Oxido-bridged neurosteroid analogues. Synthesis of 2,19-oxido-allo pregnanolone

3 α -Hydroxy-2,19-oxido-5 α H -pregnan-20-one was synthesized in 6 steps from the easily available 19-hydroxypregnenolone 3-acetate. The 2,19-oxido bridge was formed in a single step upon spontaneous cyclization and desilylation of a TBDMS protected 19-hydroxy-2 α ,3 α - epoxypregnane. The 4,19-oxido analogue was obtained as a byproduct upon cyclization of the isomeric 3 α ,4 α -epoxide.


Introduction
2][3] Such neurosteroids and their synthetic analogues can, as a result, demonstrate sedative-hypnotic, anxiolytic or anticonvulsant activities in vivo.In the search of more active, selective and water soluble steroid derivatives, several analogues with improved bioavailability having polar groups at different positions have been developed. 4ntroduction of N or O substituents at the 2β position of allopregnanolone does not change significantly the conformation of ring A and gives rise to active analogues.Thus, minaxolone (3) 5 and Org 21465 (4) 6 are highly effective general anesthetics.

H HO
The synthesis of conformationally restrained analogues of biologically active steroids, provides a way of obtaining more potent and/or selective compounds.These compounds also allow a better assessment of the contribution of the overall molecular shape in their binding to the receptor.Although the effect of conformational changes in the biological activity of steroidal hormones has been the subject of several studies, 7 little is known about their effect in the activity of neurosteroids and neuroactive steroids. 8In this regard, we 9 and others 10 have recently reported the synthesis and biological activity of several rigid derivatives.
The introduction of an oxido bridge between C-2 and C-19 of allopregnanolone, gives rise to 2,19-oxidopregnane 5, with a distorted A ring bent towards the β-face.Besides giving a rigid framework, the oxido-bridge may also mimick the apparently well-tolerated β oxygen substituent at C-2 as in minaxolone (3).A 4,19-oxidoderivative as 6 gives a similar distortion of ring A but with the oxygenated substituent at C-4. Steroids with 2,19-oxido bridges are also potentially useful drugs in breast cancer therapy as suicide substrates for aromatase, the cytochrome P-450 enzyme involved in the final biosynthetic step in the production of estrogens.

Results and Discussion
The key step in previous syntheses of substituted analogues of allopregnanolone having polar, electronegative atoms at C-2β, has been the attack of a nucleophile on a 2α,3α-epoxide. 5,6Trans diaxial opening of this epoxide accounted for the regioselectivity of the reaction, giving rise to the 3α-OH moiety, a requisite for GABA A receptor activity.The intramolecular version of this process involving a nucleophile attached to C-19, besides being energetically more favorable, would result in the formation of a bridge between positions 2β and 19.Thus, epoxide 7a (or a suitably protected precursor as 7b) was selected as the key intermediate for obtaining the 2,19oxido-bridged analogue.In a similar way, epoxide 8a should give the 4,19-oxido analogue 6.
The 19-hydroxysteroid 9 was used as starting material.It was obtained from commercially available pregnenolone acetate following essentially the procedure described previously by us for the preparation of the 20-O-acetyloxy analogue. 12The synthetic sequence is outlined in Scheme 1. Attempts to hydrogenate the 5,6-double bond in 9 were unsuccesful even at 2-3 bar.The same lack of reactivity was observed with the 19-O-formyloxy and 19-O-acetyloxy derivatives (somewhat less with the latter).However, the t-butyldimethylsilyl (TBDMS) ether 10 was smoothly hydrogenated over 10% Pd/C in ethanol (25 ºC, 1 bar); 13 reduction ocurred as expected from the least sterically demanding α-face giving rise stereoselectively to compound 11 in almost quantitative yield.The stereochemistry at C-5 of 11 was evident from the J values in the 1 H NMR spectrum.The 3α-H resonance (4.73 ppm; W 1/2 = 25 Hz) was specially diagnostic of the axial position of this hydrogen, indicative of the chair conformation of the A ring in an A/B trans-fused steroid.Double bond formation in ring A was carried out in three steps.Thus, compound 11 was deacetylated with base and treated with tosyl chloride in anhydrous pyridine giving 95% of the tosylate 12. Refluxing a solution of 12 in collidine for 3.5 h gave a mixture of the desired ∆ 2olefin 13 and the minor ∆ 3 isomer 14 (7:3 ratio by 1 H-NMR; 53% yield from 11).These compounds could not be separated by column chromatography and were used as a mixture in the following step.Treatment of the olefin mixture with m-chloroperbenzoic acid (MCPBA) in the presence of sodium carbonate gave the expected α-epoxides 7b and 8b (from 13 and 14, respectively). 6,14The 1 H NMR spectrum of the crude reaction product showed the characteristic resonances for epoxides at 3.1-3.3ppm and absence of olefinic hydrogens.The isomeric epoxides had different R f on TLC, however when separation was attempted by flash column chromatography an intramolecular attack of the C-19 oxygen occurred, with cleavage of the epoxides and concomitant de-O-silylation (probably catalyzed by the adsorbent). 15Thus, compounds 5 and 6 were obtained in one step from 13 and 14.The 7:3 ratio of the products was coincident with that of the precursor olefins.Structures of both compounds were established from 1 H and 13 C NMR spectra of 5 and 6 and the correlations in the COSY spectra.Diagnostic signals in the 1 H NMR spectra were those assigned to H-2 (for the major product 5) and H-4 (for the minor analogue, 6) at 4.144 and 3.823 ppm respectively, and their long-range correlations with the C-19 hydrogens displayed in the COSY spectra.
Figure 1 shows the AM1 calculated geometries for oxidosteroids 5 and 6 compared to that of allopregnanolone (1), showing the distortion and bending of ring A. In both steroids the 3αhydroxyl is displaced upwards and away from its axial orientation resulting in an overall curvature of the molecule towards the β-face.In the 1 H NMR spectrum of 5, H-2 appeared as a double doublet (J = 5.0 and 5.9 Hz), whereas H-4 in 6 was observed as a doublet (J = 4.8 Hz).These data are in accordance with the calculated coupling constants on the above AM1 geometries using the Altona equation (J 2,1α = 0.9 Hz, J 2,1β = 6.9 Hz and J 2,3β = 4.5 Hz for 5 and J 4,5α = 0.5 Hz and J 4,3β = 4.5 Hz for 6). 16An almost perfect "W arrangement" is predicted between one of the hydrogens at position 19 and H-5α in 5 or H-1α in 6, these are evident in the multiplicity of the H-19 signals and in the correlations observed in the COSY spectra.

Conclusions
The above procedure may be used to introduce 2,19-and 4,19-oxido bridges in a variety of 5α-H reduced steroids.Furthermore, the change of oxygen by nitrogen or sulfur at C-19, gives easy access to the aza and sulfanyl analogues, respectively.

Experimental Section
General Procedures.Melting points were determined with a Fisher-Johns apparatus and are uncorrected.NMR spectra were recorded on Bruker AC-200 (4.7 Tesla, 200.13 MHz for 1 H, 50.32 MHz for 13 C) or AM-500 (11.8 Tesla, 500.13MHz for 1 H, 125.77MHz for 13 C) spectrometers in deuteriochloroform.Chemical shifts (δ in ppm) are given from internal TMS.The J values are given in Hz.Spectra were assigned by analysis of the DEPT, and COSY 45 spectra and by comparison with those of progesterone and other oxido-bridged steroids. 9,17,18The electron impact mass spectra (EIMS) were measured on a Shimadzu QP-5000 mass spectrometer at 70 eV by direct inlet.Semiempirical AM1 calculations were performed with the MOPAC 97 module in Chem3D Ultra 5.0 (Cambridge Soft).All solvents used were reagent grade.Solvents were evaporated at ca. 45 ºC under vacuum.Flash column chromatography was carried out on silica gel Merck 9385 (0.0040-0.0063 mm).TLC analysis was performed on silica gel 60 F254 (0.2 mm thick).The homogeneity of all compounds was confirmed by TLC.