A c14-modified pleuromutilin derivative, its preparation method and use in the treatment of bacterial infections

Abstract

The present application belongs to the field of pharmaceutical chemistry and antibacterial drug technology, and particularly relates to a C14 modified pleuromutilin derivative, a preparation method thereof and application thereof in treating gram-positive bacteria, especially gram-positive drug-resistant bacterial infection. The C14 modified pleuromutilin derivative provided by the present application has a structure shown in formula I or formula II. The present application introduces a heterocyclic side chain (4-trifluoromethylphenylthiol or 1-(4,5-dihydrothiazole-2-yl)azetidine-3-thiol) at the C14 site of pleuromutilin to synthesize the C14 modified pleuromutilin derivative, which significantly enhances the antibacterial activity of the C14 modified pleuromutilin derivative on methicillin-resistant Staphylococcus aureus (MRSA) and cfr gram-positive bacterial strains, and has high drug resistance barrier, low cytotoxicity and hemolyticity. The C14 modified pleuromutilin derivative provided by the present application is suitable for treating multiple drug-resistant bacterial infection in veterinary and human medicine. Formula I, formula II.

Description

Technical Field

[0001] This invention belongs to the fields of medicinal chemistry and antibacterial drug technology, specifically relating to a C14-modified truncated pleurotin derivative, its preparation method, and its application in the treatment of bacterial infections. Background Technology

[0002] The spread of antimicrobial resistance (AMR) poses a serious challenge to global public health and veterinary clinical practice. Methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), and vancomycin-resistant Staphylococcus aureus (VISA), as representative multidrug-resistant pathogens, can cause serious diseases such as pneumonia and mastitis in animals, resulting in significant economic losses to livestock farming. Furthermore, their risk of cross-species transmission poses a public health threat.

[0003] Currently, veterinary clinics primarily rely on truncated pleurotin antibiotics such as tiamulin and voremurine to combat drug-resistant pathogens. These drugs inhibit protein synthesis by binding to the 50S subunit peptidyl transferase center (PTC) of bacterial ribosomal molecule, exhibiting low cross-resistance with traditional antibiotics. However, the application of these drugs is limited by resistance (e.g., ... cfr It has drawbacks such as gene-mediated ribosomal methylation, low oral bioavailability, and a narrow therapeutic index. Summary of the Invention

[0004] The purpose of this invention is to provide a C14-modified truncated pleurotin derivative, its preparation method, and its application in treating bacterial infections. The C14-modified truncated pleurotin derivative provided by this invention has significantly improved antibacterial activity, especially effective against drug-resistant bacteria, and has good safety. The C14-modified truncated pleurotin derivative provided by this invention can serve as a next-generation antibiotic for controlling multidrug-resistant bacterial infections in animal husbandry and human medicine, and has broad market prospects.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a C14-modified truncated pleurotin derivative having the structure shown in Formula I or Formula II: Formula I, Formula II.

[0006] This invention provides a method for preparing the C14-modified truncated pleurotin derivative described in the above technical solution, comprising the following steps: (1) The truncated pleurotin was reacted with p-toluenesulfonyl chloride in an organic solvent to obtain an intermediate with the structure shown in Formula 2; (2) React 4-trifluoromethylthiophenol or 1-(4,5-dihydrothiazo-2-yl)azacyclobutane-3-thiol with the intermediate of the structure shown in Formula 2 to obtain the C14 modified truncated pleurotin derivative. Equation 2.

[0007] Preferably, in step (1), the raw materials for the reaction further include an organic base; the molar ratio of truncated pleurotin to p-toluenesulfonyl chloride is 1:1.1~1.3; and the molar ratio of truncated pleurotin to the organic base is 1:1.8~2.2.

[0008] Preferably, in step (2), the molar ratio of the intermediate with the structure shown in Formula 2 to the 4-trifluoromethylthiophenol is 1:1.1~1.3; the molar ratio of the intermediate with the structure shown in Formula 2 to the 1-(4,5-dihydrothiazol-2-yl)azacyclobutane-3-thiol is 1:1.1~1.3.

[0009] Preferably, in step (2), the raw materials for the condensation reaction further include an organic base, and the molar ratio of the intermediate with the structure shown in Formula 2 to the organic base is 1:1.4~1.6.

[0010] Preferably, in step (2), after the reaction is completed, a reaction solution is obtained; the reaction solution is further separated into an organic phase by washing and extracting with water; the obtained organic phase is dried and purified by column chromatography to obtain the pure product of the C14 modified truncated pleurotin derivative, wherein the eluent used in the column chromatography purification is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 3~5:1.

[0011] This invention provides the use of the C14-modified truncated pleurotin derivatives or pharmaceutically acceptable salts thereof, or the C14-modified truncated pleurotin derivatives prepared by the preparation method described above, in the preparation of medicaments for treating and / or preventing bacterial infections.

[0012] Preferably, the bacteria include Gram-positive bacteria and / or Gram-positive drug-resistant bacteria, wherein the Gram-positive drug-resistant bacteria include methicillin-resistant Staphylococcus aureus (MRSA). cfr - Gene-positive methicillin-resistant Staphylococcus aureus ( cfr - One or more of the following: gene-positive MRSA strains, methicillin-resistant Staphylococcus epidermidis (MRSE), and vancomycin-intermediate Staphylococcus aureus (VISA).

[0013] The present invention provides a drug comprising the C14-modified truncated pleurotin derivative described in the above technical solution or a pharmaceutically acceptable salt thereof, or the C14-modified truncated pleurotin derivative prepared by the preparation method described in the above technical solution.

[0014] Preferably, the dosage form of the drug includes injections, tablets, or ointments; the drug is a drug used for the treatment of respiratory or skin infections in veterinary or human medicine.

[0015] This invention provides a C14-modified pleuromutilin derivative having the structure shown in Formula I or Formula II. The C14-modified pleuromutilin derivative with the structure shown in Formula I is named compound ts26. The C14-modified pleuromutilin derivative with the structure shown in Formula II is named compound ts27. The C14-modified pleuromutilin derivative provided by this invention is synthesized by introducing a heterocyclic side chain (4-trifluoromethylthiophenol or 1-(4,5-dihydrothiazol-2-yl)azacyclobutane-3-thiol) to the C14 site of pleuromutilin. This invention, through structural optimization, significantly enhances the resistance of the C14-modified pleuromutilin derivative to methicillin-resistant Staphylococcus aureus (MRSA) and... cfr Antibacterial activity of positive strains. In vitro experiments showed that the minimum inhibitory concentration (MIC) of the C14-modified truncated pleurotin derivative provided by this invention was 16-fold lower than that of the traditional drug tiamulin, and it exhibited a high resistance barrier, low cytotoxicity, and hemolytic activity. In vivo mouse models confirmed the efficacy (ED) of compound ts27. 50 =7.1 mg / kg) is superior to tiamulin (ED) 50 =10.8 mg / kg). Molecular docking showed that the C14-modified truncated pleurotin derivative provided by this invention stably binds to the ribosomal peptidyl transferase center (PTC) through hydrogen bonding and hydrophobic interactions. In summary, the C14-modified truncated pleurotin derivative provided by this invention is suitable for the treatment of multidrug-resistant bacterial infections in veterinary and human medicine.

[0016] This invention also provides a method for preparing the C14-modified truncated pleurotin derivative described in the above technical solution, comprising the following steps: (1) reacting truncated pleurotin with p-toluenesulfonyl chloride in an organic solvent to obtain an intermediate with the structure shown in Formula 2; (2) reacting 4-trifluoromethylthiophenol or 1-(4,5-dihydrothiazol-2-yl)azacyclobutane-3-thiol with the intermediate with the structure shown in Formula 2 to obtain the C14-modified truncated pleurotin derivative. The preparation method provided by this invention is simple and easy to implement, and suitable for industrial production. Attached Figure Description

[0017] Figure 1 Synthetic route diagram of C14-modified truncated pleurotin derivatives provided by the present invention; Figure 2The time-bacterial kinetics and resistance development of target compounds ts26 and ts27 against methicillin-resistant Staphylococcus aureus were investigated. Figure 3 The results of concentration-dependent cytotoxicity and erythrocyte compatibility analysis of compounds ts26, ts27 and tiamulin in human cell lines are presented. Figure 4 The dose-dependent therapeutic effects of ts27 and tiamulin in a mouse model of systemic infection with methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300) were investigated. Figure 5 The results of molecular docking analysis of ts26, ts27 and tiamulin at the peptidyl transferase center (PTC); Figure 6 The results of the study on the antibacterial mechanism of TS27 and tiamulin. Detailed Implementation

[0018] This invention provides a C14-modified truncated pleurotin derivative having the structure shown in Formula I or Formula II: Formula I, Formula II.

[0019] This invention provides a method for preparing the C14-modified truncated pleurotin derivative described in the above technical solution, comprising the following steps: (1) The truncated pleurotin was reacted with p-toluenesulfonyl chloride in an organic solvent to obtain an intermediate with the structure shown in Formula 2; (2) 4-trifluoromethylthiophenol or 1-(4,5-dihydrothiazol-2-yl)azacyclobutane-3-thiol is reacted with the intermediate of the structure shown in Formula 2 (condensation reaction) to obtain the C14 modified truncated pleurotin derivative. Equation 2.

[0020] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0021] Step (1): In this invention, truncated pleurotin is reacted with p-toluenesulfonyl chloride in an organic solvent to obtain an intermediate with the structure shown in Formula 2.

[0022] In this invention, the chemical structural formula of truncated pleurotin is shown in Formula 1: Formula 1.

[0023] In this invention, the organic solvent can be dichloromethane, specifically anhydrous dichloromethane in the embodiments. The reactants preferably also include an organic base. The organic base is preferably triethylamine. The molar ratio of truncated pleurotin to p-toluenesulfonyl chloride is preferably 1:1.1~1.3, and in the embodiments it can be 1:1.2. The molar ratio of truncated pleurotin to the organic base is preferably 1:1.8~2.2, and in the embodiments it can be 1:2. The mixing of the reactants preferably includes: dissolving the truncated pleurotin in an organic solvent, then cooling it to 0 °C in an ice-water bath, then sequentially adding p-toluenesulfonyl chloride and the organic base, and then removing the ice-water bath to proceed with the reaction. In this invention, the mixing is preferably carried out in a protective gas atmosphere, which can be nitrogen. The reaction temperature is preferably room temperature, the reaction time is preferably 12~24 h, and the reaction is carried out under stirring. The reaction is carried out in a protective gas atmosphere, which can be nitrogen. After the reaction is completed, a reaction solution is obtained. Preferably, the reaction solution is washed, extracted, and separated to obtain an organic phase. The organic phase is then dried and concentrated sequentially to obtain an intermediate with the structure shown in Formula 2. The intermediate with the structure shown in Formula 2 is a white solid. The washing and extraction are preferably performed sequentially using hydrochloric acid, a saturated NaHCO3 solution, and a saturated NaCl solution. The concentration of HCl in the hydrochloric acid is preferably 1 mol / L, and the hydrochloric acid washing is preferably performed twice. The washing with the saturated NaHCO3 solution is preferably performed twice. The washing with the saturated NaCl solution is preferably performed once. The drying reagent is preferably anhydrous sodium sulfate. The concentration can be performed under reduced pressure.

[0024] Step (2): After obtaining the intermediate with the structure shown in Formula 2, the present invention performs a condensation reaction between 4-trifluoromethylthiophenol or 1-(4,5-dihydrothiazol-2-yl)azacyclobutane-3-thiol and the intermediate with the structure shown in Formula 2 to obtain the C14 modified truncated pleurotin derivative. In the present invention, the chemical structural formulas of 4-trifluoromethylthiophenol and 1-(4,5-dihydrothiazol-2-yl)azacyclobutane-3-thiol are shown in Formulas 3 and 4, respectively: Formula 3 Formula 4.

[0025] In this invention, the organic solvent can be dichloromethane. The molar ratio of the intermediate with the structure shown in Formula 2 to the 4-trifluoromethylthiophenol is preferably 1:1.1 to 1.3, and in the examples, it can be 1:1.2. The molar ratio of the intermediate with the structure shown in Formula 2 to the 1-(4,5-dihydrothiazol-2-yl)azacyclobutane-3-thiol is preferably 1:1.1 to 1.3, and in the examples, it can be 1:1.2. In this invention, the raw materials for the condensation reaction preferably also include an organic base. The organic base can be triethylamine. The molar ratio of the intermediate with the structure shown in Formula 2 to the organic base is preferably 1:1.4 to 1.6, and in the examples, it can be 1:1.5. In this invention, the mixing of the raw materials for the condensation reaction preferably includes: dissolving the intermediate with the structure shown in Formula 2 in an organic solvent, then adding an organic base, and then adding 4-trifluoromethylthiophenol or 1-(4,5-dihydrothiazol-2-yl)azacyclobutane-3-thiol. The condensation reaction is carried out at room temperature, and the preferred reaction time is 12-15 hours. The condensation reaction is carried out under stirring.

[0026] In this invention, after the condensation reaction is completed, a condensation reaction solution is obtained. Preferably, this invention further includes separating the organic phase after washing and extraction with water from the condensation reaction solution; the obtained organic phase is then subjected to drying and column chromatography purification to obtain the pure C14-modified truncated pleurotin derivative. In this invention, the water washing and extraction are preferably performed twice. The reagent used for drying is preferably anhydrous sodium sulfate. The eluent used for column chromatography purification is preferably petroleum ether and ethyl acetate. The volume ratio of petroleum ether to ethyl acetate is preferably 3-5:1, and in the examples, it can be 4:1.

[0027] This invention provides the use of the C14-modified truncated pleurotin derivatives or pharmaceutically acceptable salts thereof, or the C14-modified truncated pleurotin derivatives prepared by the preparation method described above, in the preparation of medicaments for treating and / or preventing bacterial infections.

[0028] In this invention, the bacteria preferably include Gram-positive bacteria and / or Gram-positive drug-resistant bacteria. In this invention, the Gram-positive bacteria preferably include methicillin-sensitive Staphylococcus aureus (MSSA). In this invention, the Gram-positive drug-resistant bacteria are preferably methicillin-resistant Staphylococcus aureus (MRSA). cfr - Gene-positive methicillin-resistant Staphylococcus aureus ( cfr - One or more of the following: gene-positive MRSA strains, methicillin-resistant Staphylococcus epidermidis (MRSE), and vancomycin-intermediate Staphylococcus aureus (VISA).

[0029] The present invention provides a drug comprising the C14-modified truncated pleurotin derivative described in the above technical solution or a pharmaceutically acceptable salt thereof, or the C14-modified truncated pleurotin derivative prepared by the preparation method described in the above technical solution.

[0030] In this invention, the dosage form of the drug preferably includes injections, tablets, or ointments.

[0031] In this invention, the drug may be an antibiotic.

[0032] In this invention, the drug is preferably a drug used for the treatment of respiratory or skin infections in veterinary or human medicine.

[0033] In this invention, the drug also includes a pharmaceutically acceptable carrier.

[0034] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1: Synthesis of compounds ts26 and ts27 The synthetic routes for compounds ts26 and ts27 are as follows: Figure 1 As shown: Step 1: Synthesis of Intermediate 2 (22-O-p-toluenesulfonyl truncated pleurotin). Under nitrogen protection, truncated pleurotin (1.0 equivalent, 5.0 mmol) was dissolved in anhydrous dichloromethane (30 mL) and cooled to 0 °C in an ice-water bath. p-Toluenesulfonyl chloride (TsCl, 1.2 equivalent, 6.0 mmol) and triethylamine (Et3N, 2.0 equivalent, 10.0 mmol) were added sequentially to this solution. The ice bath was removed, and the reaction mixture was allowed to rise to room temperature and stirred for 24 hours. After the reaction was complete, the reaction solution was washed sequentially with 1M HCl (2 × 20 mL), saturated NaHCO3 solution (2 × 20 mL), and brine (20 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give intermediate 2 as a white solid, in 89% yield.

[0036] Step 2: Synthesis of compound ts26. Intermediate 2 (1.0 equivalent, 4.45 mmol) obtained above was dissolved in dichloromethane (20 mL), and triethylamine (1.5 equivalent, 6.68 mmol) was added. Subsequently, 4-trifluoromethylthiophenol (1.2 equivalent, 5.34 mmol) was added, and the reaction was stirred at room temperature for 12 hours. After the reaction was complete, the reaction solution was washed with water (2 × 20 mL), and the organic phase was dried over anhydrous Na₂SO₄. Purification by silica gel column chromatography (petroleum ether / ethyl acetate = 4:1, v / v) yielded the target compound ts26 as a white solid, in 62% yield. Its structure was confirmed by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS).

[0037] Step 3: Synthesis of compound ts27. The synthesis procedure was the same as for ts26, except that the starting material 4-trifluoromethylthiophenol was replaced with an equivalent amount of 1-(4,5-dihydrothiazol-2-yl)azacyclobutane-3-thiol. After purification by silica gel column chromatography, the target compound ts27 was obtained as a white solid in 83% yield. Its structure was confirmed by NMR and LC-MS.

[0038] Characterization data: 1. Compound ts26.

[0039] (1) 1 H NMR (500 MHz, d6 -dimethylsulfoxide [DMSO]) δ 8.66 (s, 1H), 8.02(d, J = 8.3 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 6.07 (dd, J = 17.6, 11.2 Hz, 1H), 5.55 (d, J = 7.9 Hz, 1H), 5.02 (d, J = 17.6 Hz, 1H), 4.94 (d, J = 11.1 Hz, 1H), 4.50 (d, J = 5.6 Hz, 1H), 4.02 (s, 2H), 2.16 (d, J = 11.2 Hz, 1H), 2.08 (d, J =11.5 Hz, 2H), 1.99 (s, 1H), 1.73 – 1.61 (m, 2H), 1.60 (s, 1H), 1.48 (s, 1H), 1.37 (d, J= 11.9 Hz, 2H), 1.32 (s, 3H), 1.26 (s, 1H), 1.25 (s, 1H), 1.18 (s,1H), 1.04 (d, J = 23.3 Hz, 1H), 1.01 (s, 3H), 0.80 (d, J = 6.3 Hz, 3H), 0.62 (d, J = 6.6 Hz, 3H).

[0040] (2) 13 C NMR (126 MHz, DMSO) δ 217.6, 167.5, 163.1, 146.2, 146.2, 141.2, 134.0, 133.9, 122.4, 115.5, 73.0, 70.4, 57.7, 45.4, 44.4, 43.9, 41.9, 36.8,34.5, 33.1, 31.2, 30.6, 28.8, 27.1, 24.9, 16.5, 14.9, 12.0.

[0041] (3) LC-MS (ESI) m / z: Molecular ion peak [M+H] was detected in positive ion mode. + According to the molecular formula (C 28 H 37 The calculated value of F3NO4S is 540.2; the measured mass-to-charge ratio is 540.4.

[0042] 2. Compound ts27.

[0043] (1) 1 H NMR (500 MHz, d6 -DMSO) δ 6.16 (dd, J = 17.6, 11.2 Hz, 1H), 5.56(d, J = 7.9 Hz, 1H), 5.05 (dd, J = 24.3, 14.4 Hz, 2H), 4.51 (d, J = 5.8 Hz, 1H), 3.23 (s, 2H), 2.41 (s, 1H), 2.18 (d, J = 10.9 Hz, 2H), 2.12 (s, 1H), 2.09 (s,2H), 1.98 (s, 2H), 1.77 (s, 3H), 1.67 (d, J= 14.5 Hz, 2H), 1.63 (s, 3H), 1.60(s, 1H), 1.49 (s, 1H), 1.42 – 1.38 (m, 1H), 1.37 (s, 3H), 1.30 (s, 1H), 1.27(s, 1H), 1.24 (s, 1H), 1.06 (s, 3H), 1.00 (d, J = 13.2 Hz, 1H), 0.82 (d, J = 6.6Hz, 3H), 0.63 (d, J = 6.7 Hz, 3H).

[0044] (2) 13 C NMR (126 MHz, DMSO) δ 217.6, 169.5, 141.3, 115.6, 73.1, 69.7,57.8, 45.4, 44.9, 44.3, 44.2, 43.3, 41.9, 36.9, 36.8, 36.1, 34.5, 31.2, 30.6,29.5, 29.5, 28.8, 27.1, 24.9, 16.7, 15.1, 12.0. (3) LC-MS (ESI) m / z: Molecular ion peak [M+H] was detected in positive ion mode. + According to the molecular formula (C 28 H 43 The calculated value of N2O4S2 is 535.3; the measured mass-to-charge ratio is 535.4.

[0045] Example 2: In vitro antibacterial activity assay The in vitro antibacterial spectra of ts26 and ts27 were determined using the broth microdilution method (following CLSI guidelines). The results are shown in Table 1. Both ts26 and ts27 are narrow-spectrum antibacterial compounds, exhibiting potent antibacterial activity against Gram-positive bacteria, superior to the control drug tiamulin; however, their activity against most Gram-negative bacteria is limited. They showed outstanding performance against Staphylococcus aureus, with a MIC value of 0.0313 μg / mL against methicillin-sensitive Staphylococcus aureus (MSSA, ATCC 29213), 16 times that of tiamulin (0.5 μg / mL). The MIC values ​​against MRSA standard strains (ATCC 43300, ATCC 33591) ranged from 0.0313 to 0.0625 μg / mL, also significantly lower than tiamulin (0.5 μg / mL).

[0046] The MIC value against the standard strain of Streptococcus pneumoniae (ATCC 49619) was 0.0625 μg / mL, which was 4 times that of tiamulin (0.25 μg / mL). As for Gram-negative bacteria, neither had significant inhibitory activity against Escherichia coli (K-12 MG1655) and Klebsiella pneumoniae (ATCC 13883), with MIC values ​​>64 μg / mL for both.

[0047] Based on this, both drugs also provide efficient coverage against common multidrug-resistant Gram-positive bacteria in clinical settings. The MIC value against the MRSE standard strain (ATCC 35984) is 0.0313 μg / mL, significantly lower than tiamulin (0.25 μg / mL), and against the VISA standard strain (ATCC 700699) is 0.125 μg / mL, demonstrating superior activity compared to tiamulin (0.5 μg / mL), especially against... cfr - Gene-positive MRSA bacteria ( cfr- MRSA), ts26 and ts27 were completely ineffective (MIC>64 μg / mL), while ts26 and ts27 maintained moderate activity (MIC=16 μg / mL), successfully overcoming this key resistance mechanism and making up for the core limitations of traditional truncated pleurotin antibiotics.

[0048] Table 1. MIC values ​​(μg / mL) of ts26, ts27 and tiamulin against various bacteria.

[0049] Example 3: Time-Kill Kinetics and Induction of Drug Resistance MRSA standard strain ATCC 43300 was selected as the test strain. Time-kill kinetics and continuous subculturing resistance induction tests were conducted on compounds ts26 and ts27 to assess their bactericidal rate and the risk of resistance development. First, a time-kill curve experiment was performed, with MRSA at an initial bacterial count of approximately 1 × 10⁻⁶. 6ts26 and ts27 were added to Mueller-Hinton broth at CFU / mL to achieve final concentrations of 1×, 3×, and 6× MIC, respectively. The broth was incubated at 37 °C and 180 rpm, and samples were taken at predetermined time points for serial dilution and plating for bacterial count. The results showed that both compounds exhibited significant concentration- and time-dependent bactericidal effects. ts26 reduced bacterial count by 99.9% within 6 h at 3× MIC, and achieved the same bactericidal level within 4 h at 6× MIC. In contrast, ts27 had a faster bactericidal rate, and its reduction in bacterial count at the same concentration was significantly greater than that of ts26 and the control drug tiamulin. It should be noted that the MIC of ts26 and ts27 (0.0313 μg / mL) is 1 / 16 that of tiamulin (0.5 μg / mL), showing significantly higher efficacy. At high concentrations (6×MIC), ts26 and ts27 can rapidly and continuously reduce MRSA viability, helping to reduce the bacterial survival window in the early stages of infection, thereby reducing the risk of drug resistance mutation accumulation. (See [link to relevant documentation]). Figure 2 A, B, and C in the diagram. Figure 2 The study investigated the time-killing kinetics and resistance development of target compounds ts26 and ts27 against methicillin-resistant Staphylococcus aureus (MRSA). Figure 2 In this context, A represents compound ts26. Figure 2 In this context, B represents compound ts27. Figure 2 In the figure, C represents the time-bactericidal test result of tiamulin against MRSA (ATCC 43300). Figure 2 In this context, D represents the development of resistance to MRSA after repeated treatment with compounds ts26, ts27, and tiamulin; MIC represents the minimum inhibitory concentration; and CFU represents colony-forming units.

[0050] To further verify the resistance barrier, a 21-day subinhibitory concentration induction experiment was conducted on MRSA. MRSA was cultured in 0.5 × MIC ts26, ts27 and tiamulin solutions, and the MIC was measured every 24 h, followed by 1:1000 subculture. The results showed that the tiamulin treatment group, starting from the initial MIC (0.5 μg / mL), increased to 32 μg / mL by day 19, showing a significant increase in resistance. In contrast, the resistance development in the ts26 and ts27 treatment groups was significantly slowed down. The MIC increase of ts26 was limited throughout the induction period, while ts27 showed the best resistance control effect, with its MIC remaining ≤4 μg / mL throughout the 21 days. The rate of resistance increase was approximately half that of tiamulin. Figure 2Based on the structure, it is inferred that the heterocyclic substituents introduced at C14 of ts26 and ts27 enhance their binding stability to the 50S ribosomal peptidyl transferase center, which can reduce the possibility of mutations at target sites (such as 23S rRNA or L3 protein) and weaken the induced response of bacterial efflux pumps, thereby effectively delaying the development of drug resistance. Figure 2 The bactericidal activity and resistance development of TS26, TS27 and tiamulin against MRSA were demonstrated, proving the rapid bactericidal effect and resistance barrier advantages of the present invention.

[0051] Example 4: Safety and Toxicity Assessment To evaluate the cellular safety and biocompatibility of compounds ts26 and ts27, cell viability was measured in HEK293T and HEPG2 cells using the CCK-8 assay. The results showed that both compounds exhibited dose-dependent cytotoxicity, but maintained high cell viability even at high doses. At a drug concentration of 250 μg / mL, ts26 showed cell viability of 81% and 72% in HEPG2 and HEK293T cells, respectively, while ts27 showed 89% and 75%, respectively, both significantly higher than the control drug tiamulin (HEPG2: 63%, HEK293T: 64%). Within the highest concentration range tested, ts27 exhibited the lowest toxicity, maintaining cell viability at 50–55%, superior to tiamulin (45%). Figure 3 A and B in the diagram. Further hemolysis experiments were used to evaluate the erythrocyte compatibility of ts26 and ts27. The results showed that, at 100 μg / mL, the hemolysis rates of both ts26 and ts27 were less than 15%, significantly lower than the 20% hemolysis rate of tiamulin, and also significantly lower than the absorbance values ​​(OD) of the negative control group. 450 The results were similar, with no obvious red blood cell membrane destruction. In contrast, the positive control Triton X-100 caused 100% hemolysis, demonstrating the reliability of the hemolysis test system. Figure 3 C in the middle. Figure 3 Concentration-dependent cytotoxicity and erythrocyte compatibility of compounds ts26, ts27, and tiamulin in human cell lines were analyzed. Figure 3 In the table, A and B represent the cytotoxicity assessment results of ts26, ts27, and tiamulin in HEK293T and HEPG2 cells. Figure 3 In the figure, C represents the hemolysis rate of ts26 and ts27 in rabbit erythrocytes. Figure 3 The toxicity and erythrocyte compatibility of the derivative in HEK293T and HEPG2 cells were demonstrated, confirming its safety.

[0052] Based on the minimum inhibitory concentration (MIC) of MRSA (0.0313 μg / mL), the effective antibacterial concentrations of ts26 and ts27 are 1000–3000 times lower than their cytotoxicity and hemolysis thresholds (≥100 μg / mL), indicating that both have a broad safety and therapeutic window. Conversely, tiamulin has a narrower therapeutic index (MIC = 0.5 μg / mL, corresponding to a cytotoxic threshold of approximately 50 μg / mL). These results suggest that ts26 and ts27 possess superior safety profiles and can be used for further in vivo validation.

[0053] Example 5: Evaluation of in vivo therapeutic effect To verify the anti-infective activity and therapeutic potential of compound ts27 in animals, a mouse systemic infection model based on methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300) was established for in vivo efficacy testing. Given that previous in vitro studies showed ts27 to be superior to ts26 in inhibiting resistance development and cytotoxicity, ts27 was selected as the representative compound for further in vivo validation in this study. In the experiment, immunosuppressed mice were intraperitoneally injected with an MRSA suspension, and 1 hour after infection, different doses of ts27 (e.g., 10, 20, and 40 mg / kg) were administered intraperitoneally. Survival rate and clinical manifestations were continuously observed for 7 days. The results showed that ts27 exhibited good dose-dependent therapeutic effects in all dose groups, with the 40 mg / kg dose completely protecting mouse survival (100% survival rate at 7 days), significantly superior to the tiamulin control group at the same dose. Further calculation of efficacy indicators revealed that the median effective dose (ED) of ts27 was... 50 The concentration was 7.1 mg / kg, approximately 1.5 times lower than tiamulin's 10.8 mg / kg, indicating higher therapeutic efficacy in vivo. (See [link to relevant documentation]). Figure 4 A and B in the example. Figure 4 The dose-dependent therapeutic effects of ts27 and tiamulin in a mouse model of systemic infection with methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300) were investigated. Figure 4 In this context, A represents the survival rate of mice in the ts27 treatment group. Figure 4 In the figure, B represents the survival rate of mice in the tiamulin-treated group. Figure 4 C in the figure represents the bacterial load in the mouse lungs.

[0054] In addition, lung tissue from infected mice was used to assess bacterial load and evaluate the in vivo bacterial clearance capacity of ts27. The results showed that in a mouse model of MRSA infection, ts27 exhibited a dose-dependent bacterial clearance effect in lung tissue: the 40 mg / kg dose reduced the MRSA load in mouse lung tissue by 3.5 log [missing value]. 10 CFU / g, the 20 mg / kg dose group showed a reduction of 2.8 log.10 CFU / g; Statistical analysis showed that the bacterial clearance effects of both dosage groups were significantly better than those of the same dose tiamulin group (reduced by 2.2 log...). 10 CFU / g and 1.5 log 10 (CFU / g), indicating that ts27 has superior therapeutic efficacy against pulmonary MRSA infection in vivo, see [link to article]. Figure 4 C. Throughout the experiment, no significant toxicity-related behavioral abnormalities were observed in the ts27-treated mice, indicating that ts27 has good tolerability and safety within the effective dose range. Figure 4 In vivo experimental data show that ts27 improves mouse survival and bacterial clearance rates.

[0055] Example 6: Mechanism Study To elucidate the mechanisms of action of compounds ts26 and ts27, molecular docking techniques were first employed to analyze their binding modes with the bacterial ribosomal peptidyl transferase center (PTC). This embodiment utilizes the publicly available 50S ribosome crystal structure and employs Smina software for binding site prediction and energy calculation. The results show that both ts26 and ts27 can stably bind to the same PTC region as traditional truncated pleurotin antibiotics, with ts27 exhibiting higher binding energy stability. Specifically, ts26 and ts27 can form hydrogen bonds with key residues G2088 and G2532 of 23S rRNA, respectively, and their C14 side chain is embedded in a hydrophobic pocket composed of A2089, C2090, U2612, and C2613, thereby enhancing the tightness of binding to the ribosome. Furthermore, the side chain of ts27 extends into the deep subpocket near A2451, generating additional van der Waals interactions with U2506 and C2452, resulting in significantly better binding stability than ts26. Docking results indicate that both compounds sterically hinder substrate entry at their binding sites, inhibiting peptide chain elongation, which structurally explains their strong antibacterial activity. (See [link to documentation]). Figure 5 . Figure 5 Molecular docking analysis of ts26, ts27, and lefamoline (positive control) at the peptidyl transferase center (PTC). Figure 5 In the diagram, A and C represent the superposition docking conformations of lefamoline (positive control) with ts26 (green) and ts27 (blue) in the PTC, respectively. Figure 5 B and D in the figure represent the optimal conformations of compounds ts26 (green) and ts27 (blue) obtained from the docking study. Figure 5 Molecular docking was used to elucidate the binding mode of the derivatives and explain their efficient mechanism.

[0056] To further verify the above-mentioned mechanism of action, this embodiment used *Staphylococcus aureus* (ATCC 29213) expressing green fluorescent protein (GFP) as an indicator bacterium, and evaluated the inhibitory effect of ts27 on bacterial protein synthesis by detecting GFP expression levels. The results showed that ts27 significantly reduced GFP expression intensity. At a concentration of 0.5 μg / mL, residual fluorescence was still observed in the tiamulin-treated group, while almost no GFP signal was observed in the ts27-treated group; when the drug concentration increased to 1 μg / mL, both drugs almost completely inhibited GFP expression, but the inhibitory effect of ts27 was more significant. Furthermore, the ribosome inhibition effect was further verified using an in vitro reconstructed MRSA protein synthesis system (PURE system). Experimental results showed that both ts27 and tiamulin could inhibit in vitro translation reactions, but the half-maximal inhibitory concentration (IC50) of ts27 was significantly lower. 50 =0.492±0.018 μg / mL) was significantly lower than that of tiamulin (0.612±0.047 μg / mL), indicating that ts27 has a stronger inhibitory effect on ribosome function. See Figure 6 . Figure 6 For the study of the antibacterial mechanism of TS27 and tiamulin, Figure 6 In the figures, A to E represent the inhibitory effects of different concentrations of ts27 and tiamulin on the expression of green fluorescent protein in Staphylococcus aureus; Figure 6 F in the figure represents the in vitro translation inhibition experiment, showing the effect of ts27 and tiamulin on bacterial ribosomes, with the half-maximal inhibitory concentration (IC50) being [value missing]. 50 () is the mean ± standard deviation of at least three independent experiments. Figure 6 The study demonstrated that the derivative exerts its antibacterial effect by inhibiting ribosomal translation activity.

[0057] As can be seen from the above examples, the C14-modified truncated pleurotin derivatives (such as compounds ts26 and ts27 below) provided by the present invention exhibit excellent properties by introducing specific heterocycles or substituting phenylthio side chains at the C14 position: 1. Strong antibacterial activity: The MIC values ​​against standard MRSA, MRSE, and VISA strains are as low as 0.0313~0.125 μg / mL, and the activity is 16~64 times higher than that of tiamulin; 2. Breakthrough overcoming key drug resistance: Complete inactivation of tiamulin (MIC > 64 μg / mL) cfr Positive MRSA strains still maintain effective activity (MIC=16 μg / mL). 3. High resistance barrier: In the resistance induction experiment, its MIC value increases significantly slower than that of tiamulin, and it can maintain its effectiveness for a long time. 4. Excellent safety profile: Low cytotoxicity to mammalian cells at therapeutic concentrations, low hemolytic activity, and a wide therapeutic window; 5. Significant in vivo efficacy: In a mouse model of systemic MRSA infection, its efficacy (ED) was [missing information]. 50 It is significantly superior to tiamulin, and can effectively clear lung bacteria and improve survival rate; 6. Clear antibacterial mechanism: Molecular docking studies have shown that the derivatives of this invention can bind more stably to ribosome PTC by forming more hydrogen bonds and hydrophobic interactions, which provides a structural basis for its superior performance.

[0058] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A C14-modified truncated pleurotin derivative, characterized in that, It has the structure shown in Equation I or Equation II: Formula I, Formula II.

2. The method for preparing the C14-modified truncated pleurotin derivative according to claim 1, characterized in that, Includes the following steps: (1) The truncated pleurotin was reacted with p-toluenesulfonyl chloride in an organic solvent to obtain an intermediate with the structure shown in Formula 2; (2) React 4-trifluoromethylthiophenol or 1-(4,5-dihydrothiazo-2-yl)azacyclobutane-3-thiol with the intermediate of the structure shown in Formula 2 to obtain the C14 modified truncated pleurotin derivative. Equation 2.

3. The preparation method according to claim 2, characterized in that, In step (1), the raw materials for the reaction also include an organic base; the molar ratio of the truncated pleurotin to p-toluenesulfonyl chloride is 1:1.1~1.3; the molar ratio of the truncated pleurotin to the organic base is 1:1.8~2.

2.

4. The preparation method according to claim 2, characterized in that, In step (2), the molar ratio of the intermediate with the structure shown in Formula 2 to the 4-trifluoromethylthiophenol is 1:1.1~1.3; the molar ratio of the intermediate with the structure shown in Formula 2 to the 1-(4,5-dihydrothiazol-2-yl)azacyclobutane-3-thiol is 1:1.1~1.

3.

5. The preparation method according to claim 2 or 4, characterized in that, In step (2), the raw materials for the reaction also include an organic base, and the molar ratio of the intermediate with the structure shown in Formula 2 to the organic base is 1:1.4~1.

6.

6. The preparation method according to claim 2, characterized in that, In step (2), after the reaction is completed, a reaction solution is obtained; the reaction solution is further separated into an organic phase by washing and extracting with water; the obtained organic phase is dried and purified by column chromatography to obtain the pure product of the C14 modified truncated pleurotin derivative. The eluent used in the column chromatography purification is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 3~5:

1.

7. The use of the C14-modified truncated pleurotin derivative of claim 1 or a pharmaceutically acceptable salt thereof, or the C14-modified truncated pleurotin derivative prepared by any one of claims 2 to 6, in the preparation of medicaments for treating and / or preventing bacterial infections.

8. The application according to claim 7, characterized in that, The bacteria include Gram-positive bacteria and / or Gram-positive drug-resistant bacteria, including methicillin-resistant Staphylococcus aureus. cfr - One or more of the following: methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis, and vancomycin-intermediate Staphylococcus aureus.

9. A drug, characterized in that, The product comprises the C14-modified truncated pleurotin derivative of claim 1 or a pharmaceutically acceptable salt thereof, or the C14-modified truncated pleurotin derivative prepared by the preparation method of any one of claims 2 to 6.

10. The medicament according to claim 9, characterized in that, The dosage form of the drug includes injections, tablets, or ointments; the drug is used for the treatment of respiratory or skin infections in veterinary or human medicine.

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