Sunday, May 27, 2012
The laboratory of Clayton Heathcock described the first preparation of an unsymmetrical bis-steroidal pyrazine in 1992. The Heathcock protocol combines an a-aminomethoxime with an a-acetoxyketone at high temperature (~ 140 oC) for 48 hours with modest isolated yields of the pyrazine product (< 40%). Fuchs and Guo improved upon Heathcock’s concept by coupling an a-azidoketone (e.g. 1) with an aminomethoxime (2) in the presence of dibutyltin dichloride. This reaction proceeds in only 3-6 hours under relatively mild thermal conditions (~ 80 oC). Weisheng Tian and co-workers applied the Fuchs pyrazine synthesis to the coupling of their western and eastern hemispheric cephalostatin fragments (1 and 2, respectively) and obtained a quite acceptable 67% isolated yield of the unsymmetrical bis-steroidal pyrazine product. Subsequent global deprotection then delivered the natural product (+)-cephalostatin 1. This effort constitutes only the third completed total synthesis of cephalostatin 1 since its isolation by Petit about 24 years ago.
The utilization of the a-azidoketone 1 (in place of Heathcock’s a-acetoxyketone) changes the mechanism of the condensation reaction to involve the extrusion of nitrogen gas (3 à 4) prior to an elimination/aromatization step (5 à 6). Polyvinylpyridine (PVP) is routinely used as an additive in order to suppress degradation of the acid-labile spiroketal functionality. The Fuchs-Guo protocol has been used extensively for the coupling of highly functionalized steroid spiroketals, most notably in the penultimate step of Shair’s recent synthesis of cephalostatin 1. Shair’s synthesis will be the topic of a subsequent post at this site.
Friday, May 25, 2012
The synthesis of the eastern hemisphere of the bis-steroidal pyrazine cephalostatin 1 by Tian and co-workers is notable for its efficiency on relatively large scale. The authors report that >2.5 grams of the oxygenated steroid structure shown above (right side) have been prepared as of the date of publication (2011) of their manuscript in Chemistry - An Asian Journal. The major challenges associated with the synthesis of this molecule are regio- and stereoselective oxidative manipulations of the D-ring along with ketalization to establish rings E and F. The latter task is accomplished by the Tian laboratory in an elegant way with a cascade reaction that will be discussed below.
Intermediate 1 is obtained in eight steps from the inexpensive steroidal saponin hecogenin. The hindered thioketal of 1 is then metallated with n-butyl lithium and the lithiated anion quenched at low temperature with the aldehyde 2. This provides a 68% yield of a mixture of products favoring (5-6:1) the diastereomer predicted by the Cram-Reetz model for asymmetric induction. Two additional protecting group manipulations then secured the advanced intermediate 3. The diene 3 undergoes smooth cycloaddition with singlet oxygen and reduction of the crude endoperoxide product with zinc afforded 4 with good overall stereoselectivity (d.r. 10:1).
The following step is remarkable and proceeds efficiently on gram-scale. In a cascade spiroketalization/SN2' E-ring-closure one-pot sequence, the intermediate 4 is exposed to aqueous hydrochloric acid with mild heating. This results in the removal of four acid-labile protecting groups (C3, C12, C23, C25) with concomitant assembly of the E/F ring system. The stereoselectivity of the transformation is quite good considering complexity of the system, providing 68% of the desired C22(S), C23(R) product along with about 20% of separable stereoisomers. Selective oxidation of the C3 hydroxyl of 5 with Fetizon's reagent (Ag2CO3/Celite) and acetylation of the C23 hydroxyl completes the synthesis of the eastern side of cephalostatin 1, with ~700 mg of 6 harvested from a single run. In the next post at Modern Steroid Science, we will discuss the mechanism of the Fuchs-Guo unsymmetrical pyrazine synthesis that is utilized in the endgame of all of the completed total syntheses (Tian, Shair, and Fuchs) of cephalostatin 1.
Tuesday, May 15, 2012
The potent anticancer activity of the bis-steroidal pyrazine cephalostatin 1 has been discussed here previously. The putative biological target of cephalostatin 1 and the related marine natural product ritterazine B has been identified recently by Matt Shair's laboratory. The synthetic laboratory preparation of cephalostatin 1, for obvious reasons (see structure above), is also a fascinating scientific challenge. Synthetic studies targeting the daunting dimeric pyrazine steroidal spiroketals will be the subject of a forthcoming series of posts at this site. The laboratories of Fuchs, Shair and Tian have completed syntheses of cephalostatin 1. We will begin this series by examining Weisheng Tian's preparation of the western hemispheric substructure of cephalostatin 1 starting from the inexpensive steroidal saponin hecogenin (1). The major challenges associated with this task include a selective oxidation of the unactivated angular C18 methyl group and olefin installation (dehydrogenation) on the steroidal D-ring.
The authors have developed a very useful and practical method for the degradation of a derivative of hecogenin (2) which provides the highly functionalized tetraol 3. In brief, a Baeyer-Villiger oxidation of 2 with in situ-generated performic acid followed by hydrolysis of the intermediate ester affords 3 on multi-hundred gram scale. The tetraol 3 is then advanced in four steps to 4, the substrate for remote functionalization of the C18 angular methyl group. Meystre's hypoiodite method, developed at Ciba in the 1960's, was utilized to accomplish this challenging oxidative transformation. Exposure of pregnane intermediate 4 to lead tetraacetate, iodine and light initiated a hydrogen transfer from C18 to the 20-hydroxy radical and subsequently the C18-centered radical is captured by an iodine radical (see the abbreviated mechanism below).
This oxidative process is then repeated, resulting in a second C18-centered radical and this species is eventually converted into the lactol acetate shown above. Jones oxidation of the lactol then provides the requisite lactone 5. The Ciba method of C-H activation/oxidation of the angular C18 position of pregnane derivatives was previously applied to a historic synthesis of batrachotoxin by the laboratory of Heinrich Wehrli (reviewed here).
Elaboration of the western spiroketal system of cephalostatin 1 entailed a rhodium-mediated regioselective O-alkylation of intermediate 6 followed by an efficient intramolecular Horner-Wadsworth-Emmons reaction to generate 8. Compound 8 was then converted to the western half of cephalostatin 1 by a sequence that included a tandem oxymercuration-ketalization as a key step. Epimerization of the anomeric spiroketal stereogenic position followed by global deprotection then secured the targeted advanced western intermediate (shown above, bottom right structure). Elaboration of the western substructure of cephalostatin 1 into the intact natural product by the Tian laboratory will be the subject of the next post at Modern Steroid Science.
I recently stumbled upon an outstanding review article in Natural Product Reports written by Hongbin Sun and Huaming Sheng that covers the organic chemistry and biochemistry related to the pentacyclic triterpenoids. Many organic chemists are familiar with complex natural product targets such as germanicol and lupeol (shown above) because of the pioneering total synthesis studies of Ireland, Stork, Johnson, Corey and others. This landmark synthetic work is covered in detail along with a critical analysis and comparison of retrosynthetic strategy. The pentacyclic triterpenes are also highlighted in the Review as multi-target therapeutic agents for metabolic and vascular diseases. I am currently in the process of pouring through the details of the manuscript by Sun and I would recommend it to other scientists with an interest in terpenoid research.
Thursday, May 3, 2012
A connection between hepatitis C virus (HCV) replication and sterol metabolism: A new antiviral target for HCV?
It is well known that viruses utilize host-derived lipids in order to replicate. However, the functional importance of specific lipids to the viral replication process has not been well-characterized until very recently, when a group from
uncovered a connection between hepatitis C virus (HCV) replication and cellular levels of a specific biogenic precursor to cholesterol called desmosterol. Desmosterol, shown above (middle structure), is simply D24-dehydrocholesterol, the immediate precursor to cholesterol (rightmost structure) in the Bloch branch of the cholesterol biosynthetic pathway. The Harvard group noted that HCV infection is associated with hypolipidemia that is reversible upon treatment. This provided a clue that HCV is able to manipulate host lipid metabolism to promote its replication, and thus, pharmacological inhibition of this process might represent an antiviral therapeutic strategy. LC/MS analysis of steady-state lipid abundance in an HCV cell culture model identified a lipid metabolite (later identified as desmosterol) with a 13-fold increase in abundance in infected cells relative to uninfected controls. Harvard Medical School
As depicted in the scheme above, desmosterol is produced by reduction of 7-dehydrodesmosterol, a biosynthetic transformation catalyzed by an enzyme called DHCR7. Treatment of cells with a known small molecule inhibitor of DHCR7 (AY9944) resulted in reduction of cellular desmosterol to undetectable levels. Importantly, pharmacological inhibition of DHCR7 was also associated with a dose-dependent decrease in steady-state expression of the HCV core, NS5A and NS5B proteins. This seems to indicate that DHCR7 activity is important for viral replication in the case of HCV. The therapeutic potential of targeting desmosterol metabolism as an antiviral strategy is not currently known. AY9944, like many other drugs that inhibit enzymes involved in cholesterol biosynthesis, is plagued by side effects (e.g. impairment of brain development) that prevent its clinical use. Nonetheless, the Harvard study is a unique application of lipid metabolite profiling that has clearly identified a specific sterol molecule that is required for HCV replication. Additional work in this area is warranted to elucidate the specific function (mechanism) of desmosterol and DHCR7 during HCV infection.