The Most Concise Enantioselective Approach to Fully Synthetic Steroids Reported To Date

            This is the 100^th post at Modern Steroid Science (begun in late 2011) and, likely, the last of 2015. So it is fitting that we will provide an overview of one of the most interesting organic synthesis papers from this year. It is a report from Pavel Nagorny’s group at the University of Michigan describing an asymmetric approach to fully synthetic steroids that proceeds in just two key steps (4 – 5 total steps, if we account for the preparation of the building blocks from essentially earth, wind, fire and water). To put this into context, optically active steroids of a similar level of complexity have been recently prepared in more like ~20 linear operations. Importantly, the methodology provides access to unnatural polycyclic molecules that could not be obtained from semisynthetic derivatization of an abundant sterol and the chemistry is practical. No aspect of this expedient, catalytic sequence (to my eye) seems like it would be limiting with regard to scalability. Indeed, the route has already been demonstrated on gram-scale by the academic team.

            Back in the 1980s, the laboratory of Pierre Deslongchamps at the University of Sherbrooke (Quebec) was working on a rapid approach to fully synthetic steroids. He came up with a one-step synthesis starting from a monosubstituted cyclohexenone (eventual A-ring) and an a,b-unsaturated b-keto ester (containing the D-ring) that zips up an androstane system with six contiguous stereogenic centers (see Scheme above). The chemistry, while highly expedient, has seen limited practical applications in an industrial or academic setting. A variant of this technology was used in the first total synthesis of ouabain. The anionic polycyclization of Deslongchamps furnishes racemic products, unless the starting materials are derived from chiral pool reagents, as was the case for ouabain. Control over relative stereochemistry at the C/D ring junction positions (C13/C14) in this complex annulation process is not highly predictable and seems to involve subtle structural features embedded within the starting materials. Moreover, the steroidal products thus obtained necessarily bear an ester moiety at the C6 position of the B-ring and a decarboxylation step is usually required to excise the unneeded functionality. Almost thirty years later, the anionic polycyclization chemistry first reported by Deslongchamps in 1988 was in need of a bit of a refurbishment.

            Nagorny’s two-step steroid synthesis (shown above) hinges on a challenging asymmetric, diastereoselective Michael addition between a 2-substituted b-ketoester and a b-substituted enone that delivers a conjugate addition product with vicinal quaternary and tertiary stereocenters. The reaction employs catalytic levels of inexpensive copper(II) salts with a noncoordinating counterion in combination with an optically active bis(4,5-dihydrooxazole) (Box) ligand under solvent-free (neat) conditions. The substrate scope of the enantioselective Michael addition is outstanding, generally producing enantiomeric excesses (ee’s) in the low- to mid-nineties. 

            Mechanistically, Deslongchamps’ anionic polycyclization protocol first closes the steroid B-ring by a sequential double-Michael addition. A subsequent intramolecular aldol reaction between the newly formed cesium enolate and a tethered D-ring cyclopentadione moiety yields the intact steroid skeleton. Nagorny has cleverly designed his new Michael addition such that the products obtained from the process competently participate in a sequenced intramolecular aldol reaction to construct the B-ring and produce an intermediate that is similar to the penultimate steroid precursor of Deslongchamps. Nagorny’s approach then benefits from the wealth of precedent in the steroid literature regarding final C-ring formation via an additional tandem intramolecular aldol reaction (for example), this one between the eventual C8 and C14 positions. In the event, the double Michael addition developed at the University of Michigan proceeds with outstanding efficiency and stereocontrol. Alternate reaction conditions were demonstrated that effectively control the C/D-ring junction stereocenters, providing divergent, diastereoselective formation of either a 13b- or 13a-androstanedione system. The latter annulation product possesses a C5 b-oriented hydroxyl group and the former a D^5,6 unit of unsaturation derived from elimination of water. The now readily available structures obtained in this fashion may be suitable for eventual conversion to bioactive cardenolide or limonoid natural products, classes of steroids that contain differing configurations of the angular substituent appended to C13. Notably, the modular process allows for introduction of structural variation at the C13 position, as well as alteration of the A/D ring size, which could not be achieved by semisynthetic methods.

            During my initial read of the Nagorny JACS manuscript, I was wondering if the substrate scope that is tolerated by the process would provide access to derivatives that are functionalized at the C3 position of the steroid A-ring. Nearly all bioactive sterols and synthetic derivatives thereof possess oxidative functionality at C3. Indeed, in the course of the first total synthesis of ouabain, Deslongchamps et al implemented an alkylsilane group appended to the pro-C3 position that required a late-stage Tamao-Fleming oxidation to unmask the latent C3-hydroxyl functionality. Nagorny provides a clue to the careful reader in this regard by disclosing the asymmetric Michael example depicted above. Introduction of a vinyl chloride moiety within the A-ring ketoester building block was shown to be well-tolerated. The pro-C3 vinyl chloride-containing Michael adduct is obtained in excellent yield and stereoselectivity, albeit in combination with a simplified b-substituted a,b-unsaturated ketone. It will be exciting to see if this highly expedient approach to pharmacologically privileged steroidal scaffolds will be applied in the coming new year to the production of architecturally ornate natural products or to new and improved drug candidates.

A High-Throughput Gene Signature Profiling Assay Reveals Withanolides as Lead Compounds for the Chemotherapeutic Treatment of Resistant Prostate Cancer

            Prostate cancer is a leading killer of men in North America, second only to heart disease. Proliferation and metastatic progression of prostate cancer is sensitive to the steroidal hormones testosterone and dihydrotestosterone, the principal endogenous ligands of the androgen receptor. For this reason, androgen deprivation is the standard first-line therapy used to treat the initial stages of the disease. This therapeutic regimen involves a reduction of androgen hormones using pharmaceuticals or surgery in order to prevent prostate cancer cells from growing. In response to low levels of testosterone, luteinizing hormone-releasing hormone (LHRH) is produced in the hypothalamus, which activates the synthesis of luteinizing hormone (LH) by the pituitary gland. LH then travels to the testicles, where it induces the production of testosterone. LHRH agonists and antagonists both inhibit the formation of LH in the pituitary gland and thereby lower the amount of testosterone that is made by the testicles. Reduction of androgens using drug therapy as opposed to surgery is sometimes referred to as ‘chemical castration.’
            Unfortunately, the recurrence of androgen-insensitive or ‘castration-resistant prostate cancer (CRPC)’ is often observed in patients. Current treatments for CRPC employ radiopharmaceutical, immunotherapeutic and chemotherapeutic approaches. Among the frontline chemotherapeutic agents for CRPC is a steroidal antiandrogen marketed under the trade name Zytiga. Zytiga (abiraterone) is an androgen synthesis inhibitor that is used in combination with another steroid, prednisone. The anticancer drug inhibits cytochrome P450 subtype 17A1 (CYP17A1), an enzyme with both 17a-hydroxylase and 17,20-lyase activities, which plays a critical role in the steroidogenic pathway that produces endogenous androgen hormones. Biosynthetically, CYP17A1 catalyzes the C17a-hydroxylation of pregnenolone and progesterone (see Figure above) and also acts upon those hydroxylated metabolites to excise the side-chain from the steroid nucleus (the lyase activity), furnishing the intact androgen receptor ligands. Inhibition of CYP17A1 by Zytiga effectively decreases circulating levels of androgens such as DHEA, testosterone and dihydrotestosterone. Zytiga also acts as an antagonist of the androgen receptor and as an inhibitor of the enzyme 3b-hydroxysteroid dehydrogenase (3b-HSD), further reinforcing its efficacy as an antiandrogenic treatment for metastatic CRPC. Zytiga therapy generally prolongs survival by 4.6 months when compared to placebo but durable responses are not always observed, presumably due to acquired resistance. Therefore, new therapeutic intervention strategies for resistant PC with an androgen-depletion-independent phenotype are sought.

            A team of natural products scientists at the University of Arizona and the spin-out company NuvoGen Research (Tuscon) has recently developed a unique high-throughput screening (HTS) approach that utilizes a gene expression profiling methodology. They refer to the technique as the ‘ArrayPlate’ gene signature assay and it has enabled the research team to evaluate large libraries of natural product extracts derived from local Sonoran desert plants to identify chemical agents that could effectively reverse the androgen receptor (AR) gene signature to its inactive state. They have utilized the ArrayPlate technology to analyze 18,000 samples and have recently disclosed the exciting results in the Journal of Medicinal Chemistry. Of all the samples tested, the most active was an extract derived from the flowering plant Physalis crassifolia (depicted above). The extract blocked androgen-induced expression of the gene encoding for prostate-specific antigen (or PSA), as well as others. Bioactivity-guided fractionation of the P. crassifolia extract led to the identification of molecules from a specific subclass of withanolide-type steroids, the 17b-hydroxywithanolides. These, like the classical withanolides, are ergostane-based steroidal lactones. But unlike the majority of the over 600 natural withanolides characterized to date, 17b-hydroxywithanolides possess an a-oriented C17 side chain appended to the steroid D-ring. Additionally, the side chain attachment position (C17) of all of the active compounds is hydroxylated in the b-configuration. The most active and chemically stable inhibitor of androgen-induced genes was a semisynthetic 17b-hydroxywithanolide derivative that was later shown to be structurally identical to the natural product physachenolide C (structure shown above). Physachenolides C and D displayed nanomolar cytotoxic potency against a panel of human cancer cell lines with a high degree of selectivity over normal human foreskin fibroblast cells. The molecules were effective against androgen-sensitive LNCaP cancer cells, as well as the androgen-insensitive PC-3 cell line. Given the steroidal nature of the active compounds, it was essential to show that the cytotoxic biological responses (or lack thereof) were not due to modulatory androgen receptor activity. It was confirmed that physachenolide D had no effect on androgen binding to the AR, while dihydrotestosterone inhibited binding with an IC₅₀ of 13 nM.

Adapted from: R. M. Kris, A. A. L. Gunatilaka and co-workers. J. Med. Chem. 2015, 58, 6984.

            Prior to in vivo efficacy evaluation of physachenolide C, the maximum tolerated dose (MTD) was determined and then pharmacokinetic studies were conducted using variously formulated drug substance dosed via intraperitoneal (IP), gastric gavage (GG) and subcutaneous (SC) routes of administration. Ultimately, it was determined that efficacy studies could be conducted optimally using the SC route with ‘Trappsol,’ a formulation excipient recommended for administering hydrophobic compounds and approved by the FDA for injection into humans, as the vehicle. Two human PC murine xenograft models were conducted: the first mimicked an androgen-dependent disease using mouse inoculation with cancerous LNCaP cells and the second was an androgen-independent model using PC-3 cells. The results from the LNCaP xenograft experiment (reproduced above from J. Med. Chem. 2015, 58, 6984.) showed that physachenolide C was able to reduce tumor growth in a dose-dependent manner but did not invoke a synergistic effect when applied in combination with Taxotere®, which is the current standard of care for CRPC. A similar level of efficacy was observed in the androgen-independent PC-3 xenograft model. As is often the case with cytotoxic agents, the therapeutic safety window is a key parameter. As the authors put it: “…[compound] 6 was able to reduce tumor growth…in the mice that did not die from the toxicity.” Indeed, at a dose of 25 mg/kg physachenolide C, three of the eight mice died due to drug toxicity (Figure above, lower panel). At 25 mg/kg physachenolide C, dosed in combination with Taxotere®, all eight of the mice succumbed. All other deaths observed in the course of the study were due to euthanization because of tumor size. The authors speculate on a potential mechanism of action for the observed potent and selective anti-PC activity involving TRAIL-induced apoptosis of carcinoma cells.