Sunday, October 19, 2014

Libiguin A: A Novel Phragmalin Limonoid that Induces Stimulation of Sexual Behavior in Rodents

            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[]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.

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