Synthesis and characterization of new aromatic esters based on 4,16-pregnadiene-6,20-dione skeleton

A series of new aromatic esters based on 4,16-pregnadiene-6,20-dione skeleton, namely 3β-benzoyloxy-4,16-pregnadiene-6,20-dione and 3β-furoyloxy-4,16-pregnadiene- 6,20-dione, which may be good inhibitors for the 5α-reductase enzyme and show high antiandrogenic activity, were synthesized starting from diosgenin. The structures of the steroids were characterized by elemental analysis, 1H NMR, 13C NMR, IR and mass spectrum. Single crystal X-ray diffraction measurement on one of the new compounds, 3β-(p-methoxybenzoyloxy)-4,16-pregnadiene-6,20-dione revealed that the A, B, C, and D ring adopted half chair, distorted chair, distorted chair, and distorted envelope conformation, respectively. The molecules in the crystal were packed face-to-face at the normal van der Waals distances.


Experimental
All chemical reagents were purchased from commercial sources and used as received unless stated otherwise. Melting points were determined on a XT-4 melting apparatus and the quoted temperatures were uncorrected. Elemental analysis was carried out on an Elmentar Vario EL III system. 1 H NMR and 13 C NMR spectra were recorded on a Bruker AM 400 spectrometer. CDCl 3 was used as solvent and chemical shifts recorded were internally referenced to Me 4 Si (0 ppm). IR spectra were obtained on a Thermo Electron Corporation Nicolet 380 FT-IR spectrophotometer. Mass spectra were recorded on a Shimadzu QP-2010 instrument using electron impact ionization (EI) at 70 eV. LC-MS spectra were obtained on an Agilent 6000 LC-MS instrument equipped with a SunFire C18 column (4.6 × 50 mm, 3.5 μm) under the following conditions: mobile phase: H 2 O (0.05% trifluoroacetic acid (TFA)) (A)/acetonitrile (0.05% TFA) (B); elution program: gradient from 5 to 95% of B in 1.6 min at 2.2 ml/min; temperature: 50°; detection: UV (214 nm) and MS (ESI, pos mode, 70 to 1000 amu). All of the measured samples were dissolved in methanol. X-Ray crystal structure was measured on a Bruker Smart CCD diffractometer by using Mo K α radiation at 293 K.

Results and Discussion
The synthetic route of the target steroids 3β-benzoyloxypregna-4,16-diene-6,20-dione (10a-i) and 3β-furoyloxypregna-4,16-diene-6,20-dione (10j) was showed in Figure 2. At first the starting material diosgenin was transformed by acetylation and oxidation to 3β-acetoxy-5,16-pregnadiene-20-one (16-DPA, 5). The transformation could be achieved stepwise or in a one-pot reaction [32]. We adopted the former in consideration that the purification of the product was easier to yield 16-DPA of higher purity. Epoxidation of 16-DPA to form steroid 6 was conducted by using 30% H 2 O 2 /HCO 2 H [33] or m-chloroperoxybenzoic acid (m-CPBA) [13] oxidation system in 90% or 95% yield. Conversion of compound 6 to 7 was carried out in a similar way as reported in literature [13] and the yield of steroid 7 was improved from 81% to 84%. Dehydration of 7 with thionyl chloride in pyridine resulted compound 8. We found that the quality of the solvent pyridine had an effect on the yield of the steroid 8. When dried pyridine (refluxed in the presence of potassium hydroxide for 2 h before distillation) was used instead of pyridine of analytically pure grade without treatment, the yield of 8 was improved to 73% from the literature [13] value 66%. Compound 8 was hydrolyzed with aqueous sodium hydroxide in methanol to afford 3β-hydroxypregna-4,16-diene-6,20-dione (9). The procedure for workup was improved by using extraction (with ethyl acetate) and concentration replacing filtration and subsequent recrystallization and pure product 9 was obtained in 74% yield, significantly higher than the literature [13] value 60%. The target steroids 3β-benzoyloxypregna-4,16-diene-6,20-dione (10a-i) and 3β-furoyloxypregna-4,16-diene-6,20-dione (10j) were synthesized through Steglich reaction from 9, substituted benzoic acid or furoic acid, and DCC catalyzed by DMAP in fair to good yields (45%-77%), depending upon the structure of the aryl acids. It seems that the steric hindrance from the aryl acid structure leads to decrease in the yield. Therefore the highest yield of the Steglich reaction came from furoic acid (77%) while the reactions with dinitrobenzoic acid or nitrobenzoic acid gave the lowest yield.
The chemical structures of steroids 10a-j were fully characterized by elemental analysis, 1 H NMR, 13 C NMR, IR and mass spectrum and melting point measurements. Characteristic chemical shifts in 1 H NMR spectra of steroids 10a-j were summarized in Table 1. All the chemical shifts of protons in methyl groups, OCH moieties, C = CH moieties, and aryl rings were in the reasonable range and the spectra data were in good accordance with the structures.
Single crystals of 10a suitable for X-ray crystal diffraction measurement were obtained by slow evaporation of a solution of 10a in 1:1 petroleum ether and ethyl acetate. X-Ray data for crystals of 10a were collected by graphite-monochromatized Mo K α radiation at 293 K and the crystal data and experimental details for compound 10a were summarized in Table 2. The structure was solved by direct methods and refined by full-matrix least-squares with anisotropic temperature factors for the non-hydrogen atoms. The hydrogen atoms bonded to the carbon atoms were assigned based on the expected bonding geometry. The hydrogen atoms were refined isotropically in the final least-squares cycles.
The crystal structure and molecular packing of steroid 10a were shown in Figure 3 and Figure 4 respectively. It can be seen that the molecule consists of three sixmembered rings (A, B, and C ring) and one five-membered ring (D ring), all trans fused. The six-membered rings A, B, and C occur in a half chair, distorted chair, and distorted chair conformation, respectively. Ring D adopts a distorted envelope conformation. The molecules in the crystal were packed face-to-face at the  normal van der Waals distances, similar to the crystal structure of 3β-(p-fluorobenzoyloxy)-4,16-pregnadiene-6,20-dione [34]. CCDC-787721 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac. uk/data_request/cif.

Conclusions
In summary, ten new aromatic esters based on 4,16-pregnadiene-6,20-dione skeleton, namely 3β-benzoyloxy-4,16pregnadiene-6,20-dione and 3β-furoyloxy-4,16-pregnadiene-6,20-dione were synthesized starting from diosgenin. The structures of the steroids were characterized by elemental analysis, 1 H NMR, 13 C NMR, IR and mass spectrum. Single crystal X-ray diffraction measurement on one of the new compounds, 3β-(p-methoxybenzoyloxy)-4,16-pregnadiene-6,20-dione revealed that the A, B, C, and D ring adopted half chair, distorted chair, distorted chair, and distorted envelope conformation, respectively. The molecules in the crystal were packed face-to-face at the normal van der Waals distances. These new steroids may show high antiandrogenic activity and serve as good inhibitors for the 5α-reductase enzyme. Investigation of the inhibitory activity of the new steroids for the 5α-reductase enzyme will be conducted in the near future.