Ethyl 3 b-hydroxypregna-5 , 17 ( 20 )-dien-21-oate

Recent research suggests that steroids such as ergosterol and fusidic acid display antitubercular activity (Ruggutt & Ruggutt, 2001). In fact, the minimum inhibitory concentration (MIC) for these steroids is comparable to a number of clinically used anti-TB drugs. This, as well as the structural similarity of the title compound, (II), with fusidic acid and ergosterol has prompted us to consider conjugation of this compound with a number of well known anti-TB agents (Ballell et al., 2005). Our aim is to increase the lipophilicity of the parent drug by attaching a steroid moiety which could have anti-TB activity in its own right. As part of this project, compound (II) was prepared from the commercially available ketone, dehydroandrosterone (I) (Verma et al., 2004), by the Wittig–Horner reaction (Wicha et al., 1977), and was recrystallized from methanol.

In the structure of the title steroid, C 23 H 34 O 3 , the molecules are linked in infinite chains through intermolecular C 1 1 (n) O-HÁ Á ÁO hydrogen bonds between the hydroxy proton and the ester carbonyl O atom.

Comment
Recent research suggests that steroids such as ergosterol and fusidic acid display antitubercular activity (Ruggutt & Ruggutt, 2001). In fact, the minimum inhibitory concentration (MIC) for these steroids is comparable to a number of clinically used anti-TB drugs. This, as well as the structural similarity of the title compound, (II), with fusidic acid and ergosterol has prompted us to consider conjugation of this compound with a number of well known anti-TB agents (Ballell et al., 2005). Our aim is to increase the lipophilicity of the parent drug by attaching a steroid moiety which could have anti-TB activity in its own right. As part of this project, compound (II) was prepared from the commercially available ketone, dehydroandrosterone (I) (Verma et al., 2004), by the Wittig-Horner reaction (Wicha et al., 1977), and was recrystallized from methanol.
Compound (II) crystallizes in the space group P1 with one molecule in the unit cell. The fused tetracyclic ring system adopts the expected conformations for the all-trans A/B/C/D junctions. The six-membered rings A and C adopt normal chair conformations. As previously observed in related structures (Thamotharan et al., 2004;Verma et al., 2004), the hydroxy group on C3 does not perturb the structure of ring A. The C5 C6 bond length of 1.326 (5) Å confirms the presence of the double bond in this position and imposes an 8,9-halfchair conformation on ring B. Ring D adopts the 14-envelope conformation previously observed in the dehydroandrosterone parent (Verma et al., 2004); this conformation minimizes steric interactions with the angular C18 methyl group (Fuchs, 1978).
The C17 C20 bond length of 1.331 (5) Å confirms the presence of the double bond in this position. The substituents on this bond adopt the thermodynamically favoured E configuration which, again, minimizes interactions with the angular methyl group. The C20-C21 bond length of 1.460 (5) Å suggests partial double-bond character, with the ester group adopting an s-trans configuration. The molecules in the crystal structure are linked through intermolecular C 1 1 (n) (Bernstein et al., 1995) O-HÁ Á ÁO hydrogen bonds between the O3 hydroxy proton and the O21 carbonyl O atom (Table 2), forming infinite chains along the body diagonal of the unit cell.
The 1 H and 13 C NMR spectra of the compound were fully assigned using two-dimensional gCOSY, gHSQC and gHMBC methods. The lowfield 13 C chemical shifts of the C17 (176.15 p.p.m.) and C21 (167.45 p.p.m.) C atoms were confirmed by the presence of unambiguous cross peaks in the gHMBC spectrum linking (i) the 176.15 p.p.m. resonance with the H18 and H16 resonances and (ii) the 167.45 p.p.m. resonance with the H20 and H22 resonances. The 13 C chemical shift of C20 (108.62 p.p.m.) was confirmed by direct correlation of the C20 and H20 resonances in the gHSQC matrix. The 13 C chemical shifts of the C17, C21 and C20 atoms can be compared with those previously observed for 13 C nuclei in the isolated d-ring analogue methyl 2-methylenecyclopentane acetate (Molander & Harris, 1997), i.e. 169.51, 167.31 and 111.18 p.p.m., respectively. Experimental A solution of sodium ethoxide (1.243 M, 5 ml) was added slowly to a stirred solution of dehydroisoandrosterone (600 mg, 2 mmol) and triethyl phosphonoacetate (1.6 ml, 6 mmol) in ethanol (5 ml) at room temperature, under an N 2 atmosphere. The reaction mixture was refluxed for 20 h, then cooled to room temperature and concentrated in vacuo. The residue was diluted with water and the resulting suspension acidified (acetic acid) and extracted with a mixture of ethyl acetate and tetrahydrofuran (3:1, 60 ml). The organic layer was washed with water and brine, dried (Na 2 SO 4 ) and the solvent removed by evaporation at reduced pressure. Crystallization of the residue from methanol gave the product ethyl 3-hydroxypregna-5,17(20)-dien-21-oate (525 mg, 70%) as fine colourless crystals, suitable for X-ray crystallographic analysis [m.p. 457-459 K; literature 457-458 K (Wicha et al., 1977) Table 1 Selected geometric parameters (Å , ).
Representative view of (II), with the atom-numbering scheme. Displacement ellipsoids for non-H atoms are drawn at the 30% probability level.
C-bound H atoms were constrained as riding atoms, with C-H = 0.94-0.96 Å , and with U iso (H) = 1.2U eq (parent atom). The hydroxy H atom was located in a difference Fourier synthesis and constrained as a riding atom, with O-H = 0.91 Å . In the absence of significant anomalous scattering effects, Friedel pairs were merged. The absolute configuration was assigned on the basis of the known configuration of the starting material.

S1. Comment
Recent research suggests that steroids such as ergosterol and fusidic acid display antitubercular activity (Ruggutt & Ruggutt, 2001). In fact, the minimum inhibitory concentration (MIC) for these steroids is comparable to a number of clinically used anti-TB drugs. This, as well as the structural similarity of the title compound, (II), with fusidic acid and ergosterol has prompted us to consider conjugation of this compound with a number of well known anti-TB agents (Ballell et al., 2005). Our aim is to increase the lipophilicity of the parent drug by attaching a steroid moiety which could have anti-TB activity in its own right. As part of this project, compound (II) was prepared from the commercially available ketone, dehydroandrosterone (I) (Verma et al., 2004), by the Wittig-Horner reaction (Wicha et al., 1977), and was recrystallized from methanol.
Compound (II) crystallizes in the space group P1 with one molecule in the unit cell. The fused tetracyclic ring system adopts the expected conformations for the all-transA/B/C/D junctions. The six-membered rings A and C adopt normal chair conformations. As previously observed in related structures (Thamotharan et al., 2004;Verma et al., 2004), the hydroxy group on C3 does not perturb the structure of ring A. The C5═C6 bond length of 1.326 (5) Å confirms the presence of the double bond in this position and imposes an 8β,9α-half-chair conformation on ring B. Ring D adopts the 14αenvelope conformation previously observed in the dehydroandrosterone parent (Verma et al., 2004); this conformation minimizes steric interactions with the angular C18 methyl group (Fuchs, 1978).
The C17═C20 bond length of 1.331 (5) Å confirms the presence of the double bond in this position. The substitutents on this bond adopt the thermodynamically favoured E configuration which, again, minimizes interactions with the angular methyl group. The C20-C21 bond length of 1.460 (5) Å suggests partial double-bond character, with the ester group adopting an s-trans configuration. The molecules in the crystal structure are linked through intermolecular C 1 1 (n) (Bernstein et al., 1995) O-H···O hydrogen bonds between the O3 hydroxy proton and the O21 carbonyl O atom (Table   2), forming infinite chains along the body diagonal of the unit cell.
The 1 H and 13 C NMR spectra of the compound were fully assigned using two-dimensional gCOSY, gHSQC and gHMBC methods. The lowfield 13 C chemical shifts of the C17 (176.15 p.p.m.) and C21 (167.45 p.p.m.) C atoms were confirmed by the presence of unambiguous cross peaks in the gHMBC spectrum linking (i) the 176.15 p.p.m. resonance with the H18 and H16 resonances and (ii) the 167.45 p.p.m. resonance with the H20 and H22 resonances. The 13 C chemical shift of C20 (108.62 p.p.m.) was confirmed by direct correlation of the C20 and H20 resonances in the gHSQC matrix. The 13 C chemical shifts of the C17, C21 and C20 atoms can be compared with those previously observed for 13 C nuclei in the isolated D-ring analogue methyl 2-methylenecyclopentane acetate (Molander & Harris, 1997)

S2. Experimental
A solution of sodium ethoxide (1.243 M, 5 ml) was added slowly to a stirred solution of dehydroisoandrosterone (600 mg, 2 mmol) and triethyl phosphonoacetate (1.6 ml, 6 mmol) in ethanol (5 ml) at room temperature, under an N 2 atmosphere. The reaction mixture was refluxed for 20 h, then cooled to room temperature and concentrated in vacuo. The residue was diluted with water and the resulting suspension acidified (acetic acid) and extracted with a mixture of ethyl acetate and tetrahydrofuran (3:1, 60 ml). The organic layer was washed with water and brine, dried (Na 2 SO 4 ) and the solvent removed by evaporation at reduced pressure. Crystallization of the residue from methanol gave the product ethyl
The hydroxy H atom was located in a difference Fourier synthesis and constrained as a riding atom, with O-H = 0.91 Å.
In the absence of significant anomalous scattering effects, Friedel pairs were merged. The absolute configuration is assigned on the basis of the known configuration of the starting material.

Figure 1
Representative view of (II), with the atom-numbering scheme. Displacement ellipsoids for non-H atoms are drawn at the 30% probability level.

Special details
Experimental. The scan width was (1.73 + 0.30tanθ)° with an ω scan speed of 16° per minute (up to 4 scans to achieve I/σ(I) > 10). Stationary background counts were recorded at each end of the scan, and the scan time:background time ratio was 2:1. Geometry. Bond distances, angles etc. have been calculated using the rounded fractional coordinates. All su's are estimated from the variances of the (full) variance-covariance matrix. The cell e.s.d.'s are taken into account in the estimation of distances, angles and torsion angles Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > 2σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.