Sunday, May 18, 2014

Chemical Synthesis of the Natural Appetite-Suppressant Steroid Isolated from Hoodia gordonii

            On November 21, 2004, CBS’ 60 Minutes aired a news segment that reported on the effectiveness of the Hoodia gordonii plant as a natural appetite suppressant. Hoodia gordonii (depicted below) is a leafless spiny succulent plant that grows naturally in South Africa, Namibia and Botswana. For centuries, the indigenous peoples of South Africa have used the meat of the plant to suppress appetite on long hunting trips in the Kalahari Desert. The 60 Minutes correspondent Lesley Stahl was sent to the Kalahari to try the plant herself. She reported that after chewing Hoodia gordonii, she had no desire to eat for almost 24 hours. Soon after, interest in the plant as a natural diet drug began to skyrocket and, by 2007, there were an estimated 300 products being sold by nutritional supplement companies that were touted as authentic Hoodia gordonii. Indeed, Hoodia is one the most compelling stories of ethnobotany. But is the bioactive appetite-suppressing chemical constituent present in Hoodia extracts suitable for development as an anti-obesity drug?
Hoodia gordonii
            In 1977, the South African Council for Scientific and Industrial Research (CSIR) isolated the chemical principle in Hoodia responsible for inducing its appetite-suppressant effect. They named the oxygenated pregnane glycoside P57 (structure shown below) and patented it in 1996. The CSIR then granted a British pharmaceutical company, Phytopharm, a license and, in 1997, they entered into an agreement with Pfizer to develop P57 as an oral anti-obesity drug candidate. In 2002, Pfizer released the rights to the molecule and abandoned the development program, indicating that P57 was too expensive to extract and/or synthesize. A leading researcher involved in the Hoodia program at Pfizer later disclosed that “an early clinical trial indeed showed that hoodia could be a potent appetite suppressant. But there were indications of unwanted effects on the liver caused by other components, which could not be easily removed from the supplement (The New York Times, April 26, 2005).” Phytopharm subsequently entered into a deal with Unilever to continue commercial development but, in 2008, Unilever pulled out. Unilever indicated on their website that “We stopped the project because our clinical studies revealed that products using hoodia would not meet our standards of safety and efficacy.”
            Feeding experiments in rats of the crude Hoodia extract and the pregnane glycoside P57 showed that, over an 8-day period, a decrease in food consumption and body mass was observed when compared to a control. In order to determine whether or not this effect is due to a P57-induced anorectic activity in the brain, researchers have injected the compound directly into rat brains and found a 50-60% decrease in food intake over 24 hours. Increased ATP levels were detected in the rats’ hypothalamic neurons, thereby indicating that the appetitive response of P57 likely has a central nervous system mechanism of action. The detailed mechanism of action of P57 is not currently well defined.
            P57 is not easily obtained by extraction from natural sources. The yield of extraction of oxypregnane glycosides from H. gordonii is between 0.003 – 0.02%. Moreover, H. gordonii is a protected plant and therefore of limited access. Given that a world-renowned pharmaceutical company stopped the commercial development of P57 due, at least in part, to the difficulty of synthesizing the relatively complex steroid, a new, scalable synthetic approach to P57 holds considerable value. P57 is 14-b-pregnane glycoside derivative that contains a D5,6 olefin in the B-ring and is oxygenated at the carbogenic steroidal positions 3, 12, 14 and 20. Perhaps the most challenging aspect of a chemical synthesis of P57 is the stereocontrolled assembly of the deoxytrisaccharide western substructure, wherein the establishment of the 2-deoxy-b-glycosidic linkages is notoriously difficult. In one patent that mentions the synthesis of P57, the trisaccharide was assembled onto the aglycone by means of sequential, low-yielding glycosylation reactions with fluoride glycosyl donors. Two recent innovative synthetic campaigns directed at the semisynthetic construction of P57 through the intermediacy of its aglycone, hoodigogenin A, will be the subject of this post.
            The Norrish type-I (UV-induced a-cleavage)/Prins reaction sequence, when applied to hecogenin acetate, is a long-known method for generating 14b-hydroxy steroids through the intermediacy of the photoproduct, lumihecogenin. This transformation was first reported by Bladon in the early 1960’s and the pioneering work was extended by Welzel, Winterfeldt, Fuchs and others. Fuchs’ synthetic work on the cephalostatins led to a dramatic yield improvement for the two-step tandem process (Scheme above, top panel) with the ability to modulate the stereochemical outcome at C12. The laboratory of Michel Miesch has demonstrated this process for the first time on a pregnenolone derivative (with a D5,6 double bond), prepared in seven steps from the readily available steroid precursor 1. The Norrish/Prins sequence developed by Miesch and co-workers provides access to the valuable 14b-hydroxy steroid 3 with moderate synthetic efficiency (25% isolated yield). The material balance, in this case, is largely accounted for by an accompanying spirocyclic by-product. The advanced intermediate 3 is easily converted into the P57 aglycone, hoodigogenin A, in two straightforward synthetic operations (reported in: Steroids, 2011, 76, 702 – 708).
            Biao Yu’s group at the Shanghai Institute of Organic Chemistry exploited structural similarities between the cardiotonic steroid drug, digoxin, and hoodigogenin A. Digoxin, similar to Hoodia pregnanes, is oxygenated at positions C3, C12 and C14 in the requisite stereochemical orientation. Moreover, the C17 butenolide of digoxin serves as a latent acetyl group, revealed under oxidative conditions (see Scheme above, conversion of 5 into 6). Unsaturation can be introduced into the A/B steroid ring system by a regioselective Saegusa oxidation of the silyl enol ether 7. In this example, the C3,4 enolate is oxidized by stoichiometric palladium acetate to the D4,5 enone 8 in high yield. The major oxidation product 8 is accompanied by a small amount (8%) of the corresponding D1,2 regioisomer. The alkene is then migrated to C5-C6 by deprotonation of the acidic g-position of 8 followed by kinetic re-protonation. The resultant b,g-unsaturated C3 ketone is subsequently reduced from the more accessible a-face to secure the advanced intermediate 9. The oxygenated pregnane derivative 9 could be converted into hoodigogenin A by a simple four-step sequence involving protecting group manipulation and functional group interconversion.
            Yu’s group then appended the acid-labile deoxytrisaccharide fragment in a stepwise fashion using glycosyl o-alkynylbenzoate building blocks as glycosyl donors. In the presence of a gold(I) cationic catalyst, o-alkynylbenzoate donors undergo glycosylation under relatively mild conditions via the mechanistic pathway shown above. Yu’s semisynthetic preparation of P57 underscores the synthetic challenge of achieving a high level of stereocontrol over the 2-deoxy-b-glycosidic linkages resident in the western substructure of the natural product. In the first Au(I)-mediated glycosylation reaction between P57 aglycone and the o-alkynylbenzoate monosaccharide donor 10, the condensation product 11 is obtained in good yield, but with only moderate b/a anomeric selectivity (3.5:1 in favor of the desired b-glycoside). Selective methanolysis the 4-O-acetyl group of 11 then affords the advanced oxypregnane glycoside 12. Glycosylation of 12 with the more complex donor disaccharide 13, again in the presence of a gold(I) catalyst, does indeed afford the trisaccharide 14, albeit with no control over the newly formed anomeric stereocenter (C28, highlighted below with an asterisk). Fortunately, the a- and b-anomers, formed in equal amounts, could be separated by silica gel column chromatography. Finally, successful cleavage of the O-acetyl protecting groups of 14 in the presence of the 12-O-tiglic ester furnished the valuable bioactive Hoodia saponin, P57. Yu’s synthesis of P57 proceeds in 20 linear steps with an overall yield of 2.4% from digoxin (reported in: Chem. Commun. 2012, 48, 8679 – 8681). This level of preparative efficiency compares favorably with the yield of extraction of P57 from H. gordonii by several orders of magnitude.

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