Pleuromutilin derivative capable of inhibiting biosynthesis of bacterial hydrogen sulfide as well as preparation method and application of pleuromutilin derivative

Abstract

The invention discloses a pleuromutilin derivative capable of inhibiting biosynthesis of bacterial hydrogen sulfide as well as a preparation method and application of the pleuromutilin derivative, and belongs to the field of medicinal chemistry. The derivative is introduced into an indole structure through structural modification, so that the biosynthesis of hydrogen sulfide in bacteria is effectively inhibited, and the sensitivity of bacteria to antibiotics is enhanced. Experiments of a trace double dilution method and a lead acetate test strip colorimetric method show that the derivative shows good antibacterial activity and safety on pathogenic microorganisms, especially drug-resistant bacteria and mycoplasmas, and compared with drugs on the market, the derivative has the advantages of stronger inhibition ability on the drug-resistant bacteria, better inhibition effect on hydrogen sulfide, lower toxicity, better solubility and better application prospect. The derivative and the stereoisomer, salt and other forms of the derivative are easy in preparation raw material obtaining, safe in operation, mild in reaction condition, high in yield and suitable for large-scale production, can be widely used for preventing and treating infectious diseases caused by gram-positive bacteria and gram-negative bacteria, drug-resistant bacteria or mycoplasma, and have remarkable medical development value and application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and particularly to a pleuromutilin derivative with the ability to inhibit bacterial hydrogen sulfide biosynthesis, and its preparation method and application. Background Art

[0002] In recent years, due to the abuse of antibiotics, there are more and more drug-resistant bacteria, and the problem of bacterial drug resistance has become increasingly serious. A variety of drug-resistant "super bacteria" have begun to spread globally, posing a great threat to human health. Facing the increasing number of drug-resistant bacteria, the expanding scope of drug resistance, the increasing degree of drug resistance, and the continuous emergence of multi-drug-resistant bacteria, it is of great significance to explore drugs with a new antibacterial mechanism, good bioavailability, and low toxicity. Pleuromutilin is a diterpenoid compound with a rigid 5-6-8 tricyclic carbon skeleton isolated from two natural basidiomycetes (Pleurotus mutilus, Pleurotus passeckerianus), and has potent antibacterial activity against Gram-positive bacteria and mycoplasmas. Pleuromutilin mainly acts on the peptidyl transfer center (PTC) of the bacterial ribosome, interfering with the binding of tRNA to the P site and A site, thereby inhibiting protein synthesis. Due to the high conservation of PTC, its drug resistance rate is relatively low. The antibacterial mechanism of pleuromutilin is different from that of existing antibiotics on the market, providing new ideas for finding safe and effective drugs and becoming one of the hotspots in current antibiotic research.

[0003] Hydrogen sulfide (H 2 S) is an important gaseous signaling molecule, and bacterial hydrogen sulfide (H 2 S) biosynthesis plays a key role in the bacterial metabolic process. H 2 S significantly enhances the survival ability of bacteria in adverse environments by regulating oxidative stress responses, metabolic pathways, and affecting biofilm formation. Studies have shown that the H 2 S level of persister bacteria is significantly higher than that of ordinary active cells. Inhibiting the biosynthesis of H 2 S can significantly enhance the sensitivity of bacteria to antibiotics. Therefore, inhibiting bacterial hydrogen sulfide biosynthesis is considered a potential antibacterial strategy that can enhance the efficacy of antibiotics and combat drug-resistant bacteria. Most of the existing methods for inhibiting bacterial hydrogen sulfide biosynthesis rely on small molecule inhibitors or gene editing techniques, and these methods have many limitations in practical applications. For example, small molecule inhibitors have a narrow range of action, are prone to drug resistance, have a complex R & D process and a low success rate; gene editing techniques have off-target effects, limitations in delivery systems, and ethical and legal issues, and the applicable range of clinical indications is narrow. Therefore, it is particularly important to develop new, efficient, and low-toxic antibacterial agents for inhibiting bacterial hydrogen sulfide biosynthesis. Summary of the Invention

[0004] In view of the current technical situation that existing pleuromutilin compounds have poor activity against clinically isolated drug-resistant bacteria, and thus cannot effectively treat clinical drug-resistant bacterial infections, the present invention aims to provide a pleuromutilin derivative having the inhibition of bacterial hydrogen sulfide biosynthesis, its preparation method and application. This derivative can inhibit bacterial hydrogen sulfide biosynthesis and enhance the efficacy of antibiotics.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a pleuromutilin derivative having the inhibition of bacterial hydrogen sulfide biosynthesis, which is a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer or tautomer of the compound represented by formula (I);

[0007]

[0008] wherein, R 1 is selected from any one of

[0009] R 2 is selected from any one of

[0010] The pleuromutilin derivative is selected from any one of the compounds represented by the following formulas:

[0011]

[0012] The pharmaceutically acceptable salt is a salt formed by a pleuromutilin derivative having the inhibition of bacterial hydrogen sulfide biosynthesis and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

[0013] The present invention provides a preparation method of the above-mentioned pleuromutilin derivative having the inhibition of bacterial hydrogen sulfide biosynthesis, including the following synthesis steps:

[0014] (1) Using pleuromutilin and p-toluenesulfonyl chloride as raw materials, reacting to obtain intermediate I;

[0015] The intermediate I is

[0016] (2) Using the intermediate I prepared in step (1) and SH-R 1 -NH 2 as raw materials, reacting to obtain intermediate II; The intermediate II is wherein, R1 Selected from

[0017] any one of;

[0018] (3) Using (1H-indol-3-yl)boronic acid and Br-R 2 as raw materials, reacting to obtain intermediate III;

[0019] The said intermediate III is: wherein, R 2 Selected from any one of;

[0020] (4) Reacting the intermediate III obtained in step (3) with 3-bromobenzoic acid to obtain intermediate IV;

[0021] The said intermediate IV is: wherein, R 2 Selected from any one of;

[0022] (5) Using intermediate II and intermediate IV as raw materials, reacting to obtain the pleuromutilin derivative shown in formula (I) with the function of inhibiting bacterial hydrogen sulfide biosynthesis.

[0023] In step (1), the molar ratio of the pleuromutilin to p-toluenesulfonyl chloride is 1:1 to 2, and the reaction conditions are: reacting at room temperature for 6 to 10 h;

[0024] Furthermore, the solvent is acetonitrile, and the used catalyst is a composite catalyst composed of DIEA and DMAP.

[0025] In step (2), the molar ratio of the intermediate I and SH-R 1 -NH 2 is 1:1 to 2, and the reaction conditions are: reacting at 50 to 70 °C for 5 to 7 h;

[0026] Furthermore, using N,N-dimethylformamide (DMF) as the solvent, a composite catalyst composed of K 2 CO 3 and KI.

[0027] In step (3), the molar ratio of the (1H-indol-3-yl)boronic acid and Br-R 2 is 1:1 to 2, and the reaction conditions are: reacting at 0 to 10 °C for 1 to 3 h;

[0028] Furthermore, tetrahydrofuran (THF) is the solvent, and NaH is the dehydrogenating agent.

[0029] In step (4), the molar ratio of intermediate Ⅲ to 3-bromobenzoic acid is 1:1 to 2, and the reaction conditions are to react at 100 - 140 °C for 6 - 8 h;

[0030] In step (5), the molar ratio of intermediate Ⅱ to intermediate Ⅳ is 1:1 to 2, and the reaction conditions are to react at room temperature for 1 - 3 h.

[0031] Furthermore, the solvent is N,N-dimethylformamide (DMF), the condensing agent used is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and the catalyst used is 4-dimethylaminopyridine (DMAP).

[0032] Use of the above-mentioned pleuromutilin derivatives with inhibitory effect on bacterial hydrogen sulfide biosynthesis in the preparation of antibacterial products.

[0033] The antibacterial product is a pharmaceutical preparation for treating pathogenic microorganism infectious diseases. The pharmaceutical preparation is used alone or in combination with other antibacterial drugs, or mixed with pharmaceutically acceptable excipients and diluents to form tablets, capsules, granules, syrups, premixes or pellets for oral administration, or prepared into liniments or injections for non-oral administration.

[0034] The pathogenic microorganism is Gram-positive bacteria, Gram-negative bacteria, drug-resistant bacteria or mycoplasma.

[0035] The drug for treating pathogenic microorganism infectious diseases includes a pharmaceutically acceptable carrier.

[0036] The present invention provides a pharmaceutical composition, which contains the above-mentioned pleuromutilin derivatives with inhibitory effect on bacterial hydrogen sulfide biosynthesis as an active ingredient.

[0037] Compared with the prior art, the technical solution of the present invention has achieved the following beneficial technical effects:

[0038] The pleuromutilin derivative provided by the present invention with inhibitory effect on bacterial hydrogen sulfide biosynthesis is obtained by structurally modifying pleuromutilin using an indole structure and a linker. This pleuromutilin derivative can effectively inhibit bacterial H 2The biosynthesis of S weakens the bacterial defense mechanism, thus significantly enhancing the sensitivity of bacteria to antibiotics. In vitro antibacterial tests show that this derivative exhibits good inhibitory effects on different strains, especially stronger inhibitory ability against drug-resistant bacteria than the marketed drugs valnemulin and retapamulin, indicating that this derivative has a broad antibacterial spectrum and strong antibacterial activity; through the hydrogen sulfide statistical test, this pleuromutilin derivative has a better inhibitory effect on hydrogen sulfide than tylosin and valnemulin, further demonstrating its unique advantage in inhibiting the biosynthesis of bacterial hydrogen sulfide. The hydrogen sulfide inhibition strategy not only improves the sensitivity of pleuromutilin derivatives to bacteria but also provides new ideas and methods for the development of new antibacterial drugs; in the safety evaluation experiment, compared with the positive control drugs retapamulin and valnemulin, the pleuromutilin derivative of the present invention shows lower toxicity to the test cells, indicating that this derivative has good safety while maintaining strong antibacterial activity; through structural modification and decoration, the derivative of the present invention successfully improves the solubility of pleuromutilin derivatives, making them easier to prepare and apply. The pleuromutilin derivative with the function of inhibiting the biosynthesis of bacterial hydrogen sulfide provided by the present invention not only has strong in vitro antibacterial activity and significant inhibitory ability against drug-resistant bacteria but also shows lower toxicity and better solubility, and has broad application prospects and important research value in the field of antibacterial drug research and development.

[0039] The preparation method of the novel pleuromutilin derivative with the function of inhibiting the biosynthesis of bacterial hydrogen sulfide according to the present invention has easily available raw materials, high operation safety, mild reaction conditions, low cost, and high yield, which is between 72% and 92%, and is suitable for industrial production.

[0040] The novel pleuromutilin derivative with the function of inhibiting the biosynthesis of bacterial hydrogen sulfide according to the present invention can be applied to the prevention and treatment of infectious diseases caused by Gram-positive bacteria, Gram-negative bacteria, drug-resistant bacteria or mycoplasma, and has good pharmaceutical development value. Brief Description of the Drawings

[0041] Figure 1 It is the antibacterial effect diagram of Compound 8 and Compound 9 of the present invention, where A is b Methicillin-resistant S.aureus ATCC 33591, Compound 8 (rows B, C, D), 9 (rows E, F, G); B is g S.enterica ATCC14028, Compound 8 (rows B, C, D), 9 (rows E, F, G);

[0042] Figure 2MIC heat maps (μg / mL) of the series of compounds of the present invention, valnemulin, and retapamulin. Among them, A is MRSA ATCC 33591, B is MRSA ATCC 43300, C is S. aureus ATCC 29213, D is MRSE ATCC 51625, E is A. baumannii ATCC 19606, F is S. enterica ATCC 14028, G is E. coli ATCC 25922, H is E. coli CMCC 44103, Val is valnemulin, Ret is retapamulin, and 1-12 are the series of compounds synthesized in the present invention;

[0043] Figure 3 Graph of the lead acetate test result of Compound 9 of the present invention;

[0044] Figure 4 Cytotoxicity test results of Compound 8, Compound 9, retapamulin, and valnemulin of the present invention.

[0045] Figure 5 IC 50 Inhibition activity test graph of Compound 9 of the present invention against bCSE, where A is the IC 50 Inhibition activity test graph of Compound 9 against bCSE, B is the IC 50 Inhibition activity test graph of Compound 9 against hCSE. Detailed implementation manners

[0046] The following examples are used to further illustrate the present invention, but these examples are only for better understanding of the invention and are not used to limit the scope or implementation principles of the present invention. The implementation manners of the present invention are not limited to the following content. Unless otherwise specified, the test methods used are conventional methods, and the raw materials used are commercially available products.

[0047] The Gram-positive bacteria used in the present invention are Methicillin-resistant S.aureus ATCC 33591; Methicillin-resistant S.aureus ATCC 43300; S.aureus ATCC 29213; Methicillin-resistant S.epidermidis ATCC 51625, which are purchased from the American Type Culture Collection (ATCC). The drug-resistant Gram-negative bacteria are: A.baumannii ATCC 19606; S.enterica ATCC14028; E.coli ATCC 25922, which are purchased from the American Type Culture Collection (ATCC); E.coli CMCC 44103, which is purchased from the National Center for Medical Culture Collections (CMCC); The drug-resistant strains are: MRSA-171; MRSA-575; MRSA-206; MRSA-222; MRSA-596; VRE-80; MDR-PA-126; MDR-KP-893; CR-AB-882. These 9 clinically isolated drug-resistant bacteria are from Huashan Hospital affiliated to Fudan University.

[0048] The present invention will be further described in detail below with reference to the accompanying drawings:

[0049] Example 1

[0050] This example provides a pleuromutilin derivative (Compound 1) with the ability to inhibit bacterial hydrogen sulfide biosynthesis. The specific preparation process is as follows:

[0051] (1) Preparation of Intermediate I

[0052]

[0053] Pleuromutilin (7.57 g, 20 mmol), p-toluenesulfonyl chloride (4.58 g, 24 mmol) and

[0054] 4-dimethylaminopyridine (0.25 g, 2 mmol) were added to dichloromethane (100 mL) as the solvent, stirred and dissolved, then triethylamine (8.35 mL, 60 mmol) was added, and the mixture was stirred at room temperature for 8 h for reaction; after the reaction was completed, the reaction solution was concentrated, then washed with saturated NaHCO 3 aqueous solution, and finally dried in vacuo to obtain Intermediate I with a yield of about 96.20%.

[0055] (2) Preparation of Intermediate II

[0056]

[0057] Add intermediate I (5.33 g, 10 mmol), 3-aminobenzenethiol (1.88 g, 15 mmol), potassium carbonate (2.77 g, 20 mmol) and potassium iodide (0.16 g, 1 mmol) into solvent N,N-dimethylformamide (50 mL) for dissolution, heat to 60 °C and react for 6 hours; after the reaction is completed, add 200 mL of saturated ammonium chloride aqueous solution to the reaction solution, extract with ethyl acetate, and the ethyl acetate phase is successively concentrated and separated by column chromatography to obtain intermediate II. The eluent used for column chromatography is a dichloromethane-methanol mixed solution with a volume ratio of 18:1, and the yield of intermediate II is 85.56%.

[0058] (3) Preparation of Intermediate III

[0059]

[0060] Add (1H-indol-3-yl)boronic acid (1.61 g, 10 mmol), bromoacetic acid (1.67 g, 12 mmol), sodium hydride (0.48 g, 20 mmol) into 30 mL of solvent tetrahydrofuran for dissolution, stir and react at 0 °C for 2 hours; after the reaction is completed, drop the reaction solution into 30 ml of ice water, solid precipitates, filter to obtain intermediate III, and the yield is 83.11%.

[0061] (4) Synthesis of Intermediate IV

[0062]

[0063] Add 3-bromobenzoic acid (2.01 g, 12 mmol), intermediate III (2.19 g, 10 mmol), dichlorobis(η5-cyclopentadienyl)palladium(II) (0.37 g, 0.0005 mmol), potassium carbonate (2.76 g, 20 mmol) into 30 mL of solvent N,N-dimethylformamide, under nitrogen protection, heat to 120 °C and react for 7 h; after the reaction is completed, add 200 mL of saturated ammonium chloride aqueous solution to the reaction solution, extract with ethyl acetate, and the ethyl acetate phase is successively concentrated and separated by column chromatography to obtain intermediate IV. The eluent used for column chromatography is a dichloromethane-methanol mixed solution with a volume ratio of 10:1, and the yield of intermediate IV is 82.21%.

[0064] (5) Preparation of Compound 1

[0065] Preparation of 2-(3-(3-((3-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyl-decahydro-4,9a-propacyclopenta[8]annulen-5-yl)oxy)-2-oxoethyl)thio)phenyl)carbamoyl)phenyl)-1H-indol-1-yl)acetic acid

[0066]

[0067] Dissolve intermediate Ⅳ (0.375 g, 1.5 mmol) in acetonitrile (5 mL), add EDCI (0.382 g, 2 mmol) and DMAP (0.244 g, 2 mmol), stir and react for 2 h, and monitor the carboxyl activation process by TLC. Subsequently, add intermediate Ⅱ (0.485 g, 1 mmol), continue to stir and react for 4 h, and monitor the reaction process by TLC; after the reaction is completed, add saturated sodium chloride aqueous solution to the system, extract, collect the organic phase and concentrate, and separate by flash column chromatography (eluent: dichloromethane: methanol = 20:1) to obtain compound 1 with a yield of 62.69%.

[0068] The identification data of compound 1 are as follows:

[0069] 11H NMR (600 MHz, DMSO-d6) δ 10.15 (s, 1H), 8.91 (s, 1H), 8.09 - 8.03 (m, 2H), 7.95 (ddd, J = 7.9, 2.1, 1.2 Hz, 1H), 7.81 (t, J = 2.4 Hz, 1H), 7.75 - 7.64 (m, 2H), 7.52 (ddd, J = 7.8, 1.4, 1.0 Hz, 1H), 7.41 (dd, J = 6.2, 1.4 Hz, 2H), 7.35 - 7.31 (m, 1H), 7.28 - 7.21 (m, 2H), 7.15 (ddd, J = 6.9, 2.3, 1.2 Hz, 1H), 5.79 (tdq, J = 10.9, 1.6, 0.8 Hz, 1H), 5.13 (dd, J = 10.6, 2.2 Hz, 1H), 5.00 (d, J = 0.8 Hz, 2H), 4.97 (ddq, J = 5.9, 4.3, 1.4 Hz, 1H), 4.93 (dd, J = 11.6, 2.8 Hz, 1H), 3.78 - 3.64 (m, 2H), 3.42 (dddt, J = 8.5, 6.3, 3.4, 1.2 Hz, 1H), 2.91 (d, J = 6.8 Hz, 1H), 2.34 (d, J = 3.6 Hz, 1H), 2.30 (t, J = 4.2 Hz, 2H), 2.18 (dd, J = 12.6, 5.9 Hz, 1H), 1.84 - 1.67 (m, 3H), 1.64 - 1.53 (m, 3H), 1.48 (dt, J = 12.8, 4.5 Hz, 1H), 1.41 - 1.29 (m, 2H), 1.15 - 1.07 (m, 6H), 0.94 (dd, J = 6.6, 1.4 Hz, 3H), 0.86 (d, J = 7.9 Hz, 3H).

[0070] 13 13C NMR (151 MHz, DMSO-d6) δ 218.39, 171.94, 170.08, 166.61, 146.81, 139.17, 136.62, 136.25, 133.92, 133.42, 129.77, 129.47, 129.15, 127.53, 127.12, 127.01, 125.78, 125.29, 124.69, 124.27, 120.74, 120.19, 119.49, 118.40, 112.06, 109.82, 80.79, 76.45, 58.56, 48.27, 46.73, 46.38, 44.62, 43.14, 37.90, 37.16, 35.43, 34.24, 33.89, 33.65, 26.68, 20.04, 17.91, 17.23, 12.35.

[0071] Example 2

[0072] This example provides a pleuromutilin derivative (Compound 2) with the ability to inhibit bacterial hydrogen sulfide biosynthesis. Intermediate I, Intermediate III, and Intermediate IV were prepared according to the method of Example 1. The specific preparation process is as follows:

[0073] (1) Preparation of Intermediate II

[0074]

[0075] Intermediate I (5.33 g, 10 mmol), 5-amino-2-mercaptocyclohexanol (2.20 g, 15 mmol), potassium carbonate (2.77 g, 20 mmol), and potassium iodide (0.16 g, 1 mmol) were added to the solvent N,N-dimethylformamide (50 mL) for dissolution, and the mixture was heated to 60 °C and reacted for 6 hours. After the reaction, 200 mL of saturated ammonium chloride aqueous solution was added to the reaction solution, and extraction was carried out with ethyl acetate. The ethyl acetate phase was concentrated and separated by column chromatography in turn. The eluent used for column chromatography was a dichloromethane-methanol mixed solution with a volume ratio of 18:1, and the yield of Intermediate II was 81.26%.

[0076] (2) Preparation of Compound 2

[0077] Preparation of 2-(3-(3-((3-hydroxy-4-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyl-decahydro-4,9a-propacyclopenta[8]annulen-5-yl)oxy)-2-oxoethyl)thio)cyclohexyl)carbamoyl)phenyl)-1H-indol-1-yl)acetic acid

[0078]

[0079] Intermediate IV (0.375 g, 1.5 mmol) obtained in Example 1 was dissolved in acetonitrile (5 mL), EDCI (0.382 g, 2 mmol) and DMAP (0.244 g, 2 mmol) were added, and the mixture was stirred and reacted for 2 h, and the carboxyl activation process was monitored by TLC. Subsequently, Intermediate II (0.507 g, 1 mmol) was added, and the reaction was continued with stirring for 4 h, and the reaction process was monitored by TLC. After the reaction, saturated sodium chloride aqueous solution was added to the system for extraction, the organic phase was collected and concentrated, and rapid column chromatography separation (eluent: dichloromethane: methanol = 20:1) was carried out,

[0080] Compound 2 was obtained, and the yield of Compound 2 was 54.55%. The identification data of Compound 2 are as follows:

[0081] 1 1H NMR (600 MHz, DMSO-d6) δ 10.19 (s, 1H), 8.07 - 7.94 (m, 2H), 7.81 (ddd, J = 8.0, 2.2, 1.3 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.57 (ddd, J = 7.3, 2.0, 1.2 Hz, 1H), 7.45 - 7.37 (m, 3H), 7.34 - 7.30 (m, 1H), 7.24 (ddd, J = 7.5, 6.0, 1.6 Hz, 1H), 5.69 (tdq, J = 11.2, 1.8, 0.8 Hz, 1H), 5.17 (dd, J = 10.7, 2.2 Hz, 1H), 5.08 - 4.96 (m, 3H), 4.92 (dd, J = 11.2, 2.7 Hz, 1H), 4.04 - 3.88 (m, 2H), 3.51 (dddt, J = 8.6, 6.1, 2.8, 1.4 Hz, 1H), 3.45 (s, 2H), 3.18 (d, J = 4.0 Hz, 1H), 2.99 (dt, J = 5.2, 4.1 Hz, 1H), 2.95 (d, J = 6.9 Hz, 1H), 2.38 (d, J = 3.5 Hz, 1H), 2.33 (t, J = 4.6 Hz, 2H), 2.14 (dd, J = 12.1, 5.4 Hz, 1H), 2.01 (dt, J = 12.6, 6.5 Hz, 1H), 1.98 - 1.83 (m, 2H), 1.80 - 1.64 (m, 6H), 1.59 - 1.42 (m, 4H), 1.40 - 1.27 (m, 2H), 1.15 - 1.06 (m, 6H), 0.90 (dd, J = 6.0, 1.3 Hz, 3H), 0.81 (d, J = 8.0 Hz, 3H).

[0082] 13 13C NMR (151 MHz, DMSO-d6) δ 218.42, 171.79, 171.34, 168.86, 146.98, 136.28, 133.37, 133.00, 129.11, 128.95, 128.29, 127.30, 126.81, 125.07, 124.36, 124.25, 120.67, 118.72, 112.85, 109.48, 80.79, 76.57, 72.81, 58.41, 48.11, 46.83, 46.59, 46.27, 46.05, 44.12, 43.04, 37.76, 37.45, 36.28, 34.40, 33.61, 33.02, 32.55, 28.58, 26.93, 26.75, 20.96, 17.34, 17.15, 12.93.

[0083] Example 3

[0084] This example provides a pleuromutilin derivative (Compound 3) with the ability to inhibit bacterial hydrogen sulfide biosynthesis. Intermediate I, Intermediate III, and Intermediate IV were prepared according to the method of Example 1. The specific preparation process is as follows

[0085] (1) Preparation of Intermediate II

[0086]

[0087] Intermediate I (5.33 g, 10 mmol), 2-aminothiazole-5-thiol (1.98 g, 15 mmol), potassium carbonate (2.77 g, 20 mmol), and potassium iodide (0.16 g, 1 mmol) were added to the solvent N,N-dimethylformamide (50 mL) for dissolution, and the mixture was heated to 60 °C and reacted for 6 hours. After the reaction, 200 mL of saturated ammonium chloride aqueous solution was added to the reaction solution, and extraction was carried out with ethyl acetate. The ethyl acetate phase was concentrated and separated by column chromatography in turn. The eluent used for column chromatography was a dichloromethane-methanol mixed solution with a volume ratio of 18:1, and the yield of Intermediate II was 83.33%.

[0088] (2) Preparation of Compound 3

[0089] Preparation of 2-(3-(3-((5-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyl-decahydro-4,9a-propacyclopenta[8]annulen-5-yl)oxy)-2-oxoethyl)thio)thiazol-2-yl)carbamoyl)phenyl)-1H-indol-1-yl)acetic acid:

[0090]

[0091] Intermediate IV (0.375 g, 1.5 mmol) was dissolved in acetonitrile (5 mL), and EDCI (0.382 g, 2 mmol) and DMAP (0.244 g, 2 mmol) were added. The mixture was stirred and reacted for 2 h, and the carboxyl activation process was monitored by TLC. Subsequently, Intermediate II (0.492 g, 1 mmol) was added, and the reaction was continued for 4 h while monitoring the reaction process by TLC. After the reaction, saturated sodium chloride aqueous solution was added to the system for extraction. The organic phase was collected and concentrated, and rapid column chromatography separation (eluent: dichloromethane:methanol = 20:1) was carried out to obtain Compound 3 with a yield of 60.34%. The identification data of Compound 3 are as follows

[0092] 11H NMR (600 MHz, DMSO-d6) δ 10.94 (s, 1H), 10.08 (s, 1H), 8.01 - 7.98 (m, 2H), 7.92 (ddd, J = 7.8, 1.6, 1.0 Hz, 1H), 7.78 (s, 1H), 7.65 (t, J = 7.9 Hz, 1H), 7.53 (ddd, J = 7.2, 1.6, 0.8 Hz, 1H), 7.42 (dd, J = 6.9, 1.2 Hz, 2H), 7.38 - 7.32 (m, 1H), 7.26 (ddd, J = 7.6, 6.4, 1.0 Hz, 1H), 5.72 (tdq, J = 11.2, 1.6, 1.0 Hz, 1H), 5.11 (dd, J = 10.2, 2.8 Hz, 1H), 5.04 (d, J = 0.9 Hz, 2H), 5.00 - 4.90 (m, 2H), 4.13 - 3.85 (m, 2H), 3.43 (dddt, J = 8.9, 6.4, 3.7, 1.8 Hz, 1H), 2.91 (d, J = 6.1 Hz, 1H), 2.38 (d, J = 3.9 Hz, 1H), 2.28 (t, J = 4.2 Hz, 2H), 2.14 (dd, J = 12.9, 5.1 Hz, 1H), 1.83 - 1.68 (m, 3H), 1.60 - 1.53 (m, 3H), 1.48 (dt, J = 12.0, 4.9 Hz, 1H), 1.42 - 1.31 (m, 2H), 1.19 - 1.10 (m, 6H), 0.98 (dd, J = 6.1, 1.2 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H).

[0093] 13 13C NMR (151 MHz, DMSO-d6) δ 218.70, 171.36, 170.42, 168.34, 166.04, 146.50, 144.97, 136.20, 133.83, 133.56, 129.89, 129.21, 127.85, 127.18, 127.03, 125.82, 125.20, 124.73, 124.19, 120.48, 118.61, 112.97, 109.58, 80.97, 76.75, 58.82, 48.66, 46.63, 46.19, 44.75, 43.95, 37.63, 37.12, 37.04, 34.45, 33.70, 33.65, 26.47, 20.76, 17.63, 17.09, 12.10.

[0094] Example 4

[0095] This example provides a pleuromutilin derivative (Compound 4) with the ability to inhibit bacterial hydrogen sulfide biosynthesis. Intermediate I and Intermediate II were prepared according to the method of Example 1. The specific preparation process is as follows:

[0096] (1) Preparation of Intermediate III

[0097]

[0098] (1H-Indol-3-yl)boronic acid (1.61 g, 10 mmol), (2-bromoacetyl)glycine (2.35 g, 12 mmol), and sodium hydride (0.48 g, 20 mmol) were added to 30 mL of the solvent tetrahydrofuran for dissolution, and the reaction was stirred at 0 °C for 2 hours. After the reaction was completed, the reaction solution was dropped into 30 mL of ice water, and a solid precipitated. After filtration, Intermediate III was obtained with a yield of 86.15%.

[0099] (2) Preparation of Intermediate IV

[0100]

[0101] 3-Bromobenzoic acid (2.01 g, 12 mmol), Intermediate III (2.76 g, 10 mmol), dichlorobis(triphenylphosphine)palladium(II) (0.37 g, 0.0005 mmol), and potassium carbonate (2.76 g, 20 mmol) were added to 30 mL of the solvent N,N-dimethylformamide. Under nitrogen protection, the reaction was heated to 120 °C for 7 hours. After the reaction was completed, 200 mL of saturated ammonium chloride aqueous solution was added to the reaction solution, and extraction was carried out with ethyl acetate. The ethyl acetate phase was concentrated and separated by column chromatography in turn. The eluent used for column chromatography was a dichloromethane-methanol mixed solution with a volume ratio of 10:1, and the yield was 80.53%.

[0102] (3) Preparation of Compound 4

[0103] (2-(3-(3-((3-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-Hydroxy-4,7,9,12-tetramethyl-3-oxo-7-ethenyl-decahydro-4,9a-propacyclopenta[8]annulen-5-yl)oxy)-2-oxoethyl)thio)phenyl)carbamoyl)phenyl)-1H-indol-1-yl)acetyl)glycine preparation.

[0104]

[0105] Dissolve intermediate Ⅳ (0.531 g, 1.5 mmol) in acetonitrile (5 mL), add EDCI (0.382 g, 2 mmol) and DMAP (0.244 g, 2 mmol), stir and react for 2 h, and monitor the carboxyl activation process by TLC. Subsequently, add intermediate Ⅱ (0.485 g, 1 mmol), continue to stir and react for 4 h, and monitor the reaction process by TLC. After the reaction is completed, add saturated sodium chloride aqueous solution to the system, extract, collect the organic phase and concentrate it, and separate it by flash column chromatography (the eluent is dichloromethane:methanol = 20:1) to obtain compound 4 with a yield of 52.69%. The identification data of compound 4 are as follows:

[0106] 1 H NMR (600 MHz, DMSO-d6) δ 10.21 (s, 1H), 8.80 (s, 1H), 8.12 - 8.04 (m, 2H), 7.97 (ddd, J = 8.2, 2.2, 1.4 Hz, 1H), 7.81 - 7.73 (m, 2H), 7.70 - 7.62 (m, 2H), 7.51 (ddd, J = 7.9, 1.6, 1.2 Hz, 1H), 7.43 (dd, J = 6.8, 1.5 Hz, 1H), 7.35 - 7.30 (m, 1H), 7.26 - 7.23 (m, 3H), 7.19 (ddd, J = 6.0, 2.4, 1.0 Hz, 1H), 5.64 (tdq, J = 10.2, 1.6, 1.1 Hz, 1H), 5.12 (dd, J = 11.4, 2.9 Hz, 1H), 4.98 (tq, J = 5.2, 1.9 Hz, 1H), 4.95 (dd, J = 11.8, 2.2 Hz, 1H), 4.90 (s, 2H), 3.93 (d, J = 6.0 Hz, 2H), 3.74 - 3.62 (m, 2H), 3.43 (dddd, J = 8.8, 6.2, 3.4, 1.4 Hz, 1H), 2.95 (d, J = 6.8 Hz, 1H), 2.37 (d, J = 3.4 Hz, 1H), 2.34 (t, J = 4.1 Hz, 2H), 2.15 (dd, J = 12.8, 5.2 Hz, 1H), 1.84 - 1.69 (m, 3H), 1.63 - 1.55 (m, 3H), 1.50 (dt, J = 12.0, 4.2 Hz, 1H), 1.43 - 1.31 (m, 2H), 1.18 - 1.09 (m, 6H), 0.94 (dd, J = 6.4, 1.9 Hz, 3H), 0.87 (d, J = 6.2 Hz, 3H).

[0107] 1313C NMR (151 MHz, DMSO-d6) δ 218.35, 173.76, 170.43, 169.78, 166.94, 146.69, 139.35, 136.65, 136.21, 134.53, 133.20, 129.81, 129.33, 129.05, 127.72, 127.50, 126.24, 125.48, 125.37, 124.61, 124.19, 120.12, 120.03, 119.59, 118.27, 112.46, 109.97, 80.52, 76.38, 58.72, 48.20, 46.41, 46.21, 44.16, 43.67, 41.54, 37.93, 37.18, 35.21, 34.13, 33.60, 33.18, 26.09, 20.60, 17.64, 17.15, 12.87.

[0108] Example 5

[0109] This example provides a pleuromutilin derivative (Compound 5) with the ability to inhibit bacterial hydrogen sulfide biosynthesis. Intermediate I was prepared according to the method of Example 1; Intermediate II was prepared according to the method of Example 2; Intermediate III and Intermediate IV were prepared according to the method of Example 4. The specific preparation process is as follows:

[0110] Preparation of (2-(3-(3-((3-Hydroxy-4-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyl-decahydro-4,9a-propacyclopenta[8]annulen-5-yl)oxy)-2-oxoethyl)thio)cyclohexyl)carbamoyl)phenyl)-1H-indol-1-yl)acetyl)glycine (Compound 5):

[0111]

[0112] Dissolve Intermediate IV (0.531 g, 1.5 mmol) in acetonitrile (5 mL), add EDCI (0.382 g, 2 mmol) and DMAP (0.244 g, 2 mmol), stir and react for 2 h, and monitor the carboxyl activation process by TLC. Subsequently, add Intermediate II (0.507 g, 1 mmol), continue to stir and react for 4 h, and monitor the reaction process by TLC. After the reaction is completed, add saturated sodium chloride aqueous solution to the system, extract, collect the organic phase and concentrate, and perform flash column chromatography separation (eluent: dichloromethane:methanol = 20:1) to obtain Compound 5 with a yield of 58.12%. The identification data of Compound 5 are as follows:

[0113] 11H NMR (600 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.03 - 7.96 (m, 2H), 7.87 (ddd, J = 7.9, 2.6, 1.8 Hz, 1H), 7.83 (t, J = 6.3 Hz, 1H), 7.66 (t, J = 7.2 Hz, 1H), 7.57 (ddd, J = 7.6, 2.2, 1.2 Hz, 1H), 7.49 (dd, J = 6.8, 1.6 Hz, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.36 - 7.31 (m, 1H), 7.29 - 7.24 (m, 2H), 5.63 (tdq, J = 10.1, 1.8, 0.8 Hz, 1H), 5.14 (dd, J = 11.3, 2.8 Hz, 1H), 5.06 (tq, J = 5.9, 1.7 Hz, 1H), 4.97 (dd, J = 11.2, 2.4 Hz, 1H), 4.93 (s, 2H), 4.04 - 3.96 (m, 2H), 3.91 (d, J = 5.7 Hz, 2H), 3.55 (dddt, J = 8.2, 6.2, 3.4, 1.7 Hz, 1H), 3.47 (s, 2H), 3.14 (d, J = 4.8 Hz, 1H), 2.99 (dt, J = 5.2, 4.0 Hz, 1H), 2.94 (d, J = 6.8 Hz, 1H), 2.38 (d, J = 3.4 Hz, 1H), 2.28 (t, J = 4.9 Hz, 2H), 2.11 (dd, J = 12.8, 5.2 Hz, 1H), 2.03 (dt, J = 12.4, 6.6 Hz, 1H), 1.98 - 1.83 (m, 2H), 1.81 - 1.68 (m, 6H), 1.64 - 1.53 (m, 3H), 1.46 (dt, J = 12.8, 4.9 Hz, 1H), 1.41 - 1.30 (m, 2H), 1.16 – 1.08 (m, 6H), 0.92 (dd, J = 6.8, 1.5 Hz, 3H), 0.84 (d, J = 7.2 Hz, 3H).

[0114] 1313C NMR (151 MHz, DMSO-d6) δ 218.43, 173.68, 171.20, 169.71, 168.04, 146.88, 136.14, 134.20, 133.52, 129.49, 128.23, 128.02, 126.93, 126.24, 125.36, 124.55, 124.37, 120.89, 118.13, 112.71, 109.31, 80.54, 76.19, 72.21, 58.76, 48.36, 46.89, 46.52, 46.31, 46.09, 44.67, 43.28, 41.87, 37.68, 37.23, 36.35, 34.96, 33.46, 33.17, 32.92, 28.64, 26.81, 26.34, 20.57, 17.61, 17.37, 12.94.

[0115] Example 6

[0116] This example provides a pleuromutilin derivative (Compound 6) with the ability to inhibit bacterial hydrogen sulfide biosynthesis. Intermediate I was prepared according to the method of Example 1; Intermediate II was prepared according to the method of Example 3; Intermediate III and Intermediate IV were prepared according to the method of Example 4. The specific preparation process is as follows:

[0117] Preparation of (2-(3-(3-((5-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-Hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyl-decahydro-4,9a-propacyclopenta[8]annulen-5-yl)oxy)-2-oxoethyl)thio)thiazol-2-yl)carbamoyl)phenyl)-1H-indol-1-yl)acetyl)glycine (Compound 6)

[0118]

[0119] Dissolve Intermediate IV (0.531 g, 1.5 mmol) in acetonitrile (5 mL), add EDCI (0.382 g, 2 mmol) and DMAP (0.244 g, 2 mmol), stir and react for 2 h, and monitor the carboxyl activation process by TLC. Subsequently, add Intermediate II (0.492 g, 1 mmol), continue to stir and react for 4 h, and monitor the reaction process by TLC. After the reaction is completed, add saturated sodium chloride aqueous solution to the system, extract, collect the organic phase and concentrate it, and perform rapid column chromatography separation (the eluent is dichloromethane:methanol = 20:1) to obtain Compound 6 with a yield of 50.76%. The identification data of Compound 6 are as follows:

[0120] 11H NMR (600 MHz, DMSO-d6) δ 10.87 (s, 1H), 10.13 (s, 1H), 8.06 - 8.03 (m, 2H), 7.97 (ddd, J = 7.7, 1.6, 1.2 Hz, 1H), 7.82 (t, J = 5.8 Hz, 1H), 7.73 (s, 1H), 7.65 (t, J = 7.3 Hz, 1H), 7.52 (ddd, J = 7.6, 1.4, 1.0 Hz, 1H), 7.41 (dd, J = 6.8, 1.2 Hz, 1H), 7.36 - 7.31 (m, 1H), 7.28 - 7.20 (m, 2H), 5.63 (tdq, J = 10.6, 1.7, 0.8 Hz, 1H), 5.16 (dd, J = 10.9, 2.5 Hz, 1H), 4.94 (ddt, J = 5.4, 4.6, 1.7 Hz, 1H), 4.90 (dd, J = 11.2, 2.9 Hz, 1H), 4.85 (s, 2H), 4.09 - 3.93 (m, 2H), 3.89 (d, J = 6.0 Hz, 2H), 3.49 (dddd, J = 8.0, 6.8, 3.4, 1.8 Hz, 1H), 2.97 (d, J = 6.5 Hz, 1H), 2.39 (d, J = 3.8 Hz, 1H), 2.34 (t, J = 4.7 Hz, 2H), 2.18 (dd, J = 12.5, 5.2 Hz, 1H), 1.93 - 1.69 (m, 3H), 1.65 - 1.53 (m, 3H), 1.51 - 1.47 (m, 1H), 1.46 - 1.32 (m, 2H), 1.17 - 1.08 (m, 6H), 0.93 (dd, J = 6.6, 1.2 Hz, 3H), 0.81 (d, J = 7.5 Hz, 3H).

[0121] 13 13C NMR (151 MHz, DMSO-d6) δ 218.13, 173.89, 170.26, 169.99, 168.04, 166.33, 146.08, 144.89, 136.69, 134.31, 133.71, 129.93, 129.18, 127.96, 127.52, 126.83, 125.22, 125.01, 124.68, 124.45, 120.29, 118.26, 112.65, 109.48, 80.41, 76.47, 58.18, 48.58, 46.88, 46.12, 44.87, 43.31, 41.49, 37.73, 37.14, 37.02, 34.65, 33.83, 33.48, 26.41, 20.25, 17.98, 17.65, 12.46.

[0122] Example 7

[0123] This example provides a pleuromutilin derivative (Compound 7) with the ability to inhibit bacterial hydrogen sulfide biosynthesis. Intermediate I and Intermediate II were prepared according to the method of Example 1. The specific preparation process is as follows:

[0124] (1) Preparation of Intermediate III

[0125]

[0126] (1H-Indol-3-yl)boronic acid (1.61 g, 10 mmol), 5-(bromomethyl)-2-methylfuran-3-carboxylic acid (2.62 g, 12 mmol), and sodium hydride (0.48 g, 20 mmol) were added to 30 mL of the solvent tetrahydrofuran for dissolution, and the mixture was stirred at 0 °C for 2 hours. After the reaction was completed, the reaction solution was dropped into 30 mL of ice water, and a solid precipitated. The solid was filtered to obtain Intermediate III with a yield of 83.35%.

[0127] (2) Preparation of Intermediate IV

[0128]

[0129] 3-Bromobenzoic acid (2.01 g, 12 mmol), Intermediate III (2.99 g, 10 mmol), dichlorobis(triphenylphosphine)palladium(II) (0.37 g, 0.0005 mmol), and potassium carbonate (2.76 g, 20 mmol) were added to 30 mL of the solvent N,N-dimethylformamide. Under nitrogen protection, the mixture was heated to 120 °C and reacted for 7 hours. After the reaction was completed, 200 mL of saturated ammonium chloride aqueous solution was added to the reaction solution, and extraction was carried out with ethyl acetate. The ethyl acetate phase was concentrated and separated by column chromatography in turn. The eluent used for column chromatography was a dichloromethane-methanol mixed solution with a volume ratio of 10:1. The yield of Intermediate IV was 74.33%.

[0130] (3) Preparation of Compound 7

[0131] Preparation of 5-((3-(3-((3-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-Hydroxy-4,7,9,12-tetramethyl-3-oxo-7-ethenyl-decahydro-4,9a-propacyclopenta[8]annulen-5-yl)oxy)-2-oxoethyl)thio)phenyl)carbamoyl)phenyl)-1H-indol-1-yl)methyl)-2-methylfuran-3-carboxylic acid (Compound 7)

[0132]

[0133] Intermediate Ⅳ (0.566 g, 1.5 mmol) was dissolved in acetonitrile (5 mL), and EDCI (0.382 g, 2 mmol) and DMAP (0.244 g, 2 mmol) were added. The mixture was stirred for 2 h, and the carboxyl activation process was monitored by TLC. Subsequently, Intermediate Ⅱ (0.485 g, 1 mmol) was added, and the stirring was continued for 4 h while monitoring the reaction process by TLC. After the reaction was completed, saturated sodium chloride aqueous solution was added to the system for extraction. The organic phase was collected and concentrated, and then separated by flash column chromatography (eluent: dichloromethane:methanol = 20:1) to obtain Compound 7 with a yield of 47.76%. The identification data are as follows:

[0134] 1 H NMR (600 MHz, DMSO-d6) δ 10.08 (s, 1H), 8.85 (s, 1H), 8.04 - 8.00 (m, 2H), 7.91 (ddd, J = 8.1, 2.1, 1.4 Hz, 1H), 7.82 (t, J = 2.6 Hz, 1H), 7.69 - 7.61 (m, 3H), 7.53 (ddd, J = 7.4, 1.6, 1.1 Hz, 1H), 7.44 (dd, J = 6.7, 1.4 Hz, 1H), 7.36 (ddd, J = 8.1, 7.2, 1.5 Hz, 1H), 7.24 (tt, J = 6.4, 1.9 Hz, 2H), 7.19 (ddd, J = 6.2, 2.4, 1.8 Hz, 1H), 6.64 (s, 1H), 5.63 (tdq, J = 10.2, 1.6, 0.8 Hz, 1H), 5.31 (s, 2H), 5.12 (dd, J = 10.8, 2.2 Hz, 1H), 5.03 (ddt, J = 5.8, 4.6, 1.7 Hz, 1H), 4.95 (dd, J = 11.2, 2.0 Hz, 1H), 3.83 - 3.66 (m, 2H), 3.49 (dddd, J = 8.4, 6.2, 3.1, 1.4 Hz, 1H), 2.94 (d, J = 6.9 Hz, 1H), 2.63 (s, 3H), 2.39 (d, J = 3.8 Hz, 1H), 2.34 (t, J = 4.4 Hz, 2H), 2.15 (dd, J = 12.8, 5.6 Hz, 1H), 1.79 (dt, J = 12.5, 4.8 Hz, 1H), 1.73 - 1.64 (m, 2H), 1.61 - 1.52 (m, 3H), 1.48 (dt, J = 12.8, 4.9 Hz, 1H), 1.44 - 1.29 (m, 2H), 1.16 - 1.09 (m, 6H), 0.92 (dd, J = 6.2, 1.6 Hz, 3H), 0.83 (d, J = 8.0 Hz, 3H).

[0135] 1313C NMR (151 MHz, DMSO-d6) δ 218.07, 170.76, 166.74, 166.21, 157.13, 149.80, 146.29, 139.18, 136.92, 136.76, 134.23, 133.45, 129.99, 129.37, 129.05, 127.63, 127.29, 126.68, 126.05, 125.56, 124.87, 124.23, 120.97, 120.14, 119.74, 118.50, 113.27, 112.83, 110.48, 108.65, 80.27, 76.84, 58.07, 46.68, 46.19, 44.21, 43.91, 43.25, 37.98, 37.01, 35.20, 34.90, 33.73, 33.45, 26.84, 20.57, 17.94, 17.67, 13.39, 12.10.

[0136] Example 8

[0137] This example provides a pleuromutilin derivative (Compound 8) with the ability to inhibit bacterial hydrogen sulfide biosynthesis. Intermediate I was prepared according to the method of Example 1; Intermediate II was prepared according to the method of Example 2; Intermediate III and Intermediate IV were prepared according to the method of Example 7. The specific preparation process is as follows:

[0138] Preparation of 5-((3-(3-((3-hydroxy-4-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyl-decahydro-4,9a-propacyclopenta[8]annulen-5-yl)oxy)-2-oxoethyl)thio)cyclohexyl)carbamoyl)phenyl)-1H-indol-1-yl)methyl)-2-methylfuran-3-carboxylic acid (Compound 8)

[0139]

[0140] Dissolve Intermediate IV (0.566 g, 1.5 mmol) in acetonitrile (5 mL), add EDCI (0.382 g, 2 mmol) and DMAP (0.244 g, 2 mmol), stir and react for 2 h, and monitor the carboxyl activation process by TLC. Subsequently, add Intermediate II (0.507 g, 1 mmol), continue to stir and react for 4 h, and monitor the reaction process by TLC. After the reaction is completed, add saturated sodium chloride aqueous solution to the system, extract, collect the organic phase and concentrate it, and perform flash column chromatography separation (the eluent is dichloromethane:methanol = 20:1). The yield is 47.76%. The identification data are as follows:

[0141] 1 1H NMR (600 MHz, DMSO-d6) δ 10.01 (s, 1H), 8.06 - 8.02 (m, 2H), 7.88 (ddd, J = 8.0, 2.6, 1.5 Hz, 1H), 7.65 - 7.59 (m, 2H), 7.52 (ddd, J = 7.2, 2.0, 1.1 Hz, 1H), 7.46 (dd, J = 6.8, 1.4 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H), 7.34 (ddd, J = 8.2, 7.4, 1.6 Hz, 1H), 7.29 - 7.22 (m, 1H), 6.73 (s, 1H), 5.54 (tdq, J = 10.2, 1.5, 0.6 Hz, 1H), 5.25 (s, 2H), 5.16 (dd, J = 11.3, 2.6 Hz, 1H), 5.03 (tq, J = 5.4, 1.7 Hz, 1H), 4.97 (dd, J = 12.1, 2.6 Hz, 1H), 4.01 - 3.93 (m, 2H), 3.48 (dtt, J = 6.4, 3.2, 1.4 Hz, 1H), 3.42 (s, 2H), 3.17 (d, J = 4.8 Hz, 1H), 2.93 (dt, J = 5.2, 3.4 Hz, 1H), 2.87 (d, J = 6.8 Hz, 1H), 2.66 (s, 3H), 2.35 (d, J = 3.4 Hz, 1H), 2.28 (t, J = 4.6 Hz, 2H), 2.12 (dd, J = 12.8, 5.2 Hz, 1H), 2.04 (dt, J = 12.0, 6.4 Hz, 1H), 1.96 - 1.83 (m, 2H), 1.81 - 1.65 (m, 6H), 1.62 - 1.50 (m, 3H), 1.46 (dt, J = 12.2, 4.9 Hz, 1H), 1.41 - 1.30 (m, 2H), 1.16 - 1.08 (m, 6H), 0.92 (dd, J = 6.8, 1.6 Hz, 3H), 0.84 (d, J = 7.8 Hz, 3H).

[0142] 1313C NMR (151 MHz, DMSO-d6) δ 218.52, 171.86, 168.32, 166.98, 157.70, 149.07, 146.43, 136.74, 133.61, 133.23, 129.38, 128.57, 128.09, 126.36, 126.12, 126.03, 124.47, 124.39, 120.25, 118.81, 113.52, 112.77, 110.16, 108.71, 80.23, 76.45, 72.80, 58.25, 46.91, 46.56, 46.32, 46.04, 44.53, 43.79, 43.11, 37.95, 37.13, 36.52, 34.92, 33.65, 33.17, 32.51, 28.39, 26.61, 26.32, 20.18, 17.50, 17.26, 13.53, 12.72.

[0143] Example 9

[0144] This example provides a pleuromutilin derivative (Compound 9) with the ability to inhibit bacterial hydrogen sulfide biosynthesis. Intermediate I was prepared according to the method of Example 1; Intermediate II was prepared according to the method of Example 3; Intermediate III and Intermediate IV were prepared according to the method of Example 7. The specific preparation process is as follows:

[0145] Preparation of 5 - ((3 - (3 - ((5 - ((2 - (((3aR,4R,5R,7S,8S,9R,9aS,12R) - 8 - hydroxy - 4,7,9,12 - tetramethyl - 3 - oxo - 7 - vinyl - decahydro - 4,9a - propacyclopenta[8]annulen - 5 - yl)oxy) - 2 - oxoethyl)thio)thiazol - 2 - yl)carbamoyl)phenyl) - 1H - indol - 1 - yl)methyl) - 2 - methylfuran - 3 - carboxylic acid (Compound 9):

[0146]

[0147] Dissolve Intermediate IV (0.566 g, 1.5 mmol) in acetonitrile (5 mL), add EDCI (0.382 g, 2 mmol) and DMAP (0.244 g, 2 mmol), stir and react for 2 h, and monitor the carboxyl activation process by TLC. Subsequently, add Intermediate II (0.492 g, 1 mmol), continue to stir and react for 4 h, and monitor the reaction process by TLC. After the reaction is completed, add saturated sodium chloride aqueous solution to the system, extract, collect the organic phase and concentrate it, and perform flash column chromatography separation (the eluent is dichloromethane:methanol = 20:1). The yield of Compound 9 is 59.16%, and the identification data are as follows:

[0148] 1 1H NMR (600 MHz, DMSO-d6) δ 10.91 (s, 1H), 10.16 (s, 1H), 8.02 - 7.97 (m, 2H), 7.92 (ddd, J = 7.7, 1.8, 1.2 Hz, 1H), 7.78 (s, 1H), 7.74 - 7.66 (m, 2H), 7.51 (ddd, J = 7.8, 1.9, 1.4 Hz, 1H), 7.47 (dd, J = 6.8, 1.7 Hz, 1H), 7.39 (ddd, J = 8.2, 7.4, 1.6 Hz, 1H), 7.26 (ddd, J = 7.8, 6.4, 1.6 Hz, 1H), 6.63 (s, 1H), 5.62 (tdq, J = 10.2, 1.6, 0.9 Hz, 1H), 5.48 (s, 2H), 5.21 (dd, J = 10.8, 2.7 Hz, 1H), 4.97 (tq, J = 5.6, 1.2 Hz, 1H), 4.93 (dd, J = 11.2, 2.8 Hz, 1H), 4.02 - 3.98 (m, 2H), 3.46 (dddd, J = 8.9, 6.1, 3.4, 1.3 Hz, 1H), 2.97 (d, J = 6.6 Hz, 1H), 2.69 (s, 3H), 2.37 (d, J = 3.2 Hz, 1H), 2.34 (t, J = 4.8 Hz, 2H), 2.12 (dd, J = 12.7, 5.6 Hz, 1H), 1.89 - 1.70 (m, 3H), 1.68 - 1.54 (m, 3H), 1.50 (dt, J = 12.9, 4.8 Hz, 1H), 1.46 - 1.33 (m, 2H), 1.20 - 1.11 (m, 6H), 0.98 (dd, J = 6.9, 1.3 Hz, 3H), 0.90 (d, J = 7.4 Hz, 3H).

[0149] 1313C NMR (151 MHz, DMSO-d6) δ 218.12, 170.45, 168.53, 166.67, 166.13, 157.80, 149.66, 146.75, 144.38, 136.25, 134.79, 133.67, 129.46, 129.15, 127.29, 127.08, 126.59, 126.02, 125.71, 124.74, 124.28, 120.14, 118.05, 113.37, 112.84, 110.68, 108.47, 80.56, 76.85, 58.98, 46.95, 46.27, 44.11, 43.20, 43.02, 37.54, 37.12, 37.03, 34.61, 33.76, 33.25, 26.39, 20.86, 17.93, 17.08, 13.16, 12.46.

[0150] Example 10

[0151] This example provides a pleuromutilin derivative (Compound 10) with the ability to inhibit bacterial hydrogen sulfide biosynthesis. Intermediate I and Intermediate II were prepared according to the method of Example 1. The specific preparation process is as follows:

[0152] (1) Preparation of Intermediate III

[0153]

[0154] (1H-Indol-3-yl)boronic acid (1.61 g, 10 mmol), 3-(bromomethyl)-1H-pyrazole-5-carboxylic acid (2.46 g, 12 mmol), and sodium hydride (0.48 g, 20 mmol) were added to 30 mL of the solvent tetrahydrofuran for dissolution, and the mixture was stirred at 0 °C for 2 hours. After the reaction was completed, the reaction solution was dropped into 30 mL of ice water, and a solid precipitated. The solid was filtered to obtain Intermediate III with a yield of 77.37%.

[0155] (2) Preparation of Intermediate IV

[0156]

[0157] 3-Bromobenzoic acid (2.01 g, 12 mmol), Intermediate III (2.85 g, 10 mmol), palladium dichloride dicyclopentadienyl (0.37 g, 0.0005 mmol), and potassium carbonate (2.76 g, 20 mmol) were added to 30 mL of the solvent N,N-dimethylformamide. Under nitrogen protection, the mixture was heated to 120 °C and reacted for 7 h. After the reaction, 200 mL of saturated ammonium chloride aqueous solution was added to the reaction solution, and extraction was carried out with ethyl acetate. The ethyl acetate phase was concentrated and separated by column chromatography in turn to obtain Intermediate IV. The eluent used for column chromatography was a dichloromethane-methanol mixed solution with a volume ratio of 10:1, and the yield was 85.52%.

[0158] (3) Preparation of Compound 10

[0159] Preparation of 3-((3-(3-((3-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyl-decahydro-4,9a-propacyclopenta[8]annulen-5-yl)oxy)-2-oxoethyl)thio)phenyl)carbamoyl)phenyl)-1H-indol-1-yl)methyl)-1H-pyrazole-5-carboxylic acid (Compound 10):

[0160]

[0161] Intermediate IV (0.545 g, 1.5 mmol) was dissolved in acetonitrile (5 mL), and EDCI (0.382 g, 2 mmol) and DMAP (0.244 g, 2 mmol) were added. The mixture was stirred and reacted for 2 h, and the carboxyl activation process was monitored by TLC. Subsequently, Intermediate II (0.485 g, 1 mmol) was added, and the reaction was continued for 4 h while monitoring the reaction process by TLC. After the reaction, saturated sodium chloride aqueous solution was added to the system for extraction. The organic phase was collected and concentrated, and rapid column chromatography separation was carried out (the eluent was dichloromethane:methanol = 20:1). The yield of Compound 10 was 56.76%, and the identification data was as follows:

[0162] 11H NMR (600 MHz, DMSO-d6) δ 10.99 (s, 1H), 10.18 (s, 1H), 8.88 (s, 1H), 8.25 (s, 1H), 8.03 - 7.98 (m, 2H), 7.92 (ddd, J = 8.2, 2.3, 1.4 Hz, 1H), 7.83 (t, J = 2.8 Hz, 1H), 7.75 - 7.61 (m, 2H), 7.52 (ddd, J = 7.9, 1.6, 1.4 Hz, 1H), 7.46 (dd, J = 6.8, 1.4 Hz, 1H), 7.38 (ddd, J = 8.2, 7.3, 1.4 Hz, 1H), 7.31 - 7.27 (m, 2H), 7.22 (ddd, J = 6.6, 2.0, 1.3 Hz, 1H), 6.85 (s, 1H), 5.63 (tdq, J = 10.2, 1.7, 0.8 Hz, 1H), 5.49 (d, J = 4.8 Hz, 2H), 5.17 (dd, J = 10.2, 2.6 Hz, 1H), 4.98 (tq, J = 5.9, 1.7 Hz, 1H), 4.92 (dd, J = 11.4, 2.8 Hz, 1H), 3.91 - 3.62 (m, 2H), 3.43 (dddt, J = 8.3, 6.0, 3.7, 1.4 Hz, 1H), 2.95 (d, J = 6.8 Hz, 1H), 2.36 (d, J = 3.2 Hz, 1H), 2.32 (t, J = 4.6 Hz, 2H), 2.16 (dd, J = 12.8, 5.6 Hz, 1H), 1.79 (dt, J = 12.4, 4.4 Hz, 1H), 1.71 - 1.66 (m, 2H), 1.63 - 1.54 (m, 3H), 1.50 (dt, J = 12.5, 4.6 Hz, 1H), 1.45 - 1.33 (m, 2H), 1.18 - 1.09 (m, 6H), 0.95 (dd, J = 6.3, 1.5 Hz, 3H), 0.88 (d, J = 7.7 Hz, 3H).

[0163] 1313C NMR (151 MHz, DMSO-d6) δ 218.38, 170.08, 166.26, 162.68, 146.18, 143.96, 139.91, 138.71, 138.24, 136.59, 134.68, 133.14, 129.64, 129.19, 129.05, 127.43, 127.21, 127.01, 126.42, 125.33, 124.75, 124.29, 120.90, 120.43, 119.75, 118.37, 112.92, 110.18, 107.34, 80.53, 76.15, 58.96, 46.94, 46.21, 44.82, 43.51, 42.75, 37.54, 37.29, 35.38, 34.76, 33.65, 33.08, 26.53, 20.25, 17.71, 17.22, 12.41.

[0164] Example 11

[0165] This example provides a pleuromutilin derivative (Compound 11) with the ability to inhibit bacterial hydrogen sulfide biosynthesis. Intermediate I was prepared according to the method of Example 1; Intermediate II was prepared according to the method of Example 2; Intermediate III and Intermediate IV were prepared according to the method of Example 10. The specific preparation process is as follows:

[0166] Preparation of 3 - ((3 - (3 - ((3 - hydroxy - 4 - ((2 - (((3aR,4R,5R,7S,8S,9R,9aS,12R) - 8 - hydroxy - 4,7,9,12 - tetramethyl - 3 - oxo - 7 - vinyl - decahydro - 4,9a - propacyclopenta[8]annulen - 5 - yl)oxy) - 2 - oxoethyl)thio)cyclohexyl)carbamoyl)phenyl) - 1H - indol - 1 - yl)methyl) - 1H - pyrazole - 5 - carboxylic acid

[0167]

[0168] Dissolve Intermediate IV (0.545 g, 1.5 mmol) in acetonitrile (5 mL), add EDCI (0.382 g, 2 mmol) and DMAP (0.244 g, 2 mmol), stir and react for 2 h, and monitor the carboxyl activation process by TLC. Subsequently, add Intermediate II (0.507 g, 1 mmol), continue to stir and react for 4 h, and monitor the reaction process by TLC. After the reaction is completed, add saturated sodium chloride aqueous solution to the system, extract, collect the organic phase and concentrate it, and perform flash column chromatography separation (the eluent is dichloromethane:methanol = 20:1). The yield of Compound 10 is 51.26%. The identification data are as follows:

[0169] 11H NMR (600 MHz, DMSO-d6) δ 11.03 (s, 1H), 10.14 (s, 1H), 8.29 (s, 1H), 8.05 - 7.96 (m, 2H), 7.83 (ddd, J = 8.2, 2.4, 1.3 Hz, 1H), 7.62 (t, J = 7.9 Hz, 1H), 7.51 (ddd, J = 7.9, 2.6, 1.5 Hz, 1H), 7.43 (dd, J = 6.9, 1.2 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.34 (ddd, J = 8.0, 7.4, 1.2 Hz, 1H), 7.23 (ddd, J = 7.6, 6.2, 1.4 Hz, 1H), 6.81 (s, 1H), 5.62 (tdq, J = 10.2, 1.6, 0.7 Hz, 1H), 5.49 (d, J = 4.8 Hz, 2H), 5.17 (dd, J = 10.6, 2.8 Hz, 1H), 5.06 (tq, J = 5.9, 1.3 Hz, 1H), 4.97 (dd, J = 11.2, 2.8 Hz, 1H), 3.96 - 3.88 (m, 2H), 3.49 (dddt, J = 8.7, 6.9, 3.2, 1.7 Hz, 1H), 3.41 (s, 2H), 3.16 (d, J = 4.5 Hz, 1H), 2.95 (dd, J = 5.8, 4.0 Hz, 1H), 2.87 (d, J = 6.4 Hz, 1H), 2.34 (d, J = 3.2 Hz, 1H), 2.27 (t, J = 4.8 Hz, 2H), 2.16 (dd, J = 12.4, 5.9 Hz, 1H), 2.00 (dt, J = 12.6, 6.4 Hz, 1H), 1.94 - 1.87 (m, 1H), 1.86 - 1.81 (m, 1H), 1.79 - 1.67 (m, 6H), 1.62 - 1.51 (m, 3H), 1.47 (dt, J = 12.5, 4.2 Hz, 1H), 1.42 - 1.28 (m, 2H), 1.14 - 1.06 (m, 6H), 0.95 (dd, J = 6.9, 1.8 Hz, 3H), 0.87 (d, J = 7.1 Hz, 3H).

[0170] 1313C NMR (151 MHz, DMSO-d6) δ 218.63, 171.80, 168.81, 162.99, 146.02, 143.40, 138.25, 138.04, 133.89, 133.53, 129.71, 128.36, 128.09, 127.91, 126.24, 126.11, 124.69, 124.13, 120.38, 118.93, 112.57, 110.01, 107.95, 80.87, 76.42, 72.20, 58.51, 46.81, 46.67, 46.32, 46.07, 44.80, 43.57, 42.68, 37.73, 37.25, 36.29, 34.78, 33.61, 33.15, 32.66, 28.84, 26.31, 26.06, 20.16, 17.63, 17.29, 12.77.

[0171] Example 12

[0172] This example provides a pleuromutilin derivative (Compound 12) with the ability to inhibit bacterial hydrogen sulfide biosynthesis. Intermediate I, Intermediate III, and Intermediate IV were prepared according to the method of Example 1. The specific preparation process is as follows:

[0173] Preparation of 3 - ((3 - (3 - ((5 - ((2 - (((3aR,4R,5R,7S,8S,9R,9aS,12R) - 8 - hydroxy - 4,7,9,12 - tetramethyl - 3 - oxo - 7 - vinyl - decahydro - 4,9a - propacyclopenta[8]annulen - 5 - yl)oxy) - 2 - oxoethyl)thio)thiazol - 2 - yl)carbamoyl)phenyl) - 1H - indol - 1 - yl)methyl) - 1H - pyrazole - 5 - carboxylic acid

[0174]

[0175] Dissolve Intermediate IV (0.545 g, 1.5 mmol) in acetonitrile (5 mL), add EDCI (0.382 g, 2 mmol) and DMAP (0.244 g, 2 mmol), stir and react for 2 h, and monitor the carboxyl activation process by TLC. Subsequently, add Intermediate II (0.492 g, 1 mmol), continue to stir and react for 4 h, and monitor the reaction process by TLC. After the reaction is completed, add saturated sodium chloride aqueous solution to the system, extract, collect the organic phase and concentrate it, and perform flash column chromatography separation (the eluent is dichloromethane:methanol = 20:1). The yield of Compound 12 is 47.27%. The identification data are as follows:

[0176] 11H NMR (600 MHz, DMSO-d6) δ 11.07 (s, 1H), 10.95 (s, 1H), 10.09 (s, 1H), 8.31 (s, 1H), 8.07–8.02 (m, 2H), 7.96 (ddd, J = 7.6, 1.4, 1.0 Hz, 1H), 7.75 (s, 1H), 7.65 (t, J = 7.9 Hz, 1H), 7.51 (ddd, J = 7.4, 1.6, 1.0 Hz, 1H), 7.43 (dd, J = 6.2, 1.1 Hz, 1H), 7.39 (ddd, J = 8.4, 7.2, 1.6 Hz, 1H), 7.22 (ddd, J = 7.4, 6.2, 1.4 Hz, 1H), 6.81 (s, 1H), 5.63 (tdq, J = 10.2, 1.6, 0.8 Hz, 1H), 5.49 (d, J = 4.7 Hz, 2H), 5.12 (dd, J = 10.2, 2.6 Hz, 1H), 4.97 (tq, J = 5.4, 1.3 Hz, 1H), 4.89 (dd, J = 11.0, 2.6 Hz, 1H), 4.10 - 3.79 (m, 2H), 3.48 (dddt, J = 8.3, 6.8, 3.0, 1.7 Hz, 1H), 2.97 (d, J = 6.8 Hz, 1H), 2.31 (d, J = 3.2 Hz, 1H), 2.28 (t, J = 4.6 Hz, 2H), 2.16 (dd, J = 12.1, 5.8 Hz, 1H), 1.82 - 1.66 (m, 3H), 1.64 - 1.51 (m, 3H), 1.46 (dt, J = 12.5, 4.1 Hz, 1H), 1.42 - 1.30 (m, 2H), 1.14 - 1.05 (m, 6H), 0.90 (dd, J = 6.6, 1.8 Hz, 3H), 0.81 (d, J = 6.8 Hz, 3H).

[0177] 1313C NMR(151MHz,DMSO-d6)δ218.94,170.49,168.12,166.10,162.02,146.59,144.22,143.67,138.25,138.07,134.56,133.30,129.43,129.11,127.87,127.25,126.51,126.14,125.29,124.85,124.03,120.57,118.84,112.34,110.76,107.65,80.58,76.23,58.03,46.52,46.04,44.86,43.27,42.98,37.38,37.18,37.02,34.51,33.96,33.08,26.42,20.13,17.90,17.62,12.93.

[0178] Example 13

[0179] On the basis of Examples 1 to 12, the antibacterial activity in vitro of the compounds obtained in the present invention was determined.

[0180] Using the microbroth dilution method, tiamulin, valnemulin, and retapamulin were used as positive control drugs to test the minimum inhibitory concentration (Minimum Inhibitory Concentration, MIC) of pleuromutilin derivatives with the ability to inhibit bacterial hydrogen sulfide biosynthesis and their raw material pleuromutilin.

[0181] The experimental strains included drug-resistant Gram-positive bacteria: Methicillin-resistant S.aureus ATCC33591; Methicillin-resistant S.aureus ATCC 43300; S.aureus ATCC 29213; Methicillin-resistant S.epidermidis ATCC 51625. Drug-resistant Gram-negative bacteria: A.baumanniiATCC 19606; S.enterica ATCC14028; E.coli ATCC 25922; E.coli CMCC 44103. Nine clinically isolated drug-resistant strains: MRSA-171; MRSA-575; MRSA-206; MRSA-222; MRSA-596; VRE-80; MDR-PA-126; MDR-KP-893; CR-AB-882. The experimental strains were all provided by Huashan Hospital Affiliated to Fudan University (Institute of Antibiotics, Fudan University) and used after being identified by conventional methods.

[0182] The specific operation steps are as follows:

[0183] (1) Preparation of MHB medium: Weigh 18.0 g of MHB medium, add it to 1 L of distilled water, heat to boiling until completely dissolved, dispense into conical flasks, sterilize at 121 °C under high pressure for 15 min, and reserve for use.

[0184] (2) Culturing the experimental strain to the logarithmic growth phase: Under aseptic conditions, inoculate the experimental strain into fresh MHB medium, place it in a constant temperature and humidity incubator at 37 °C for 24 h, adjust the bacterial liquid concentration to 1.5×10⁸ CFU / mL, and then dilute it 200 times for reserve.

[0185] (3) Preparation of stock solution: Weigh the test sample, dissolve it with 1% DMSO solution to prepare a stock solution with a concentration of 2560 μg / mL; weigh the positive control product, dissolve it with sterile distilled water to prepare a stock solution with a concentration of 2560 μg / mL.

[0186] (4) Preparation of bacterial suspension: Under aseptic conditions, adjust the experimental strain cultured to the logarithmic growth phase to a turbidity standard of 0.5 McFarland unit with MHB medium and then dilute it at a ratio of 1:200. Finally, prepare a bacterial suspension with a concentration of 5×10 5 CFU / mL for reserve.

[0187] (5) Determination of MIC by microdilution method: Take a sterile 96-well plate, add 10 μL of the test compound with a concentration of 2560 μg / mL, and perform serial dilution by the two-fold dilution method. At the same time, set up a negative control group without adding the drug. Then add 190 μL of the diluted bacterial liquid to each well to make the final bacterial liquid concentration in each well 5×10⁵ CFU / mL, and incubate in a constant temperature and humidity incubator at 37 °C for 24 h.

[0188] (6) Reading the MIC endpoint: Observe with the naked eye against a black background. The concentration that can completely inhibit bacterial growth in the 96-well plate is the minimum inhibitory concentration of the sample against this kind of bacteria. Record the results in Table 1.

[0189] Table 1: Minimum inhibitory concentrations of the drug of the present invention and the positive control drug (μg·mL -1 )

[0190]

[0191]

[0192] b Methicillin-resistant S. aureus ATCC 33591. c Methicillin-resistant S. aureus ATCC 43300. d S. aureus ATCC 29213. e Methicillin-resistant S. epidermidis ATCC 51625. f A. baumannii ATCC 19606. g S. enterica ATCC 14028. h E. coli ATCC 25922. i E. coli CMCC 44103.

[0193] Table 2: Antibacterial activities of Compounds 8 and 9 against 9 clinically isolated strains

[0194]

[0195] b MRSA: methicillin-resistant Staphylococcus aureus - 171; c MRSA: methicillin-resistant Staphylococcus aureus - 575; d MRSA: methicillin-resistant Staphylococcus aureus - 206; e MRSA: methicillin-resistant Staphylococcus aureus - 222; f MRSA: methicillin-resistant Staphylococcus aureus - 596; g VRE: vancomycin-resistant Enterococcus - 80; h MDR-PA: multidrug-resistant Pseudomonas aeruginosa - 126; i MDR-KP: multidrug-resistant Klebsiella pneumoniae - 893; j CR-AB: carbapenem-resistant Acinetobacter baumannii - 882.

[0196] As can be seen from Table 1, the minimum values of the 12 target compounds (1 - 12), retapamulin, and valnemulin. The research results show that after introducing different R1 and R2 structures, the pleuromutilin derivatives containing indole structures exhibit excellent activities; compared with valnemulin and retapamulin, Compound 5, Compound 6, and Compound 7 of the present invention show relatively similar potencies and nearly comparable antibacterial activities, indicating that these compounds are comparable to the control drugs in terms of antibacterial effects and are potential antibacterial candidate drugs; in the comparison of antibacterial activities with valnemulin and retapamulin, Compound 8 shows good antibacterial activity, while Compound 9 shows the most advantageous antibacterial activity. See Figure 1 As shown, for both Gram-positive and Gram-negative bacteria, Compound 8 and Compound 9 have quite good efficacies and broad-spectrum antibacterial characteristics; after introducing the indole structure of 5-(bromomethyl)-2-methylfuran-3-carboxylic acid into Compound 7, Compound 8, and Compound 9, the antibacterial efficacy against Gram-positive bacteria is enhanced, and the antibacterial activities against Gram-negative bacteria such as Escherichia coli and Acinetobacter baumannii are also significantly improved, and the activities are better than those of the positive control drug, indicating that the indole structure modification can significantly enhance the antibacterial activity of the compounds. The MIC heat map of the compounds obtained from the data in Table 1 is as Figure 2 shown.

[0197] Compound 8 and Compound 9 were further tested against 9 clinical isolates including MRSA (methicillin-resistant Staphylococcus aureus), VRE (vancomycin-resistant Enterococcus), and MDRPA (multidrug-resistant Pseudomonas aeruginosa). The results are shown in Table 2.

[0198] Compound 9 shows effective anti-VRE and anti-CR-AB (carbapenem-resistant Acinetobacter baumannii) activities, and the minimum inhibitory concentrations (MICs) against VRE and CR-AB are 8 and 4 μg / mL respectively, indicating the potent antibacterial effect of Compound 9 against these two refractory pathogens; the activities of Compound 9 against clinically common pathogenic Gram-negative bacteria such as MDR-PA, MDR-KP (multidrug-resistant Klebsiella pneumoniae), and CR-AB are better than those of retapamulin, indicating that Compound 9 is superior to the control drug retapamulin in terms of antibacterial effects against these Gram-negative bacteria; Compound 9 shows antibacterial activities against clinical MRSA isolates (MRSA-171, MRSA-575, MRSA-206, MRSA-222, MRSA-596), and the MIC is 2 - 8 μg / mL. Against the MRSA-206 strain, the MIC of Compound 9 is 2 μg / mL, showing excellent antibacterial activity.

[0199] Example 14

[0200] On the basis of Examples 1 to 12, this example examines the effect of the pleuromutilin derivatives obtained by the present invention on the biosynthesis of bacterial hydrogen sulfide.

[0201] Use lead acetate test strips to measure gaseous H 2 S in the solution. Fix the strip soaked with 2% lead acetate on the inner wall of the culture tube, higher than the liquid culture level of wild-type or mutant bacteria; dilute the overnight culture 1:50 in LB (Lysogeny broth), add an appropriate amount of L-cysteine (Staphylococcus aureus S.aureus is ATCC29213) (concentration range is 200 - 500 μM) and TSB (Pseudomonas aeruginosa) to the diluted culture medium, and incubate the culture medium aerobically at 37 °C for 12 - 20 hours. After the experiment, observe the color change of the test strip. The color depth of the test strip is directly related to the content of H 2 S in the sample. The darker the color, the higher the content of H 2 S. See the appendix for the results Figure 3 as shown.

[0202] From the appendix Figure 3 data, it can be seen that as the concentration of compound 9 increases, the color of the test strip gradually fades, indicating that the content of H 2 S in the sample gradually decreases. Compound 9 has an obvious inhibitory effect on the biosynthesis of bacterial hydrogen sulfide. As the concentration of compound 9 increases, the amount of H 2 S produced by bacteria significantly decreases, verifying the inhibitory effect of compound 9 of the present invention on the biosynthesis of bacterial hydrogen sulfide, and providing important data for further research on the biological activity of the compound.

[0203] Example 15

[0204] Use the CCK-8 (Cell Counting Kit-8) method to evaluate the cytotoxicity of the compound:

[0205] (1) Prepare PBS buffer: Dissolve 8.00 g of NaCl, 0.20 g of KCl, 1.44 g of Na 2 HPO 4 and 0.24 g of KH 2 PO 4 in 800 mL of distilled water, adjust the pH value to 7.4, make up the volume to 1 L with distilled water, and sterilize for 40 min to obtain 10 mM PBS buffer for standby.

[0206] (2) Preparation of the bottom agar medium: Weigh 6.00 g of agar powder and dissolve it in 400 mL of distilled water. Stir well and autoclave for 40 min to ensure the sterility of the medium. When the solution cools to 60 - 70 °C, add 8 mL of vitamin VS solution and 8 mL of glucose GS solution, and mix well to ensure uniform distribution. Pour the mixed agar solution into petri dishes to prepare the bottom agar medium.

[0207] (3) Preparation of the top agar medium: Weigh 1.20 g of agar powder and 1.00 g of NaCl, dissolve them in 200 mL of distilled water, mix and autoclave for 40 min to ensure the sterility of the medium. When the solution cools to 60 °C, add 1.00 mL of HBT solution, mix well to obtain a mixed solution. Take 2 mL of the mixed solution and dispense it into sterile test tubes, and keep it at 45 °C for later use.

[0208] (4) Cell culture: HepG2, HEK293, and A549 cells are evenly dispersed in the medium, inoculated into 96 - well plates, and cultured under suitable environmental conditions to keep the cells fully attached to the well walls.

[0209] (5) Compound treatment: Add compounds 8 and 9 with different concentrations (the concentrations are 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 μM in sequence, and conduct 6 parallel experiments) to each well; set up a blank control group and a normal control group; after incubating for 24 h, observe the cell situation using a microscope.

[0210] (6) CCK - 8 solution treatment: Add CCK - 8 solution to each well and incubate for 4 h to allow it to react with the cells.

[0211] (7) Absorbance measurement: Use a full - wavelength scanning multi - functional reader. Place the 96 - well plate in the instrument, shake and mix to make the color evenly distributed, and measure the absorbance value at 490 nm.

[0212] (8) Data analysis: Calculate the cell viability at different concentrations based on the measured absorbance values. According to the dose - response curve, calculate the median cytotoxic concentration (CC50) to evaluate the cytotoxicity of the compounds against A549, HEK293, and HepG2 cells. The experimental results are shown in Table 3 and Appendix Figure 4 .

[0213] Table 3: Test results of the cytotoxicity experiments of compounds 8, 9, retamolin, and valnemulin

[0214]

[0215] As can be seen from Table 4, in the safety evaluation experiment, at a concentration of 100 μM, Compound 8 of Example 8 and Compound 9 of Example 9 of the present invention showed low toxicity to HepG2, HEK293 and A549 cells, because their CC50 values were higher than or much greater than 100 μM (especially on A549 cells). The test cells included: HepG2 cells, HEK293 cells and A549 cells. Compared with the control drugs retapamulin and valnemulin, the CC50 values of Compound 8 of Example 8 and Compound 9 of Example 9 of the present invention on the same cell line were generally higher, indicating that they had lower toxicity to the test cells and had better safety prospects in drug development.

[0216] Example 16

[0217] In this example, the IC 50 inhibitory activity test of the candidate drug was carried out. bCSE (bacterial source CSE enzyme) and hCSE (human source CSE enzyme) were successfully recombinantly expressed in Escherichia coli, and the IC 50 inhibitory activity test of the candidate compound was carried out. The inhibitory activity of the compounds of the present invention on the purified bacterial source CSE enzyme (bCSE) and human source CSE enzyme (hCSE) was determined by fluorescence spectrophotometry, and their IC 50 values were calculated. A buffer solution was prepared. 10 μM pyridoxal phosphate and 1.2 or 2.0 μM WSP5 fluorescent probe (excitation wavelength: 500 nm, emission wavelength: 530 nm) were added to the sodium phosphate buffer solution (where the concentration of bCSE or hCSE was 0.1 μM, pH 8.0). In a 96-well plate, different concentrations of Compound 9 of Example 9 of the present invention to be tested and 50 mM of the prepared buffer solution were added and incubated together for 20 minutes. 100 μM L-cysteine was added to the mixture to initiate the enzymatic reaction, and the fluorescence signal was recorded using a cell imaging microplate reader, and the relative fluorescence intensity was calculated to evaluate the enzyme activity. The IC 50 curve was plotted using GraphPad Prism 21 software, with the concentration as the abscissa and the relative fluorescence intensity as the ordinate. As Figure 5 shown, the results showed that the IC 50 of Compound 9 on bCSE was 1.73 μM, and the IC 50 on hCSE was 37.24 μM, indicating that Compound 9 had higher activity on bCSE and weaker inhibitory activity on hCSE, showing good selectivity.

[0218] The above content is only an example and illustration of the concept of the present invention. Those skilled in the art of the present technology can make various modifications or supplements to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all belong to the protection scope of the present invention.

Claims

1. A pleuromutilin derivative having the effect of inhibiting bacterial hydrogen sulfide biosynthesis, characterized in that: The pleuromutilin derivative is a compound as shown in formula (I), or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer or tautomer of the compound as shown in formula (I); Wherein R1 is selected from Any of the following: R2 is selected from Any one of .

2. A pleuromutilin derivative having the effect of inhibiting bacterial hydrogen sulfide biosynthesis according to claim 1, characterized in that: The pleuromutilin derivative is selected from any one of the compounds shown in the following formula:

3. The pleuromutilin derivative having the effect of inhibiting bacterial hydrogen sulfide biosynthesis according to claim 1, characterized in that: The pharmaceutically acceptable salt is a salt formed by a pleuromutilin derivative having the function of inhibiting bacterial hydrogen sulfide biosynthesis and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

4. A method for preparing a pleuromutilin derivative having the ability to inhibit bacterial hydrogen sulfide biosynthesis according to any one of claims 1 to 3, characterized in that: The synthesis steps include: (1) Using pleuromutilin and p-toluenesulfonyl chloride as raw materials, the intermediate I is obtained by reaction; The intermediate I is (2) using the intermediate I obtained in step (1) and SH-R1-NH2 as raw materials, reacting to obtain intermediate II; The intermediate II is Wherein R1 is selected from Any of the following: (3) Using (1-hydrogen-indol-3-yl)boric acid and Br-R2 as raw materials, the intermediate III is obtained by reaction; The intermediate III is Wherein R2 is selected from Any of the following: (4) reacting the intermediate III obtained in step (3) with 3-bromobenzoic acid to obtain intermediate IV; The intermediate IV is: Wherein R2 is selected from Any of the following: (5) Intermediate II and intermediate IV are used as raw materials to react to obtain a pleuromutilin derivative having the function of inhibiting bacterial hydrogen sulfide biosynthesis as shown in formula (I).

5. The method for preparing a pleuromutilin derivative having the ability to inhibit bacterial hydrogen sulfide biosynthesis according to claim 4, characterized in that: In step (1), the molar ratio of pleuromutilin to toluenesulfonyl chloride is 1:1-2, and the reaction conditions are: reaction at room temperature for 6-10 hours; in step (2), the molar ratio of intermediate I to SH-R1-NH2 is 1:1-2, and the reaction conditions are: reaction at 50-70°C for 5-7 hours.

6. The method for preparing a pleuromutilin derivative having the ability to inhibit bacterial hydrogen sulfide biosynthesis according to claim 4, characterized in that: In step (3), the molar ratio of (1-hydrogen-indol-3-yl)boric acid to Br-R2 is 1:1-2, and the reaction conditions are: reaction at 0-10°C for 1-3 hours; in step (4), the molar ratio of intermediate III to 3-bromobenzoic acid is 1:1-2, and the reaction conditions are reaction at 100-140°C for 6-8 hours.

7. The method for preparing a pleuromutilin derivative having the ability to inhibit bacterial hydrogen sulfide biosynthesis according to claim 4, characterized in that: In step (5), the molar ratio of intermediate II to intermediate IV is 1:1-2, and the reaction conditions are room temperature for 1-3 hours.

8. Use of the pleuromutilin derivatives according to any one of claims 1 to 3 that have the ability to inhibit bacterial hydrogen sulfide biosynthesis in the preparation of antibacterial products.

9. The use according to claim 6, characterized in that: The antibacterial product is a pharmaceutical preparation for treating infectious diseases caused by pathogenic microorganisms. The pharmaceutical preparation is used alone or in combination with other antibacterial drugs, or is mixed with pharmaceutically acceptable excipients and diluents to form tablets, capsules, granules, syrups, premixes or pellets for oral administration, or is prepared as ointments or injections for non-oral administration; the pathogenic microorganisms are Gram-positive bacteria, Gram-negative bacteria, drug-resistant bacteria or mycoplasma; the drug for treating infectious diseases caused by pathogenic microorganisms includes a pharmaceutically acceptable carrier.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the pleuromutilin derivative having the ability to inhibit bacterial hydrogen sulfide biosynthesis according to any one of claims 1 to 3 as an active ingredient.

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