Sunday, August 23, 2015

Application of Catalytic Asymmetric Olefin Hydroamination to Estrone Conjugation

            The synthesis of optically active a-branched chiral aliphatic amines often relies on enzymatic resolutions or stoichiometric auxiliaries, such as the Davis/Ellman tert-butylsulfinimine. Fukuyama’s nosylate alkylation/de-sulfonylation sequence is another effective way to access secondary amines in a stereocontrolled fashion and can also be used to construct medium-sized rings. However, the Fukuyama secondary amine synthesis is a multistep approach requiring unproductive protecting group manipulations to access chiral amines.
            Recently, Stephen Buchwald’s group has developed a copper-catalyzed intermolecular hydroamination reaction that effectively converts unactivated internal olefins (Science 2015) and styrenes (JACS 2015) into enantioenriched a-branched amines. The catalytic cycle of this powerful transformation is initiated by enantiodetermining hydrocupration involving the addition of copper(I) hydride across the double bond of the olefinic substrate. This furnishes a secondary alkylcopper intermediate in an enantioselective fashion. The intermediary organocopper compound is susceptible to a chain-walking mechanism consisting of iterative b-hydride elimination/migratory insertion steps that lead to the formation of undesired achiral by-products. This pathway is completely suppressed under the new Buchwald conditions. Rather, electrophilic interception of the transiently generated alkylcopper species with a customized hydroxylamine ester furnishes the intended amine product with release of copper benzoate. The latter is reconverted to ligated copper hydride by a stoichiometrically applied hydrosilane reagent. The electronic properties of the amine transfer reagent used in the process were optimized for electrophilic reactivity toward the secondary alkylcopper intermediate and resistance against reductive N-O bond cleavage upon exposure to hydrosilane. The axially-chiral ligand, DTBM-SEGPHOS (structure shown above), is highly active for promoting hydrocupration, yet effectively discerns between prochiral faces of a minimally differentiated olefin such as 2-butene. The method has been integrated into a cascade sequence that generates optically active pyrrolidines in one step from readily available precursors. The chemistry was also successfully demonstrated in the context of conjugation of chemically complex API-type substrates. For example, a vinylarene prepared in one step from Claritin (loratidine) could be coupled with an amine transfer reagent derived from estrone benzyl ether to afford the conjugated steroidal adduct (structure shown in the Scheme above) in good yield and with a high level of stereocontrol. The new Buchwald protocol has enormous potential for future pharmaceutical applications and provides and answer to the long-standing synthetic challenge of forging nitrogen-carbon bonds from abundant alkene precursors and amines.

Monday, August 17, 2015

The Sterol-Binding Fungicidal Mechanism of Action of Amphotericin B

            Amphotericin B (AmB) is a chemically complex polyene-macrolide natural product that has been used continuously since the 1960s as a last line of defense against systemic fungal infections. Unfortunately, AmB is plagued by an extrememly narrow therapeutic index. Indeed, because of the often dose-limiting toxicity of the antifungal drug, mortality rates for systemic fungal infections still hover near 50%.
            Martin Burke’s laboratory at the University of Illinois at Urbana-Champaign has taken a keen interest in the fungicidal mode of action of AmB, which has managed to evade significant microbial resistance for over 50 years. A better understanding of the precise nature of AmB’s ‘resistance-refractory’ mechanism might lead to new antimicrobial agents with reduced toxicity. The prevailing model used to interpret biophysical studies of AmB’s role within living systems is the ‘artificial ion channel model.’ According to this theory, AmB exists in the form of small ion channel aggregates that are inserted into lipid bilayers with membrane sterols oriented in between vertically-stacked molecules of AmB, arranged in a circular pore or cavity (see Figure below, top left). The artificial channels are thought to permeabilize and ultimately kill cells. The ion channel model dictates that the strategic pathway to improving therapeutic index is by achieving selective formation of ion channels in yeast over human cells.

            Burke has recently advanced an alternate mechanistic scenario to explain AmB's potent fungicidal properties. In the 'sterol sponge model' (Figure above, top right), AmB forms large extramembranous aggregates that extract the essential sterol ergosterol from phospholipid bilayers. The coincidental extraction of membrane cholesterol (unselectively with ergosterol) might then be primarily responsible the observed toxicity of AmB to human cells. This insight, if proven correct, could guide the development of novel AmB derivatives with an improved therapeutic index. With regard to evasion of resistance, AmB may simultaneously disrupt all of the cellular processes that depend on membrane ergosterol. Simultaneous mutation of all requisite fungal proteins in order to alleviate the ergosterol dependence of the pathogenic organism is then improbable and, hence, no significant resistance emerges.
            In 2014, Burke’s group presented evidence from biophysical, cell-based experiments that uniformly supports the new sterol sponge model. As such, an improvement in the relative binding affinity of AmB aggregates for ergosterol over cholesterol was expected to translate to antimicrobial agents with lower toxicity towards human cells. The Illinois team approached this challenge by initially examining a recent crystal structure of an AmB derivative that reveals an intramolecular salt bridge between the C16 carboxylate and the C3’ ammonium moiety of AmB’s mycosamine subunit (See AmB molecular structure, depicted above). They hypothesized that a complex network of intramolecular noncovalent interactions, including the aforementioned salt bridge, orients the mycosamine in a conformation that is capable of forming an H-bond to the 3b-hydroxyl of both ergosterol and cholesterol (diagrammed below). The mycosamine C2’-hydroxyl was implicated in this network of interactions by previous studies which demonstrated that deletion of the C2’-hydroxyl group (in semisynthetic C2’deOAmB) leads to differential binding of ergosterol and cholesterol. A high-resolution structure of the AmB aggregate (the functional ‘sponge’), with and without bound sterol, would enable rational design of new synthetic molecules. However, in its absence, a practical and highly modular semisynthetic route to derivatize the ‘eastern’ mycosamine-bearing substructural framework of AmB was sought. Newly generated analogs would then be evaluated for sterol binding selectivity and correlation to selective toxicity to yeast.
            A remarkably concise strategy for AmB derivatization was developed by Burke and co-workers. The route (shown below) involves only three reaction ‘pots’ and requires one chromatographic separation to produce pure material on gram-scale. The exceptionally mild oxazolidinone ring-opening by amine nucleophiles is noteworthy. The facile nature of this transformation, which introduces a new point of diversity for SAR studies, likely reflects inherent ring-strain residing within the trans-fused 6/5 heterocyclic ring system of the bracketed intermediate. The tetrahydropyran methyl ketal of the penultimate species is converted into its corresponding hemiacetal during the course of HPLC purification, which uses aqueous formic acid in the mobile phase. An ultracentrifugation-based membrane isolation assay indicated that the new C16-urea derivatives exhibit binding selectivity for ergosterol over cholesterol. Moreover, the sterol binding selectivity was generally associated with an improvement in therapeutic index, as indicated by a comparison of the minimum inhibitory concentration (MIC) against S. cerevisiae with the minimum hemolytic concentration (MHC) determined against human red blood cells. This project is a great example of a practical and well-executed organic chemistry strategy, applied to a long-standing pharmacological problem with profound human clinical implications. The emerging AmB mechanistic picture is an example of a rarely encountered pharmacological phenomenon – that is, noncovalent binding between a small molecule drug and an endogenous small molecule. The seemingly ‘resistance-refractory’ nature of this type of mechanism renders it a highly desirable platform for pharmacological intervention aimed at anti-infective drug development with low tox and evasion of resistance. The work also illuminates to the ubiquity and essential nature of sterols such as cholesterol and ergosterol to living systems within the cells of both humans as well as pathogenic organisms.

Friday, August 7, 2015

Glycosylation Reactions Involving a High Degree of Chemical Complexity: Nicolaou’s Total Synthesis of Shishijimicin A

            Naturally occurring glycosylated steroids and triterpenoids often exhibit pharmacological bioactivity superior to that of their corresponding aglycones. Prominent examples include the immunostimulatory adjuvant QS-21-apiose and the cardiotonic glycoside drug digoxin. Limonin glucoside, unlike its parent aglycone, limonin, is freely water-soluble and has been shown to lower levels of circulating biomarkers of chronic inflammatory diseases when administered orally in beverages. Moreover, while limonin glucoside is almost tasteless, low concentrations of its aglycone impart an extreme bitterness that is unacceptable to consumers.
            A number of fully carbohydrate-based polysaccharide drugs have been registered to date, but most of these active pharmaceutical ingredients (APIs) originate from natural sources. Synthetic oligosaccharides are rare, due to a reliance of carbohydrate synthesis on linear strategies involving elaborate, orthogonal protecting group schemes and cumbersome purifications. Fondaparinux (molecular structure depicted below) is a unique synthetic pentasaccharide API that was approved in 2001 for the prevention of venous thromboembolism. It has been obtained from total synthesis by a route that requires approximately 50 chemical steps. In view of the examples noted above, it is not surprising that the development of efficient and scalable chemical glycosylation protocols is of great interest to the pharmaceutical industry.
            Total synthesis studies targeting bioactive natural products often stimulate the invention of novel bond-forming methodologies that enable the eventual chemical synthesis of increasingly complex classes of drug candidates. This important area of research also provides an arena to evaluate the effectiveness of existing synthetic technologies, when applied in the context of molecular architectures at the height of chemical complexity. The endiyne antitumor antibiotics fall into this latter category. Natural products from the endiyne class such as calicheamicin g1 (structure shown below) exhibit exquisitely potent cytotoxicity against cancer cell lines due to a fascinating mechanism of action involving Bergman cycloaromatization. This phenomenon gives rise to a 1,4-benzenoid diradical that can abstract two hydrogens from DNA, causing strand cleavage. Calicheamicin g1 serves as the ‘warhead’ or ‘payload’ that is conjugated to an antibody in the approved antibody-drug conjugate (ADC), Mylotarg. The advent of ADCs has prompted renewed interest in high-potency cytotoxic agents that were previously unsuitable for clinical applications due to severe side effects. Recently, the total synthesis of a related endiyne natural product, shishijimicin A, was reported by K. C. Nicolaou’s laboratory at Rice University in Houston, Texas. The inarguably impressive synthesis reported by Nicolaou’s group employs a challenging glycosylative coupling reaction between two highly elaborated fragments that calls attention to a limitation of the classical Schmidt trichloroacetimidate protocol. The daunting glycosylation chemistry outlined below highlights the synthetic difficulties associated with endiyne production in the laboratory and underscores the need for new and robust oligosaccharide coupling technologies.
            In the course of Nicolaou and co-workers’ total synthesis of shishijimicin A, the two advanced intermediates, a trichloroacetimidate glycosyl donor and a hydroxy-endiyne glycosyl acceptor (Scheme below, lower panel), were prepared by a lengthy but reasonably efficient synthetic sequence of reactions. The two fragments were subjected to a standard variant of the Schmidt glycosylation reaction, wherein exposure to the Lewis acid BF3-OEt2 promotes formation of a transient oxonium species derived from the glycosyl donor, ultimately furnishing the b-glycoside adduct in a meager 26% yield. The reaction was exemplified on 40-milligram scale. The authors attributed the low yield to steric hindrance, although both coupling partners contain a diverse array of functionality that could potentially engage in unproductive pathways under Lewis acidic conditions. The condensation product was shown to be suitable for subsequent conversion to the natural product. Shishijimicin A is expected to serve as a uniquely powerful payload for ADC biomedical applications due to its extremely potent antitumor properties (IC50 = 0.48 pM against leukemia cells). A similarly complex endiyne family member, maduropeptin, was synthesized by Masahiro Hirama’s group at Tohoku University in Japan in 2009. In the case of maduropeptin, an embedded tertiary alcohol serves as the glycosyl acceptor in the critical glycosylative fragment-coupling step. A Schmidt glycosylation using 6.4 milligrams of the endiyne glycosyl acceptor afforded 4.9 milligrams (40% yield) of the advanced maduropeptin synthetic intermediate, as a mixture of two atropisomers. Hirama et al achieved the first total synthesis and structure revision of the maduropeptin chromophore. The chemical synthesis of endiyne targets residing at the frontier of chemical complexity is instructive to organic chemists and generates precious material for biochemical investigations and ADC-related conjugation studies. However, the yields achieved in the requisite late-stage glycosylative coupling steps clearly illustrate a need for further methodological refinement within this important and medicinally relevant area of synthetic chemistry.

Sunday, July 19, 2015

On Biosynthetic and Biomimetic Steroid Chemistry: The Polyene Cyclization Mechanism of Lanosterol


            The biogenetic mechanism for the formation of the 6-6-6-5 tetracyclic steroid lanosterol (outlined below), an intermediate in the biosynthesis of cholesterol, has been studied by chemists for over half a century. Enzyme crystallographic data, together with other bioorganic and mutational studies, have yielded a biomolecular mechanism for enzymatic triterpene cyclization starting from acyclic 2,3-oxidosqualene. The mechanism involves initiation through protonation by a specific aspartic acid residue. The polycyclization cascade then proceeds away from the catalytic acid, with intermediates shielded by aromatic amino acid residues that line the active site cavity with p-electron-rich faces. However, this biosynthetic conversion is incredibly complicated and many of its precise details have not been fully resolved. One key question that has remained unanswered until recently concerns the extent to which ring formation is concerted with the initial protonation step. Earlier this year, in an effort to sort out uncertainties associated with this complex mechanism, Ruibo Wu’s group in Guangzhou, China, in collaboration with a team at Vanderbilt University led by the theoretical computational chemist Andes Hess, successfully characterized the cyclization of squalene oxide at the highest computational level carried out to date.
            The investigators used quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) simulations to construct the overall cyclization free-energy profile (reproduced below) for this fascinating biosynthetic transformation. The study shows that the entirety of the process (acyclic substrate √† protosterol cation) entails two concerted, asynchonous reactions which proceed through two metastable states (the A and B states) and one stable intermediate, under oxidosqualene cyclase (OSC) enzyme catalysis. The overall cyclization can be considered a carbocation migration and is exergonic by ~4.4 kcal/mol. The rate-limiting step is the concerted formation of the A-ring, with opening of the epoxide ring. The stable intermediate (the C’ state) is a tricyclic 6-6-5 species that is subsequently converted to the tetracyclic protosterol cation (the D state) by a ring expansion with concomitant (concerted) formation of the D-ring. Importantly, all previous experimental mutagenic data is consistent with the identified reaction mechanism.

Hess and Wu. ACIE 2015, 54, 8693.
            The 2015 computational mechanistic study of Hess and Wu will no doubt inspire the synthetic enthusiasts among us to reflect on the myriad of creative ways in which organic chemists have tried to mimic the action of the enzyme OSC in a laboratory flask or reactor. This area of so-called ‘biomimetic’ research was pioneered by W. S. Johnson at Stanford University in the 1960’s and 70’s. Johnson’s efforts were later extended by countless others. In his 1976 Review Article in Angewandte Chemie, Johnson elegantly summarizes the synthetic strategy: “Certain polyenic substances having trans olefinic bonds in a 1,5 relationship can be induced to undergo stereospecific, non-enzymic, cationic cyclization to give polycyclic products with all-trans (“natural”) configuration. These transformations appear to mimic in principle the biogenetic conversion of squalene into polycyclic triterpenoids…” A highlight from Johnson’s impressive body of research is a remarkably short synthesis of racemic progesterone, starting from the cyclopentenol shown below. Cyclization under Br√łnsted acidic conditions produces a crystalline tetracyclic product in 71% yield. Subsequent zonolysis followed by intramolecular aldol condensation then furnishes synthetic progesterone. A related process (lower panel below) was also conceived by Johnson to generate a valuable 11a-substituted intermediate that intersects with a known commercial route for the production of hydrocortisone acetate. More recently, ‘biomimetic’ polyolefin carbocyclization has been elegantly applied by E. J. Corey’s laboratory to the chemical synthesis of complex limonoid steroidal systems (highlighted here).