PANAXADIOL GLYCOSIDE DERIVATIVE AND PREPARATION METHOD AND APPLICATION THEREOF
20210054019 ยท 2021-02-25
Assignee
Inventors
Cpc classification
International classification
Abstract
The invention discloses a panaxadiol glycoside derivative and a preparation method and application thereof. Such compounds show strong anti-inflammatory effects in vitro and in animal model experiments, and thus can be used to prepare anti-inflammatory drugs, especially can be used for treating asthma and COPD. In the experiment, the above-mentioned compounds have obvious effects on asthma and COPD, and the efficacy of the high-dose group is superior than that of dexamethasone and budesonide. Even under the dose much exceeding the therapeutic dose, no obvious affect on blood routine and blood glucoseis observed. It has high industrial prospects in the field of anti-inflammatory drugs, especially in the field for treating asthma and COPD.
Claims
1-10. (canceled)
11. A panaxadiol glycoside derivative represented by the structure of general formula (I) or a pharmaceutically acceptable salt thereof, ##STR00041## wherein, R.sub.1 is selected from hydroxyl or non-glucose pyranosyl or ##STR00042## R.sub.2 together with R.sub.3 represents O or NOR.sub.8; or R.sub.2 is hydrogen and R.sub.3 is hydroxyl; R.sub.4 and R.sub.6 combine to form a bond, and R.sub.5 and R.sub.7 are independently selected from hydrogen, C.sub.1-6 alkoxy, hydroxyl, cyano group, C.sub.1-6 ester group, and glycosyl; or R.sub.6 together with R.sub.7 represents O or NOH, and R.sub.5 and R.sub.4 are independently selected from hydrogen, C.sub.1-6 alkoxy, hydroxyl, and cyano group; or R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are independently selected from hydrogen, C.sub.1-6 alkoxy, hydroxyl, cyano group, C.sub.1-6 ester group, and glycosyl; R.sub.8 is selected from hydrogen or C.sub.1-6 alkyl.
12. The panaxadiol glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 11, wherein, R.sub.2 together with R.sub.3 represents NOH.
13. The panaxadiol glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 11, wherein, R.sub.4 and R.sub.6 combine to form a bond.
14. The panaxadiol glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 13, wherein, R.sub.5 is selected from glycosyl; and R.sub.1 represents hydroxyl.
15. The panaxadiol glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 11, wherein, R.sub.6 together with R.sub.7 represents NOR.sub.8; and R.sub.8 represents hydrogen or methyl.
16. The panaxadiol glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 11, wherein, said pyranosyl of non-glucose in R.sub.1 is selected from rhamnopyranosyl, fucosyl, arabinosyl, xylosyl, ribosyl, quinovosyl, galactosyl, aminoglucosyl, 6-deoxy-6-aminoglucosyl, lactosyl and cellobiosyl.
17. The panaxadiol glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 11, wherein, said glycosyl is independently selected from deoxy glycosyl or a five-carbon glycosyl.
18. The panaxadiol glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 12, wherein, said glycosyl is independently selected from deoxy glycosyl or a five-carbon glycosyl.
19. The panaxadiol glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 13, wherein, said glycosyl is independently selected from deoxy glycosyl or a five-carbon glycosyl.
20. The panaxadiol glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 14, wherein, said glycosyl is independently selected from deoxy glycosyl or a five-carbon glycosyl.
21. The panaxadiol glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 15, wherein, said glycosyl is independently selected from deoxy glycosyl or a five-carbon glycosyl.
22. The panaxadiol glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 16, wherein, said glycosyl is independently selected from deoxy glycosyl or a five-carbon glycosyl.
23. The panaxadiol glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 11, wherein the structure of the panaxadiol glycoside derivative is as follows: ##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050##
24. A pharmaceutical composition containing the panaxadiol glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 11 and a pharmaceutically acceptable excipient.
25. An application of the panaxadiol glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 11 in the preparation of the drug for preventing and/or treating asthma and COPD.
26. The application according to claim 25, wherein the panaxadiol glycoside derivative or a pharmaceutically acceptable salt is further the panaxadiol glycoside derivative or a pharmaceutically acceptable salt of claim 12.
27. The application according to claim 25, wherein the panaxadiol glycoside derivative or a pharmaceutically acceptable salt is further the panaxadiol glycoside derivative or a pharmaceutically acceptable salt of claim 13.
28. The application according to claim 25, wherein the panaxadiol glycoside derivative or a pharmaceutically acceptable salt is further the panaxadiol glycoside derivative or a pharmaceutically acceptable salt of claim 14.
29. The application according to claim 25, wherein the panaxadiol glycoside derivative or a pharmaceutically acceptable salt is further the panaxadiol glycoside derivative or a pharmaceutically acceptable salt of claim 15.
30. The application according to claim 25, wherein the panaxadiol glycoside derivative or a pharmaceutically acceptable salt is further the panaxadiol glycoside derivative or a pharmaceutically acceptable salt of claim 23.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0089]
[0090]
DETAILED DESCRIPTION
Example 1 3-O-acetyl-20(S)O--D-glucopyranosyl Dammarane-24-ene-12-one (IA)
1.1 Synthesis of 3,12-di-O-acetyl-20(S)-panaxadiol Glycoside (I-1a)
[0091] ##STR00009##
[0092] 20(S)-Panaxadiol glycoside (120.0 g, 0.26 mol) was dissolved in dry pyridine (750.0 mL), DMAP in a catalytic amount was added, Ac2O (99.1 mL, 1.04 mol) was dripped under ice bath, and the mixture was naturally restored to room temperature and reacted for 6.0 h. The reaction mixture was concentrated under reduced pressure, then diluted with ethyl acetate (2.0 L), washed successively with dilute hydrochloric acid, saturated NaHCO.sub.3 aqueous solution and saturated NaCl aqueous solution. Organic layer was dried with anhydrous Na2SO4, after filtered and concentrated under reduced pressure, light yellow solid was obtained. After recrystallized (ethyl acetate/petroleum ether), white crystal I-la (110.3 g, 77.8% yield) was obtained. 1H NMR (CDCl3) 5.15 (t, J=6.6 Hz, 1H, H-24), 4.72 (td, J=10.8, 4.8 Hz, 1H, H-12), 4.48 (dd, J=12.0, 4.2 Hz, 1H, H-3), 2.04 (s, 3H, HCOCH3), 2.03 (s, 3H, HCOCH3), 1.70 (s, 3H), 1.63 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 0.94 (s, 3H), 0.87 (s, 3H), 0.85 (s, 3H), 0.84 (s, 3H).
1.2 Synthesis of 3--O-acetyl-20(S)-panaxadiol glycoside (I-2a)
[0093] ##STR00010##
[0094] Compound I-1a (110.0 g, 0.20 mol) was dissolved in 100.0 mL of CH2Cl2, 400.0 mL methanol and MeONa (1.1 g, 0.02 mol) were added and reacted for 3.0 h at room temperature. Appropriate amount of cation resin was added to adjust pH to 7, and then the resin was removed through filtration. After concentration, light yellow solid I-2a (91.7 g, 90.3%) was obtained. 1H NMR (CDCl.sub.3) 5.15 (t, J=6.6 Hz, 1H, H-24), 4.47 (dd, J=11.0, 5.5 Hz, 1H, H-3), 3.60 (td, J=10.5, 5.0 Hz, 1H, H-12), 2.04 (s, 3H, HCOCH3), 1.69 (s, 3H), 1.63 (s, 3H), 1.19 (s, 3H), 0.98 (s, 3H), 0.90 (s, 3H), 0.87 (s, 3H), 0.85 (s, 6H).
1.3 Synthesis of 3--O-acetyl-20(S)-hydroxyl dammarane-24-ene-12-one (I-3a)
[0095] ##STR00011##
[0096] Compound I-2a (91.0 g, 0.18 mol) was dissolved in 500.0 mL of dried CH2Cl2, PDC (101.6 g, 0.27 mol) and acetic anhydride (34.0 mL, 0.36 mol) were added and reacted for about 5.0 h at room temperature. The insoluble substance was removed through suction filtration. The filtrate was concentrated and separated by column chromatography (ethyl acetate/n-hexane= 1/10), and white crystal I-3a (63.4 g, 70.0%) was obtained. 1H NMR (CDCl3) 5.10 (s, 1H, H-24), 4.48 (dd, J=11.6, 4.4 Hz, 1H, H-3), 2.85 (d, J=10.2 Hz, 1H, H-13), 2.44-2.37 (m, 1H, H-17), 2.28 (d, J=14.3 Hz, 1H), 2.23 (d, J=14.0 Hz, 1H), 2.05 (s, 3H), 1.68 (s, 3H), 1.62 (s, 3H), 1.17 (s, 3H), 1.12 (s, 3H), 0.95 (s, 3H), 0.87 (s, 6H), 0.80 (s, 3H).
1.4 Synthesis of 3--O-acetyl-20(S)O--D-glucopyranosyl dammarane-24-ene-12-one (IA)
[0097] ##STR00012##
[0098] I-3a (25.0 g, 49.92 mmol) and 2,3,4,6-tetra-O-acetyl glucosamine trichloroimine ester (36.9 g, 74.88 mmol) were dissolved in dried CH2Cl2, appropriate amount of 4 molecular sieve was added, protected by argon, stirred for 30 min at room temperature, and then the temperature of the reaction system was reduced to 40 C., TMSOTf (901.69 L, 4.99 mmol) was dripped, and reaction at 40 C. After TLC detection, Et3N was added to stop the reaction, restored to room temperature, the molecular sieve was removed by suction filtration, and the reaction solution was concentrated into solid. A quarter of the concentrate was dissolved in a mixed solvent of dichloromethane and methanol (400.0 mL, v:v=1:1), sodium methoxide was added to make pH=9-10, and then the mixture was reacted for 1.0 h at room temperature. The reaction was detected through TLC until it was completed. Cation resin was added to neutralize the reaction solution. After filtration, concentration and column chromatography, white solid IA (6.7 g, 81.0% two-step yield) was obtained. 1H NMR (400 MHz, CD3OD) 5.09 (t, J=6.5 Hz, 1H, H-24), 4.47-4.43 (m, 1H), 4.44 (d, J=7.6 Hz, 1H, H-1), 3.80 (dd, J=11.7, 1.8 Hz, 1H), 3.64 (dd, J=11.8, 5.3 Hz, 1H), 3.36-3.32 (m, 2H), 3.27 (t, J=8.8 Hz, 1H), 3.22-3.18 (m, 1H), 3.10 (t, J=8.2 Hz, 1H), 2.51 (dd, J=9.7, 4.4 Hz, 1H), 2.44 (t, J=13.2 Hz, 1H), 2.11 (dd, J=12.8, 3.3 Hz, 1H), 2.02 (s, 3H), 1.66 (s, 3H), 1.62 (s, 3H), 1.27 (s, 3H), 1.11 (s, 3H), 1.02 (s, 3H), 0.91 (s, 3H), 0.88 (s, 3H), 0.76 (s, 3H); 13C NMR (150 MHz, CD3OD) 215.2, 172.8, 132.0, 126.0, 98.3, 82.5, 82.1, 78.8, 77.4, 75.7, 71.8, 62.9, 57.5, 57.2, 57.1, 56.1, 43.0, 41.9, 40.7, 40.7, 39.4, 39.0, 38.8, 35.5, 33.0, 28.5, 25.9, 25.0, 24.7, 24.6, 22.9, 21.2, 19.4, 17.8, 17.2, 16.9, 16.7, 16.3. MALDI-HRMS calcd for C38H62NaO9 [M+Na]+685.4286, found 685.4293.
Example 2 Preparation of 3--O-acetyl-20(S)O--D-glucopyranuronic acid methyl ester dammarane-24-ene-12-one (IA-1)
[0099] ##STR00013##
[0100] I-3a (4.4 g, 8.79 mmol) and 2,3,4-tri-O-acetyl glucuronide methyl trichloroimine ester (5.0 g, 10.44 mmol) were dissolved in dried CH2Cl2, appropriate amount of 4 molecular sieve was added, protected by argon, stirred for 30 min at room temperature, then the temperature of the reaction system was reduced to 40 C., and TMSOTf (157.21 L, 0.87 mmol) was dripped for reaction at 40 C. The reaction was detected through TLC until it was completed. Et3N was added to stop the reaction and is the mixture was restored to room temperature, the molecular sieve was removed through suction filtration, and the reaction solution was concentrated into solid. The concentrate was dissolved in a mixed solvent of dichloromethane and methanol (150.0 mL, v:v=1:1), sodium methoxide was added to make pH=9-10, then reaction was preformed for 1.0 h at room temperature. The reaction was detected through TLC until it was completed. Cation resin was added to neutralize the reaction solution, after filtration, concentration and column chromatography, white solid IA-1 (3.3 g, 54.1% two-step yield) was obtained. The 1H NMR (400 MHz, DMSO-D6) is shown in
Example 3-hydroxyl-20(S)O--D-glucopyranosyl dammarane-24-ene-12-one (IB)
[0101] ##STR00014##
[0102] Two quarters of the concentrate in step 1.4 of the example was dissolved in the mixed solvent of dichloromethane and methanol (400.0 mL, v:v=1:1), sodium methoxide was added to make pH=9-10. The reaction was allowed to take place for 6.0 h at 50 C. and detected through TLC until it was completed. Cation resin was added to neutralize the reaction solution, after filtration, concentration and column chromatography, white solid IB (11.6 g, 74.8% two-step yield) was obtained. 1H NMR (400 MHz, CD3OD) 5.09 (t, J=6.2 Hz, 1H), 4.44 (d, J=7.6 Hz, 1H, H-1), 3.80 (d, J=11.5 Hz, 1H), 3.64 (dd, J=11.7, 5.3 Hz, 1H), 3.34-3.31 (m, 2H), 3.28 (t, J=8.8 Hz, 1H), 3.21-3.18 (m, 1H), 3.16-3.08 (m, 2H), 2.52-2.40 (m, 2H), 1.67 (s, 3H), 1.62 (s, 4H), 1.27 (s, 3H), 1.11 (s, 3H), 0.98 (s, 3H), 0.97 (s, 3H), 0.79 (s, 4H), 0.74 (s, 3H).
Example 4 3-hydroxyl-20(S)O--D-glucopyranosyl dammarane-12-one (IB-1)
[0103] ##STR00015##
[0104] IB (7.5 g, 12.08 mmol) was dissolved in MeOH (200.0 mL), and then Pd/C (750.0 mg) was added. After H2 replacement, the reaction was allowed to take place for 2 h at room temperature and then detected through TLC until it was completed. Pd/C was removed through diatomite filtration, and after concentration and column chromatography (CH2Cl2/MeOH=10/1), white solid IB-1 (4.6 g, 61.3%) was obtained. 1H NMR (400 MHz, CD3OD) 4.43 (d, J=7.7 Hz, 1H), 3.80 (dd, J=11.6, 1.5 Hz, 1H), 3.64 (dd, J=11.6, 5.4 Hz, 1H), 3.36-3.31 (m, 2H), 3.27 (d, J=8.8 Hz, 1H), 3.21-3.17 (m, 1H), 3.15 (t, J=6.4, 4.8 Hz, 1H), 3.09 (t, J=8.4 Hz, 1H), 2.50-2.40 (m, 2H), 2.11 (dd, J=12.7, 3.5 Hz, 1H), 1.9-1.91 (m, 1H), 1.84-1.78 (m, 1H), 1.27 (s, 3H), 1.08 (s, 3H), 0.98 (s, 3H), 0.97 (s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.79 (s, 3H), 0.74 (s, 3H); 13C NMR (150 MHz, CD3OD) b 215.6, 98.3, 82.6, 79.3, 78.8, 77.4, 75.6, 71.8, 62.9, 57.5, 57.2, 57.1, 56.3, 42.9, 41.9, 41.0, 40.7, 40.0, 40.0, 38.8, 35.6, 33.0, 29.0, 28.6, 27.9, 24.9, 23.8, 23.0, 22.9, 19.5, 17.1, 16.7, 16.3, 16.0. MALDI-HRMS calcd for C36H62NaO8 [M+Na]+645.4337, found 645.4354.
Example 5 3, 25-dihydroxyl-20(S)O--D-glucopyranosyl dammarane-12-one (IB-2)
5.1 Synthesis of 24-Br-25-hydroxyl-3-O-acetyl-20(S)O-(2,3,4,6-tetra-O-acetyl--D-glucopyranosyl) dammarane-12-one (I-3A-2)
[0105] ##STR00016##
[0106] I-3A (7.5 g, 9.02 mmol) was dissolved in 150 mL of tetrahydrofuran, 15 mL of water was added, N-bromosuccinimide (2.4 g, 13.53 mmol) was added in batches under ice bath, and the reaction was continued for 1 h under ice bath. After the reaction was detected through TLC until it was completed, 200 mL of ethyl acetate was added to dilute the reaction solution, then washed with 5% sodium thiosulfate solution and saturated salt water in turn. The organic layer was dried with anhydrous sodium sulfate, and after filtration, concentration and column chromatography (ethyl acetate/petroleum ether=), white solid I-3A-2 (7.6 g, 90.5%) was obtained. 1H NMR (400 MHz, CDCl3) 5.20 (t, J=9.4 Hz, 1H, H-3), 5.01 (t, J=9.8 Hz, 1H, H-4), 4.95 (t-like, J=9.0, 8.2 Hz, 1H, H-2), 4.68 (d, J=7.8 Hz, 1H, H-1), 4.46 (dd, J=11.4, 4.7 Hz, 1H, H-3), 4.12-4.18 (m, 2H, H-6), 3.89 (d, J=9.8 Hz, 1H, H-24), 3.70-3.68 (m, 1H, H-5), 2.97 (d, J=9.8 Hz, 1H, H-13), 2.38-2.44 (m, 1H, H-17), 2.16 (d, J=8.6 Hz, 2H, H-11), 2.10 (s, 3H, HAc), 2.04 (s, 3H, HAc), 2.02 (s, 3H, HAc), 1.98 (s, 6H, HAc*2), 1.35 (s, 3H, H-26, H-27), 1.19 (s, 3H, H-20), 1.04 (s, 3H, H-Me), 0.95 (s, 3H, H-Me), 0.87 (s, 3H, H-Me), 0.85 (s, 3H, H-Me), 0.72 (s, 3H, H-Me); 13C NMR (125 MHz, CDCl.sub.3) 211.3 (C-12), 170.9, 170.7, 170.2, 169.5, 169.1, 94.7 (C-1), 81.8, 80.3, 73.1, 72.6, 72.1, 71.8, 71.7, 68.6, 62.4, 56.2, 55.8, 55.7, 54.3, 41.4, 40.5, 39.7, 38.6, 38.2, 37.8, 37.5, 34.2, 31.6, 29.1, 27.9, 26.5, 26.2, 23.7, 23.4, 22.8, 21.3, 20.8, 20.6, 18.2, 16.7, 16.4, 16.2, 15.6. MALDI-HRMS calcd for C46H71014BrNa [M+Na]+949.3919, found 949.3919.
5.2 Synthesis of 25-hydroxyl-3-O-acetyl-20(S)O-(2,3,4,6-tetra-O-acetyl--D-glucopyranosyl) dammarane-12-one (I-3A-3)
[0107] ##STR00017##
[0108] I-3A-2 (7.2 g, 7.76 mmol) was dissolved in 150 mL of EtOAc, DIEA 3 mL and Pd/C 720.0 mg was added, and after H2 replacement, the reaction was allowed to take place for 2 h at room temperature. The reaction was detected through TLC until it was completed. Pd/C was removed by diatomite filtration, the reaction solution was concentrated, and white solid I-3A-3 (6.0 g, 90.9%) was obtained by column chromatography (ethyl acetate/petroleum ether=1/1). 1H NMR (400 MHz, CDCl3) b 5.17 (t, J=9.4 Hz, 1H, H-3), 5.00 (t-like, J=9.9, 9.3 Hz, 1H, H-4), 4.93 (t, J=8.8 Hz, 1H, H-2), 4.61 (d, J=7.7 Hz, 1H, H-1), 4.46 (dd, J=11.5, 4.4 Hz, 1H, H-3), 4.16 (dd, J=12.1, 6.1 Hz, 1H, H-6-1), 4.09 (dd, J=12.1, 2.0 Hz, 1H, H-6-2), 3.64 (ddd, J=9.9, 6.1, 2.2 Hz, 1H, H-5), 2.98 (d, J=9.9 Hz, 1H, H-13), 2.45 (td, J=10.4, 5.5 Hz, 1H, H-17), 2.07 (s, 3H, HAc), 2.03 (s, 3H, HAc), 2.01 (s, 3H, HAc), 1.98 (s, 6H, HAc, HAc), 1.21 (s, 6H, H-26, H-27), 1.19 (s, 3H, H-21), 1.02 (s, 3H, H-Me), 0.95 (s, 3H, H-Me), 0.87 (s, 3H, H-Me), 0.85 (s, 3H, H-Me), 0.72 (s, 3H, H-Me); 13C NMR (125 MHz, CDCl.sub.3) 211.6 (C-12), 170.8 (CAc), 170.6 (CAc), 170.2 (CAc), 169.5 (CAc), 169.0 (CAc), 94.6 (C-1), 82.3, 80.4, 73.2, 71.9, 71.5, 70.7, 68.8, 62.6, 56.1, 55.8, 55.6, 54.5, 44.7, 41.2, 40.5, 39.7, 39.6, 38.3, 37.9, 37.5, 34.3, 29.4, 29.3, 27.9, 23.5, 23.5, 23.2, 21.2, 20.8, 20.6, 19.6, 18, 2, 16.8, 16.4, 16.2, 15.6. MALDI-HRMS calcd for C46H72014Na [M+Na]+871.4820, found 871.4824.
5.3 Synthesis of 3 (3, 25-dihydroxyl-20(S)O--D-glucopyranosyl dammarane-12-one (IB-2)
[0109] ##STR00018##
[0110] I-3A-3 (6.0 g, 7.07 mmol) was dissolved in dry CH2Cl2 (60 mL), 60 mL of CH3OH was added, and then MeONa was added to adjust pH to 10. The reaction was allowed to take place for about 6 h at 48 C. Appropriate amount of cation resin was added to adjust pH to neutral, then the resin was removed through filtering, and after concentrating and column chromatography (CHCl3/CH3OH=15/1), white solid IB-2 (3.9 g, 86.6%) was obtained. 1H NMR (400 MHz, CD3OD) 4.44 (d, J=7.7 Hz, 1H), 3.81 (dd, J=11.8, 1.8 Hz, 1H), 3.63 (dd, J=11.8, 5.5 Hz, 1H), 3.34-3.31 (m, 2H), 3.27 (t, J=9.0 Hz, 1H), 3.23-3.18 (m, 1H), 3.15 (t, J=6.0, 4.8 Hz, 1H), 3.09 (t, J=8.4 Hz, 1H), 2.50 (dd, J=9.5, 4.3 Hz, 1H), 2.43 (t, J=13.2 Hz, 1H), 2.11 (dd, J=12.7, 3.3 Hz, 1H), 1.95-1.90 (m, 1H), 1.27 (s, 3H), 1.17 (s, 6H), 1.11 (s, 3H), 0.98 (s, 3H), 0.97 (s, 3H), 0.79 (s, 3H), 0.75 (s, 3H); 13C NMR (150 MHz, CD3OD) 215.6, 98.3, 82.7, 79.3, 78.7, 77.5, 75.6, 71.8, 71.5, 62.9, 57.5, 57.2, 57.2, 56.3, 45.5, 43.3, 41.9, 41.3, 40.7, 40.0, 39.9, 38.8, 35.6, 33.0, 29.4, 29.2, 28.6, 27.9, 25.0, 22.9, 20.6, 19.5, 17.1, 16.7, 16.3, 16.0. MALDI-HRMS calcd for C36H6209Na [M+Na]+661.4292, found 661.4304.
[0111] According to the same methods above, other panaxadiol glycoside derivatives having different glycosyl groups were prepared.
Example 6 3p-hydroxyl-20(S)O--D-galactopyranosyl dammarane-24-ene-12-one(IC)
[0112] White solid, 5.2 g, two-step yield is 70.3%. 1H NMR (400 MHz, CD3OD) 5.08 (t, J=6.8 Hz, 1H, H-24), 4.39 (d, J=6.2 Hz, 1H, H-1), 3.82 (s, 1H), 3.71 (dd, J=10.6, 6.6 Hz, 1H), 3.64 (dd, J=10.8, 6.4 Hz, 1H), 3.46-3.42 (m, 3H), 3.35 (d, J=9.4 Hz, 1H), 3.14 (dd, J=10.7, 4.5 Hz, 1H), 2.52-2.40 (m, 2H), 2.11 (dd, J=12.7, 2.3 Hz, 1H), 1.66 (s, 4H), 1.62 (s, 3H), 1.27 (s, 3H), 1.11 (s, 3H), 0.98 (s, 3H), 0.97 (s, 3H), 0.80 (s, 3H), 0.74 (s, 3H); 13C NMR (150 MHz, CD3OD) 215.6, 131.9, 126.0, 98.8, 82.4, 79.3, 76.1, 75.6, 73.1, 70.1, 62.1, 57.5, 57.2, 57.1, 56.4, 43.0, 41.9, 40.7, 40.6, 40.0, 39.9, 38.8, 35.6, 33.0, 28.6, 27.9, 25.9, 24.9, 24.7, 22.9, 19.5, 17.8, 17.1, 16.7, 16.3, 16.0.
[0113] MALDI-HRMS calcd for C36H60NaO8 [M+Na]+643.4180, found 643.4190.
Example 7 3-hydroxyl-20(S)O--D-mannopyranosyl dammarane-24-ene-12-one (ID)
[0114] White solid, 4.9 g, two-step yield is 66.2%, whose 1H NMR ((400 MHz, DMSO-D6)) is showed in
Example 8 3p-hydroxyl-20(S)O--D-xylopyranosyl dammarane-24-ene-12-one (IE)
[0115] White solid, 4.5 g, two-step yield is 63.4%. 1H NMR (400 MHz, CD3OD) 5.08 (t, J=6.4 Hz, 1H), 4.39 (d, J=7.3 Hz, 1H, H-1), 3.77 (dd, J=11.2, 5.2 Hz, 1H), 3.49-3.42 (m, 1H), 3.33 (d, J=9.4 Hz, 1H), 3.27 (t, J=8.8 Hz, 1H), 3.16-3.11 (m, 2H), 3.07 (t, J=8.8 Hz, 1H), 2.49 (dd, J=10.0, 4.4 Hz, 1H), 2.43 (t, J=13.2 Hz, 1H) 2.11 (dd, J=12.8, 2.4 Hz, 1H), 1.67 (s, 4H), 1.61 (s, 5H), 1.26 (s, 3H), 1.09 (s, 3H), 0.97 (s, 6H), 0.79 (s, 3H), 0.74 (s, 3H); 13C NMR (150 MHz, CD3OD) 215.5, 132.1, 125.8, 98.9, 82.4, 79.3, 78.4, 75.5, 71.3, 66.5, 57.5, 57.2, 57.1, 56.3, 42.9, 41.9, 40.7, 40.7, 40.0, 39.9, 38.8, 35.6, 33.0, 28.6, 27.9, 25.9, 24.9, 24.7, 23.0, 19.5, 17.7, 17.1, 16.7, 16.3, 16.0. MALDI-HRMS calcd for C35H58NaO7 [M+Na]+613.4075, found 613.4078.
Example 9 3-hydroxyl-20(S)O--L-rhamnopyranosyl dammarane-24-ene-12-one (IF)
[0116] White solid, 4.8 g, two-step yield is 66.7%. 1H NMR (400 MHz, (CD3)2SO) 5.05 (t, J=6.4 Hz, 1H), 4.78 (s, 1H), 4.74 (s, 1H), 4.60 (s, 1H), 4.50 (s, 1H), 4.35 (s, 1H), 3.55-3.48 (m, 2H), 3.44 (d, J=8.4 Hz, 1H), 3.15 (t, J=9.2 Hz, 1H), 2.98 (m, 1H), 2.94 (d, J=9.2 Hz, 1H), 2.36 (t, J=12.8 Hz, 2H), 1.64 (s, 4H), 1.57 (s, 3H), 1.14 (s, 3H), 1.07 (d, J=6.4 Hz, 3H), 0.96 (s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.70 (s, 3H), 0.66 (s, 3H); 13C NMR (150 MHz, (CD3)2SO) 201.4, 130.5, 124.6, 94.0, 79.9, 76.5, 72.2, 72.0, 71.0, 68.6, 55.3, 55.3, 55.1, 53.9, 40.7, 38.9, 38.6, 38.1, 37.1, 33.9, 31.7, 28.1, 27, 25.5, 23.7, 23.1, 21.0, 18.1, 17.9, 17.45, 16.4, 15.7, 15.7, 15.3. MALDI-HRMS calcd for C37H62NaO6 [M+Na]+625.4439, found 625.4444.
Example 10 3--methoxyl-20(S)O--D-glucopyranosyl dammarane-24-ene-12-one (IG)
10.1 Synthesis of 3--hydroxyl-20(S)-hydroxyl dammarane-24-ene-12-one (I-4)
[0117] ##STR00019##
[0118] Compound I-3a (16.0 g, 31.96 mmol) was dissolved in 80.0 mL of CH2Cl2, 80.0 mL of methanol was added, and then sodium methoxide was added to make pH=9-10. The reaction was allowed to take place under 50 C. for 6.0 h and detected through TLC until it was completed. Cation resin was added to neutralize the reaction solution, and after filtration, concentration and column chromatography, light yellow solid I-4 (14.1 g, 96.2%) was obtained. 1H NMR (400 MHz, CDCl3) 5.10 (s, 1H), 3.20 (d, J=9.0 Hz, 1H), 2.85 (d, J=10.3 Hz, 1H), 2.40 (q, J=9.0 Hz, 1H), 2.28 (d, J=13.6 Hz, 1H), 2.21 (t, J=14.4 Hz, 1), 1.68 (s, 3H), 1.62 (s, 3H), 1.17 (s, 3H), 1.11 (s, 3H), 0.99 (s, 3H), 0.93 (s, 3H), 0.80 (s, 6H).
10.2 Synthesis of 3--methoxyl-20(S)-hydroxyl dammarane-24-ene-12-one (I-5a)
[0119] ##STR00020##
[0120] 1-4 (7.3 g, 15.91 mmol) was dissolved in dried DMF (200.0 mL), iodomethane (1.99 mL, 31.96 mmol) was added, 60% sodium hydride (1.9 g, 47.94 mmol) was added in batch under ice bath, and the reaction was allowed to take place at room temperature. After the reaction, water was slowly dripped to quench the reaction, diluted with ethyl acetate, washed successively with 1 mol/L hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride, the organic layer was dried with anhydrous sodium sulfate, concentrated under reduced pressure, and separated with column chromatography (EA/PE, 1:6), and light yellow liquid I-5a (5.9 g, 78.4%) was obtained. 1H NMR (400 MHz, CDCl3) 5.61 (s, 1H), 5.06 (t, J=7.1 Hz, 1H), 3.52 (s, 3H), 3.23 (dd, J=11.0, 4.4 Hz, 1H), 2.94-2.86 (m, 1H), 1.66 (s, 3H), 1.60 (s, 3H), 1.17 (s, 3H), 1.00 (s, 3H), 0.99 (s, 3H), 0.97 (s, 3H), 0.84 (s, 3H), 0.80 (s, 3H); 13C NMR (150 MHz, CDCl.sub.3) 214.3, 142.7, 125.7, 79.0, 74.5, 55.8, 55.2, 54.0, 52.4, 48.9, 39.0, 38.9, 38.5, 37.3, 36.7, 34.6, 31.6, 28.3, 28.2, 27.3, 26.8, 25.9, 25.2, 22.7, 22.2, 18.3, 17.9, 16.9, 15.8, 15.7.
10.3 3--methoxyl-20(S)O--D-glucopyranosyl dammarane-24-ene-12-one (IG)
[0121] ##STR00021##
[0122] I-5a (5.8 g, 12.27 mmol) and 2,3,4,6-tetra-O-acetyl glucosamine trichloroimine ester (9.1 g, 18.41 mmol) were dissolved in dried CH2Cl2, appropriate amount of 4 molecular sieve was added, protected by argon, stirred for 30 min at room temperature, then the temperature of the reaction system was reduced to 40 C., and TMSOTf (222.3 L, 1.23 mmol) was dripped for reaction at 40 C. After the reaction was detected through TLC until it was completed, Et3N was added to stop the reaction and the mixture was restored to room temperature. The molecular sieve was removed by suction filtration, and the reaction solution was concentrated into solid. The concentrate was dissolved in a mixed solvent of dichloromethane and methanol (150.0 mL, v:v=1:1), sodium methoxide was added to make pH=9-10, then reaction for 1.0 h at room temperature. The reaction was detected through TLC until it was completed. Cation resin was added to neutralize the reaction solution, and after filtration, concentration and column chromatography, white solid IG (4.0 g, 52.0% two-step yield) was obtained. 1H NMR (400 MHz, CD3OD) 5.09 (t, J=6.7 Hz, 1H), 4.48 (d, J=7.6 Hz, 1H), 3.81 (d, J=11.3 Hz, 1H), 3.64 (dd, J=11.8, 7.6 Hz, 1H), 3.48 (s, 3H), 3.38 (t, J=8.9 Hz, 1H), 3.23-3.14 (m, 4H), 3.09-3.07 (m, 1H), 2.18-1.95 (m, 5H), 1.66 (s, 3H), 1.62 (s, 3H), 1.33 (s, 3H), 1.03 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H), 0.88 (s, 3H), 0.79 (s, 3H).
Example 11 20(S)O--D-glucopyranosyl dammarane-3, 24-diene-12-one (IH, i.e. RSM-17)
11.1 Synthesis of 3--O-p-toluene sulfonyl-20(S)-hydroxyl dammarane-24-ene-12-one (I-6)
[0123] ##STR00022##
[0124] I-4 (3.3 g, 7.19 mmol) was dissolved in dried CH2Cl2 and pyridine, p-toluenesulfonyl chloride (13.7 g, 71.90 mmol) was added under ice bath, and the reaction was allowed to take place at 80 C. for 6.0 h. After the reaction, water was slowly dripped to quench the reaction, diluted with ethyl acetate, washed successively with 1 mol/L hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride, the organic layer was dried with anhydrous sodium sulfate, concentrated under reduced pressure, and separated with column chromatography, and white solid I-6 (3.8 g, 87.5%) was obtained. 1H NMR (400 MHz, CDCl3) 7.79 (d, J=7.3 Hz, 2H), 7.33 (d, J=7.4 Hz, 2H), 5.09 (s, 1H), 4.18 (d, J=11.2 Hz, 1H), 3.18 (s, 1H), 2.83 (d, J=10.0 Hz, 1H), 2.44 (s, 3H), 2.41-2.34 (m, 1H), 2.20 (d, J=11.2 Hz, 2H), 1.68 (s, 3H), 1.61 (s, 3H), 1.15 (s, 3H), 1.10 (s, 3H), 0.90 (s, 3H), 0.83 (s, 6H), 0.77 (s, 3H); 13C NMR (150 MHz, CDCl.sub.3) 213.8, 144.5, 134.9, 131.7, 129.8, 127.8, 125.0, 90.3, 73.3, 56.3, 56.0, 54.8, 53.3, 46.2, 40.3, 39.3, 38.8, 38.4, 38.0, 37.3, 33.9, 30.9, 28.0, 26.5, 25.9, 24.8, 24.6, 22.6, 21.8, 18.5, 17.8, 17.6, 16.3, 16.0, 15.9. MALDI-HRMS calcd for C37H57O5S [M+H]+ 613.3921, found 613.3927.
11.2 Synthesis of 20(S)-hydroxyl dammarane-3, 24-diene-12-one (I-7)
[0125] ##STR00023##
[0126] I-6 (3.8 g, 6.20 mmol) was dissolved in DMF (50.0 mL), lithium bromide (5.2 g, 49.60 mmol) and lithium carbonate (3.7 g, 49.60 mmol) were added. the reaction was allowed to take place under 153 C. for 1.5 h. The reaction solution was cooled to room temperature, water was added to stop the reaction, diluted with ethyl acetate, washed successively with 1 mol/L hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride, the organic layer was dried with anhydrous sodium sulfate, concentrated under reduced pressure, and separated with column chromatography(EA/PE, 1:6), I-7 (2.3 g, 84.2%) was obtained. 1H NMR (400 MHz, CDCl3) 5.40 (s, 2H), 5.11 (s, 1H), 2.89 (d, J=9.9 Hz, 1H), 2.42 (s, 1H), 2.32-1.96 (m, 5H), 1.69 (s, 3H), 1.62 (s, 3H), 1.21 (s, 3H), 1.13 (s, 3H), 0.96 (s, 6H), 0.92 (s, 3H), 0.81 (s, 3H); 13C NMR (150 MHz, CDCl3) 214.4, 138.2, 131.6, 125.0, 121.1, 73.3, 56.3, 55.1, 52.5, 52.4, 46.2, 40.9, 40.4, 39.4, 37.9, 36.8, 34.8, 33.3, 31.8, 30.9, 26.5, 25.9, 24.8, 22.7, 22.6, 19.6, 17.8, 17.5, 16.2, 15.5. MALDI-HRMS calcd for C30H48NaO2 [M+Na]+463.3547, found 463.3543.
11.3 20(S)O--D-glucopyranosyl dammarane-3, 24-diene-12-one (IH)
[0127] ##STR00024##
[0128] I-7 (2.3 g, 5.22 mmol) and 2,3,4,6-tetra-O-benzoyl glucose trichloroimine ester (4.6 g, 6.26 mmol) were dissolved in dried CH2Cl2 (60.0 mL), and appropriate amount of 4 molecular sieve was added, protected by argon. It was stirred for 30 min at room temperature, then the temperature of the reaction system was reduced to 40 C., and TMSOTf (94.3 L, 0.52 mmol) was dripped and the reaction was allowed to take place under 40 C. After the reaction was detected through TLC until it was completed, Et3N was added to stop the reaction, the reaction system was restored to room temperature, the molecular sieve was removed by suction filtration, and the reaction solution was concentrated into solid. The concentrate was dissolved in a mixed solvent of dichloromethane and methanol (50.0 mL, v:v=1:1), sodium methoxide was added to make pH=9-10, then reaction was allowed to take place for 4.0 h at room temperature. The reaction was detected through TLC until it was completed. Cation resin was added to neutralize the reaction solution, and after filtration, concentration and column chromatography, white solid IH (2.5 g, 79.5% two-step yield) was obtained. 1H NMR (400 MHz, CD3OD) 5.44-5.36 (m, 2H), 5.09 (t, J=6.6 Hz, 1H), 4.44 (d, J=7.6 Hz, 1H), 3.80 (dd, J=11.7, 1.5 Hz, 1H), 3.65 (dd, J=11.7, 5.5 Hz, 1H), 3.37-3.34 (m, 2H), 3.28 (t, J=8.8 Hz, 1H), 3.20 (dd, J=8.3, 6.0 Hz, 1H), 3.10 (t, J=8.1 Hz, 1H), 2.54-2.45 (m, 2H), 2.08 (dd, J=12.8, 3.2 Hz, 1H), 1.66 (s, 3H), 1.62 (s, 3H), 1.30 (s, 3H), 1.12 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H), 0.93 (s, 3H), 0.76 (s, 3H); 13C NMR (150 MHz, CD3OD) 215.5, 139.2, 131.9, 126.0, 122.2, 98.3, 82.5, 78.7, 77.4, 75.6, 71.7, 62.9, 57.8, 57.1, 55.1, 53.9, 42.9, 42.2, 41.9, 40.8, 40.7, 38.0, 35.7, 34.8, 33.0, 32.1, 25.9, 24.9, 24.7, 23.0, 22.9, 20.7, 17.8, 17.1, 16.9, 15.7. MALDI-HRMS calcd for C36H58NaO7 [M+Na]+625.4075, found 625.4080.
Example 12 20-O--D-xylopyranosyl-20(S)-panaxadiol glycoside(IJ)
[0129] White solid. 1H NMR (CD3OD): 5.09 (t, J=7.1 Hz, 1H, H-24), 4.52 (d, J=7.7 Hz, 1H, H-1), 3.78 (dd, J=11.5, 5.5 Hz, 1H, H-5-2), 3.68 (td, J=10.4, 4.9 Hz, 1H, H-12), 3.45 (ddd, J=10.4, 8.8, 5.5 Hz, 1H, H-4), 3.29 (t, J=8.8 Hz, 1H, H-3), 3.14 (dd, J=11.5, 10.4 Hz, 1H, H-5-1), 3.13 (dd, J=11.0, 4.4 Hz, 1H, H-3), 3.07 (dd, J=8.8, 7.7 Hz, 1H, H-12), 1.67 (s, 3H), 1.61 (s, 3H), 1.32 (s, 3H), 1.00 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.90 (s, 3H), 0.70 (s, 3H); .sup.1C NMR (CDCl.sub.3): 132.3 (C-25), 128.2 (C-24), 98.9 (C-1), 84.8 (C-20), 79.6 (C-3), 78.4 (C-3), 75.3 (C-2), 71.8 (C-12), 71.1 (C-4), 66.8 (C-5), 57.3, 53.1, 52.4, 51.0, 40.9, 40.2, 40.0, 38.1, 36.7, 35.9, 31.5, 30.8, 28.6, 28.0, 27.2, 25.9, 23.9, 22.4, 19.4, 18.3, 17.8, 17.3, 16.7, 16.3, 16.1.
Example 13 20-O--L-rhamnopyranosyl-20(S)-panaxadiol glycoside(IK)
[0130] White solid. 1H NMR (CD3OD): 5.13 (d, J=1.4 Hz, 1H, H-1), 5.13 (t, J=7.1 Hz, 1H, H-24), 3.79 (m, 1H, H-2), 3.79 (m, 1H, H-5), 3.60 (td, J=10.1, 5.5 Hz, 1H, H-12), 3.56 (dd, J=9.6, 3.2 Hz, 1H, H-3), 3.38 (t-like, J=9.6, 9.2 Hz, 1H, H-4), 3.13 (dd, J=11.5, 4.6 Hz, 1H, H-3), 1.69 (s, 3H), 1.62 (s, 3H), 1.36 (s, 3H), 1.24 (d, J=6.0 Hz, 3H, H-5), 1.00 (s, 3H), 0.96 (s, 3H), 0.93 (s, 3H), 0.91 (s, 3H), 0.77 (s, 3H); MS: 629[M+Na]+, 607[M+H]+, 589.5[M-OH]+, 443.4, 425.4, 407.4.
Example 14 20-O--L-arabinopyrauosyl-20(S)-panaxadiol glycoside (IL)
[0131] White solid. 1H NMR (CD3OD): 5.10 (d, J=7.3 Hz, 1H, H-24), 4.50 (d, J=7.3 Hz, 1H, H-1), 3.84 (dd, J=12.4, 1.4 Hz, 1H, H-5-1), 3.79 (brs, 1H, H-4), 3.71 (td, J=10.6, 5.5 Hz, 1H, H-12), 3.53 (dd, J=12.4, 1.4 Hz, 1H, H-5-2), 3.51 (dd, J=6.4, 3.2 Hz, 1H, H-3), 3.45 (dd, J=9.1, 7.3 Hz, 1H, H-2), 3.14 (dd, J=11.5, 4.6 Hz, 1H, H-3), 1.67 (s, 3H), 1.62 (s, 3H), 1.34 (s, 3H), 1.01 (s, 3H), 0.96 (s, 3H), 0.92 (s, 3H), 0.91 (s, 3H), 0.78 (s, 3H).
Example 15 Synthesis of 16 20(S)O--D-glucopyranosyl dammarane-24-ene-3, 12-dione (IIA) and 20(S)-hydroxyl-3-O--D-glucopyranosyl dammarane-3, 24-diene-12-one (IIA-1)
15.1 Synthesis of 20(S)-hydroxyl dammarane-24-ene-3, 12-dione (II-1)
[0132] ##STR00025##
[0133] PPD (40.0 g, 86.82 mmol) was dissolved in dried dichloromethane (500.0 mL), PDC (98.0 g, 260.46 mmol) and acetic anhydride (32.8 mL, 347.28 mmol) were added, reaction was allowed to take place for 5.0 h at room temperature, insoluble substance was removed through suction filtration, filtrate was concentrated and separated by column chromatography(EA/PE, 1:8), and light yellow foamed solid II-1 (24.3 g, 61.2%) was obtained. 1H NMR (400 MHz, CDCl3) 5.11 (t, J=7.1 Hz, 1H, H-24), 2.90 (d, J=9.6 Hz, 1H, H-13), 2.55-2.40 (m, 3H), 2.29 (d, J=7.7 Hz, 2H), 1.69 (s, 3H), 1.62 (s, 3H), 1.23 (s, 3H), 1.11 (s, 6H) 1.07 (s, 3H), 1.04 (s, 3H), 0.81 (s, 3H).
15.2 Synthesis of 20(S)O--D-glucopyranosyl dammarane-24-ene-3, 12-dione (2A) and 20(S)-hydroxyl-3-O--D-glucopyranosyl dammarane-3, 24-diene-12-one (2A-1)
[0134] ##STR00026##
[0135] II-1 (11.0 g, 24.08 mmol) and 2,3,4,6-tetra-O-acetyl glucose trichloroimine ester (17.8 g, 36.12 mmol) were dissolved in dried CH2Cl2, and appropriate amount of 4 molecular sieve was added, protected by argon. It was stirred for 30 min at room temperature, then the temperature of the reaction system was reduced to 40 C., and TMSOTf (435.5 L, 2.41 mmol) was dripped for reaction at 40 C. After the reaction was detected through TLC until it was completed, Et3N was added to stop the reaction, the reaction system was restored to room temperature, the molecular sieve was removed through suction filtration, and the reaction solution was concentrated into solid. The concentrate was dissolved in a mixed solvent of dichloromethane and methanol (150.0 mL, v:v=1:1), sodium methoxide was added to make pH=9-10, and then reaction was allowed to take place for 1.0 h at room temperature. The reaction was detected through TLC until it was completed. Cation resin was added to neutralize the reaction solution, and after filtration, concentration and column chromatography, white solid IIA (5.8 g, 38.9% two-step yield) and IIA-1 (5.6 g, 37.6% two-step yield) were obtained. IIA: 1H NMR (400 MHz, CD3OD) 5.10 (t, J=6.4 Hz, 1H), 4.45 (d, J=7.7 Hz, 1H), 3.80 (dd, J=11.8, 1.4 Hz, 1H), 3.65 (dd, J=11.7, 5.4 Hz, 1H), 3.40-3.33 (m, 2H), 3.28 (d, J=8.9 Hz, 1H), 3.21 (dd, J=7.4, 5.1 Hz, 1H), 3.11 (t, J=8.2 Hz, 1H), 2.54-2.48 (m, 4H), 2.14 (dd, J=12.8, 3.2 Hz, 1H), 1.67 (s, 3H), 1.62 (s, 3H), 1.31 (s, 3H), 1.12 (s, 3H), 1.09 (s, 3H), 1.07 (s, 6H), 0.77 (s, 3H); 13C NMR (150 MHz, CD3OD) 220.0, 214.8, 131.9, 126.0, 98.3, 82.5, 78.8, 77.4, 75.6, 71.8, 62.9, 57.5, 57.2, 56.1, 55.4, 43.0, 41.8, 40.8, 40.7, 40.3, 38.5, 34.8, 33.0, 27.1, 25.9, 24.9, 24.7, 22.9, 21.4, 20.9, 17.8, 17.1, 16.3, 15.8. MALDI-HRMS calcd for C36H58NaO8 [M+Na]+641.4024, found 625.4041.
[0136] IIA-1:1H NMR (400 MHz, CD3OD) 5.09 (t, J=6.7 Hz, 1H), 4.94 (d, J=7.3 Hz, 1H), 4.59 (d, J=8.1 Hz, 1H), 3.83 (d, J=12.2 Hz, 1H), 3.66 (dd, J=12.1, 4.7 Hz, 1H), 3.39-3.29 (m, 3H), 3.07 (d, J=9.5 Hz, 1H), 2.46-2.35 (m, 2H), 2.14 (d, J=14.8 Hz, 1H), 2.06-1.92 (m, 3H), 1.66 (s, 3H), 1.61 (s, 3H), 1.27 (s, 3H), 1.15 (s, 3H), 1.01 (s, 6H), 0.99 (s, 3H), 0.77 (s, 3H); 13C NMR (150 MHz, CD3OD) 215.2, 160.6, 132.1, 125.8, 102.1, 97.2, 78.3, 77.9, 75.1, 75.0, 71.4, 62.5, 57.5, 57.3, 55.0, 54.6, 44.4, 42.1, 41.6, 40.7, 40.6, 38.6, 37.6, 34.7, 32.7, 28.8, 25.9, 25.7, 24.9, 23.9, 20.6, 20.0, 17.7, 17.3, 16.4, 15.7. MALDI-HRMS calcd for C36H58NaO8 [M+Na]+641.4024, found 625.4040.
Example 17 20(S)O--D-glucopyranosyl dammarane-1, 24-diene-3, 12-dione (IIB)
17.1 Synthesis of 20(S)-hydroxyl dammarane-1, 24-diene-3, 12-dione (II-2)
[0137] ##STR00027##
[0138] I-1 (12.0 g, 26.27 mmol) was dissolved in DMSO (88.0 mL), and IBX (24.0 g, 39.41 mmol) was added. The reaction was allowed to take place under 70 C. for 24.0 h. The reaction solution was cooled to room temperature, water was added to stop the reaction, diluted with ethyl ether, washed successively with saturated sodium bicarbonate and saturated sodium chloride, the organic layer was dried with anhydrous sodium sulfate, concentrated under reduced pressure, and separated with column chromatography (EA/PE, 1:7), 11-2 (8.4 g, 70.5%) was obtained. 1H NMR (400 MHz, CDCl3) 6.98 (d, J=10.0 Hz, 1H), 5.81 (d, J=9.9 Hz, 1H), 5.07 (s, 1H), 2.88 (d, J=10.8 Hz, 1H), 2.47 (d, J=13.8 Hz, 1H), 2.38 (t, J=12.5 Hz, 2H), 1.65 (s, 3H), 1.59 (s, 3H), 1.23 (s, 3H), 1.13 (s, 6H), 1.09 (s, 6H), 0.78 (s, 3H).
17.2 Synthesis of 20(S)O--D-glucopyranosyl dammarane-1, 24-diene-3, 12-dione (IIB)
[0139] ##STR00028##
[0140] II-2 (8.3 g, 18.25 mmol) and 2,3,4,6-tetra-O-acetyl glucose trichloroimine ester (13.5 g, 27.37 mmol) were dissolved in dried CH2Cl2, appropriate amount of 4 molecular sieve was added, protected by argon, stirred for 30 min at room temperature, then the temperature of the reaction system was reduced to 40 C., and TMSOTf (330.7 L, 1.83 mmol) was dripped for reaction at 40 C. After the reaction was detected through TLC until it was completed, Et3N was added to stop the reaction, the reaction system was restored to room temperature, the molecular sieve was removed by suction filtration, and the reaction solution was concentrated into solid. The concentrate was dissolved in a mixed solvent of dichloromethane and methanol (100.0 mL, v:v=1:1), sodium methoxide was added to make pH=9-10, and then reaction was allowed to take place for 1.0 h at room temperature. The reaction was detected through TLC until it was completed. Cation resin was added to neutralize the reaction solution, and after filtration, concentration and column chromatography, white solid 2B (5.1 g, 45.3% two-step yield) was obtained. 1H NMR (400 MHz, CD3OD) 7.21 (d, J=10.1 Hz, 1H), 5.81 (d, J=10.1 Hz, 1H), 5.10 (t, J=6.3 Hz, 1H), 4.45 (d, J=7.4 Hz, 1H), 3.80 (d, J=12.0 Hz, 1H), 3.65 (dd, J=12.0, 4.9 Hz, 1H), 3.40 (d, J=9.7 Hz, 1H), 3.35 (t, J=9.2 Hz, 1H), 3.27 (d, J=9.6 Hz, 1H), 3.22-3.19 (m, 1H), 3.11 (t, J=7.9 Hz, 1H), 2.64 (t, J=13.0 Hz, 1H), 2.54-2.49 (m, 2H), 2.39 (d, J=12.4 Hz, 1H), 1.66 (s, 3H), 1.62 (s, 3H), 1.35 (s, 3H), 1.21 (s, 3H), 1.14 (s, 6H), 1.11 (s, 4H), 0.76 (s, 3H); 13C NMR (150 MHz, CD3OD) 213.8, 207.3, 160.6 131.9, 126.1, 126.0, 98.3, 82.4, 78.7, 77.4, 75.6, 71.7, 62.8, 57.3, 55.0, 50.1, 45.9, 42.9, 42.7, 41.3, 40.7, 40.4, 34.9, 32.8, 28.1, 25.9, 24.9, 24.7, 22.9, 21.8, 20.2, 19.7, 17.8, 17.1, 16.5. MALDI-HRMS calcd for C36H56NaO8 [M+Na]+639.3867, found 639.3873.
Example 18 20(S)O--D-glucopyranosyl dammarane-3-methoxy imino-24-ene-12-one (IIIA)
18.1 Synthesis of 3--hydroxyl-12--O-trimethylacetyl-20(S)-panaxadiol glycoside
[0141] ##STR00029##
[0142] PPD (45.0 g, 97.68 mmol) was dissolved in 500.0 mL of dichloromethane, triethylamine (27.1 mL, 195.36 mmol) was added, the temperature of the reaction system was reduced to 5 C., and trimethylacetyl chloride (24.1 mL, 195.36 mmol) was dripped under ice bath. The reaction was allowed to take place for 3.0 h at 5 C. Water was added to stop the reaction, washed successively with water and saturated sodium chloride, the organic layer was dried with anhydrous sodium sulfate, and concentrated under reduced pressure. 36.6 g of crude product was obtained, which was directly used for the next reaction.
18.2 Synthesis of 12--O-trimethylacetyl-20(S)-hydroxyl dammarane-24-ene-3-one (III-2)
[0143] ##STR00030##
[0144] II-1 (36.6 g, 67.17 mmol) was dissolved in 600.0 mL of dried dichloromethane, PDC (37.9 g, 100.76 mmol) and acetic anhydride (19.0 mL, 201.51 mmol) were added, reacted at room temperature for about 5.0 h. The insoluble substance was removed by suction filtration. The filtrate was concentrated and directly used for the next reaction. 1H NMR (400 MHz, CDCl.sub.3) 5.15 (t, J=7.2 Hz, 1H), 4.82 (t, J=10.4 Hz, 1H), 2.47-2.45 (m, 2H), 2.22 (s, 1H), 1.71 (s, 3H), 1.63 (s, 3H), 1.21 (s, 9H), 1.12 (s, 3H), 1.09 (s, 3H), 1.06 (s, 3H), 1.04 (s, 3H), 0.98 (s, 3H), 0.93 (s, 3H).
18.3 Synthesis of 12-p-hydroxyl-20(S)-hydroxyl dammarane-24-ene-3-one (III-3)
[0145] ##STR00031##
[0146] The concentrate previous-step was dissolved in the mixed solvent of dichloromethane and methanol (400.0 mL, v:v=1:1), sodium methoxide was added to make pH=9-10, and then reaction was allowed to take place for 6.0 h at 50 C. The reaction was detected through TLC until it was completed. Cation resin was added to neutralize the reaction solution, and after filtration, concentration and column chromatography, white solid III-3 (17.9 g, 39.9% three-step yield) was obtained. 1H NMR (400 MHz, CDCl3) 5.16 (s, 1H, H-24), 3.62-3.57 (m, 1H, H-3), 2.54-2.41 (m, 2H), 1.70 (s, 3H), 1.64 (s, 3H), 1.20 (s, 3H), 1.08 (s, 3H), 1.04 (s, 3H), 1.03 (s, 3H), 0.98 (s, 3H), 0.89 (s, 3H).
18.4 Synthesis of 12-p-hydroxyl-20(S)-hydroxyl dammarane-3-methoxy imino-24-ene (III-4a)
[0147] ##STR00032##
[0148] III-3 (2.3 g, 5.01 mmol) was dissolved in pyridine (60.0 mL), and O-methyl hydroxylamine hydrochloride (628.1 mg, 7.52 mmol) was added. The reaction was allowed to take place under 80 C. for 4.0 h. The reaction solution was cooled to room temperature, water was added to stop the reaction, diluted with ethyl acetate, washed successively with 1 mol/L hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride, the organic layer was dried with anhydrous sodium sulfate, concentrated under reduced pressure, and directly used for the next reaction.
18.5 Synthesis of 20(S)-hydroxyl dammarane-3-methoxy imino-24-ene-12-one (III-5a)
[0149] ##STR00033##
[0150] III-4a was dissolved in 120 mL of dry dichloromethane, PDC (2.8 g, 7.52 mmol) and acetic anhydride (1.4 mL, 15.03 mmol) were added. The reaction was allowed to take place under room temperature for about 5.0 h. The insoluble substance was removed by suction filtration. The filtrate was concentrated and separated by column chromatography, light yellow foamed solid III-5a (1.1 g, 45.2% two-step yield) was obtained. 1H NMR (400 MHz, CDCl3) 5.10 (s, 1H, H-24), 3.81 (s, 3H), 3.25 (s, 1H), 2.92 (d, J=14.8 Hz, 1H), 2.86 (d, J=10.4 Hz, 1H), 2.40 (d, J=8.0 Hz, 1H), 1.69 (s, 3H), 1.62 (s, 3H), 1.20 (s, 3H), 1.16 (s, 3H), 1.12 (s, 3H), 1.07 (s, 3H), 1.01 (s, 3H), 0.78 (s, 3H); 13C NMR (150 MHz, CDCl3) 214.0, 165.2, 131.7, 125.0, 73.2, 61.2, 56.4, 56.0, 54.9, 53.1, 46.2, 40.3, 40.2, 39.3, 38.5, 37.9, 37.5, 33.7, 30.9, 27.5, 26.5, 25.9, 24.8, 23.2, 22.6, 19.2, 17.8, 17.5, 15.9, 15.6. MALDI-HRMS calcd for C31H51NNaO3 [M+Na]+508.3761, found 508.3760.
18.6 Synthesis of 20(S)O--D-glucopyranosyl dammarane-3-methoxy imino-24-ene-12-one (IIIA)
[0151] ##STR00034##
[0152] III-5a (1.1 g, 2.26 mmol) and 2,3,4,6-tetra-O-acetyl glucosamine trichloroimine ester (1.3 g, 2.71 mmol) were dissolved in dried CH2Cl2, appropriate amount of 4 molecular sieve was added, protected by argon, stirred for 30 min at room temperature, then the temperature of the reaction system was reduced to 40 C., and TMSOTf (40.8 L, 0.23 mmol) was dripped for reaction at 40 C. After the reaction was detected through TLC until it was completed, Et3N was added to stop the reaction, the reaction system was restored to room temperature, the molecular sieve was removed by suction filtration, and the reaction solution was concentrated into solid. The concentrate was dissolved in a mixed solvent of dichloromethane and methanol (50.0 mL, v:v=1:1), sodium methoxide was added to make pH=9-10, then reaction for 1.0 h at room temperature. When the reaction was complete by TLC detection, cation resin was added to neutralize the reaction solution, and after filtration, concentration and column chromatography, white solid IIIA (860.0 mg, 58.5% two-step yield) was obtained. 1H NMR (400 MHz, CD3OD) 5.09 (t, J=6.7 Hz, 1H), 4.44 (d, J=7.7 Hz, 1H), 3.80 (d, J=11.7 Hz, 1H), 3.76 (s, 3H), 3.64 (dd, J=11.7, 5.3 Hz, 1H), 3.36-3.31 (m, 2H), 3.27 (t, J=8.9 Hz, 1H), 3.20 (dd, J=7.3, 5.1 Hz, 1H), 3.10 (t, J=8.2 Hz, 1H), 2.88 (dt, J=8.5, 4.8 Hz, 1H), 2.52-2.43 (m, 2H), 2.31-2.22 (m, 1H), 2.10 (dd, J=12.7, 2.9 Hz, 1H), 1.66 (s, 3H), 1.62 (s, 3H), 1.29 (s, 3H), 1.14 (s, 3H), 1.11 (s, 3H), 1.07 (s, 3H), 1.05 (s, 3H), 0.74 (s, 3H); 13C NMR (150 MHz, CD3OD) 215.1, 166.5, 131.9, 126.0, 98.3, 82.5, 78.7, 77.4, 75.6, 71.7, 62.8, 61.3, 57.5, 57.2, 57.1, 55.8, 42.9, 41.9, 41.1, 40.8, 40.7, 39.6, 38.7, 35.1, 33.0, 28.1, 25.9, 24.9, 24.7, 23.5, 22.9, 20.3, 18.4, 17.8, 17.1, 16.2, 16.1. MALDI-HRMS calcd for C37H61NNaO8 [M+Na]+670.4289, found 670.4294.
Example 19 20(S)O--D-glucopyranosyl dammarane-3-hydroxyl imino-24-ene-12-one (IIIB)
19.1 Synthesis of 12--hydroxyl-20(S)-hydroxyl dammarane-3-allyloxyimido-24-ene (III-4b)
[0153] ##STR00035##
[0154] III-3 (5.0 g, 10.90 mmol) was dissolved in pyridine (120.0 mL), and O-allyl hydroxylamine hydrochloride (1.8 g, 16.35 mmol) was added. The reaction was allowed to take place under 80 C. for 4.0 h. The reaction solution was cooled to room temperature, water was added to stop the reaction, diluted with ethyl acetate, washed successively with 1 mol/L hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride, the organic layer was dried with anhydrous sodium sulfate, concentrated under reduced pressure, and directly used for the next reaction.
19.2 Synthesis of 20(S)-hydroxyl dammarane-3-allyloxyimido-24-ene-12-one (III-5b)
[0155] ##STR00036##
[0156] III-4b was dissolved in 120 mL of dry dichloromethane, PDC (6.2 g, 16.35 mmol) and acetic anhydride (3.1 mL, 32.70 mmol) were added. The reaction was performed under room temperature for about 5.0 h. The insoluble substance was removed by suction filtration. The filtrate was concentrated and separated by column chromatography, and light yellow foamed solid III-5b (3.6 g, 64.6% two-step yield) was obtained. 1H NMR (400 MHz, CDCl3) 5.99 (dd, J=17.4, 11.4 Hz, 1H), 5.26 (d, J=17.3 Hz, 1H), 5.17 (d, J=10.5 Hz, 1H), 5.09 (t, J=6.8 Hz, 1H), 4.52 (d, J=2.6 Hz, 2H), 3.24 (s, 1H), 2.97 (d, J=15.8 Hz, 1H), 2.86 (d, J=10.3 Hz, 1H), 2.43-2.36 (m, 1H), 1.69 (s, 3H), 1.62 (s, 3H), 1.20 (s, 3H), 1.16 (s, 3H), 1.12 (s, 3H), 1.06 (s, 3H), 1.01 (s, 3H), 0.79 (s, 3H); 13C NMR (150 MHz, CDCl3) 214.0, 165.3, 134.9, 131.7, 125.0, 117.0, 74.4, 73.2, 56.4, 56.0, 54.9, 53.1, 46.2, 40.3, 40.3, 39.3, 38.4, 37.9, 37.5, 33.7, 30.9, 27.6, 26.5, 25.9, 24.8, 23.2, 22.6, 19.2, 17.8, 17.8, 17.5, 15.9, 15.5. MALDI-HRMS calcd for C33H53NNaO3 [M+Na]+534.3918, found 534.3921.
19.3 Synthesis of 20(S)O--D-glucopyranosyl dammarane-3-hydroxyl imino-24-ene-12-one (IIIB)
[0157] ##STR00037##
[0158] III-5b (3.6 g, 7.03 mmol) and 2,3,4,6-tetra-O-acetyl glucose trichloroimine ester (4.2 g, 8.44 mmol) were dissolved in dry CH.sub.2Cl.sub.2, appropriate amount of 4 molecular sieve was added, protected by argon, stirred for 30 min at room temperature, then the temperature of the reaction system was reduced to 40 C., and TMSOTf (127.0 L, 0.70 mmol) was dripped for reaction at 40 C. After the reaction was detected through TLC until it was completed, Et.sub.3N was added to stop the reaction, the reaction system restored to room temperature, the molecular sieve was removed by suction filtration, and the reaction solution was concentrated into solid. The concentrate was dissolved in a mixed solvent of dichloromethane and methanol (100.0 mL, v:v=1:1), sodium methoxide was added to make pH=9-10, and then reaction was allowed to take place for 1.0 h at room temperature. When the reaction was complete by TLC detection, cation resin was added to neutralize the reaction solution, after filtration and concentration, the concentrate was dissolved in the mixed solvent of ethanol and water (50.0 mL, v:v=4:1), triphenylphosphine (256.5 mg, 0.98 mmol), palladium acetate (73.2 mg, 0.33 mmol), triethylamine (4.1 mL, 29.34 mmol) and formic acid (1.1 mL, 29.34 mmol) were added, heated and refluxed for 1.5 h. The reaction solution was concentrated and purified by column chromatography, white solid IIIB (1.8 g, 40.4% three-step yield) was obtained. .sup.1H NMR (400 MHz, CD.sub.3OD) 5.09 (t, J=6.5 Hz, 1H), 4.44 (d, J=7.5 Hz, 1H), 3.80 (d, J=11.6 Hz, 1H), 3.65 (dd, J=11.8, 5.3 Hz, 1H), 3.36-3.31 (m, 2H), 3.28 (t, J=9.2 Hz, 1H), 3.22-3.19 (m, 1H), 3.10 (t, J=8.2 Hz, 1H), 2.96 (dt, J=14.6, 3.9 Hz, 1H), 2.53-2.44 (m, 2H), 2.33-2.24 (m, 1H), 2.11 (dd, J=12.2, 2.4 Hz, 1H), 1.66 (s, 3H), 1.62 (s, 3H), 1.30 (s, 3H), 1.14 (s, 3H), 1.11 (s, 3H), 1.07 (s, 3H), 1.06 (s, 3H), 0.74 (s, 3H); .sup.13C NMR (150 MHz, CD.sub.3OD) 215.2, 166.5, 131.9, 126, 98.3, 82.5, 78.7, 77.4, 75.6, 71.7, 62.8, 57.5, 57.2, 57.1, 55.9, 42.9, 41.9, 41.1, 40.8, 40.7, 39.6, 38.8, 35.1, 33.0, 28.1, 25.9, 24.9, 24.7, 23.4, 22.9, 20.3, 17.8, 17.7, 17.1, 16.12, 16.1. MALDI-HRMS calcd for C36H59NNaO8 [M+Na]+656.4133, found 656.4139.
Example 20 20(S)O--D-glucopyranosyl dammarane-3-hydroxyl-2-cyano-2, 24-diene-12-one (IIIC)
20.1 Synthesis of 20(S)-hydroxyl dammarane-3-hydroxyl-2-cyano-2, 24-diene-12-one (III-9)
[0159] ##STR00038##
[0160] III-3 (10.0 g, 21.80 mmol) was dissolved in dried ethyl formate (150.0 mL), 30% sodium methoxide (30 mL) was added, and reacted at room temperature for 3.0 h. After reaction, diluted with ethyl acetate, washed successively with 1 mol/L hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride, the organic layer was dried with anhydrous sodium sulfate, and concentrated under reduced pressure. The concentrate was dissolved in the mixed solvent of ethanol (150.0 mL) and water (26.4 mL), hydroxylamine hydrochloride (3.0 g, 43.60 mmol) and triethylamine (3.0 mL, 21.80 mmol) were added. The reaction was allowed to take place at 55 C. for 10.0 h. After reaction, diluted with ethyl acetate, washed successively with 1 mol/L hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride, the organic layer was dried with anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was dissolved in 150.0 mL of dry dichloromethane, PDC (12.3 g, 32.70 mmol) and acetic anhydride (4.1 mL, 43.60 mmol) were added to react at room temperature for 6.0 h, the insoluble substance was removed by suction filtration, the filtrate was concentrated, the concentrate was dissolved in dry methanol (130.0 mL), and 30% sodium methoxide (5.4 mL) was added to react at 55 C. for 3.5 h. After reaction, diluted with ethyl acetate, washed successively with 1 mol/L hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride, the organic layer was dried with anhydrous sodium sulfate, concentrated under reduced pressure, separated by column chromatography, white solid III-9 (3.0 g, 28.6% four-step yield) was obtained. .sup.1H NMR (600 MHz, (CD.sub.3).sub.2SO) 9.78 (s, 1H), 5.06 (brs, 1H), 3.92 (s, 1H), 2.97 (d, J=9.3 Hz, 1H), 2.40 (t, J=13.1 Hz, 1H), 2.20 (brs, 1H), 1.63 (s, 3H), 1.57 (s, 3H), 1.17 (s, 3H), 1.10 (s, 3H), 1.01 (s, 3H), 0.90 (s, 3H), 0.87 (s, 3H), 0.66 (s, 3H); .sup.13C NMR (150 MHz, (CD.sub.3).sub.2SO) 210.3, 171.5, 130.1, 125.1, 119.9, 77.9, 72.3, 55.4, 55.4, 51.8, 51.7, 42.3, 41.5, 40.5, 38.1, 35.9, 32.8, 31.4, 26.9, 25.5, 25.3, 23.3, 22.6, 19.0, 17.5, 16.5, 15.2, 14.8. MALDI-HRMS calcd for C31H47NNaO3 [M+Na]+504.3448, found 504.3452.
20.2 20(S)O--D-glucopyranosyl dammarane-3-hydroxyl-2-cyano-2,24-diene-12-one (IIIC)
[0161] ##STR00039##
[0162] III-9 (2.0 g, 4.15 mmol) and 2,3,4,6-tetra-O-benzoyl glucose trichloroimine ester (3.7 g, 4.98 mmol) were dissolved in dried CH2Cl2 (90.0 mL), and appropriate amount of 4 molecular sieve was added, protected by argon. It was stirred for 30 min at room temperature, then the temperature of the reaction system was reduced to 0 C., and TMSOTf (75.0 L, 0.42 mmol) was dripped for reaction at 0 C. After the reaction was detected through TLC until it was completed, Et3N was added to stop the reaction, the reaction system was restored to room temperature, the molecular sieve was removed by suction filtration, and the reaction solution was concentrated into solid. The concentrate was dissolved in a mixed solvent of dichloromethane and methanol (50.0 mL, v:v=1:1), sodium methoxide was added to make pH=9-10, and then reaction was allowed to take place for 4.0 h at room temperature. When the reaction was complete by TLC detection, cation resin was added to neutralize the reaction solution, after filtration, concentration and column chromatography, white solid IIIC (1.6 g, 59.9% two-step yield) was obtained. 1H NMR (400 MHz, CD3OD) 5.09 (t, J=6.5 Hz, 1H), 4.44 (d, J=7.7 Hz, 1H), 3.80 (dd, J=11.8, 1.9 Hz, 1H), 3.64 (dd, J=11.9, 5.3 Hz, 1H), 3.38-3.34 (m, 2H), 3.27 (d, J=8.8 Hz, 1H), 3.22-3.17 (m, 1H), 3.10 (t, J=8.2 Hz, 1H), 2.56-2.48 (m, 2H), 1.66 (s, 3H), 1.62 (s, 3H), 1.29 (s, 3H), 1.16 (s, 3H), 1.12 (s, 3H), 1.09 (s, 4H), 1.02 (s, 3H), 0.76 (s, 3H); 13C NMR (150 MHz, CD3OD) 214.7, 173.6, 131.9, 126.0, 120.6, 98.3, 82.5, 79.4, 78.7, 77.4, 75.6, 71.7, 62.8, 57.6, 57.1, 54.4, 53.9, 42.9, 42.4, 41.6, 40.7, 40.6, 39.7, 37.7, 34.5, 33.0, 27.9, 25.9, 24.9, 24.7, 22.9, 20.6, 19.8, 17.8, 17.0, 16.0, 15.7. MALDI-HRMS calcd for C37H57NNaO8 [M+Na]+666.3976, found 666.3973.
Example 21 20(S)O--D-glucopyranosyl dammarane-3,12-hydroxyl imino-24-ene-12-one (IVA)
[0163] ##STR00040##
[0164] IIA (1.25 g, 2.02 mmol) was dissolved in pyridine (60.0 mL), hydroxylamine hydrochloride (421.1 mg, 6.06 mmol) was added. The reaction was allowed to take place at 80 C. for 4.0 h. The reaction solution was cooled to room temperature, water was added to stop the reaction, diluted with ethyl acetate, washed successively with 1 mol/L hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride, the organic layer was dried with anhydrous sodium sulfate, concentrated under reduced pressure, purified by column chromatography, and white solid IVA (913.0 mg, 69.7%) was obtained. 1H NMR (400 MHz, CD3OD) 5.09 (t, J=6.8 Hz, 1H), 4.46 (d, J=7.6 Hz, 1H), 3.80 (d, J=11.4 Hz, 1H), 3.65 (dd, J=11.5, 5.1 Hz, 1H), 3.37-3.33 (m, 2H), 3.30-3.28 (m, 1H), 3.20 (dd, J=14.6, 7.8 Hz, 1H), 3.11 (t, J=8.0 Hz, 1H), 2.94-2.88 (m, 1H), 2.85 (d, J=9.6 Hz, 1H), 2.59 (dd, J=11.0, 6.9 Hz, 1H), 2.38-2.30 (m, 1H), 1.66 (s, 3H), 1.60 (s, 3H), 1.22 (s, 3H), 1.18 (s, 3H), 1.13 (s, 3H), 1.06 (s, 3H), 1.03 (s, 3H), 0.77 (s, 3H); 13C NMR (150 MHz, CD3OD) 166.9, 161.8, 131.8, 126.2, 98.4, 83.4, 78.6, 77.3, 75.4, 71.8, 62.9, 57.0, 55.5, 53.2, 43.4, 41.9, 41.1, 40.5, 39.9, 38.8, 35.6, 32.5, 28.2, 25.9, 24.4, 23.4, 22.7, 20.2, 17.8, 17.3, 16.3, 15.8. MALDI-HRMS calcd for C36H60N2NaO8 [M+Na]+671.4242, found 671.4246.
Example 22 the Research of the Effect of Compounds on Asthma Induced by Ovalbumin in Balb/c Mice
[0165] In this experiment, the above-mentioned ginsenoside derivatives (hereinafter referred to as GR derivatives) and ginsenoside CK were selected.
[0166] Drug preparation: the corresponding amount of sample was ground in a mortar, and then 0.5% CMCNa was used to prepare the corresponding volume by the method of equivalent multiplication.
[0167] Positive Control:
[0168] This series involve oral administration. Dexamethasone acetate tablets (manufacturer: Shanghai Xinyi Pharmaceutical Co., Ltd., batch number, specification: 015150901, 0.75 mg) were selected as positive drugs.
[0169] Ginsenoside CK (manufacturer: Shanghai Standard Biotech Co. Ltd, batch number, specification: 3690/20548, 5000.0 mg, purity: 92%)
[0170] Reagents
[0171] OVA (ovalbumin): batch No. SLBF4846V, specification 500 g/bottle, Sigma-Aldrich (USA).
[0172] Mouse IgE ELISA Kit: Cat EK2752, Lot 227570132, valid until July 2018, Multi Sciences (Lianke) Biotech.
[0173] Mouse IgE ELISA Kit: Cat EK2752, Lot 227570341, valid until Sep. 2018 09, Multi Sciences (Lianke) Biotech.
[0174] Mouse IgE ELISA Kit: Cat EK2752, Lot 227570842, valid until Feb. 2019 02, Multi Sciences (Lianke) Biotech.
[0175] Preparation of aluminum hydroxide adjuvant: under strong agitation, 100 mL of 5% sodium hydroxide solution was added into 250 mL of 5% aluminum sulfate solution. The precipitates were centrifuged and washed twice with normal saline, and then suspended into normal saline to make it reach 250 mL.
[0176] Equipment
[0177] Nebulizer: model: 403C household air compression nebulizer, manufacturer: Yuwell medical.
[0178] Laboratory Animals
[0179] Animals were purchased from Shanghai Sippr-BK laboratory animal Co., Ltd. License No.: SCXK (Shanghai) 2013-0016.
[0180] Experimental Method
[0181] Groups and Dosages
[0182] The 1st time: Balb/c mice, female, weight 18-20 g, were divided into blank group, model group, dexamethasone 0.6 mg/kg group, CK group, GR derivative group, 5 in each group, oral administration.
[0183] The 2nd time: Balb/c mice, female, weight 18-20 g, were divided into blank group, model group, dexamethasone 0.6 mg/kg group, CK group, GR derivative group, 5 in each group, oral administration.
[0184] Experimental Method:
[0185] In addition to the blank group, the mice were sensitized by intraperitoneal injection of OVA (20 g OVA/mice) on day 0 and day 14. Stimulated by atomization administration of OVA on day 21-25. 24 hours after the last stimulation, blood was collected from the canthus of mice, serum was taken, and the level of IgE in serum was determined by ELISA kit.
[0186] Data Analysis
[0187] The data were represented by mean and standard deviation (
[0188] 6 Results
TABLE-US-00001 TABLE 1-1 Effect of samples on the concentration of IgE in serum of mouse asthma model induced by OVA Groups Concentration (ng/mL) blank group 176.70 36.97** model group 2244.05 429.07 Dexamethasone acetate tablets 0.6 mg/kg 1131.05 211.85** CK-20 mg/kg 1701.85 284.66* IC 20 mg/kg 975.82 260.32** ID 20 mg/kg 1336.96 211.85** IB 20 mg/kg 1337.11 216.28** IVA 20 mg/kg 1358.27 248.70** IH 20 mg/kg 1191.08 107.59** IJ 20 mg/kg 1250.07 144.38** IK 20 mg/kg 1020.78 136.94** IL 20 mg/kg 996.36 102.45** Compared to model group: *p < 0.05, **p < 0.01
TABLE-US-00002 TABLE 1-2 Effect of samples on the concentration of IgE in serum of mouse asthma model induced by OVA Groups Concentration (ng/mL) blank group 80.59 10.16** model group 2322.52 296.04 Dexamethasone acetate tablets 0.6 mg/kg 1367.07 103.80** CK 15 mg/kg 1921.25 160.12* CK 30 mg/kg 1676.69 159.13* CK 60 mg/kg 1501.85 110.68* IB 15 mg/kg 2023.33 203.47 IB 30 mg/kg 1909.19 121.73 IB 60 mg/kg 1769.13 164.34* IC 15 mg/kg 1908.70 221.74 IC 30 mg/kg 1738.93 124.22 IC 60 mg/kg 1575.92 148.33* ID 15 mg/kg 1571.67 134.04* ID 30 mg/kg 1427.83 164.28** ID 60 mg/kg 1264.95 127.60** IVA 15 mg/kg 2026.13 273.15 IVA 30 mg/kg 1927.60 151.61 IVA 60 mg/kg 1723.86 167.59* IH 15 mg/kg 1682.40 182.76* IH 30 mg/kg 1311.37 165.93* IH 60 mg/kg 1150.59 129.55** IJ 15 mg/kg 2068.41 153.29 IJ 30 mg/kg 1824.85 155.74 IJ 60 mg/kg 1622.18 130.38* IK 15 mg/kg 1720.54 165.49* IK 30 mg/kg 1406.28 171.05** IK 60 mg/kg 1217.43 162.75** IL 15 mg/kg 1548.67 190.16* IL 30 mg/kg 1311.37 154.37** IL 60 mg/kg 1080.12 131.23** Compared to model group: *p < 0.05, **p < 0.01
[0189] Conclusion: in the model of mouse asthma induced by OVA, Samples ID, IH, IK and IL can significantly reduce the content of IgE in mice serum, and 0.6 mg/kg dexamethasone can also significantly reduce the content of IgE in mice serum. ID, IH, IK and IL have obvious therapeutic effect on bronchial asthma induced by OVA.
Example 23 Determination of Inflammatory Cells in Bronchoalveolar Lavage Fluid of Mice
[0190] 144 6-week-old BALB/C mice (18-20 g) were divided into 24 groups: control group, placebo group, dexamethasone group (3 mg/kg) and the drug groups. There were 6 in each group. The control group was given normal saline without any treatment. The other groups were sensitized by intraperitoneal injection of OVA 20 g and aluminum hydroxide 200 L PBC (2 mg, prepared into emulsion). Each animal was given sensitization stimulation by inhalation of 3% OVA 30 minutes on day 0 and day 14. On day 21, 22 and 23, the therapeutic agent (i.e. 3 mg/kg dexamethasone in the positive control group and oral administration in the drug group) was given orally. From day 17 to 23, PBS was given to the control group and placebo group respectively, PBS (without OVA) was given to the control group. On day 0 and day 14, the normal saline was nebulized for 30 minutes to remove the aluminum hydroxide on day 21, 22 and 23 See in Table 2.
TABLE-US-00003 TABLE 2 The results of determination of inflammatory cells in bronchoalveolar lavage fluid of mice Dosage Total Acidic Neutral lymphocyte Model group 72.36 33.42 38.08 15.16 14.08 6.15 12.71 7.12 Sample IB 2 mg/kg 46.76 25.18* 31.19 8.46 12.11 7.22 10.07 6.12 groups 20 mg/kg 25.18 13.12** 9.35 4.16*** 7.53 4.95* 200 mg/kg 12.53 6.23** 5.14 3.08*** 3.49 2.37*** 3.75 2.18*** IA 2 mg/kg 40.75 26.14* 29.23 10.29 10.40 6.48 11.81 5.97 20 mg/kg 26.93 15.01** 11.46 4.12*** 6.38 3.45** 8.84 5.63 200. mg/kg 10.85 4.62*** .sup.7.37 5.5.13*** 2.08 2.01*** 4.27 3.16*** IA-1 2 mg/kg 43.42 26.14* 26.55 13.23* 10.21 6.07 13.22 8.27 20 mg/kg 28.11 14.91** 13.26 3.92*** 3.95 2.41*** 4.76 3.18*** 200 mg/kg 11.48 4.39*** 5.22 3.31*** 3.75 2.46*** 3.82 2.66*** IVA 2 mg/kg 51.47 3172 29.09 4.83 9.15 5.68 15.35 9.23 20 mg/kg 32.46 17.21** 15.83 2.88** 7.38 3.09** 5.24 3.34** 200 mg/kg 14.69 5.77*** 4.45 2.14*** 6.17 4.09** 4.19 3.48** IC 2 mg/kg 50.77 32.49 31.51 16.14 13.29 7.12 9.30 6.85 20 mg/kg 32.95 21.07** 18.44 7.78** 8.16 5.03** 6.49 5.28** 200 mg/kg 18.25 9.48*** 8.72 3.31*** 5.17 3.19** 3.77 2.81*** ID 2 mg/kg 46.39 22.74 34.15 18.88 13.18 7.76 14.38 8.71 20 mg/kg 29.46 13.56** 15.17 8.34** 6.26 4.73** 8.92 6.89 200 mg/kg 13.81 6.76*** 6.79 3.87*** 4.39 3.13*** .sup.4.16 317*** CK 2 mg/kg 52.29/34.92 32.47 20.33 15.24 7.94 16.81 9.88 20 mg/kg 24.87 20.58** 16.39 9.67** 8.78 5.33** 10.15 7.34 200 mg/kg 22.38 12.69** 13.12 7.38** 4.32 3.16*** 7.42 4.53 DEX 3 mg/kg 36.12 14.89** 7.49 5.25*** 9.64 5.16** 8.46 5.73
[0191] The results showed that: the inflammatory cells in bronchoalveolar lavage fluid of the mice in asthma model group showed a large number of inflammatory cells converged, while the inflammatory cells in the bronchoalveolar lavage fluid of the mice in GR derivative group showed that the number of inflammatory cells in GR derivative group was significantly lower than that in model group.
Example 24 Study on Effects of GR Derivatives on COPD Induced by Smoke Exposure in Rats
[0192] Budesonide suspension for inhalation (batch No. 318205, manufacturer: AstraZeneca Pty Ltd.)
[0193] Huangguoshu cigarette, tar content 11 mg/cigarette, Guizhou Zhongyan Industry Co., Ltd.
[0194] Nebulizer: model: 403C household air compression nebulizer, manufacturer: Yuwell medical.
[0195] Animal pulmonary function analysis system: model: AniRes2005. Manufacturer: Beijing belanbo Technology Co., Ltd.
[0196] Passive smoking animal exposure system: model: PAB-S200. Manufacturer: Beijing belanbo Technology Co., Ltd.
[0197] Animal Wistar rats, male, weight 160 g, clean grade, purchased from Shanghai SLAC Laboratory Animal Co., Ltd, production license No.: SCXK (Shanghai) 2013-0016.
[0198] Experimental Method:
[0199] (1) Smoke generation and inhalation: In addition to the blank group, the cigarettes were put into the smoke generator (20 cigarettes/time), the rats were placed in the nebulization inhalation box, the size of the nebulization box is 60 cm60 cm80 cm, after the cigarettes were lighted, the smoke was injected into the poisoning box through the automatic suction function of the syringe, and all the cigarettes were burned out within five minutes. Twice a day in the morning and evening, 30 minutes each time, more than 4 hours apart, 180 consecutive days.
[0200] (2) Budesonide administration:
[0201] Budesonide formulation was diluted with normal saline and then put into an nebulizing cup for administration. The concentration of nebulizing solution in 0.25 mg/mL group was 0.25 mg/mL, 4 mL each time. Each nebulization administration was last for 30 minutes.
[0202] (3) CK and GR derivative administration:
[0203] It was divided into blank group, model group, budesonide, CK and GR derivatives: IB, IC, ID, IVA, IH, IJ, IK, IL oral administration groups, the dosage gradient of CK and GR derivatives was 20 mg/kg, 40 mg/kg, 80 mg/kg. 5 animals in each group, and the drugs were administered continuously for 180 days after grouping. The lung function was measured after the experiment. The rats were anesthetized with pentobarbital sodium and then intubated. The related indexes of forced pulmonary ventilation (FVC) in each animal. were measured by AniRes2005 pulmonary function tester. The related indexes of lung function of rats were analyzed by software.
[0204] Experimental Results:
TABLE-US-00004 TABLE 3 Effects of tested substance on FEV0.2/FVC % (forced expiratory index within 200 ms) of COPD rats Groups FEV2/FVC % Blank 72.23 15.92* Model 55.45 9.11 Budesonide 79.47 12.39** CK 20 mg/kg 61.29 11.76 CK 40 mg/kg 70.89 10.56** CK 80 mg/kg 80.24 13.73** IB 20 mg/kg 65.14 12.78 IB 40 mg/kg 73.59 10.81* IB 80 mg/kg 82.16 9.01** IC 20 mg/kg 65.45 14.17 IC 40 mg/kg 76.64 7.98** IC 80 mg/kg 83.15 8.42** ID 20 mg/kg 66.28 13.54 ID 40 mg/kg 75.23 10.71* ID 80 mg/kg 86.34 8.60** IVA 20 mg/kg 66.25 11.49 IVA 40 mg/kg 75.98 9.26* IVA 80 mg/kg 88.33 12.49** IH 20 mg/kg 63.27 14.65 IH 40 mg/kg 73.59 12.14* IH 80 mg/kg 85.51 15.27** IJ20 mg/kg 65.78 12.35 IJ 40 mg/kg 77.57 9.69* IJ 80 mg/kg 84.36 7.10** IK 20 mg/kg 67.67 11.39 IK 40 mg/kg 78.59 6.12* IK 80 mg/kg 85.56 10.07** IL 20 mg/kg 71.15 10.34* IL 40 mg/kg 80.25 8.79** IL 80 mg/kg 89.62 5.14** Compared to model group: *p < 0.05, **p < 0.01
[0205] Conclusion: in the model of COPD rats induced by smoke exposure, budesonide was inhaled and the other compounds were orally administered. The high dose group of CK, IB, IC, ID, IVA, IH, IJ, IK and IL was superior to budesonide. Therefore, GR derivatives have a good effect on COPD.
Example 25 Blood Routine
[0206] 33 ICR mice were randomly divided into 11 groups, i.e. normal saline group, 1.8 mg/kg dexamethasone acetate group (Dex), CK, IB, IC, ID, IVA, IH, IJ, IK and IL were all given 225 mg/kg. Mice were intragastric administration for 6 consecutive days, and sufficient blood samples were taken for blood routine test 1 hour after the last dose.
TABLE-US-00005 TABLE 4 Blood test data of GR series compounds WBC*10.sup.2/mm.sup.3 Ne % Ly % Eo % Mo % Ba % normal saline 6.13 1.52 28.21 3.64 62.34 20.72 0.83 0.79 7.55 3.62 0.07 0.03 group dexamethasone 1.26 0.49** 45.17 16.22** 42.59 28.17** 0.09 0.02 12.08 6.28** 0.04 0.03 acetate group CK 4.96 2.14 31.39 2.72 62.54 30.29 0.75 0.71 6.01 8.91 0.01 0.02 IB 5.27 0.98 37.81 10.97 51.35 16.33 0.05 0.06 10.75 1.93 0.05 0.04 IC 6.39 2.27 35.01 10.73 56.37 29.01 0.10 0.33 8.52 2.30 0.01 0.11 ID 4.92 1.45 37.89 12.34 53.03 15.16 0.10 0.03 7.95 2.88 0.08 0.02 IVA 6.48 1.34 22.80 13.42 70.15 19.03 0.07 0.02 7.87 1.02 0.08 0.04 IH 4.92 2.07 33.45 9.64 60.41 12.90 0.11 0.23 8.74 3.07 0.09 0.10 IJ 4.76 1.12 34.17 8.37 61.68 13.75 0.12 0.05 9.55 2.12 0.07 0.02 IK 6.37 0.73 30.89 12.03 59.04 12.36 0.08 0.04 11.27 1.82 0.02 0.02 IL 4.25 1.35 26.28 10.92 62.12 2.18 0.15 0.13 10.56 2.44 0.03 0.01 Note: compared to blank group, *P < 0.05, **P < 0.01
[0207] The hematological data showed that compared to the blank control group, the lymphocyte percentage of 1.8 mg/kg dexamethasone group decreased significantly, the neutrophil percentage increased significantly, the leukocyte count decreased significantly, the monocyte percentage increased significantly; while no hematological related changes were caused by CK and GR derivatives.
Example 26 Blood Glucose of Caudal Vein
[0208] 33 ICR mice were randomly divided into 11 groups, i.e. normal saline group, 1.8 mg/kg dexamethasone acetate group (Dex), CK, IB, IC, ID, IVA, IH, IJ, IK and IL were respectively given 225 mg/kg. The mice were intragastric administration for 6 consecutive days, fasting was started at about 8:00 in the morning on the sixth day, and blood glucose in caudal vein was measured at about 4:00 in the next day.
TABLE-US-00006 TABLE 5 Blood glucose data of GR series compounds Gruop Blood glucose (mmol/L) Blank group 3.05 0.11 Dex 5.78 0.36** CK 3.18 0.28 IB 3.10 0.19 IC 3.25 0.26 ID 2.79 0.56 IVA 3.02 0.23 IH 2.98 0.37 IJ 3.11 0.43 IK 3.09 0.28 IL 3.03 0.21 Note: compared to blank group, * P < 0.05, **P < 0.01
[0209] Blood glucose data showed that compared with the blank control group, dexamethasone could increase blood glucose in mice, while no blood glucose related changes were caused by CK and GR derivatives.