Tuesday, February 17, 2015

On the Biosynthesis of the Phainanoids, A New Class of Immunosuppressive Steroids

            The phainanoids are a recently reported class of oxidatively modified triterpenoids isolated from Phyllanthus hainanensis, a lovely shrub that is indigenous to the Hainan island of China. The architectural framework of the phainanoids includes two rare and intricate spirocyclic motifs. One of those, an eastern spiro-fused benzofurancyclobutanone, is unprecedented amongst naturally occurring steroids. Plants in the genus Phyllanthus have long been used used in traditional Chinese and Ayurvedic medicine for the treatment of infections, diabetes and hepatitis B. The phainanoids were shown to exhibit potent immunosuppressive bioactivity in in vitro experiments that tested the compounds’ abilities to inhibit the proliferation of T and B lymphocytes. New immunosuppressants that are devoid of liver and renal toxicity offer tremendous therapeutic potential as experimental candidates for the treatment of organ transplant and other immunological-associated ailments such as rheumatoid arthritis. Phainanoid F displayed impressive single-digit nanomolar potency against both T and B cells, which far exceeds the immunosuppressive activity of the well-known transplant drug, cyclosporin A.
            While the western A-ring-annulated cyclobutane system is quite unique, particularly amongst steroidal natural products, the right-hand side of the phainanoids shares significant structural homology with a known class of triterpenoids isolated from the Dichapetalaceae family of plants. The dichapetalins are dammarane-type triterpenoids that contain a 2-phenylpyrano system fused to the steroid A-ring. The C17 side chain of dichapetalin M (shown below), comprised of a gamma-butyrolactone-spiroketal, is very similar, in terms of atom connectivity, to the eastern substructure of the six newly identified phainanoid natural products. This leads one to postulate about a potential biogenic relationship between the two triterpenoid structural classes. Indeed, in 2008, five highly complex dichapetalin-type triterpenoids, coined the ‘acutissimatriterpenes,’ were isolated from a plant of the genus Phyllanthus (The phainanoids were also isolated from a Phyllanthus species). The dichapetalins were shown to exhibit cytotoxic activity against several cancer cell lines, but no immunosuppressive activity was reported.
            The eastern gamma-butyrolactone-spiroketal moiety of the phainanoids and dichapetalins is likely derived from enzymatic oxidation of several positions along the prototypical dammarane/cholestane C17 side chain. Oxidation of the C20 substituent to an ester sets up a hypothetical spiroketalization/lactonization event (shown below) that ultimately furnishes this ornate spirocyclic structural motif.
            The biosynthetic origin of the novel western spiro-fused benzofurancyclobutanone is less obvious. However, given the high degree of structural similarity between dichapetalin M and the phainanoid class of triterpenoids, along with their common occurrence in Phyllanthus species of flowering plants, it is tempting to propose dichapetalin M as a biogenetic precursor to the phainanoids. Hypothetically speaking, oxidation of the ortho-position of the eastern phenyl ring of dichapetalin M could produce the benzofuran moiety of the phainanoids. Subsequently, an allylic radical adjacent to the benzofuran heteroatom could induce fragmentation of the carbon-oxygen bond of the pyran system, resulting in a ring contraction to fashion the unique phainanoid cyclobutane. It is noteworthy that all of the carbon atoms of the phainanoid benzofurancyclobutanone are contained within the 2-phenylpyrano substructure of the dichapetalins. Installation of a single oxygen atom, along with the aforementioned radical-induced ring contraction and C-O bond fragmentation (shown below) seems like a plausible biosynthetic pathway leading to the new triterpenoid class of immunosuppressive compounds. Hopefully, future biosynthetic studies will be conducted to reveal the precise nature of the biogenetic origin of the phainanoids.

Sunday, February 8, 2015

Carl Djerassi (1923 - 2015): The Story of a Drug Needs to Start with Chemistry

The steroid pioneer Carl Djerassi, often referred to as "the father of the birth control pill," passed away in January of this year. Not much can be said about Djerassi's accomplishments that has not been discussed at length elsewhere. An account of his momentous impact on the post-World War II era race to synthesize cortisone is provided here. The indefatigable Djerassi, even at the age of 91, was still writing seething letters to C&EN about objectionable work published by the science magazine. In the October 27, 2014 issue (page 3), Djerassi made the following comments regarding Jonathan Eig's book about the development of the birth control pill:


"I find it remarkable that a chemical science magazine would publish a glib review of a glib journalistic rehashed account by Jonathan Eig of the origin of the Pill, which ignores that the work, as with any other synthetic drug, needs to start with chemistry (C&EN, Oct. 27, 2014, page 3 - 4)..."

Steroid Total Synthesis: New Transition Metal-Catalyzed Methods for Closure of the Steroid B-Ring

            Phil Baran’s group has recently disclosed a de novo synthetic approach for construction of the tetracyclic carbogenic steroid framework. Their methodology features (arguably underutilized) quinone diazide functionality as a latent cyclohexadienone carbene that readily undergoes intramolecular olefin cyclopropanation to close the steroid B-ring. The authors note that this unique carbene renders a phenol electrophilic at its normally nucleophilic para position. The quinone diazide cyclopropanation reaction exhibits quite broad substrate scope in an intermolecular sense as well.
            The advanced triazene intermediate (5) was designed by the Scripps team to serve as the masked quinone diazide that would, in the key synthetic operation (conversion of 6 à 7), undergo B-ring annulation. The synthesis of intermediate 5 involves a standard Sonogashira coupling to introduce the two carbon atoms of the requisite tether, followed by reduction of the nitro group. The resultant aniline 2 is then diazotized and trapped with diisopropylamine to generate the triazene functionality. The two-carbon tether is next elaborated to a vinyl cuprate species which engages the bicyclic enone 4 in a conjugate addition reaction. Preparation of the racemic steroidal C/D substructure 4 required six non-trivial synthetic operations starting from 2-methylcyclopentenone. Installation of an exocyclic olefin onto 5 is then required for the critical steroid annulation step. This is achieved by treatment of the silyl enol ether 5 with the well-known one-carbon electrophile, Eschenmoser’s salt. The critical rhodium-catalyzed cyclopropanation operation proceeds through the intermediacy of the transiently generated carbenoid shown above in the bracketed [TS-I]. The polycyclic steroid derivative 7 is used in crude form in a subsequent carbon-carbon bond fragmentation step (vida infra).
            As one would expect, cyclopropane ring-opening by nucleophilic chloride ion leads to cleavage of the C10-C19 bond, furnishing the dione 8 in acceptable yield over the previous five steps. The authors noted that much time and effort was invested in unsuccessful attempts to break the C9-C19 bond. A fragmentation of this nature would provide access to a synthetically useful prednisone-type carbocyclic connectivity. Unfortunately, in the case of substrate 7, the A-ring dienone acts as an efficient electron-sink and concomitant A-ring aromatization provides a strong thermodynamic driving force for cleavage of C10-C19. Alternate reductive conditions were developed to break the C9-C10 bond, expanding ring B to a seven-membered system reminiscent of the cortistatins. The Baran route to prepare the dione 8 should be considered the successful execution of a conceptually interesting retrosynthetic hypothesis for steroid synthesis. However, in terms of practicality, their protocol falls a bit short. The route requires a total of 18 synthetic operations (longest linear sequence of 13), beginning from a relatively costly trisubstituted nitrophenol (1). Moreover, the chemistry produces a racemic 11-oxo-estrone variant bearing a chloromethyl substituent at C9. This specific molecular architecture is non-naturally occurring and possesses (to my knowledge) no known pharmacologic or therapeutic medicinal value.
            On the contrary, novel methods for direct halogenation of the steroid C9 position offer tremendous value due to the demonstrated anti-inflammatory bioactivity of the 9-halo-corticosteroid series (e.g. fludrocortisone). Assuming that a 9-hydroxymethyl derivative of Baran’s advanced intermediate 8 could be accessed via cyclopropane cleavage with an O-nucleophile, a subsequent base-mediated retro-aldol process would afford an enolate (Int-I shown above) that could be trapped at C9 with an electrophilic source of fluorine. A proton quench of the same enolate, followed by equilibration to the thermodynamically favored natural estrone configuration would also be of interest.
            A related methodology for steroid total synthesis was recently reported by Wenjun Tang’s group. They have developed a new P-chiral biaryl monophosphine ligand (L*) that, in combination with a palladium catalyst, mediates the enantioselective dearomative cyclization of phenols that are tethered to enol triflates or aryl halides. For example, cyclization of the enol triflate 13, following initial oxidative addition, proceeds through the intermediacy of TS-I, depicted above in brackets. Nucleophilic substitution at the 4-position of the phenol moiety, followed by reductive elimination, then closes the steroid B-ring and furnishes 14 in a highly stereocontrolled fashion. Tang’s asymmetric synthesis of 14 (or 15, using the opposite antipode of the phosphine) proceeds in only four steps (!!!) starting from the readily available and optically active Hajos-Parrish ketone. This level of efficiency and practicality could potentially become competitive with analogous microbial biofermentation processes, particularly if the unique P-chiral catalyst were to become commercially available.