Novel pleuromutilin derivative containing aniline amide phenyl side chain as well as synthesis method and application of novel pleuromutilin derivative

Abstract

The invention belongs to the field of medicinal chemistry, and discloses a novel pleuromutilin derivative containing an aniline amide phenyl side chain, a synthesis method of the novel pleuromutilin derivative and application of the novel pleuromutilin derivative to preparation of a medicine for preventing and treating mycoplasma. The derivative is a compound with a structure as shown in a formula 2 or a pharmaceutically acceptable salt thereof, and a solvent compound, an enantiomer, a diastereoisomer, a tautomer or a mixture of the compound with the structure as shown in the formula 2 or the pharmaceutically acceptable salt thereof in any proportion, including a racemic mixture. The pleuromutilin derivative has good antibacterial and anti-mycoplasma activity and low toxicity, and is especially suitable for being used as a novel antibacterial agent for systemic system infection of animals or human beings. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a novel pleuromutilin derivative containing an aniline amide phenyl side chain and a synthesis method and application thereof, in particular to an application in preparing a drug for preventing and treating mycoplasma. Background Art

[0002] The nitro group is one of the common and unique functional groups in medicinal chemistry. It can be found in many anti-tumor drugs, antibiotics, anti-tuberculosis drugs, anti-parasitic drugs, sedatives, pesticides and herbicides. The nitro group has a strong electron-withdrawing ability, which can produce local electron-deficient sites in the molecule and interact with biological nucleophiles such as proteins, amino acids, nucleic acids and enzymes in living systems through nucleophilic addition, electron transfer or complexation. However, the nitro group is a recognized structural warning and a well-known toxic group in drug design. Because of the toxicity of the nitro group, experienced medicinal chemists mostly avoid the nitro group. Many nitro-containing drugs are mutagenic, genotoxic and myelosuppressive. Therefore, structural modification of nitro-containing derivatives is beneficial to reduce their toxic side effects and increase the possibility of drug development (such as replacing the nitro group with a heterocyclic aromatic group or reducing the nitro group to reduce its toxic side effects).

[0003] Mycoplasma is the smallest and simplest prokaryotic organism discovered so far. The only organelle in mycoplasma cells is the ribosome. Mycoplasma has multiple pathogenicity, such as community-acquired pneumonia caused by Mycoplasma pneumoniae, and urogenital tract infections caused by Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma genitalium. Animal pathogenic mycoplasma often causes respiratory infections in livestock and poultry in veterinary clinics. Because it has no cell wall, antibiotics such as penicillin and cephalosporin that act on bacterial cell walls are ineffective against it. In human clinical medicine, macrolide antibacterial drugs such as erythromycin, roxithromycin and azithromycin are the first choice for the treatment of mycoplasma infections. Fluoroquinolones and tetracyclines are also used to treat mycoplasma pneumonia. In veterinary clinics, in addition to the above drugs, pleuromutilin drugs such as tylosin and valnemulin are also widely used in the treatment of various mycoplasma infections such as respiratory diseases of livestock and poultry caused by mycoplasma infections.

[0004] Pleuromutilin (Formula 1) is a natural tricyclic diterpenoid compound produced by the cultivation of Pleurotus mutilus and Pleurotus Passeckerianus. It has a structure such as a 5-6-8 tricyclic carbon skeleton with eight stereocenters and a glycolic acid chain at C(14). Studies have shown that pleuromutilin and its derivatives exhibit effective antibacterial activity against Gram-positive bacteria. Unlike other antibacterial drugs widely used in clinical practice, pleuromutilin and its derivatives can inhibit bacterial protein synthesis by binding to the V region of the peptidyl transferase center (PTC) of the 23s RNA of the bacterial 50s ribosomal subunit.

[0005]

[0006] It is reported that pleuromutilin drugs have a strong killing effect on mycoplasma, can kill pathogens quickly and efficiently, and the treatment cycle is short. Therefore, the development of new pleuromutilin derivatives for the prevention and treatment of mycoplasma infection has broad prospects. Summary of the invention

[0007] In order to overcome the shortcomings and deficiencies in the prior art, the primary purpose of the present invention is to provide a novel truncated pleuromutilin derivative containing an aniline amide phenyl side chain, which has both antibacterial and antimycoplasma activities and is particularly suitable as a novel antibacterial drug for systemic infections in animals or humans.

[0008] Another object of the present invention is to provide a method for synthesizing the above-mentioned pleuromutilin derivative containing anilinoamide phenyl side chain.

[0009] Another object of the present invention is to provide the application of the above-mentioned pleuromutilin derivative containing aniline amide phenyl side chain.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] A novel pleuromutilin derivative containing an aniline amide phenyl side chain, wherein the derivative is a compound having a structure shown in Formula 2 or a pharmaceutically acceptable salt thereof, and a solvent compound, an enantiomer, a diastereomer, a tautomer or a mixture thereof in any proportion, including a racemic mixture, of the compound having a structure shown in Formula 2 or a pharmaceutically acceptable salt thereof:

[0012]

[0013] Wherein, R1 is hydrogen or alkyl such as C1-8 alkyl, preferably hydrogen;

[0014] X is O, S or NR7, R7 is hydrogen or C 1-4 Alkyl, X is preferably O;

[0015] Wherein, R2, R3, R4, R5, and R6 are one of hydrogen and amino;

[0016] Preferably, R2 is hydrogen, R3 is amino, R4 is hydrogen, R5 is amino, and R6 is hydrogen;

[0017] Or R2 is hydrogen, R3 is hydrogen, R4 is amino, R5 is hydrogen, and R6 is hydrogen;

[0018] Or R2 is amino, R3 is hydrogen, R4 is hydrogen, R5 is hydrogen, and R6 is hydrogen.

[0019] The specific groups of the compounds of the above preferred structures are summarized in Table 1:

[0020] Table 1 Compound numbers and specific groups

[0021]

[0022] The pharmaceutically acceptable salt is a salt formed by the compound of the structure shown in Formula 2 and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

[0023] The pharmaceutically acceptable salt preferably has the following structural formula 3:

[0024]

[0025] The synthesis method of the novel pleuromutilin derivative containing an aniline amide phenyl side chain comprises the following steps:

[0026] (1) Add pleuromutilin and p-toluenesulfonyl chloride to solvent 1, mix, add triethylamine dropwise while stirring, react at room temperature for 3-4 hours, then add 4-aminothiophenol dropwise, continue to react at room temperature for 3-4 hours, then add water dropwise, filter, wash, and dry, and the resulting crude product is added to solvent 2 for pulping purification, filter, wash, and dry to obtain an intermediate I with a structure shown in formula 4;

[0027]

[0028] (2) adding the intermediate I obtained in step (1), a nitro-containing phenylacyl chloride and a base to a solvent 3, stirring at room temperature for 4 to 12 hours to carry out an amidation reaction, and extracting, drying and purifying the obtained reaction product to obtain an intermediate-II as shown in formula 5;

[0029]

[0030] wherein R2' is nitro, and R3', R4', R5' and R6' are hydrogen; or R6' is nitro, and R2', R3', R4' and R5' are hydrogen; or R4' is nitro, and R2', R3', R5' and R6' are hydrogen; or R3' and R5' are nitro, and R2', R4' and R6' are hydrogen;

[0031] (3) The intermediate II obtained in step (2), iron powder and acid are added to solvent 4 and stirred at room temperature for 4 to 12 hours. The reaction solution is filtered, water is added, filtered, washed, dried, mixed and purified by column to obtain a truncated pleuromutilin derivative with a structure shown in Formula 2.

[0032] The solvent 1 in step (1) is any one of acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, and 1,4-dioxane; the most preferred solvent is acetonitrile;

[0033] The solvent 2 is any one of methyl tert-butyl ether, tetrahydrofuran, isopropyl ether and diethyl ether; the most preferred solvent is methyl tert-butyl ether;

[0034] The step of adding water dropwise, filtering, washing and drying is as follows: adding water dropwise to the reaction solution, precipitating solid, filtering, washing with water for three times and drying under vacuum.

[0035] The pulping purification, filtration, washing and drying are specifically as follows: the crude product is added to methyl tert-butyl ether and stirred at room temperature for 1 to 2 hours, filtered, washed with methyl tert-butyl ether for 3 times, and dried under vacuum.

[0036] The molar volume ratio of the intermediate I, the nitro-containing phenylacyl chloride, the base and the solvent 3 in step (2) is 1-3 mmol: 1-6 mmol: 1-10 mmol: 3-10 ml;

[0037] The solvent 3 is any one of dichloromethane, acetonitrile, tetrahydrofuran, dioxane, and toluene; the most preferred solvent is dichloromethane;

[0038] The base is any one of sodium hydroxide, potassium hydroxide, lithium hydroxide, DIPEA, triethylamine, DBU, potassium carbonate, cesium carbonate, and sodium carbonate; the most preferred is potassium carbonate;

[0039] The extraction is performed using dichloromethane, the drying is performed using anhydrous sodium sulfate, and the purification is performed using column chromatography.

[0040] The molar volume ratio of the intermediate II, iron powder, acid and solvent 4 described in step (3) is 1mmol:1-12mmol:2-24mmol:1-10ml;

[0041] The solvent 4 is one of methanol, ethanol and isopropanol, and methanol is the most preferred;

[0042] The steps of filtering, dripping water, filtering, washing, drying, mixing, and column purification are as follows: filtering the reaction solution through diatomaceous earth, dripping water into the filtrate, precipitating solids, filtering to obtain a crude product, dissolving the crude product in ethyl acetate, adding silica gel to dryness, and column purification.

[0043] The application of the novel pleuromutilin derivative containing anilinoamide phenyl side chain in the preparation of drugs for treating drug-resistant Staphylococcus aureus, multidrug-resistant bacteria or mycoplasma infectious diseases.

[0044] The application of the novel pleuromutilin derivative containing anilinoamide phenyl side chain in the preparation of drugs for simultaneously treating drug-resistant Staphylococcus aureus, multidrug-resistant bacteria and mycoplasma infectious diseases.

[0045] The medicament may contain one or more pharmaceutically acceptable carriers, excipients or diluents;

[0046] The drug preparations include a variety of clinical drug dosage forms, such as tablets, injections, liposome nanoparticles, controlled release agents, etc.

[0047] The drug may refer to an antibiotic drug, a novel pleuromutilin derivative containing an effective amount of anilamide phenyl side chain, and the remainder is a pharmaceutical excipient or other compatible drug;

[0048] The pharmaceutical excipients refer to conventional pharmaceutical excipients, such as solvents, disintegrants, flavoring agents, preservatives, colorants and adhesives;

[0049] The other compatible drugs refer to the effective dose of pleuromutilin derivatives containing aniline amide phenyl in the side chain as drug raw materials, and then combined with other natural drugs or chemical drugs;

[0050] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0051] (1) The pleuromutilin derivatives synthesized in the present invention are based on the structural formulas of compound 5, compound 6 and compound 14 disclosed in the inventor's previous research results (patent application with publication number CN114940671A) (abbreviated as P5, P6, P14 for the convenience of discussion and analysis in this patent application). After further optimization, three new types of compounds P5-1, P6-1 and P14-1 that have not been reported before were obtained.

[0052] (2) After extensive and in-depth research on the background of knowledge, the present invention synthesized three novel truncated pleuromutilin derivatives P5-1, P6-1, and P14-1 containing aniline amide phenyl side chains after reduction modification of the nitro group recognized in medicinal chemistry research, and conducted extensive in vitro antibacterial activity evaluations. It was found that the compounds still had good anti-infection ability after reduction modification, with no significant difference compared with those before modification, and were significantly better than tylosin, and were therefore particularly suitable as new antibacterial drugs for the prevention and treatment of infectious diseases in humans or animals.

[0053] (3) The antibacterial ability of the pleuromutilin derivatives P5-1, P6-1, and P14-1 prepared by the present invention is much better than that of tylosin; and there is no significant difference in their antibacterial activity compared with some compounds disclosed in the patent application with publication number CN114940671A, so they also have the potential to be developed into new antibiotics.

[0054] (4) The inhibitory effects of the pleuromutilin derivatives P5-1, P6-1, and P14-1 prepared by the present invention and the compounds (P5, P6, and P14) disclosed in the patent application with publication number CN114940671A on the growth of human Mycoplasma pneumoniae ATCC15531 and ATCC29342 are not significantly different compared with those before modification, and therefore they also have the potential to be developed as drugs against Mycoplasma pneumoniae infection.

[0055] (5) The truncated pleuromutilin derivatives P5-1, P6-1, and P14-1 containing anilinoamide phenyl prepared by the present invention and the compounds (P5, P6, and P14) disclosed in the patent application with publication number CN114940671A have inhibitory effects on the growth of Mycoplasma synoviae MS, CH13, CH18, CH19, and CH20 of Gallinarum far superior to that of tylosin before modification, and therefore have the potential to be developed as drugs against livestock and poultry Mycoplasma.

[0056] (6) Compared with the compounds disclosed in the patent application with publication number CN114940671A, the P5-1, P6-1 and P14-1 prepared by the present invention have no obvious toxic inhibitory effect on primate-derived cells such as human lung cancer cells (A549), human cervical cancer cells (HeLa), African green monkey kidney cells (Vero), and human liver cancer cells (HepG2), indicating that they are safer and more conducive to drug development.

[0057] (7) The predicted clogp value of the pleuromutilin derivatives containing aniline amide phenyl in the side chain prepared by the present invention is closer to 5 than some compounds disclosed in the patent application with publication number CN114940671A, so the drugability of the compounds of the present invention is improved.

[0058] Chart Description

[0059] Figure 1 It is the hydrogen nuclear magnetic resonance spectrum of compound P5-1.

[0060] Figure 2 This is the carbon NMR spectrum of compound P5-1.

[0061] Figure 3 This is the H NMR spectrum of compound P6-1.

[0062] Figure 4 This is the carbon NMR spectrum of compound P6-1.

[0063] Figure 5 This is the H NMR spectrum of compound P14-1.

[0064] Figure 6 This is the carbon NMR spectrum of compound P14-1.

[0065] Figure 7 The present invention relates to the effects of tylosin, compounds P5, P5-1, P6, P6-1, P14 and P14-1 on the cell viability of A549 (human lung cancer cells) at different concentrations of 10 μg / mL to 50 μg / mL.

[0066] Figure 8 The present invention relates to the effects of tylosin, compounds P5, P5-1, P6, P6-1, P14, and P14-1 on the viability of HeLa (human cervical cancer cell) cells at different concentrations of 10 μg / mL to 50 μg / mL.

[0067] Fig. 9 The present invention relates to the effects of tylosin, compounds P5, P5-1, P6, P6-1, P14 and P14-1 on the cell viability of Vero (African green monkey kidney cells) at different concentrations of 10 μg / mL to 50 μg / mL.

[0068] Fig.10 The present invention relates to the effects of tylosin, compounds P5, P5-1, P6, P6-1, P14, and P14-1 on the viability of HepG2 (human liver cancer cells) cells at different concentrations of 10 μg / mL to 50 μg / mL.

[0069] Fig.11 It is the structural formula of compounds P5, P6, and P14 disclosed in the patent application with publication number CN114940671A and the structural formula of P5-1, P6-1, and P14-1 of the present invention. DETAILED DESCRIPTION

[0070] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0071] Example 1

[0072] 1. Preparation of Intermediate I:

[0073] 10 g of pleuromutilin and 5.27 g (27.73 mmol) of p-toluenesulfonyl chloride were dissolved in 50 mL of acetonitrile, and 6.69 g (66.10 mmol) of triethylamine was added dropwise under stirring in an ice bath. After the addition was completed, the mixture was stirred at room temperature for 3 h. Then, 3.97 g (31.75 mmol) of 4-aminothiophenol was added dropwise under stirring at room temperature. After the addition was completed, the mixture was stirred at room temperature for 12 h. Then, 250 mL of deionized water was added dropwise to precipitate a solid. The solid was stirred for 30 min, filtered, and washed with 250 mL of deionized water. The crude product was dried under vacuum. The crude product was added into 50 mL of methyl tert-butyl ether, stirred at room temperature for 2 h, filtered, washed with 50 mL of methyl tert-butyl ether, and dried to obtain an intermediate I having a structure shown in Formula 4, with a yield of 95%.

[0074]

[0075] Example 2

[0076] 1. Preparation of pleuromutilin derivative intermediates II (P5, P6, P14)

[0077] 4.94 mmol of the intermediate I prepared in Example 1 was dissolved in 5 mL of dichloromethane and 8.24 mmol of potassium carbonate was added, 4.12 mmol of nitro-containing benzoyl chloride was added dropwise, and the mixture was stirred at room temperature for 4 h. 50 mL of dichloromethane and 100 mL of deionized water were added, and the mixture was transferred to a separatory funnel and shaken, and allowed to stand for stratification; the organic phase was taken and washed again with 100 mL of dilute hydrochloric acid; dried over anhydrous sodium sulfate, and purified by column chromatography (petroleum ether: ethyl acetate = 2:1 ~ dichloromethane: methanol = 10:1), and concentrated to obtain the intermediate II with the structure shown in Formula 5;

[0078]

[0079] Example 3

[0080] 1. Preparation of pleuromutilin derivatives (P5-1, P6-1, P14-1)

[0081] 7.88 mmol of the intermediate II (P5, P6, P14) prepared in Example 2 was dissolved in 25 mL of methanol, 4.728 mmol of iron powder and 4.728 mmol of acetic acid were added, and the mixture was heated at 50°C overnight for reaction. The mixture was filtered with diatomaceous earth and rinsed with 25 mL of methanol. The filtrate was added dropwise to 150 mL of water and filtered with suction. The filter cake was washed with 50 mL of water, dried in vacuo, and purified by column chromatography (petroleum ether: ethyl acetate = 2:1 to dichloromethane: methanol = 10:1). After concentration, the pleuromutilin derivatives P5-1, P6-1, and P14-1 having the structure shown in formula (2) were obtained.

[0082] The H NMR spectra of the obtained pleuromutilin derivatives P5-1, P6-1 and P14-1 are as follows: Figure 1 , Figure 3 and Figure 5 As shown, the C NMR spectra of pleuromutilin derivatives P5-1, P6-1, and P14-1 are respectively as shown in Figure 2 , Figure 4 and Figure 6 As shown, the structures of the three derivatives are shown in Formula 2, the groups are shown in Table 1, and the specific structures are shown in Fig.11 .

[0083] The above-mentioned preparation synthetic route is shown in the following formula:

[0084]

[0085] The yields of the above compounds are summarized in Table 2.

[0086] Table 2 Compound numbers and yields

[0087]

[0088] Effect Example

[0089] (1) In vitro antibacterial test

[0090] The experiment used the broth dilution method. The experimental control drug was tylosin. Tylosin is a pleuromutilin antibiotic and one of the top ten veterinary antibiotics in the world.

[0091] The strains used in the experiment were methicillin-resistant Staphylococcus aureus ATCC43300 and Staphylococcus aureus ATCC29213, clinical Staphylococcus aureus AD3 and clinical Staphylococcus aureus 144 (clinical bacteria AD3 and 144 were isolated and identified in the Pharmacology Laboratory of the School of Veterinary Medicine of South China Agricultural University, and have been published in the literature Zhe ZA, Kang LA, Gyz A, et al. Design, synthesis and biological activities of novel pleuromutilin derivatives with a substituted triazole moiety as potent antibacterial agents-Science Direct [J]. European Journal of Medicinal Chemistry, 2020.).

[0092] Preparation of target compound stock solution: 3.2 mg of target compound (compounds P5, P6, P14, P5-1, P6-1, P14-1 and tiamulin prepared in Examples 2 and 3) were accurately weighed and placed in a 10 mL volumetric flask, dissolved with 0.25 mL of DMSO, added with 9.5 mL of distilled water, 0.25 mL of Tween 80 to volume, and shaken well to obtain a stock solution (3.2 mg / mL), sterilized with a 0.22 μm filter membrane, divided into small tubes, and stored at -20°C. The control drug tiamulin was also prepared according to the above method.

[0093] Preparation of bacterial solution: Take out the strains that are well preserved at -20℃ and inoculate them on new MH plates. After culturing at 37℃ for 24 hours, pick out a single colony and inoculate it in MH medium for another 24 hours. Select a single colony and transfer it to sterile physiological saline and adjust its turbidity to 0.6McF. At this time, the concentration of bacterial solution is 10 6 CFU / mL.

[0094] MIC plate preparation: dilute the target compound stock solution (3.2 mg / mL) 20 times to obtain a target compound solution with a concentration of 160 μg / mL; take a sterile 96-well plate, add 180 μL MH broth medium to the first well, add 100 μL MH broth medium to the second to tenth wells, add 20 μL of antibacterial drugs with a concentration of 160 μg / mL to the first well, mix well, take 100 μL and add it to the second well, mix well, and then draw 100 μL to the third well, and so on, draw 100 μL from the 12th well and discard it. At this time, the drug concentrations of each well are: 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03 μg / mL, and three groups of parallel drugs are made for each concentration.

[0095] Inoculation: Add 100 μL of bacterial solution to each well from 1 to 12, so that the final bacterial concentration in each well is about 5×10 5 CFU / mL, the drug concentrations in wells 1 to 12 were 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015, 0.00725, 0.003125, and 0.001 μg / mL, respectively. The inoculated 96-well plate was placed in a 37°C incubator for culture, and the growth of the bacterial solution was observed for 24 hours. The control drug tylosin was determined in the same way, and the lowest drug concentration that completely inhibited bacterial growth in the small well was MIC. The bacteria in the positive control well (i.e., without drug) needed to grow significantly. When a single jump hole appeared in the microbroth dilution method, the highest drug concentration that inhibited the bacteria was recorded. If multiple jump holes appeared, the test needed to be repeated.

[0096] Table 3 shows the MIC results, which shows that the target compound has good antibacterial activity against the selected strains and has good inhibitory activity against drug-resistant Staphylococcus aureus. It is particularly suitable as a new antibacterial drug for the prevention and treatment of infectious diseases caused by human or animal or drug-resistant Staphylococcus aureus or multidrug-resistant bacteria.

[0097] Table 3 In vitro antibacterial data

[0098]

[0099]

[0100] (2) In vitro mycoplasma activity inhibition test

[0101] The experiment used the agar double dilution method to determine the minimum inhibitory concentration (MIC) of some of the series of compounds obtained in Example 2 of the present invention.

[0102] The mycoplasma strains selected in the experiment were Mycoplasma pneumoniae ATCC15531, Mycoplasma pneumoniae ATCC29342, Mycoplasma gallinarum MS, Mycoplasma gallinarum CH13, Mycoplasma gallinarum CH18, Mycoplasma gallinarum CH19, and Mycoplasma gallinarum CH20.

[0103] Preparation of test compound stock solution: All test compounds were dissolved in 1 mL of DMF, and then diluted to an initial concentration of 5120 μg / mL in a 250 mL volumetric flask as a stock solution. The stock solution was diluted in culture dishes by doubling dilution method, with each culture dish containing 2 mL of the drug solution, and diluted to 20 mL with melted MH medium, so that the final concentrations of the test compounds in the series of culture dishes were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.0313, 0.0152, 0.0078, 0.0031, 0.0017, 0.0009 μg / mL, respectively.

[0104] Arrange 15 sterile 2mL EP tubes per row, add 0.9mL of liquid culture medium to the first tube and 0.5mL to the remaining tubes, take 0.1mL of the prepared liquid and add it to the first tube and mix thoroughly, then take 0.5mL and add it to the second tube, and so on. Finally, discard the 0.5mL that is aspirated out, then add 0.5mL of the diluted bacterial solution to each tube, cover the lid, make a blank control, and culture at 37℃ to observe the growth of mycoplasma.

[0105] Table 4-1 and Table 4-2 show the results of the in vitro inhibition MIC of the compounds against mycoplasma (unit: μg / mL).

[0106] Table 4-1 In vitro mycoplasma activity inhibition data

[0107]

[0108]

[0109] Table 4-2 In vitro mycoplasma activity inhibition data

[0110]

[0111] The data in Table 4 show that the compounds of the present invention have the same good inhibitory effect on the growth of human Mycoplasma pneumoniae ATCC15531 as some compounds disclosed in the patent application with publication number CN114940671A, so the modified compounds also have the potential to be developed as drugs against Mycoplasma pneumoniae infection.

[0112] (3) Cytotoxicity assay

[0113] The experiment used the MTT method.

[0114] Preparation of target compound stock solution: accurately weigh 50 mg of target compound (compound obtained in Examples 2 and 3), dissolve in 500 μL of DMSO, shake well to obtain stock solution (100 mg / mL), sterilize with 0.22 μm filter membrane, and store at -20°C for future use.

[0115] Cell recovery, culture and passaging:

[0116] After the frozen A549, Hela, Vero, and HepG2 cells were taken out of liquid nitrogen, they were quickly thawed in a 37°C water bath, and 4 mL of preheated 10% DMEM complete medium (10% (v / v) fetal bovine serum) was added and mixed, and the centrifuge tube was moved to a centrifuge for centrifugation for 3 min (speed was 1000 r / min). The supernatant was discarded, and the cell pellet was resuspended in DMEM complete medium and 1640 complete medium respectively, and the cell suspension was moved to a dedicated 90 mm cell culture dish (Gibco), and then placed in a cell-specific incubator for culture.

[0117] Subculturing: The cells in the culture dish are cultured in the incubator until the growth confluence rate reaches 90%, and then subculture is performed. First, the original culture medium is discarded, and the cells are rinsed with PBS (0.01M, pH 7.2-7.4), and the PBS is discarded. Complete culture medium is added to the adherent cells and gently blown to make them fall off. After fully mixing, the cell suspension is transferred to the culture dish at a ratio of 1:3. After shaking to evenly distribute the cells, it is placed in a constant temperature incubator with 5% CO2 for continued static culture.

[0118] Cryopreservation: Select cells with good morphology and in the logarithmic growth phase for cryopreservation. First, discard the old culture medium of the culture dish, rinse the cells with PBS and discard the PBS, add 3mL of complete culture medium and gently blow to make the adherent cells fall off and mix, transfer the cell suspension to a centrifuge tube, and centrifuge at 1000r / min for 3min. After centrifugation, discard the upper liquid, add the prepared cell freezing solution (DMSO: FBS = 1:9), blow the cell pellet evenly and transfer it to a cell freezing tube, put it in a -80℃ refrigerator for 24h, and finally store it in liquid nitrogen.

[0119] MTT incubation and OD value determination: Collect cells in good growth state and add 100 μL of each well of a 96-well plate, about 5×10 5 Cells with a density of 100 μg / mL were plated. The 96-well plate was placed in a constant temperature incubator with 5% CO2 overnight. The complete culture medium was discarded, and the test compound was added with a final concentration of 10μg / mL, 20μg / mL, 30μg / mL, 40μg / mL, and 50μg / mL, and incubated in a constant temperature incubator with 5% CO2 for 24 hours. The supernatant was discarded, and freshly prepared MTT with a concentration of 0.5mg / mL (dissolved in the basal culture medium to prepare a 5mg / mL mother solution, and stored in aliquots at -20℃) was added and incubated in the dark for 2 hours. The supernatant was discarded, 150μL DMSO was added, and the plate was placed in an oscillator at room temperature and oscillated at 70r / min for 10 minutes. The absorbance value was measured at 490nm with an enzyme reader, and the cell survival rate was calculated. The experiment was repeated three times independently. The formula is as follows:

[0120] Cell survival rate (%) = OD drug group / OD control group × 100%

[0121] The toxicity test results of the compounds are as follows Figures 7-10 As shown, compounds P5-1, P6-1, and P14-1 had no obvious toxic inhibitory effects on the selected cell lines at concentrations of 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL, and some compounds had certain growth-promoting effects, and were suitable as new antibacterial drugs for the prevention and treatment of infectious diseases caused by humans or animals or drug-resistant Staphylococcus aureus or multidrug-resistant bacteria.

[0122] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0123] (5) Prediction of lipid-water partition coefficient of compounds

[0124] The clogP value of each compound was predicted using Chem3D. The experimental results are shown in Table 5

[0125] Table 5 clogP values ​​of pleuromutilin derivatives

[0126]

[0127] The data in the above table show that the predicted clogP value of the compound of the present invention is closer to 5 than some compounds disclosed in the patent application with publication number CN114940671A, and therefore the compound of the present invention is more drugable.

[0128] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A novel pleuromutilin derivative containing an aniline amide phenyl side chain, characterized in that: The derivative is a compound of the structure shown in Formula 2 or a pharmaceutically acceptable salt thereof, and a solvent compound, enantiomer, diastereomer, tautomer or a mixture thereof in any proportion, including a racemic mixture, of the compound of the structure shown in Formula 2 or a pharmaceutically acceptable salt thereof: Wherein, R1 is hydrogen or C1-8 alkyl; X is O, S or NR7; R7 is hydrogen or C 1-4 alkyl; Among them, R2, R3, R4, R5, and R6 are one of hydrogen and amino.

2. The novel pleuromutilin derivative containing an aniline amide phenyl side chain according to claim 1, characterized in that: R1 is hydrogen, X is oxygen, R2 is hydrogen, R3 is amino, R4 is hydrogen, R5 is amino, and R6 is hydrogen; Or R1 is hydrogen, X is oxygen, R2 is hydrogen, R3 is hydrogen, R4 is amino, R5 is hydrogen, and R6 is hydrogen; Alternatively, R1 is hydrogen, X is oxygen, R2 is amino, R3 is hydrogen, R4 is hydrogen, R5 is hydrogen, and R6 is hydrogen.

3. The novel pleuromutilin derivative containing an aniline amide phenyl side chain according to claim 1, characterized in that: The pharmaceutically acceptable salt is a salt formed by the compound of the structure shown in Formula 2 and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

4. The method for synthesizing a novel pleuromutilin derivative containing an aniline amide phenyl side chain according to any one of claims 1 to 2, characterized in that The following steps are included: (1) Add pleuromutilin and p-toluenesulfonyl chloride to solvent 1, mix, add triethylamine dropwise while stirring, react at room temperature for 3-4 hours, then add 4-aminothiophenol dropwise, continue to react at room temperature for 3-4 hours, then add water dropwise, filter, wash, and dry, and the resulting crude product is added to solvent 2 for pulping purification, filter, wash, and dry to obtain an intermediate I with a structure shown in formula 4; (2) adding the intermediate I obtained in step (1), a nitro-containing phenylacyl chloride and a base to a solvent 3, stirring at room temperature for 4 to 12 hours to carry out an amidation reaction, and extracting, drying and purifying the obtained reaction product to obtain an intermediate-II as shown in formula 5; wherein R2' is nitro, and R3', R4', R5' and R6' are hydrogen; or R6' is nitro, and R2', R3', R4' and R5' are hydrogen; or R4' is nitro, and R2', R3', R5' and R6' are hydrogen; or R3' and R5' are nitro, and R2', R4' and R6' are hydrogen; (3) The intermediate II obtained in step (2), iron powder and acid are added to solvent 4 and stirred at room temperature for 4 to 12 hours. The reaction solution is filtered, water is added, filtered, washed, dried, mixed and purified by column to obtain a truncated pleuromutilin derivative with a structure shown in Formula 2.

5. The synthesis method according to claim 4, characterized in that: The solvent 1 in step (1) is any one of acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide and 1,4-dioxane; The solvent 2 is any one of methyl tert-butyl ether, tetrahydrofuran, isopropyl ether, and diethyl ether; The step of adding water dropwise, filtering, washing and drying is as follows: adding water dropwise to the reaction solution, precipitating solid, filtering, washing with water three times, and drying under vacuum; The pulping purification, filtration, washing and drying are specifically as follows: the crude product is added to methyl tert-butyl ether and stirred at room temperature for 1 to 2 hours, filtered, washed with methyl tert-butyl ether for 3 times, and dried under vacuum.

6. The synthesis method according to claim 4, characterized in that: The molar volume ratio of the intermediate I, the nitro-containing phenylacyl chloride, the base and the solvent 3 in step (2) is 1-3 mmol: 1-6 mmol: 1-10 mmol: 3-10 ml; The solvent 3 is any one of dichloromethane, acetonitrile, tetrahydrofuran, dioxane, and toluene; The base is any one of sodium hydroxide, potassium hydroxide, lithium hydroxide, DIPEA, triethylamine, DBU, potassium carbonate, cesium carbonate, and sodium carbonate; The extraction is performed using dichloromethane, the drying is performed using anhydrous sodium sulfate, and the purification is performed using column chromatography.

7. The synthesis method according to claim 4, characterized in that: The molar volume ratio of the intermediate II, iron powder, acid and solvent 4 described in step (3) is 1mmol:1-12mmol:2-24mmol:1-10ml; The solvent 4 is one of methanol, ethanol and isopropanol; The process of filtration, dripping water, filtration, washing, drying, sample mixing and column purification is as follows: the reaction solution is filtered through diatomaceous earth, water is dripped into the filtrate, solid is precipitated, and the crude product is obtained by filtration. The crude product is dissolved in ethyl acetate, silica gel is added and spin-dried, and the product is purified by column purification.

8. Use of the novel pleuromutilin derivative containing anilinoamide phenyl side chain according to claim 1 in the preparation of drugs for treating drug-resistant Staphylococcus aureus, multidrug-resistant bacteria or mycoplasma infectious diseases.

9. Use of a novel pleuromutilin derivative containing an aniline amide phenyl side chain according to claim 1 in the preparation of a drug for simultaneously treating drug-resistant Staphylococcus aureus, multidrug-resistant bacteria and mycoplasma infectious diseases.

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