids showed any disruption (data not shown).
Deguelin and related rotenoids are attractive pharmaco-
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breast, colon and skin cancer[51–53] and preventing growth in
a mouse xenograft bearing a lung tumor (H1299).[11] While
deguelin has been shown to disrupt some of Hsp90’s func-
tion, it does not bind to the N-terminal domain nor C-termi-
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Acknowledgements
This work was supported in part by a grant from Institut Universitaire de
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Keywords: deguelin · Hsp90 · natural products · tephrosin ·
total synthesis
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Received: April 23, 2010
Published online: June 22, 2010
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9771