Pleuromulin lipoic acid ester with antibacterial activity and a method of preparing the same

Preparation of the pleuromulin lipoic acid ester (2-(((3aS,4R,5S,6S,8R,9R,9aR,12R)-5-hydroxy-4,6,9,12-tetramethyl-1-oxo-6-vinyldecahydro-3a, 9-propanocyclopenta[8]annulen-8-yl)oxy)-2-oxoethyl 5-(1,2-dithiolan-3-yl)pentanoate)

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 49.5 mg (0.24 mmol) lipoic acid and 2.4 mg (0.02 mmol) 4-DMAP (4-dimethylamino)pyridine were dissolved in 20 mL dichloromethane. After stirring for five minutes at 0° C., 41.2 mg (0.20 mmol) of condensing agent DCC (N,N′-Dicyclohexylcarbodiimide) was added under nitrogen atmosphere. The reaction mixture was stirred at 25° C. for 10 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was washed with water, extracted with ethyl acetate, concentrated and dried to give a crude product. The crude product was purified by silica gel column chromatography, with dichloromethane: methanol=20:1 as eluent, and the eluent containing the product was combined, concentrated under reduced pressure, and dried to obtain 85.5 mg of the pleuromulin lipoic acid ester, a total yield of 75.49%.

1 H-NMR (400 MHz, chloroform-d) δ (ppm): 6.51(1H, m), 5.84 (1H, d), 5.34 (2H, s), 4.58 (1H, d), 3.61(1H, t), 3.40 (1H, s), 3.21 (1H, d), 2.54 (1H, m), 2.47 (2H, t), 2.35 (2H, t), 2.25 (1H, s), 2.00-1.91 (6H, m), 1.72 (2H, m), 1.62-1.50 (8H, m), 1.48 (2H, m), 1.43 (3H, m), 1.22 (2H, m), 1.17 (3H, m), 0.93 (3H, d), 0.80 (3H, d); 13 C-NMR (400 MHz, chloroform-d) δ (ppm): 216.8, 172.6, 166.7, 138.8, 117.3, 74.6, 69.6, 61.2, 58.0, 56.3, 45.4, 44.6, 44.0, 41.9, 40.2, 38.5, 36.0, 34.5, 33.5, 30.4, 28.6, 26.5, 24.8, 24.5, 16.6, 14.8, 11.4.

Preparation of the pleuromulin lipoic acid ester

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 49.5 mg (0.24 mmol) lipoic acid and 2.4 mg (0.02 mmol) 4-DMAP (4-dimethylamino)pyridine were dissolved in 20 mL toluene. After stirring for five minutes at 0° C., 41.2 mg (0.20 mmol) of condensing agent DCC (N,N′-Dicyclohexylcarbodiimide) was added under nitrogen atmosphere. The reaction mixture was stirred at 20° C. for 12 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was washed with water, extracted with ethyl acetate, concentrated and dried to give a crude product. The crude product was purified by silica gel column chromatography, with dichloromethane: methanol=20:1 as eluent, and the eluent containing the product was combined, concentrated under reduced pressure, and dried to obtain 72.9 mg of the pleuromulin lipoic acid ester, a total yield of 64.35%.

Preparation of the pleuromulin lipoic acid ester

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 45.4 mg (0.24 mmol) lipoic acid and 2.4 mg (0.02 mmol) 4-DMAP (4-dimethylamino)pyridine were dissolved in 20 mL DMF. After stirring for five minutes at 0° C., 41.2 mg (0.20 mmol) of condensing agent DCC (N,N′-Dicyclohexylcarbodiimide) was added under nitrogen atmosphere. The reaction mixture was stirred at 30° C. for 10 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was washed with water, extracted with ethyl acetate, concentrated and dried to give a crude product. The crude product was purified by silica gel column chromatography, with dichloromethane: methanol=20:1 as eluent, and the eluent containing the product was combined, concentrated under reduced pressure, and dried to obtain 76.1 mg of the pleuromulin lipoic acid ester, a total yield of 67.19%.

Preparation of the pleuromulin lipoic acid ester

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 45.4 mg (0.22 mmol) lipoic acid and 2.4 mg (0.02 mmol) 4-DMAP (4-dimethylamino)pyridine were dissolved in 20 mL dichloromethane. After stirring for five minutes at 0° C., 41.2 mg (0.20 mmol) of condensing agent DCC (N,N′-Dicyclohexylcarbodiimide) was added under nitrogen atmosphere. The reaction mixture was stirred at 35° C. for 8 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was washed with water, extracted with ethyl acetate, concentrated and dried to give a crude product. The crude product was purified by silica gel column chromatography, with dichloromethane: methanol=20:1 as eluent, and the eluent containing the product was combined, concentrated under reduced pressure, and dried to obtain 78.4 mg of the pleuromulin lipoic acid ester, a total yield of 69.25%.

Preparation of the pleuromulin lipoic acid ester

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 49.5 mg (0.24 mmol) lipoic acid and 2.4 mg (0.02 mmol) 4-DMAP (4-dimethylamino)pyridine were dissolved in 20 mL toluene. After stirring for five minutes at 0° C., 41.2 mg (0.20 mmol) of condensing agent DCC (N,N′-Dicyclohexylcarbodiimide) was added under nitrogen atmosphere. The reaction mixture was stirred at 40° C. for 8 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was washed with water, extracted with ethyl acetate, concentrated and dried to give a crude product. The crude product was purified by silica gel column chromatography, with dichloromethane: methanol=20:1 as eluent, and the eluent containing the product was combined, concentrated under reduced pressure, and dried to obtain 71.8 mg of the pleuromulin lipoic acid ester, a total yield of 63.41%.

Preparation of the pleuromulin lipoic acid ester

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 53.6 mg (0.26 mmol) lipoic acid and 2.4 mg (0.02 mmol) 4-DMAP (4-dimethylamino)pyridine were dissolved in 20 mL DMF. After stirring for five minutes at 0° C., 41.2 mg (0.20 mmol) of condensing agent DCC (N,N′-Dicyclohexylcarbodiimide) was added under nitrogen atmosphere. The reaction mixture was stirred at 20° C. for 9 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was washed with water, extracted with ethyl acetate, concentrated and dried to give a crude product. The crude product was purified by silica gel column chromatography, with dichloromethane: methanol=20:1 as eluent, and the eluent containing the product was combined, concentrated under reduced pressure, and dried to obtain 75.2 mg of the pleuromulin lipoic acid ester, a total yield of 66.41%.

Preparation of the pleuromulin lipoic acid ester

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 47.5 mg (0.23 mmol) lipoic acid and 2.4 mg (0.02 mmol) 4-DMAP (4-dimethylamino)pyridine were dissolved in 20 mL dichloromethane. After stirring for five minutes at 0° C., 41.2 mg (0.20 mmol) of condensing agent DCC (N,N′-Dicyclohexylcarbodiimide) was added under nitrogen atmosphere. The reaction mixture was stirred at 35° C. for 9 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was washed with water, extracted with ethyl acetate, concentrated and dried to give a crude product. The crude product was purified by silica gel column chromatography, with dichloromethane: methanol=20:1 as eluent, and the eluent containing the product was combined, concentrated under reduced pressure, and dried to obtain 79.9 mg of the pleuromulin lipoic acid ester, a total yield of 70.56%.

Preparation of the pleuromulin lipoic acid ester

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 47.5 mg (0.23 mmol) lipoic acid and 2.4 mg (0.02 mmol) 4-DMAP (4-dimethylamino)pyridine were dissolved in 20 mL toluene. After stirring for five minutes at 0° C., 41.2 mg (0.20 mmol) of condensing agent DCC (N,N′-Dicyclohexylcarbodiimide) was added under nitrogen atmosphere. The reaction mixture was stirred at 30° C. for 11 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was washed with water, extracted with ethyl acetate, concentrated and dried to give a crude product. The crude product was purified by silica gel column chromatography, with dichloromethane: methanol=20:1 as eluent, and the eluent containing the product was combined, concentrated under reduced pressure, and dried to obtain 71.3 mg of the pleuromulin lipoic acid ester, a total yield of 62.95%.

Preparation of the pleuromulin lipoic acid ester

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 47.5 mg (0.23 mmol) lipoic acid and 2.4 mg (0.02 mmol) 4-DMAP (4-dimethylamino)pyridine were dissolved in 20 mL DMF. After stirring for five minutes at 0° C., 41.2 mg (0.20 mmol) of condensing agent DCC (N,N′-Dicyclohexylcarbodiimide) was added under nitrogen atmosphere. The reaction mixture was stirred at 40° C. for 11 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was washed with water, extracted with ethyl acetate, concentrated and dried to give a crude product. The crude product was purified by silica gel column chromatography, with dichloromethane: methanol=20:1 as eluent, and the eluent containing the product was combined, concentrated under reduced pressure, and dried to obtain 76.3 mg of the pleuromulin lipoic acid ester, a total yield of 67.41%.

Preparation of the pleuromulin lipoic acid ester

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 53.6 mg (0.26 mmol) lipoic acid and 2.4 mg (0.02 mmol) 4-DMAP (4-dimethylamino)pyridine were dissolved in 20 mL dichloromethane. After stirring for five minutes at 0° C., 41.2 mg (0.20 mmol) of condensing agent DCC (N,N′-Dicyclohexylcarbodiimide) was added under nitrogen atmosphere. The reaction mixture was stirred at 20° C. for 12 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was washed with water, extracted with ethyl acetate, concentrated and dried to give a crude product. The crude product was purified by silica gel column chromatography, with dichloromethane: methanol=20:1 as eluent, and the eluent containing the product was combined, concentrated under reduced pressure, and dried to obtain 81.6 mg of the pleuromulin lipoic acid ester, a total yield of 72.05%.

Preparation of the pleuromulin lipoic acid ester

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 49.5 mg (0.24 mmol) lipoic acid and 2.4 mg (0.02 mmol) 4-DMAP (4-dimethylamino)pyridine were dissolved in 20 mL dichloromethane. After stirring for five minutes at 0° C., 41.2 mg (0.20 mmol) of condensing agent DCC (N,N′-Dicyclohexylcarbodiimide) was added under nitrogen atmosphere. The reaction mixture was stirred at 30° C. for 11 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was washed with water, extracted with ethyl acetate, concentrated and dried to give a crude product. The crude product was purified by silica gel column chromatography, with dichloromethane: methanol =20:1 as eluent, and the eluent containing the product was combined, concentrated under reduced pressure, and dried to obtain 79.5 mg of the pleuromulin lipoic acid ester, a total yield of 70.16%.

Preparation of the pleuromulin lipoic acid ester

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 45.4 mg (0.22 mmol) of lipoic acid and 3.7 mg (0.002 mmol) silicomolybdic acid were dissolved in 30 mL of 1-octyl-3-methylimidazolium hexafluorophosphate under nitrogen atmosphere. After full dissolution, the reaction mixture was stirred at 20° C. for 6 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was allowed to separate into layers to give a crude product. The crude product was recrystallized with 10 mL methanol and dried to obtain 96.0 mg of the pleuromulin lipoic acid ester, a total yield of 84.79%.

Preparation of the pleuromulin lipoic acid ester

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 45.4 mg (0.22 mmol) of lipoic acid and 3.7 mg (0.002 mmol) silicomolybdic acid were dissolved in 30 mL of 1-ethyl-3-methylimidazolium tetrachloroferrate under nitrogen atmosphere. After full dissolution, the reaction mixture was stirred at 10° C. for 8 hours. Thin layer chromatography was used to track the reaction to completion, and the protective device was removed. The reaction mixture was allowed to separate into layers to give a crude product. The crude product was recrystallized with 10 mL methanol and dried to obtain 90.5 mg of the pleuromulin lipoic acid ester, a total yield of 79.959%.

Preparation of the pleuromulin lipoic acid ester

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 45.4 mg (0.22 mmol) of lipoic acid and 3.7 mg (0.002 mmol) silicomolybdic acid were dissolved in 30 mL of 1-butyl-3-methylimidazolium tetrafluoroborate under nitrogen atmosphere. After full dissolution, the reaction mixture was stirred at 50° C. for 4 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was allowed to separate into layers to give a crude product. The crude product was recrystallized with 10 mL methanol and dried to obtain 92.2 mg of the pleuromulin lipoic acid ester, a total yield of 81.43%.

Preparation of the pleuromulin lipoic acid ester

In a 100 mL three-necked flask, 75.7 mg (0.20 mmol) of pleuromutilin, 45.4 mg (0.22 mmol) of lipoic acid and 3.7 mg (0.002 mmol) silicomolybdic acid were dissolved in 30 mL of 1-octyl-3-methylimidazolium hexafluorophosphate under nitrogen atmosphere. After full dissolution, the reaction mixture was stirred at 20° C. for 8 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was allowed to separate into layers to give a crude product. The crude product was recrystallized with 10 mL methanol and dried to obtain 92.9 mg of the pleuromulin lipoic acid ester, a total yield of 82.07%.

Antibacterial activity test of the compounds of the invention

The antimicrobial efficacy was determined by a paper diffusion drug sensitivity test.

Experimental strains: multi-resistant Staphylococcus aureus 18-206, multi-resistant Staphylococcus aureus 18-222, multi-resistant Staphylococcus aureus 18-596. The experimental strain was identified by Huashan Hospital Affiliated to Fudan University (Institute of Antibiotic of Fudan University).

Drug sensitive paper: The drug sensitive paper is a special drug sensitive paper with a diameter of 6.35 mm and a water absorption of 0.02 mL. The control drug was vancomycin (30 μg/tablet); the test drugs were pleuromulin (30 μg/tablet), lipoic acid (30 μg/tablet) and pleuromulin lipoic acid ester (30 μg/tablet).

Reagents: LB agar medium, LA broth medium, 0.5% DMSO solution.

Equipment: Ultra-clean workbench, high-pressure sterilization pot, gas bath constant temperature shaking incubator.

Preparation of bacterial suspension:

The experimental strains were inoculated in non-selective medium and placed in air at 37° C. for 24 h. Pick a single colony that grows well and inoculate it into broth medium, incubate at 35° C.±2° C. for 6 hours, and use LA broth medium to calibrate the concentration of the bacterial solution to 0.5 Mie turbidimetric tube (1.5×10 8 CFU/mL). A bacterial suspension is obtained.

Paper diffusion method drug sensitivity test:

Weigh the LB dry powder, sterilize at 103.4 Kpa, 121.3° C. high-pressure steam for 15 min, and then put it in a 40° C.-50° C. water bath. Place a sterile empty plate (inner diameter 9 cm) on the surface of the ultra-clean table water table, shake and shake LB, and then pour the plate. The thickness of each plate is 3 mm to 4 mm. After the plate is cooled at room temperature, store it in the refrigerator at 2° C.-8° C. Use a sterile cotton swab to dip the bacterial solution, and evenly coat the surface of the LB plate 3 times. After inoculation of the bacterial suspension, the LB plate was dried at room temperature for 3 min to 5 min. Use sterile forceps to closely attach the antibacterial drug paper to the dish. Put the dish upside down and place it in a 37° C. incubator for 24 h. Observe the result and measure the diameter. Taking 0.5% DMSO solution as a negative control, the antibacterial activity is expressed by the diameter of the inhibition zone. The inhibition zone 17 mm, sensitive; the inhibition zone is 15 mm-16 mm, intermediary; the inhibition zone 14 mm, drug resistance.

In FIGS. 1 - 3 , the pleuromulin lipoic acid ester is represented by the number six. FIG. 1 shows the antibacterial effect of the pleuromulin lipoic acid ester on multi-resistant Staphylococcus aureus 18-206. FIG. 2 shows the antibacterial effect of the pleuromulin lipoic acid ester on multi-resistant Staphylococcus aureus 18-222. FIG. 3 shows the antibacterial effect of the pleuromulin lipoic acid ester on multi-resistant Staphylococcus aureus 18-596. The results are shown in Table 1.

The results in FIGS. 1 - 3 and Table 1 show that the starting materials pleuromutilin and lipoic acid have no inhibitory effect on drug-resistant bacteria. Pleuromutilin lipoic acid ester has strong inhibitory effects on multi-drug resistant Staphylococcus aureus 18-206, 18-222, 18-596, and the diameter of bacteriostatic zone against multidrug resistant Staphylococcus aureus 18-596 was up to 21 mm. In summary, the pleuromutilin lipoic acid ester of the present invention can be used as an antibacterial drug candidate for multi-drug resistant Staphylococcus aureus , and further preclinical studies will be conducted.