CN119192119A

CN119192119A - Coumarin thioether pleuromutilin derivatives and uses thereof

Info

Publication number: CN119192119A
Application number: CN202411708796.6A
Authority: CN
Inventors: 张园园; 商凤; 管梦霞; 刘东芳; 罗晓俊; 吴彩骏; 刘阳; 付欢; 吴春霞; 王梦倩; 能比阿呷; 李筱丹; 任诗妍
Original assignee: Xihua University
Current assignee: Xihua University
Priority date: 2024-11-27
Filing date: 2024-11-27
Publication date: 2024-12-27

Abstract

The invention belongs to the technical field of antibacterial medicines, and discloses a coumarin thioether pleuromutilin derivative and application thereof, wherein the coumarin thioether pleuromutilin derivative is a compound with a structure shown in a formula I and pharmaceutically acceptable salts thereof, and R ₁、R₂ is H, OMe, NO ₂、NH₂, me, F, cl or Br. The invention has novel structure and excellent antibacterial activity. The in vitro antibacterial experiment results show that all compounds have antibacterial effects superior to those of tiamulin, most of the compounds have antibacterial effects equivalent to those of control valnemulin, and the antibacterial activities of 9 compounds are smaller than or equal to 0.062 mug.mL ^‑1 on 5 gram positive bacteria staphylococcus aureus resistant strains ATCC 33591 and ATCC 43300, staphylococcus aureus sensitive strain ATCC 2913, staphylococcus epidermidis resistant strain ATCC 51625 and staphylococcus epidermidis sensitive strain ATCC 12228. Is expected to be applied to the treatment of bacterial infection caused by staphylococcus aureus and provides a new choice for anti-infective drugs.

Description

Coumarin thioether pleuromutilin derivative and application thereof

Technical Field

The invention belongs to the technical field of antibacterial medicines, and particularly relates to a coumarin thioether pleuromutilin derivative and application thereof.

Background

The discovery of antibiotics has great significance in the development history of human beings, but bacterial resistance is increasingly improved nowadays due to the wide and long-term use of antibiotics in clinic, which definitely forms a great threat to the life safety of human beings. In order to cope with the dilemma of year-by-year reduction of effective antibacterial agents and increasing bacteria resistance, it is highly desirable to find an effective way to solve this troublesome global problem. The united states Food and Drug Administration (FDA) initiated an incentive program (GENERATING ANTIBIOTICS INCENTIVES NOW, GAIN) to promote antibiotic development in 2012. Of the antiinfective (antibacterial, antifungal, antiparasitic and antiviral) agents on the market, nearly 70% are either directly derived from natural products or obtained by modification of their structure.

Pleuromutilin (Pleuromutilin) is a tricyclic diterpenoid compound with good antibacterial activity isolated from higher fungi Pleurotus multilus (Fr.) sacc. And Pleurotus Passecke-rianus Pilat in 1951 by Kavanagh and his colleagues. Studies have shown that pleuromutilin binds to the 23S rRNA of the ribosomal 50S subunit of bacteria, a tight pocket is formed at the A site by its tricyclic parent nucleus located in the centre of the Peptidyl Transferase (PTC) of the ribosomal 50S subunit, and at the same time, its C14 side chain moiety covers the P site to which tRNA binds, preventing the aminoacyl transferase from binding to the P site and inhibiting the activity of the peptidyl transferase, further preventing the translation process of bacterial proteins, thereby inhibiting bacterial growth. The compounds only selectively inhibit the synthesis of prokaryotic cell proteins, have no influence on eukaryotic cells, and do not interact with ribosomes of mammals, and are not easy to generate cross drug resistance of other antibiotics due to the special action mechanism. Although pleuromutilins have a certain antibacterial activity, they are difficult to develop into effective antibacterial drugs due to their low in vivo absorption rate by being slightly soluble in water. Therefore, since the discovery of pleuromutilin, researchers have conducted structure-activity relationship research and structural modification on pleuromutilin, and attempt to introduce various polar groups so as to find pleuromutilin derivatives with high antibacterial activity, good water solubility and high bioavailability.

The structure-activity relationship (SAR) research shows that the tricyclic structure of the mother nucleus, the ester group at the C-14 position, the carbonyl group at the C-2 position and the hydroxyl group at the C-11 position are all functional groups necessary for antibacterial activity. In recent years, many researchers have achieved better results in the derivatization of the side chain at the C14 position. In 1979, the first marketed drug of pleuromutilin derivatives, tiamulin (Tamulin), was successfully approved as a veterinary antibiotic. Valnemulin (Valnemulin) was subsequently successfully marketed in 1999 as the second derivative approved as a veterinary antibiotic. In addition, azamorline (Azamulin) entered the clinical stage in 1980, has excellent antibacterial effect, but was declared to terminate due to its strong inhibitory effect on human cytochrome P450 and its low oral bioavailability and too short half-life. In 2007, the first topical pleuromutilin derivative, ritamillin (Ratapamulin), for the treatment of human skin infections was developed by the company GlaxoSmithKline. In 2006, nabriva developed Lafemulin (BC-3781), a novel semisynthetic pleuromutilin antibacterial agent, mainly for the treatment of Acute Bacterial Skin and Skin Structure Infections (ABSSSI) and community-acquired bacterial pneumonia (CABP). On day 19, 8 in 2019, the FDA passed Nabriva's filed new drug application for Lafemulin to treat CABP both orally and intravenously (NDAs).

The structural formulas of pleuromutilin (Pleuromutilin), tiamulin (Tamulin), valnemulin (Valnemulin), azamolin (Azamulin), ritaline (Ratapamulin) and Lafemulin (BC-3781) are as follows:

Pleuromutilins develop relatively rapidly in veterinary medicine, but their role in the course of human antibacterial drug research is not fully demonstrated. Based on unmet clinical needs, there is an urgent need to develop drugs with novel structures and unique mechanisms of action that have higher antimicrobial activity.

The benzoheterocyclic compound is widely applied to the field of medicinal chemistry, wherein coumarin is a bisfuran ring compound, and can be combined with various active sites in organisms through non-covalent bond action forms (hydrophobic action, pi-pi electron interaction, hydrogen bond and the like) because the structure of the benzoheterocyclic compound comprises pi-pi conjugated systems, so that the coumarin compound has very wide biological activity and has the effects of resisting bacteria, inflammation, HIV, depression, oxidation, anticoagulation, anticancer, virus, fungi, asthma and the like in the field of medicines. Based on the above, a plurality of researchers introduce coumarin derivatives into the C-14 side chain of pleuromutilin to improve the activity and pharmacokinetic properties of the pleuromutilin.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Disclosure of Invention

The object of the present invention is to provide pleuromutilin derivatives based on coumarin thioethers and their use, which overcome the above-mentioned drawbacks of the prior art.

In order to achieve the above object, the present invention provides a coumarin thioether-type pleuromutilin derivative, wherein the coumarin-type pleuromutilin derivative is a compound having a structure shown in formula I and pharmaceutically acceptable salts thereof:

(formula I)

Wherein R ₁、R₂ is H, OMe, NO ₂、NH₂, me, F, cl or Br.

The invention also provides a method for preparing the coumarin thioether pleuromutilin derivative, which comprises the following steps of:

(1) Reacting tosyl chloride with pleuromutilin to obtain pleuromutilin intermediate 2 of the formula:

(2) Reacting intermediate 2 with potassium thioacetate to obtain pleuromutilin intermediate 3 of the formula:

(3) Reacting intermediate 3 with sodium methoxide to obtain pleuromutilin intermediate 4 of the formula:

(4) Reacting substituted acetophenone a0 with sodium hydride diethyl carbonate turbid liquid to obtain a substituted 4-hydroxycoumarin intermediate a1 shown in the following formula:

(5) Reacting the substituted 4-hydroxycoumarin a1 with tosyl chloride to obtain a 4-hydroxycoumarin derivative intermediate a2 shown in the following formula:

(6) Reacting the 4-hydroxycoumarin derivative intermediate a2 with the pleuromutilin intermediate 4 to obtain the coumarin thioether pleuromutilin derivative shown in the following formula I:

(formula I)

Further, preferably, the reaction condition of the step (1) is that p-toluenesulfonyl chloride and pleuromutilin are dissolved in a mixed solution of methyl tertiary butyl ether and water, a sodium hydroxide solution is slowly dripped into the mixed solution under the ice bath, the mixed solution is reacted for about 0.5 to 1.5 hours under the condition of 45 to 65 ^o ℃, water is added after the reaction is finished, the pressure is reduced, the filtration is carried out, and a filter cake is washed by water and dried to obtain the pleuromutilin intermediate 2.

Further, as a preferable reaction condition of the step (2), the intermediate 2 and potassium thioacetate are dissolved in EA, reacted at 20-30 ℃, washed with saturated NaHCO ₃ solution after the reaction is completed, and the organic layer is collected by separating liquid, dried by anhydrous Na ₂SO₄ and concentrated under reduced pressure to obtain yellow oily substance, namely pleuromutilin intermediate 3.

Further, preferably, the reaction condition of the step (3) is that the intermediate 3 is dissolved in methanol under the protection of N ₂, then sodium methoxide is added into the reaction solution, the pH of the reaction solution is 9-11, the reaction is carried out for 2-4 hours, the reaction is carried out under reduced pressure and concentrated, and the white solid target compound pleuromutilin intermediate 4 is obtained after purification by a column chromatography.

Further, preferably, the reaction condition of the step (4) is that the substituted acetophenone a0 is dissolved in toluene and placed in ice bath, sodium hydride is taken in diethyl carbonate, then sodium hydride diethyl carbonate turbid liquid is slowly dripped into the substituted acetophenone a0 solution, the ice bath is taken out after dripping is finished and the temperature is raised to 100-120 ℃, the magnetic stirring reaction is carried out for 3-5 h, after the reaction is finished, the cooling is carried out to room temperature, the unreacted sodium hydride is quenched by water, the excessive diethyl carbonate is extracted by methyl tertiary butyl ether, the pH=3 is regulated by 10% diluted hydrochloric acid, a large amount of white solid is precipitated at the moment, the white solid is obtained after suction filtration and drying, and the crude product is purified by column chromatography to obtain the substituted 4-hydroxycoumarin intermediate a1.

Further, preferably, the reaction condition of the step (5) is that the substituted 4-hydroxycoumarin a1 is dissolved in acetonitrile, triethylamine is added, after stirring at room temperature for 25-35 min ℃, p-toluenesulfonyl chloride is added, the temperature is raised to 75-85 ℃, the magnetic stirring reaction is carried out for 3-5 hours, after the reaction is finished, the reaction solution is cooled to room temperature, the reaction solution is dried by spin, absolute ethyl alcohol is then added, the reaction solution is left at room temperature for overnight until solid precipitation, filter residues are collected by filtration, and the 4-hydroxycoumarin derivative intermediate a2 is obtained after drying.

Further, as preferable reaction conditions in the step (6), the 4-hydroxycoumarin derivative a2 and the pleuromutilin intermediate 4 are dissolved in N, N-dimethylformamide, triethylamine is added under stirring at room temperature, the reaction is carried out at 20-30 ℃ under the protection of N ₂, water is added until solid is separated out after the reaction is finished, filter residues are collected after suction filtration and washing, a crude product is obtained, and the crude product is purified by a column chromatography method, thus obtaining the coumarin thioether pleuromutilin derivative shown in the formula I.

The invention also provides a stereoisomer or pharmaceutically acceptable salt of the coumarin thioether pleuromutilin derivative.

Wherein the pharmaceutically acceptable salts include, but are not limited to, salts of the compounds 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 and the like, preferably the pharmaceutically acceptable salts are selected from the group consisting of hydrochloride, fumarate, malate, hydrobromide, succinate, phosphate, methanesulfonate and benzoate.

The invention also provides application of the coumarin thioether pleuromutilin derivative, a stereoisomer or a pharmaceutically acceptable salt thereof in preparing medicines for treating infectious diseases, wherein the infectious diseases are infectious diseases caused by drug-resistant bacteria.

The invention also provides a pharmaceutical composition for treating infectious diseases, which comprises a therapeutically effective amount of coumarin thioether pleuromutilin derivative, a stereoisomer or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable auxiliary material.

Further, preferably, the adjuvants 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, polyvinylpyrrolidone, cellulose substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, lanolin, and the like.

Compared with the prior art, the invention has the following beneficial effects:

The coumarin thioether-type pleuromutilin derivative has novel structure and excellent antibacterial activity, all compounds have antibacterial effects superior to those of tiamulin, and most compounds have antibacterial effects equivalent to those of control valnemulin;

the compound is a derivative with PA-7 to PA-12 being different halogen substitutions in coumarin benzene ring region, wherein fluorine substitution derivatives (PA-7, PA-8) have better activity, under the same substituent, 7-position substitution pleuromutilin derivatives of coumarin are slightly better than 6-position substitution pleuromutilin derivatives of coumarin, derivatives (PA-2, PA-3, PA-5, PA-6) obtained by introducing methoxy and methyl into coumarin benzene ring region are also found to have better activity by introducing substituent at 7-position of coumarin benzene ring region.

The compound has the advantages that the MBC/MIC value of most of 13 compounds is larger than 4, the antibacterial effect is shown, the activity of the compound PA-2 is optimal, the MIC value of the compound PA-2 is 4-64 times higher than that of tiamulin based on the data of MIC and MBC, the MIC and MBC values of the compound PA-2 are even better than those of contrast valnemulin and ritapaline, and the compound PA-2 is expected to be applied to the treatment of bacterial infection caused by staphylococcus aureus and provides a new choice for anti-infective drugs.

Drawings

FIG. 1 is a time-sterilization graph of tiamulin versus MRSA 33591 in the present invention;

FIG. 2 is a graph showing the time-sterilization profile of the compound PA-2 of the present invention against MRSA 33591;

FIG. 3 is a graph showing the kinetics of bacterial growth following exposure of MRSA ATCC33591 to tiamulin 2h in accordance with the present invention;

FIG. 4 is a graph showing the kinetics of bacterial growth following exposure of MRSA ATCC33591 to compound PA-2 of 2h in accordance with the present invention;

FIG. 5 is a graph showing the results of cytotoxicity test after MRSA ATCC33591 is exposed to compound PA-2 of 2h in the present invention;

FIG. 6 is a macroscopic wound surface diagram of wound healing in an infection model according to the invention, wherein 'R' represents ritaparine;

FIG. 7 is a schematic representation of the invention in which 6D after infection is stained by HE, wherein A is model group, B is blank group, C is 1% Ruitaloplin group, D is 3% Ruitaloplin group, E is 1% PA-2 group, F is 3% PA-2 group;

FIG. 8 is a schematic representation of the invention in which 6D was stained by Masson after infection, wherein A is the model group, B is the blank group, C is the 1% Ruitaloplin test group, D is the 3% Ruitaloplin test group, E is the 1% PA-2 test group, and F is the 3% PA-2 test group.

Detailed Description

The following detailed description of specific embodiments of the invention is, but it should be understood that the invention is not limited to specific embodiments.

The following presents a simplified summary of one or more aspects in order to provide a basic understanding of such aspects. This summary is not an extensive overview of all contemplated aspects, and is intended to neither identify key or critical elements of all aspects nor delineate the scope of any or all aspects. Its sole purpose is to present some concepts of one or more aspects in a simplified form as a prelude to the more detailed description that is presented later.

The invention provides a coumarin thioether type pleuromutilin derivative, which has a structural formula shown in the following table:

The preparation method of the coumarin thioether pleuromutilin derivative comprises the following steps:

1. The pleuromutilin intermediate was synthesized according to the following synthetic route:

reagents and reaction conditions ：( = 1 \* ROMAN I) TsCl, NaOH, MTBE / H₂O, 60℃; (Ⅱ) Potassium thioacetate, EA, rt;(Ⅲ) Sodium methoxide, MeOH, rt.

Step of synthesizing intermediate 4:

1.1 dissolving tosyl chloride (4.3 g,22.7 mmol) and pleuromutilin (7.8 g,20.6 mmol) in a mixed solution of 25mL methyl tert-butyl ether and water (v/v=4:1), slowly dropwise adding 5mL sodium hydroxide solution (10M) into the solution under ice bath, reacting at 60 ^o C for about 1h, adding a proper amount of water after the reaction is finished, filtering under reduced pressure, washing a filter cake with water, and drying to obtain an intermediate 2, confirming the structure by nuclear magnetism, wherein 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).

1.2 intermediate 2 (7.20 g,13.52 mmol) and potassium thioacetate (1.54 g,13.52 mmol) were dissolved in EA and reacted at 25℃with TLC to check the progress of the reaction, washed with saturated NaHCO ₃ solution (50 mL X3), the organic layer was collected by separation, dried over anhydrous Na ₂SO₄ and concentrated under reduced pressure to give a yellow oil, intermediate 3.

1.3, Dissolving intermediate 3 (1.00 g,2.26 mmol) in methanol under the protection of N ₂, adding sodium methoxide into the reaction solution, adding the pH of the reaction solution to about 10, reacting for about 3 hours, detecting the reaction progress by TLC, concentrating under reduced pressure, purifying by column chromatography to obtain a white solid target compound intermediate 4, confirming the structure by nuclear magnetism, and the data are as follows:

White power; Yield: 87 %; ¹H NMR (400 MHz, CDCl₃) δ 6.46 (dd, J = 17.2, 11.2 Hz, 1H), 5.73 (d, J = 8.4 Hz, 1H), 5.34 (dd, J = 11.2, 1.2 Hz, 1H), 5.20 (dd, J = 17.2, 1.2 Hz, 1H), 3.36 (dd, J = 8.8, 7.0 Hz, 1H), 3.23 – 3.11 (m, 2H), 2.39 – 2.01 (m, 6H), 1.82 – 1.36 (m, 9H), 1.17 (d, J = 2.4 Hz, 3H), 1.14 – 1.07 (m, 1H), 0.88 (d, J = 6.8 Hz, 3H), 0.73 (d, J = 6.8 Hz, 3H).

2. coumarin intermediates were synthesized according to the following synthetic route:

Reagents and reaction conditions (I) Diethyl carbonate, naH, toluene, 120 ℃, (II) TsCl, TRIETHYLAMINE, acetonitrile, 82 ℃.

2.1, Step of synthesizing intermediate a 1:

Dissolving substituted acetophenone a0 (6.25 mmol) in toluene (10 mL), magnetically stirring to dissolve, and placing in ice bath;

Sodium hydride (0.80 g,33.00 mmol) is taken and added into diethyl carbonate (1.60 mL,13.00 mmol), then sodium hydride diethyl carbonate turbid liquid is slowly added into a round-bottom flask, after the dripping is finished, ice bath is taken out and heated to 110 ℃, magnetic stirring is carried out for reaction 4h, and TLC is used for monitoring the reaction progress;

After the reaction is finished, cooling to room temperature, quenching unreacted sodium hydride with 10mL water, extracting excessive diethyl carbonate with methyl tertiary butyl ether for multiple times until an diethyl ether layer is colorless, leaving a layer of water phase, and adjusting pH=3 with 10% dilute hydrochloric acid, wherein a large amount of white solid is precipitated;

the white solid obtained after suction filtration and drying was the crude product purified by column chromatography (DCM: meoh=50:1), with a molar yield of 72%, the structure confirmed by nuclear magnetism, data as follows:

4-hydroxycoumarin (a1-1): white powder; yield: 70%; mp: 209.6-212.4℃;¹H NMR (400 MHz, DMSO) δ (ppm): 12.50 (s, 1H), 7.81 (dd, J = 8.0, 1.2 Hz, 1H), 7.66 – 7.59 (m, 1H), 7.39 – 7.29 (m, 2H), 5.59 (s, 1H).

7-Methoxy-4-hydroxycoumarin (a1-2): white powder; yield: 66%; ¹H NMR (400 MHz, DMSO) δ (ppm): 12.34 (s, 1H), 7.71 (d, J = 8.8 Hz, 1H), 6.97 – 6.89 (m, 2H), 5.44 (s, 1H), 3.84 (s, 3H).

6-Methoxy-4-hydroxycoumarin (a1-3): white powder; yield: 66%; ¹H NMR (400 MHz, DMSO) δ (ppm): 12.49 (s, 1H), 7.33 – 7.29 (m, 1H), 7.25–7.20 (m, 2H), 5.59 (s, 1H), 3.81 (s, 3H).

7-Methyl-4-hydroxycoumarin (a1-5): white powder; yield: 68%; ¹H NMR (400 MHz, DMSO) δ (ppm): 12.38 (s, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.20–7.13 (m, 2H), 5.53 (s, 1H), 2.40 (s, 3H).

6-Methyl-4-hydroxycoumarin (a1-6): white powder; yield: 76%; ¹H NMR (400 MHz, DMSO) δ (ppm): 12.37 (s, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.21 – 7.14 (m, 2H), 5.53 (s, 1H), 2.40 (s, 3H).

7-Fluoro-4-hydroxycoumarin (a1-7): white powder; yield: 78%; ¹H NMR (400 MHz, DMSO) δ (ppm): 12.62 (s, 1H), 7.84 (dd, J = 8.8, 6.4 Hz, 1H), 7.33 (dd, J = 9.6, 2.4 Hz, 1H), 7.21 (td, J = 8.8, 2.4 Hz, 1H), 5.55 (s, 1H).

6-Fluoro-4-hydroxycoumarin (a1-8): white powder; yield: 72%; ¹H NMR (400 MHz, DMSO) δ (ppm): 12.68 (s, 1H), 7.54 – 7.46 (m, 2H), 7.45 – 7.39 (m, 1H), 5.62 (s, 1H).

7-Chloro-4-hydroxycoumarin (a1-9): white powder; yield: 84%; ¹H NMR (400 MHz, DMSO) δ (ppm): 12.65 (s, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.40 (dd, J = 8.4, 2.0 Hz, 1H), 5.59 (s, 1H).

6-Chloro-4-hydroxycoumarin (a1-10): white powder; yield: 81%; ¹H NMR (400 MHz, DMSO) δ (ppm): 12.73 (s, 1H), 7.76 (d, J = 2.4 Hz, 1H), 7.67 (dd, J = 8.8, 2.4 Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H), 5.64 (s, 1H).

7-Bromo-4-hydroxycoumarin (a1-11): white powder; yield: 74%; ¹H NMR (400 MHz, DMSO) δ (ppm): 12.70 (s, 1H), 8.02 – 7.62 (m, 2H), 7.61 – 7.35 (m, 1H), 5.64 (s, 1H).

6-Bromo-4-hydroxycoumarin (a1-12): white powder; yield: 72%;¹H NMR (400 MHz, DMSO) δ (ppm): 12.72 (s, 1H), 7.89 (d, J = 2.0 Hz, 1H), 7.79 (dd, J = 8.8, 2.4 Hz, 1H), 7.35 (d, J = 8.8 Hz, 1H), 5.62 (s, 1H).

2.1.1 Steps for synthesizing a1-4

KNO ₃ (0.52 g,6.17 mmol) was dissolved in ice-cold sulfuric acid (20.00 mL), and 4-hydroxycoumarin (1.00 g,6.17 mmol) was added to the solution;

after stirring at 0 ℃ for 12 h, the mixture was poured into ice with a large amount of solids generated, the filter residue was collected by filtration, and the crude product was purified by column chromatography to give the objective compounds a1-4, the structure was confirmed by nuclear magnetism, and the data were as follows:

6-nitro-4-hydroxycoumarin (a1-4): light yellow powder; yield: 35%; ¹H NMR (400 MHz, CDCl₃) δ 8.04 (d, J = 8.4 Hz, 1H), 7.46 – 7.36 (m, 1H), 7.36 – 7.26 (m, 2H), 7.17 (dd, J = 7.6, 1.2 Hz, 1H), 2.74 (s, 3H).

2.2, Step of synthesizing intermediate a 2:

Dissolving substituted 4-hydroxycoumarin a1 (6.17 mmol) in acetonitrile (20 mL), magnetically stirring to dissolve, adding triethylamine (1.71 mL, 12.34 mmol) into the solution, stirring at room temperature for 30min, adding p-toluenesulfonyl chloride (1.29 g, 6.78 mmol), heating to 82 ℃, magnetically stirring to react for 4h, and monitoring the reaction progress by TLC;

After the reaction is finished, cooling to room temperature, spin-drying the reaction liquid, then adding absolute ethyl alcohol (20 mL) into a reaction bottle, standing overnight at room temperature, separating out solid, filtering, collecting filter residues, drying to obtain a crude product a2, and directly using the crude product a2 in the next reaction without purification.

3. Coumarin thioether pleuromutilin derivatives PA-1 to PA-13 were synthesized according to the following synthetic route:

reagents and conditions (= 1\ ROMAN I) TRIETHYLAMINE, DMF, N2/rt.

4-Hydroxycoumarin derivative a2 (0.5 mmol) and pleuromutilin intermediate 4 (0.2 g,0.5 mmol) were dissolved in a small amount of N, N-dimethylformamide (2 mL), triethylamine (0.14 mL,1.0 mmol) was added with stirring at room temperature and reacted under nitrogen at 25 ℃. The progress of the reaction was monitored by TLC, after the completion of the reaction, a large amount of water (50 mL) was added, and solids were precipitated, suction filtered, and the residue was washed with water, and the residue was collected to give a crude product. The crude product was purified by column chromatography (PE: ea=1:1) to give the pleuromutilin derivatives PA-1 to PA-12.

Preparation of the Compound PA-13

PA-4 (0.1 g,0.17 mmol) was weighed, ammonium chloride (0.02 g,0.34 mmol) was added to ethanol (5 mL), glacial acetic acid (0.3 mL) was added with stirring, and reduced iron powder (0.05 g,0.86 mmol) was added and the temperature was raised to 80℃and 3h was reached. The progress of the reaction was monitored by TLC, after the reaction was completed, the reaction solution was filtered while it was still hot, the residue was washed with hot ethanol (50 ℃ C., 10mL X2), the filtrate was concentrated under reduced pressure to give a crude product, which was then purified by column chromatography (PE: EA=1:1) to give the derivative PA-13.

All compounds were confirmed by nuclear magnetism, high resolution mass spectrometry as follows:

(PA-1): White power; Yield: 38%; mp: 200.0-202.1℃; ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.74 (dd, J = 8.0, 1.6 Hz, 1H), 7.56 (dt, J = 8.4 , 7.2, 1.6 Hz, 1H), 7.33 (dd, J = 8.0, 0.8 Hz, 1H), 7.32 – 7.27 (m, 1H), 6.40 (dd, J = 17.2, 11.2 Hz, 1H), 6.14 (s, 1H), 5.82 (d, J = 8.4 Hz, 1H), 5.32 (dd, J = 10.8, 1.2 Hz, 1H), 5.18 (dd, J = 17.2, 1.2 Hz, 1H), 3.78 –3.73 (m, 2H), 3.37–3.33 (m, 1H), 2.38 – 2.08 (m, 6H), 1.86 – 1.35 (m, 9H), 1.16 (s, 3H), 1.14 – 1.06 (m, 1H), 0.88 (d, J = 6.8 Hz, 3H), 0.75 (d, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ (ppm)216.7, 165.9, 158.7, 154.2, 152.2, 138.6, 132.4, 124.2, 123.8, 117.7, 117.6, 117.3, 108.1, 74.6, 71.1, 58.1, 45.4, 45.1, 44.0, 41.9, 36.7, 36.0, 34.4, 34.0, 30.4, 26.8, 26.4, 24.8, 16.8, 14.9, 11.5. HRMS: calculated for C₃₁H₃₉O₆S ([M + H]⁺):539.2462; found 539.2469.

(PA-2): White power; Yield: 54%; mp:193.2-195.1℃;¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.63 (d, J = 8.8 Hz, 1H), 6.85 (dd, J = 8.8, 2.4 Hz, 1H), 6.80 (d, J = 2.4 Hz, 1H), 6.40 (dd, J = 17.2, 10.8 Hz, 1H), 5.98 (s, 1H), 5.82 (d, J = 8.4 Hz, 1H), 5.32 (dd, J = 10.8, 0.8 Hz, 1H), 5.19 (dd, J = 17.2, 0.8 Hz, 1H), 3.88 (s, 3H), 3.79 –3.70(m, 2H), 3.37 –3.33 (m, 1H), 2.37 – 2.07 (m, 6H), 1.83 – 1.35 (m, 9H), 1.16 (s, 3H), 1.14 – 1.09 (m, 1H) , 0.89 (d, J = 6.8 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) 216.8, 166.1, 163.2, 159.2, 154.3, 154.0, 138.6, 124.8, 117.6, 112.5 111.3, 105.0, 100.9, 74.6, 71.0, 58.1, 55.9, 45.5, 45.0, 44.0, 41.9, 36.7, 36.0, 34.4, 33.9, 30.4, 26.8, 26.4, 24.8, 16.9, 14.9, 11.5. HRMS: calculated for C₃₂H₄₁O₇S ([M + H]⁺):569.2568; found 569.2567.

(PA-3): White power; Yield: 51%; mp: 106.5-108.9℃;:¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.27 – 7.25 (m, 1H), 7.14 (s, 1H), 7.12 (d, J = 2.8 Hz, 1H), 6.40 (dd, J = 17.2, 10.8 Hz, 1H), 6.14 (s, 1H), 5.82 (d, J = 8.4 Hz, 1H), 5.32 (dd, J = 10.8, 1.2Hz, 1H), 5.18 (dd, J = 17.2, 1.2 Hz, 1H), 3.86 (s, 3H), 3.81 –3.71(m, 2H), 3.37 –3.33 (m, 1H), 2.37 – 2.07 (m, 6H), 1.82 – 1.35 (m, 9H), 1.16 (s, 3H), 1.14 – 1.10 (m, 1H), 0.88 (d, J = 6.8 Hz, 3H), 0.75 (d, J = 6.8 Hz, 3H).¹³C NMR (101 MHz, CDCl₃): δ (ppm) 216.8, 166.0, 159.0, 155.9, 153.7, 146.5, 138.6, 120.0, 118.3, 118.0, 117.6, 108.3, 106.3, 74.6, 71.1, 58.1, 55.9, 45.4, 45.0, 43.9, 41.9, 36.6, 36.0, 34.4, 34.0, 30.4, 26.8, 26.4, 24.8, 16.8, 14.8, 11.5. HRMS: calculated for C₃₂H₄₁O₇S ([M + H]⁺):569.2568; found 569.2573.

(PA-4): White power; Yield: 46%; mp: 198.1-201.0℃;¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.66 (d, J = 2.4 Hz, 1H), 8.42 (dd, J = 9.2, 2.4 Hz, 1H), 7.46 (d, J = 9.2 Hz, 1H), 6.39 (dd, J = 17.2, 11.2 Hz, 1H), 6.27 (s, 1H), 5.84 (d, J = 8.4 Hz, 1H), 5.32 (dd, J = 10.8, 1.2 Hz, 1H), 5.19 (dd, J = 17.2, 1.2 Hz, 1H), 3.83–3.78 (m, 2H), 3.38–3.34 (m, 1H), 2.37 – 2.10 (m, 6H), 1.82 – 1.36 (m, 9H), 1.17 (s, 3H), 1.15 – 1.10 (m, 1H), 0.89 (d, J = 6.8 Hz, 3H), 0.75 (d, J = 6.8 Hz, 3H).¹³C NMR (101 MHz, CDCl₃): δ (ppm)216.6, 165.5, 156.8, 155.7, 153.3, 143.8, 138.6, 127.1, 120.1, 118.4, 117.9, 117.3, 109.5, 74.5, 71.3, 57.9, 45.3, 45.0, 43.9, 41.8, 36.5, 36.0, 34.3, 34.0, 30.3, 26.7, 26.4 ,24.7, 16.7, 14.7, 11.4. HRMS: calculated for C₃₁H₃₈NO₈S ([M + H]⁺):584.2313; found 584.2319.

(PA-5): White power; Yield: 44%; mp: 104.2-106.8℃;¹H NMR (400 MHz, CDCl₃) : δ (ppm) 7.60 (d, J = 8.0 Hz, 1H), 7.13 (s, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.40 (dd, J = 17.2, 10.8 Hz, 1H), 6.07 (s, 1H), 5.82 (d, J = 8.4 Hz, 1H), 5.32 (dd, J = 10.8, 1.2 Hz, 1H), 5.18 (dd, J = 17.2, 1.2 Hz, 1H), 3.79 –3.70(m, 2H), 3.37 –3.33 (m, 1H), 2.45 (s, 3H), 2.38 – 2.05 (m, 6H), 1.82 – 1.34 (m, 9H), 1.16 (s, 3H), 1.14 – 1.09 (m, 1H), 0.88 (d, J = 6.8 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ (ppm)216.8, 166.0, 159.1, 154.3, 152.2, 143.8, 138.6, 125.4, 123.5, 117.7, 117.4, 115.3, 107.0, 74.6, 71.0, 58.1, 45.5, 45.0, 44.0, 41.9, 36.7, 36.0, 34.4, 33.9, 30.4, 26.8, 26.4, 24.9, 21.7, 16.9, 14.9, 11.5. HRMS: calculated for C₃₂H₄₁O₆S ([M + H]⁺):553.2618; found 553.2632.

(PA-6): White power; Yield: 47%; mp: 168.2-169.7℃; ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.51 (s, 1H), 7.36 (dd, J = 8.4, 1.6 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 6.40 (dd, J = 17.2, 10.8 Hz, 1H), 6.12 (s, 1H), 5.82 (d, J = 8.4 Hz, 1H), 5.32 (dd, J = 10.8, 1.2 Hz, 1H), 5.19 (dd, J = 17.2, 1.2 Hz, 1H), 3.78 –3.72 (m, 2H), 3.36 –3.33 (m, 1H), 2.42 (s, 3H), 2.38 – 2.07 (m, 6H), 1.82 – 1.35 (m, 9H), 1.16 (s, 3H), 1.14 – 1.10 (m, 1H), 0.89 (d, J = 6.8 Hz, 3H), 0.75 (d, J = 6.8 Hz, 3H).¹³C NMR (101 MHz, CDCl₃): δ (ppm) 216.7, 166.0, 158.9, 154.0, 150.2, 138.6, 134.0, 133.4, 123.5, 117.5, 117.4, 117.0, 107.9, 74.6, 71.0, 60.4, 58.1, 45.4, 45.0, 43.9, 41.9, 36.6, 36.0, 34.4, 34.0, 30.4, 26.8, 26.4, 24.8, 21.0, 16.8, 14.8, 11.5.HRMS: calculated for C₃₂H₄₁O₆S ([M + H]⁺):553.2618; found 553.2623.

(PA-7): White power; Yield: 50%; mp: 77.2-79.5℃;¹H NMR (400 MHz, CDCl₃) δ 7.73 (dd, J = 8.8, 5.6 Hz, 1H), 7.07 – 7.04 (m, 1H), 7.04 – 7.02 (m, 1H), 6.39 (dd, J = 17.2, 10.8 Hz, 1H), 6.09 (s, 1H), 5.82 (d, J = 8.4 Hz, 1H), 5.32 (dd, J = 10.8, 1.2 Hz, 1H), 5.18 (dd, J = 17.2, 1.2 Hz, 1H), 3.80 –3.71(m, 2H), 3.36 –3.35 (m, 1H), 2.39 – 2.07 (m, 6H), 1.82 – 1.34 (m, 9H), 1.16 (s, 3H), 1.14 – 1.10 (m, 1H), 0.88 (d, J = 6.8 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H).¹³C NMR (101 MHz, CDCl₃) : δ (ppm)216.8, 166.0, 165.8, 163.5, 158.4, 153.9, 153.6, 153.4, 138.5, 125.7, 125.6, 117.7, 114.4, 112.5, 112.3, 106.9, 104.8, 104.6, 74.6, 71.1, 58.1, 45.4, 45.0, 43.9, 41.9, 36.6, 36.0, 34.4, 34.0, 30.4, 26.8, 26.3, 24.8, 16.9, 14.8, 11.5 HRMS: calculated for C₃₁H₁₈FO₆S ([M + H]⁺):557.2368; found 557.2374.

(PA-8): White power; Yield: 48%; mp: 72.4-75.0℃; ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.40 (dd, J = 8.4, 2.4 Hz, 1H), 7.32 – 7.24 (m, 2H), 6.38 (dd, J = 17.2, 10.8 Hz, 1H), 6.17 (s, 1H), 5.81 (d, J = 8.4 Hz, 1H), 5.30 (dd, J = 10.8, 1.2 Hz, 1H), 5.17 (dd, J = 17.2, 1.2 Hz, 1H), 3.81 –3.72(m, 2H), 3.36 –3.33 (m, 1H), 2.34 – 2.10 (m, 6H), 1.83 – 1.33 (m, 9H), 1.15 (s, 3H), 1.13 – 1.07 (m, 1H), 0.87 (d, J = 6.8 Hz, 3H), 0.73 (d, J = 6.8 Hz, 3H).¹³C NMR (101 MHz, CDCl₃) : δ (ppm) δ 216.7, 165.8, 159.8, 158.4, 157.4, 153.3, 148.4, 138.6, 120.1, 119.8, 119.0, 118.9, 118.6, 118.5, 117.7, 109.9, 109.6, 109.0, 74.6, 71.2, 58.1, 45.5, 45.1, 44.0, 41.9, 36.7, 36.0, 34.4, 34.1, 30.4, 26.9, 26.4, 24.9, 16.9, 14.9, 11.6. HRMS: calculated for C₃₁H₁₈FO₆S ([M + H]⁺):557.2368; found 557.2374.

(PA-9): White power; Yield: 49%; mp: 118.4-120.1℃;¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.63 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 2.0 Hz, 1H), 7.23 (dd, J = 8.4, 2.0 Hz, 1H), 6.36 (dd, J = 17.2, 10.8 Hz, 1H), 6.10 (s, 1H), 5.79 (d, J = 8.4 Hz, 1H), 5.28 (dd, J = 10.8, 1.2 Hz, 1H), 5.15 (dd, J = 17.2, 1.2 Hz, 1H), 3.79 –3.71(m, 2H), 3.36 –3.32 (m, 1H), 2.35 – 2.04 (m, 6H), 1.86 – 1.31 (m, 9H), 1.13 (s, 3H), 1.11 – 1.06 (m, 1H), 0.86 (d, J = 6.8 Hz, 3H), 0.71 (d, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) 216.7, 165.8, 152.6, 138.6, 138.5, 124.8, 124.8, 117.7, 117.5, 107.9, 74.6, 71.2, 58.1, 45.5, 45.1, 44.0, 41.9, 36.6, 36.0, 34.4, 34.0, 30.4, 26.8, 26.4, 24.9, 16.9, 14.9, 11.6. HRMS: calculated for C₃₁H₃₈ClO₆S ([M + H]⁺):573.2072; found 573.2086.

(PA-10): White power; Yield: 55%; mp: 104.6-107.0℃;¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.68 (d, J = 2.4 Hz, 1H), 7.48 (dd, J = 8.8, 2.4 Hz, 1H), 7.24 (s, 1H), 6.37 (dd, J = 17.2, 10.8 Hz, 1H), 6.14 (s, 1H), 5.80 (d, J = 8.4 Hz, 1H), 5.29 (dd, J = 10.8 1.2 Hz, 1H), 5.16 (dd, J = 17.2, 1.2 Hz, 1H), 3.78 –3.70 (m, 2H), 3.35 –3.31 (m, 1H), 2.37 – 2.05 (m, 6H), 1.80 – 1.32 (m, 9H), 1.14 (s, 3H), 1.12 – 1.07 (m, 1H), 0.86 (d, J = 6.8 Hz, 3H), 0.72 (d, J = 6.8 Hz, 3H).¹³C NMR (101 MHz, CDCl₃): δ (ppm)216.7, 165.7, 158.0, 153.1, 150.6, 138.6, 132.4, 129.8, 123.4, 118.8, 118.7, 117.6, 109.0, 74.6, 71.2, 58.1, 45.4, 45.1, 44.0, 41.9, 36.6, 36.0, 34.4, 34.0, 30.4, 26.8, 26.4, 24.8, 16.8, 14.8, 11.5. HRMS: calculated for C₃₁H₃₈ClO₆S ([M + H]+):573.2072; found 573.2085.

(PA-11): White power; Yield: 44%; mp: 108.1-109.4℃;¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.56 (d, J = 8.4 Hz, 1H), 7.47 (d, J = 1.6 Hz, 1H), 7.38 (dd, J = 8.4, 2.0 Hz, 1H), 6.36 (dd, J = 17.2, 10.8 Hz, 1H) , 6.12 (s, 1H), 5.79 (d, J = 8.4 Hz, 1H), 5.28 (dd, J = 10.8, 1.2 Hz, 1H), 5.15 (dd, J = 17.2, 1.2 Hz, 1H), 3.79 –3.70(m, 2H), 3.36 –3.32 (m, 1H), 2.37 – 2.04 (m, 6H), 1.83 – 1.31 (m, 9H), 1.13 (s, 3H), 1.11 – 1.06 (m, 1H), 0.86 (d, J = 6.8 Hz, 3H), 0.71 (d, J = 6.8 Hz, 3H).¹³C NMR (101 MHz, CDCl₃) : δ (ppm) 216.7, 165.8, 158.0, 153.8, 152.4, 138.6, 127.7, 126.5, 124.9, 120.5, 117.7, 116.7, 108.1, 74.6, 71.2, 58.1, 45.5, 45.1, 44.0, 41.9, 36.6, 36.0, 34.4, 34.0, 30.4, 26.8, 26.4, 24.8, 16.9, 14.9, 11.5. HRMS: calculated for C₃₁H₃₈BrO₆S ([M + H]⁺):617.1567; found 617.1578.

(PA-12): White power; Yield: 56%; mp: 105.8-108.0℃;¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.84 (d, J = 2.0 Hz, 1H), 7.64 (dd, J = 8.8, 2.0 Hz, 1H), 7.21 (d, J = 8.8 Hz, 1H), 6.39 (dd, J = 17.2, 10.8 Hz, 1H), 6.16 (s, 1H), 5.82 (d, J = 8.4 Hz, 1H), 5.31 (dd, J = 10.8, 1.2 Hz, 1H), 5.18 (dd, J = 17.2, 1.2 Hz, 1H), 3.81 –3.72(m, 2H), 3.37 –3.33 (m, 1H), 2.40 – 2.07 (m, 6H), 1.83 – 1.34 (m, 9H), 1.16 (s, 3H), 1.14 – 1.10 (m, 1H), 0.88 (d, J = 6.8 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ (ppm)216.6, 165.6, 157.8, 152.9, 150.9, 138.6, 135.1, 126.3, 119.1, 118.9, 117.3, 116.9, 108.8, 74.5, 71.1, 57.9, 45.3, 44.9, 43.9, 41.8, 36.5, 35.9, 34.3, 33.9, 30.3, 26.7, 26.4, 24.7, 16.7, 14.7, 11.4. HRMS: calculated for C₃₁H₃₈BrO₆S ([M + H]⁺):617.1567; found 617.1574.

(PA-13): White power; Yield: 70%; mp: 150.2-160.4℃;¹H NMR (400 MHz, CDCl₃): δ (ppm)7.13 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 2.4 Hz, 1H), 6.88 (dd, J = 8.4, 2.6 Hz, 1H), 6.39 (dd, J = 17.2, 10.8 Hz, 1H), 6.09 (s, 1H), 5.81 (d, J = 8.4 Hz, 1H), 5.31 (dd, J = 10.8, 1.2 Hz, 1H), 5.18 (dd, J = 17.2, 1.2 Hz, 1H), 3.78 –3.72(m, 2H), 3.35 (d, J = 6.4 Hz, 1H), 2.37 – 2.06 (m, 6H), 1.81 – 1.34 (m, 9H), 1.15 (s, 3H), 1.13 – 1.09 (m, 1H), 0.88 (d, J = 6.8 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 216.8, 166.0, 159.2, 153.6, 145.4, 143.0, 138.6, 120.3, 118.2, 118.0, 117.6, 108.3, 74.6, 71.0, 58.1, 45.4, 45.0, 44.0, 41.9, 36.7, 36.0, 34.4, 34.0, 30.4, 26.8, 26.4, 24.8, 16.8, 14.9, 11.5. HRMS: calculated for C₃₁H₄₀NO₆S ([M + H]⁺):554.2571; found 554.2576.

4. In vitro antibacterial Activity Studies

4.1 Minimum Inhibitory Concentration (MIC) test methods

(1) The test strain is determined by selecting gram positive bacteria MRSA ATCC 33591,MRSA ATCC 43300,MSSA ATCC 29213,MRSE ATCC 51625,MSSE ATCC 12228 and gram negative bacteria E.coli ATCC 25922 as MIC values.

(2) And (3) diluting the medicine, namely respectively dissolving and diluting the target compound and the tiamulin by taking DMSO as a solvent to prepare mother solution with the concentration of 12800 mug.mL ^-1, and placing the mother solution in a refrigerator for light-shielding sealing and storage for standby.

(3) The preparation of bacterial liquid comprises the steps of activating each tested bacterium, picking a monoclonal colony in 0.9% physiological saline, preparing the bacterial liquid into 0.5 McP (1.5×10 ⁸ CFU·mL^-1), and diluting the bacterial liquid with Mueller-Hinton sterile broth (MHB) for 10 times for later use.

(4) Positive control tiamulin, valnemulin and ritaparine were selected as positive controls.

(5) The MIC measurement comprises the steps of adding 100 mu L of MHB into the wells except the edge well and the second row well, adding 196 mu L of MHB and 4 mu L of mother solution into the second well, respectively diluting the compound and the positive control by a double dilution method, diluting the compound and the positive control into diluent with concentration gradient of 128-0.25 mu g.mL ^-1, adding 100 mu L of bacterial floating solution into each well except the edge well, fully mixing the bacterial floating solution, finally adding 200 mu L of sterile water into each well except the edge well, culturing at a constant temperature of 37 ℃ for 18-24 h, observing the growth condition of the test bacteria, taking the minimum concentration of the non-growing drug as the MIC value of the drug on the test bacteria, taking the ethanol solution with the concentration of the compound being equal as the negative control, carrying out 3 parallel experiments on each strain of the test bacteria, and repeating the experiments for 3 times. The growth conditions of the test bacteria of the negative control group are all good, and the rest experimental results are shown in table 1.

TABLE 1

Note that "R" represents ritapalin, "T" represents tiamulin fumarate and "V" represents valnemulin hydrochloride

The MIC value of the compound PA of the coumarin thioether pleuromutilin derivative is 0.002-0.25 mu g.mL ^-1, and all the compounds have antibacterial effect superior to tiamulin. However, the desired bacteriostatic activity against gram-negative bacteria E.coli ATCC 25922 was not achieved. The bacteriostatic activity of the compound is more than or equal to 0.062 mug.mL ^-1 on 9 compounds of 5 gram positive bacteria MRSA ATCC 33591,MRSA ATCC 43300,MSSA ATCC 29213,MRSE ATCC 51625,MSSE ATCC 12228,PA, wherein most of the compounds have the same bacteriostatic effect as control valnemulin. The compounds are derivatives with different halogen substitutions in the benzene ring region of coumarin, wherein the fluorine substitution derivatives (PA-7, PA-8) have better activity, and under the same substituent, the 7-substituted pleuromutilin derivative of coumarin is slightly better than the 6-substituted pleuromutilin derivative of coumarin. Derivatives (PA-2, PA-3, PA-5, PA-6) obtained by introducing methoxy and methyl into coumarin benzene ring region have better activity by introducing substituent at 7 position of coumarin benzene ring region. Meanwhile, MBC values of the targets were measured to determine whether they have antibacterial or bactericidal effects (table 1). As shown in the table, among the 13 compounds, most compounds have MBC/MIC values greater than 4, and exhibit antibacterial effects. From the data of MIC and MBC, the activity of the compound PA-2 is optimal, the MIC value of the compound PA-2 is 4-64 times higher than that of tiamulin, and the MIC and MBC values of the compound PA-2 are even better than those of the control valnemulin and Ruitapalene.

The coumarin thioether pleuromutilin derivative compounds of the invention all show excellent antibacterial effect, and are expected to treat bacterial infection caused by staphylococcus aureus resistance.

4.2 Time-Sterilization Curve determination of pleuromutilin derivative PA-2

(1) The test strain is methicillin-resistant staphylococcus aureus ATCC 33591

(2) In the experiment, bacteria to be detected are recovered, passaged and diluted to about 10 ⁵ CFU· mL^-1, inoculated onto MHB culture medium with corresponding medicine concentration for culture, wherein the medicine concentration comprises 1,2,4 XMIC tiamulin, 1,2,4 XMIC compound to be detected and negative control, the sampling time is 0,2,4,6,8,12,24 h, the bacteria to be detected are sampled from the culture medium (10 mu L) at each designated sampling time point, corresponding multiples are diluted in saline, then 25 mu L of diluted solution is uniformly inoculated on an MHA plate, after incubation is carried out at 37 ^o C for 20 h, the total number of bacteria (CFU.ML ^-1) on the plate is measured, the bacteria are measured three times in parallel, an average value is obtained, a sterilization curve is drawn by drawing the relation between log ₁₀ CFU·mL^-1 and time, and the experimental result is shown in figure 1 and figure 2.

Therefore, the compound PA-2 can kill bacteria in a shorter time, the sterilization rate is far faster than that of tiamulin, and both the compound PA-2 and the tiamulin show the time dependence and the concentration dependence of the medicine.

4.3 Post-antibiotic Effect determination of pleuromutilin derivative PA-2

(1) The test strain is methicillin-resistant staphylococcus aureus ATCC 33591

(2) Experimental protocol in this experiment, the test bacteria were resuscitated, passaged, and then diluted to about 10 ⁶CFU·mL^-1 in MHB as inoculum, then the test compound or tiamulin was added to the tubes containing the inoculum (final drug concentration of 2,4×mic tiamulin and 2,4×mic test compound and negative control), these tubes were incubated at 37 ℃ for 2h, after 2h the cultures in the tubes were diluted 1000-fold into preheated MH broth, then incubated in new tubes, samples (100 μl) were extracted in the tubes at 0,1,2,4 and 6 h, the samples were diluted by corresponding fold with sterile saline and plated on preheated MHA agar plates, plates were counted after incubation at 37 ℃ 22-26 h, the results of Post Antibiotic Effect (PAE) were calculated by the formula pae=ta-TC, as shown in the following table, (time required for increasing 1 log ₁₀ CFU/mL for negative control bacteria in the experiment, TC is time required for increasing log ₁₀ CFU/mL for the control group) as shown in fig. 3.

PAEs value of tiamulin and Compound PA-2 on MRSA ATCC33591

The results show that the PAE duration of the compound PA-2 is longer than that of tiamulin under 2 xMIC and 4 xMIC, and that Tiamulin has PAE which is independent of concentration and the PAE of the compound PA-2 shows a certain concentration dependence from the table, and the prolongation of the PAE of the compound PA-2 also promotes us to further research and develop the potential application of the compound in treating the infection caused by MRSA.

4.4 Cytotoxicity experiments of pleuromutilin derivative PA-2

(1) The experimental cell strain is two kinds of HepG2 and A549 cells.

(2) The experimental method comprises the steps of counting and plating cells in a logarithmic growth phase when the cells are recovered to a good growth condition, inoculating the cells in a logarithmic growth phase into a 96-well plate (about 7×10 ³ cells per well), observing the morphology and the number of the cells by an inverted microscope, culturing 24h under a constant temperature condition of 37 ℃ and CO ₂ (5%), replacing old culture medium by adding DMEM culture medium containing compound solution to be tested with the concentration of 0, 5, 10, 20 and 40 mug.mL ^-1 respectively, continuously culturing 24h, adding 150 mu L of MTT (0.5 mg.mL ^-1) into each well under a light-resistant condition, continuously culturing 4h in an incubator, removing MTT in the well after the culturing is finished, adding 150 mu L of DMSO into each well, placing 10min under the temperature condition of 37 ℃, sufficiently dissolving the crystal, firstly vibrating 15 s on an enzyme-label instrument, measuring the absorbance value of each well, measuring the detection wavelength to be 570 nm, finally calculating the relative growth rate of the cells according to the OD value ratio between a test group and a blank group, and evaluating the toxicity of the compound to be tested, and taking the results of the test in parallel to the graph as shown in the figure 5.

Cell survival and growth conditions are detected by an MTT method, specific tetrazolium salts are converted by utilizing activity of enzymes in mitochondria of living cells to generate blue-violet crystal formazan sediment, and cytotoxicity of the compound PA-2 to cancer cells HepG2 and A549 is measured by an enzyme-labeled instrument, as shown in figure 5, the influence on the activity of the compound PA-2 in a concentration range of 0-40 mug.mL ^-1 is small, the cell survival rate is above 85%, and the compound PA-2 shows lower cytotoxicity to test cell strains in the concentration range.

4.5 Test of the treatment effect of the model of wound inflammation in mice of the pleuromutilin derivative PA-2

(1) Experimental animals clean-up male BALB/c 28 day old mice, body weight 23-25 g.

(2) The test strain is methicillin-resistant staphylococcus aureus ATCC 33591.

(3) The experimental method comprises the following steps:

MRSA stored at-70 ℃ is inoculated on a flat plate, after rejuvenation is carried out three times, single colony is selected to be inoculated on TSB culture medium again, bacteria in the logarithmic phase are cultivated for 12-16 h, the obtained bacterial liquid is centrifuged, supernatant is removed, PBS is used for adjusting the bacteria to 10 ¹² CFU/mL after PBS is resuspended.

The method comprises the steps of injecting chloral hydrate into abdominal cavity of 10% of mice for anesthesia and fixing, shearing and exposing an operation area on the back of the mice, disinfecting skin of the operation area by alcohol, gently clamping the skin of the operation area by forceps to avoid clamping muscles, making a vertical incision with a length of about 1.5 cm depth to a myomembrane at about 0.5 cm of the spine of the back of the mice by using sterile scissors, after the incision is formed, treating blood stains of the wounds by cotton balls for standby, taking MRSA 33591 bacterial liquid 0.1 mL (10 ¹² CFU/mL) in a logarithmic growth phase to uniformly smear the whole wound (a blank control group is not smeared with bacteria), taking 1% and 3% of ointment as an experiment group after bacterial infection is 4h, smearing 15 mg/each time, smearing the blank matrix by using ointment matrix without medicines, 15 mg/time, using 7 days for continuous use, feeding all treated experimental animals in a single cage to avoid mutual biting wounds, recording the wound on each day, recording the day, and storing the wound after HE dyeing, and carrying out the results of Mason dyeing experiments as shown in FIG. 8, and MAS dyeing results shown in FIG. 6, and MAS dyeing results are shown in FIG. 8 after the map are observed.

The results show that the mice in the blank group have good wound healing capacity and almost complete healing at 6d, the mice in the model group show some symptoms (such as non-crusting of the wound and partial purulent exudation), compared with the model group, the PA-2 and Retapamulin experimental groups can enhance wound healing, the wounds of 1% of the mice in the PA-2 group gradually crusted after 4d but not healed at 6d, the wounds of 1% Retapamulin group gradually healed after 4d, the wound healing condition of the mice in the 6d is greatly improved, the wound healing condition of the 3% of the mice in the PA-2 and 3% Retapamulin groups is greatly improved, the wound starts to heal and crusts, the wound healing condition of the wounds of the 6d is greatly healed, the residual small crusts are obviously smaller than that of the model group, and the wound healing condition of the wounds of the 3% of the PA-2 experimental group is good, the wound almost complete healing condition of the wounds is close to the blank group.

Histopathological analysis was performed by HE and Masson staining. 6 d after infection, severe inflammatory cell infiltration, inflammatory cell accumulation, muscle fiber swelling and granule degeneration were found on the wound skin of model group mice. Notably, myofibroblast proliferation and new capillary increase with significant relief of inflammation following treatment with 3% of compound PA-2 and 3% Retapamulin ointment. In addition, after treatment with 3% PA-2 and 3% Retapamulin ointment, the wound showed significant blue collagen deposition.

The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims

1. A coumarin thioether pleuromutilin derivative, characterized in that the coumarin pleuromutilin derivative is a compound having a structure shown in Formula I and a pharmaceutically acceptable salt thereof:

(Formula I)

In the formula, R ₁ and R ₂ are H, OMe, NO ₂ , NH ₂ , Me, F, Cl or Br.

2. The coumarin thioether 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 and 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 the coumarin thioether pleuromutilin derivative according to any one of claims 1 to 2, characterized in that it 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 thioacetate to obtain the pleuromutilin intermediate 3 shown in the following formula:

(3) The intermediate 3 is reacted with sodium methoxide to obtain the pleuromutilin intermediate 4 shown in the following formula:

(4) The substituted acetophenone a0 is reacted with sodium hydride diethyl carbonate turbid solution to obtain the substituted 4-hydroxycoumarin intermediate a1 shown in the following formula:

(5) The substituted 4-hydroxycoumarin a1 is reacted with toluenesulfonyl chloride to obtain a 4-hydroxycoumarin derivative intermediate a2 as shown in the following formula:

(6) reacting the 4-hydroxycoumarin derivative intermediate a2 with the pleuromutilin intermediate 4 to obtain a coumarin thioether pleuromutilin derivative having the following formula I:

(Formula I) .

4. A method for preparing a coumarin sulfide 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 an ice bath, and the mixture is placed at 45-65 ^° C for reaction for about 0.5-1.5h, and after the reaction is completed, water is added and filtered under reduced pressure, the filter cake is washed with water, and dried to obtain the pleuromutilin intermediate 2.

5. A method for preparing a coumarin thioether pleuromutilin derivative according to claim 3, characterized in that the reaction conditions of step (2) are as follows: intermediate 2 and potassium thioacetate are dissolved in EA, reacted at 20-30°C, washed with saturated _NaHCO3 solution after the reaction is completed, the organic layer is separated and collected, and then dried over anhydrous _Na2SO4 , and concentrated _under reduced pressure to obtain a yellow oily substance, i.e. pleuromutilin intermediate 3.

6. A method for preparing a coumarin thioether pleuromutilin derivative according to claim 3, characterized in that the reaction conditions of step (3) are: dissolving the intermediate 3 in methanol under _N2 protection, then adding sodium methoxide to the reaction solution until the pH of the reaction solution is 9-11, reacting for 2-4 hours, and concentrating under reduced pressure after the reaction is completed, and purifying by column chromatography to obtain a white solid target compound pleuromutilin intermediate 4.

7. A method for preparing a coumarin sulfide pleuromutilin derivative according to claim 3, characterized in that the reaction conditions of step (4) are as follows: dissolving substituted acetophenone a0 in toluene and placing it in an ice bath; taking sodium hydride in diethyl carbonate, and then slowly dropping the sodium hydride diethyl carbonate turbid solution into the substituted acetophenone a0 solution, after the dropwise addition is complete, taking out the ice bath and heating to 100-120°C, stirring magnetically to react for 3-5 hours, after the reaction is completed, cooling to room temperature, quenching the unreacted sodium hydride with water, extracting the excess diethyl carbonate with methyl tert-butyl ether, and then adjusting the pH to 3 with 10% dilute hydrochloric acid, at this time a large amount of white solid precipitates, and filtering and drying to obtain a white solid as a crude product, which is purified by column chromatography to obtain the substituted 4-hydroxycoumarin intermediate a1.

8. A method for preparing a coumarin thioether pleuromutilin derivative according to claim 3, characterized in that the reaction conditions of step (5) are as follows: dissolving substituted 4-hydroxycoumarin a1 in acetonitrile, adding triethylamine, stirring at room temperature for 25-35 min, adding p-toluenesulfonyl chloride, heating to 75-85°C, and reacting under magnetic stirring for 3-5h. After the reaction is completed, cooling to room temperature, spinning the reaction solution to dryness, and then adding anhydrous ethanol, leaving it at room temperature overnight until solid precipitates, filtering and collecting the residue, and drying to obtain the 4-hydroxycoumarin derivative intermediate a2.

9. A method for preparing a coumarin sulfide truncated pleuromutilin derivative according to claim 3, characterized in that the reaction conditions of step (6) are as follows: dissolving 4-hydroxycoumarin derivative a2 and truncated pleuromutilin intermediate 4 in N,N-dimethylformamide, adding triethylamine under stirring at room temperature, reacting at 20-30°C under _N2 protection, adding water after the reaction until solid precipitates, filtering, washing, collecting the filter residue to obtain a crude product, and purifying the crude product by column chromatography to obtain a coumarin sulfide truncated pleuromutilin derivative having formula I.

10. Use of the coumarin thioether pleuromutilin derivative according to any one of claims 1 to 2, characterized in that it is used for preparing a drug for treating infectious diseases, wherein the infectious diseases are infectious diseases caused by drug-resistant bacteria.