Regioselective Semisynthetic Modification of a Cardiotonic Steroid Glycoside

Semisynthetic derivatization of complex natural products can often be an effective medicinal chemistry strategy to improve upon the potency/selectivity profile or pharmacokinetic properties of a naturally occurring bioactive molecule. For example, the potent and broad-spectrum antifungal drug CANCIDAS is a synthetic derivative of the echinocandin macrocyclic lipopeptide natural product, pneumocandin Bo. A significant challenge to this type of approach is the development and implementation of highly selective synthetic methods that can effectively manipulate a single part of a molecule in the presence of other functionality with similar reactivity. Along these lines, the regioselective acylation of polyol natural products is an active area of academic research that could eventually impact the very nature of the drug development process by rendering previously inaccessible semisynthetic compounds readily available. Takeo Kawabata's research group at Kyoto University has recently described the regioselective acylation of the cardiotonic steroid glycoside, lanatoside C, which is composed of the aglycone digoxigenin, linked via the steroidal C3-oxygen to a complex tetrasaccharide (structure shown above). Both the closely related steroid digoxin (1), as well as lanatoside C (2), are clinically used cardiotonic chemical entities. The Kawabata group has developed conditions to selectively monoacylate either the C(3'''')-OH or the C(4'''')-OH of the terminal glucopyranoside of the western tetrasaccharide of 2.

N,N-dimethylaminopyridine (DMAP)-catalyzed acylation of lanatoside C gives the C(3'''')-O-acylate (3) with 97% regioselectivity due to the inherent nucleophilic reactivity of the substrate. On the other hand, acylation of lanatoside C in the presence of the complex organocatalyst (A) affords the C(4'''')-O-acylated product with about 90% catalyst-controlled regioselectivity. The authors provide a very interesting hypothetical transition state assembly model to account for the observed outcome. In this picture, the catalyst-substrate interaction involves hydrogen bonds between (C6'''')-OH (to a carbonyl) and C(3'''')-oxygen (to indole N-H) and the catalyst. A molecular modeling conformational analysis of lanatoside C was also conducted. It is quite remarkable that Kawabata's organocatalyst (A) can distinguish between the no less than eight free hydroxy groups within 2 to provide essentially a single acylation product (4), an outcome that is completely divergent from the inherent reactivity patterns of the natural product. This outstanding effort is reminiscent of the now classic pioneering work done by Scott Miller's group on erythromycin A, apoptolidin A and vancomycin.

The Cycloalkenone Diels-Alder Approach to Substituted Hydrindenes

          Hydrindane and hydrindene structures such as 7 are important synthetic building blocks for the stereocontrolled assembly of complex carbocyclic frameworks found in triterpenoids and steroids. For example, an optically active hydrindane structure served as an important advanced intermediate in Corey's synthesis of the sesterpenoid retigeranic acid. Danishefsky's laboratory at Columbia University has recently described a general approach to the hydrindane system that exploits a unique intramolecular Diels-Alder reaction (IMDA) followed by an oxidative fragmentation of the tricyclic cycloadduct (JACS 2012). 

          As depicted in the scheme above, a diene tethered to a highly dienophilic cyclobutenone (4) was synthesized in three straightforward steps from 1 and 2. Upon exposure of this substrate to a catalytic amount of Lewis acid, IMDA reaction proceeded smoothly to provide 5 (92% yield) with a high level of stereoselectivity, wherein an endo transition state arrangement (shown above in brackets) gave rise to the cis ring junction between the six- and four-membered rings. The suprafacial [4+2] cycloaddition reaction also established a trans relationship between the six- and five-membered rings of the tricyclic adduct (5). Subsequent Baeyer-Villiger oxidation of 5 led to a ring-expanded lactone structure (6) without epoxidation of the C-C double bond. Finally, reductive cleavage of the lactone with lithium aluminum hydride secured the unique, angularly substituted trans hydrindene 7, a synthon that is inaccessible by a conventional Diels-Alder strategy due to its ring junction stereochemistry and double bond position. The olefin functionality also affords the opportunity for additional diversification and elaboration. The trans hydrindenoid 7 will likely serve as a useful intermediate in future syntheses of architecturally complex carbocyclic structures.

David Gin's Synthesis of the Investigational Immunostimulatory Adjuvant QS-21(Api)

The 21^st fraction from the reverse-phase HPLC purification of the extract of the South American tree, Quillaja saponaria Molina, was shown to contain the complex triterpene QS-21-Apiose (1). The natural product 1 is comprised of a central quillaic acid triterpene core, flanked on both sides by complex oligosaccharides. As we discussed here previously, a family of immunostimulatory natural products structurally related to 1 have emerged as promising new adjuvants for immune response potentiation. Notably, scientists at Memorial Sloan-Kettering Cancer Center have synthesized several of these complex isomeric saponins, permitting, for the first time, access to pure samples of homogenous composition. In an effort to understand the structure-activity relationships (SARs) associated with this family of investigational adjuvants, the same group has also prepared non-natural analogues of 1. Synthetic derivatives such as 2 provide improved chemical stability and favorable toxicity profiles relative to QS-21-Apiose (1). Naturally-derived QS-21 has been used in more than 100 clinical trials to augment the human immune response to vaccine antigens targeting disorders including cancer, HIV and hepatitis. We will examine here the chemical synthesis of QS-21-Apiose conducted by the laboratory of the late David Gin.

The de novo synthesis of the complex fatty acid chain of QS-21 relies on the pseudosymmetry of this substructural fragment. The propionaldehyde derivative 3 first undergoes asymmetric diastereoselective crotylation under conditions developed by H. C. Brown. The enantioenriched crotylation product 5 is then elaborated in two steps to the aldehyde 6, which next undergoes a diastereoselective aldol reaction with a chiral enolate (shown in the scheme above) to afford the beta-hydroxy ester 7. Subsequent functional group interconversions lead to the secondary alcohol 9, the substrate for a dehydrative glycosylation reaction with an arabinofuranose-derived glycosyl donor, ultimately providing the glycoconjugate 10. Finally, barium hydroxide-mediated ester hydrolysis followed by a  Yamaguchi-type esterification/condensation were successfully implemented to assemble the intact fatty acyl moiety of QS-21 in protected form (11).

The construction of the requisite linear tetrasaccharide fragment involves another stereocontrolled dehydrative glycosylation reaction to give  14, which is immediately used as a glycosyl acceptor in a subsequent glycosylation event. This product (not shown) is then elaborated to the linear trisaccharide 15 in three additional synthetic operations. A final dehydrative glycosylation reaction then secures the fully protected tetrasaccharide 17 following a standard acetate methanolysis (of the acetate 16).

The advanced fragments 11 and 17 are condensed via the intermediacy of a mixed anhydride derived from 2,4,6-trichlorobenzoyl chloride. Subsequent cleavage of the anomeric TIPS silyl ether then reveals a hemiacetal group which is converted to a competent glycosyl donor, the alpa-trichloroacetimidate 18, by condensation with trichloracetonitrile under basic conditions. The triterpene-trisaccharide substructure 19, obtained by a lengthy semisynthetic protocol starting from natural semi-purified Quillaja extracts, efficiently couples with the acylated tetrasaccharide 18 upon exposure to a catalytic amount of boron trifluoride diethyl etherate. Finally, fully protected QS-21(Api) readily undergoes global deprotection under the conditions specified above to generate, in useful quantities, structurally homogenous samples of this clinically-relevant immunostimulatory natural product (1). The elegant work of the Gin laboratory expands the availability a precious bioactive triterpenoid natural product and provides access to nonnatural synthetic derivatives of 1 (e.g. 2) with improved drug properties. 

Shair’s Synthesis of (+)-Cephalostatin 1 from Hecogenin Acetate and trans-Androsterone

The architectural complexity of the bis-steroidal pyrazine cephalostatin 1, along with its potent antiproliferative activity and unique biological mechanism of action, have inspired organic chemists since its isolation by Petit in the early 1990s. We have noted previously that three laboratories have completed chemical syntheses of 1. In this post, we will examine the enantioselective synthesis of cephalostatin 1 by the Shair group at Harvard.  Shair and co-workers dissected 1 into two hemispheric fragments: the eastern substructure 2 and a western half (10) derived from hecogenin acetate. The fragment 2 was prepared from trans-androsterone, a readily available 17-keto steroid. The synthetic challenges dictated by the use of this starting material are (1) the remote oxidation of the C12 position to install the 12-hydroxy functionality within 1 and (2) installation and elaboration of a stereochemically complex spiroketal system (rings E & F) wherein the spiro-fused ring junction position (C22) is in a relatively sensitive kinetic configuration. The C22-epimeric diastereomer (of 2) is stabilized by two anomeric effects as compared with the requisite structure 2 (three-dimensional structure shown above on bottom right), which contains a monoanomeric spiroketal linkage.

Remote oxidation of the C12 position is accomplished by implementation of the copper-mediated methodology of Schonecker. We have previously discussed the mechanism of this stereoselective oxidation in the context of the synthesis of cyclopamine by Giannis and co-workers. Next, a Sonogashira coupling reaction between an optically active alkyne (which contains seven of the eight carbons of the E/F spiroketal) and a C17 enol triflate generates the advanced intermediate 3, shown above. Meinwald and Liu pioneered this type of Pd-catalyzed D-ring functionalization in 1996 and an advancement was later published by Jones and co-workers in 2001. Sharpless dihydroxylation of 3 led to a cis-diol intermediate (not shown) with a-hydroxy functionality at C17. A unique oxidation of this diol with benzeneselinic anhydride then generated an a-hydroxy cyclopentenone that was reduced with triacetoxyborohydride to produce 4. The stereochemical outcome of the latter transformation is due to directing group participation of the C17 hydroxyl group, resulting in a trans-diol (4) with a C16 hydroxyl in the b-configuration. Subsequent exposure of 4 to a gold(I)-cataytic system promoted a 5-endo-dig cyclization to forge the dihydrofuran 5 with outstanding efficiency. Next, the C17 hydroxyl group is used again, in this instance to direct a Simmons-Smith cyclopropanation reaction that stereoselectively introduces the final carbon atom of the eventual spiroketal E/F system. Desilylation then precedes the critical oxidative spiroketalization reaction using NBS to deliver predominantly the desired (kinetically favored) C22-(S) isomer 7 under pH-neutral, non-equilibrating reaction conditions. Debromination under free-radical conditions gave the advanced spiroketal 8 that was finally converted to 2 by a three-step sequence that included silylation of the hindered C17 hydroxyl and adjustment of the oxidation state of C3.

Shair’s synthesis of the western half of cephalostatin 1 from hecogenin acetate will not be discussed here in great detail except to mention that the methods used to selectively oxidize the angular C18 methyl group are indeed very interesting from a mechanistic perspective. Installation of the D(14,15) olefin was accomplished concurrently in the course the C18 oxidation reaction sequence. The reorganization of the hecogenin spiroketal system involved implementation of a well-established Marker degradation procedure followed by elongation and elaboration of a functionalized pregnane (progesterone-type) system that eventually led to 10. Overall, Shair’s preparation of 10 from hecogenin acetate is conceptually interesting but somewhat linear and lengthy as compared to that of the Tian laboratory, which has reportedly synthesized multigram quantities of a related cephalostatin western domain intermediate by a relatively concise synthetic protocol. Similar to Tian’s synthesis of 1, Shair and co-workers, in their endgame, apply the Fuchs unsymmetrical pyrazine synthesis to condense the advanced steroidal precursors 9 and 10. We recently discussed the detailed mechanism of this transformation here. Finally, in a single global deprotection step, fluoride-mediated desilylation at four positions is accompanied by hydrolysis of the C12 acetate to furnish synthetic cephalostatin 1. Completed in 2009, this landmark effort constitutes the second of only three chemical syntheses of 1 that have been disclosed to date. As a reminder, the Fuchs group at Purdue University reported the first synthesis of cephalostatin 1 in 1998 and, as noted above, the most recent chemical preparation of the complex bis-steroidal pyrazine was reported by the laboratory of Weisheng Tian.

Chemical Synthesis of the Immunostimulatory Adjuvant QS-7-Api

An adjuvant is an immunological agent that can be included in a vaccine to enhance the recipient's immune response to a supplied antigen. Adjuvants are often added to vaccines to enhance the immune system's response to the target antigen, but do not, in and of themselves, confer immunity. The adjuvant component enables dose-sparing of precious antigens and extends immunotherapeutic benefits to poor responders. Aluminum salts (Alum) are common adjuvants in vaccines sold in the United States and so it is not surprising that new small molecule entities, devoid of neurotoxicity or related side effects, are sought after as novel immunostimulatory agents for use in vaccine therapies. The various triterpenoidal molecular constituents of the extracts from the bark of the Quillaja saponaria tree, found in the desert regions of Chile, Bolivia and southern Peru, are considered, in aggregate, one such promising adjuvant which is currently undergoing clinical investigation. The immunoactive components of this material contain a central triterpene (quillaic acid), a branched trisaccharide at the C3 position of the triterpene and a complex polysaccharide attached at C28 of the quillaic acid. The laboratory of the late David Gin (Memorial Sloan-Kettering Cancer Center) accomplished a phenomenal chemical synthesis of QS-7-Apiose (1) in 2008. This tour de force synthetic campaign will be subject of the current post.

It was shown by Gin and co-workers that a commercially available semipurified extract from Quillaja saponaria could be converted into the polysilylated monodesmoside saponin 2 in only 3 steps with useful synthetic efficiency (257 mg of 2 obtained from 1.15 grams of extract). This triterpene-trisaccharide conjugate would serve as a glycosyl acceptor in a subsequent C28-carboxylate glycosylation reaction with a complex hexasaccharide donor component (vida infra).

To begin, monosaccharides 3 and 4 were subjected to dehydrative glycosylation conditions developed by the Gin laboratory to afford the beta-disaccharide 5 in excellent yield. Direct glycosylation of 5 with an apiose-derived donor (shown above) gave the trisaccharide 6. Subsequent functional group interconversions then led to 7, which underwent Schmidt glycosylation with a glucosyl imidate to provide the tetrasaccharide intermediate. A benzoate ester (required for neighboring group participation in the previous operation) was next converted to a silyl ether (in 2 steps) and then the anomeric TIPS group was transformed into the requisite trichloroacetimidate of the intact glycosyl donor fragment. The glycosyl acceptor coupling partner (shown above in the bottom-right box) was synthesized independently in an unremarkable fashion.

The aforementioned donor and acceptor were exposed to TMSOTf to generate a hexasaccharide advanced intermediate whose TIPS-acetal was subsequently cleaved with a fluoride source and then transformed into a trichloroacetimidate (8). Ultimately, the triterpene-trisaccharide conjugate (2) served as an effective glycosyl acceptor in the critical C28-carboxylate glycosylation reaction with the complex glycosyl donor 8. Finally, global deprotection of the resultant adduct afforded the natural adjuvant QS-7-Api (1) in a synthetic campaign that can only be considered a heartbreaking work of staggering genius. Subsequently, the design and synthesis of simplified semisynthetic congeners of the immunostimulatory natural product 1 was accomplished. These efforts resulted in the development of an adjuvant candidate molecule with enhanced chemical stability and aqueous solubility along with diminished levels of toxicity. These relatively recent medicinal chemistry studies (JACS, 2012) will be the topic of a forthcoming post at Modern Steroid Science.