Tuesday, April 21, 2015

Baran’s Semisynthetic Work on Ouabagenin and Related Bioactive Glucocorticoid Derivatives

            Phil Baran’s laboratory at The Scripps Research Institute recently disclosed a full account of their 20-step synthesis of the aglycone of ouabain starting from a derivative of cortisone. Their general strategy for introduction of the dense hydroxylation pattern of the target molecule was to ‘redox-relay’ pre-existing oxidation states that are naturally present within the cortisone framework to neighboring and distal carbon atoms. For example, using Norrish type II photochemistry, the C11 ketone oxidation state was effectively relayed to the angular C19 methyl group through the intermediacy of a cyclobutanol. Oxidative fragmenation of the cyclobutanol then furnishes the C19-functionalized product. Baran’s optimization and extension of the Norrish protocol to the problem of functionalization of the unactivated steroid C19 substituent provides a useful and complementary alternative to more classical tactics, such as those reported years ago by Barton, as well as Meystre and Heusler from Ciba. A more detailed analysis of Baran’s partial synthesis of ouabagenin can be found here.

            Interestingly, Baran’s group describes a foray into the realm of medicinal chemistry in their recent JACS full article. They report the application of the Norrish type II photochemical C-H functionalization methodology to the synthesis of C19-hydroxylated analogues of corticosteroid drugs such as clobetasol propionate. The authors indicate a motivation to minimize the undesired biological effects of glucocorticoid receptor (GR) agonists stemming from off-target mineralocorticoid antagonism. Unfortunately, the novel clobetasol analogue 1, synthesized in eleven steps from cortisone acetate, exhibits binding affinity to GR that is inferior to clobetasol propionate by about an order of magnitude. While selectivity against the mineralocorticoid receptor is not reported, 1 does exhibit moderate anti-inflammatory efficacy in a cellular assay. Baran’s analogues are clearly lacking 9a-halogenation. The 9a-halogenated glucocorticoid series was first described in 1953 and has been shown to provide a >10-fold increase in binding affinity to GR, relative to parent C9-proteo hormones. The synthetic route used to prepare clobetasol analogue 1 is shown below.
            Arguably, the most robust and practical methodology reported by Baran for semisynthetic cardenolide modification with applications to therapeutically relevant corticosteroids was disclosed much earlier. In the Journal of Organic Chemistry, Baran reports a strategy for systematic deoxygenation of ouabain as a means to obtain important 17b-hydroxyacetyl pregnane-type steroids. Ouabain, by virtue of its pre-built oxygenation at the C11 position and latent side chain derivable from the C17 butenolide, is a logical candidate for a synthetic precursor to various glucocorticoids.
            As proof of this principle, ouabagenin 1,19-acetonide was reductively deoxygenated (see below) at the C1 position via the intermediacy of a 3,11-dione. Subsequently, upon exposure of an 11,19-methyl-ketal to thionyl chloride and pyridine at low temperature, the tertiary hydroxyl group at C14 eliminates to generate an olefinic intermediate. Catalytic hydrogenation of this intermediate results in a saturated system with b-hydrogen atoms located at the newly formed stereogenic ring junction positions. Finally, oxidative manipulation of the a,b-unsaturated lactone moiety affords a 20-oxo-14-iso-pregnane that can be considered a 19-hydroxylated congener of cortisone acetate. Most of the synthetic operations described in this manuscript were demonstrated on decagram scale. For example, the hydrogenation depicted below (step 3) was conducted on 73-gram scale and, ultimately, 4 grams of the 14-isopregnane glucocorticoid derivative were generated from a single batch run.
            This latter synthetic work was conducted by John S. Baran (i.e. not Phil) of The Laboratories of G. D. Searle and Co., Chicago, Illinois. The single author manuscript (see excerpt below) was received by JOC on August, 29, 1963. During this time, the post-World War II era race to synthesize cortisone was in full swing. Ouabain and strophanthidin were two cardenolides that were evaluated as feedstock precursors to the newly discovered and highly sought-after corticosteroid wonder drugs of the 1950s – 1960s. G. D. Searle, much like other major players in the cortisone race - Merck, Syntex and Upjohn - figured prominantly in the vitally important steroid research that was ongoing during this time period.

Saturday, April 11, 2015

Stereocontrolled Syntheses of Dafachronic Acid Starting from Abundant Sterol Precursors

            Dafachronic acid, much like glycinoeclepin A and solanoeclepin A (structures shown above), is an important hormonal signaling molecule that regulates nematode development and lifespan. In order to facilitate the investigation of biochemical mechanisms that influence the longevity of organisms such as Caenorhabditis elegans, it is critical that synthetic technologies provide access to substantial quantities of dafachronic acid, which is available from natural sources only in trace amounts. Moreover, non-naturally occurring chemical tools to enable monitoring and quantification of dafachronic acid levels in whole organisms are also urgently needed. The challenges inherent to a chemical synthesis of dafachronic acid include establishment of the (25S)-configuration within the bile acid-like side chain, along with installation of the embedded D7 unit of unsaturation. Several inventive synthetic studies targeting dafachronic acid have been reported in the literature. We provide an overview of this important body of work below.
            One of the first syntheses of (25S)-D7-dafachronic acid was reported by Sharma, Mangelsdorf and co-workers in 2009. Their route highlights a key synthetic challenge associated with the target molecule, that being stereocontrolled elaboration of the steroid side chain. This issue is further confounded by the fact that most readily available plant-derived sapogenins (e.g. diosgenin) bear the (25R)-configuration within the spiroketalized side chain. Therefore, reductive opening of the sapogenin spiroketal affords a dafachronic acid precursor that requires a difficult epimerization of the C25 position, if this synthetic approach is to be successfully employed.
            The team of Sharma and Mangelsdorf began with a hypothesis that dafachronic acid biosynthesis is the result of cytochrome P450-mediated oxidation of a precursor sterol. They carried out a series of bioassays of many synthetic sterol derivatives and eventually identified (25S)-D7-dafachronic acid as the natural ligand for the DAF-12 receptor based on its potency in transactivation and dauer rescue assays. Their synthetic route (shown above) to this important endogenous ligand begins with a Clemmensen reduction of diosgenin leading to intermediate 2, which bears the unnatural C25 stereochemical configuration, as compared to dafachronic acid. The (25S)-isomer is eventually obtained by enzyme-mediated kinetic resolution of a diastereomeric mixture. A final redox adjustment within the steroid side chain then furnishes (25S)-D7-dafachronic acid.
            E. J. Corey’s 2007 synthesis of (25S)-D7-dafachronic acid (shown above, adapted from Org. Biomol. Chem., 2010) begins with protection of the steroid A-ring by rearrangement to a bicyclo[3.1.0]hexane, followed by oxidative degradation of the b-stigmasterol side chain. Subsequent homologation and elaboration of the side chain included a stereocontrolled homogenous hydrogenation with Ru(OAc)2[(S)-H8-BINAP] which set the (25S)-configuration with 8:1 diastereoselectivity. Recrystallization of the resultant mixture of stereoisomers then provided stereochemically homogenous material. Reestablishment of the sterol A-ring (with 3a-acetate) was proceeded by a commonly employed allylic oxidation (CrO3)/dehydration (Burgess reagent) sequence to install the D7 double bond. Three additional synthetic operations were finally required to complete the synthesis of (25S)-D7-dafachronic acid. Corey’s synthesis proceeds in a total of 16 steps and in 13% overall yield.
            Corey’s laboratory has also developed a concise synthesis of diastereomeric (25R)-D7-dafachronic acid (reproduced above from Org. Lett., 2008starting from abundant b-ergosterol, which conveniently bears a preinstalled D7 olefinic linkage. The key transformation in this process is a stereocontrolled Claisen [3,3]-sigmatropic rearrangement (shown below) of a silylketene acetal that sets the C25 configuration with concomitant installation of the requisite carboxylic acid functionality. The route leading to the (25R)-diastereomer requires only 10 synthetic steps and proceeds in 13% overall yield.
            Hans-Joachim Knölker’s group in Dresden, Germany has worked extensively on steroid ligands for the nuclear receptor DAF-12 of C. elegans. His elegant and expedient synthesis of (25S)-D7-dafachronic acid (shown below) features an auxiliary-mediated Evans aldol reaction. The stereoselective condensation furnishes an adduct (10) that bears the desired (25S)-configuration, along with an extraneous hydroxyl group at C24. The latter is removed using the classical Barton-McCombie deoxygenation procedure. The route requires an additional 7 steps to complete the target molecule (15 linear steps overall), yet proceeds with outstanding overall efficiency. The overall yield of 27% reported by Knölker and co-workers is unsurpassed by other known synthetic protocols.
            In 2015, the synthesis of a dueterated derivative of (25S)-D7-dafachronic acid (5, 24, 25-D3) was disclosed by Xiaoguang Lei’s laboratory in Beijing, China. This unique, isotopically labeled chemical probe (see below, structure 21) is effective for sensitive and robust absolute quantification of endogenous dafachronic acid during the reproductive development of C. elegans using mass spectrometry. Their biogenetically inspired synthetic approach begins from cholesterol (13) and, much like the biosynthesis of dafachronic acid, involves sequential, site-selective C-H oxidative functionalizations. For example, upon exposure of the cholesterol derivative 15 to methyl(trifluoromethyl)dioxirane (TFDO), a selective C-H hydroxylation of the unactivated tertiary C25 position ensues with good efficiency (90% b.r.s.m.) and regiocontrol. An iridium/phosphine-oxazoline-catalyzed late-stage asymmetric deuterium reduction employing a catalyst system recently developed by Zhou’s group completed the synthesis of the isotope-labeled dafachronic acid tool compound.
            Access to meaningful amounts of dafachronic acid and related unnatural derivatives will facilitate studies to elucidate the precise molecular mechanisms involved in modulation of the C. elegans life cycle. Investigative efforts of this nature, directed towards basic developmental and metabolic processes, will likely provide new avenues for drug discovery and biotechnology research.