Straightforward synthesis of unnatural poly-oxygenated steroid sapogenins

Saponins and their biosynthetic intermediates, sapogenins, display a variety of biological activities of interest to the pharmaceutical, cosmetic and food sectors. Three unnatural steroid sapogenins bearing oxygenated functions in rings A, B and F were prepared following a straightforward synthetic protocol that comprises the installation of a carbonyl function in ring F, a two-step, one-pot generation of a ketol in A,B rings


Introduction
Saponins are secondary metabolites found widespread in nature.2][3] These kinds of compounds have shown a wide spectrum of properties that include sweet 4 or bitter taste, 5 and antimicrobial, insecticidal, and molluscicidal activities 6 as well as hemolytic 7 and cytotoxic 8 properties amongst many others. 1,9In addition to the pharmacological and medicinal importance, 10 the usefulness of saponins in the food industry is well recognised. 11aponins are traditionally classified as triterpene or steroid glycosides according to the aglycone (nonsaccharide) part.The wide diversity of these compounds lays in the structural diversity of the less polar aglycone portion that is coupled to one or more units of different carbohydrates. 12n particular, steroid saponins have attracted significant attention since the mid part of the last century due to their utility as raw materials for the production of steroid sapogenins.The aglycone part, obtained by hydrolysis, serves as the classical starting material for the synthesis of steroid hormones 13 and other biologically active compounds.  Addionally, the recognition of biological activity and pharmacological importance of steroid saponins, brought attention of chemists, biochemists and biologists to this family of compounds.][23] Consequently, multiple efforts directed towards the synthesis of naturally occurring steroid saponins have been described. 24,25Such efforts comprise both the installation of the required functionality in the aglycone part, and synthesis of the carbohydrate building blocks.The last steps of the synthetic sequence are generally the coupling of both units and deprotection of the functional groups.
As a part of our program on the synthesis of biologically active steroids, we have become interested in the synthesis of unnatural steroid sapogenins that could serve as aglycones for the construction of steroid glycosides that may be interesting candidates for biological-activity screening.Herein, we describe the synthesis and characterization of three unnatural steroid sapogenins bearing oxygenated functions in rings A, B and F.

Results and Discussion
The synthetic protocol started with the introduction of a carbonyl function at position C-23 of the side chain of diosgenin acetate (1) employing a methodology described by Barton. 26 According to that report, treatment of a steroid sapogenin (I) with NaNO2 and BF3•Et2O produces a 23E-oximino sapogenin (II) that is hydrolyzed to a 23-keto sapogenin (III) by column chromatography in neutral alumina (Brokmann activity III).Our detailed study of this reaction showed that the compound formed by the treatment with NaNO2 and BF3•Et2O is the 23E-nitroimino derivative (IV) (Scheme 1). 27AUTHOR(S) Scheme 1. Introduction of a carbonyl group at position C-23 of the side chain of steroid sapogenin.Thus, treatment of diosgenin acetate (1) with NaNO2 and BF3•Et2O in acetic acid followed by column chromatography in neutral alumina (Brokmann activity III) following Barton´s procedure afforded the ketone 2 that was subsequently converted into the acetylated ketol 3 employing our simple one-pot procedure which includes epoxidation with meta-chloroperoxybenzoic acid (mCPBA) followed by oxidative cleavage of the oxirane ring with CrO3. 28ydrolysis of the acetate at position C-3 afforded the target dihydroxylated ketone 4 that was converted into the unsaturated ketol 5 by tosylation in pyridine followed by elimination using treatment with LiBr and Li2CO3 in refluxing DMF.Subsequent dihydroxylation employing OsO4 and N-methylmorpholine N-oxide (NMO) as co-oxidant in TMF afforded a mixture of the target trihydroxylated diketone 6 as the maJor compound, accompanied by its 2,3-diastereomer 7 that were separated by column chromatography (Scheme 2).Scheme 2. Synthesis of the polyoxygenated steroid sapogenins 4, 6 and 7. 1 and 2) were carried out with the aid of a combination of 1D and 2D NMR techniques that included 1 H, 13 C, 1H-1H COSY, Nuclear Overhauser Effect Spectroscopy (NOESY), Heteronuclear Single Quantum Correlation (HSQC) and Heteronuclear Multiple Bond Correlation (HMBC).The observed NOEs allowed the determination of the orientations of the 2,3 and 2,3 diols introduced into the A rings of compounds 6 and 7, respectively (Figure 1).

Conclusions
We have set up procedures for the synthesis and unambiguous characterization of unnatural steroid sapogenins that will serve as aglycones for the construction of steroid glycosides which may be interesting candidates for biological-activity screening.

Experimental Section
General.(25R)-3-acetoxy-5-hydroxy-5-spirostan-6,23-dione (3).meta-Chloroperoxybenzoic acid (mCPBA) (414.5 mg) was added to a solution of the ketone 2 (798.4 mg, 1.70 mmol) in CH2Cl2 (10 mL) and the mixture was stirred until the starting material disappeared (1 h, TLC).Acetone (20 mL) was added, and the mixture was cooled to 0° C in an ice bath before the dropwise addition of a solution of CrO3 (303.4 mg) in water (1 mL).The ice bath was removed, the mixture was stirred at room temperature for 20 min and cooled to 0° C in the ice bath prior to dropwise addition of a solution of CrO3 (773.6 mg) in water (2.3 mL).The ice bath was removed, and the mixture stirred for 2.5 h, before addition of (20 mL) and extraction with ethyl acetate (2 × 25 mL).organic layer was washed sequentially with water (10 × 20 mL), a 10% NaHCO3 solution (5 × 20 mL), water (2 × 20 mL) and brine (20 mL), then dried (anh.Na2SO4) and evaporated to afford 787.(25R)-5-Hydroxy-5α-espirost-2-en-6,23-dione (5).Tosyl chloride (672 mg) was added to a solution of the dihydroxylated diketone 4 (638.3mg, 1.39 mmol) in dry pyridine (8 mL) and the mixture was stirred for 24 h before pouring into a 3% HCl/ice bath.The produced solid was filtered off and dissolved in ethyl acetate (60 mL), and the organic solution was washed with water (2 × 20 mL), dried (anh.Na2SO4) and evaporated.The residue was stirred with LiBr (713 mg) and Li2CO3 (590 mg) under reflux in dry DMF (12 mL) for 3 h and the mixture was cooled to room temperature.Ethyl acetate (60 mL) was added, the mixture was filtered, the inorganic salts were washed with ethyl acetate (4 × 5 mL) and the organic solution was washed with water (6 × 20 mL) dried (anh.Na2SO4) and evaporated.The produced residue was purified in a chromatographic column