Pleuromutilin derivative having 2-amino phenyl mercaptan side chain as well as preparation method and application thereof

Abstract

A pleuromutilin derivative having a 2-amino phenyl mercaptan side chain as well as a preparation method and application thereof are provided. The derivative has a structure represented by formula 2 or formula 3, wherein, R1, R2 and R3 are each independently selected from a hydrogen atom, hydroxyl, amino, sulfydryl, hydroxymethyl, amine methyl, nitro, halogen, trihalogenated methyl, methyl, natural amino acid acylamino and C1-6 alkoxy. The plueuromutilin derivative in the disclosure has good activity of inhibiting drug-resistant Staphylococcus aureus and mycoplasma, and is especially suitable for preventing and treating infectious diseases caused by human or animal mycoplasma or drug-resistant Staphylococcus aureus or multidrug resistant bacteria as a novel antibacterial drug.

Claims

1. A method for preparing a pleuromutilin derivative having a 2-amino phenyl mercaptan side chain, comprising the following steps: (1) reacting pleuromutilin with paratoluensulfonyl chloride to obtain an intermediate I having a structure of formula 4; ##STR00008## (2) reacting the intermediate I with sodium iodide to be further activated, and then reacting a resulting product with 2-amino phenyl mercaptan under an alkaline condition to obtain an intermediate II having the structure of formula 3; and ##STR00009## (3) reacting the intermediate II with various substituted benzoic acids in the presence of a condensing agent to obtain the pleuromutilin derivative having the structure of formula 2; ##STR00010## wherein, R.sub.1 R.sub.2 and R.sub.3 are each independently selected from the group consisting of hydrogen atom, hydroxyl, amino, sulfydryl, hydroxymethyl, amine methyl, nitro, halogen, trihalogenated methyl, methyl, and C1-6 alkoxy.

2. The method according to claim 1, wherein, the reaction in step (1) is carried out for 3 hours at 0 C. with pyridine as a solvent; a molar ratio of paratoluensulfonyl chloride to pleuromutilin is 1.1:1.

3. The method according to claim 1, wherein, the reaction in step (2) is carried out in a non-protonic solvent, and the intermediate I is dissolved into the non-protonic solvent, wherein, an amount of the solvent is 30 times the mass of the intermediate I; then anhydrous sodium iodide is added, and heating reflux is carried out for 1 hour, wherein, a molar ratio of the intermediate I to anhydrous sodium iodide is 1:1.1; prior to the reaction, 2-amino phenyl mercaptan and alkali are dissolved into water at first, and then subjected to heating reflux for 2 hours together with a product obtained by activation, wherein, a molar ratio of the intermediate I to 2-amino phenyl mercaptan is 1:1.1, and a molar ratio of 2-amino phenyl mercaptan to alkali is 1:2; the alkali is sodium hydroxide, potassium hydroxide, cesium hydroxide, sodium carbonate, potassium carbonate or cesium carbonate; and the non-protonic solvent is N,N-dimethylformamide.

4. The method according to claim 1, wherein, the reaction in step (3) is carried out in a non-protonic solvent, substituted benzoic acid is added and then condensed for 1-36 hours with the intermediate II at 0-70 C. in the presence of the condensing agent while heating and stirring are carried out, and recrystallization or column chromatography purification is carried out; wherein, the non-protonic solvent is dichloromethane, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide or pyridine; the condensing agent is methyl chloroformate, ethyl chloroformate, ter-butyl chloroformate, carbonyl diimidazole (CDI), sulfonyl chloride, Boc anhydride, N,N-dicyclohexyl carbodiimide (DCC), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate, O-benzotriazole-N,N,NN-tetramethylurea tetrafluoroboric acid, 2-(7-oxidized benzotriazole)-N,N,NN-tetramethylurea hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxyl-benzotriazole, 1H-benzotriazole-1-oxotripyrrolidyl hexafluorophosphate, oxalyl chloride and thionyl chloride.

Description

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(1) The disclosure will be further described in combination with examples, but embodiments of the disclosure are not limited thereto. Numbers of compounds obtained in the following examples 1-12 are as shown in claims.

(2) An Example of Preparation of an Intermediate I

(3) 5.4 g (14.27 mmol) of pleuromutilin was dissolved into 30 ml of pyridine and subjected to ice bath until a temperature reaches about 0 C., and 8.6 g (45.11 mmol) of paratoluensulfonyl chloride was added. After the reaction was carried out for 3 h on ice bath under the condition of stirring, 50 ml of ice water was added for quenching reaction. A reaction liquid was poured into a separating funnel, 50 ml of chloroform was added at first for layering, a water phase was removed, subsequently, 100 ml of 2 mol/L sulfuric acid solution was used to wash an organic phase twice, then 50 ml of saturated sodium bicarbonate solution was used to wash the organic phase twice, and finally, 100 ml of deionized water was used to wash the organic phase twice and then the organic phase was dried using anhydrous sodium sulfate. Rotary evaporation was carried out on the organic phase, the residual solid was added with 10 ml of isopropanol and dissolved by heating, a large amount of white powders were separated out after cooling, suction filtration was carried out, a filtrate was washed with isopropanol, and the residual liquid of the product while powder was naturally volatilized to be dried. The obtained white powder was the intermediate I. The yield was 88.84%.

(4) An Example of Preparation of an Intermediate II (a Compound of Formula 3, a Compound 13)

(5) 1 g (1.88 mmol) of intermediate I was dissolved into 35 ml of ethyl acetate, 0.31 g (2.07 mmol) of anhydrous sodium iodide was added, and heating and stirring were carried out at about 70 C. to react for 1 h. 0.25 g (2.04 mmol) of 2-amino phenyl mercaptan was taken and placed into 10 ml of water, 0.08 g (2.04 mmol) of sodium hydroxide was added into aqueous solution, the above aqueous solution was added into a reaction system, and heating and stirring were carried out at about 70 C. to react for 2 h. A reaction liquid was poured into a separating funnel, 30 ml of chloroform was added for extraction, and an organic phase was taken. Rotary evaporation was carried out on the obtained organic phase to obtain a mixture, the obtained mixture was redissolved with dichloromethane, 1 g of silica gel having 100-200 meshes was added and sufficiently mixed, the above crude product-silica gel powder mixture is purified via column chromatography (silica gel powder having 200-300 meshes was a stationary phase, and a mobile phase was composed of petroleum ether and ethyl acetate in a ratio of 1:2) after the solvent is completely volatilized, so as to obtain a purified product of the intermediate II having a structure of formula 3. The yield was 81.22%.

Example 1: synthesis of 22-O-[2-(2-methylbenzamido) phenyl] thioacetyl valnemulin (compound 1)

(6) 0.82 g (1.88 mmol) of intermediate II was dissolved into 35 ml of ethyl acetate, 2-methyl benzoic acid (2.07 mmol) and 2.07 mmol of oxalyl chloride were added, and heating and stirring were carried out at about 70 C. to react for 1 h to obtain a target product. Rotary evaporation was carried out on the obtained mixed solution to obtain a mixture, the obtained mixture was redissolved with dichloromethane, 1 g of silica gel having 100-200 meshes was added and sufficiently mixed, and the above crude product-silica gel powder mixture was purified via column chromatography (silica gel powder having 200-300 meshes was a stationary phase, and a mobile phase was composed of petroleum ether and ethyl acetate in a ratio of 1:1) after the solvent was completely volatilized, so as to obtain a purified product of 22-O-[2-(2-methylbenzamide) phenyl] thioacetyl valnemulin (compound 1). The yield was 86.55%. HR-MS (ESI): Cal: 604.3091; Found: 604.3117.

Example 2: synthesis of 22-O-[2-(3-methylbenzamnido) phenyl] thioacetyl valnemulin (compound 2)

(7) 0.82 g (1.88 mmol) of intermediate II was dissolved into 35 ml of dichloromethane, 3-methyl benzoic acid (2.07 mmol) and 2.07 mmol of ter-butyl chloroformate were added, and heating and stirring were carried out at about 70 C. to react for 1 h to obtain a target product. Rotary evaporation was carried out on the obtained mixed solution to obtain a mixture, the obtained mixture was redissolved with dichloromethane, 1 g of silica gel having 100-200 meshes was added and sufficiently mixed, and the above crude product-silica gel powder mixture was purified via column chromatography (silica gel powder having 200-300 meshes was a stationary phase, and a mobile phase was composed of petroleum ether and ethyl acetate in a ratio of 1:1) after the solvent was completely volatilized, so as to obtain a purified product of 22-O-[2-(3-methylbenzamide) phenyl] thioacetyl valnemulin (compound 2). The yield was 85.75%. HR-MS (ESI): Cal: 604.3091; Found: 604.3110.

Example 3: synthesis of 22-O-[2-(4-methylbenzamido) phenyl] thioacetyl valnemulin (compound 3)

(8) 0.82 g (1.88 mmol) of intermediate II was dissolved into 35 ml of dichloromethane, 4-methyl benzoic acid (2.07 mmol) and 2.07 mmol of thionyl chloride were added, and heating and stirring were carried out at about 70 C. to react for 1 h to obtain a target product. Rotary evaporation was carried out on the obtained mixed solution to obtain a mixture, the obtained mixture was redissolved with dichloromethane, 1 g of silica gel having 100-200 meshes was added and sufficiently mixed, and the above crude product-silica gel powder mixture was purified via column chromatography (silica gel powder having 200-300 meshes was a stationary phase, and a mobile phase was composed of petroleum ether and ethyl acetate in a ratio of 1:1) after the solvent was completely volatilized, so as to obtain a purified product of 22-O-[2-(4-methylbenzamido) phenyl] thioacetyl valnemulin (compound 3). The yield was 83.09%. HR-MS (ESI): Cal: 604.3091; Found: 604.3110.

Example 4: synthesis of 22-O-[2-(2-fluorobenzamido) phenyl] thioacetyl valnemulin (compound 4)

(9) 0.82 g (1.88 mmol) of intermediate II was dissolved into 35 ml of N,N-dimethylformamide, 2.06 mmol of 2-fluorobenzoic acid, 2.06 mmol of 1H-benzotriazole-1-oxytripyrrolidyl hexafluorophosphate and 6 mmol of N,N-diisopropylethylamine were added, and heating and stirring were carried out at about 70 C. to react for 1 h to obtain a target product. Rotary evaporation was carried out on the obtained mixed solution to obtain a mixture, the obtained mixture was redissolved with dichloromethane, 1 g of silica gel having 100-200 meshes was added and sufficiently mixed, and the above crude product-silica gel powder mixture was purified via column chromatography (silica gel powder having 200-300 meshes was a stationary phase, and a mobile phase was composed of petroleum ether and ethyl acetate in a ratio of 1:1) after the solvent was completely volatilized, so as to obtain a purified product of 22-O-[2-(2-fluorobenzamido) phenyl] thioacetyl valnemulin (compound 4). The yield was 91.47%. HR-MS (ESI): Cal: 608.2840; Found: 608.2867.

Example 5: synthesis of 22-O-[2-(3-fluorobenzamido) phenyl] thioacetyl valnemulin (compound 5)

(10) 0.82 g (1.88 mmol) of intermediate II was dissolved into 35 ml of ethyl acetate, 3-fluorobenzoic acid (2.06 mmol), 2.06 mmol of 6-chlorobenznotriazole-1,1,3,3-tetramethylurea hexafluorophosphate and 6 mmol of triethylamine were added, and heating and stirring were carried out at about 70 C. to react for 1 h to obtain a target product. Rotary evaporation was carried out on the obtained mixed solution to obtain a mixture, the obtained mixture was redissolved with dichloromethane, 1 g of silica gel having 100-200 meshes was added and sufficiently mixed, and the above crude product-silica gel powder mixture was purified via column chromatography (silica gel powder having 200-300 meshes was a stationary phase, and a mobile phase was composed of petroleum ether and ethyl acetate in a ratio of 1:1) after the solvent was completely volatilized, so as to obtain a purified product of 22-O-[2-(3-fluorobenzamido) phenyl] thioacetyl valnemulin (compound 5). The yield was 87.39%. HR-MS (ESI): Cal: 608.2840; Found: 608.2856.

Example 6: synthesis of 22-O-[2-(4-fluorobenzamido) phenyl] thioacetyl valnemulin (compound 6)

(11) 0.82 g (1.88 mmol) of intermediate II was dissolved into 35 ml of dichloromethane, 4-fluorobenzoic acid (2.06 mmol) and 2.06 mmol of carbonyldiimidazole were added, and stirring was carried out at room temperature to react for 1 h to obtain a target product. Rotary evaporation was carried out on the obtained mixed solution to obtain a mixture, the obtained mixture was redissolved with dichloromethane, 1 g of silica gel having 100-200 meshes was added and sufficiently mixed, and the above crude product-silica gel powder mixture was purified via column chromatography (silica gel powder having 200-300 meshes was a stationary phase, and a mobile phase was composed of petroleum ether and ethyl acetate in a ratio of 1:1) after the solvent was completely volatilized, so as to obtain a purified product of 22-O-[2-(4-fluorobenzamido) phenyl] thioacetyl valnemulin (compound 6). The yield was 84.91%. HR-MS (ESI): Cal: 608.2840; Found: 608.2836.

Example 7: synthesis of 22-O-[2-(2-chlorobenzamido) phenyl] thioacetyl valnemulin (compound 7)

(12) 0.82 g (1.88 mmol) of intermediate II was dissolved into 35 ml of ethyl acetate, 2-chlorobenzoic acid (2.06 mmol) and 2.06 mmol of 1-(3-dimethyl amino propyl)-3-ethyl carbondiimine were added, and heating and stirring were carried out at 50 C. to react for 1 h to obtain a target product. Rotary evaporation was carried out on the obtained mixed solution to obtain a mixture, the obtained mixture was redissolved with dichloromethane, 1 g of silica gel having 100-200 meshes was added and sufficiently mixed, and the above crude product-silica gel powder mixture was purified via column chromatography (silica gel powder having 200-300 meshes was a stationary phase, and a mobile phase was composed of petroleum ether and ethyl acetate in a ratio of 1:1) after the solvent was completely volatilized, so as to obtain a purified product of 22-O-[2-(2-chlorobenzamido) phenyl] thioacetyl valnemulin (compound 7). The yield was 87.86%. HR-MS (ESI): Cal: 624.2545; Found: 624.2575.

Example 8: synthesis of 22-O-[2-(3-chlorobenzamido) phenyl] thioacetyl valnemulin (compound 8)

(13) 0.82 g (1.88 mmol) of intermediate II was dissolved into 35 ml of N,N-dimethylformamide, 2.06 mmol of 3-chlorobenzoic acid and 2.06 mmol of N,N-bicyclohexylcarbondiimine were added, and heating and stirring were carried out at 25 C. to react for 1 h to obtain a target product. Rotary evaporation was carried out on the obtained mixed solution to obtain a mixture, the obtained mixture was redissolved with dichloromethane, 1 g of silica gel having 100-200 meshes was added and sufficiently mixed, and the above crude product-silica gel powder mixture was purified via column chromatography (silica gel powder having 200-300 meshes was a stationary phase, and a mobile phase was composed of petroleum ether and ethyl acetate in a ratio of 1:1) after the solvent was completely volatilized, so as to obtain a purified product of 22-O-[2-(3-chlorobenzamido) phenyl] thioacetyl valnemulin (compound 8). The yield was 85.04%. HR-MS (ESI): Cal: 624.2545; Found: 624.2562.

Example 9: synthesis of 22-O-[2-(4-chlorobenzamido) phenyl] thioacetyl valnemulin (compound 9)

(14) 0.82 g (1.88 mmol) of intermediate II was dissolved into 35 ml of N,N-dimethylacetamide, 2.06 mmol of 4-chlorobenzoic acid and 2.06 mmol of carbonyldiimidazole were added, and heating and stirring were carried out at about 30 C. to react for 1 h to obtain a target product. Rotary evaporation was carried out on the obtained mixed solution to obtain a mixture, the obtained mixture was redissolved with dichloromethane, 1 g of silica gel having 100-200 meshes was added and sufficiently mixed, and the above crude product-silica gel powder mixture was purified via column chromatography (silica gel powder having 200-300 meshes was a stationary phase, and a mobile phase was composed of petroleum ether and ethyl acetate in a ratio of 1:1) after the solvent was completely volatilized, so as to obtain a purified product of 22-O-[2-(4-chlorobenzamido) phenyl] thioacetyl valnemulin (compound 9). The yield was 89.66%. HR-MS (ESI): Cal: 624.2545; Found: 624.2540.

Example 10: synthesis of 22-O-[2-(2-methoxybenzamido) phenyl] thioacetyl valnemulin (compound 10)

(15) 0.82 g (1.88 mmol) of intermediate II was dissolved into 35 ml of ethyl acetate, 2.05 mmol of 2-methoxybenzoic acid, 2.05 mmol of methyl chloroformate and 2.5 mmol of morpholine were added, and stirring were carried out at 25 C. to react for 1 h to obtain a target product. Rotary evaporation was carried out on the obtained mixed solution to obtain a mixture, the obtained mixture was redissolved with dichloromethane, 1 g of silica gel having 100-200 meshes was added and sufficiently mixed, and the above crude product-silica gel powder mixture was purified via column chromatography (silica gel powder having 200-300 meshes was a stationary phase, and a mobile phase was composed of petroleum ether and ethyl acetate in a ratio of 1:1) after the solvent was completely volatilized, so as to obtain a purified product of 22-O-[2-(2-methoxybenzamido) phenyl] thioacetyl valnemulin (compound 10). The yield was 92.24%. HR-MS (ESI): Cal: 620.3040; Found: 620.3037.

Example 11: synthesis of 22-O-[2-(3-methoxybenzamido) phenyl] thioacetyl valnemulin (compound 11)

(16) 0.82 g (1.88 mmol) of intermediate II was dissolved into 35 ml of N,N-dimethylacetamide, 2.05 mmol of 3-methoxybenzoic acid and 2.05 mmol of O-benzotriazole-N,N,N,N-tetramethylurea tetraboric acid and 6 mmol of N-methyl morpholine were added, and stirring were carried out at 0 C. to react for 3 h to obtain a target product. Rotary evaporation was carried out on the obtained mixed solution to obtain a mixture, the obtained mixture was redissolved with dichloromethane, 1 g of silica gel having 100-200 meshes was added and sufficiently mixed, and the above crude product-silica gel powder mixture was purified via column chromatography (silica gel powder having 200-300 meshes was a stationary phase, and a mobile phase was composed of petroleum ether and ethyl acetate in a ratio of 1:1) after the solvent was completely volatilized, so as to obtain a purified product of 22-O-[2-(3-methoxybenzamido) phenyl] thioacetyl valnemulin (compound 11). The yield was 94.82%. HR-MS (ESI): Cal: 620.3040; Found: 620.3062.

Example 12: synthesis of 22-O-[2-(4-methoxybenzamido) phenyl] thioacetyl valnemulin (compound 12)

(17) 0.82 g (1.88 mmol) of intermediate II was dissolved into 35 ml of ethyl acetate, 2.05 mmol of 4-methoxybenzoic acid and 2.05 mmol of ethyl chloroformate were added, and heating and stirring were carried out at 70 C. to react for 36 h to obtain a target product. Rotary evaporation was carried out on the obtained mixed solution to obtain a mixture, the obtained mixture was redissolved with dichloromethane, 1 g of silica gel having 100-200 meshes was added and sufficiently mixed, and the above crude product-silica gel powder mixture was purified via column chromatography (silica gel powder having 200-300 meshes was a stationary phase, and a mobile phase was composed of petroleum ether and ethyl acetate in a ratio of 1:1) after a solvent was completely volatilized, so as to obtain a purified product of 22-O-[2-(4-methoxybenzamido) phenyl] thioacetyl valnemulin (compound 11). The yield was 87.07%. HR-MS (ESI): Cal: 620.3040; Found: 620.3067.

(18) Experiment Example: In-Vitro Bacteriostatic Experiment

(19) Experimental Method

(20) Minimum inhibitory concentrations (MIC) of a series of compounds obtained in the disclosure were measured by using a two-fold agar dilution method. An experimental control drug selected valnemulin, and valnemulin belongs to pleuromutilin antibiotics and is the most widely applied veterinary antibiotic in the pleuromutilin antibiotics at present. By using a multi-point inoculator, bacteria solution was inoculated into a culture dish containing drugs having different concentrations at a bacteria inoculating amount of 10.sup.6 CFU/ml. A result was observed after incubation for 24 hours at 37 C., and the concentration of a compound in a culture dish without bacteria served as the minimum inhibitory concentration (MIC) of this compound.

(21) Strains used in the experiment were E. Coli ATCC25922, Staphylococcus aureus ATCC29213, methicillin-resistant staphylococcus ATCC43300 and mycoplasma gallisepticum S6.

(22) 25.6 mg of synthesized target compound was precisely weighed to be placed in 10 mL of volumetric flask and dissolved with a small amount of N,N-dimethylformamide and then a volume was metered with N,N-dimethylformamide to 10 mL, so as to prepare 2560 g/mL stock solution. In addition, 25.6 mg of pleuromutilin, tiamulin, valnemulin and retapamulin were respectively and precisely weighed to be placed in 10 ml of volumetric flask, and a volume was metered with N,N-dimethylformamide to 10 mL, so as to prepare 2560 g/mL control stock solution.

(23) The control stock solution was diluted in a culture dish with a doubling dilution method, each culture dish contained 1 ml of medicine liquid, the medicine liquid is diluted to 20 ml with molten MH agar, so that final concentrations of subject compounds in a series of culture dishes were respectively 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.0625 g/ml.

(24) MIC results are shown in Table 3 below.

(25) TABLE-US-00003 TABLE 3 in-vitro bacteriostasis data Compound/ E. coli S. aureus MRSA material ATCC25922 ATCC29213 ATCC43300 S6 1 >64 0.25 0.25 0.125 2 >64 1 0.5 0.25 3 >64 0.5 0.5 0.25 4 >64 0.5 0.5 0.25 5 >64 0.125 0.125 0.125 6 >64 1 1 0.5 7 >64 0.5 0.5 0.25 8 >64 0.25 0.25 0.125 9 >64 0.5 0.5 0.25 10 >64 1 0.5 0.25 11 >64 1 1 0.25 12 >64 1 1 0.5 13 >64 0.0156 0.0156 0.0156 Pleuromutilin, >64 1 0.5 0.0625 Tiamulin >64 0.5 0.5 0.0156 Valnemulin 32 0.0625 0.0625 0.0039 Retapamulin 0.0625 0.0625

(26) All of target compounds exhibit good antibacterial activity on staphylococcus, the antibacterial activity of most compounds on staphylococcus and methicillin-resistant staphylococcus is close to or superior to that of tiamulin widely used in clinic, the bacterial activity of a compound 13 on staphylococcus and methicillin-resistant staphylococcus is obviously superior to that of valnemulin and retapamulin widely used in clinic.

(27) The above examples are preferred embodiments of the disclosure, but embodiments of the disclosure are not limited by the above examples, and changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the disclosure are all equivalent replacement modes and are included within the protective scope of the disclosure.