17α-Acetoxy-11β-hydroxy-6α-methylpregn-4-ene-3,20-dione

The title compound, C24H34O5, a fungal-transformed metabolite of the injectable contraceptive medroxyprogesterone acetate, consists of four fused rings (A, B, C and D; steroid labelling). Ring A exists in a half-chair conformation while trans-fused rings B and C adopt chair conformations. The five-membered ring D adopts an envelope conformation with the C atom bound to the methyl group at the flap. In the crystal, adjacent molecules are linked by O—H⋯O and C—H⋯O hydrogen bonds, forming infinite chains along the a axis.

The title compound, C 24 H 34 O 5 , a fungal-transformed metabolite of the injectable contraceptive medroxyprogesterone acetate, consists of four fused rings (A, B, C and D; steroid labelling). Ring A exists in a half-chair conformation while trans-fused rings B and C adopt chair conformations. The fivemembered ring D adopts an envelope conformation with the C atom bound to the methyl group at the flap. In the crystal, adjacent molecules are linked by O-HÁ Á ÁO and C-HÁ Á ÁO hydrogen bonds, forming infinite chains along the a axis.

Comment
Biotransformation has been extensively applied in the production of several therapeutically important steroids on commercial scale. Such studies are done by utilizing the capability of microorganisms to convert a wide range of organic compounds into their modified derivatives and are very much useful in the production of hydoxylated metabolites (Manosroi et al., 2006;Choudhary et al., 2005). In the current biotransformational study of commonly used injectable contraceptive medroxyprogesterone acetate (17α-acetoxy-6α-methylpregn-4-ene-3,20-dione; MPA), was carried out by using Cunninghamella blakesleeana to obtain the title compound.

Fementation of medroxyprogesterone acetate:
The fungal media were transferred into 60 conical flasks (100 ml each) and autoclaved at 394 K. Seed flasks were prepared from three-day old slants of Cunninghamella blakesleeana (ATCC 9244) and fermentation was allowed for 4 days on a rotary shaker at 299 K. The remaining flasks were inoculated from the seed flasks. After sufficient growth of culture, medroxyprogesterone acetate (0.9 g) was dissolved in acetone (60 ml) and transferred into each flask (15 mg ml -1 ) and kept for 10 days. The culture media were filtered and extracted with dichloromethane. The extract was dried over anhydrous Na 2 SO 4 and evaporated under reduced pressure to get brown gummy material (1.2 g) which was subjected to fractionation on silica gel column with petroleum ethe r-ethyl acetate with increasing polarity. The fraction obtained using 45% ethyl acetate in petroleum ether was finally purified by using Reversed Phase -High Performance compound which was recrystalized from methanol.

Refinement
H atoms on methyl, methylene, methine and oxygen were positioned geometrically with C-H = 0.96 Å, 0.97 Å, 0.93 Å and O-H = 0.82 Å, respectively, and constrained to ride on their parent atoms with U iso (H) = 1.2U eq (CH 2 , CH and OH) and 1.5U eq (CH 3 ). A rotating group model was applied to the methyl groups. An absolute structure could not be established due to lack of anomalous dispersion effects. Therefore, 1684 Friedel pairs were merged.

Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. 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 > σ(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.