Patent Application
20220348539
The present invention relates to the field of medicinal chemistry, and in particular, to pleuromutilin (phenylithio)acetic acid ester with anti-drug resistant bacteria activity and a method of preparing the same.
With the widespread use of antibiotics around the world, the overuse of antibiotics has become more and more common, and microorganisms that are resistant to the corresponding antibiotics have emerged, posing new threats to human health. Multidrug-resistant bacteria (MDROs) are bacteria that are resistant to three or more types of antibacterial drugs. In recent years, with the unreasonable use of antibiotics, pathogenic bacteria with multi-drug resistance have gradually emerged, posing a serious risk to public health. It is estimated that 10 million people in the world die each year due to bacterial resistance, and by 2050, bacterial resistance will cause 1 to 3.4 trillion U.S. dollars in economic losses worldwide each year. As people pay more attention to food safety and environmental protection, it will become a new direction for the research and development of new antibacterial drugs. The most common way to develop new drugs that can deal with multi-drug resistant bacteria is to modify the chemical structure of existing antibacterial drugs under the guidance of structure-activity relationship research.
Pleuromutilin (compound of formula II) is a tricyclic diterpenoid compound produced by the metabolism of Pleurotus mutilus. Using it as a precursor for structural modification has obtained Tiamulin, Valnemulin, and other highly active semi-synthetic antibiotics. Pleuromutilin and its derivatives can interfere with the binding of tRNA to the P-site and A-site by interacting with the peptide acyltransferase center of the bacterial ribosome, thereby inhibiting protein synthesis. Due to the special antibacterial mechanism and slow resistance of pleuromutilin and its derivatives, it has become one of the focus areas of current antibiotic research. Although pleuromutilin has good antibacterial activity, its bioavailability is low. Therefore, the design and synthesis of pleuromutilin derivatives with novel structure, strong antibacterial activity, good water solubility, and high bioavailability is of great significance for the treatment of multidrug-resistant bacterial infections.
(Phenylithio)acetic acid (CAS: 103-04-8) (compound of formula IV) is a pharmaceutical intermediate, which is a reactant for preparing benzoxazole, benzimidazole and benzothiazole with antibacterial activity.
The invention modifies pleuromutilin through (phenylithio)acetic acid structure to obtain a pleuromutilin (phenylithio)acetic acid ester. Preliminary antibacterial activity experiment shows the compound has excellent antibacterial activity and has high medical research and application value in the treatment of infectious diseases caused by multi-drug-resistant bacteria.
In one embodiment, the present invention provides a compound with anti-drug resistant bacteria activity having the following formula (I):
In another embodiment, a method of preparing the compound of formula (I) includes: reacting a compound of formula (II) with a compound of formula (III) to obtain the compound of formula (I):
In another embodiment, the reaction of the compound of formula (II) with the compound of formula (III) comprises the following steps: placing the compound of formula (II) and the compound of formula (III), in a molar ratio of 1:1 to 1:1.3, in a reactor; adding an organic solvent and a catalytic amount of triethylamine under nitrogen atmosphere to obtain a reaction mixture; heating the reaction mixture at 20-60° C. for 3-6 hours; extracting the reaction mixture with ethyl acetate to obtain a crude product; and purifying the crude product on a silica gel fresh chromatography column with petroleum ether and ethyl acetate as an eluent to obtain the compound of formula (I).
In another embodiment, the organic solvent is toluene, dichloromethane or N,N-dimethylformamide.
In another embodiment, the organic solvent is dichloromethane.
In another embodiment, the molar ratio of the compound of formula (II) and the compound of formula (III) is 1:1.1.
In another embodiment, the reaction mixture is heated at 25° C.
In another embodiment, the reaction mixture is heated for 5 hours.
In another embodiment, the eluent is petroleum ether:ethyl acetate=1:1.
In another embodiment, a method of preparing the compound of formula (I) includes: reacting a compound of formula (II) with a compound of formula (IV) to obtain the compound of formula (I):
In another embodiment, the reaction of the compound of formula (II) with the compound of formula (IV) comprises the following steps: placing the compound of formula (II), a catalyst, and an ionic liquid in a reactor under nitrogen atmosphere, the catalyst being 12-molybdosilicic acid hydrate (H6Mo12O41Si); adding the compound of formula (IV) to the reactor to form a reaction mixture; heating the reaction mixture at 20-50° C. for 4-8 hours; placing the reaction mixture in a separating funnel to separate a crude product; purifying the crude product by recrystallization in methanol to obtain the compound of formula (I); and recycling the ionic liquid.
In another embodiment, the ionic liquid is 1-octyl-3-methylimidazolium hexafluorophosphate, 1-hexyl-3-methylimidazolium tetrafluoroborate, or 1-butyl-3-methylimidazolium tetrafluoroborate ([BMM][BF4]).
In another embodiment, the ionic liquid is the ionic liquid is 1-butyl-3-methylimidazolium tetrafluoroborate.
In another embodiment, the compound of formula (II) and the compound (IV) have a molar ratio of 1:1 to 1:1.3.
In another embodiment, the molar ratio of the compound of formula (II) and the compound of formula (IV) is 1:1.1.
In another embodiment, the reaction mixture is heated at 30° C.
In another embodiment, the reaction mixture is heated for 6 hours.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention.
In the drawings:
Reference will now be made in detail to embodiments of the present invention, example of which is illustrated in the accompanying drawings. The following examples illustrate the present invention, but the present invention is not limited to the following examples.
Preparation of pleuromutilin (phenylithio)acetic acid ester (the compound of formula I) ((3 aS,4R,5S,6S,8R,9R,9aR,12R)-5-hydroxy-4,6,9,12-tetramethyl-1-oxo-6-vinyl-decahydro-3a,9-propanocyclopenta[8]annulen-8-yl 2-(2-(phenylthio)acetoxy)acetate)
In a 100 mL three-necked flask, 246.0 mg (0.65 mmol) and 6.1 mg (0.06 mmol) triethylamine were dissolved in 15 mL of dichloromethane under nitrogen atmosphere. 134.4 mg (0.72 mmol) of (phenylithio)acetic acid chloride (compound of formula III) was dissolved in 10 mL of dichloromethane, and added dropwise to the reaction mixture by a separatory funnel. After the completion of the dropwise addition, the reaction was carried out at 25° C. for 5 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 concentrated, washed in water, extracted with ethyl acetate, concentrated and dried to obtain a crude product. The crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate 1:1 as eluent, and the eluent was combined and concentrated under reduced pressure to obtain 246.2 mg of the title compound, a yield of 71.64%.
1-NMR (400 MHz, Chloroform-d) δ (ppm): 7.42(3H, d), 6.45 (2H, d), 5.79 (1H, t), 5.23 (2H, d), 5.07 (2H, s), 4.54 (2H, t), 3.96 (1H, t), 3.73 (1H, d), 3.35 (1H, d), 2.31 (1H, s), 2.20 (4H, t), 1.47-1.13 (11H, t), 1.13 (3H, t), 0.89 (3H, s), 0.72 (3H, s); 13C-NMR (100 MHz, Chloroform-d) δ (ppm): 216.7, 168.9, 166.1, 138.7, 134.7, 130.0, 129.0, 127.1, 117.3, 74.5, 70.6, 69.8, 61.9, 58.0, 44.7, 44.0, 41.4, 36.0, 26.8, 24.8, 16.7, 16.5, 14.7, 11.4.
In a 100 mL three-necked flask, 246.0 mg (0.65 mmol) and 6.1 mg (0.06 mmol) triethylamine were dissolved in 15 mL of N,N-dimethylformamide under nitrogen atmosphere. 134.4 mg (0.72 mmol) of (phenylithio)acetic acid chloride was dissolved in 10 mL of N,N-dimethylformamide, and added dropwise to the reaction mixture by a separatory funnel. After the completion of the dropwise addition, the reaction was carried out at 25° 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 concentrated, washed in water, extracted with ethyl acetate, concentrated and dried to obtain a crude product. The crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate 2:1 as eluent, and the eluent was combined and concentrated under reduced pressure to obtain 220.4 mg of the title compound, a yield of 64.13%.
In a 100 mL three-necked flask, 246.0 mg (0.65 mmol) and 6.1 mg (0.06 mmol) triethylamine were dissolved in 15 mL of toluene under nitrogen atmosphere. 134.4 mg (0.72 mmol) of (phenylithio)acetic acid chloride was dissolved in 10 mL of toluene, and added dropwise to the reaction mixture by a separatory funnel. After the completion of the dropwise addition, the reaction was carried out at 30° 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 concentrated, washed in water, extracted with ethyl acetate, concentrated and dried to obtain a crude product. The crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate 1:1 as eluent, and the eluent was combined and concentrated under reduced pressure to obtain 223.63 mg of the title compound, a yield of 65.08%.
In a 100 mL three-necked flask, 246.0 mg (0.65 mmol) and 6.1 mg (0.06 mmol) triethylamine were dissolved in 15 mL of dichloromethane under nitrogen atmosphere. 145.6 mg (0.78 mmol) of (phenylithio)acetic acid chloride was dissolved in 10 mL of dichloromethane, and added dropwise to the reaction mixture by a separatory funnel. After the completion of the dropwise addition, the reaction was carried out 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 concentrated, washed in water, extracted with ethyl acetate, concentrated and dried to obtain a crude product. The crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate 1:1 as eluent, and the eluent was combined and concentrated under reduced pressure to obtain 230.7 mg of the title compound, a yield of 67.13%.
In a 100 mL three-necked flask, 246.0 mg (0.65 mmol) and 6.1 mg (0.06 mmol) triethylamine were dissolved in 15 mL of N,N-dimethylformamide under nitrogen atmosphere. 145.6 mg (0.78 mmol) of (phenylithio)acetic acid chloride was dissolved in 10 mL of N,N-dimethylformamide, and added dropwise to the reaction mixture by a separatory funnel. After the completion of the dropwise addition, the reaction was carried out at 40° 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 concentrated, washed in water, extracted with ethyl acetate, concentrated and dried to obtain a crude product. The crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate 1:1 as eluent, and the eluent was combined and concentrated under reduced pressure to obtain 223.0 mg of the title compound, a yield of 64.89%.
In a 100 mL three-necked flask, 246.0 mg (0.65 mmol) and 6.1 mg (0.06 mmol) triethylamine were dissolved in 15 mL of dichloromethane under nitrogen atmosphere. 158.7 mg (0.85 mmol) of (phenylithio)acetic acid chloride was dissolved in 10 mL of dichloromethane, and added dropwise to the reaction mixture by a separatory funnel. After the completion of the dropwise addition, the reaction was carried out at 40° C. for 5 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 concentrated, washed in water, extracted with ethyl acetate, concentrated and dried to obtain a crude product. The crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate 1:1 as eluent, and the eluent was combined and concentrated under reduced pressure to obtain 233.1 mg of the title compound, a yield of 67.83%.
In a 100 mL three-necked flask, 246.0 mg (0.65 mmol) and 6.1 mg (0.06 mmol) triethylamine were dissolved in 15 mL of toluene under nitrogen atmosphere. 134.4 mg (0.72 mmol) of (phenylithio)acetic acid chloride was dissolved in 10 mL of toluene, and added dropwise to the reaction mixture by a separatory funnel. After the completion of the dropwise addition, the reaction was carried out at 60° C. for 3 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 concentrated, washed in water, extracted with ethyl acetate, concentrated and dried to obtain a crude product. The crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate 1:1 as eluent, and the eluent was combined and concentrated under reduced pressure to obtain 215.2 mg of the title compound, a yield of 62.62%.
In a 100 mL three-necked flask, 246.0 mg (0.65 mmol) and 6.1 mg (0.06 mmol) triethylamine were dissolved in 15 mL of dichloromethane under nitrogen atmosphere. 145.6 mg (0.7 8 mmol ) of (phenylithio)acetic acid chloride was dissolved in 10 mL of dichloromethane, and added dropwise to the reaction mixture by a separatory funnel. After the completion of the dropwise addition, the reaction was carried out at 50° C. for 5 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 concentrated, washed in water, extracted with ethyl acetate, concentrated and dried to obtain a crude product. The crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate 1:1 as eluent, and the eluent was combined and concentrated under reduced pressure to obtain 231.7 mg of the title compound, a yield of 67.42%.
In a 100 mL three-necked flask, 246.0 mg (0.65 mmol) and 6.1 mg (0.06 mmol) triethylamine were dissolved in 15 mL of toluene under nitrogen atmosphere. 145.6 mg (0.78 mmol) of (phenylithio)acetic acid chloride was dissolved in 10 mL of toluene, and added dropwise to the reaction mixture by a separatory funnel. After the completion of the dropwise addition, the reaction was carried out at 50° C. for 5 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 concentrated, washed in water, extracted with ethyl acetate, concentrated and dried to obtain a crude product. The crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate 2:1 as eluent, and the eluent was combined and concentrated under reduced pressure to obtain 220.8 mg of the title compound, a yield of 64.25%.
Preparation of pleuromutilin (phenylithio)acetic acid ester
In a 100 mL three-necked flask, 246.0 mg (0.65 mmol) and 6.1 mg (0.06 mmol) triethylamine were dissolved in 15 mL of toluene under nitrogen atmosphere. 121.3 mg (0.65 mmol) of (phenylithio)acetic acid chloride was dissolved in 10 mL of toluene, and added dropwise to the reaction mixture by a separatory funnel. After the completion of the dropwise addition, the reaction was carried out 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 concentrated, washed in water, extracted with ethyl acetate, concentrated and dried to obtain a crude product. The crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate 1:1 as eluent, and the eluent was combined and concentrated under reduced pressure to obtain 218.15 mg of the title compound, a yield of 63.48%.
In a 100 mL three-necked flask, 246.0 mg (0.65 mmol) and 6.1 mg (0.06 mmol) triethylamine were dissolved in 15 mL of dichloromethane under nitrogen atmosphere. 134.4 mg (0.72 mmol) of (phenylithio)acetic acid chloride was dissolved in 10 mL of dichloromethane, and added dropwise to the reaction mixture by a separatory funnel. After the completion of the dropwise addition, the reaction was carried out at 35° 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 concentrated, washed in water, extracted with ethyl acetate, concentrated and dried to obtain a crude product. The crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate 1:1 as eluent, and the eluent was combined and concentrated under reduced pressure to obtain 226.2 mg of the title compound, a yield of 65.82%.
In a 100 mL three-necked flask, 246.0 mg (0.65 mmol) and 6.1 mg (0.06 mmol) triethylamine were dissolved in 15 mL of dichloromethane under nitrogen atmosphere. 121.3 mg (0.65 mmol) of (phenylithio)acetic acid chloride was dissolved in 10 mL of dichloromethane, and added dropwise to the reaction mixture by a separatory funnel. After the completion of the dropwise addition, the reaction was carried out at 30° 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 concentrated, washed in water, extracted with ethyl acetate, concentrated and dried to obtain a crude product. The crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate 1:1 as eluent, and the eluent was combined and concentrated under reduced pressure to obtain 231.8 mg of the title compound, a yield of 67.45%.
In a 250 mL three-necked flask, 246.0 mg (0.65 mmol) of pleuromutilin, 121.1 mg (0.72 mmol) of (phenylithio)acetic acid and 12.0 mg (0.007 mmol) silicomolybdic acid were dissolved in 100 mL of 1-butyl-3-methylimidazolium tetrafluoroborate under nitrogen atmosphere. After full dissolution, the temperature of the reaction mixture was raised to 30° C. and the reaction was carried out 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 system was allowed to separate into layers to give a crude product. 1-Butyl-3-methylimidazolium tetrafluoroborate was recovered. The crude product was recrystallized with 30 mL methanol and dried to obtain 297.5 mg of the title compound, a yield of 86.57%.
In a 250 mL three-necked flask, 246.0 mg (0.65 mmol) of pleuromutilin, 121.1 mg (0.72 mmol) of (phenylithio)acetic acid and 12.0 mg (0.007 mmol) silicomolybdic acid were dissolved in 100 mL of 1-butyl-3-methylimidazolium tetrafluoroborate under nitrogen atmosphere. After full dissolution, the temperature of the reaction mixture was raised to 20° C. and the reaction was carried out 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 system was allowed to separate into layers to give a crude product. 1-Butyl-3-methylimidazolium tetrafluoroborate was recovered. The crude product was recrystallized with 30 mL methanol and dried to obtain 273.3 mg of the title compound, a yield of 79.53%.
In a 250 mL three-necked flask, 246.0 mg (0.65 mmol) of pleuromutilin, 121.1 mg (0.72 mmol) of (phenylithio)acetic acid and 12.0 mg (0.007 mmol) silicomolybdic acid were dissolved in 100 mL of 1-octyl-3-methylimidazolium hexafluorophosphate under nitrogen atmosphere. After full dissolution, the temperature of the reaction mixture was raised to 40° C. and the reaction was carried out 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 system was allowed to separate into layers to give a crude product. 1-Octyl-3-methylimidazolium hexafluorophosphate was recovered. The crude product was recrystallized with 30 mL methanol and dried to obtain 266.3 mg of the title compound, a yield of 77.49%.
In a 250 mL three-necked flask, 246.0 mg (0.65 mmol) of pleuromutilin, 121.1 mg (0.72 mmol) of (phenylithio)acetic acid and 12.0 mg (0.007 mmol) silicomolybdic acid were dissolved in 100 mL of 1-hexyl-3-methylimidazolium tetrafluoroborate under nitrogen atmosphere. After full dissolution, the temperature of the reaction mixture was raised to 40° C. and the reaction was carried out 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 system was allowed to separate into layers to give a crude product. 1-Hexyl-3-methylimidazolium tetrafluoroborate was recovered. The crude product was recrystallized with 30 mL methanol and dried to obtain 257.8 mg of the title compound, a yield of 75.02%.
The antimicrobial efficacy was determined by a paper diffusion method drug sensitivity test.
Experimental strains: multi-resistant Staphylococcus aureus 171, multi-resistant Staphylococcus aureus 222, multi-resistant Staphylococcus aureus 596. The experimental strains were 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 pleuromutilin (phenylithio)acetic acid ester (30 μg/tablet).
Reagents: LB agar medium, LA broth medium, dichloromethane.
Equipment: Ultra-clean workbench, high-pressure sterilization pot, gas bath constant temperature shaking incubator.
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 (1.5×108 CFU/mL). A bacterial suspension is obtained.
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 (1.5×158 CFU/mL). A bacterial suspension is obtained.
LB dry powder was weighed, sterilized at 103.4 Kpa, 121.3° C. high-pressure steam for 15 min, and then placed it in a 40° C.-50° C. water bath. A sterile empty plate (inner diameter 9 cm) was placed on the surface of the ultra-clean table water table, and LB dry powder was poured to the plate. The thickness of each plate was 3 mm to 4 mm. After the plate was cooled at room temperature, it was stored in the refrigerator at 2° C.-8° C. A sterile cotton swab was used to dip the bacterial solution and to 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. Sterile forceps were used to closely attach the antibacterial drug paper to the dish. The dish was put upside down and placed in a 37° C. incubator for 24 h. The results were observed by measuring 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 of 15 mm-16 mm, intermediary; the inhibition zone ≤14 mm, drug resistance.
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