OSW-1, a Naturally Occurring Antiproliferative Steroidal Saponin

          OSW-1, a cholestane glycoside isolated from the bulbs of the garden plant, Ornithogalum saudersiae, exhibits potent in vitro cytotoxicity against several malignant tumor cell lines. For example, the IC₅₀ against human leukemia (HL-60) cells ranges from 0.1-0.3 nM. However, the mode of action (more on this in a subsequent post: stay tuned) that elicits this very promising anticancer activity is poorly understood. It has been noted that the cytotoxicity pattern of OSW-1 against the NCI-60, a collection of human cancer cell lines, resembles that of ritterazine B, a complex antiproliferative dimeric steroid. This suggests that the two steroid derivatives share a similar mechanism. In an effort to elucidate the precise cellular target and MOA of OSW-1, organic chemists have developed efficient strategies to synthesize the complex steroidal saponin, recently on multigram scale. A selection of this research effort is highlighted below.

          The structure of OSW-1 suggests a logical retrosynthetic disconnection into two hemispheres, the cholestane aglycone and the beta-disaccharide moiety. The synthesis of the disaccharide from L-arabinose and D-xylose, is relatively straight-forward, involving Schmidt glycosylation methodology to control stereochemistry at the anomeric positions. We will focus instead on expedient methods to assemble the aglycone portion from commercially available steroids.

          The first synthesis of OSW-1 was completed in 1999 by the laboratory of Biao Yu at the Shanghai Institute of Organic Chemistry. Yu’s method generates the steroidal aglycone in 12 synthetic operations from dehydroisoandrosterone. Zhongwu Guo's group has recently (JOC 2008) adapted and optimized Yu’s route in the disclosure of a 10 step synthesis of OSW-1 (6.4% overall yield) that has generated gram quantities of the precious lead compound for the development of novel anti-tumor drugs. According to the Guo protocol, the side chain is introduced to C17 of dehydroisoandrosterone in a modified fashion. An initial aldol condensation of the 17-oxo steroid functionality with propanenitrile delivers an excellent yield of epimeric beta-hydroxy (C20-) nitrile products that are conveniently isolated by recrystallization. The side chain is then elongated by addition of a Grignard reagent to the C20 nitrile, and this procedure is accompanied by elimination of water and isomerization of the resultant olefin to the endocyclic (delta^16,17) D-ring position. Silylation of the C3 beta-hydroxyl followed by protection of the C22 carbonyl as the cyclic ketal then secures Yu’s advanced intermediate in only four scalable operations. Finally, regio- and stereoselective dihydroxylation of the D-ring double bond with stoichiometric osmium tetroxide followed by inversion of C16 stereochemistry provides the OSW-1 aglycone that was successfully advanced by Yu and Guo to the natural product OSW-1.

          Zhendong Jin’s group at the University of Iowa has described, arguably, the most innovative and efficient preparation of OSW-1. The route proceeds in ten linear synthetic steps with 28% overall yield. An inventive initial transformation developed by Jin and co-workers is a regio- and stereoselective SeO₂-mediated allylic oxidation that provides access to a key enone intermediate. Jin's olefination/allylic oxidation protocol offers a facile entry into C16-oxygenated steroid systems and may be of broad utility to the synthetic community.  In a subsequent operation, a stereoselective 1,4-addition of an alpha-alkoxy vinyl cuprate (acyl anion equivalent) to the aforementioned enone incorporates the cholestane side chain with outstanding control of C20 stereochemistry. Enolate oxidation with Davis’ reagent then affords an alpa-hydroxy C16 ketone that is stereospecifically reduced from the alpha-face due to the directing effect of the C17 hydroxyl group. The synthetic technologies described Jin and the other outstanding organic chemists mentioned above will facilitate the semisynthetic construction of designed non-naturally occurring analogues of OSW-1 that may eventually inspire the invention of novel, effective and relatively nontoxic anticancer agents.

The Synthesis of Cyclopamine by Giannis et al.

          The synthesis of cyclopamine by the laboratory of Giannis begins with the remote stereoselective C-H functionalization of the steroidal C12 position according to a modification of the protocol of Schonecker. It seems initially surprising that the attack of the oxygen occurs on the hindered (by the pseudoaxial 13beta-methyl) beta-side of the steroid. This indicates that in the active copper complex (shown), the copper and oxygen atoms must lie in the same plane as the 12beta-hydrogen such that the oxygen can abstract the 12betaH more readily than the axial 12alphaH. In this way, hydroxylation of the readily available dehydroepiandrosterone secures the corresponding 12beta-hydroxy steroid derivative in a very good yield, when one considers the relative challenges associated a total synthesis approach to this type of functionalization.

          After a straight-forward installation of the C17 spiro-lactone, the steroid skeletal framework was rearranged to the C-nor-D-homo system of cyclopamine. A very interesting cationic ring contraction/expansion was developed for this purpose. The reaction involves triflation under thermal conditions and the detailed mechanism has not been described. Interestingly, the 12beta-triflate does not seem to be an intermediate in the formation of the rearrangement products of the reaction, which contain an exocyclic double bond (major) along with an endo isomer (minor). Clearly, a cationic mechanism involving C14 bond migration is operative in the rearrangement process. The reaction is effective on a variety of different steroidal systems including 17-keto steroids (separate methodology paper: JACS 2010).

          Subsequently, a tandem Horner-Wadsworth-Emmons (HWE) olefination/intramolecular oxy-Michael addition was successfully conducted on an advanced azidolactol intermediate. Diastereoselectivity in the case is probably derived from the relative thermodynamic stability of the equatorially disposed furan substituent in the observed stereoisomer. Next, the piperidine is constructed in four steps as shown above. Given the presence of three double bonds (two exocyclic) in the advance piperidine intermediate, regioselective olefin functionalization was required to complete the synthesis of cyclopamine. First, regio- and stereoselective hydrogenation of the C25-27 olefin was achieved with Wilkinson’s rhodium(I) catalyst. The C13-18 exocyclic double bond was then selectively functionalized by execution of an intramolecular ene reaction which generated an N-sulfinylated intermediate that was desulfurized with Raney nickel. Consecutive reductive deprotections completed a concise (20 steps) and efficient synthesis of the biomedically useful modified steroid, cyclopamine.

Cyclopamine, a Potent Antagonist of the Hedgehog Signaling Pathway

       The connection between the natural product cyclopamine and research into the basic developmental biology associated with the hedgehog pathway is a very interesting story (indeed!) that has been eloquently relayed in detail by Giannis, a leading researcher in the field. These investigations have recently culminated in the disclosure of small-molecule inhibitors of the hedgehog signaling pathway that show great promise as clinically relevant therapeutic agents for the treatment of certain types of cancer. In this post, we’ll look briefly at the role of the hedgehog pathway in tumorigenesis and examine two medicinal chemistry efforts to develop potent and orally active antagonists of the hedgehog pathway.

Overview of the Hedgehog Signaling Pathway

          The term ‘hedgehog’ is derived from the discovery of a gene in the fruit fly that caused larvae to grow a coat of spines on their undersides when mutated. These larvae apparently resembled the spiny mammals for which the hedgehog gene was eventually named. The hedgehog gene encodes for three varieties of hedgehog proteins (Sonic or SHH is the best studied), all of which are ligands for the membrane-bound receptor Patched (PTCH). Binding of a doubly lipid-modified SHH ligand to PTCH induces sequestration and degradation of the receptor, which leads to release of the 7-transmembrane domain protein, Smoothened (SMO), from intracellular compartments. In the absence of a hedgehog ligand, the role of PTCH is to block the function of SMO. With PTCH internalized, SMO can now enter the primary cilia where it stimulates the translocation and activation of transcription factors (Gli1-3) that control expression of Sonic hedgehog target genes and are implicated in the development of some cancers. Loss-of-function mutations in PTCH and activating mutations in SMO have been identified in patients with basal cell carcinoma.
          The precise molecular mechanism by which the 12-transmembrane-spanning receptor Patched suppresses the activity of Smoothened is not well characterized. Scott and Corcoran noted that PTCH is related structurally to a cholesterol transporter and provided evidence to suggest that this receptor modulates SMO activity by acting like a sterol pump that regulates the distribution of some unidentified endogenous oxysterol SMO ligand. They show that specific oxysterols are required for SHH pathway signal transduction and that a subset of these can maximally activate SHH target gene transcription through SMO. However, the identity of the putative endogenous SMO ligand remains unknown. Moreover, Beachy and co-workers have shown with competition and binding studies that oxysterols indirectly activate SMO and that the regulatory event probably occurs upstream of SMO. Delineation of the precise molecular mechanism that connects SMO to Gli’s remains an exciting and active area of biochemical research.

          Since aberrant activation of the hedgehog pathway is associated with malignancy, inhibition of this signaling network is a potential pharmacological intervention against cancer. Cyclopamine is a naturally occurring antagonist of the hedgehog pathway that has been shown to directly bind to SMO and inhibit its activity by a conformational change. However, the development of cyclopamine as an anticancer therapy is hampered by its thermodynamically favorable conversion to veratramine under acidic conditions. This transformation involves ring opening of the spirotetrahydrofuran followed by aromatization of the D-ring. Veratramine is inactive as a SMO antagonist and is known to induce side-effects stemming from serotonergic and other off-target activities. In order to overcome this obstacle and to generally improve cyclopamine’s drug properties, researchers at Infinity Pharmaceuticals homologated the steroidal D-ring by one methylene unit. This modification, in conjunction with A-ring derivatization to incorporate a sulfonamide or a fused pyrazole, delivered a chemically stable and orally active SMO antagonist (IPI-926) that completely regressed tumor formation in a medulloblastoma allograft model after daily oral administration (40 mg/kg). The partial chemical synthesis of IPI-926 and related analogues from cyclopamine, along with the semisynthesis of cyclopamine by the laboratory of Giannis, will be highlighted in subsequent posts.

          Additional chemical classes of SMO inhibitors have also appeared in the literature. These synthetic non-steroidal SMO ligands are exemplified by Genentech’s GDC-0449 (vismodegib) and most are comprised of a biaryl framework substituted with a benzamide. Published clinical results indicate that vismodegib exhibits antitumor activity in patients afflicted with basal-cell carcinoma. However, this class of compounds generally exhibits low aqueous solubility and is plagued by high plasma protein binding that can be attributed to high lipophilicity. Solubility/lipophilicity issues were overcome by medicinal chemists at Pfizer who recently disclosed the structure of PF-4449913, an orally bioavailable inhibitor of SMO that has been advanced to human clinical studies. Collectively, these favorable results encourage the pursuit of SMO antagonists as a means to discover and invent novel anticancer therapies.