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. 1H NMR and 13C NMR spectra were recorded on a Bruker AM 400 spectrometer. CDCl3 was used as solvent and chemical shifts recorded were internally referenced to Me4Si (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: H2O (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 Kα radiation at 293 K.
Pseudodiosgenin diacetate (4) [32]
To a solution of diosgenin 3 (16.0 g, 38.6 mmol) and ammonium chloride (2.5 g, 46.7 mmol) in acetic anhydride (82 ml) was added pyridine (1.5 ml) and the mixture was heated at 140°C for 8-9 h. The resulting solution was cooled to room temperature and poured into ice water. The precipitate was filtered and recrystallized from methanol to give the product pseudodiosgenin diacetate (4) as yellow solid (16.6 g, 86%). M.p. 70-72°C.
3β-Acetoxy-5,16-pregnadiene-20-one (5)
A solution of pseudodiosgenin diacetate (4) (15.4 g, 30.8 mmol) in acetic anhydride (76 ml) was diluted with water (4.8 ml) and acetic acid (50 ml). The mixture was cooled to 0°C then a solution of chromium(VI) oxide (8.9 g, 89.0 mmol) in acetic acid (25 ml) was added dropwise in 1 h. After the addition the solution was allowed to warm to 10-16°C and the stirring was continued for 5 h at this temperature. Sodium bisulphite (9.3 g, 89.4 mmol) in water (30 ml) was added then the mixture was refluxed for 3 h, cooled and poured into water to give sticky solids. The crude product was crystallized from methanol then purified by means of column chromatography on silica gel with petroleum ether (b.p. 60-90°C) and ethyl acetate (4:1, v/v) as eluent to afford product 5 (4.5 g, 41%) as yellow needles. M.p. 170-172°C (literature [32] value: 171-172°C). Rf 0.58 (petroleum ether/ethyl acetate, 4:1, v/v).
Compound 6 was synthesized as described in literature [33] in 90% yield and as described in literature [13] in 95% yield. M.p. 170-172°C (literature [13] value: 170-172°C). Rf 0.57 (petroleum ether/ethyl acetate, 4:1, v/v).
Compound 7 was synthesized as described in literature [13] in 84% yield. M.p. 243-245°C (literature [13] value: 244-245°C). Rf 0.56 (petroleum ether/ethyl acetate, 4:1, v/v).
Compound 8 was synthesized as described in literature [13] in 73% yield. M.p. 192-194°C (literature [13] value: 193-195°C). Rf 0.58 (petroleum ether/ethyl acetate, 4:1, v/v).
3β-Hydroxypregna-4,16-diene-6,20-dione (9)
A solution of steroid 8 (0.3 g, 0.8 mmol) in methanol (45 ml) and 2% aqueous sodium hydroxide (3 ml) was stirred for 30 min at room temperature. The resulting solution was poured into ice water (45 ml) then extracted with ethyl acetate (3 × 30 ml). The organic phase was dried over anhydrous magnesium sulfate and the solvent was removed in vacuum. The residue was purified by column chromatography on silica gel with petroleum ether and ethyl acetate (1:1, v/v) as eluent to afford product 9 (0.25 g, 74%) as pale yellow needles. M.p. 168-170°C (literature [13] value: 168-170°C). Rf 0.52 (petroleum ether/ethyl acetate, 4:1, v/v).
3β-(p-Methoxybenzoyloxy)pregna-4,16-diene-6,20-dione (10a)
A solution containing steroid 9 (0.10 g, 0.3 mmol), p-methoxybenzoic acid (0.13 g, 0.7 mmol), 1,3-dicyclohexylcarbodiimide (DCC, 0.10 g, 0.5 mmol) and 4-dimethylaminopyridine (DMAP, 0.06 g, 0.5 mmol) in methylene dichloride (6 ml) was stirred for 2 h at about 10°C. Ice water was added and the reaction mixture was extracted with chloroform (3 × 20 ml). The organic phase was dried over anhydrous magnesium sulfate and the solvent was removed in vacuum. The crude product was dissolved in ethyl acetate and filtered through a column containing silica gel. The organic solvent was removed in vacuum to give a white crystalline solid 10a (0.14 g, 71%). M.p. 244-246°C. Rf 0.50 (petroleum ether/ethyl acetate, 4:1, v/v). 1H NMR (CDCl3): 0.94 (s, 3 H, H-C(18)); 1.10 (s, 3 H, H-C(19)); 2.28 (s, 3 H, H-C(21)); 3.87 (s, 3 H, OMe); 5.56 (m, 1 H, H-C(3)); 6.23 (m, 1 H, H-C(4)); 6.71 (t, 1 H, J = 1.3 Hz, H-C(16)); 6.93 (m, 2 H, H-Ph); 8.02 (m, 2 H, H-Ph). 13C NMR (CDCl3): 15.8 (C(18)); 19.7 (C(19)); 27.1 (C(21)); 69.5 (C(3)); 131.8 (C(4)); 143.8 (C(16)); 147.9 (C(5)); 154.9 (C(17)); 165.9 (ester C = O); 196.6 (C(20)); 201.9 (C(6)). IR (KBr): 3075, 2958, 1706, 1690, 1656, 1629. EI-MS: 312 (12, M+ + 1 - p-MeO-C6H4COO), 175 (31), 157 (38), 121 (30), 105 (41), 93 (33), 91 (66), 79 (51), 77 (46), 43 (100). LC-MS: 485.4 (M+ + Na, retention time 2.20 min). Anal. Calcd. for C29H34O5: C, 75.30; H, 7.41; Found: C, 75.12; H, 7.37.
Other steroidal compounds 10b-j were prepared in a similar procedure and the physical data of the new steroids were as follows.
3β-(o-Methoxybenzoyloxy)pregna-4,16-diene-6,20-dione (10b)
White powder. Yield 45%. M.p. 240-242°C. Rf 0.42 (petroleum ether/ethyl acetate, 4:1, v/v). 1H NMR (CDCl3): 0.93 (s, 3 H, H-C(18)); 1.07 (s, 3 H, H-C(19)); 2.27 (s, 3 H, H-C(21)); 3.89 (s, 3 H, OMe); 5.56 (m, 1 H, H-C(3)); 6.23 (m, 1 H, H-C(4)); 6.73 (t, J = 1.6 Hz, H-C(16)); 6.96 (m, 2 H, H-Ph); 7.46 (m, 1 H, H-Ph); 7.80 (m, 1 H, H-Ph). 13C NMR (CDCl3): 17.5 (C(18)); 22.0 (C(19)); 26.5 (C(21)); 71.8 (C(3)); 136.0 (C(4) and C(16)); 146.1 (C(5)); 149.9 (C(17)); 167.0 (ester C = O); 198.9 (C(20)); 203.8 (C(6)). IR (KBr): 3040, 2946, 1688. LC-MS: 485.4 (M+ + Na, retention time 2.13 min). Anal. Calcd. for C29H34O5: C, 75.30; H, 7.41; Found: C, 75.04; H, 7.46.
3β-(o-Methylbenzoyloxy)pregna-4,16-diene-6,20-dione (10c)
White powder. Yield 62%. M.p. 182-184°C. Rf 0.39 (petroleum ether/ethyl acetate, 3:1, v/v). 1H NMR (CDCl3): 0.94 (s, 3 H, H-C(18)); 1.08 (s, 3 H, H-C(19)); 2.28 (s, 3 H, H-C(21)); 2.60 (s, 3 H, Me-Ph); 5.56 (m, 1 H, H-C(3)); 6.24 (m, 1 H, H-C(4)); 6.72 (t, 1 H, J = 1.8 Hz, H-C(16)); 7.23 (m, 2 H, H-Ph); 7.40 (m, 1 H, H-Ph); 7.93 (m, 1 H, H-Ph). 13C NMR (CDCl3): 15.8 (C(18)); 19.7 (C(19)); 27.1 (C(21)); 69.6 (C(3)); 132.1 (C(4)); 143.9 (C(16)); 148.0 (C(5)); 154.9 (C(17)); 168.4 (ester C = O); 196.7 (C(20)); 201.8 (C(6)). IR (KBr): 3070, 2946, 1688. LC-MS: 469.4 (M+ + Na, retention time 2.28 min). Anal. Calcd. for C29H34O4: C, 78.00; H, 7.67; Found: C, 77.81; H, 7.63.
3β-(m-Methylbenzoyloxy)pregna-4,16-diene-6,20-dione (10d)
White powder. Yield 69%. M.p. 138-140°C. Rf 0.42 (petroleum ether/ethyl acetate, 3:1, v/v). 1H NMR (CDCl3): 0.95 (s, 3 H, H-C(18)); 1.11 (s, 3 H, H-C(19)); 2.27 (s, 3 H, H-C(21)); 5.60 (m, 1 H, H-C(3)); 6.24 (m, 1 H, H-C(4)); 6.71 (t, 1 H, J = 1.8 Hz, H-C(16)); 7.32 (m, 2 H, H-Ph); 7.44 (m, 1 H, H-Ph); 7.84 (m, 1 H, H-Ph). 13C NMR (CDCl3): 15.8 (C(18)); 19.7 (C(19)); 27.1 (C(21)); 69.8 (C(3)); 138.0 (C(4)); 143.9 (C(16)); 148.0 (C(5)); 154.9 (C(17)); 166.4 (ester C = O); 196.6 (C(20)); 201.9 (C(6)). IR (KBr): 3060, 2945, 1688, 1589, 1457. LC-MS: 469.4 (M+ + Na, retention time 2.29 min). Anal. Calcd. for C29H34O4: C, 78.00; H, 7.67; Found: C, 78.18; H, 7.71.
3β-(p-Nitrobenzoyloxy)pregna-4,16-diene-6,20-dione (10e)
Pale yellow powder. Yield 46%. M.p. 174-176°C. Rf 0.36 (petroleum ether/ethyl acetate, 3:1, v/v). 1H NMR (CDCl3): 0.95 (s, 3 H, H-C(18)); 1.12 (s, 3 H, H-C(19)); 2.28 (s, 3 H, H-C(21)); 5.62 (m, 1 H, H-C(3)); 6.21 (m, 1 H, H-C(4)); 6.72 (t, 1 H, J = 1.8 Hz, H-C(16)); 8.23 (m, 2 H, H-Ph); 8.28 (m, 2 H, H-Ph). 13C NMR (CDCl3): 15.8 (C(18)); 19.6 (C(19)); 27.1 (C(21)); 71.0 (C(3)); 130.8 (C(4)); 135.4 (C(16)); 148.6 (C(5)); 154.9 (C(17)); 164.3 (ester C = O); 196.6 (C(20)); 201.8 (C(6)). IR (KBr): 3062, 2946, 1688, 1528, 1456. LC-MS: 500.3 (M+ + Na, retention time 2.17 min). Anal. Calcd. for C28H31NO6: C, 70.42; H, 6.54; N, 2.93; Found: C, 70.57; H, 6.52; N, 2.87.
3β-(o-Nitrobenzoyloxy)pregna-4,16-diene-6,20-dione (10f)
Pale yellow powder. Yield 55%. M.p. 158-161°C. Rf 0.29 (petroleum ether/ethyl acetate, 3:1, v/v). 1H NMR (CDCl3): 0.92 (s, 3 H, H-C(18)); 1.05 (s, 3 H, H-C(19)); 2.27 (s, 3 H, H-C(21)); 5.60 (m, 1 H, H-C(3)); 6.17 (m, 1 H, H-C(4)); 6.70 (t, 1 H, J = 1.8 Hz, H-C(16)); 7.63-7.68 (m, 2 H, H-Ph); 7.72 (m, 1 H, H-Ph); 7.95 (m, 1 H, H-Ph). 13C NMR (CDCl3): 15.8 (C(18)); 19.6 (C(19)); 27.1 (C(21)); 71.5 (C(3)); 133.0 (C(4)); 143.8 (C(16)); 148.6 (C(5)); 154.9 (C(17)); 165.1 (ester C = O); 196.6 (C(20)); 201.6 (C(6)). IR (KBr): 3033, 2945, 1732, 1687, 1656, 1628, 1587, 1540, 1456. LC-MS: 500.3 (M+ + Na, retention time 2.11 min). Anal. Calcd. for C28H31NO6: C, 70.42; H, 6.54; N, 2.93; Found: C, 70.25; H, 6.49; N, 2.90.
3β-(m-Nitrobenzoyloxy)pregna-4,16-diene-6,20-dione (10g)
Pale yellow powder. Yield 47%. M.p. 176-178°C. Rf 0.38 (petroleum ether/ethyl acetate, 3:1, v/v). 1H NMR (CDCl3): 0.94 (s, 3 H, H-C(18)); 1.12 (s, 3 H, H-C(19)); 2.27 (s, 3 H, H-C(21)); 5.63 (m, 1 H, H-C(3)); 6.20 (m, 1 H, H-C(4)); 6.71 (t, J = 1.2 Hz, H-C(16)); 7.66 (m, 1 H, H-Ph); 8.36-8.43 (m, 2 H, H-Ph); 8.86 (m, 1 H, H-Ph). 13C NMR (CDCl3): 15.8 (C(18)); 19.6 (C(19)); 27.1 (C(21)); 71.0 (C(3)); 135.4 (C(4)); 143.8 (C(16)); 148.6 (C(5)); 154.9 (C(17)); 164.1 (ester C = O); 196.6 (C(20)); 201.7 (C(6)). IR (KBr): 3040, 2945, 1689, 1526, 1456. LC-MS: 500.3 (M+ + Na, retention time 2.17 min). Anal. Calcd. for C28H31NO6: C, 70.42; H, 6.54; N, 2.93; Found: C, 70.27; H, 6.58; N, 2.88.
3β-(3,5-Dinitrobenzoyloxy)pregna-4,16-diene-6,20-dione (10h)
Pale yellow powder. Yield 45%. M.p. 168-170°C. Rf 0.59 (petroleum ether/ethyl acetate, 2:1, v/v). 1H NMR (CDCl3): 0.95 (s, 3 H, H-C(18)); 1.15 (s, 3 H, H-C(19)); 2.29 (s, 3 H, H-C(21)); 5.70 (m, 1 H, H-C(3)); 6.20 (m, 1 H, H-C(4)); 6.72 (t, 1 H, J = 1.4 Hz, H-C(16)); 9.16 (m, 2 H, H-Ph); 9.24 (m, 1 H, H-Ph). 13C NMR (CDCl3): 15.8 (C(18)); 19.5 (C(19)); 27.1 (C(21)); 72.2 (C(3)); 133.8 (C(4)); 143.8 (C(16)); 148.6 (C(5)); 154.8 (C(17)); 162.2 (ester C = O); 196.6 (C(20)); 201.6 (C(6)). IR (KBr): 3037, 2946, 1689, 1543, 1457. LC-MS: 500.3 (M+ + Na, retention time 3.14 min). Anal. Calcd. for C28H30N2O8: C, 64.36; H, 5.79; N, 5.36; Found: C, 64.22; H, 5.75; N, 5.19.
3β-(o-Chlorobenzoyloxy)pregna-4,16-diene-6,20-dione (10i)
White powder. Yield 64%. M.p. 192-194°C. Rf 0.32 (petroleum ether/ethyl acetate, 3:1, v/v). 1H NMR (CDCl3): 0.95 (s, 3 H, H-C(18)); 1.11 (s, 3 H, H-C(19)); 2.27 (s, 3 H, H-C(21)); 5.59 (m, 1 H, H-C(3)); 6.24 (m, 1 H, H-C(4)); 6.71 (t, 1 H, J = 1.8 Hz, H-C(16)); 7.32 (m, 1 H, H-Ph); 7.36 (m, 2 H, H-Ph); 7.87 (m, 1 H, H-Ph). 13C NMR (CDCl3): 15.8 (C(18)); 19.6 (C(19)); 27.1 (C(21)); 70.5 (C(3)); 133.9 (C(4)); 143.8 (C(16)); 148.2 (C(5)); 154.9 (C(17)); 165.2 (ester C = O); 196.6 (C(20)); 201.7 (C(6)). IR (KBr): 3040, 2946, 1688, 1522, 1456. LC-MS: 490.2 (M+ + Na, retention time 2.23 min). Anal. Calcd. for C28H31ClO4: C, 72.01; H, 6.69; Found: C, 71.83; H, 6.65.
3β-(2-Furoyloxy)pregna-4,16-diene-6,20-dione (10j)
White powder. Yield 77%. M.p. 180-182°C. Rf 0.40 (petroleum ether/ethyl acetate, 2:1, v/v). 1H NMR (CDCl3): 0.94 (s, 3 H, H-C(18)); 1.09 (s, 3 H, H-C(19)); 2.28 (s, 3 H, H-C(21)); 5.57 (m, 1 H, H-C(3)); 6.20 (m, 1 H, H-C(4)); 6.52 (t, 1 H, J = 1.8 Hz, H-C(16)); 6.71 (m, 1 H, H-Ar); 7.20 (m, 1 H, H-Ar); 7.59 (m, 1 H, H-Ar). 13C NMR (CDCl3): 15.8 (C(18)); 19.6 (C(19)); 27.1 (C(21)); 69.9 (C(3)); 128.7 (C(4)); 143.8 (C(16)); 148.1 (C(5)); 154.8 (C(17)); 158.3 (ester C = O); 196.6 (C(20)); 201.8 (C(6)). IR (KBr): 3042, 2946, 1720, 1689, 1657, 1580, 1523, 1457. LC-MS: 445.2 (M+ + Na, retention time 2.06 min). Anal. Calcd. for C26H30O5: C, 73.91; H, 7.16; Found: C, 73.73; H, 7.21.