NMR and X-ray characterization of steroids with furospirostane side chains

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Introduction
Steroids bearing oxygenated spiroketal side chains are widespread in both the natural and synthetic domains.Steroid sapogenins, which have produced a large variety of interesting reactions and rearrangements, 1 are characterized by the presence of a 16,22:22,26-diepoxy moiety in side chain and can be regarded as 1,6-dioxaspiro [4.5]decane derivatives (Figure 1).This kind of compound has been subject of much research due to their biological activity 2 and their usefulness as starting materials for the synthesis of different bioactive compounds as sexual or adrenocortical hormones, 1a ecdysteroids, 3 plant growth stimulators, 4 and cytotoxic steroids 5 among others.Furospirostanes, a smaller and relatively less known family of steroids bearing a 16,22:22,25-diepoxy moiety in the chain, may be considered 1,6-dioxaspiro [4.4]nonane derivatives, and include compounds with antitumor activity as the ritterazines 4, 5f cephalostatines 5, 5f or hippuristanols 6-8, 6 among others (see Figure 2).As a part of our project to explore the reactivity of steroid bearing spiroketals side chains, we have recently focused our attention on the 16,22:22,25-diepoxy moiety derived from our recently reported hypervalent iodine-induced F-ring contraction (see Scheme 1).1f  In this context and for structural reference purposes, we decided to carry out a detailed characterization of compounds bearing such functionality that serve as materials for our biological, synthetic and chemical reactivity studies.In this communication we describe the unambiguous assignment of all 1 H and 13 C chemical shifts of spirocyclic steroids bearing the 16,22:22,25-diepoxy moiety in the side chain.In addition a detailed analysis of the crystal structures of such compounds is also provided.

Results and Discussion
Since compound 10 persisted in crystallizing in needles not suitable or X-Ray studies, derivatization of 10 was necessary.Hence the acetate 11 was prepared in 74.8% yield by the standard Ac2O/pyridine procedure.LiAlH4 reduction of 10 afforded the diol 12 in 84.9 yield (see Scheme 2).The reduction of the carboxymethyl group in 10 to produce the diol 12 results in the downfield shift of H-20 and the upfield shift of H-23.The 1 H spectrum of the reduced compound 12 also shows two new signals corresponding to the diasterotopic hydrogens attached to C-23´ which shows the typical AB-X coupling pattern.Table 2 shows the key 2D homo-and heteronuclear correlations that allow the unambiguous assignment of most of the 1 H signals associated to the (23S)-23-hydroxymethylene-16,22:22,25-diepoxy side chain of compound 12, (see Figure 4 for key H-H correlations and supplementary information file for annotated spectra).While the comparison between the coupling constants of the protons in the side chain of the spirostane derivative 9 with those of the furospirostanes 10 to 12 indicates that the conversion of 6-membered F-ring into a 5-membered ring results in the expected and evident conformational changes, the coincidence of the coupling constants of H-23 pro-R in compounds 11 and 12 suggests that the conversion of the carboxymethyl moiety of 11 into the exocyclic hydroxymethylene of 12 does not induce significant conformational changes in the side chain. 13C NMR discussion.In addition to the extensive collection of the 13 C chemical shifts of around 400 steroids built up by Blunt and Stothers,8a Agrawal published an excellent review which comprises the assignment of the 13 C chemicals shifts of 66 steroid sapogenins bearing the 16,22:22,26-diepoxy in the side chain.8b Some of our previous works on this subject have also provided rules for the systematization of the effects exerted by different functional groups in NMR signals of the 16,22:22,26-diepoxy side chain.8c-e Consequently, the discussion here will be will be focused in the main NMR characteristic associated with the 5-membered 16,22:22,25-diepoxy side chain result of the F-ring contraction (see crystallographic studies).The conversion of the F-ring of 9 into the 5-membered ring of 10 produces the downfield shift of C-25 (formerly C-26 in 9) that now resonates at between 71.8 and 72.5 ppm as a result of the lost of the 1,3 shielding interactions present in the 6-membered F-ring of compound 9.In addition, the 16,22:22,25-diepoxy side chains of compounds 10 to 12 are characterized by the presence of a deshielded signal corresponding to the spiroketal carbon C-22 between 119 and 120 ppm (See Table 4).
In particular the 13 C spectra of compounds 10 and 11 are characterized by the presence of the signal corresponding to C-23 around 59.  51.8 51.8 (0) -*Signals of compound 9 8e are included for reference.**Δ ppm associated to the changes in the side chain are given in parentheses and were calculated using the synthetic precursor as reference.
The absolute configuration of the studied compounds was considered and corroborated as that from the starting steroid sapogenin.8e Studied compounds 11 and 12 contain the steroid ABCD fused ring framework with the axial methyl groups attached to C-10 and C-13 cis A/B, trans B/C, and trans C/D rings junctions, they also bear 16,22:22,25-diepoxy moiety characteristic of the furospirostane side chain.Puckering parameters were calculated as described by Cremer and Pople. 9a In compound 11 the equatorial acetoxy group bonded to C-3 is arranged in such a way that the carbonyl O atom nearly eclipses the axial H atom attached to C-3 and does not disturb ring A, which present a chair conformation with a puckering amplitude (Q) 0.555(2) Å, θ = 178.0(2)º,  = 242(5) º, if the calculation starts from C-1 to C-10 and proceeds in a counterclockwise direction.Ring B assumes a conformation very similar to a chair form with a puckering amplitude Puckering Amplitude (Q) = 0.578(2) Å, θ = 3.2(2) º,  = 226(4) º, if the calculation starts from C-5 to C-10 and proceeds in a counterclockwise direction.Ring C assumes a conformation close to a chair with a puckering amplitude (Q) = 0.569(2) Å, θ = 9.6(2) º,  = 254.9(14)º, if the calculation starts from C-8 to C-14 and proceeds in a clockwise direction.Ring D can be described as cyclopentane twisted on C-13-C-14 with puckering parameters of Q( 2 5).The crystal structure of compound 11 is shown in Figure 5, with the ellipsoid drawn at the 50% of probability.9b   As expected, in compound 12, the presence of the smaller equatorial hydroxyl substituent attached to C-3 does not disturb the chair conformation of ring A, which shows a puckering amplitude (Q) = 0.560(2) Å, θ = 175.0(2)º,  = 282(2) º, if the calculation starts from C-1 to C-10 and proceeds in the counterclockwise direction.Ring B assumes a conformation very similar to a chair form with a puckering amplitude Puckering Amplitude (Q) = 0.573(2) Å, θ = 4.5(2) º,  = 227(2) º, if the calculation starts from C-5 to C-10 and proceeds in a counterclockwise direction.Ring C assumes a conformation close to a chair with a puckering amplitude (Q) = 0.5550(19) Å, θ = 10.7(2)º,  = 247.5(11)º, if the calculation starts from C-8 to C-14 and proceeds in a clockwise direction.Ring D can be described as cyclopentane twisted on C-13-C-14 with a conformation very similar to a half chair with puckering parameters of Q( 2 6).The crystal structure of compound 12 is shown in Figure 7, with the ellipsoid drawn at the 50% of probability.9b   In order to establish quantitative differences between the studied compounds 11 and 12, a least-squares overlay analysis of the structures is performed.Table 7 shows the results of leastsquares overlay analysis obtained with the Mercury program.9c From the obtained data it is clear that, as expected, the modifications introduced in the side chain do not produce significant conformational changes in the steroid framework, at least qualitatively.Interestingly, when F-rings of 11 and 12 are compared, it becomes evident that the envelope configuration shifts from C(25) in 11 to C(22) in 12, making the overlay between these two rings very bad and indicating evident differences in the conformations adopted by the side chains of 11 and 12. Table 8 shows the puckering descriptors as defined by Cremer and Pople 9a and calculated using the PLATON program.9d-e The minor differences observed amongst rings A to D across all compounds may be attributed to the different intermolecular interactions that are, of course, influenced by the changes introduced in the side chain.While the quite similar coupling constants of F-ring protons of compounds 11 and 12 (see Table 3) indicate that, in solution, both side chains adopt very similar conformations, the important -and somehow unexpected-conformational changes observed between both side chains in the solid state, should be associated with the different crystal packing of compounds 11 and 12, (see Figures 6 and 8).

Experimental Section
General.Reactions were monitored by TLC on Alugram SIL G/UV254 plates from Macherey-Nagel.Chromatographic plates were sprayed with a 1% solution of vanillin in 50% HClO4 and heated until color developed.Melting points were measured on a Melt-Temp II equipment.Mass spectra were registered in a Thermo-Electron spectrometer model DFS (Double Focus Sector).NMR spectra were recorded in CDCl3 solution in a Varian Inova 400 spectrometer using the solvent signal 7.26 ppm for 1 H and 77.00 ppm for 13 C as references.NMR signals assignment was made using a combination of 2D homonuclear and heteronuclear correlation techniques, which included 1 H-1 H COSY, 1 H-1 H Nuclear Overhauser effect (NOESY), Heteronuclear Single Quantum Correlation (HSQC) and Heteronuclear Multiple Bond Correlation (HMBC).All 2D NMR spectra were recorded using the standard pulse sequences and parameters recommended by the manufacturer and processed using Mestrenova (http://mestrelab.com).
Copies of the spectra can be found in the supplementary information file.
(22S,23R,24R)-16, 22:22, 25-diepoxy-23-carboxymethyl-24-methyl-26,27-dinor-5-cholestan-3-ol acetate (11).Acetic anhydride 0.8 mL was added to a solution of 10 1f in pyridine (8 mL), the mixture was stirred overnight, poured into a mixture of ice and 50 mL of 10% HCl.The mixture was extracted with ethyl acetate (2x40 mL) and the organic layer was washed with 10% HCl solution (2x25 mL), 5% CuSO4 solution (3x25 mL), water (2x30 mL), dried (anh.Na2SO4) and evaporated to afford the acetylated product 11 (1.06 g, 2.11 mmol, 75%).m.p. 172-174 °C from acetone/hexane.See tables 3 and 4 for  A solution of the rearranged product 10 1f (643 mg, 1.39 mmol) in THF (15 ml) was slowly added to a refluxing suspension of LiAlH4 (643 mg, 1.39 mmol) in THF (25 mL) under nitrogen and the mixture was refluxed for 2 hours.Ethyl acetate (30 mL) was carefully added to destroy the excess of LiAlH4 (caution !!) followed by water (30 mL) and the mixture was extracted with ethyl acetate (2x50 mL).The organic layer was washed with water (5x30 mL) dried (anh.Na2SO4) and evaporated to afford the reduced compound 12, (512.5 mg, 1.18 mmol, 85%).m.p. 177-178 °C from ethyl acetate/hexane.See tables 3 and 4 for 1 H and 13 C NMR signals.MS (EI, 70e V): 433 MH + ,415,397,385,367,285,255,155,137, (MH + C27H45O4 requires 433.3318)Suitable crystals for X-Ray diffraction studies were obtained from the analytical samples as follows: crystals of 11 from a 10/1 mixture of hexane/ethyl acetate by slow evaporation; and crystals of 12 from an acetone solution left at 4º C for several days.Compounds 11 and 12 crystallized as orthorhombic systems with space group P212121.X-Ray diffraction measurements were performed at 293 (2) K on an Oxford Diffraction Atlas (Gemini) diffractometer with Mo K radiation  = 0.71073 Å.Data collection routine and data reduction were carried out with CrysAlisPro, Oxford Diffraction Ltd.Oxford Diffraction.10a The structures of both molecules were solved using SIR2004 10b and refined using SHELXL-97.10c All non-hydrogen atoms were refined anisotropically and the hydrogen atoms were found in the difference Fourier maps, placed at geometrically calculated positions and refined using the riding model.The obtained bond lengths and angles of each compound are normal and are available from the electronic supporting information (CIF file) for the structure.Crystallographic data have been deposited with the Cambridge Crystallographic Data Center as supplementary material numbers CCDC 773328 (11) and CCDC 773330 (12).Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK.E-mail: deposit@ccdc.cam.ac.uk.

Figure 2 .
Figure 2. Some furospirostanes with cytotoxic activity against cancer cells.

Figure 4 .
Figure 4. Selected H-H and NOE correlations in the side chain of compound 12.
7 ppm and the those corresponding to the exocyclic carboxymethyl group attached to C-23 around 172 ppm (C=O) and 51.8 (OCH3).The F-ring contraction also produces a slight upfield shift on C-24´ (formerly C-27 in compound 9) as well as slight downfield shifts on C-20, C-21 and C-24 (formerly C-25 in compound 9).As expected, the reduction of the exocyclic carboxylic ester moiety to produce an exocyclic hydroxy methylene results on the upfield shifts of the signals of C-23 and C-23´.Provided that the reduction of the C-23´carboxyl ester moiety attached to C-23 does not produce significant conformational changes in the side chain (vide supra), the slight upfield and downfield shifts observed in other C nuclei of the side chain should be attributed to field changes associated to the conversion of the magnetically anisotropic carboxymethyl moiety into a hydroxymethylene group.

Figure 6 .
Figure 6.View of the unit cell and the main contacts of 11.

Figure 8 .
Figure 8.View of the main contacts of 12.

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Main hetero-and homonuclear correlations of the side chain of compound 10 Figure 3. Selected H-H and NOE correlations in the side chain of compound 10.

Table 2 .
Main hetero-and homonuclear correlations of the side chain of compound 12

Table 3 .
1H chemical shifts of the compounds 9 to 12

Table 5 .
Calculated puckering parameters for compound 11

Table 6 .
Calculated puckering parameters for compound 12

Table 7 .
Least squares overlay analysis of the rings

Table 8 .
Puckering descriptors obtained using the PLATON program