Saturday, October 3, 2015

Dionicio Siegel’s ‘Nonbiomimetic’ Polyene Cyclization Process Enables the Total Synthesis of Celastroid Pentacyclic Triterpenoids

            The natural product celastrol exhibits diverse biological properties including anti-inflammatory and anti-cancer, as well as suppression of phenotypes associated with neurodegenerative disorders. For example, the unique quinone methide triterpenoid acts as a downregulator of mediators of anti-inflammatory responses such as interleukin-1a, TNF-a and nuclear factor kB (NF-kB). It also inhibits human prostate tumor growth and human glioma xenografts in mice. Recently, celastrol was identified as an inhibitor of heat shock protein 90 (Hsp90), which is over-expressed in cancer cells and plays a role in activation of certain pro-oncogenic signaling molecules. Celastrol is a non-ATP-competitive inhibitor of Hsp90 and acts via a mechanism that likely involves (in some as yet undefined fashion) conjugate addition of cysteine residues within biological nucleophile[s] to the electrophilic quinone methide substructure. In 2011, Richard Silverman’s laboratory at Northwestern University demonstrated that a range of soft nucleophiles add to the pharmacophore of celastrol in a highly stereospecific fashion. It is interesting to note that a related triterpene derivative, bardoxolone methyl (or CDDO, structure shown above), also contains a reactive cyanoenone Michael acceptor motif embedded in the western A-ring of its pentacyclic architecture. Its ability to generate reversible Michael adducts with biological sulfur nucleophiles is also speculated to be relevant to the molecular mechanism of action of CDDO. Bardoxolone methyl is currently in phase III clinical trials for the treatment of severe chronic kidney disease in type 2 diabetes mellitus patients.
            The total synthesis of celastrol was recently reported by Dionicio Siegel’s laboratory at the University of Texas at Austin. The ultimate success of Siegel’s synthetic approach to celastroid pentacyclic triterpenoids hinged on a polyene cyclization reaction that is described by the authors as ‘nonbiomimetic.’ Siegel and co-workers note that “biological polyene cyclization leading to celastrol is exceedingly difficult to reproduce in the laboratory due to a set of complex and energetically unfavorable methyl and hydride shifts.” The UT Austin team references synthetic work targeting alnusenone and friedelin, conducted in the late 1960’s and 70’s by Bob Ireland’s group. At this juncture, it may be instructive for readers to revisit the enzyme-catalyzed p-cation polyene cyclization of oxidosqualene into the ornate pentacyclic carbon skeleton of b-amyrin (detailed mechanism shown below) in order to understand why Siegel chooses to characterize his own cyclization reaction as ‘nonbiomimetic.’
            Tsutomu Hoshino’s laboratory at Niigata University in Japan has shown that oxidosqualene (and related unnatural polyene substrates) must be correctly ‘folded’ into a chair-chair-chair-boat-boat conformation in order to facilitate polycyclization leading to the intact b-amyrin carbocyclic scaffold. Hoshino’s group conducted studies involving incubation of synthetic derivatives of oxidosqualene in the presence of b-amyrin synthase derived from the African plant, Euphorbia tirucalli. They demonstrated that the methyl group at carbon position 30 of oxidosqalene plays an important role in binding to a hydrophobic recognition site of the enzyme, leading to appropriate construction of the ordered architectural conformation of the polyene substrate. Hoshino's studies suggest that a correct folding conformation of the polyene strongly influences the success of the polycyclization cascade. Oxidosqualene analogues that possess an intact Me-30 are more efficiently converted into pentacyclic terpenoid systems as compared to those lacking a terminal (Z)-Me group. Substrates that do not contain Me-30 generate far more abortive cyclization products. It is clear from the enzymatic mechanism depicted above, as well as the Hoshino group’s recent biosynthetic studies (outlined below), that the chemical polyene cyclization process developed by Siegel and co-workers at UT Austin is indeed ‘nonbiomimetic.’
            The UT Austin total synthesis of celastrol is initiated by execution of a two-directional approach starting from 2,3-dimethylbutadiene. In relatively short order, an advanced polyene-aldehyde intermediate (structure depicted below) is obtained by an expedient sequence of reactions featuring a tin-lithium exchange/alkylation to install the aromatic moiety. Stork-enamine Robinson annulation with methyl vinyl ketone (MVK) was successfully conducted on multigram scale to generate a critical cyclohexenone intermediate and subsequent lithium aluminum hydride reduction furnished the polyene cyclization substrate as an inconsequential diastereomeric mixture. Remarkably, exposure of a dilute solution of the cyclohexenol intermediate to the Lewis acid ferric chloride at low temperature promoted the stereocontrolled formation of the desired pentacycle with useful efficiency, in light of the complexity of the overall transformation. Siegel’s ‘nonbiomimetic’ polycyclization reaction was demonstrated on 1-gram scale. The Jones reagent was then used to oxidize the benzylic position located in the B-ring and subsequent selenoxide elimination installed the requisite enone. Demethylation afforded the catechol natural product wilforol A, which served as an intermediate that was suitable for eventual conversion into celastrol and its corresponding methyl ester, pristimerin. The total synthesis of racemic celastrol (obtained as a red-orange solid) was achieved in a total of 31 linear operations, starting from 2,3-dimethylbutadiene. The work is extremely important because it provides synthetic access to a medicinally relevant quinone methide triterpenoid that could not be easily obtained by a more cost-effective semisynthetic approach. For example, it is difficult to envision a method by which one might oxidatively convert a readily available pentacyclic triterpenoid such as oleanolic acid (the starting material for bardoxolone methyl) or b-amyrin into a sensitive quinone methide derivative. Hopefully, synthetic access to meaningful quantities of celastrol will facilitate studies to elucidate the precise biochemical mode of action leading to the diverse biological activies exhibited by this unique natural product.

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