Formation of the steroidal 3 β-hydroxy-6-oxo-moiety . Synthesis and cytotoxicity of glucolaxogenin

An efficient alternative route to the synthesis of the steroidal 3β-hydroxy-6-oxo moiety starting from diosgenin and cholesterol is described. The sequential tosylation, oxidative hydroboration, and selective reduction steps provided the target 3β-hydroxy-6-oxo moiety, yielding laxogenin 1 in 82% and 3β-hydroxycholestan-6-one 8 in 83% overall yield. The cleavage of the S-O bond of 3β-tosylate-6-oxo intermediates was succeeded by means of sodium naphthalenide at -80 °C; when the reduction was explored at room temperature the cleavage of the C-O bond was favored and the corresponding i-steroids were observed. Glucolaxogenin 15 was synthesized in 68% from 1, and its antiproliferative activity was evaluated in cervical cancer cells HeLa, CaSki and ViBo. The effect on peripheral blood lymphocytes was assessed founding that the cell growth was unaffected showing therefore high selectivity.


Introduction
The 3β-hydroxy-6-oxo moiety has been found in several naturally occurring steroids such as laxogenin 1 and several brassinosteroids (growth phytohormones) i.e. teasterone 2, cathasterone 3 and 6-oxocampestanol (4, Figure 1). 1 In the general brassinosteroid biosynthetic pathway, the 3β-hydroxy-6-oxo moiety is early introduced in 6-oxocampestanol 4, 2 inducing enzymes to hydroxylate the campestanic side chain to afford teasterone 2, one of the most distributed brassinosteroids in plant kingdom. 3Laxogenin is a naturally occurring compound which was  4 Laxogenin became important due to its growth promoting activity in plants, 5,3b as an analogue of brassinosteroids, and recently because of its cytotoxic activity. 6Several pathways for the synthesis of laxogenin have been reported, in moderate to good yields.Iglesias-Arteaga et al. reported the synthesis of 1 by means of a selective oxidation of 6β-hydroxytigogenin (β-chlorogenin); 3b Yu et al. reported a protocol based on the hydroboration of benzylated diosgenin 7 and more recently, our group reported a route supported on the transformation of the 5β,6β-epoxide of the diosgenin acetate. 8Since some brassinosteroids have recently shown antiproliferative activity; 9 herein we report an alternative route for the synthesis of laxogenin glucoside and the determination of its antiproliferative activity.

Results and Discussion
In order to insert an electron-withdrawing protecting group at C-3 (instead of electron-donating substituents such as ethers), cholesterol 5 was transformed into cholesteryl tosylate 5a. 10 In our experience using tosylates in the hydroboration-oxidation reaction, better yields have been obtained.The 6-oxo functionality was inserted following the Brown's oxidative hydroboration procedure: 11,3b,7 the double bond of 5a was treated by diborane (prepared in situ by means of NaBH 4 and BF 3 •OEt 2 ), and the corresponding steroidal boranes were oxidized with 35% H 2 O 2 in the presence of methanolic KOH.Subsequent PDC oxidation of the introduced hydroxyl groups at C-6 provided the ketone 6 in good yield.All these steps have been commonly followed by other authors and in our case, they were achieved successfully.
To recover the hydroxyl group at C-3, we envisaged the reductive cleavage of tosylates by means of sodium naphthalenide.The radical anion sodium naphthalenide (SN) is a one electron donor, easy to prepare. 12The SN promotes reactions as the reductive cyclization of steroidal acetylenic ketones, 13 coupling of ketones 14 and thiocarbonyl functionalities; 15 converts vicinal cyanohydrins and geminal dihalides into olefins, 16 and removes p-toluensulfonamide, mesylate and tosylate protecting groups among others. 17The reaction conditions in the reductive cleavage of tosylates by means of SN have been widely studied as aforementioned, finding that vicinal groups have influence in the reaction pathway.In this report we determined the reaction conditions for the reduction of steroidal tosylates located at C-3, bearing at C-6 a carbonyl function.
The reductive cleavage of a great variety of simple tosylates using SN has been previously reported at room temperature as described by Closson.17e However, under similar conditions, the reduction of 6 with SN afforded the 3α,5-cyclo-5α-steroid 7 instead of the desired 3β-hydroxy-6oxo-5α-steroid 8 (Scheme 1).The synthesis of the A-cyclosteroid 7 starting from 6 has been previously reported by means of KOH/MeOH, under reflux, in 85% yield. 18The SN treatment of 6 directed to 7 instantaneously, at room temperature in a better yield (95%).When the SN treatment was carried out at 0 °C compound 8 was disclosed, and a practically quantitative yield of 8 was obtained working at -80 °C.In this manner, compound 8 was obtained in 83% overall yield from 5; the 5α stereochemistry was confirmed by 1 H-NMR.The same sequence of reactions was applied to diosgenin 9 to yield the corresponding laxogenin 1 in 82% overall yield (Scheme 2).It is important to note that the labile spiroketal side chain of diosgenin resulted unchanged either under borane or SN reductive reaction conditions.On the other hand, 3α,5-cyclo-5α-steroids such as 7 and 11, were transformed into the 3βhydroxy-6-oxosteroids (8 and 1 respectively) by acidic hydrolysis as reported. 18,19 plausible reaction mechanism is proposed (Scheme 3) to explain the results under the action of SN: in Path A, one electron from SN promotes the formation of the radical anion i that losses a sulfinate moiety through the homolytic cleavage of the S-O bond.One more electron from SN transforms the steroidal radical ii, into the alkoxide iii which traps a proton during the quenching to yield the 3β-hydroxy-6-oxo moiety (compounds 8 and 1).In Path B the electron from SN is trapped by the carbonyl group obtaining the radical anion iv which through the elimination of the hydrogen atom at C-5 generates the enolate v; the latter undergoes the elimination of a tosylate group (C-O bond cleavage) 17c affording the A-cyclosteroid 7 or 11.Scheme 3. Plausible mechanisms for SN reduction of steroidal 6-oxotosylates.
Laxogenin 1 showed to be poorly soluble in EtOH, EtOAc or DMSO; therefore, in order to evaluate 1 in cell cultures, to determine its antiproliferative effect we proceeded to obtain its glycosylated derivative.The glucolaxogenin was prepared under the standard glycosylation procedure (Scheme 4).The glucosyl donor was prepared from commercially available glucose 12, which was firstly peracetylated and then selectively deprotected at the anomeric center by means of hydrazine acetate.The inhibitory effect of compound 15 (Table 1, Figure 2) on the proliferation of cervical cancer cells HeLa, CaSki and ViBo was determined.Cells were cultured in 96 well-plates and treated with 15.Antiproliferative activity (IC 50 ) was determined after 24 hours by staining with crystal violet dye.It is well known that some compounds used in chemotherapy such as cisplatin present antiproliferative activity and apoptosis in cervical cancer cells like HeLa and Caski in a nM range (around 40). 20 However, major compounds used currently in chemotherapy present problems for selective activity towards malignant cells and produce undesirable secondary effects, and therefore immune system is usually affected.For this reason, glucolaxogenin effect on the proliferation of enriched lymphocyte population (ELP) was assessed.ELPs from normal blood donor were labeled with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), stimulated with phytohemagglutinin (PHA), or treated with glucolaxogenin 15, and cultured for 72 hours.Cells were harvested and analyzed by flow cytometry; data were processed through CellQuest software.The effect of 15 on proliferative potential of ELPs is shown in Figure 3, expressing that in normal conditions, proliferating cells were 49.26% (Figure 3-B).When lymphocytes were treated with the higher concentration of 15 (0.169 mM for ViBo), proliferating cells were 55.78% (Figure 3-F).At lower concentrations (0.094 mM for HeLa and 0.120 mM for CaSki) cells were surprisingly induced to proliferate, reaching 85.17 and 66.96% (Figures 3-D

Conclusions
The 3β-hydroxy-6-oxo steroidal moiety was obtained starting from diosgenin and cholesterol, using a three-step procedure in high overall yield.The reported conditions for the oxidativehydroboration and the subsequent oxidation of the resulting alcohols were optimized and the reduction of tosylates was afforded by sodium naphthalenide.We achieved the selective fission of either the S-O or C-O bond in dependence of the temperature.A likely reaction mechanism to explain the generation of all products is proposed.When this procedure was applied to diosgenin 9, the naturally occurring laxogenin 1 was obtained in 82% overall yield.Glucolaxogenin 15 was synthesized in order to evaluate its antiproliferative activity in cervical cancer cell lines HeLa, CaSki and ViBo and in non tumoral cells (lymphocytes).Remarkably, while 15 affected the proliferation of these cell cultures, normal cells were induced to proliferate at the same concentrations.This selective action gives to glucolaxogenin a high potential for therapeutic use.

Experimental Section
General.Melting points were obtained on a Melt-temp apparatus and were not corrected. 1H and 13 C NMR spectra were recorded at 400 and 100 MHz respectively on a Varian Mercury spectrometer.The spectra were registered in CDCl 3 and referenced to TMS.The chemical shift values are reported as ppm units and coupling constants are expressed in Hertz (Hz).All assignments were confirmed with the aid of two dimensional experiments (COSY, HSQC and HMBC).HRMS data were taken on a JEOL The MStation spectrometer.Optical rotations were measured in a Perkin Elmer 241 polarimeter.

Antiproliferative activity
Three different cervical cancer cell lines were used: HeLa, CaSki and ViBo.Cells were obtained from American Type Culture Collection (ATCC Rockville, MD).The cervical cancer cell lines were cultured in RPMI-1640 medium containing 5% NCS (Newborn Calf Serum).Assays were performed by seeding 7500 cells in 96-well tissue culture plates in a volume of 100 µL of RPMI-1640 medium supplemented with 5% NCS per well.The antiproliferative effect was evaluated 24 hours after the addition of compound 15 by crystal violet staining.