Saturday, November 22, 2014
Potato cyst nematodes (Globodera rostochiensis and G. pallida) are destructive parasites that feed on host potato plants. Oddly enough, the potato plant itself secretes a small molecule that stimulates the juvenile nematodes to hatch from the flask-shaped shell structure of the cyst. The parasitic nematodes then live and feed on the potatoes of the host plant, reducing crop yields dramatically. The hatching stimulus for potato cyst nematodes is the plant natural product, solanoeclepin A (chemical structure depicted above).
Outside the firm protective covering of the cyst, the hatched juvenile nematodes die within a few weeks in the absence of potatoes. One strategy for extermination of the potato cyst nematode, which inflicts serious damage upon crop plants in numerous countries, is to spread a dilute solution of the hatching stimulus over potato fields after the harvest. This induces the nematodes to hatch and then die shortly thereafter due to the lack of host plants. For this reason, solanoeclepin A is in high demand as an investigational agrochemical that may help to address food scarcity issues of the 21st century. For use in this type of application, a robust and scalable chemical synthesis of a hatch-stimulating substance will be required, due to the low abundance of solanoeclepin A that is accessible from natural sources.
The hatching stimulus for the potato cyst nematode was first isolated in 1986 and the absolute chemical structure of solanoeclepin A was unambiguously determined in 1999 by X-ray crystallographic analysis. The rarely encountered structural motifs embedded within the solanoeclepin A fused-ring skeleton pose significant challenges for synthetic chemists. For example, the highly strained D/E/F tricyclo[188.8.131.52]decane ring system encompasses four-, five- and six-membered carbocyclic rings. In addition, the unique western A/B/C framework consists of an oxabicyclo[2.2.1]heptan-2-one fused to a seven-membered C-ring. Indeed, all of the carbocyclic ring permutations from size three (cyclopropane) to size seven (cycloheptane) can be found within the daunting architecture of solanoeclepin A. The exquisite bioactivity, scarcity in nature and fascinating molecular structure of solanoeclepin A has made it an attractive target for chemical synthesis. A leading reference into this body of synthetic literature is Minoru Isobe’s recent disclosure in Organic Letters. The laboratories of Hiemstra and Nishikawa are also actively pursuing synthetic solanoeclepin A. In 2011, Tanino and Miyashita reported the first and only total synthesis of this extraordinarily complex, plant-derived nortriterpenoid. An overview of their tour de force 52-step chemical synthesis is provided below.
Very little is known about the biosynthesis of solanoeclepin A. However, in order to envision a plausible biosynthetic hypothesis for solanoeclepin A, it is instructive to first consider the plant natural product, glycinoeclepin A. Glycinoeclepin A happens to be the hatch-stimulating substance for the soybean cyst nematode and could potentially serve as a relevant biosynthetic intermediate en route to solanoeclepin A. Tadashi Masamune first noted in 1985 the structural similarities between glycinoeclepin A and cycloartane steroids such as cimigenol. Indeed, oxidative cleavage of the cycloartenol B-ring (shown above) with loss of one carbon atom, along with migration of two methyl groups in rings C and D leads to the pentanortriterpene, glycinoeclepin A. Additional metabolic processing and oxidative elaboration of this seco steroid or a similar biosynthetic precursor might then culminate in the biogenesis of solanoeclepin A. In 1994, Corey and Hong were able to "chemically emulate" the cycloartenol biosynthetic hypothesis by preparing 12-desoxyglycinoeclepin A in a semisynthetic fashion from the cycloartane steroid abietospiran. A practical semisynthetic approach to solanoeclepin A from a readily available plant sterol has not been investigated to date, but would undoubtedly be of great interest to the agrochemical industry.
As noted above, solanoeclepin A was synthesized in 52 synthetic operations from 3-methylcyclohexenone (1) by the laboratory of Masaaki Miyashita in Tokyo, Japan. Their overall synthetic strategy was to first assemble the eastern D/E/F substructure (e.g. 6), a trans-fused indane bearing three contiguous quaternary asymmetric carbon centers, two of which are at the bridge heads. This building block is then appended to a furanyl diene moiety, which is used to construct the western A/B/C framework by means of an intramolecular Diels-Alder (IMDA) reaction.
The synthesis is initiated by a five-step cyclopentene annulation which is based on a conjugate addition reaction and lipase-mediated optical resolution of the annulation product. The details of this method were communicated back in 2006 by Tanino and Miyashita. Stereocontrolled elaboration of the cyclopentene 2 led to the a-epoxy alcohol 3, which, upon exposure to trimethylsilyl trifluormethanesulfonate, underwent 1,2-migration of the vinyl group with concomitant epoxide ring-opening to furnish the hydroxyketone 4 with excellent efficiency (97% yield). The angular vinyl moiety served as a latent epoxide that would be utilized to forge the requisite four-membered carbocycle. Thus, as described in the mid-1970s by Gilbert Stork, treatment of the epoxy nitrile 5 with lithium diisopropylamide induced an intramolecular cyclization reaction that fashioned the cyclobutane portion of the highly strained eastern D/E/F bridged substructure. A few subsequent functional group interconversion steps provided the allylic alcohol 7 and then a stereoselective Simmons-Smith cyclopropanation, conducted in the presence of an asymmetric ligand, was utilized to install the fourth and final carbocyclic ring of the eastern D/E/F/G polycyclic system.
From the cyclopropanation product 8, ten additional operations were required to advance to intermediate 9, bearing a slightly modified protecting group array along with an exocyclic methylene projected from the cyclobutane E-ring, obtained from the selenoxide elimination protocol of Grieco. The crucial enol triflate coupling partner 11 was later obtained from 9 via the intermediacy of the keto enamine/vinylogous amide 10. Nucleophilic addition to the carboxaldehyde moiety by a substituted 2-furyl lithium species was accomplished in the presence of the neighboring enol triflate at low temperature to afford quantitatively the a-alcohol 12, which was elaborated in two steps to intermediate 13, the immediate precursor to the crucial IMDA substrate.
The enone system that would serve as the eventual dienophile component in the forthcoming IMDA step was appended by means of a palladium-catalyzed cross coupling reaction between the enol triflate 13 and the methyl enol ether, 4-methyl-2-trimethylsilyloxy-1,3-pentadiene, to generate the adduct 14 in moderate yield. Subsequent exposure of 14 to Lewis acidic conditions promoted an intramolecular [4+2] cycloaddition between the electron-rich furanyl moiety and deficient enone to furnish the endo product as the predominant stereoisomer. A two-step hydrolysis/oxidation sequence then secured the advanced trione intermediate 16 that was eventually converted into fully synthetic solanoeclepin A in 13 additional operations. It should be noted that 9 of the final 13 synthetic steps entailed late-stage redox adjustments occurring at the carbon positions denoted below in structure 16 with asterisks.
In total, 52 steps were required to convert 3-methylcyclohexenone into solanoeclepin A in the course of the first and only chemical synthesis of the hatching stimulus for the potato cyst nematode. While solanoeclepin A exhibits the appropriate biological properties for potential downstream agrochemical applications, this synthetic approach exceeds the limits of industrial manufacturing practicality by orders of magnitude. A realistic chemical solution that addresses food scarcity problems related to the increasing global population and parasite-induced crop damage can only be found in the form of structurally simplified solanoeclepin A derivatives which retain hatch-stimulating bioactivity. Along these lines, Corey’s laboratory has already synthesized a simplified benzenoid derivative of glycinoeclepin A, but the detailed biological properties associated with the analogue were not disclosed. Alternatively, and as emphasized above, a practical semisynthetic approach to solanoeclepin A production beginning from an abundant cycloartane-type plant sterol would be of great interest in this regard.
Sunday, October 19, 2014
Limonoid natural products have been reported to possess a wide range of biological activities including antimalarial, anti-HIV and, most notably, insect antifeedant. Phragmalin limonoids are oxidatively modified B,D-seco limonoids that are biosynthetically derived from their relatively less oxidized progenitors, the mexicanolides (representative structures depicted above). The architectural relationship between the carbon skeletons of typical limonoids, mexicanolides and phragmalins has been a previous topic of discussion at this site (here and here). Another very good introduction to synthetic aspects related to the limonoid molecular framework is this Scripps presentation. In brief, mexicanolides possess an A/B-bicyclo[3.3.1]-ring system while phragmalin limonoids contain an additional 1,8,9-orthoester functionality (two hydroxyls condensed with an O-acetyl carbonyl group) across the bottom (a-) face of the B-ring. The phragmalin western A/B substructural domain is referred to as a tricyclo[184.108.40.206]decane due to the additional bridging oxidative functionality. Phragmalin limonoids are found only in the genera of the Meliaceae family of flowering trees and shrubs. Libiguin A is a phragmalin-type B,D-seco limonoid isolated from the Madagascarian meliaceae species, Neobeguea mahafalensis. The major structural difference between phragmalin and libiguin A (highlighted above in blue) is the location of the eastern lactone ring. The 17-oxo functionality, which is cyclized into the lactone ring of phragmalin, is oxidized to a ketone in libiguin A. For this reason, trans-lactonization of phragmalin was explored as a practical semisynthetic approach (see below) to provide access to quantities of libiguin A that will facilitate investigation of the pharmacological properties of this new limonoid, as well as the biochemical mechanism(s) of action that underlie its bioactivity.
The laboratory of Jarl Wikberg at Uppsala University in Sweden discovered that libiguin A, extracted and purified from natural sources, induces a profound stimulation of sexual behavior in rodents at dosage levels in the low ug/kg range. A subset of representative data from Wikberg and co-workers’ 2014 Planta Medica report is reproduced above. The bar graph depicts rodent mounting behavior corresponding to the different subcutaneous doses of libiguin A, indicated in mg/kg. The data set actually describes the number of ‘mounts’ observed during the third hour after introduction of the female to the male mouse. The normal pattern of rodent ‘mounting’ is that initially the mounting activity is very high but then almost totally ceases during the third hour. By contrast, libiguin A elicited dose-dependent and sustained sexual activity over a long period of time after the introduction of the mating partner to the male. The authors note that a central mechanism of action is likely, in view of the unique behavioral patterns induced.
Curiously, another complex steroid found in plant species such as the Tribulus and Dioscorea families, namely protodioscin (molecular structure depicted above), is reported to elicit sexual-enhancing ‘aphrodisiac’ effects. Protodioscin most likely exerts its ‘proerectile’/aphrodisiac effects because it is metabolized to bioactive androgenic steroids such as [dihydro]testosterone and dehydroepiandrosterone. Protodioscin has also been demonstrated to trigger the release of nitric oxide in corpus cavernosum tissue. Regrettably, studies in humans involving this intriguing plant-derived steroidal saponin have failed to show efficacy.
Isolation of libiguin A from N. mahafalensis is plagued by low natural abundance as well as the presence of many related compounds with similar chromatographic properties. In order to obtain sufficient quantities for the detailed biochemical characterization of the sexual enhancing effects of the natural product, a semisynthetic process to generate libiguin-type molecules was developed by Wikberg’s laboratory. Phragmalin was identified as a raw material for the semisynthesis of libiguin A due to its availability in large quantities from commercially cultivated species of the Meliaceae family. For example, phragmalin can be obtained from seeds of Chukrasia tabularis at a yield of 3.52 g/kg of seeds. Access to gram-quantities of this complex limonoid allowed Wikberg and co-workers to explore the critical trans-lactonization transformation required to construct the skeletal connectivity of the libiguins. First, a selective monoacylation of the C3-hydroxyl of phragmalin with isobutyryl chloride afforded intermediate 1. Next, reaction of the lactone 1 with MeONHMe-HCl promoted by trimethylaluminum accomplished the desired lactone ring opening. The authors note that a number of alternate hydrolytic, reductive and aminolytic conditions met with limited success. The effectiveness of the Weinreb amidation relied on careful time control in order to avoid the formation of unwanted by-products derived from ester aminolysis. The unblocked C17-hydroxyl group could then be selectively oxidized to the requisite ketone using Dess-Martin periodinane. In the penultimate step, lactone ring closure with the C30-hydroxyl was achieved upon exposure of the advanced intermediate 3 to the Lewis acid, TMSOTf. Finally, acylation of the remaining C2-hydroxyl, again under Lewis acidic conditions, secured semisynthetic libiguin A in excellent yield. In spite of its demonstration on relatively small scale (8 mg of libiguin A were synthesized), the route will allow the authors to conduct more advanced pharmacological profiling of the natural product, as well as analogues, in order to better characterize the biochemical origins of the sexual stimulating activities that were previously observed.