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.

Wednesday, September 9, 2015

Recent Synthetic Work on the Triterpenoid Biopesticide Azadirachtin

            Azadirachtin is a complex tetranortriterpenoid limonoid natural product isolated from the neem tree Azadirachta indica, of the Meliaceae family of flowering plants. The neem tree is evergreen and produces white and fragrant flowers as well as a fruit that is smooth and olive-like. The neem is native to India and the Indian subcontinent including Nepal, Pakistan, Bangladesh and Sri Lanka. The azadirachtin content of neem oil pressed from fruits and seeds varies from 300 to 2500 ppm, depending on the extraction method and quality of the neem seeds used to produce the oil. Nimbin was the first limonoid extracted from the neem tree. Subsequently, more than 150 bioactive chemical constituents were isolated from neem oil and various neem tissues. Aside from triterpenoids, neem is known to contain sterols such as b-sitosterol and stigmasterol, as well as polyunsaturated fatty acids.
            Azadirachtin is a powerful insect repellent/anti-feedant with low mammalian toxicity (LD50 in rats is >3.5 g/kg). A 1.2% formulated solution of azadirachtin is marketed as Azatrol, an insecticide that provides broad-spectrum insect control and is non-toxic to honeybees. Azadirachtin is the active ingredient in a number of other pesticides including TreeAzin, AzaMax and AzaGuard. The enormous commercial potential of new azadirachtin analogues as safe anti-insect agents has inspired/funded organic chemists for decades. The sole chemical synthesis of azadirachtin, reported in 2007 by Steven Ley’s group at the University of Cambridge, relied on a relay approach and required >70 synthetic operations. Ley’s retrosynthetic disconnection of the crowded vicinal quaternary carbon atoms located at the C8 and C14 positions, a tactic that was adopted by numerous contemporary research groups (most notably, Nicolaou’s), generates two advanced synthetic intermediates: a highly oxygenated western decalin substructure and an eastern furopyran oxabicycle. Zhen Yang’s team in Shenzhen, China has recently disclosed remarkably concise protocols for the chemical synthesis of both of these fragments. A discussion of Yang’s synthetic studies of the azadirachtin-type limonoids is provided below.
            Upon preliminary examination of the molecular architecture of azadirachtin, the structural relationship between this oxidatively rearranged tetranortriterpenoid and more simplified C13a/17-furyl-androstane limonoids is perhaps not immediately intuitive (otherwise put, what does azadirachtin have to do with a steroid?!). Identification of the genes and proteins involved in the neem biosynthetic machinery is currently the subject of intense investigation due to the impracticalities associated with commercial production of azadirachtin via chemical synthesis. The prevailing biogenetic theory is that azadirachtin arises from C-seco-limonoids derived from Norrish a-cleavage of an oxidation product of azadirone or azadiradione. So, yes, azadirachtin is likely a biosynthetic descendant of azadirone, which bears an androstane/steroidal core carbon skeleton. Cyclization of a cyclopentenol precursor (bracketed intermediate shown above) onto the heteroaryl furan system likely forges the eastern, bridged hydroxyfuran acetal motif. Evidence for this type of biogenetic pathway is found in the chemical composition of neem oil, which is known to contain relatively high levels of putative biosynthetic intermediates including azadirone, azadiradione, nimbin, nimbidinin, salannin, salannol, along with trace amounts of other structurally related limonoids.
            Yang’s synthesis of azadirachtin’s western A/B decalin fragment begins from the spearmint oil-derived enantiomer of carvone, which was converted in two steps into the substituted cyclohexene derivative depicted in the Scheme above. This early-stage intermediate was subjected to palladium-catalyzed oxyalkynylation, furnishing the requisite [6,5]-oxabicycle in good yield under optimized conditions. The reaction is completely regio- and diastereoselective, with the relative stereochemical outcome presumably dictated by minimization of steric interactions between the methyl and vinyl substituents. Subsequent ozonolysis of the propenyl group then delivers an allylic acetate intermediate bearing an intact endocyclic olefin. This latter reaction was developed in the early 1980s by Stuart Schreiber’s group, then at Yale University. Schreiber showed that the intermediary carbonyl oxide derived from retro-[3+2] of the initially formed molozonide could be trapped by 1,3-addition of methanol to generate an a-methoxy hydroperoxide species. Upon acylation of the hydroperoxide in the presence of base, a Criegee rearrangement involving alkyl migration (similar to the Baeyer-Villiger mechanism) ensues to provide the acetate as an inconsequential mixture of diastereomers. Seven additional operations were required to eventually produce the critical 1,7-diyne that was designed to serve as the substrate for a remarkable gold-catalyzed tandem cyclization reaction, described below.
            The gold-catalyzed cascade reaction proceeded under mild reaction conditions, generating the densely functionalized trans-decalin product as a single stereoisomer in 49% yield. This stereocontrolled alkyne metathesis-type cyclization reaction forms two rings and two new stereocenters in a single operation. The fully intact azadirachtin western decalin substructure was synthesized from the cascade cyclization product in eight additional steps. The overall route to Yang’s decalin requires 20 total synthetic operations, starting from (-)-carvone. It should be noted that a similar advanced decalin intermediate was synthesized in the early 2000’s by Nicolaou’s group by a sequence involving a total of 47 linear operations.
            Yang’s synthesis of the eastern furo[2,3b]pyran azadirachtin fragment relies on silylglyoxylate-based synthetic technology developed by Jeff Johnson’s group at UNC Chapel Hill. Yang’s recent limonoid work extends Johnson’s methodology to accommodate the use of allylzinc bromide in place of Reformatsky’s reagent in the diastereoselective three-component coupling process (see Scheme above). The reaction proceeds by addition of the allylzinc reagent to the silylglyoxylate benzyl ester, followed by [1,2]-Brook rearrangement to afford a zinc-chelated enolate. The latter nucleophile approaches the b-lactone coupling partner from its less hindered convex side to furnish the condensation product with exquisite 1,4-stereoinduction. The remaining stages of the route involve initial g-lactonization followed by ozonolysis of a lactol intermediate to construct the oxabicyclic ring system. A bis-TBS-protected furo[2,3b]pyran building block was prepared in a total of nine steps and a differentially protected system, similar to Ley’s advanced intermediate, was generated in 15 steps. Those familiar with the Ley group’s endgame know all too well that the completion of azadirachtin from the advanced intermediates described above is far from trivial. It will be interesting to see the extent to which Yang’s expedited syntheses of the eastern and western fragments will enable methodological improvements in critical aspects of the azadirachtin endgame strategy, such as construction of the notoriously challenging C8-C14 bond which links the two substructural hemispheres of the natural product.