3.2. General procedure for the synthesis of N-[3β-acetoxyurs-11-oxo-12-en -28-acyl]-amine compounds 5,6
A solution of UA (300 mg, 0.658 mmol) in THF (10 mL), pyridine (1 mL), acetic anhydride (1 mL) and a small amount of DMAP was stirred for 4 h at room temperature. When the reaction was complete, the solvent was cncentrated in vacuo and the solids were dispersed in water, then acidified to pH 3–4 with HCl, filtered, washed with water to neutrality, and dried at room temperature to give white solid compound 2. A mixture of compound 2 (50 mg, 0.1 mmol) with acetic anhydride (0.75 mL), acetic acid (14.25 mL) and CrO3 (99.6 mg, 0.1 mmol) was stirred for 4–6 h at room temperature. Water (20 mL) and CH2Cl2 (20 mL) were added. When stratified, the water layer was extracted twice with CH2Cl2, combined organic phase, washed with saturated NaHCO3, then washed with water to neutrality. Dried over Na2SO4, filtered, and concentrated in vacuo. Compound 3 was obtained as a light green oil, which was dissolved in CH2Cl2 (10 mL), oxalyl chloride (0.2 mmol) was added and the mixture stirred 20 h to give compound 4. Reaction solvent and unreacted oxalyl chloride were eliminated by vacuum. Then the residue was dissolved in cyclonexane (5 mL). This was done twice. Acyl chloride was mixed with CH2Cl2 (5 mL) and Et3N to adjust the pH to 9–10. The solution was stirred for 5 min and amine (0.3 mmol) was added in at room temperature. The reaction completion was detected by TLC. The dichloromethane was eliminated by vacuum, brine (5 mL) was added and the mixture was acidified with concentrated HCl to pH 3–4. A white solid was precipitated, filtered, the filter cake was washed to neutrality with water and dried to give a white solid.
3.2.1. N-[3β-Acetoxy-urs-11-oxo-12-en-28-acyl]aniline (5)
Compound 4 was reacted with aniline (0.3 mmol) using the general procedure to give compound 5 (27.5 mg, yield: 43%); m.p. 215–217 °C; IR (KBr): 3389, 2930, 1712, 1658, 1610, 1599, 1534, 1499, 1439, 1373, 1260, 754, 691 cm-1; 1H-NMR (CDCl3): 7.10–7.45 (m, 5H, Ar-H), 5.75 (s, 1H, NH), 5.65 (s, 1H, H-12), 4.50 (m, 1H, H-3), 2.06 (s, 3H, CH3CO), 0.86, 0.90, 0.95, 0.97, 1.03 (s, each 3H), 0.81 (d, 3H, CH3), 0.87 (d, 3H, CH3); MS m/z: 605.0 [M+18]+; Elemental analysis (%, found) C, 77.68 (77.64); H,8.98 (9.09); N, 2.29 (2.38).
3.2.2. N-[3-Acetoxy-urs-11-oxo-12-en-28-acyl]-4-methylpiperazine (6)
Compound 4 was reacted 2-amino-1-propanol (0.3 mmol) using the general procedure to afford compound 6 (28mg, yield: 50.3%); m.p. 203–204 °C; IR (KBr): 3441, 2929, 1732, 1657, 1630, 1457, 1366, 1246 cm-1; 1H-NMR (CDCl3): 5.636 (s, 1H, H-12), 5.845–5.861 (d, 1H, NH), 4.537 (m, 1H, H-3), 3.502–3.615 (m, 2H, CH2OH), 3.982 (m, H, NCH), 2.063 (s, 3H, CH3CO), 1.102 (s, 3H, CH3), 1.089 (s, 3H, CH3), 0.953 (s, 3H, CH3), 0.938 (s, 3H, CH3), 0.900 (d, 3H, CH3) ,0.882 (s, 3H, CH3), 0.798–0.820 (d, 3H, CH3), 0.813 (d, 3H, CH3); MS m/z: 570.5 [M+1]+; Elemental analysis (%, found) C,73.71 (73.77); H, 9.73 (9.61); N, 2.46 (2.52).
3.3. General procedure for the synthesis of N-[3-oxo-urs-12-en-28-oyl]-amine compounds 9,10
To a solution of compound 1 (100 mg, 0.22 mmol) in acetone (1.5 mL) Jones’ reagent (0.4 mL) was added dropwise in an ice-salt bath. The reaction mixture was allowed to warm up to room temperature and stirred for 1 hour. After cooling to 0 ºC, 2-propanol (5 mL) was added and the solution stirred at room temperature for 30 minutes. The green precipitate was collected and washed well with acetone. The acetone solution from the combined filtrates were concentrated and dried. By purification on a silica gel column compound 7 was obtained as a white solid. A mixture of compound 7 (50 mg, 0.11 mmol) and oxalyl chloride (0.04 mL) in CH2Cl2 (2 mL) was stirred at room temperature for 20 h. The mixture was concentrated to dryness under reduced pressure. Cyclohexane (3 × 1 mL) was added to the residue, then concentrated to dryness to yield crude 3-oxoursolyl chloride 8. To a CH2Cl2 (4 mL) solution of 8 was added an amine compound (0.44 mmol). The reaction mixture was stirred in presence of Et3N at room temperature. The resultant residue was partitioned with 3 mL water, then treated with 2N HCl to pH 3, CH2Cl2 was removed under vacuum to precipitate a white solid which was filtered and the filter cake was washed with water to pH 7, and dried. The crude was purified on a silica gel column with petroleum ether/ethyl acetate to yield a white powder.
3.3.1. N-[3-Oxo-urs-12-en-28-oyl]-3-amino-1-propanol (9)
Compound 8 was reacted by using general procedure with 3-amino-1-propanol to give compound 9. The reaction mixture was stirred at room temperature for 5 h. Elution with petroleum ether/ethyl acetate (v/v) = 3:1; Yield: 45.1%; m.p. 112–114 °C; IR (KBr): 3398, 2927, 1704, 1634, 1527, 1456, 1382, 1077cm-1; 1H-NMR (CDCl3): δ6.23 (s, 1H, NH), 5.33(s, 1H, H-12), 3.60(m, 2H, CH2OH), 3.59(br, 1H, NHCHa), 3.17(br, 1H, NHCHb), 2.52 (m, 1H, H-2b), 2.42 (m, 1H, H-2a), 1.12 (s, 3H, CH3), 1.10(s, 3H, CH3), 0.97 (s, 3H, CH3), 0.90 (d, 3H, CH3), 0.84 (s, 3H, CH3); ESI-MS: 512.5 (M+H)+; Elemental analysis (%, found) C, 77.38 (77.45); H,10.41 (10.44); N, 2.81 (2.74).
3.3.2. N-[3-Oxo-urs-12-en-28-oyl]-4-methoxyaniline (10)
Compound 8 was reacted with p-methoxyaniline using the general procedure to give compound 10. The reaction mixture was stirred at room temperature for 5 h. Eluted with petroleum ether/ethyl acetate (v/v) = 3:1; Yield: 45.1%; m.p. 112–114 °C; IR (KBr): 3420, 2897, 1650, 1600, 1589, 1490, 1298, 977cm-1; 1H-NMR (CDCl3): δ6.23 (s, 1H, NH), 5.33(s, 1H, H-12), 3.60(m, 2H, CH2OH), 3.59(br, 1H, NHCHa), 3.17(br, 1H, NHCHb), 2.52 (m, 1H, H-2b), 2.42 (m, 1H, H-2a), 1.12 (s, 3H, CH3), 1.10(s, 3H, CH3), 0.97 (s, 3H, CH3), 0.90 (d, 3H, CH3), 0.84 (s, 3H, CH3); ESI-MS: 560.5 (M+H)+; Elemental analysis (%, found) C, 78.99 (79.10); H, 9.79 (9.87); N, 2.54 (2.49).
3.4. General procedure for the synthesis of N-[3β-acetoxyurs-12-en-28-oyl]-amines 12,13
A mixture of compound 2 (50 mg, 0.11 mmol) and oxalyl chloride (0.04 mL) in CH2Cl2 (2 mL) was stirred at room temperature for 20 hours. The mixture was concentrated to dryness under reduced pressure. Cyclohexane (3 × 1 mL) was added to the residue, then concentrated to dryness to yield crude 3-O-acetylursolyl chloride 11. To a CH2Cl2 (4 mL) solution of 11 was added the appropriate amine compound (0.44 mmol). The reaction mixture was stirred in the presence of Et3N at room temperature. The resultant residue was partitioned in 3 mL water, then treated with 2N HCl to pH 3, CH2Cl2 was removed under vacuum to precipitate a white solid, that was filtered and the filter cake was washed with water to pH 7, and dried. The crude was purified on a silica gel column with petroleum ether/ ethyl acetate to yield a white powder.
3.4.1. N-[3β-Acetoxyurs-12-en-28-oyl]-3’,4’-difluorobenzylamine (12)
Compound 11 was reacted with 3’,4’-difluorobenzylamine using the general procedure to give compound 12. Eluted by petroleum ether/ethyl acetate (v/v) = 8:1; Yield: 50.1%; m.p. 110–112 °C; IR (KBr): 3538, 3002, 1694, 1594, 1497, 1410, 1352, 1035 cm-1; 1H-NMR (CDCl3): δ 6.965–7.108 (m, 3H, Ph-H) 6.194 (d, 1H, NH), 5.251–5.262 (m, 1H, H-12), 4.471–4.504 (br, 1H, NHCH2), 4.099–4.132 (m, 1H, H-3), 2.052 (s, 3H, CH3CO), 1.089 (s, 3H, CH3), 0.925 (s, 6H, CH3×2), 0.867 (m, 9H, CH3×3), 0.676 (s, 3H, CH3); ESI-MS: 624.4 (M+H)+; Elemental analysis (%, found) C, 74.71 (74.60); H, 9.00 (9.06); N, 2.34 (2.29).
3.4.2. N-[3β-Acetoxyurs-12-en-28-oyl]-3-morpholin-4-yl-1-propylamine (13)
Compound 11 was reacted with 3-morpholin-4-yl-propylamine using the general procedure to give compound 13. Eluted by petroleum ether/ethyl acetate (v/v) = 1:6; Yield: 42.4%; m.p. 98–100 °C; IR (KBr): 3468, 2998, 1694, 1593, 1497, 1421, 1311, 977cm-1;1H-NMR (CDCl3): δ6.369 (d, 1H, NH), 5.286 (s, 1H, H-12), 4.500 (t, 1H, H-3), 3.431 (m, 1H, NCH), 2.051 (s, 3H, CH3CO), 1.089 (s, 3H, CH3), 0.942 (s, 6H, CH3×2), 0.856–0.889 (m, 6H, CH3×2), 0.772 (s, 3H, CH3); ESI-MS: 626.1(M+H)+; Elemental analysis (%, found) C, 74.85 (74.71); H, 10.69 (10.61); N, 4.38 (4.47).
3.5. General procedure for the synthesis of N-[3β- butyryloxyloxy-urs-12-ene-28-oyl] amines 16,17
Compound 14 was obtained from ursolic acid 1 (100 mg, 0.22 mmol) and butyric anhydride by using the same method described for the preparation of compound 2. A mixture of compound 14 (50 mg, 0.11 mmol) and oxalyl chloride (0.04 mL) in CH2Cl2 (2 mL) was stirred at room temperature for 20 h. The mixture was concentrated to dryness under reduced pressure. Cyclohexane (3 × 1 mL) was added to the residue, which was then concentrated to dryness to yield crude 3-O-butyryloxyl chloride 15. To a CH2Cl2 (4 mL) solution of 15 was added an amine compound (0.44 mmol). The reaction mixture was stirred in the presence of Et3N at room temperature (TLC control). The resulting residue was partitioned in 3 mL water, then treated with 2N HCl to pH 3, CH2Cl2 was removed under vacuum to precipitate white solid that was filtered and the filter cake was washed with water to pH 7, and dried. The crude was purified on a silica gel column with petroleum ether/ethyl acetate as eluents to yield a white powder.
3.5.1. Methyl N-[3β-butyryloxyl-urs-12-en-28-oyl]-2-amine acetate (16)
Compound 14 was reacted with glycine methyl ester hydrochloride using the general procedure to give compound 16. Eluted by petroleum ether/ethyl acetate (v/v) = 5:1; Yield: 69.5%; m.p. 79–84 °C; IR (KBr): 3388, 2897, 1724, 1664, 1532, 1467, 1311, 1025cm-1; 1H-NMR (CDCl3):δ 6.517 (m, 1H, NH), 5.397 (t-like, 1H, H-12), 4.488 (m, 1H, H-3), 4.075–4.115 (br, 1H, NHCHa), 3.809–3.846 (br, 1H, NHCHb), 3.753 (s, 3H, O CH3), 2.273 (s, 3H, CH3CO), 1.090 (s, 3H, CH3), 0.943 (s, 9H, CH3×3), 0.852 (m, 12H, CH3×4), 0.701 (s,3H, CH3); ESI-MS m/z: 598.6 (M+H)+; Elemental analysis (%, found) C, 73.96 (74.08); H, 10.15 (10.25); N, 2.45 (2.33).
3.5.2. N-[3β- butyryloxyl-urs-12-en-28-oyl]-benzyl amine (17)
Compound 14 was reacted with benzylamine using the general procedure to give compound 17. Eluted by petroleum ether/ acetone (v/v) = 7:1; 61.8%. m.p. 92–94 °C; IR (KBr): 3498, 2826, 1684, 1612, 1487, 1398, 1108cm-1; 1H-NMR (CDCl3), δ 7.233–7.337 (m, 5H, Ar-H), 6.151 (t, 1H, NH), 5.208 (t, 1H, H-12), 4.552 (d, 1H, Ar-CHa), 4.482 (m, 1H, H-3), 4.155(d, 1H, Ar-CHb), 2.288 (m, 2H, CH2CO), 1.078 (s, 3H, CH3), 0.945 (m, 9H, CH3×3), 0.853 (m, 9H, CH3×3), 0.701 (s, 3H, CH3); ESI-MS: 616.8 (M+H)+; Elemental analysis (%, found) C, 79.73(79.69); H, 10.33(10.28); N, 2.21(2.27).