Pleuromutilin derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antibacterial drugs, and discloses a pleuromutilin derivative and a preparation method and application thereof.The pleuromutilin derivative is a compound with the structure shown in the formula I and pharmaceutically acceptable salt of the compound, and in the formula, R1, R2 and R3 are H, OMe, NO2, Me, F, Cl or Br. According to the present invention, the structure is novel, the antibacterial activity is excellent, and the in vitro antibacterial experiment result shows that the antibacterial activity of more than 90% of the compound on the Gram-positive bacteria staphylococcus aureus drug-resistant strains ATCC 33591 and ATCC 43300 and the staphylococcus aureus sensitive strain ATCC 2913 is superior to the antibacterial activity of tiamulin, such that the tiamulin is expected to be used in the treatment of the bacterial infection caused by the staphylococcus aureus, and the application prospect is broad. And a new choice is provided for anti-infection drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial drugs, and particularly relates to a pleuromutilin derivative and a preparation method and use thereof. Background Art

[0002] The discovery of antibiotics is of great significance in the history of human development. However, due to the widespread and long-term use of antibiotics in clinical practice, bacterial resistance is becoming increasingly serious, which undoubtedly poses a huge threat to human life and safety. In order to cope with the dilemma of the decreasing number of effective antimicrobial drugs and the increasing number of drug-resistant bacteria, it is urgent to find an effective way to solve this thorny global problem. In 2012, the U.S. Food and Drug Administration (FDA) launched an incentive program to promote the development of antibiotics (generating antibiotics incentives now, GAIN). Among the anti-infective (antibacterial, antifungal, antiparasitic and antiviral) drugs on the market, nearly 70% are directly derived from natural products or obtained by modifying their structures.

[0003] Pleuromutilin is a tricyclic diterpenoid compound with good antibacterial activity isolated from higher fungi Pleurotus multilus (Fr.) Sacc. and Pleurotus Passecke- rianus Pilat by Kavanagh and his colleagues in 1951. Studies have shown that pleuromutilin binds to the 23S rRNA of the 50S subunit of the bacterial ribosome, and locates in the peptidyl transferase (PTC) center of the 50S subunit of the ribosome through its tricyclic mother nucleus, forming a tight pocket at the A site. At the same time, its C14 side chain partially covers the P site of tRNA binding, which can prevent aminoacyl transfer RNA from binding to the P site and inhibit the activity of peptidyl transferase, further preventing the translation process of bacterial proteins, thereby inhibiting bacterial growth. This type of compound selectively inhibits the synthesis of prokaryotic cell proteins, has no effect on eukaryotic cells, and does not interact with mammalian ribosomes. It is precisely because of this special mechanism of action that it is not easy to produce cross-resistance with other antibiotics. Although pleuromutilin has certain antibacterial activity, it is difficult to develop into an effective antibacterial drug because it is slightly soluble in water and has a low absorption rate in the body. Therefore, since the discovery of pleuromutilin, researchers have been studying its structure-activity relationship and structural modification, trying to introduce various polar groups in order to find pleuromutilin derivatives with high antibacterial activity, good water solubility and high bioavailability.

[0004] Structure-activity relationship (SAR) studies have shown that the tricyclic structure of its parent nucleus, the ester group at C-14, the carbonyl group at C-2, and the hydroxyl group at C-11 are all functional groups necessary for antibacterial activity. In recent years, many researchers have achieved good results in the derivatization of its C14 side chain. In 1979, the first marketed drug of pleuromutilin derivatives, Tamulin, was successfully approved as a veterinary antibiotic. Subsequently, in 1999, Valnemulin was successfully launched and became the second derivative approved as a veterinary antibiotic. In addition, in 1980, Azamulin entered the clinical stage. Azamulin has excellent antibacterial effects, but it was terminated due to its strong inhibition of human cytochrome P450, its low oral bioavailability and short half-life. In 2007, GlaxoSmithKline developed the first topical pleuromutilin derivative for the treatment of human skin infections, Ratapamulin. In 2006, Nabriva developed Lafemulin (BC-3781), a new semi-synthetic pleuromutilin antibacterial agent, mainly used to treat acute bacterial skin and skin structure infections (ABSSSI) and community-acquired bacterial pneumonia (CABP). On August 19, 2019, the FDA approved Nabriva's new drug application (NDAs) for Lafemulin for the treatment of CABP by oral and intravenous injection.

[0005] The structural formulas of Pleuromutilin, Tamulin, Valnemulin, Azamulin, Ratapamulin and Lafemulin (BC-3781) are as follows: Pleuromutilin has been developing rapidly in veterinary drugs, but its role in the research of human antibacterial drugs has not yet been fully reflected. Based on unmet clinical needs, it is urgent to develop drugs with novel structures, unique mechanisms of action and higher antibacterial activity.

[0006] Benzoheterocyclic compounds are widely used in the field of pharmaceutical chemistry, among which coumarin is a bifuran ring compound. Because its structure contains a π-π conjugated system, it can bind to various active sites in the body through non-covalent bonding (hydrophobic interaction, π-π electronic interaction, and hydrogen bonding, etc.), which makes coumarin compounds have a very wide range of biological activities. In the field of medicine, it has antibacterial, anti-inflammatory, anti-HIV, anti-depression, antioxidant, anticoagulant, anticancer, antiviral, antifungal and anti-asthma effects. Based on this, many researchers have introduced coumarin derivatives into the C-14 side chain of truncated pleuromutilin to improve its activity and pharmacokinetic properties.

[0007] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0008] The object of the present invention is to provide a pleuromutilin derivative and a preparation method and use thereof, thereby overcoming the defects in the above-mentioned prior art.

[0009] In order to achieve the above object, the present invention provides a pleuromutilin derivative, wherein the pleuromutilin derivative is a compound having a structure shown in Formula I and a pharmaceutically acceptable salt thereof: (Formula I) Wherein R1, R2, R3 are H, OMe, NO2, Me, F, Cl or Br.

[0010] Further, as a preference, the pharmaceutically acceptable salt is a salt formed by a compound having a structure as shown in Formula I and hydrochloric acid, sulfuric acid, phosphoric acid, phosphorous acid, hydrobromic acid, nitric acid, malic acid, maleic acid, citric acid, fumaric acid, tartaric acid, succinic acid, acetic acid, lactic acid, benzoic acid, p-toluenesulfonic acid, methanesulfonic acid or palmitic acid.

[0011] The above method for producing a pleuromutilin derivative comprises the following steps: (1) Tosyl chloride and pleuromutilin are reacted to obtain pleuromutilin intermediate 2 as shown in the following formula: (2) The intermediate 2 is reacted with potassium carbonate and mercaptoethylamine to obtain the pleuromutilin intermediate 3 shown in the following formula: (3) Substituted salicylaldehyde b0 is reacted with diethyl malonate, glacial acetic acid and piperidine to obtain an intermediate b1 as shown in the following formula: (4) The intermediate b1 is reacted with sodium hydroxide to obtain the coumarin intermediate b2 shown in the following formula: (5) reacting the pleuromutilin intermediate 3 with the coumarin intermediate b2 to obtain a pleuromutilin derivative having the following formula I: (Formula I) Furthermore, as a preferred embodiment, the reaction conditions of step (1) are as follows: p-toluenesulfonyl chloride and pleuromutilin are dissolved in a mixed solution of methyl tert-butyl ether and water, sodium hydroxide solution is slowly added dropwise to the solution under an ice bath, and the mixture is placed at 50-70 o C for about 0.5-1.5h. After the reaction is completed, water is added and the mixture is filtered under reduced pressure. The filter cake is washed with water and dried to obtain intermediate 2.

[0012] Furthermore, as a preference, the reaction conditions of step (2) are as follows: intermediate 2, potassium carbonate and mercaptoethylamine are dissolved in acetonitrile solution, placed in an oil bath at 45-55°C for reaction, and after the reaction is complete, the reaction solution is concentrated and purified by column chromatography to obtain white powdery pleuromutilin intermediate 3.

[0013] Furthermore, as a preference, the reaction conditions of step (3) are as follows: dissolving the substituted salicylaldehyde b0 in ethanol, stirring magnetically to dissolve, adding diethyl malonate, a drop of glacial acetic acid and piperidine to the solution, condensing and refluxing to react, cooling to room temperature after the reaction, adding ethanol to the reaction solution, placing the reaction solution on a low-temperature stirrer and stirring for 25-35 minutes, a large amount of solid precipitates in the solution, filtering and collecting the residue, washing the solid with glacial ethanol, and drying to obtain the intermediate b1.

[0014] Furthermore, as a preference, the reaction conditions of step (4) are as follows: intermediate b1 is dissolved in ethanol, sodium hydroxide solution is added, and the reaction is carried out at 75-85°C. After the reaction is completed, the reaction is cooled to room temperature, the reaction flask is placed in ice water, and the pH is adjusted to 2-4 with dilute hydrochloric acid solution. At this time, solids precipitate in the solution, the residue is collected by filtration, the solids are washed with ice water, and dried to obtain coumarin intermediate b2.

[0015] Furthermore, preferably, the reaction conditions of step (5) are as follows: dissolving the coumarin intermediate b2 and HATU in acetonitrile, adding triethylamine under stirring, reacting for 25-35 min under ice bath, adding the pleuromutilin intermediate 3, and then continuing the reaction at room temperature. After the reaction is completed, the reaction solution is concentrated under reduced pressure and purified by column chromatography to obtain the pleuromutilin derivative shown in formula I.

[0016] The present invention also provides stereoisomers or pharmaceutically acceptable salts of the above pleuromutilin derivatives.

[0017] Among them, pharmaceutically acceptable salts include, but are not limited to, salts of the compound of formula I with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, phosphorous acid, hydrobromic acid and nitric acid, and salts with various organic acids such as malic acid, maleic acid, citric acid, fumaric acid, tartaric acid, succinic acid, acetic acid, lactic acid, benzoic acid, p-toluenesulfonic acid, methanesulfonic acid, palmitic acid, etc.; preferably, the pharmaceutically acceptable salt is selected from hydrochloride, fumarate, malate, hydrobromide, succinate, phosphate, methanesulfonate or benzoate.

[0018] The present invention also provides a use of the above-mentioned pleuromutilin derivative, its stereoisomer or pharmaceutically acceptable salt for preparing a drug for treating infectious diseases, wherein the infectious diseases are infectious diseases caused by drug-resistant bacteria.

[0019] The present invention also provides a pharmaceutical composition for treating infectious diseases, which comprises a therapeutically effective amount of a pleuromutilin derivative, a stereoisomer or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient.

[0020] Further, as a preference, the auxiliary materials include but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum proteins, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium oxide, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, lanolin, etc.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The coumarin-type pleuromutilin derivatives connected with mercaptoethylamine of the present invention have novel structures and excellent antibacterial activity; all the compounds have antibacterial effects superior to tylosin, and most of the compounds have antibacterial effects equivalent to those of the control valnemulin. DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0023] A brief summary of one or more aspects is given below to provide a basic understanding of these aspects. This summary is not an exhaustive overview of all conceived aspects, and is neither intended to identify the key or critical elements of all aspects nor to define the scope of any or all aspects. Its only purpose is to give some concepts of one or more aspects in a simplified form as a prelude to a more detailed description that will be given later.

[0024] The present invention provides a coumarin pleuromutilin derivative connected with mercaptoethylamine, and the structural formula of the coumarin pleuromutilin derivative is shown in the following table: The preparation method of the above pleuromutilin derivative is as follows: 1. Synthesize the pleuromutilin intermediate according to the following synthetic route: Reagents and conditions: ( = 1 \* ROMAN I) TsCl, NaOH, MTBE / H2O, 60℃. (Ⅱ) Mercapto-ethylamine, Potassium carbonate, DMF, 50℃.

[0025] Steps for synthesizing intermediate 3: 1.1. Dissolve p-toluenesulfonyl chloride (4.3 g, 22.7 mmol) and pleuromutilin (7.8 g, 20.6 mmol) in 25 mL of a mixed solution of methyl tert-butyl ether and water (v / v=4:1). Slowly add 5 mL of sodium hydroxide solution (10 M) to the solution under ice bath and place at 60 o The reaction was carried out under C conditions for about 1 h. After the reaction was completed, an appropriate amount of water was added and the mixture was filtered under reduced pressure. The filter cake was washed with water and dried to obtain intermediate 2. The structure was confirmed by NMR. The data are as follows: White powder; yield: 92.3%; 1H NMR (400 MHz, CDCl3): δ (ppm) 7.81 (d,J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 6.41 (dd, J = 17.2, 11.2 Hz, 1H), 5.76 (d, J = 8.4 Hz, 1H), 5.33 (dd, J = 11.2, 1.2 Hz, 1H), 5.19 (dd, J =17.2, 1.2 Hz, 1H), 4.48 (s, 2H), 3.34 (d, J = 6.4 Hz, 1H), 2.45 (s, 3H), 2.33– 1.99 (m, 5H), 1.81 – 1.41 (m, 8H), 1.40 (s, 3H), 1.38 – 1.30 (m, 1H), 1.29 – 1.20 (m, 1H), 1.15 (s, 3H), 1.13 – 1.05 (m, 1H), 0.87 (d, J = 6.8 Hz, 3H), 0.62 (d, J = 6.8 Hz, 3H).

[0026] 1.2. Pleuromutilin p-toluenesulfonate (532.2 mg, 1.0 mmol), i.e., intermediate 2, potassium carbonate (276.4 mg, 2.0 mmol) and mercaptoethylamine (92.6 mg, 1.2 mmol) were dissolved in acetonitrile solution and placed in an oil bath at 50°C for reaction. After TLC detection showed that the reaction was complete, the reaction solution was concentrated and purified by column chromatography to obtain white powder 3 with a yield of 75.2%. The structure was confirmed by NMR. The data are as follows: White power; Yield: 75.2 %; 1H NMR (400 MHz, CDCl3) δ 6.48 (dd, J =17.2, 11.2 Hz, 1H), 5.76 (d, J = 8.4 Hz, 1H), 5.35 (d, J = 11.2 Hz, 1H), 5.21(d, J = 17.2 Hz, 1H), 3.36 (d, J = 6.4 Hz, 1H), 3.14 (s, 2H), 2.89 (t, J =6.4 Hz, 2H), 2.71 (t, J = 6.4 Hz, 2H), 2.41 – 1.98 (m, 6H), 1.83 – 1.44 (m,9H), 1.18 (s, 3H), 1.12 (dd, J = 14.0, 4.4 Hz, 1H), 0.89 (d, J = 6.8 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H).

[0027] 2. Synthesize coumarin intermediates according to the following synthetic route: Reagents and conditions: ( = 1 \* ROMAN I) Diethyl malonate, Piperidine, Acetic acid, Ethanol, 78℃. (Ⅱ) NaOH, Ethanol / H2O, 80℃.

[0028] 2.1. Steps for synthesizing intermediate b1: Dissolve substituted salicylaldehyde b0 (7.14 mmol) in ethanol (5 mL), stir magnetically to dissolve, add diethyl malonate (1.14 mL, 7.49 mmol), a drop of glacial acetic acid and piperidine (0.2 mL) to the solution, condense and reflux to react, monitor the reaction progress by TLC, after the reaction is completed, cool to room temperature, add 20 mL of ethanol to the reaction solution, stir the reaction solution on a low-temperature stirrer for 30 min, a large amount of solid precipitates in the solution, filter and collect the residue, wash the solid twice with glacial ethanol, and dry the solid to obtain intermediate b1. The structure was confirmed by NMR, and the data are as follows: 6-Fluoro-3-carboxylic acid ethyl ester coumarin (b1-2): grey powder; yield: 46%; 1H NMR (400MHz, DMSO) δ 8.71 (s, 1H), 7.81 (dd, J = 8.4, 2.8 Hz, 1H), 7.62 (td, J = 8.4,2.8 Hz, 1H), 7.50 (dd, J = 9.2, 4.4 Hz, 1H), 4.31 (q, J = 7.2 Hz, 2H), 1.32(t, J = 7.2 Hz, 3H). 7-Fluoro-3-carboxylic acid ethyl ester coumarin (b1-3): white powder; yield: 54%; 1 H NMR (400MHz, DMSO) δ 8.75 (s, 1H), 8.00 (dd, J = 8.8, 6.4 Hz, 1H), 7.41 (dd, J = 9.8,2.4 Hz, 1H), 7.30 (td, J = 8.8, 2.4 Hz, 1H), 4.29 (q, J = 7.2 Hz, 2H), 1.31(t, J = 7.2 Hz, 3H). 8-Fluoro-3-carboxylic acid ethyl ester coumarin (b1-4): Grey powder; yield: 41%; 11 H NMR (400MHz, DMSO) δ 8.78 (d, J = 1.6 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.69 (ddd, J= 10.8, 8.4, 1.2 Hz, 1H), 7.38 (td, J = 8.0, 4.8 Hz, 1H), 4.31 (q, J = 7.2Hz, 2H), 1.32 (t, J = 7.2Hz, 3H). 6-Chloro-3-carboxylic acid ethyl ester coumarin (b1-5): white powder; yield: 74%; 1 H NMR (400MHz, DMSO) δ 8.70 (s, 1H), 8.04 (d, J = 1.6 Hz, 1H), 7.76 (ddd, J = 8.8, 2.4,0.8 Hz, 1H), 7.47 (d, J = 8.8 Hz, 1H), 4.31 (q, J = 7.2 Hz, 2H), 1.32 (t, J =7.2 Hz, 3H). 7-Chloro-3-carboxylic acid ethyl ester coumarin (b1-6): pale yellow powder; yield: 64%; 1 H NMR (400 MHz, DMSO) δ 8.75 (s, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 2.0Hz, 1H), 7.47 (dd, J = 8.4, 2.0 Hz, 1H), 4.30 (q, J = 7.2 Hz, 2H), 1.31 (t, J= 7.2 Hz, 3H). 8-Chloro-3-carboxylic acid ethyl ester coumarin (b1-7): white powder; yield: 55%; 1 H NMR (400MHz, DMSO) δ 8.75 (s, 1H), 7.86 (ddd, J = 9.6, 8.0, 1.2 Hz, 2H), 7.39 (t, J =8.0 Hz, 1H), 4.31 (q, J = 7.2 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H). 6-Bromo-3-carboxylic acid ethyl ester coumarin (b1-8): grey powder; yield: 64%; 1 H NMR (400 MHz, DMSO) δ 8.70 (s, 1H), 8.17 (d, J = 2.4 Hz, 1H), 7.87 (dd, J = 8.8, 2.4 Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H), 4.31 (q, J = 7.2 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H). 7-Bromo-3-carboxylic acid ethyl ester coumarin (b1-9): yellow powder; yield: 58%; 1 H NMR (400MHz, DMSO) δ 8.70 (s, 1H), 8.17 (d, J = 2.4 Hz, 1H), 7.87 (dd, J = 8.8, 2.4Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H), 4.31 (q, J = 7.2 Hz, 2H), 1.32 (t, J = 7.2Hz, 3H). 8-Bromo-3-carboxylic acid ethyl ester coumarin (b1-10): white powder; yield: 60%; 1H NMR (400MHz, DMSO) δ 8.75 (s, 1H), 8.00 (dd, J = 8.0, 1.6 Hz, 1H), 7.93 (dd, J = 8.0,1.6 Hz, 1H), 7.34 (s, 1H), 4.31 (q, J = 7.2 Hz, 2H), 1.32 (t, J = 7.2 Hz,3H). 6-Methyl-3-carboxylic acid ethyl ester coumarin (b1-11): white powder; yield: 65%; 1 H NMR (400MHz, DMSO) δ 8.63 (s, 1H), 7.66 (d, J = 1.2 Hz, 1H), 7.53 (dd, J = 8.4, 1.6Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 4.29 (q, J = 7.2 Hz, 2H), 2.36 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H). 7-Methyl-3-carboxylic acid ethyl ester coumarin (b1-12): white powder; yield: 67%; 1 H NMR (400MHz, DMSO) δ 8.68 (s, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.23 (s, 1H), 7.21 (d, J= 8.0 Hz, 1H), 4.28 (q, J = 7.2 Hz, 2H), 2.43 (s, 3H), 1.30 (t, J = 7.2 Hz,3H). 8-Methyl-3-carboxylic acid ethyl ester coumarin (b1-13): white powder; yield: 75%; 1 H NMR (400MHz, DMSO) δ 8.69 (s, 1H), 7.71 (dd, J = 7.6, 0.8 Hz, 1H), 7.59 (dd, J = 7.6,0.8 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H), 4.30 (q, J = 7.2 Hz, 2H), 2.35 (s,3H), 1.31 (t, J = 7.2 Hz, 3H). 6-Methoxy-3-carboxylic acid ethyl ester coumarin (b1-14): yellow powder; yield: 54%; 1 H NMR(400 MHz, DMSO) δ 8.66 (s, 1H), 7.45 (d, J = 2.8 Hz, 1H), 7.28 – 7.37 (m,2H), 4.29 (q, J = 7.2 Hz, 2H), 3.80 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H). 7-Methoxy-3-carboxylic acid ethyl ester coumarin (b1-15): grey powder; yield: 68%; 1 H NMR(400 MHz, DMSO) δ 8.68 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 6.97 – 7.00 (m,2H), 4.27 (q, J = 7.2 Hz, 2H), 3.88 (s, 3H), 1.30 (t, J = 7.2 Hz, 3H). 8-Methoxy-3-carboxylic acid ethyl ester coumarin (b1-16): yellow powder; yield: 59%; 1 H NMR (400 MHz, DMSO) δ 8.71 (s, 1H), 7.44 (dd, J =8.0, 1.6 Hz, 1H), 7.40 (dd, J =8.0, 1.6 Hz, 1H), 7.35 – 7.29 (m, 1H), 4.29 (q, J = 7.2 Hz, 2H), 3.91 (s,3H), 1.31 (t, J = 7.2 Hz, 3H). 6-Nitro-3-carboxylic acid ethyl ester coumarin (b1-17): dark yellow powder; yield: 42%; 1 HNMR (400 MHz, DMSO) δ 8.91- 8.92 (m, 2H), 8.49 (dd, J = 9.2, 2.8 Hz, 1H), 7.64 (d, J = 9.2 Hz, 1H), 4.33 (q, J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H). 7-Nitro-3-carboxylic acid ethyl ester coumarin (b1-18): brown powder; yield: 49%; 1H NMR (400MHz, DMSO) δ 8.83 (s, 1H), 8.24 – 8.14 (m, 3H), 4.33 (q, J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H). 2.2. Steps for synthesizing intermediate b2: Weigh the intermediate b1 (1 g) in ethanol (4 mL), add 5 mL of 10% sodium hydroxide solution, react at 80°C, monitor the reaction progress by TLC, and after the reaction is completed, cool to room temperature, place the reaction bottle in ice water, and adjust the pH to about 3 with 10% dilute hydrochloric acid solution. At this time, a large amount of solid precipitates in the solution, filter and collect the residue, wash the solid twice with ice water, and dry the solid to obtain intermediate b2. The structure was confirmed by NMR, and the data are as follows: 3-Carboxycoumarin (b2-2) is a purchased drug.

[0029] 6-Fluoro-3-carboxycoumarin (b2-2): grey powder; yield: 70%; 1 H NMR (400 MHz, DMSO) δ 8.37 (s, 1H), 7.91 (dd, J = 8.0, 1.2 Hz, 1H), 7.83 (dd, J = 8.0, 1.2Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H). 7-Fluoro-3-carboxycoumarin (b2-3): yellow powder; yield: 68%; 1 H NMR (400 MHz, DMSO) δ 13.21 (s, 1H), 8.76 (s, 1H), 7.99 (dd, J = 8.8, 6.4 Hz, 1H), 7.42 (dd, J = 9.6, 2.4 Hz, 1H), 7.30 (td, J = 8.8, 2.4 Hz, 1H). 8-Fluoro-3-carboxycoumarin (b2-4): grey powder; yield: 66%; 1 H NMR (400 MHz, DMSO) δ 8.75 (d, J = 1.6 Hz, 1H), 7.71 (dd, J = 6.8, 1.2 Hz, 1H), 7.67 (ddd,J = 10.8, 8.4, 1.2 Hz, 1H), 7.38 (td, J = 8.4, 4.8 Hz, 1H). 6-Chloro-3-carboxycoumarin (b2-5): white powder; yield: 77%; 1 H NMR (400 MHz, DMSO) δ 8.73 (s, 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.91 (d, J = 7.6 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H). 7-Chloro-3-carboxycoumarin (b2-6): yellow powder; yield: 74%; 1 H NMR (400 MHz, DMSO) δ 8.71 (s, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 2.0 Hz, 1H), 7.46 (dd, J = 8.4, 2.0 Hz, 1H). 8-Chloro-3-carboxycoumarin (b2-7): white powder; yield: 69%; 1 H NMR (400 MHz, DMSO) δ 8.75 (s, 1H), 7.86 (ddd, J = 8.0, 7.2, 1.2 Hz, 2H), 7.39 (t, J = 8.0Hz, 1H). 6-Bromo-3-carboxycoumarin (b2-8): white powder; yield: 74%; 1H NMR (400 MHz,DMSO) δ 8.65 (s, 1H), 8.15 (d, J = 2.4 Hz, 1H), 7.86 (dd, J = 8.8, 2.4 Hz,1H), 7.40 (d, J = 8.8 Hz, 1H). 7-Bromo-3-carboxycoumarin (b2-9): yellow powder; yield: 67%; 1 H NMR (400 MHz, DMSO) δ 13.27 (s, 1H), 8.72 (s, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.74 (d, J =1.6 Hz, 1H), 7.59 (dd, J = 8.4, 1.6 Hz, 1H). 8-Bromo-3-carboxycoumarin (b2-10): white powder; yield: 65%; 1H NMR (400 MHz, DMSO) δ 8.73 (s, 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.91 (d, J = 7.6 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H). 6-Methyl-3-carboxycoumarin (b2-11): white powder; yield: 59%; 1 H NMR (400MHz, DMSO) δ 13.17 (s, 1H), 8.63 (s, 1H), 7.65 (d, J = 1.6 Hz, 1H), 7.53 (dd,J = 8.4, 1.6 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 2.36 (s, 3H). 7-Methyl-3-carboxycoumarin (b2-12): white powder; yield: 74%; 1 H NMR (400MHz, DMSO) δ 13.10 (s, 1H), 8.69 (s, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.25 (s,1H), 7.22 (d, J = 8.0 Hz, 1H), 2.43 (s, 3H). 8-Methyl-3-carboxycoumarin (b2-13): white powder; yield: 72%; 1 H NMR (400MHz, DMSO) δ 8.70 (s, 1H), 7.71 (d, J = 6.8 Hz, 1H), 7.58 (d, J = 7.6 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H), 2.36 (s, 3H). 6-Methoxy-3-carboxycoumarin (b2-14): dark yellow powder; yield: 77%; 1 H NMR(400 MHz, DMSO) δ 13.18 (s, 1H), 8.66 (s, 1H), 7.43 (d, J = 2.8 Hz, 1H), 7.36(d, J = 9.2 Hz, 1H), 7.29 (dd, J = 9.2, 2.8 Hz, 1H), 3.80 (s, 3H). 7-Methoxy-3-carboxycoumarin (b2-15): pale yellow powder; yield: 64%; 1 H NMR (400 MHz, DMSO) δ 12.94 (s, 1H), 8.70 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.00 (dt, J = 8.4, 2.4 Hz, 2H), 3.89 (s, 3H). 8-Methoxy-3-carboxycoumarin (b2-16): pale yellow powder; yield: 66%; 1 H NMR(400 MHz, DMSO) δ 13.22 (s, 1H), 8.69 (s, 1H), 7.42 (dd, J = 7.6, 1.2 Hz,1H), 7.39 (dd, J = 8.0, 1.2 Hz, 1H), 7.34 – 7.28 (m, 1H), 3.91 (s, 3H). 6-Nitro-3-carboxycoumarin (b2-17): yellow powder; yield: 74%; 1 H NMR (400MHz, DMSO) δ 13.47 (s, 1H), 8.90 (d, J = 2.8 Hz, 1H), 8.88 (s, 1H), 8.49 (dd,J = 9.2, 2.8 Hz, 1H), 7.64 (d, J = 9.2 Hz, 1H). 7-Nitro-3-carboxycoumarin (b2-18): brown powder; yield: 79%; 1 H NMR (400MHz, DMSO) δ 8.80 (s, 1H), 8.14 – 8.23 ​​(m, 3H). 3. The coumarin derivatives PB-1 to PB-18 connected with mercaptoethylamine were synthesized according to the following synthetic route: Reagents and conditions: ( = 1 \* ROMAN I) ( = 1 \* ROMAN I) HATU, Triethylamine,Acetonitrile, rt.

[0030] Weigh substituted 3-carboxyl coumarin (0.1 g, 0.48 mmol), i.e., coumarin intermediate b2 and HATU (0.18 g, 0.48 mmol) in acetonitrile (4 mL), add triethylamine (67 μL, 0.48 mmol) under stirring, react for 30 min under ice bath, add pleuromutilin intermediate 3 (0.21 g, 0.48 mmol), and then continue the reaction at room temperature. Monitor the reaction progress by TLC. After the reaction is completed, concentrate the reaction solution under reduced pressure, and purify it by column chromatography (PE:EA=2:1) ​​to obtain PB derivatives.

[0031] The structures of all compounds were confirmed by NMR and high-resolution mass spectrometry. The data are as follows: (PB-1): White power; Yield: 52%; mp: 80.0-81.5℃; 1 H NMR (400 MHz, CDCl3) δ 9.04 (s, 1H), 8.89 (s, 1H), 7.68 (s, 2H), 7.40 (d, J = 7.6 Hz, 2H), 6.55 – 6.36 (m, 1H), 5.75 (d, J = 6.8 Hz, 1H), 5.33 (d, J = 10.8 Hz, 1H), 5.19 (d, J = 17.2 Hz, 1H), 3.68 (s, 2H), 3.36 (s, 1H), 3.21 (s, 2H), 2.86 (s,2H), 2.41 – 1.90 (m, 6H), 1.84 – 1.41 (m, 9H), 1.17 (s, 3H), 1.10 (d, J =13.6 Hz, 1H), 0.87 (s, 3H), 0.74 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 217.2168.9, 161.8, 161.5, 154.6, 148.6, 139.2, 134.3, 123.0, 125.5, 118.7, 118.4,117.4, 116.8, 74.8, HRMS: calculatedfor C 34 H 42FNO7S ([M + H] + ):610.2833; found 610.2839. (PB-2): White power; Yield: 47%; mp: 71.4-73.2℃; 1 H NMR (400 MHz,CDCl3) δ 9.00 (s, 1H), 8.83 (s, 1H), 7.43 – 7.33 (m, 3H), 6.46 (dd, J = 17.2,11.2 Hz, 1H), 5.75 (d, J = 8.4 Hz, 1H), 5.33 (dd, J = 11.2, 1.2 Hz, 1H), 5.19(dd, J = 17.2, 1.2 Hz, 1H), 3.67 (q, J = 6.4 Hz, 2H), 3.35 (s, 1H), 3.20 (s,2H), 2.86 (t, J = 6.8 Hz, 2H), 2.42 – 1.99 (m, 6H), 1.81 – 1.42 (m, 9H), 1.16(s, 3H), 1.11 (dd, J = 14.0, 4.0 Hz, 1H), 0.87 (d, J = 6.8 Hz, 3H), 0.74 (d,J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 217.1, 168.9, 161.4, 161.0, 160.4,158.0, 150.8, 147.6, 147.6, 139.2, 122.0, 121.7, 119.5, 119.4, 119.4, 118.5,118.4, 117.3, 115.0, 114.7, 74.8, 69.6, 58.3, 45.6, 45.0, 44.1, 41.9, 38.8,36.9, 36.2, 34.6, 34.1, 32.2, 30.6, 27.0, 26.5, 25.0, 17.0, 15.0, 11.6. HRMS:calculated for C 34 H 42 FNO7S ([M + H] + ):628.2739; found 628.2741. (PB-3): White power; Yield: 61%; mp: 73.8-74.1℃;1 H NMR (400 MHz,CDCl3) δ 8.92 (t, J = 5.2 Hz, 1H), 8.85 (s, 1H), 7.69 (dd, J = 9.2, 6.0 Hz,1H), 7.15 – 7.08 (m, 2H), 6.46 (dd, J = 17.2, 11.2 Hz, 1H), 5.75 (d, J = 8.4Hz, 1H), 5.33 (dd, J = 11.2, 1.6 Hz, 1H), 5.18 (dd, J = 17.2, 1.6 Hz, 1H),3.66 (q, J = 6.4 Hz, 2H), 3.35 (d, J = 5.6 Hz, 1H), 3.19 (s, 2H), 2.85 (t, J= 6.8 Hz, 2H), 2.42 – 2.01 (m, 6H), 1.80 – 1.42 (m, 9H), 1.16 (s, 3H), 1.10(dd, J = 14.0, 4.0 Hz, 1H), 0.87 (d, J = 6.8 Hz, 3H), 0.73 (d, J = 6.8 Hz,3H). 13 C NMR (101 MHz, CDCl3) δ 217.1, 168.9, 167.4, 164.8, 161.6, 161.0,156.0, 155.8, 148.0, 139.2, 131.9, 131.8, 117.3, 115.5, 115.5, 114.1, 113.9,104.6, 104.4, 74.8, 69.6, 58.3, 45.6, 45.0, 44.1, 41.9, 38.8, 36.9, 36.2,34.6, 34.1, 32.3, 30.6, 27.0, 26.5, 25.0, 17.0, 15.0, 11.6. HRMS: calculatedfor C 34 H 42 FNO7S ([M + H] + ):628.2739; found 628.2739. (PB-4): White power; Yield: 68%; mp: 92.1-93℃; 1H NMR (400 MHz,CDCl3) δ 8.94 (s, 1H), 8.87 (s, 1H), 7.50 – 7.37 (m, 2H), 7.30 (td, J = 8.0,4.4 Hz, 1H), 6.44 (ddd, J = 17.2, 10.8, 2.0 Hz, 1H), 5.73 (d, J = 8.4 Hz,1H), 5.30 (d, J = 10.8 Hz, 1H), 5.16 (d, J = 17.2 Hz, 1H), 3.71 – 3.59 (m,2H), 3.34 (s, 1H), 3.18 (s, 2H), 2.84 (t, J = 6.8 Hz, 2H), 2.39 – 1.93 (m,6H), 1.80 – 1.40 (m, 9H), 1.14 (s, 3H), 1.08 (d, J = 14.4 Hz, 1H), 0.85 (d, J= 6.8 Hz, 3H), 0.72 (d, J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 217.1,168.8, 161.2, 160.0, 150.4, 148.0, 147.9, 139.1, 125.3, 125.2, 125.0, 124.9,120.4, 120.3, 120.2, 119.2, 117.2, 74.6, 69.4, 58.2, 45.5, 44.8, 43.9, 41.8,38.7, 36.8, 36.0, 34.5, 33.9, 32.1, 30.4, 26.9, 26.4, 24.9, 16.9, 14.9, 11.5.HRMS: calculated for C 34 H 42 FNO7S ([M + H] + ):628.2739; found 628.2749. (PB-5): White power; Yield: 70%; mp: 91.6-92.7℃; 1H NMR (400 MHz,CDCl3) δ 8.98 (t, J = 5.6 Hz, 1H), 8.81 (s, 1H), 7.66 (d, J = 2.4 Hz, 1H),7.60 (dd, J = 8.8, 2.4 Hz, 1H), 7.35 (d, J = 8.8 Hz, 1H), 6.47 (dd, J = 17.2,11.2 Hz, 1H), 5.75 (d, J = 8.4 Hz, 1H), 5.33 (dd, J = 11.2, 1.2 Hz, 1H), 5.19(dd, J = 17.2, 1.2 Hz, 1H), 3.67 (q, J = 6.4 Hz, 2H), 3.35 (dd, J = 9.6, 6.8Hz, 1H), 3.20 (s, 2H), 2.85 (t, J = 6.8 Hz, 2H), 2.42 – 2.01 (m, 6H), 1.81 –1.43 (m, 9H), 1.16 (s, 3H), 1.10 (dd, J = 13.6, 4.0 Hz, 1H), 0.87 (d, J = 6.8Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 217.1, 168.9,161.3, 160.9, 152.9, 147.3, 139.2, 134.2, 130.8, 128.9, 119.7, 119.5, 118.3,117.4, 74.8, 69.5, 58.3, 45.6, 45.0, 44.1, 41.9, 38.8, 36.9, 36.1, 34.6,34.1, 32.2, 30.6, 27.0, 26.5, 25.0, 17.0, 15.0, 11.6. HRMS: calculated forC 34 H 42 ClNO7S ([M + H] + ):644.2443; found 644.2440. (PB-6): White power; Yield: 43%; mp: 58.8-60.4℃; 1H NMR (400 MHz,CDCl3) δ 8.91 (t, J = 5.2 Hz, 1H), 8.81 (s, 1H), 7.60 (d, J = 8.4 Hz, 1H),7.37 (s, 1H), 7.32 (dd, J = 8.4, 1.6 Hz, 1H), 6.42 (dd, J = 17.2, 11.2 Hz,1H), 5.71 (d, J = 8.4 Hz, 1H), 5.28 (d, J = 11.2 Hz, 1H), 5.15 (d, J = 17.2Hz, 1H), 3.63 (q, J = 6.4 Hz, 2H), 3.33 (s, 1H), 3.17 (s, 2H), 2.82 (t, J =6.6 Hz, 2H), 2.35 – 1.95 (m, 6H), 1.78 – 1.39 (m, 9H), 1.13 (s, 3H), 1.06(dd, J = 13.6, 3.6 Hz, 1H), 0.84 (d, J = 6.8 Hz, 3H), 0.70 (d, J = 6.8 Hz,3H). 13 C NMR (101 MHz, CDCl3) δ 217.1, 168.8, 161.3, 160.7, 154.6, 147.6,140.4, 139.1, 130.6, 126.1, 118.2, 117.2, 117.0, 74.6, 69.5, 58.2, 45.5,44.9, 44.0, 41.8, 38.7, 38.6, 36.8, 36.0, 34.5, 33.9, 32.1, 30.5 26.9, 26.5,24.9, 16.9, 14.9, 11.5. HRMS: calculated for C 34 H 42 ClNO7S ([M + H] + ):644.2443;found 644.2448. (PB-7): White power; Yield: 52%; mp: 76.1-77.2℃; 1H NMR (400 MHz,CDCl3) δ 8.96 (t, J = 5.2 Hz, 1H), 8.86 (s, 1H), 7.70 (dd, J = 8.0, 1.2 Hz,1H), 7.60 (dd, J =8.0, 1.2 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 6.46 (dd, J =17.2, 11.2 Hz, 1H), 5.75 (d, J = 8.4 Hz, 1H), 5.33 (dd, J = 11.2, 1.2 Hz,1H), 5.18 (dd, J = 17.2, 1.2 Hz, 1H), 3.67 (q, J = 6.4 Hz, 2H), 3.39 – 3.32(m, 1H), 3.20 (s, 2H), 2.85 (t, J = 6.8 Hz, 2H), 2.43 – 2.01 (m, 6H), 1.84 –1.42 (m, 9H), 1.16 (s, 3H), 1.10 (dd, J = 13.6, 4.0 Hz, 1H), 0.87 (d, J = 6.8Hz, 3H), 0.73 (d, J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 217.2, 168.9,161.3, 160.4, 150.2, 148.2, 139.2, 134.4, 128.4, 125.6, 121.8, 119.9, 119.1,117.3, 74.7, 69.5, 58.3, 45.6, 44.9, 44.0, 41.9, 38.8, 38.7, 36.9, 36.1,34.6, 34.1, 32.2, 30.6, 27.0, 26.5, 25.0 17.0, 15.0, 11.6. HRMS: calculatedfor C 34 H 42 ClNO7S ([M + H] + ):644.2443; found 644.2447. (PB-8): White power; Yield: 61%; mp: 97.0-99.2℃; 1H NMR (400 MHz,CDCl3) δ 8.97 (t, J = 5.6 Hz, 1H), 8.81 (s, 1H), 7.82 (d, J = 2.4 Hz, 1H),7.74 (dd, J = 8.8, 2.4 Hz, 1H), 7.29 (d, J = 8.8 Hz, 1H), 6.47 (dd, J = 17.2,11.2 Hz, 1H), 5.76 (d, J = 8.4 Hz, 1H), 5.34 (d, J = 11.2 Hz, 1H), 5.19 (d, J= 17.2 Hz, 1H), 3.67 (q, J = 6.4 Hz, 2H), 3.35 (s, 1H), 3.20 (s, 2H), 2.86(t, J = 6.4 Hz, 2H), 2.41 – 2.01 (m, 6H), 1.81 – 1.43 (m, 9H), 1.17 (s, 3H),1.11 (dd, J = 14.0, 4.0 Hz, 1H), 0.87 (d, J = 6.8 Hz, 3H), 0.74 (d, J = 6.8Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 217.1, 168.9, 161.3, 160.8, 153.4, 147.2,139.2, 137.0, 132.0, 120.2, 119.5, 118.5, 118.1, 117.4, 74.8, 69.5, 58.3,45.6, 45.0, 44.1, 41.9, 38.8, 36.9, 36.1, 34.6, 34.1, 32.2, 30.6, 27.0, 26.5,25.0, 17.0, 15.0, 11.6. HRMS: calculated for C 34 H 42 BrNO7S ([M + H] + ):688.1938;found 688.1939. (PB-9): White power; Yield: 66%; mp: 139.4-140.5℃; 1H NMR (400 MHz,CDCl3) δ 8.92 (t, J = 5.2 Hz, 1H), 8.81 (s, 1H), 7.59 – 7.43 (m, 3H), 6.44(dd, J = 17.2, 11.2 Hz, 1H), 5.72 (d, J = 8.4 Hz, 1H), 5.30 (d, J = 11.2 Hz,1H), 5.16 (d, J = 17.2 Hz, 1H), 3.64 (q, J = 6.4 Hz, 2H), 3.33 (s, 1H), 3.17(s, 2H), 2.83 (t, J = 6.4 Hz, 2H), 2.37 – 1.98 (m, 6H), 1.79 – 1.40 (m, 9H),1.14 (s, 3H), 1.08 (dd, J = 13.6, 4.0 Hz, 1H), 0.85 (d, J = 6.8 Hz, 3H), 0.71(d, J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 217.2, 168.9, 161.5, 160.7,154.6, 147.9, 139.1, 130.7, 129.1, 128.7, 120.2, 118.5, 117.6, 117.4, 74.7,69.5, 58.3, 45.6, 44.9, 44.0, 41.9, 38.7, 36.9, 36.1, 34.6, 34.0, 32.2, 30.5,27.0, 26.4, 25.0, 17.0, 15.0, 11.7. HRMS: calculated for C 34 H 42 BrNO7S ([M + H] + ):688.1938; found 688.1943. (PB-10): White power; Yield: 58%; mp: 60.1-62.0℃; 1H NMR (400 MHz,CDCl3) δ 8.96 (t, J = 5.2 Hz, 1H), 8.84 (s, 1H), 7.87 (dd, J = 8.0, 1.2 Hz,1H), 7.64 (dd, J = 8.0, 1.2 Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 6.47 (dd, J =17.2, 11.2 Hz, 1H), 5.75 (d, J = 8.4 Hz, 1H), 5.33 (dd, J = 11.2, 1.2 Hz,1H), 5.18 (dd, J = 17.2, 1.2 Hz, 1H), 3.68 (q, J = 6.4 Hz, 2H), 3.35 (s, 1H),3.20 (s, 2H), 2.86 (t, J = 6.8 Hz, 2H), 2.43 – 2.04 (m, 6H), 1.84 – 1.42 (m,9H), 1.16 (s, 3H), 1.10 (dd, J = 13.6, 4.0 Hz, 1H), 0.87 (d, J = 6.8 Hz, 3H),0.73 (d, J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 217.1, 168.9, 161.3,160.5, 151.2, 148.2, 139.2, 137.5, 129.1, 126.1, 119.9, 119.1, 117.3, 110.3,74.7, 69.5, 58.3, 45.6, 44.9, 44.0, 41.9, 38.8, 38.7, 36.9, 36.1, 34.6, 34.1,32.2, 30.5, 27.0, 26.5, 25.0, 17.0, 15.0, 11.6. HRMS: calculated forC 34 H 42 BrNO7S ([M + H] + ):688.1938; found 688.1925. (PB-11): White power; Yield: 42%; mp: 65.5-66.7℃; 1H NMR (400 MHz,CDCl3) δ 9.05 (s, 1H), 8.79 (s, 1H), 7.49 – 7.19 (m, 3H), 6.43 (dd, J = 17.2,11.2 Hz, 1H), 5.73 (d, J = 8.4 Hz, 1H), 5.28 (t, J = 11.2 Hz, 1H), 5.16 (d, J= 17.2 Hz, 1H), 3.65 (dd, J = 12.4, 6.4 Hz, 2H), 3.40 – 3.30 (m, 1H), 3.18(s, 2H), 2.84 (t, J = 6.4 Hz, 2H), 2.40 (d, J = 7.2 Hz, 3H), 2.37 – 1.97 (m,6H), 1.81 – 1.37 (m, 9H), 1.14 (s, 3H), 1.08 (d, J = 13.6 Hz, 1H), 0.86 (d, J= 6.8 Hz, 3H), 0.71 (d, J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 217.1,172.6, 168.8, 161.9, 161.5, 152.7, 148.5, 139.1, 135.4, 135.2, 129.4, 118.3,118.0, 117.2, 116.4, 74.6, 69.4, 58.2, 45.5, 44.9, 44.0, 41.8, 38.7, 36.8,36.0, 34.5, 34.0, 32.1, 30.5, 26.9, 26.5, 24.9, 20.8, 16.9, 14.9, 11.5. HRMS:calculated for C 35 H 45 NO7S ([M + H] + ):624.2990; found 624.2983. (PB-12): White power; Yield: 46%; mp: 70.5-72.1℃; 1H NMR (400 MHz,CDCl3) δ 9.01 (s, 1H), 8.80 (s, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.15 (d, J =7.2 Hz, 2H), 6.43 (dd, J = 17.2, 11.2 Hz, 1H), 5.72 (d, J = 8.4 Hz, 1H), 5.29(d, J = 11.2 Hz, 1H), 5.15 (d, J = 17.2 Hz, 1H), 3.64 (dd, J = 12.4, 6.0 Hz,2H), 3.38 – 3.27 (m, 1H), 3.17 (s, 2H), 2.82 (t, J = 6.4 Hz, 2H), 2.46 (s,3H), 2.35 – 1.96 (m, 6H), 1.80 – 1.39 (m, 9H), 1.13 (s, 3H), 1.07 (d, J =14.0 Hz, 1H), 0.84 (d, J = 6.8 Hz, 3H), 0.71 (d, J = 6.8 Hz, 3H). 13 C NMR (101MHz, CDCl3) δ 217.1, 172.6, 168.8, 162.0, 161.6, 154.6, 148.4, 146.2, 139.1,129.5, 126.7, 117.2, 117.0, 116.7, 116.3, 74.62, 69.4, 60.4, 58.2, 45.5,44.8, 43.9, 41.8, 38.6, 36.8, 36.0, 34.5, 34.0, 32.1, 30.4, 26.9, 26.5, 24.9,22.2, 21.1, 16.8, 14.9, 14.2, 11.5. HRMS: calculated for C 35 H 45 NO7S ([M + H] + ):624.2990; found 624.2990. (PB-13): White power; Yield: 53%; mp: 69.5-71.2℃; 1H NMR (400 MHz,CDCl3) δ 9.06 (t, J = 5.6 Hz, 1H), 8.85 (s, 1H), 7.57 – 7.41 (m, 2H), 7.26(t, J = 7.6 Hz, 1H), 6.46 (dd, J = 17.2, 11.2 Hz, 1H), 5.75 (d, J = 8.4 Hz,1H), 5.33 (dd, J = 11.2, 1.2 Hz, 1H), 5.19 (dd, J = 17.2, 1.2 Hz, 1H), 3.68(q, J = 6.4 Hz, 2H), 3.36 (dd, J = 9.6, 6.8 Hz, 1H), 3.21 (s, 2H), 2.86 (t, J= 6.8 Hz, 2H), 2.48 (s, 3H), 2.39 – 1.98 (m, 6H), 1.80 – 1.43 (m, 9H), 1.16(d, J = 6.8 Hz, 3H), 1.15 – 1.06 (m, 1H), 0.87 (d, J = 6.8 Hz, 3H), 0.74 (d,J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 217.1, 168.9, 161.9, 161.5, 152.9,148.9, 139.2, 135.5, 127.6, 126.3, 125.0, 118.4, 117.9, 117.3, 74.7, 69.5,58.3, 45.5, 44.9, 44.0, 41.9, 38.7, 36.9, 36.1, 34.5, 34.0, 32.2, 30.5, 26.9,26.5, 24.9, 16.9, 15.4, 15.0, 14.3, 11.6. HRMS: calculated for C 35 H 45 NO7S ([M +H] + ):624.2990; found 624.2991. (PB-14): White power; Yield: 71%; mp: 75.0-76.2℃; 1H NMR (400 MHz,CDCl3) δ 9.06 (s, 1H), 8.80 (s, 1H), 7.29 (d, J = 9.2 Hz, 1H), 7.21 (d, J =9.2 Hz, 1H), 7.04 (s, 1H), 6.44 (dd, J = 17.2, 11.2 Hz, 1H), 5.73 (d, J = 8.0Hz, 1H), 5.30 (d, J = 10.8 Hz, 1H), 5.16 (d, J = 17.2 Hz, 1H), 3.84 (s, 3H),3.69 – 3.60 (m, 2H), 3.34 (s, 1H), 3.18 (s, 2H), 2.83 (t, J = 6.0 Hz, 2H),2.38 – 1.89 (m, 6H), 1.80 – 1.40 (m, 9H), 1.14 (s, 3H), 1.08 (d, J = 14.0 Hz,1H), 0.85 (d, J = 6.0 Hz, 3H), 0.72 (d, J = 6.0 Hz, 3H). 13 C NMR (101 MHz,CDCl3) δ 217.1, 168.8, 161.8, 161.5, 156.7, 149.1, 148.3, 139.1, 122.8,119.0, 118.4, 117.8, 117.3, 110.8, 74.7, 69.5, 58.3, 56.0, 45.5, 44.9, 44.0,41.8, 38.7, 36.9, 36.1, 34.5, 34.0, 32.2, 30.5, 26.9, 26.5, 24.9, 16.9, 15.0,14.3, 11.6. HRMS: calculated for C 35 H 45 NO8S ([M + H] + ):640.2939; found640.2931. (PB-15): White power; Yield: 67%; mp: 66.0-67.2℃; 1H NMR (400 MHz,CDCl3) δ 8.95 (t, J = 5.6 Hz, 1H), 8.77 (s, 1H), 7.54 (d, J = 8.8 Hz, 1H),6.90 (dd, J = 8.8, 2.4 Hz, 1H), 6.82 (d, J = 2.4 Hz, 1H), 6.43 (dd, J = 17.2,11.2 Hz, 1H), 5.72 (d, J = 8.4 Hz, 1H), 5.30 (dd, J = 11.2, 1.2 Hz, 1H), 5.16(dd, J = 17.2, 1.2 Hz, 1H), 3.88 (s, 3H), 3.63 (q, J = 6.4 Hz, 2H), 3.33 (s,1H), 3.18 (s, 2H), 2.82 (t, J = 6.8 Hz, 2H), 2.39 – 1.94 (m, 6H), 1.79 – 1.39(m, 9H), 1.13 (s, 3H), 1.07 (dd, J = 13.6, 4.0 Hz, 1H), 0.84 (d, J = 6.8 Hz,3H), 0.71 (d, J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 217.1, 168.8, 165.0,162.2, 161.7, 156.7, 148.4, 139.1, 131.0, 117.2, 114.6, 114.1, 112.4, 100.3,74.7, 69.4, 58.2, 56.1, 45.5, 44.9, 44.0, 41.8, 38.6, 38.6, 36.8, 36.1, 34.5,34.0, 32.2, 30.5, 26.9, 26.5, 24.9, 16.9, 14.9, 11.6. HRMS: calculated forC 35 H 45 NO8S ([M + H] + ):640.2939; found 640.2926. (PB-16): White power; Yield: 60%; mp: 79.7-81.4℃; 1H NMR (400 MHz,CDCl3) δ 9.03 (s, 1H), 8.85 (s, 1H), 7.31 - 7.18 (m, 3H), 6.46 (dd, J = 17.2,11.2 Hz, 1H), 5.75 (d, J = 8.4 Hz, 1H), 5.33 (d, J = 11.2 Hz, 1H), 5.19 (d, J= 17.2 Hz, 1H), 3.98 (s, 3H), 3.67 (dd, J = 12.8, 6.4 Hz, 2H), 3.36 (d, J =6.4 Hz, 1H), 3.21 (s, 2H), 2.86 (t, J = 6.8 Hz, 2H), 2.42 – 1.99 (m, 6H),1.81 – 1.43 (m, 9H), 1.17 (s, 3H), 1.11 (dd, J = 13.6, 3.6 Hz, 1H), 0.88 (d,J = 6.8 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 217.1,168.8, 161.7, 160.9, 148.7, 147.1, 144.1, 139.1, 125.2, 121.0, 119.3, 118.5,117.3, 115.7, 74.7, 69.4, 58.2, 56.4, 45.5, 44.9, 44.0, 41.8, 38.8, 36.8,36.1, 34.5, 34.0, 32.1, 30.5, 26.9, 26.5, 24.9, 16.9, 15.0, 11.6. HRMS:calculated for C 35 H 45 NO8S ([M + H] + ):640.2939; found 640.2923. (PB-17): White power; Yield: 71%; mp: 81.2-83.4℃; 1H NMR (400 MHz,CDCl3) δ 9.00 (s, 1H), 8.85 (t, J = 5.6 Hz, 1H), 8.65 (d, J = 2.8 Hz, 1H),8.49 (dd, J = 9.2, 2.8 Hz, 1H), 7.54 (d, J = 9.2 Hz, 1H), 6.45 (dd, J = 17.2,11.2 Hz, 1H), 5.73 (d, J = 8.4 Hz, 1H), 5.30 (d, J = 11.2 Hz, 1H), 5.17 (d, J= 17.2 Hz, 1H), 3.68 (q, J = 6.4 Hz, 2H), 3.35 (d, J = 6.4 Hz, 1H), 3.19 (s,2H), 2.85 (t, J = 6.4 Hz, 2H), 2.37 – 1.97 (m, 6H), 1.79 – 1.41 (m, 9H), 1.15(s, 3H), 1.09 (dd, J = 13.6, 4.0 Hz, 1H), 0.86 (d, J = 6.8 Hz, 3H), 0.72 (d,J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 217.1, 168.8, 160.6, 159.9, 157.5,147.3, 144.7, 139.2, 128.5, 125.7, 120.5, 118.7, 118.0, 117.2, 74.7, 69.6,60.5, 58.2, 45.5, 44.9, 44.0, 41.8, 38.8, 36.8, 36.1, 34.5, 34.0, 32.2, 30.5,26.9, 26.5, 24.9, 21.1, 16.9, 15.0, 14.3, 11.6. HRMS: calculated forC 34 H 42 N2O9S ([M + H] + ):655.2684; found 655.2680. (PB-18): White power; Yield: 56%; mp: 92.0-93.2℃; 1H NMR (400 MHz, CDCl3) δ 8.98 – 8.85 (m, 2H), 8.25 – 8.17 (m, 2H), 7.89 (d, J = 8.4 Hz, 1H), 6.46 (dd, J = 17.2, 11.2 Hz, 1H), 5.74 (d, J = 8.4 Hz, 1H), 5.32 (dd, J =11.2, 0.8 Hz, 1H), 5.18 (dd, J = 17.2, 0.8 Hz, 1H), 3.68 (q, J = 6.4 Hz, 2H), 3.35 (t, J = 7.2 Hz, 1H), 3.19 (s, 2H), 2.86 (t, J = 6.4 Hz, 2H), 2.41 – 1.97(m, 6H), 1.81 – 1.42 (m, 9H), 1.16 (s, 3H), 1.10 (dd, J = 14.0, 4.2 Hz, 1H), 0.86 (d, J = 6.8 Hz, 3H), 0.73 (d, J = 6.8 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ217.1, 168.8, 160.6, 160.1, 154.0, 150.5, 146.5, 139.2, 130.9, 123.2, 121.4,119.9, 117.3, 112.5, 74.7, 69.6, 58.3, 45.6, 44.9, 44.0, 41.9, 38.9, 36.9,36.1, 34.6, 34.0, 32.1, 30.5, 27.0, 26.5, 24.9, 17.0, 15.0, 11.6. HRMS:calculated for C 34 H 42 N2O9S ([M + H] + ):655.2684; found 655.2682. 4. In vitro antibacterial activity study 4.1 Minimum inhibitory concentration (MIC) test method (1) Experimental strains: Gram-positive bacteria MRSA ATCC 33591, MRSA ATCC 43300, MSSA ATCC29213, MRSE ATCC 51625, MSSE ATCC 12228, and Gram-negative bacteria E. coli ATCC 25922 were selected as strains for MIC value determination.

[0032] (2) Drug dilution: Dissolve and dilute the target compound and tylosin in DMSO to a concentration of 12800 μg mL -1 The mother liquor was sealed and stored in a refrigerator away from light for later use.

[0033] (3) Preparation of bacterial solution: Activate each test bacteria, pick a single clone colony and place it in 0.9% saline. Prepare the bacterial solution to a concentration of 0.5 McFarland (1.5×10 8 CFU·mL -1 ), and then diluted 10 times with Mueller-Hinton sterile broth (MHB) for later use.

[0034] (4) Positive control: tylosin, valnemulin and retapalin were selected as positive controls.

[0035] (5) MIC determination: 100 μL of MHB was added to each well of the 96-well plate except the edge wells and the second column wells. 196 μL of MHB and 4 μL of stock solution were added to the second well. The compound and positive control were diluted to 128 – 0.25 μg mL using the two-fold dilution method. -1 10 dilutions with different concentration gradients were added, and then 100 μL of bacterial suspension was added to each well except the edge wells, and mixed thoroughly. Finally, 200 μL of sterile water was added to each edge well. The test bacteria were cultured at 37°C for 18-24 h, and the growth of the test bacteria was observed. The lowest concentration of the drug with no growth was taken as the MIC value of the drug for the test bacteria; tylosin, valnemulin and retapalin were used as positive controls, and the ethanol solution with the same concentration of the prepared compound was used as the negative control. Three parallel experiments were performed for each test bacteria, and the experiment was repeated 3 times. The growth of the test bacteria in the negative control group was good. The other experimental results are shown in Table 1.

[0036] Note: “R” stands for retapalene, “T” stands for tylosin fumarate, and “V” stands for valnemulin hydrochloride For the mercaptoethylamine-linked derivative PB, the MIC value was 0.008-0.25 μg·mL -1, the antibacterial activity is slightly weaker than that of PA compounds. For Gram-positive bacteria MRSA ATCC 33591, MRSA ATCC 43300, and MSSA ATCC 29213, more than 90% of PB compounds have better antibacterial activity than tylosin. Compounds PB-2 to 10 are derivatives of coumarin with different halogen substitutions in the benzene ring region, among which the derivatives substituted with fluorine at the 8th position have the best activity; under the same substituents, the derivatives obtained by introducing substituents at the 8th position of coumarin have better activity. The derivatives obtained by introducing methyl and methoxy groups into the benzene ring region of coumarin (PB-11 to 16) also found that the derivatives obtained by introducing substituents at the 8th position of the benzene ring region of coumarin have better activity. At the same time, the MBC values ​​of the targets were determined. Among these 18 compounds, the MBC / MIC values ​​of most compounds were greater than 4, showing antibacterial effects. Based on the data of MIC and MBC, compound PB-16 has the best activity. In addition, mercaptoethylamine derivatives had no inhibitory effect on Gram-negative bacteria Escherichia coli ATCC 25922. Based on the above results, the coumarin-type pleuromutilin compounds linked with mercaptoethylamine in the present invention all showed excellent antibacterial effects and are expected to treat bacterial infections caused by resistant Staphylococcus aureus.

[0037] Based on the above results, the coumarin-type pleuromutilin compounds linked with mercaptoethylamine in the present invention all showed excellent antibacterial effects and are expected to treat bacterial infections caused by resistant Staphylococcus aureus.

Claims

1. A pleuromutilin derivative, characterized in that: The pleuromutilin derivative is a compound having a structure shown in Formula I and a pharmaceutically acceptable salt thereof: (Formula I) Wherein R1, R2, R3 are H, OMe, NO2, Me, F, Cl or Br.

2. A pleuromutilin derivative according to claim 1, characterized in that: The pharmaceutically acceptable salt is a salt formed by a compound having a structure as shown in Formula I and hydrochloric acid, sulfuric acid, phosphoric acid, phosphorous acid, hydrobromic acid, nitric acid, malic acid, maleic acid, citric acid, fumaric acid, tartaric acid, succinic acid, acetic acid, lactic acid, benzoic acid, p-toluenesulfonic acid, methanesulfonic acid or palmitic acid.

3. A method for preparing a pleuromutilin derivative according to any one of claims 1 to 2, characterized in that: The following steps are involved: Tosyl chloride and pleuromutilin are reacted to obtain pleuromutilin intermediate 2 as shown in the following formula: The intermediate 2 is reacted with potassium carbonate and mercaptoethylamine to obtain the pleuromutilin intermediate 3 shown in the following formula: (3) Substituted salicylaldehyde b0 is reacted with diethyl malonate, glacial acetic acid and piperidine to obtain an intermediate b1 as shown in the following formula: (4) The intermediate b1 is reacted with sodium hydroxide to obtain the coumarin intermediate b2 shown in the following formula: (5) reacting the pleuromutilin intermediate 3 with the coumarin intermediate b2 to obtain a pleuromutilin derivative having the following formula I: (Formula I).

4. The method for preparing a pleuromutilin derivative according to claim 3, characterized in that: The reaction conditions of step (1) are as follows: p-toluenesulfonyl chloride and pleuromutilin are dissolved in a mixed solution of methyl tert-butyl ether and water, sodium hydroxide solution is slowly added dropwise to the mixed solution under ice bath, and the mixture is placed at 50-70 o C for about 0.5-1.5h. After the reaction is completed, water is added and the mixture is filtered under reduced pressure. The filter cake is washed with water and dried to obtain intermediate 2.

5. The method for preparing a pleuromutilin derivative according to claim 3, characterized in that: The reaction conditions of step (2) are as follows: intermediate 2, potassium carbonate and mercaptoethylamine are dissolved in acetonitrile solution, and placed in an oil bath at 45-55° C. for reaction. After the reaction is complete, the reaction solution is concentrated and purified by column chromatography to obtain white powdery pleuromutilin intermediate 3.

6. A method for preparing a pleuromutilin derivative according to claim 3, characterized in that: The reaction conditions of step (3) are as follows: dissolving substituted salicylaldehyde b0 in ethanol, stirring magnetically to dissolve, adding diethyl malonate, a drop of glacial acetic acid and piperidine to the solution, condensing and refluxing to react, cooling to room temperature after the reaction, adding ethanol to the reaction solution, stirring the reaction solution on a low-temperature stirrer for 25-35 minutes, a large amount of solid precipitates in the solution, filtering and collecting the residue, washing the solid with glacial ethanol, and drying to obtain the intermediate b1.

7. A method for preparing a pleuromutilin derivative according to claim 3, characterized in that: The reaction conditions of step (4) are as follows: intermediate b1 is dissolved in ethanol, sodium hydroxide solution is added, and the reaction is carried out at 75-85°C. After the reaction is completed, the reaction bottle is cooled to room temperature, and the reaction bottle is placed in ice water. The pH is adjusted to 2-4 with dilute hydrochloric acid solution. At this time, solids precipitate in the solution, and the residue is collected by filtration. The solids are washed with ice water and dried to obtain coumarin intermediate b2.

8. The method for preparing a pleuromutilin derivative according to claim 3, characterized in that: The reaction conditions of step (5) are as follows: dissolving the coumarin intermediate b2 and HATU in acetonitrile, adding triethylamine under stirring, reacting in an ice bath for 25-35 min, adding the pleuromutilin intermediate 3, and then continuing the reaction at room temperature. After the reaction is completed, the reaction solution is concentrated under reduced pressure and purified by column chromatography to obtain a pleuromutilin derivative having formula I.

9. Use of the pleuromutilin derivative according to any one of claims 1 to 2, characterized in that: Used for preparing medicines for treating infectious diseases, wherein the infectious diseases are infectious diseases caused by drug-resistant bacteria.

10. A pharmaceutical composition for treating infectious diseases, characterized in that: A composition comprising a therapeutically effective amount of the pleuromutilin derivative, stereoisomer or pharmaceutically acceptable salt according to any one of claims 1 to 2.

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